Eur. J. Immunol. 2008. 38: 273–282
Clinical immunology
Clinical immunology
Characterization of a subset of antigen-specific human central memory CD4+ T lymphocytes producing effector cytokines Muriel Stubbe1, Nathalie Vanderheyde1, Hanspeter Pircher2, Michel Goldman1 and Arnaud Marchant1 1 2
Institute for Medical Immunology, Universit Libre de Bruxelles, Charleroi, Belgium Institute of Medical Microbiology and Hygiene, Department of Immunology, University of Freiburg, Freiburg, Germany
CCR7+ central memory (TCM) CD4+ T cells play a central role in long-term immunological memory. Recent reports indicate that a proportion of CD4+ TCM is able to produce effector cytokines. The phenotype and the role of this subset remain unknown. We characterized cytokine-producing human CD4+ TCM specific for cleared protein and persistent viral Ag. Our results demonstrate that the type of Ag stimulation is a major determinant of CD4+ TCM differentiation. CMV-specific TCM were significantly more differentiated than protein Ag-specific TCM and included higher proportions of IFN-c-producing cells. The expression of killer cell lectin-like receptor G1 (KLRG1) by protein Ag- and CMV-specific TCM was associated with increased production of effector cytokines. KLRG1+ TCM expressed high levels of CD127, suggesting that they can survive long term under the influence of IL-7. The induction of KLRG1+ TCM may therefore represent an important target of vaccination against pathogens controlled by cellular immune responses.
Received 27/6/07 Revised 20/9/07 Accepted 8/11/07 [DOI 10.1002/eji.200737611]
Key words: Helper T cells Memory cells Vaccination
See accompanying commentary: http://dx.doi.org/10.1002/eji.200738044 Supporting information for this article is available at http://www.wiley-vch.de/contents/jc_2040/2007/37611_s.pdf
Introduction After pathogen or vaccine Ag encounter, emergence of memory T lymphocytes able to mount a rapid effective secondary response and to survive long term is the cornerstone of protective immunological memory [1, 2].
Correspondence: Arnaud Marchant, Institute for Medical Immunology, Rue Adrienne Bolland 8, 6041, Charleroi, Belgium Fax: +32-2-650-9563 e-mail:
[email protected] Abbreviations: HBs: hepatitis B surface Ag KLRG1: killer cell lectin-like receptor G1 SEB: staphylococcal enterotoxin B TCM: central memory T cell TEM: effector memory T cell TT: tetanus toxod f 2008 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
Memory T lymphocytes have been divided in effector and central memory subsets [3]. In the classical model, effector memory T cells (TEM) lack the expression of the CCR7 LN homing receptor and display rapid effector functions. The central memory T cells (TCM) express CCR7 and thereby migrate to the LN. TCM produce IL-2 and have a high proliferative capacity but low effector functions. Studies in mice have shown that CD4+ and CD8+ TCM persist longer in vivo and are more efficient at mediating protective immunity than TEM [4–7]. On the other hand, the induction of CD4+ TCM after SIV challenge correlates with prolonged survival [8]. Riou et al. [9] demonstrated that the longevity and persistence of CD4+ TCM in humans depends, at least in part, on the signal transducer and activator of transcription 5a (STAT5a) www.eji-journal.eu
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Eur. J. Immunol. 2008. 38: 273–282
and forkhead box O3a (FOXO3a). Thus, TCM have a selective advantage for survival and could be more efficient at mediating protective immune responses against infectious diseases [10]. Recent studies indicate that TCM do not represent a homogenous subset of memory lymphocytes but rather include cells with diverse effector functions. Rivino et al. [11] have shown that human CD4+ TCM include precursors of Th1 and Th2 cells expressing distinct chemokine receptors. In addition, several studies in mice demonstrated that the pool of CCR7+ lymphocytes includes cells rapidly expressing effector functions upon activation [12–14]. In line with these observations, we recently reported that human CD4+ TCM specific for protein Ag include cells producing multiple effector cytokines upon short-term stimulation ex vivo [15]. The relationship between the functional heterogeneity of TCM and their capacity to survive and to mediate protective immune responses is unclear. TCM producing effector cytokines may represent a subset of embellished memory cells with increased capacity to mediate protective immunity [16]. To test this hypothesis, it will be important to determine whether TCM producing cytokines represent a distinct subset of memory cells expressing a specific differentiation phenotype. The differentiation markers of CD4+ T cells currently available have not allowed the identification of TCM subsets [17–19]. In this study, we evaluated whether CD4+ TCM producing effector cytokines can be distinguished on the basis of their expression of the IL-7 receptor alpha-chain (CD127) and the differentiation marker killer cell lectinlike receptor G1 (KLRG1). CD127 plays a critical role in the survival of memory CD4+ T cells in mice [20–23] and humans [9, 24]. The expression of CD127 was shown to be inversely correlated with the differentiation of memory CD8+ T lymphocytes [25, 26]. KLRG1 is an inhibitory NK receptor expressed by human CD8+ T cells specific for persistent viruses [27, 28]. KLRG1 expression is associated with poor proliferative capacity and increased IFN-c production. Interestingly, recent data suggest that KLRG1 can be expressed at an early stage of
CD8+ T cell differentiation [27, 28]. Using CD154 production as a marker of Ag-specific CD4+ T cells [15, 29, 30], we tested the hypothesis that the phenotype and function of TCM are influenced by the type of Ag they recognize. TCM specific for two cleared protein Ag, TT and HBs, inducing an early stage of CD4+ T cell differentiation were compared to TCM specific for CMV, a persistent virus inducing an advanced stage of CD4+ T cell differentiation [31].
