Thierry Chardès

Nicolas Chapal

Damien Bresson

Cédric Bès

Véronique Giudicelli

Marie-Paule Lefranc

Sylvie Peraldi-Roux

Sylvie Péraldi-Roux

M.-P Lefranc

Marie-Paule Lefranc

The human anti-thyroid peroxidase autoantibody repertoire in

Keywords: Thyroid peroxidase, Autoantibody, Phage display, Variable gene, IMGT database

Introduction

The anti-thyroid peroxidase (TPO) autoantibodies (aAb) are the most frequently represented aAb in the sera of patients suffering from autoimmune thyroid disease (AITD); they are present in 90% of Hashimoto's thyroiditis and 75% of Graves' disease patients [START_REF] Mariotti | Antithyroid peroxidase autoantibodies in thyroid diseases[END_REF]. In vitro cytotoxic effector functions mediated by TPO-specific aAb, such as C3 complement activation [START_REF] Chiovato | Antibodies producing complement-mediated thyroid cytotoxicity in patients with atrophic or goitrous autoimmune thyroiditis[END_REF][START_REF] Parkes | The role of complement in the pathogenesis of postpartum thyroiditis[END_REF][START_REF] Wadeleux | Cytotoxic assay of circulating thyroid peroxidase antibodies[END_REF]) and antibody-dependent cell cytotoxicity [START_REF] Bogner | Thyroid cytotoxic antibodies in atrophic and goitrous autoimmune thyroiditis[END_REF][START_REF] Guo | Recombinant thyroid peroxidase-specific Fab converted to immunoglobulin G (IgG) molecules: evidence for thyroid cell damage by IgG1, but not IgG4, autoantibodies[END_REF][START_REF] Metcalfe | Analysis of antibody-dependent cell-mediated cytotoxicity in autoimmune thyroid disease[END_REF][START_REF] Rodien | Antibody-dependent cell-mediated cytotoxicity in autoimmune thyroid disease: relationship to antithyroperoxidase antibodies[END_REF][START_REF] Weetman | Affinity purification of IgG subclasses and the distribution of thyroid auto-antibody reactivity in Hashimoto's thyroiditis[END_REF], trigger thyroid cell destruction. Moreover, it has been suggested that thyroidinfiltrating B lymphocytes as antigen-presenting cells through membrane-bound anti-TPO antibodies modulate antigen processing [START_REF] Guo | Autoantibody-mediated capture and presentation of autoantigen to T cells via the Fc epsilon receptor by a recombinant human autoantibody Fab converted to IgE[END_REF][START_REF] Mclachlan | The molecular biology of thyroid peroxidase: cloning, expression and role as autoantigen in autoimmune thyroid disease[END_REF][START_REF] Rapoport | Combinatorial libraries: new insights into human organ-specific autoantibodies[END_REF].

Only one human anti-TPO antibody was obtained by cell immortalization [START_REF] Horimoto | Production and characterisation of a human monoclonal thyroid peroxidase autoantibody[END_REF]). However, McLachlan and Rapoport's group pioneered the application of combinatorial libraries to the study of aAb in thyroid diseases [START_REF] Portolano | A human Fab fragment specific for thyroid peroxidase generated by cloning thyroid lymphocytederived immunoglobulin genes in a bacteriophage lambda library[END_REF], and a large number of human anti-TPO aAb have since been isolated by this group and others [START_REF] Chazenbalk | Human organ-specific autoimmune disease. Molecular cloning and expression of an autoantibody gene repertoire for a major autoantigen reveals an antigenic immunodominant region and restricted immunoglobulin gene usage in the target organ[END_REF][START_REF] Hexham | Cloning and characterisation of TPO autoantibodies using combinatorial phage display libraries[END_REF]Jaume et al. 1994a, b;[START_REF] Jaume | The epitopic fingerprint of thyroid peroxidase-specific Fab isolated from a patient's thyroid gland by the combinatorial library approach resembles that of autoantibodies in the donor's serum[END_REF][START_REF] Mcintosh | Analysis of immunoglobulin G kappa antithyroid peroxidase antibodies from different tissues in Hashimoto's thyroiditis[END_REF][START_REF] Portolano | Recognition by recombinant autoimmune thyroid disease-derived Fab fragments of a dominant conformational epitope on human thyroid peroxidase[END_REF]Portolano et al. , 1993a, b;, b;1995;Prummel et al. 1994a, b). In the last 2 years, about 100 anti-TPO aAb directed against immunodominant or non-immunodominant epitopes have been described [START_REF] Chapal | Human anti-thyroid peroxidase single-chain fragment variable of Ig isolated from a combinatorial library assembled in-cell: insights into the in vivo situation[END_REF][START_REF] Lefranc | IMGT, the international ImMunoGeneTics database[END_REF][START_REF] Guo | Rarity of autoantibodies to a major autoantigen, thyroid peroxidase, that interact with denatured antigen or with epitopes outside the immunodominant region[END_REF][START_REF] Pichurin | Human monoclonal autoantibodies to B-cell epitopes outside the thyroid peroxidase autoantibody immunodominant region[END_REF]. Given this enlarged TPO-specific repertoire, and particularly the numerous Ig gene sequences published to date, we compiled and analyzed the genes encoding these aAb using the international ImMunoGeneTics database (IMGT) (http://imgt.cines.fr), an integrated information system devoted to the study of immunoglobulins, T-cell receptors, and major histocompatibility molecules of several vertebrate species [START_REF] Giudicelli | IMGT, the international ImMunoGeneTics database[END_REF][START_REF] Lefranc | The immunoglobulin Factsbook[END_REF].

TPO-specific heavy chain gene usage in AITD

Ig variable domain sequences encoding TPO aAb have been obtained from Fab and single chain variable fragment (scFv) combinatorial libraries, mainly derived from thyroid-infiltrating B cells of Graves' disease patients [START_REF] Chapal | Human anti-thyroid peroxidase single-chain fragment variable of Ig isolated from a combinatorial library assembled in-cell: insights into the in vivo situation[END_REF][START_REF] Lefranc | IMGT, the international ImMunoGeneTics database[END_REF][START_REF] Chazenbalk | Human organ-specific autoimmune disease. Molecular cloning and expression of an autoantibody gene repertoire for a major autoantigen reveals an antigenic immunodominant region and restricted immunoglobulin gene usage in the target organ[END_REF]Jaume et al. 1994a[START_REF] Jaume | The epitopic fingerprint of thyroid peroxidase-specific Fab isolated from a patient's thyroid gland by the combinatorial library approach resembles that of autoantibodies in the donor's serum[END_REF][START_REF] Portolano | Recognition by recombinant autoimmune thyroid disease-derived Fab fragments of a dominant conformational epitope on human thyroid peroxidase[END_REF]Portolano et al. , 1993a[START_REF] Portolano | Molecular cloning and characterization of human thyroid peroxidase autoantibodies of lambda light chain type[END_REF]Prummel et al. 1994a, b). Only two libraries constructed from thyroid-infiltrating B cells or lymph node B lymphocytes of Hashimoto's patients have been described [START_REF] Hexham | Cloning and characterisation of TPO autoantibodies using combinatorial phage display libraries[END_REF][START_REF] Mcintosh | Analysis of immunoglobulin G kappa antithyroid peroxidase antibodies from different tissues in Hashimoto's thyroiditis[END_REF]. Although we cannot formally exclude that differences observed in IGV gene usage of TPO-specific aAb obtained from the libraries cited in Table 1 (consisting of parts a, b and c) are due to preferential primer amplification of certain IGV genes or gene families, we consider that the data reflect the reality in vivo since the analyses were carried out on more than 180 human anti-TPO aAb obtained from four laboratories that used different primers. Analysis of the heavy chain variable domains of the anti-TPO aAb shows a restriction in the IGHV gene usage in both Graves' and Hashimoto's AITD (Table 1, consisting of parts a, b and c) [START_REF] Mcintosh | Somatic hypermutation in autoimmune thyroid disease[END_REF][START_REF] Mclachlan | Autoimmune response to the thyroid in humans: thyroid peroxidase -the common autoantigenic denominator[END_REF]. The heavy chains of the anti-TPO aAb are mainly encoded by genes of the IGHV1 (75.4%) and IGHV3 (21.2%) subgroups, with a large predominance of the IGHV1-3 gene in thyroid diseases.

Interestingly, IGHV gene analysis of anti-TPO aAb from patients with Graves' disease or with Hashimoto's hypothyroiditis clearly indicates a discrimination in IGHV subgroup usage (Table 2). In Graves' disease, the anti-TPO aAb mainly use IGHV1 subgroup genes (88.9%), with overrepresentation of IGHV1-3 (50.4%) and IGHV1-2 (25.5%). In Hashimoto's thyroiditis, the IGHV3 subgroup (71%) is dominant among the anti-TPO aAb, with a large predominance of IGHV3-21 (47.4%) and IGHV3-23 (18.4%) (Table 1 (consisting of parts a, b and c) and 2). Preferential use of IGHV4, IGHV5, and IGHV6 genes by aAb in autoimmune diseases was suggested by several studies [START_REF] Dijk-Hard I Van, Feld | Increased utilization of the VH6 gene family in patients with autoimmune idiopathic thrombocytopenic purpura[END_REF][START_REF] Melero | The frequent expansion of a subpopulation of B cells that express RF-associated cross-reactive idiotypes: evidence from analysis of a panel autoreactive monoclonal antibodies[END_REF]Pascual and Capra 1992;Pascual et al. 1992a, b, c;[START_REF] Roben | Repertoire cloning of lupus anti-DNA autoantibodies[END_REF]. On the other hand, underexpression of the IGHV1 subgroup in aAb is a very common feature in autoimmune diseases, as demonstrated for numerous autoantigens [START_REF] Bona | Expression and pairing of VH and VL families in murine and human self-reactive antibodies[END_REF]. The overexpression of the IGHV3 subgroup in Hashimoto's thyroiditis and that of the IGHV1 subgroup in Graves' disease seems to be a characteristic of the anti-TPO aAb repertoire, and suggests that there is a skewing of IGHV gene usage in TPO-specific aAb in the sera of patients suffering from autoimmune thyroid diseases.

