MSc Fabienne Venet

MD, PhD; Julien Bohé

Pharm Anne-Lise Debard

Pharm D, PhD; Jacques Bienvenu

MD Alain Lepape

PharmD Guillaume Monneret

Both percentage of ␥␦ T lymphocytes and CD3 expression are reduced during septic shock

D

espite the great advances of modern medicine, septic shock remains a major cause of death in intensive care units, with a mortality rate that is regularly reported as high as 40 -50% [START_REF] Annane | Septic shock[END_REF]. It is now agreed that septic shock deeply perturbs immune homeostasis by inducing an initial tremendous systemic inflammatory response that is rapidly followed by an anti-inflammatory process, acting as a negative feedback [START_REF] Munford | Normal responses to injury prevent systemic inflammation and can be immunosuppressive[END_REF][START_REF] Hotchkiss | The pathophysiology and treatment of sepsis[END_REF]. These inhibitory mechanisms may become deleterious as nearly all immune functions are compromised. It is thus hypothesized that these mechanisms play a major role in the decreased resistance to nosocomial infections in patients who survived initial resuscitation [START_REF] Munford | Normal responses to injury prevent systemic inflammation and can be immunosuppressive[END_REF][START_REF] Hotchkiss | The pathophysiology and treatment of sepsis[END_REF]. The mechanistic bases for how septic shock might induce immune dysfunctions are far from being extensively established. A better understanding of the complexity of sepsis immunology may reveal new insights into ways in which immune functions could be restored during this phase of immunosuppression.

Recent evidence supports a role for ␥␦ T lymphocytes during sepsis, especially in potentiating the early inflammatory response (4 -5). One important result of the Matsushima et al. study [START_REF] Matsushima | Early activation of ␥␦ T lymphocytes in patients with severe systemic inflammatory response syndrome[END_REF] is the report of decreased percentage of ␥␦ T lymphocytes in patients with sepsis (although not significant). It is established that septic patients have features consistent with depressed innate immunity (2), in which ␥␦ lymphocytes are thought to participate by having a role in the first line of defense in epithelial tissues [START_REF] Jameson | T-cell effector mechanisms: ␥␦ and CD1d-restricted subsets[END_REF]. Another characteristic of these cells is that they express high levels (two-fold more than ␣␤ cells) of CD3/TCR complex, the lymphocyte counterpart of monocyte human leukocyte antigen (HLA)-DR [START_REF] Thibault | Compared TCR and CD3 epsilon expression on alpha beta and gamma delta T cells. Evidence for the association of two TCR heterodimers with three CD3 epsilon chains in the TCR/CD3 complex[END_REF][START_REF] Nicolas | Human ␥␦ T cells express a higher TCR/CD3 complex density than ␣␤ T cells[END_REF]. Interestingly, monocyte HLA-DR expression is known to be severely diminished during septic shock, which constitutes an impor-tant feature of monocyte paralysis (9 -10). On the basis of these data, we hypothesized that the percentage of circulating ␥␦ T lymphocytes might be also decreased during septic shock as well as CD3 expression on T cells, which would constitute additional characteristics of sepsis-induced immunosuppression.

METHODS

Patients and Study Design. The study group consisted of 21 consecutive patients with septic shock according to the diagnostic criteria of the American College of Chest Physicians/Society of Critical Care Medicine [START_REF] Bone | Definitions for sepsis and organ failure and guidelines for the use of innovative therapies in sepsis. The ACCP/SCCM Consensus Conference Committee[END_REF]. These patients belong to a larger group of patients examined for their percentage of HLA-DR-positive monocytes (ongoing study, first part of results reported elsewhere) [START_REF] Lepape | Monitoring of sepsis-induced immunoparalysis by measuring monocytic HLA-DR in patients with septic shock[END_REF]. Septic shock was defined by an identifiable site of infection and evidence of a systemic inflammatory response manifested by at least two of the following criteria: a) temperature Ͼ38°C or Ͻ36°C; b) heart rate Ͼ90 beats/min; c) respiratory rate Ͼ20 breaths/min; d) white blood cell count Ͼ12,000/mm 3 or Ͻ4 00/mm 3 and hypotension persisting despite fluid resuscitation and requiring vasopressor therapy.