Results Size and phenotype of Ag-specific memory CD4+ T cells populations The proportion of individuals with detectable Agspecific memory cells and the mean frequencies are described in Table 1. All donors responded to stimulation with staphylococcal enterotoxin B (SEB), which was used as positive control. Five (28%), 17 (94%) and 7 (39%) of the 18 donors had detectable hepatitis B surface Ag- (HBs-), tetanus toxod- (TT-) and CMVspecific cells, respectively. The mean frequencies SEM were 1.04 0.48/1000 CD4+ T cells (HBs), 2.59 0.47/1000 CD4+ T cells (TT) and + 10.54 4.16/1000 CD4 T cells (CMV). Dot plots of representative donors are available as Supporting Information Fig. 1. All CMV-seropositive donors had detectable CD4+ T cell responses to CMV Ag, whereas no CD154+ cells were detected among CMV-seronegative donors, further supporting the sensitivity and specificity of the method to identify Ag-specific cells. The phenotype of CD154+CD4+ memory T cells following TT (n=17 responders), HBs (n=5) and CMV lysate (n=7) short-term in vitro stimulation was analyzed and compared to the phenotype of the total memory CD4+ T cells (Fig. 1A). Previous reports have proposed a linear model of CD4+ T cells differentiation involving the sequential loss of the expression of CD27 and CD28 [17–19]. We observed that nearly all TT- and HBs-specific cells expressed CD28 but a proportion of
Table 1. Frequencies of memory CD154+CD4+ T cells Stimulus
Donors with detectable memory CD154+ CD4+ T cells
Frequency of memory CD154+ cells/ 1000 CD4+ T cells (mean SEM)
HBs protein vaccinated donors with anti-HBs IgG>10 UI/mL
5/18
1.04 0.48
Tetanus toxoid vaccinated donors
17/18
2.59 0.47
CMV lysate CMV-seropositive donors CMV-seronegative donors
7/7 0/11
10.54 4.16 0.06 0.05
Staphylococcal enterotoxin B
18/18
191.42 23.98
f 2008 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
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Eur. J. Immunol. 2008. 38: 273–282
them had lost the expression of CD27. A higher frequency of HBs-specific cells were CD27– as compared to TT-specific cells but this difference was not significant (p=0.09). The majority of CMV-specific cells were CD27– and, within this subset, a large proportion of cells had lost CD28 expression (Fig. 1B). These results indicate that TT- and HBs-specific cells accumulate at an intermediate stage of differentiation, whereas a more advanced stage of differentiation characterizes CMV-
Clinical immunology
specific cells, confirming recently reported observations [31]. As shown in Fig. 1A, the large majority of TT- and HBs-specific cells expressed CD127, whereas more than the half of CMV-specific cells had lost CD127 expression. The differences in CD127 expression between TT-/HBsand CMV-specific cells were also apparent when the mean fluorescence intensities (MFI) were compared (Supporting Information Fig. 2). Co-analysis of CD127 and CD27 expression indicated that, for the three Ag
Figure 1. Phenotype of Ag-specific memory CD4+ T cells. PBMC were obtained from healthy donors (n=5–17) and were stimulated in vitro with TT (10 lg/mL), HBs (10 lg/mL) or CMV lysate (5 lg/mL) for 18 h. Ag-specific cells were identified by intracytoplasmic CD154 staining and flow cytometry. Expression of CD28, CD27, CD127 and KLRG1 was measured at the surface of Ag-specific or total memory CD4+ T cell populations (A); * p