With regard to the organization of the human IGH locus (Fig. 1), TPO-specific aAb from patients with Graves' disease and from Hashimoto's hypothyroiditis preferentially use D proximal IGHV1 genes and D distal IGHV3 genes, respectively. Two different hypotheses can explain the preferential expression and/or selection of a particular IGHV gene: (1) selection derived from preferential rearrangement due to the gene position in the IGH locus and/or accessibility to the recombinase machinery and (2) functional selection based on the recognition of defined epitopes on the TPO molecule [START_REF] Sasso | Human IgM molecules that bind staphylococcal protein A contain VHIII H chains[END_REF]). The preferential use of the D proximal IGHV5 subgroup gene previously designated 7183 is well documented in mice [START_REF] Bona | Expression and pairing of VH and VL families in murine and human self-reactive antibodies[END_REF]), but the fact that genes from IGHV subgroups are scattered throughout the IGH locus (Fig. 1) does not support the "position" hypothesis. On the other hand, the fact that non-IDR (immunodominant region) TPO-specific aAb show a restricted IGHV1-69 gene usage [START_REF] Pichurin | Human monoclonal autoantibodies to B-cell epitopes outside the thyroid peroxidase autoantibody immunodominant region[END_REF] argues in favor of the second hypothesis.

The D genes used by these aAb show a high diversity with a large number of genes in an inverted orientation of transcription (38%) (Table 1, consisting of parts a, b and c). Inverted D genes are rarely used by aAb, and this event seems to be a peculiarity of anti-TPO aAb. This observation suggests the possible involvement of particular mechanisms such as the use of D genes with irregular spacers (DIR elements) [START_REF] Tuaillon | Use of D gene segments with irregular spacers in terminal deoxynucleotidyltransferase (TdT)+/+ and TdT-/-mice carrying a human Ig heavy chain transgenic minilocus[END_REF], preferential V-D rearrangements (Tuaillon and Capra 2000b), or modulation of terminal deoxynucleotidyltransferase activity (Tuaillon and Capra 2000a) to generate heavy chain diversity in the TPO repertoire. Analysis of D gene usage suggests that there is no apparent restriction in D gene use, whereas IGHJ4 (61.6%) and IGHJ6 (29.9%) are preferentially rearranged among the TPO-directed aAb (Tables 1 (consisting of parts a, b and c), 2) in Graves' disease.

TPO-specific light chain gene usage in AITD

J distal IGKV1 and IGLV1 genes (Fig. 1) are preferentially rearranged in TPO-specific recombinant aAb (Tables 1 (consisting of parts a, b and c) and 2). Within 142 Fig. 1 Germline gene usage of human anti-thyroid peroxidase (TPO) antibodies in relation to their position on the immunoglobulin heavy (IGH), kappa (IGK), and lambda (IGL) variable gene loci. Percentage of anti-TPO clones derived from the corresponding germline gene of patients with Graves' disease (solid bars), and Hashimoto's thyroiditis (open bars). Genes IGKVI-12 and IGKV1-39 could not be differentiated from their duplicated genes IGKV1D-12 and IGK1D-39, respectively. The loci representations were recovered and simplified from the IMGT database and the legend may be found at http://imgt.cines.fr v Table 1a Human anti-thyroid peroxidase (TPO) antibody fragments isolated from combinational libraries. Antibodies showing in-cell H/L associations are boxed 2000). Concerning the TPO-specific IGL repertoire, few anti-TPO recombinant Fab expressing a λ light chain have been characterized and sequenced. This is probably due to the fact that only a few libraries have been constructed using λ-specific amplification primers [START_REF] Jaume | The epitopic fingerprint of thyroid peroxidase-specific Fab isolated from a patient's thyroid gland by the combinatorial library approach resembles that of autoantibodies in the donor's serum[END_REF][START_REF] Mcintosh | Analysis of immunoglobulin G kappa antithyroid peroxidase antibodies from different tissues in Hashimoto's thyroiditis[END_REF]Prummel et al. 1994b). The decision by other authors to use only κ-specific amplification primers for library construction was based on the fact that κ-chain TPO aAb predominated in the sera of the thyroid disease patients from whom the library originated [START_REF] Chazenbalk | Human organ-specific autoimmune disease. Molecular cloning and expression of an autoantibody gene repertoire for a major autoantigen reveals an antigenic immunodominant region and restricted immunoglobulin gene usage in the target organ[END_REF][START_REF] Guo | Rarity of autoantibodies to a major autoantigen, thyroid peroxidase, that interact with denatured antigen or with epitopes outside the immunodominant region[END_REF][START_REF] Hexham | Cloning and characterisation of TPO autoantibodies using combinatorial phage display libraries[END_REF][START_REF] Pichurin | Human monoclonal autoantibodies to B-cell epitopes outside the thyroid peroxidase autoantibody immunodominant region[END_REF][START_REF] Portolano | A human Fab fragment specific for thyroid peroxidase generated by cloning thyroid lymphocytederived immunoglobulin genes in a bacteriophage lambda library[END_REF][START_REF] Portolano | Recognition by recombinant autoimmune thyroid disease-derived Fab fragments of a dominant conformational epitope on human thyroid peroxidase[END_REF]Portolano et al. , 1993a, b), b). Using a mixture of κand λspecific primers, we recently obtained numerous λ anti-TPO scFv by an in-cell library and random combinatorial libraries (Table 1, consisting of parts a, b and c) [START_REF] Chapal | Human anti-thyroid peroxidase single-chain fragment variable of Ig isolated from a combinatorial library assembled in-cell: insights into the in vivo situation[END_REF][START_REF] Lefranc | IMGT, the international ImMunoGeneTics database[END_REF]. Analysis of this enlarged λ-derived TPO repertoire revealed a dominant use of the IGLV1 subgroup in thyroid diseases, with two genes mainly found, IGLV1-51 (47.4%) and IGLV1-40 (26.3%) (Tables 1 (consisting of parts a, b and c), 2). Autoantibodies with λ light chains have been described in various autoimmune diseases [START_REF] Cairns | Analysis of variable region genes encoding a human anti-DNA antibody of normal origin. Implications for the molecular basis of human autoimmune responses[END_REF]Prummel et al. 1994a, b;[START_REF] Ravirajan | Genetic, structural and functional properties of an IgG DNA-binding monoclonal antibody from a lupus patient with nephritis[END_REF][START_REF] Serrano | Nucleotide sequences of variable regions of an human anti-acetylcholine receptor autoantibody derived from a myasthenic patient[END_REF][START_REF] Song | Molecular and genetic characterization of two anti-DNA antibodies derived from patients with systemic lupus erythematosus[END_REF]; in particular, λ anti-TSHr aAb are involved in thyroid stimulation in patients with Graves' disease [START_REF] Knight | Thyroidstimulating autoantibodies usually contain only lambda-light chains: evidence for the "forbidden clone" theory[END_REF][START_REF] Williams Rc | Kappa/lambda immunoglobulin distribution in Graves' thyroid-stimulating antibodies. Simultaneous analysis of C lambda gene polymorphisms[END_REF][START_REF] Zakarija | Thyroid-stimulating antibody (TSAb) of Graves' disease[END_REF]. Moreover, five IGLV1-40-and one IGLV1-51-derived anti-Tg aAb have been isolated from a combinatorial library constructed from a patient with Hashimoto's thyroiditis [START_REF] Mcintosh | Cloning and analysis of Igκ and Igλ anti-thyroglobulin autoantibodies from a patient with Hashimoto's thyroiditis: evidence for in vivo antigen-driven repertoire selection[END_REF][START_REF] Mcintosh | Somatic hypermutation in autoimmune thyroid disease[END_REF].

H/L pairing of TPO aAb

Chain pairing in a TPO-selected random library can contain in vivo H/L combinations as suggested by "roulette" studies [START_REF] Costante | Recombinant thyroid peroxidase-specific autoantibodies. II. Role of individual heavy and light chains in determining epitope recognition[END_REF]Portolano et al. 1993a). This was demonstrated by comparison of H/L combinations obtained from an in-cell library with those obtained from various random libraries [START_REF] Chapal | Thyroid peroxidase autoantibodies obtained from random single chain Fv libraries contain the same heavy/light chain combinations as occur in vivo[END_REF]). However, only TPO-directed aAb from an in-cell combinatorial library [START_REF] Chapal | Human anti-thyroid peroxidase single-chain fragment variable of Ig isolated from a combinatorial library assembled in-cell: insights into the in vivo situation[END_REF] and clone 2G4 obtained from cell fusion [START_REF] Horimoto | Production and characterisation of a human monoclonal thyroid peroxidase autoantibody[END_REF]) formally reflect the in vivo situation (Table 1, consisting of parts a, b and c). Although a previous study described the lack of promiscuity between TPO-specific heavy and light chains (Portolano et al. 1993a), an extensive analysis of H/L rearrangements of anti-TPO aAb does not show apparent restriction in H/L pairing (Table 1, consisting of parts a, b and c). Indeed, the heavy chains encoded by the dominant IGHV1-3 gene are associated with light chains encoded by 11 of 18 different IGKV or IGLV genes (Table 1, consisting of parts a, b and c). Reciprocally, the most frequently used light chain genes, i.e., IGKV1-39, IGLV1-40, and IGLV1-51, are combined with around 50% of the IGHV genes used by TPO aAb. Overrepresentation of IGHV1-3/IGKV1-39, IGHV1-3/IGHLV1-51, and IGHV1-3/IGLV1-40 pairings probably reflects the predominance of the expressed IGHV, IGKV, and IGLV genes in the TPO antibody repertoire. The clones resulting from an in-cell library and from cell fusion show the IGHV1-3/IGLV1-51, IGHV1-69/IGLV1-40, and IGHV3-53/IGKV3-20 associations found respectively in 14, 1, and none of the anti-TPO aAb obtained from random combinatorial libraries (Table 1, consisting of parts a, b and c). These observations indicate the need to enlarge the number of in vivo clones to definitively conclude that there is a restricted H/L pairing in TPOspecific aAb, even though it is possible to obtain at least part of the in vivo anti-TPO repertoire with combinatorial libraries.