KEY WORDS: T cells; ␥␦ T lymphocytes; CD3 expression; human leukocyte antigen-DR

Severity was assessed by the Simplified Acute Physiology Score II [START_REF] Gall | A new Simplified Acute Physiology Score [SAPS II] based on a European/North American multicenter study[END_REF]. Mortality was defined as death occurring within 28 days after the onset of shock. To provide a panel of values from healthy donors, we also included 21 individuals from laboratory staff of our hospital (aged 23-59 yrs, 13 females, eight males, without known comorbidity). Cell phenotyping was assessed at days 3-5 after the onset of shock when immunoparalysis is known to be established (on the basis of low monocyte HLA-DR expression). This prospective study was reviewed by the institutional ethics committee. This committee waived the need for informed consent because measurements were performed when residual blood was avail-able after completing routine follow-up performed in our intensive care units.

Flow Cytometry. Flow cytometric (EPICS XL, Beckman-Coulter, Hialeah, FL) expression of cell-surface markers was assessed on peripheral whole blood collected on EDTA anticoagulant tubes. Monoclonal antibodies were used according to the manufacturer's Monocytes were characterized as previously described (9 -10). Results are expressed as percentages of HLA-DR-positive monocytes. We isolated T lymphocytes (CD3 positive) on a biparametric histogram on the basis of side-scatter and CD3 staining (Fig. 1a). Then the percentages of ␣␤and ␥␦-T lymphocytes among T cells were defined on monoparametric histograms (Fig. 1b and1c, left). The thresholds were easily set up between negative and positive cell populations. Last, on these selected T cells, we examined CD3 expression (means of fluorescence intensities reflecting the CD3 density per cell) related to the entire gated population (Fig. 1b and1c, right).

Statistical Study. Due to the sample size, comparisons between groups were made using the nonparametric Mann-Whitney test. We considered p Ͻ .05 as significant. Results are expressed as median and interquartile range.

RESULTS AND DISCUSSION

In parallel with our ongoing study on the modulation of HLA-DR expression on monocytes [START_REF] Lepape | Monitoring of sepsis-induced immunoparalysis by measuring monocytic HLA-DR in patients with septic shock[END_REF], we examined 21 septic shock patients for ␥␦ and ␣␤ percentages and CD3 expression. Their clinical characteristics are presented in Table 1; mortality rate was 24%. At days 3-5, patients presented with decreased monocyte HLA-DR expression: 41% (31-55), control Ͼ90% (9), which is in accordance with a depressed immune status. Regarding T-lymphocyte count, we observed significantly decreased values in patients (590/L [(230 -840]) in comparison with healthy donors (1230/L [1140 -1600], p Ͻ .0001). We then established the significantly reduced percentage of ␥␦ T cells in peripheral blood among total T lymphocytes (Fig. 2a). Conversely, we observed an increase in ␣␤ T-lymphocyte percentage. This reinforces the results of Matsushima et al. ( 4), suggesting a reduced percentage of circulating ␥␦ T cells in patients with sepsis. Nevertheless, it is necessary to note that ␥␦ T cells present with several impairments in elderly ( 14). Although we did not find any correlation between age and percentage of ␥␦ T cells in our patients (r ϭ .02, p ϭ .7), these first results remain to be confirmed in a larger study including age-matched control individuals. As ␥␦ T cells play a role in early host defense against bacterial pathogens by providing a link between innate and specific immune responses through the production of interferon-␥ (6), their diminished percentage after septic shock may be partly responsible for the elevated incidence of secondary infections during sepsis-induced immunodepression, especially in a situation of decreased circulating T lymphocytes [START_REF] Hotchkiss | The pathophysiology and treatment of sepsis[END_REF]. Another scenario may be concomitantly envisaged. As inflammatory response is compartmentalized during sepsis, we may also hypothesize that ␥␦ T cells are rapidly recruited in inflamed tissues to contribute to the killing of invading pathogens. It constitutes one of their major characteristics, and consequently, we cannot rule out the idea that their absolute number in the body could remain normal whereas the balance between circulating and tissue ␥␦ T cells would be modified. Nevertheless, the consequence could be the same. Indeed, ␥␦ T would be concentrated in the initially infected organ whereas absent (or inadequately numerous) in another tissue where a putative nosocomial infection would be secondarily starting. These hypotheses remain to be demonstrated.