Amino acid multi-sequence alignment of TPO-specific aAb

Whereas numerous somatic hypermutations are observed in TPO-specific heavy chains whatever the library origin (Table 3, consisting of parts a, b and c)), there is no or only limited amino acid replacement in most TPO-specific light chains, particularly those encoded by the J proximal IGLV2-14, IGKV1-9, IGKV3-11, IGKV3-15, IGKV3-20, and IGKV4-1 genes (Tables 1 (consisting of parts a and b), 5). The pattern of mutations in IGHV genes from anti-TPO aAb is typical of an antigen-driven selection during AITD. On the other hand, preferential usage of J proximal IGLV or IGKV genes for some TPO aAb, with little or no residue mutations, strongly suggests a defect in receptor editing of the light chain during aAb generation in AITD, as demonstrated for lupus-associated anti-DNA aAb [START_REF] Bensimon | Human lupus anti-DNA autoantibodies undergo essentially primary V kappa gene rearrangements[END_REF][START_REF] Chen | Editing disease-associated autoantibodies[END_REF]. In this case, certain TPO-specific B cells might have been blocked in their capacity to turn off their autoreactivity by light chain replacement, leading to the acquisition of a new specificity.

As previously suggested by others [START_REF] Mcintosh | Analysis of immunoglobulin G kappa antithyroid peroxidase antibodies from different tissues in Hashimoto's thyroiditis[END_REF]Portolano et al. 1993b[START_REF] Portolano | Molecular cloning and characterization of human thyroid peroxidase autoantibodies of lambda light chain type[END_REF] and confirmed by our recent publications [START_REF] Chapal | Human anti-thyroid peroxidase single-chain fragment variable of Ig isolated from a combinatorial library assembled in-cell: insights into the in vivo situation[END_REF][START_REF] Lefranc | IMGT, the international ImMunoGeneTics database[END_REF], extensive analysis of somatic hypermutations among IGHV1-3, IGHV1-2, and IGKV1-39 dominant-derived aAb indicate that certain residue replacements (e.g., Ile39 and Thr95 for IGHV1 genes) are systematically found in the majority of TPO-specific aAb independently of the library, but other amino acid mutations are mostly library or patient specific (Tables 3 (consisting of parts a, b and c), 4 (consisting of parts a and b), and 5). These observations support the hypothesis that the hypermutation process could be the hallmark of the TPO aAb repertoire.

Table 3a

Amino acid sequences of human anti-TPO antibody IGHV chains aligned with the closest putative germline genes. Designation of the complementarity determining regions (CDR) and framework regions (FR) are according to IMGT [START_REF] Lefranc | The immunoglobulin Factsbook[END_REF][START_REF] Lefranc | IMGT, the international ImMunoGeneTics database[END_REF]. Only substituted amino acids are shown. Antibody sequences were obtained from databases except antibodies WR1.223, KMI, WR1.102, WR1.107, and WR1.112. Boxed amino acids at the N-terminus correspond to possible primer-derived sequences

Table 3b

Table 4a Amino acid sequences of human anti-TPO antibody IGKV chains aligned with the closest putative germline genes. Designation of the complementarity determining regions (CDR) and framework regions (FR) are according to IMGT [START_REF] Lefranc | The immunoglobulin Factsbook[END_REF][START_REF] Lefranc | IMGT, the international ImMunoGeneTics database[END_REF]. Only substituted amino acids are shown. Antibody sequences were obtained from databases except antibodies WR1.223 and KM1. Boxed amino acids at the N-terminus correspond to possible primer-derived sequences

Table 4b

Table 5 Amino acid sequences of human anti-TPO antibody IGLV chains aligned with the closest putative germline genes. Designation of the complementarity determining regions (CDR) and framework regions (FR) are according to IMGT [START_REF] Lefranc | The immunoglobulin Factsbook[END_REF][START_REF] Lefranc | IMGT, the international ImMunoGeneTics database[END_REF]. Only substituted amino acids are shown. Antibody sequences were obtained from databases except antibodies WR1.102, WR1.107, and WRI.112.

Boxed amino acids at the N-terminus correspond to possible primer-derived sequences

Correlation between Ig gene usage and TPO-specific antibody epitopes

Pairing of one defined heavy chain with different light chains does not alter antigen binding (Burton andBarbas 1992, 1994). This observation strongly suggests that the heavy chain initiates the formation of the antigen/antibody complex and thereby provides the specificity of the interaction, whereas its light chain counterpart stabilizes the interaction with subsequent affinity modulation [START_REF] Noel | Analysis of the individual contributions of immunoglobulin heavy and light chains to the binding of antigen using cell transfection and plasmon resonance analysis[END_REF]. Such an effect of the anti-TPO aAb light chain on affinity is less conclusive, since neither IGKV nor IGLV gene usage of anti-TPO aAb has been shown to modulate antigen affinity [START_REF] Chapal | Human anti-thyroid peroxidase single-chain fragment variable of Ig isolated from a combinatorial library assembled in-cell: insights into the in vivo situation[END_REF][START_REF] Chapal | Thyroid peroxidase autoantibodies obtained from random single chain Fv libraries contain the same heavy/light chain combinations as occur in vivo[END_REF][START_REF] Mcintosh | Analysis of immunoglobulin G kappa antithyroid peroxidase antibodies from different tissues in Hashimoto's thyroiditis[END_REF][START_REF] Portolano | A human Fab fragment specific for thyroid peroxidase generated by cloning thyroid lymphocytederived immunoglobulin genes in a bacteriophage lambda library[END_REF][START_REF] Portolano | Recognition by recombinant autoimmune thyroid disease-derived Fab fragments of a dominant conformational epitope on human thyroid peroxidase[END_REF]Portolano et al. , 1993b)). On the other hand, several groups have pointed out that domain A of the TPO immunodominant region (IDR/A) is preferentially recognized by TPO-specific aAb with the IGKV1-39 light chain, whereas TPOspecific aAb showing other IGKV light chains map in domain B of the IDR (IDR/B) (Table 1, consisting of parts a, b and c) [START_REF] Chazenbalk | Human organ-specific autoimmune disease. Molecular cloning and expression of an autoantibody gene repertoire for a major autoantigen reveals an antigenic immunodominant region and restricted immunoglobulin gene usage in the target organ[END_REF][START_REF] Costante | Recombinant thyroid peroxidase-specific autoantibodies. II. Role of individual heavy and light chains in determining epitope recognition[END_REF][START_REF] Guo | Relationship between autoantibody epitopic recognition and immunoglobulin gene usage[END_REF][START_REF] Jaume | Influence of the light chain repertoire on immunoglobulin genes encoding thyroid autoantibody Fab from combinatorial libraries[END_REF][START_REF] Jaume | The epitopic fingerprint of thyroid peroxidase-specific Fab isolated from a patient's thyroid gland by the combinatorial library approach resembles that of autoantibodies in the donor's serum[END_REF][START_REF] Mcintosh | Analysis of immunoglobulin G kappa antithyroid peroxidase antibodies from different tissues in Hashimoto's thyroiditis[END_REF][START_REF] Portolano | Molecular cloning and characterization of human thyroid peroxidase autoantibodies of lambda light chain type[END_REF]. This IDR/B has been at least partially identified even though the location of the IDR on the TPO molecule is still under debate. Region 713-721 is located on the C-terminal myeloperoxidase-like domain of the TPO molecule; this region, recognized by murine Mab 47/C21 antibody [START_REF] Finke | Determination at the molecular level of a B-cell epitope on thyroid peroxidase likely to be associated with autoimmune thyroid disease[END_REF][START_REF] Libert | Thyroperoxidase, but not the thyrotropin receptor, contains sequential epitopes recognized by autoantibodies in recombinant peptides expressed in the pUEX vector[END_REF] and by serum polyclonal TPO aAb [START_REF] Libert | Thyroperoxidase, but not the thyrotropin receptor, contains sequential epitopes recognized by autoantibodies in recombinant peptides expressed in the pUEX vector[END_REF][START_REF] Ruf | Relationship between immunological structure and biochemical properties of human thyroid peroxidase[END_REF], was initially thought to be outside the IDR [START_REF] Chazenbalk | Human organ-specific autoimmune disease. Molecular cloning and expression of an autoantibody gene repertoire for a major autoantigen reveals an antigenic immunodominant region and restricted immunoglobulin gene usage in the target organ[END_REF]. Furthermore, mutations in the 713-721 region do not affect the recognition of aAb directed against IDR [START_REF] Nishikawa | The quest for the autoantibody immunodominant region on thyroid peroxidase: guided mutagenesis based on a hypothetical threedimensional model[END_REF]. On the other hand, high concentrations of IDR/B-specific aAb TR1.9 inhibited the binding of Mab47/C21 to TPO [START_REF] Guo | Relationship between autoantibody epitopic recognition and immunoglobulin gene usage[END_REF] and mapped an epitope comprising amino acid residue K713 (Guo et al. 2001), suggesting that region 713-721 is located on the fringe of an IDR. The crystal structure of the Fab TR1.9 has been solved [START_REF] Chacko | Structural studies of human autoantibodies[END_REF], but in the absence of the three-dimensional structure for the complex of TR1.9 with TPO, it is difficult to determine the structural details of the binding. The role IGLV genes play in affecting anti-TPO specificity remains to be elucidated. The initially described λ-derived anti-TPO aAb had low affinity and were directed against TPO-IDR/B [START_REF] Portolano | Molecular cloning and characterization of human thyroid peroxidase autoantibodies of lambda light chain type[END_REF]Prummel et al. 1994b). In contrast, some of our λ-derived aAb demonstrated high affinity to TPO and inhibited the binding of a majority of the serum aAb to TPO [START_REF] Bresson | Pertinence of kappa and lambda recombinant antibodies directed against thyroid peroxidase in thyroid autoimmune disease[END_REF][START_REF] Chapal | Thyroid peroxidase autoantibodies obtained from random single chain Fv libraries contain the same heavy/light chain combinations as occur in vivo[END_REF], suggesting that these aAb recognized the IDR (defined by epitope mapping using BIACORE as regions II, VI, and VIII) (Table 1, consisting of parts a, b and c). Future studies involving λ-derived aAb such as T13/VI, B4/VIII, or ICA5/II and Fab defining IDR/A and /B (WR1-7, SP1-4, TR1-8, and TR1-9) could shed new light on the epitope specificity and gene usage of these aAb that recognize IDR.

Recently, [START_REF] Pichurin | Human monoclonal autoantibodies to B-cell epitopes outside the thyroid peroxidase autoantibody immunodominant region[END_REF] produced and characterized human recombinant aAb by phage display technology binding outside the TPO-IDR (defined as non-IDR). All these heavy chains are encoded by IGHV1-69, with an extremely long CDR3, and paired with different types of light chains, suggesting that non-IDR specificity is determined primarily by a common heavy chain. Interestingly, almost all IDR-specific aAb obtained in the same experiment use IGHV1-2 and IGHV1-3, as is also the case for a majority of the IDR aAb previously described (Table 1, consisting of parts a, b and c). Does IGHV1-2 or IGHV1-3 gene usage reflect a particular TPO-IDR specificity of recombinant aAb? Even though the methodologies used to define epitope recognition of anti-TPO recombinant aAb are different, these results reveal the difficulty of correlating gene usage with epitope recognition of TPO-specific aAb.

Conclusion

Several laboratories have produced and characterized numerous human anti-TPO aAb, leading to an enlarged autoantibody repertoire. Analysis of these antibodies using the IMGT database [START_REF] Giudicelli | IMGT, the international ImMunoGeneTics database[END_REF][START_REF] Lefranc | IMGT, the international ImMunoGeneTics database[END_REF][START_REF] Lefranc | The immunoglobulin Factsbook[END_REF][START_REF] Lefranc | IMGT, the international ImMunoGeneTics database[END_REF]) reveals several characteristics of the TPO-specific aAb repertoire: (1) a restriction in the IGV gene usage to generate anti-TPO aAb in AITD, (2) a VDJ recombination process using preferentially inverted D genes, (3) limited somatic mutations of J proximal light chain genes suggesting a defect in receptor editing in AITD, and (4) presence of certain somatic mutations systematically in the anti-TPO aAb repertoire. The annotations described in this paper and the protein display will soon be available as a new specialized IMGT page on human anti-TPO aAb genes. This page will evolve with time and integrate all the sequences devoted to autoantibodies that are published in the future.

Table 1b Table 2

 1b2 Germline genes used by the human TPO-specific autoantibody repertoire (ND not determined by IMGT/V-QUEST) N=38 for IGKV and for IGKL the IGKV1 subgroup, a strong restriction is observed: 72.8% of the κ anti-TPO aAb are encoded by genes derived from the IGKV1-39 (or IGKV1D-39) gene in Graves' disease (Tables 1 (consisting of parts a, b and c) and 2)[START_REF] Mcintosh | Somatic hypermutation in autoimmune thyroid disease[END_REF] McLachlan and Rapoport

			% b		34.2		26.3		36.8		2.6
			IGLJ gene n		IGLJ1 13		IGLJ2 10		IGLJ3 14		ND 1
			% b IGLV gene n % b		17.4 IGLV1-40 10 26.3	IGLV1-44 2 5.2	36.9 IGLV1-51 18 47.4		1 2.6 2.9 IGLV2-8	3 7.9 IGLV2-14	14.5	1 2.6 IGLV3-21	2 5.2 2.9 IGLV3-25	1 2.6 2.9 IGLV7-43	22.3			13.1		2.6		10.5		63.0	10.5
			% b IGKJ gene n		0.9 IGKJ1 18	2.9	1.9 IGKJ2 38	72.8	IGKJ3 3	2.7	IGKJ4 15	9.7	6.8 IGKJ5 3	1.9 ND 3	-d 23			42.1 IGKJ1 5	2.6	2.6 IGKJ2 1	44.7	IGKJ3 4	2.6	5.2 IGKJ4 24	IGKJ5 4
	Thyroid IG variable gene usage a	disease	IGHV gene n % b % b IGKV gene n IGHJ gene n	Graves' disease c	2 1.4 IGKV1-5 1 IGHV1-2 35 25.5 IGHJ1	IGKV1-12 3 IGHV1-3 69 50.4	7.5 5.4 IGKV1-13 2 IGHV1-8 2 1.4 IGHJ3	IGKV1-39 75 IGHV1-69 16 11.6	84.5 61.6 IGHJ4	IGKV2-28 3 IGHV3-23 2 1.4	29.9 41 IGHV3-30 2 1.4 IGHJ6	IGKV3-11 10 IGHV3-53 3 2.2	1.4 IGKV3-20 7 2 IGHV3-64 3 2.2 -d	IGKV4-1 2 IGHV4-30-4 1 0.7	IGHV5-51 2 1.4	-d 2 1.4	Hashimoto's disease	5.2 IGKV1-9 16 2 IGHV1-3 9 23.7 IGHJ4	IGKV1-12 1 IGHV1-8 1 2.6	1 47.4 IGKV1-27 18 IGHJ5	IGKV1-39 17 IGHV3-21 18 47.4	47.4 18 IGHV3-23 7 18.4 IGHJ6	1 IGKV3-15 IGHV3-48 1 2.6	2 IGKV3-20 IGHV3-53 1 2.6	IGHV4-31 1 2.6

a IGHD gene usage is not indicated since numerous anti-TPO antibody gene sequences present the same alignment score with different germline genes b %=n/N×100, where n=number of anti-TPO IGHV genes in the IGHV subgroup and N=total number of anti-TPO IGHV genes studied c N=37 for IGHV and for IGHJ; N=103 for IGKV and for IGKJ; N=38 for IGLV and for IGLJ d Nucleotide sequences not annotated by IMGT/V-QUEST e N=35 for IGHV and for IGHJ;

Acknowledgements We thank Dr. Sharon Lynn Salhi for critical comments and help in preparing the manuscript. We are indebted to Misses Nathalie Bosc, Géraldine Folch, Christèle Jean, and Dominique Scairiner for the annotation of the anti-TPO sequences in IMGT/LIGM-DB.

Table 1c

Table 3c


Caroline Angleraux

Matteo Mossio
email: matteo.mossio@univ-paris1.fr

Special Issue, "Single-cell analysis: Epistemological inquiries"

des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

5

This special issue gathers four articles that aim at making explicit and discussing some epistemological issues arising from single-cell analysis.

What is single-cell analysis? The label does not refer to the general practice of observing and describing individual cells, which is something that biologists have been doing for a long time. Single-cell analysis consists more specifically in a set of recent -omics sequencing techniques that enable scientists to study the different so-called "-omes" of single cells, and thereby to describe their "molecular profile". In doing so, the aim is to have a better and a more comprehensive view about how each individual cell works within a biotic or abiotic environment.

Single-cell analysis involves different techniques for isolating cells. Depending on the abundance of cells within the sample, the cells' shape, the accuracy and the granularity of the requisite results or the available funds, single-cell isolation techniques encompass serial dilution, robotic micromanipulation, microfluid platforms, laser-capture microdissection and others. Then, following the kind of target -omes (whole genome, transcriptome, epigenome, etc.), there is also a variety of methods for amplifying them (degenerative-oligonucleotide-PCR [DOP-PCR]) and multiple-displacement-amplification [MDA] or Oligo dT-anchoring) as well as for sequencing the molecular material (like SMART-Seq or using unique molecular identifiers). A huge amount of data results from the sequencing procedures. This is usually categorized within barcoded libraries (Wang & Song 2017).

These overall steps (isolating, amplifying, sequencing, and categorizing) are usually common to any single-cell analysis, whatever the field involved-carcinogenesis, immunology, microbiology, neurobiology, etc. And yet, the variety of techniques and methods used at each step, together with a lack of standardized practices between laboratories and fields, affects collaborations between research infrastructure and communication of data (Lähnemann et al. 2020). In this respect, single-cell analysis may be an interesting object for sociology of sciences. In this special issue, we leave this range of questions aside and focus on epistemological issues.

Generally, there is a huge enthusiasm by biologists' communities that employ these techniques. The promise of a high degree of precision in the data collected, and the ultimate ambition of connecting different explanatory levels in order to achieve a broader understanding of living beings are the main reasons why single-cell analysis is so widespread in laboratories today. The questions addressed in this special issue concern the contribution of single-cell analysis to the advancement of biological knowledge. Is biologists' enthusiasm vis-à-vis single-cell analysis epistemologically justified? To what extent does single-cell analysis contribute to provide a more adequate explanation of biological phenomena?