On the other hand, we measured a highly significant decrease of CD3 expression on both ␣␤ and ␥␦ T lymphocytes from patients (Fig. 2b). Interestingly, we also observed that the CD3 mean fluorescence intensities ratio (i.e., ␥␦-CD3/␣␤-CD3) was not affected. It is known that ␥␦ T lymphocytes express two-fold more CD3 molecules on their surface than ␣␤ T lymphocytes [START_REF] Thibault | Compared TCR and CD3 epsilon expression on alpha beta and gamma delta T cells. Evidence for the association of two TCR heterodimers with three CD3 epsilon chains in the TCR/CD3 complex[END_REF][START_REF] Nicolas | Human ␥␦ T cells express a higher TCR/CD3 complex density than ␣␤ T cells[END_REF]. We found the ratio to be 2.2 (2.1-2.4) and 2.1 (1.9 -2.3) in healthy individuals and septic patients, respectively. These ratios suggested that the magnitude in the decrease of CD3 expression was similar in ␣␤ and ␥␦ cells. Hence, we may hypothesize that CD3 expressions are down-regulated by a common mechanism in both T-cell lineages. CD3 is mandatory for lymphocyte activation during antigen presentation. Consequently, combined with a reduced percentage of monocyte expressing HLA-DR, a reduced CD3 expression on T lymphocytes may be involved in the failure of a Comorbidities are chronic obstructive pulmonary disease (n ϭ 2), malignant diseases (n ϭ 3), chronic heart failure (n ϭ 1), and chronic renal failure (n ϭ 1). Median and interquartile range were used for continuous variables.

antigen presentation depicted during septic shock [START_REF] Manjuck | Decreased response to recall antigens is associated with depressed costimulatory receptor expression in septic critically ill patients[END_REF]. Despite overlapping values between healthy individuals and septic patients, we found a very significant difference between groups, and CD3 expression may thus deserve further assessment as a marker of immunoparalysis. Furthermore, although not significant, survivors' values were higher than those of nonsurvivors (data not shown, this tendency was also observed for the percentage of circulating ␥␦ T cells that was higher in survivors).

CONCLUSIONS

The present study, which focused on T-cell lineages in septic shock patients, illustrates some modifications that could affect immune responses. These results extend those of Matsushima et al. ( 4) observed in patients with systemic inflammatory response syndrome. Although the consequences of those changes remain to be demonstrated, these preliminary observational results could constitute additional pieces of the complex puzzle of the immunosuppressive state affecting patients after surgery, trauma, burns, or sepsis [START_REF] Munford | Normal responses to injury prevent systemic inflammation and can be immunosuppressive[END_REF]. An improved understanding of the causes and consequences of immune alterations may reveal suitable molecules for restoring the host defense response in case of persisting immunosuppression in critically ill patients.