In the study of multicellular systems, sequencing a cell sample usually implies a global genetic knowledge of the tissue, which hides the intrinsic heterogeneity of molecular profiles within the sample. The rationale behind the use of single-cell analysis might be described as follows [START_REF] Qian | Cellular heterogeneity and single-cell omics[END_REF]:

-Understanding multicellular organization requires understanding the different functions exerted by different types of cells, assembled in tissues and organs;

-Cell types can be identified by a certain molecular profile. By hypothesis, all the cells belonging to the same type share the same molecular profile; -Single-cell analysis allows biologists to detect different molecular profiles within a single tissue, and to distinguish between different cell types, beyond global "means" established over populations of cells.

In the case of unicellular systems, see for instance (Ku & Sebé-Pedros 2019), single-cell analysis is used within an evolutionary approach:

-Understanding how unicellular organisms interact with their environment requires understanding how they adapt or are more specified (for studying symbiosis, ecological or evolutionary processes); -Types can be identified and clustered into phyla depending on the molecular profiles; -Single-cell analysis allows biologists to track the specification within different unicellular phyla, by detecting different molecular patterns.

Overall, this rationale relies on a twofold background presupposition, according to which:

-Molecular characteristics are more reliable than other criteria (like metabolic behavior) to typify cells; -The functional role of cells is subtended by a combination of molecular characteristics, i.e. their "molecular profile".

Such a presupposition is theoretically loaded, and raises an epistemological problem that might be broken down into three related issues.

(1) Single-cell analysis is claimed to provide a global and even holistic approach, insofar as it can combine datasets obtained through various -omics technologies, and referring to different biological objects such as mRNA, DNA, ribosomal RNA, etc. (Anam et al. 2019). Yet, the question is how single-cell analysis would realize a more comprehensive view of living beings. Indeed, the very idea according to which the molecular profile of a cell subtends its functional role is consistent with a reductionist approach to biological phenomena. And it might be argued that looking at the various molecular characteristics of a cell does not inform as such about its dynamic organization. Understanding how a cell works would imply a wider vision that does not focus on the molecular level alone. If so, then single-cell analysis provides data that are not sufficient to make sense of cell functions.

(2) The presupposition that the molecular profile is relevant (and sufficient) to categorize cells into types seems to overlook the processual dimension of biological phenomena. Cells are dynamic entities that undergo a life cycle, during which their molecular profile changes over ontogenetic time. Cells are plastic, some can dedifferentiate or transdifferentiate; ontogeny is quite reversible. In contrast, single-cell analysis provides stable data, that describe a biological mapping at a given moment (a "snapshot"), with a given set of spatial interactions. Single-cell analysis (for now) is only able to take snapshots of cells' life cycles, which means that it cannot produce any description of individual trajectories and it cannot determine whether a certain snapshot is representative of a certain cell type [START_REF] Trapnell | The dynamics and regulators of cell fate decisions are revealed by pseudotemporal ordering of single cells[END_REF]. This snapshot has to be put into perspective and compared either with other snapshots of the same biological process at a different time, or stated as the representative of a given cell type, based on previous knowledge. For now, the description of developmental or transitional dynamics in cells relies on a pseudo-time derived from a comparison of quantitative measurements between proximate moments in different cells in order to infer the states that precede or follow each other.

These limitations raise several questions: (a) To what extent does the pseudo-time account for the developmental time of a living organism? (b) By relying on molecular patterns only, how distinguishing between two different cell types, on the one hand, and the same cell at two different moments, on the other hand? (c) How determining which snapshot better characterizes a cell type? As a consequence of the dynamic nature of living processes, a (theoretical) choice should be made regarding what "moment" in the cell's life cycle is the relevant one to determine its type.

(3) More generally, single-cell analysis claims to be data-driven only. In fact, it seems to rely rather on epistemological choices, which are not always explicit [START_REF] Leonelli | The challenges of big data biology[END_REF]. There is nowadays an increasing acknowledgement of the "heterogeneity" of data across individual cells. Cells that supposedly belong to the same type and perform the same function (because they are in the same tissue for instance) do not exhibit the same molecular profile. In general, molecular data exhibit a continuum among the various categories of cell rather than sharp discontinuities. Instead of finding similarities, single-cell analysis finds differences. Such heterogeneity pushes biologists to multiply categories ("rare" cell types), which risks to be a useless process that would result in obtaining as many types as individual cells.

Therefore, single-cell analysis requires making epistemological choices while structuring categories.

It is what happens when bio-analysts define clusters by selecting a set of features, and measure to what extent each cell possess such features. As Gross (this issue) puts it, "membership is based on overall similarity, that is, the degree to which objects share a set of properties". Clustering requires to make a choice about what features are considered as relevant, and how the "distance" or "score" is measured. Depending on these choices, different categories emerge. Single-cell analysis is not (and cannot be) entirely data-driven: categorization does not emerge from data themselves. Categories depend on choices, which in turn depend on previous knowledge about cell types, background theories and assumptions.

The issue of identifying and making explicit epistemological choices also raises a number of questions: (a) What criteria should determine the features by which categories and clusters are elaborated? And how do these criteria promote different research directions? (b) How does background (structural, functional and genealogical) knowledge affect the elaboration of cells categories? (c) How do background epistemological choices impact information sharing and communication? A research team produces databases that can be hard to understand by a different team. The way to classify and identify nomenclatures (which exacerbates the variable number and the tendency towards multiplications of categories, as we mentioned before) may complexify the adequation between different databases. It also questions, in another way, the ability to reproduce results. In this sense, making explicit epistemological choices is an absolute necessity for securing disclosable data.

In a word, the focus of single-cell analysis on cells molecular profile raises a number of questions about reductionism, cell dynamics and implicit epistemological choices. More generally, single-cell analysis can be critically examined in terms of the characterization of cells and different biological processes that it puts forward, as well as the criteria for biological identity that it adopts. During its history, biology has oscillated between structural, functional and genealogical criteria, and the debate about their relation is a never-ending one. The epistemological enquiries about single-cell analysis should also be located within this larger and fascinating debate.

The four contributions to this special issue, authored by biologists and philosophers alike, examine the above questions from different, and yet complementary perspectives.

In his contribution, Fridolin Gross examines how single-cell analysis impacts the very concept of cell type. He emphasizes the tension existing between the idea of using single-cell analysis to elaborate more solid cell types and the recognition of huge spatial and temporal cellular heterogeneity. He describes what might possibly be labelled a "molecular pheneticist account" to cell types, and focuses on (and questions) the claim that such account might be theory-free. Gross shows that fundamental steps in single-cell analysis (as dimensionality reduction and clustering methods) do require to make choices (based on theories or at least on background knowledge) about the number of dimensions and the parameter values. Above all, clustering methods are "importantly driven by the concern to reproduce previously accepted cell type classifications".

Gross concludes by claiming "it seems inappropriate to refer to them as 'theory-free' or purely data-driven as this would ignore the clearly theory-guided process of method selection". Gross generalizes his argument. According to him, thinking that, in principle, the more data are added, the more they would converge in creating stable categories in a theory-free manner is delusional. Even more generally, Gross mentions the fact that focusing on "structures" is not a straightforward choice, because of the everlasting tension with functional and genealogical criteria. So Gross asks: "Why then should biologists focus so much on a theory-free classification approach if that approach misses the central goal that cell type classifications are meant to achieve?".

Racine and Paldi also underscore that single-cell analysis is supposed to contribute to our understanding of cells identity and differentiation. Classifications based on origins vs. similarities have always co-existed, but the more recent idea is that "cells of the same type must express the same genes and can be identified on the basis of the transcriptional regulator (transcription factors) they express".

As Gross, they emphasize the strong heterogeneity in gene expression, and in the resulting molecular profile. So, while cells belonging to different tissues or organs tend to exhibit distinguishable gene expression patterns, the same is not true for supposedly different cells belonging to the same tissue, for instance. According to them, the continuous nature of gene expression in cell population makes that "If one picks up randomly a cell from the population, there are good chances that it is impossible to say on the basis of its gene expression pattern to which type it belongs". Again, clustering methods, no matter how powerful they are, do not produce types by themselves, but depend on several background choices made by the biologist (about p-values, thresholds, filters, the presumed number of clusters one expects, etc.).

Racine and Paldi also suggest that the more our analysis is fine-grained, the more we find molecular differences. We find "rare cell types", and "cell states" which, according to the authors, do not change anything to the initial problem. The identity of cells is contextual and dynamic, and any search by single-cell analysis should rely on a definition elaborated beforehand. This means in particular deciding what counts as relevant stability in cell life cycles, which are profoundly variable and dynamic. We should not forget that "stability" itself is a scale-dependent notion, with respect to which a decision also has to be taken. The Authors conclude by calling for "a new interpretation framework based on solid theoretical ground", possibly centered on the organicist tradition.

Heams' article agrees with the previous ones about the fact that the questions raised by single-cell analysis are not just technical, but profoundly epistemological and theoretical. Heams underscores that singlecell analysis is mainly used within the Genetic Determinism Paradigm, according to which (among other things) similarities and differences among cells should unambiguously correspond to similarity and differences in their gene expression and in their overall molecular profile. Yet, single-cell analysis has shown for 20 years now that even a population of clonal cell shows very heterogeneous gene expression, to the point that the idea of stochastic gene expression was proposed, and it constitutes now a solid hypothesis in molecular biology. Single-cell analysis, in this sense, contributed not to find stability and categories, but to shake the very foundations of GDP. Heams discusses the various ways of interpreting unpredictable variability, ranging from the more conservative and GDP-related to the more original and alternative one, which Heams calls the "probabilistic alternative framework (PAF)". PAF claims that gene expression is fundamentally stochastic and incompatible with GDP. Heams discusses the strength and weaknesses of PAF, and in particular the extent to which it is at odds with some of the theoretical pillar of evolutionary theory, i.e. the necessity of cooperative behavior. On this crucial point, Heams argues that PAF does not exclude cooperative behavior, but that this very notion should be reconceptualized within a probabilistic framework. Heams goes farther in discussing how both GDP and PAF are challenged by the discovery that cells constituting a multicellular system are not genetically homogeneous, although he argues that the challenge is not the same. GDP "is affected at its very core", while "there is nothing to prevent genetically different cells in a clonal population from also exhibiting stochastic behaviour".

The upshot of his analysis is that while singlecell analysis has shaken the GDP at its foundations, the mainstream paradigm is still… mainstream. This raises the question of how experimental results (in this case, obtained by single-cell analysis) can actually falsify a paradigm, and open the way to innovative research directions.

Angleraux's article (which will be published in the following issue, because of editorial reasons) follows the same line of Heams, Racine and Paldi regarding the need to clarify the theoretical ground of single-cell analysis. She questions the type of biological explanation underlying single-cell sequencing, and she applies general frameworks in philosophy of biology (especially new mechanism and systems biology) to specify how these techniques explain biological phenomena. She comes to the conclusion of a gap between the scientific narrative and what single-cell sequencing de facto produces.

Indeed, by combining different databases, singlecell analysis (as a kind of -omics sequencing techniques) aims to achieve a comprehensive and a more integrative explanation of biological phenomena, which matches with the zeitgeist against reductionism of current theoretical and philosophical perspectives in biology. New mechanism, on the one hand, claims to be nonreductionist because it takes into account emergent properties of organisms and explains them by integrating elements at different levels of description. Systems biology, on the other hand, also alleges a holistic view of life by combining biological subsystems. Angleraux examines to what extent single-analysis embraces new mechanism's and systems biology's zeitgeist. However, this is the case also because single-cell analysis shares the same theoretical and philosophical limits with these perspectives. In particular, both mechanism and system biology keep favoring bottom-up, rather than top-down explanations of living phenomena. As a consequence, Angleraux underscores the hiatus between the scientific narrative-what single-cell analysis declares to accomplish-and what it actually does for now.

We would like to thank the "Who am I? Single Cell Initiative" (WISCI), led by Pierre-Antoine Defossez at Paris Cité, and funded by the Labex "Who am I?", which provided financial support for the workshop "Enjeux épistémologiques de l'approche «single cell»" held in Paris in May 31-June 1, 2022. The contributions to this special issue were initially presented at the workshop. We thank in particular Pierre-Antoine Defossez and Nikos Konstantinides who also attended the workshop and hosted Caroline Angleraux in their team as a postdoc. Their work provided a relevant case study to work on and think about single-cell analysis. Our warmest thanks go to the editors of the journal Organisms for accepting to publish this special issue, and to the reviewers of the articles for their rigorous and constructive critical comments.


Mathilde Schoenauer Sebag

Zhelu Hu

Karmel De

Oliveira Lima

Hengyang Xiang

Patrick Gredin

Michel Mortier

Laurent Billot

Lionel Aigouy

Zhuoying Chen

Microscopic Evidence of Upconversion-Induced Near-Infrared Light Harvest in Hybrid Perovskite Solar Cells

Keywords: photon upconversion, upconversion solar cells, perovskite solar cells, upconversion nanoparticles, light-beam induced current

Photon upconversion represents a promising avenue to reduce the spectral mismatch losses limiting the efficiency of solar cells. Here we studied in detail the impact of inserting Yb 3+ /Er 3+ co-doped KY7F22 upconversion nanoparticles (UCNPs) into the different interfaces of a solution-processed mixed-cation lead mixed-halide perovskite solar cell. Besides macroscopic photovoltaic characteristics, we quantify the upconversion contribution by the light-beam induced

current/fluorescence mapping technique on devices with only half of their interfaces decorated by UCNPs. Such mapping experiments offer a detailed microscopic and spectroscopic picture allowing a correlation of the electrical and optical contribution of UCNPs together with the solar cell morphology.

TEXT. Photon upconversion, a phenomenon to convert low energy photons into high energy ones, has been a fascinating topic since its discovery. [START_REF] Bloembergen | Solid State Infrared Quantum Counters[END_REF] Over the last decades various material systems have been identified among which fluoride, 2 oxide 3 and oxysulfide [START_REF] Yocom | Rare-Earth-Doped Oxysulfides for GaAs-Pumped Luminescent Devices[END_REF] matrixes doped or co-doped by trivalent lanthanide ions are major examples capable to perform upconversion through a twoor multi-photon mechanism. [START_REF] Auzel | Upconversion and Anti-Stokes Processes with F and D Ions in Solids[END_REF] Extensive investigation has since been carried out to achieve upconversion systems in the form of nanoparticles (NPs), [START_REF] Schäfer | Synthesis of Hexagonal Yb3+,Er3+-Doped NaYF4 Nanocrystals at Low Temperature[END_REF][START_REF] Ghosh | Tuning of Crystal Phase and Luminescence Properties of Eu3+ Doped Sodium Yttrium Fluoride Nanocrystals[END_REF][START_REF] Zhao | Controlled Synthesis, Formation Mechanism, and Great Enhancement of Red Upconversion Luminescence of NaYF4:Yb3+, Er3+ Nanocrystals/submicroplates at Low Doping Level[END_REF][START_REF] Wang | Simultaneous Phase and Size Control of Upconversion Nanocrystals through Lanthanide Doping[END_REF] facilitating their application in different fields including biomedical engineering, [START_REF] Idris | Upconversion Nanoparticles as Versatile Light Nanotransducers for Photoactivation Applications[END_REF][START_REF] Sedlmeier | Surface Modification and Characterization of Photon-Upconverting Nanoparticles for Bioanalytical Applications[END_REF][START_REF] Wang | Upconversion Nanoparticles in Biological Labeling, Imaging, and Therapy[END_REF] optical amplification, [START_REF] Zhang | Optical Gain at 1535 Nm in LaF3: Er,Yb Nanoparticle-Doped Organic-Inorganic Hybrid Material Waveguide[END_REF] thermal sensing, [START_REF] Aigouy | Scanning Thermal Imaging of Microelectronic Circuits with a Fluorescent Nanoprobe[END_REF] photodetection, [START_REF] Yan | Near-IR Photoresponse in New up-Converting CdSe/NaYF4:Yb,Er Nanoheterostructures[END_REF] and solar energy harvest. [START_REF] Van Der Ende | Lanthanide Ions as Spectral Converters for Solar Cells[END_REF][START_REF] Goldschmidt | Upconversion for Photovoltaics -a Review of Materials, Devices and Concepts for Performance Enhancement[END_REF] In particular, for photovoltaics, upconversion materials have been considered a promising avenue to reduce the spectral mismatch losses limiting the efficiency of solar cells. [START_REF] Van Der Ende | Lanthanide Ions as Spectral Converters for Solar Cells[END_REF][START_REF] Goldschmidt | Upconversion for Photovoltaics -a Review of Materials, Devices and Concepts for Performance Enhancement[END_REF][START_REF] De Wild | Upconverter Solar Cells: Materials and Applications[END_REF] Spectral mismatch losses, also called transmission or sub-bandgap losses, originate from the fact that photons of energies below the absorption threshold of the photovoltaic absorber cannot be absorbed and cannot contribute to the current generation in the solar cell. Such sub-bandgap losses can be quite significant: For single-junction crystalline silicon solar cells with a bandgap of 1.1 eV, sub-bandgap losses account for more than 19% of the power incident from the sun. [START_REF] Goldschmidt | Upconversion for Photovoltaics -a Review of Materials, Devices and Concepts for Performance Enhancement[END_REF] This percentage is even more elevated, reaching 39%, for solutionprocessed organic-inorganic hybrid perovskite halide solar cells due to their larger bandgap (e.g. about 1.5 eV for methylammonium lead iodide perovskite). [START_REF] Goldschmidt | Upconversion for Photovoltaics -a Review of Materials, Devices and Concepts for Performance Enhancement[END_REF] While low-bandgap semiconducting materials can allow an enlarged solar spectrum harvest, single-junction solar cells based on lowbandgap materials will however exhibit a reduced open-circuit voltage. [START_REF] Kim | Origin of Open-Circuit Voltage Loss in Polymer Solar Cells and Perovskite Solar Cells[END_REF] Various forms of upconversion materials have therefore been applied into solar cells based on different semiconducting materials. Yb 3+ and Er 3+ co-doped materials represent the most widely investigated upconversion systems while other rare-earth other activators (such as Tm 3+ and/or Ho 3+ together with Yb 3+ ) can also be used to achieve various unconverted fluorescence in the visible spectrum. [START_REF] Gouveia-Neto | Generation of Wide Color Gamut Visible Light in Rare-Earth Triply Doped Tantalum Oxide Crystalline Ceramic Powders[END_REF] They are applied in a position either before or after the illumination reaches the semiconducting absorber (termed below as "front" or "rear" side respectively). For example, a Yb 3+ and Er 3+ co-doped vitroceramic layer has been placed on the rear side of a GaAs solar cell capable to harvest a sub-bandgap illumination (1.39 eV) and generate a photovoltaic efficiency of 2.5%. [START_REF] Gibart | Below Band-Gap IR Response of Substrate-Free GaAs Solar Cells Using Two-Photon Up-Conversion[END_REF] Polycrystalline 20% Er 3+ -doped NaYF4, mixed with near-infrared (NIR) transparent white oil, was adhered to the rear side of a crystalline silicon solar cell exhibiting an external quantum efficiency (EQE) close to 3.4% at 1523 nm (at 6mW). [START_REF] Shalav | Improvements of an up-Conversion NaYF/sub 4/:Er/sup 3+/ Phosphor/silicon Solar Cell System for an Enhanced Response in the near-Infrared[END_REF] On solution-processed solar cells, a frontside application of upconversion materials is the main configuration used by different works: For example, a Yb 3+ and Er 3+ co-doped TiO2 layer has led to a 2.4% increase of short-circuit current in dye-sensitized solar cells. [START_REF] Shan | Near-Infrared Sunlight Harvesting in Dye-Sensitized Solar Cells via the Insertion of an Upconverter-TiO2 Nanocomposite Layer[END_REF] Similarly, Ho 3+ , Yb 3+ and F -tridoped TiO2 NPs were applied in dyesensitized solar cells leading to a 37% enhancement of power conversion efficiency (PCE) compared to pristine device without any upconversion layer. [START_REF] Yu | Enhanced near-Infrared to Visible Upconversion Nanoparticles of Ho(3)(+)-Yb(3)(+)-F(-) Tri-Doped TiO(2) and Its Application in Dye-Sensitized Solar Cells with 37% Improvement in Power Conversion Efficiency[END_REF] On P3HT:PCBM organic solar cells, an upconversion quantum efficiency of 0.19% was observed under the illumination of a 975 nm laser diode (at 250 mW cm -2 ) when both a Yb 3+ and Er 3+ co-doped yttrium fluoride upconversion phosphor and a NIR filter was placed in the front side of the cell. [START_REF] Wang | Rare-Earth Ion Doped up-Conversion Materials for Photovoltaic Applications[END_REF] On methylammonium lead iodide perovskite solar cells, by a front-side configuration, He et al. applied Yb 3+ and Er 3+ co-doped NaYF4 NPs as the mesoporous scaffold directly on the compact TiO2 layer and observed an increase of PCE from 17.8% to 18.1% upon 975 nm laser illumination (at 28 W cm -2 ). [START_REF] He | Monodisperse Dual-Functional Upconversion Nanoparticles Enabled Near-Infrared Organolead Halide Perovskite Solar Cells[END_REF] While these results are highly encouraging, the application of upconversion NPs (UCNPs) in the front side of the solar cell (before the illumination reaches the semiconducting layer) can render the mechanistic study very complex due to the possible structural/morphological modification of the absorber materials deposited on top and the optical scattering induced by the UCNPs. In addition, unless an NIR filter was applied together with the upconversion layer, defect [START_REF] Ball | Defects in Perovskite-Halides and Their Effects in Solar Cells[END_REF] and/or charge-transfer complex-induced 28 sub-bandgap states may also contribute to the observed photocurrents under NIR illumination leading to an over-estimation of upconversion contribution.