Objective:

  The mechanisms involved during sepsis-induced immunosuppression are far from being extensively established. The objective of the present study was to investigate whether two characteristics of T cells were altered in this situation: the percentage of circulating ␥␦ T lymphocytes and the level of CD3 expression on T lymphocytes. Design: Observational study. Setting: Adult intensive care units in a university hospital. Patients: Patients with septic shock (n ؍ 21) and healthy individuals (n ؍ 21). Interventions: None. Measurements and Main Results: In patients, we first observed the decreased percentage of ␥␦ T lymphocytes in peripheral blood (1% [0.7-3.1], median [interquartile range]) in comparison with healthy individuals (3.5% [2.1-4.8]). Regarding CD3, we measured a highly significant decrease of its expression on both ␣␤ and ␥␦ T lymphocytes from patients (p < .005), whereas the CD3 mean fluorescence intensities ratio (␥␦/␣␤) was not affected: 2.2 [2.1-2.4] and 2.1 [1.9 -2.3] in healthy individuals and septic patients, respectively. The magnitude in the decrease of CD3 expression was thus similar in ␣␤ and ␥␦ cells, suggesting a common down-regulation mechanism for both T-cell lineages. Conclusions: Combined with a reduced percentage of monocytes expressing human leukocyte antigen-DR, a reduced CD3 expression may be involved in the failure of antigen presentation depicted after septic shock, whereas the diminished percentage of circulating ␥␦ T cells could be partly responsible for the elevated incidence of secondary infections. These two observations constitute additional pieces of the complex puzzle of sepsisinduced immunosuppression. (Crit Care Med 2005; 33:2836-2840)

Figure 1 .

 1 Figure 1. Identification of ␣␤ and ␥␦ T lymphocytes by flow cytometry. a, ungated leukocyte biparametric representation on the basis of side-scatter characteristics (SSC, y-axis) and CD3 expression (ECD-CD3, x-axis). CD3-expressing population (T lymphocytes) is easily distinguishable: gating region T lymphocytes. b, characteristics of ␣␤ lymphocytes. Left, gated cells from T lymphocytes expressed on the basis of ␣␤ expression. The threshold for positivity is easily set up between the two peaks of fluorescence; it provides the percentage of ␣␤ϩ cells. Right, gated cells from T lymphocytes and ␣␤ϩ cells expressed on the basis of CD3 expression; it provides the means of CD3 fluorescence intensities (MFI) related to the entire ␣␤ϩ T lymphocyte population. c, characteristics of ␥␦ lymphocytes. Left, gated cells from T lymphocytes expressed on the basis of ␥␦ expression. The threshold for positivity is easily set up between the two peaks of fluorescence; it provides the percentage of ␥␦ϩ cells. Right, gated cells from T lymphocytes and ␥␦ϩ cells expressed on the basis of CD3 expression; it provides the means of CD3 MFI related to the entire ␥␦ϩ T-lymphocyte population.

Figure 2 .

 2 Figure 2. a, percentages of ␣␤ and ␥␦ T lymphocytes after septic shock. Percentages were determined among total T lymphocytes in whole blood by flow cytometry. Circulating lymphocytes from septic patients were phenotyped once between day 3 and day 5 after shock (open boxes). As control, values from healthy individuals are represented (filled boxes). Results are shown as box-plot representations. The nonparametric Mann-Whitney test was used to assess significances between groups (*p Ͻ .05, **p Ͻ .01). b, CD3 expression on ␣␤ and ␥␦ T lymphocytes after septic shock. CD3 expression, represented as mean of fluorescence intensity (MFI), was measured in septic patients (open boxes) and healthy volunteers (filled boxes). Results are shown as box-plot representations. The nonparametric Mann-Whitney test was used to assess significances between groups (***p Ͻ .005).

Table 1 .

 1 Demographic and clinical characteristics of patients (n ϭ 21)

	No. (Range or %)

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ACKNOWLEDGMENTS

We thank J. Kingsley for greatly assisting in the preparation of the manuscript. This work was conducted thanks to the logistic support (H. Thizy, F. Gueyffier) from the Centre d'Investigation Clinique (Clinical Research Centre) of INSERM and Hospices Civils de Lyon.