Most above-mentioned studies are based on current-voltage characterizations, a more detailed photovoltaic-microscopic correlation characterization would be valuable to reveal the intrinsic upconversion contribution.

In this work, we present both a macroscopic and microscopic characterization of the upconversion-induced NIR light harvest in solution-processed hybrid perovskite solar cells. Uniform and well-crystalline KY7F22 UCNPs co-doped by 20 mol% Yb 3+ and 5 mol% Er 3+ were synthesized by a co-precipitation method. [START_REF] Assy | Nanoscale Thermometry with Fluorescent Yttrium-Based Er/Yb-Doped Fluoride Nanocrystals[END_REF] Detailed synthesis procedure and characterization methods are described in the supporting information (SI). The nature and crystallinity of the as-synthesized UCNPs were controlled using powder XRD. The as-recorded XRD pattern was carefully compared with that of a reference (SI Figure S1). The good agreement between both patterns and the absence of extra-diffraction lines indicated clearly that the synthesized compound was KY7F22. Moreover, the high intensity and the rather sharp profile of the diffraction peaks indicated a good crystallinity of the final product. As synthesized UCNPs were stable below about 400°C based on thermogravimetric analysis/differential thermal analysis (TGA/DTA) exhibiting a strong sharp peak at around 400°C (SI Figure S2). The XRD pattern obtained after annealing at above 400°C exhibited a different pattern composed of diffraction peaks due to KY3F10 and YF3. KY7F22 is thus advantageous by comparison to the more well-known upconversion host NaYF4, where cubic and hexagonal phases often co-exist but only the hexagonal ones favour efficient luminescence properties. [START_REF] Schäfer | Synthesis of Hexagonal Yb3+,Er3+-Doped NaYF4 Nanocrystals at Low Temperature[END_REF] As indicated in the inset of Figure 1a, the upconversion processes in KY7F22:20% Yb 3+ /5% Er 3+ is believed to be similar as those in Yb 3+ /Er 3+ co-doped NaYF4, involving the excitation of Yb 3+ by the NIR excitation, followed by energy transfer from Yb 3+ to Er 3+ and the radiative decays of Er 3+ . [START_REF] Heer | Highly Efficient Multicolour Upconversion Emission in Transparent Colloids of Lanthanide-Doped NaYF4 Nanocrystals[END_REF] Under 975 nm NIR excitation, three strong upconversion fluorescence emission peaks were observed at about 525 nm, 543 nm, and 655 nm corresponding to the 2 H11/2® 4 I15/2, 4 S3/2® 4 I15/2, and 4 F9/2® 4 I15/2 transitions of Er 3+ (Figure 1a). A relatively weak upconversion fluorescence at about 408 nm was also observed (not shown in Figure 1a). KY7F22:20% Yb 3+ /5% Er 3+ UCNPs exhibit rather long lifetimes of excited states (for example, approximately 300 µs for the 4 S3/2® 4 I15/2 transition as shown in Figure 1b). These KY7F22:20% Yb 3+ /5% Er 3+ UCNPs thus possess the required optical properties to serve as good upconversion candidates for the hybrid solar cells studied in this work. KY7F22:20% Yb 3+ /5% Er 3+ UCNPs were then inserted at the interface between the different stacking layers of a functional hybrid perovskite halide solar cell based on FA0.83Cs0.17Pb(I0.6Br0.4)3 (FA = formamidinium [HC(NH2)2]) (Figure 2). Here, a mixed-cation lead mixed-halide perovskite component was chosen due to their improved photostability compared to other prototype perovskite halides such as methylammonium lead iodide. 31 An electron transport layer (ETL)-free solar cell structure 32 was applied to decouple the influence of UCNP insertion and the experimental variation of ETL deposition. As synthesized KY7F22:Yb 3+ /Er 3+ UCNPs were dispersed in anhydrous isopropanol to form a NP suspension of a certain concentration (2 wt%, 5 wt%, 7 wt%, and 10 wt%). Each UCNP isopropanol solution was then deposited on top of either the fluorine doped tin oxide (FTO) or the perovskite layer by spin-coating. As indicated in Figure 2, this allows us to achieve the insertion of UCNPs either at the interfaces between FTO and the perovskite ("front-side" insertion) or between the perovskite and the hole-transport polymer layer ("rear-side" insertion). Scanning electron microscopy (SEM) characterizations, carried out after the UCNP deposition on top of the FTO (Fig. 2b and2c) or the perovskite layer (Fig. 2e and2f), revealed a globally uniform distribution of UCNPs on their substrates with an average particle diameter of 183 nm ± 8 nm. The use of a UCNP isopropanol suspension of a higher concentration indeed results an increase density of UCNPs decorated on FTO or perovskite. Even with a relatively dilute UCNP solution (2 wt%, Fig. 2b and2e), the density of UCNPs on their substrates is not negligible with at least one UCNP within the perimeter of slightly less than one micron. Applying a UCNP solution of a higher concentration (7 wt%, Fig. 2c and2f), some large UCNP clusters containing more than 10 NPs start to form. Various UCNP solution concentrations (2 wt%, 5 wt%, 7 wt%, and 10 wt%) were therefore applied to control the density of UCNPs during the optimization of UCNP/perovskite solar cell performance. Solar cell fabrication was then completed after the deposition of UCNPs according to the device structures shown in Figure 2. The evolution of solar cell characteristics of UCNP-incorporated FA0.83Cs0.17Pb(I0.6Br0.4)3 perovskite solar cells fabricated by the dispersion of UCNP isopropanol suspensions of different concentrations is shown in Figure 3. Here we aim to obtain the optimal amount of UCNPs to incorporate into such perovskite solar cells allowing a macroscopic photovoltaic characteristic enhancement before applying this optimal condition for microscopic studies. We compared solar cells fabricated from the same batch on the same day to avoid batchto-batch device variations. By comparison to the control device without any UCNPs, both front and rear-side UCNP insertion can lead to a noticeable enhancement of short-circuit current (Jsc) when a UCNP solution of a 5-7 wt% concentration was applied (Fig. 3a). Specifically, about 5% of averaged Jsc enhancement was obtained when a 5 wt% UCNP solution was applied under the front-or rear-side NP insertion configuration. Such Jsc enhancement, however, starts to decrease with the use of a UCNP solution of a higher concentration (> 5 wt%). While a larger amount of UCNPs can result in a higher upconversion current contribution, the formation of large insulating 33 UCNP clusters can increase the solar cell series resistance as well as the surface roughness of the interface. These factors thus lead to the observed progressive reduction of Jsc when the UCNP solution concentration was beyond the optimal value (~ 5 wt%). Concerning the open-circuit voltage (Voc), on both front-and rear-side insertion configurations we observed negligible effect when the UCNP concentration is small or moderate (£ 7 wt%) and a reduction of Voc when a 10 wt% UCNP concentration was applied. Such a reduction of Voc can be due to the formation of large UCNP clusters at the interface which acts as charge recombination centers and/or perturbs the morphology of the perovskite or HTL layer formed on top. In terms of fill factor (FF), similar FF values as the control device were observed when the UCNP concentrations were £ 7 wt% for both front-and read-side configurations, suggesting minor deterioration of series and shunt resistances due to the insertion of UCNPs as long as their concentration was relatively low. At a higher UCNP concentration (≥7 wt%), however, a reduction of FF can be observed in both device configurations, suggesting increased series resistance and recombination losses due to the formation of large insulating UCNP clusters. Overall, by comparison to the control device, about 5.7% and 7.4% of averaged PCE enhancement was observed respectively in the front-and rearside UCNP insertion configuration when an optimal UCNP solution concentration (5 wt%) was applied. The current-voltage J-V and external quantum efficiency (EQE) characteristics of the bestperforming solar cells, with and without UCNP insertion by the two device architectures are summarized in the SI (Figure S3, S4 and Table S1). Besides the above-mentioned ETL-free perovskite solar cells based on FA0.83Cs0.17Pb(I0.6Br0.4)3, with the obtained optimal UCNP solution concentration (5 wt%), we equally experimented planar Cs0.05FA0.79MA0.16PbI2.5Br0.5 perovskite solar cells with a compact TiO2 as the electron transport layer. Macroscopic J-V characteristics of these solar cells revealed similar effects of UCNP incorporation as those observed in ETL-free FA0.83Cs0.17Pb(I0.6Br0.4)3 perovskite solar cells (Figure S5 and Table S2). In the following discussion, we will focus our microscopic LBIC/fluorescence mapping experiments on ETL-free FA0.83Cs0.17Pb(I0.6Br0.4)3 perovskite solar cells due to the simplicity in device fabrication facilitating one to decouple the influence of UCNP incorporation and the experimental variation of ETL deposition.

We note that the results from the macroscopic solar cell characterization described above represent a global consequence of UCNP insertion on the perovskite solar cells where multiple effects (e.g. upconversion effect, impact on the electrical and structural characteristics) play together. In order to quantify the upconversion contribution, here we further performed a microscopic solar cell characterizations by the LBIC technique to correlate UCNP emission, upconversion-contributed photocurrent, together with the morphology of the solar cell under the excitation of a NIR laser (at 975 nm). From the results described above, we observed that the rearside UCNP insertion configuration allowed for slightly more optimized UCNP-incorporated perovskite solar cells than the front-side insertion configuration. We therefore focused on the rearside UCNP insertion configuration to carry out the following microscopic studies. In addition, as the UCNPs were deposited after the perovskite crystallization, the structural impacts on perovskite crystallization due to the existence of UCNPs can be minimized. In order to facilitate LBICmicrostructure correlation, half of the perovskite/HTL interface was masked on purpose during the deposition of UCNPs (5 wt% solution used here) and thus creating solar cells with half of their perovskite/HTL interface decorated by UCNPs (Fig. 4a and4b). From the gold contact side of such a solar cell, the two parts of the same device (with and without UCNPs) show different optical contrast under an optical microscope due to the increased surface roughness induced by the existence of UCNPs (Fig. 4c).

The LBIC/fluorescence mapping in this work was achieved by focusing a 975 nm NIR laser through the transparent side (FTO) of the solar cell at the perovskite/HTL interface where UCNPs were inserted for half of the device (Fig. 4a). The laser spot diameter was about 30 -50 µm and the total power of the laser spot was about 100 mW. While the laser spot was fixed during mapping experiments, the position of the solar cell was controlled by a piezoelectric scanning stage. On each pixel, we recorded simultaneously the short-circuit current Jsc of the solar cell together with a fluorescence spectrum. A rectangular area (100 µm by 25 µm) containing both the parts with and without UCNPs was chosen for LBIC/fluorescence mapping experiments (indicated as the dark dashed line square in Fig. 4b). A typical LBIC and fluorescence mapping results were shown in Figure 4e, 4g, and4h. When the laser spot mapped from the part of device without UCNPs to the part with UCNPs, we observed ~ 3-fold increase of Jsc which represented directly the upconversion contribution from UCNPs excited by the 975 nm laser. It is worth to note that, during LBIC/fluorescence mapping experiments, as the laser was shrined from the FTO side (Figure 4a), if there exists any defect-induced photocurrent under NIR excitation, it should be identical for both parts of the (same) device with or without UCNPs. Therefore, the observed increase of photocurrent under NIR excitation when we mapped from the part without UCNPs to the part with UCNPs can represent well the upconversion contribution. In coherence with the Jsc mapping, an appearance of fluorescence of UCNPs was observed when the laser spot travelled from the part without to the part with UCNPs. These trends are clearly visible from the dashed line profile of the mapping results, plotted in Fig. 4f. The fluorescence spectrum recorded on the part with UCNP (e.g. at the point A of Figure 4e) exhibited two major fluorescence peaks located respectively between 517 nm and 560 nm (green fluorescence) and between 630 nm and 680 nm (red fluorescence). By comparison to the fluorescence spectrum recorded from stand-alone UCNPs deposited on a glass substrate (Fig. 1a), the green/red fluorescence intensity ratio recorded in LBIC/fluorescence mapping experiment is largely reduced. This is due to the fact that the fluorescence signal must travel through the perovskite layer before being detected during LBIC/fluorescence mapping. As the perovskite layer has a larger absorption coefficient at the green fluorescence wavelengths than red fluorescence ones, the fluorescence signal detected exhibited a reduced proportion of green/red fluorescence. This reduced UCNP green fluorescence observed therefore originated from the optical contribution of the UCNPs in a perovskite solar cell where upconversion fluorescence was absorbed by the perovskite layer. The above-described LBIC/fluorescence mapping experiments thus offer a detailed microscopic picture, by which one can find the electrical and optical contribution of UCNPs together in excellent agreement with the solar cell morphology (i.e. the parts with and without UCNPs).

In summary, in this work we studied in detail macroscopically and microscopically the impact of inserting UCNPs into a functional solar cell based on solution-processed mixed-cation lead mixed-halide perovskite FA0.83Cs0.17Pb(I0.6Br0.4)3. Uniform and well-crystalline Yb 3+ /Er 3+ codoped KY7F22 UCNPs were applied onto either the FTO/perovskite or the perovskite/HTL interface of a perovskite solar cell (the front-or rear-side configuration) while the density of UCNPs were controlled by their concentration in solution during the deposition. By comparison to the control device, about 6.1% and 6.5 % of averaged PCE enhancement were observed respectively in the front-and rear-side NP insertion configuration when an optimal UCNP solution concentration was applied. To quantify the upconversion contribution, microscopic solar cell characterizations by the LBIC/fluorescence mapping technique were carried out on devices with only half of their perovskite/HTL interface decorated by UCNPs fabricated on purpose. These mapping experiments offered a detailed microscopic picture allowing a correlation of the electrical and optical contribution of UCNPs together with the solar cell morphology: when the laser spot goes from the part of device without UCNPs to the part with UCNPs, together with the appearance of UCNP fluorescence, a ~ 3-fold increase of Jsc was observed attributed directly from the upconversion contribution. The reduced green/red ratio of the upconversion fluorescence spectra observed during mapping experiments were associated with the higher perovskite absorbance at the green fluorescence wavelength range. This served as a direct evidence for the optical contribution of the UCNPs in a perovskite solar cell where the upconversion fluorescence was harvested by the perovskite layer.

ASSOCIATED CONTENT

Supporting Information.

The supporting information is available free of charge on the ACS Publications website. Detailed experimental procedures and methods, XRD pattern of KY7F22 NPs, TGA/DTA analyses of Yb 3+ and Er 3+ co-doped KY7F22 NPs, the J-V characteristics of the best-performing planar ETL-free FA0.83Cs0.17Pb(I0.6Br0.4)3 solar cells with and without UCNP insertion, EQE characteristics, J-V characteristics of the best-performing planar Cs0.05FA0.79MA0.16PbI2.5Br0.5 perovskite solar cells with a compact TiO2 (with and without UCNP insertion), summary of the photovoltaic parameters from the best-performing planar ETL-free FA0.83Cs0.17Pb(I0.6Br0.4)3 perovskite solar cells and planar Cs0.05FA0.79MA0.16PbI2.5Br0.5 perovskite solar cells with a compact TiO2 as ETL, with and without Yb 3+ and Er 3+ co-doped KY7F22 NPs inserted (PDF).

  Uniform and well-crystalline KY7F22 UCNPs with an average diameter of 183 nm co-doped by 20 mol% Yb 3+ and 5 mol% Er 3+ were inserted into the different interfaces of a functional solar cell based on mixed-cation lead mixed-halide perovskite FA0.83Cs0.17Pb(I0.6Br0.4)3 (FA = formamidinium [HC(NH2)2]). Besides the optimizations on the amount of UCNPs based on macroscopic photovoltaic characteristics, we performed detailed microscopic characterizations by the light-beam induced current (LBIC)/fluorescence mapping technique on solar cells with only half of their interface decorated by UCNPs fabricated on purpose. Such mapping experiments provided detailed microscopic and spectroscopic evidence allowing one to correlate the electrical and optical contribution of UCNPs together with the solar cell morphology.

FIGURESFigure 1 .

 1 FIGURES

Figure 2 .

 2 Figure 2. (a) and (d) Schematics displaying the solar cell architectures and the interface where

Figure 4 .

 4 Figure 4. (a) Schematic of the LBIC/fluorescence mapping experimental set-up and the cross-

This work was supported in part by the UPNAN project (in the framework of IDEX with the reference of ANR-10-IDEX-0001-02 PSL*) and the PROCES project (ANR-17-CE05-0028-01).

Z. Hu and H. Xiang acknowledge the Chinese Scholarship Council for their PhD scholarships.

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