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International Immunology Advance Access originally published online on June 23, 2006
International Immunology 2006 18(8):1337-1345; doi:10.1093/intimm/dxl066
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© The Japanese Society for Immunology. 2006. All rights reserved. For permissions, please e-mail: journals.permissions@oxfordjournals.org

Human germinal center T cells are unique Th cells with high propensity for apoptosis induction

Ekaterina Marinova, Shuhua Han and Biao Zheng

Department of Immunology, Baylor College of Medicine, M929, One Baylor Plaza, Houston, TX 77030, USA

Correspondence to: B. Zheng; E-mail: bzheng{at}bcm.tmc.edu


   Abstract
Top
 Abstract
Introduction
 Methods
 Results
 Discussion
 References
 
Collaborative interactions between Th cells and B cells are necessary for the production of antibody responses to most protein antigens and for the generation of memory B cells in germinal centers (GCs). Although it is well established that Th cells are pivotal for the GC reaction, the mechanisms that control the homeostasis of Th cells during the GC response remain largely unknown. Here we show that, unlike other effector T cells, a significant number of CD4+CD45RO+CD57+ T cells, which are the major Th cells residing in the GCs, are undergoing apoptosis in vivo. CD4+CD45RO+CD57+ GC T cells exhibit similar sensitivities to apoptotic signals and to caspase inhibitors as immature thymocytes. Moreover, CD4+CD45RO+CD57+ GC T cells express a unique profile of genes that control apoptosis and cell cycle, providing possible molecular mechanisms for the high rates of apoptotic death of these Th cells in the GCs.

Keywords: apoptosis, germinal center, helper T cells


   Introduction
Top
 Abstract
 Introduction
Methods
 Results
 Discussion
 References
 
Germinal center (GC) reaction is a T cell-dependent process that drives a series of important events including clonal expansion and selection of antigen-specific B cells, somatic hypermutation (SHM) of Ig genes, affinity maturation of antibody responses and generation of B cell memory (1, 2). Critical to the maintenance of immunological homeostasis, GC response is self-limiting and exhibits a major contraction of GC cell populations in ~3 weeks after antigenic stimulation (1, 2). GC Th cells are a requisite component of the GC response; in the absence of T cell help, functional GCs are not formed (3, 4). Importantly, GCs elicited in the absence of Th cells or induced by T-independent antigens are short lived and functionally defective in Ig SHM and B cell memory development (5, 6). Despite their central role in humoral immunity, little is known about the Th populations in GCs. It has been shown that the intrinsic properties of GC B cells may partly contribute to the decadence of GC reaction in vivo (7, 8). However, how the wax and wane of Th population in the GC are regulated remains largely unknown. To date, only a few studies have addressed the phenotypes and functions of follicular and/or GC Th cells (9–13). Our earlier in vivo work in a mouse model has shown a distinctive kinetics of GC T cell response and suggested that GC T cells may represent an alternative pathway for the selection of antigen-specific peripheral T cells (14).

In the current study, we have investigated the mechanisms that control the apoptosis induction in GC Th cells by studying the dynamics and property of human tonsillar GC T cells. We show that human CD4+CD45RO+CD57+ T cells, which are the major Th cells found in the GCs, are constantly undergoing apoptosis. The most striking difference between CD4+CD45RO+CD57+ GC T cells and other mature peripheral T cells, including naive and memory/effector T cells, is that CD4+CD45RO+CD57+ GC T cells exhibit high sensitivity to apoptosis induction, similar to that of developing thymocytes. Moreover, CD4+CD45RO+CD57+ GC T cells express unique profiles of apoptotic antagonists and agonists that distinguish these Th cells from other effector T cells in the periphery. These findings demonstrate that CD4+CD45RO+CD57+ GC T cells possess unique propensity to apoptotic death, which may represent a mechanism of controlling homeostasis of Th cells during the GC response.


   Methods
Top
 Abstract
 Introduction
 Methods
Results
 Discussion
 References
 
Cell preparation
Human tonsillar cells were prepared from tonsils removed during routine tonsillectomy as previously described (15). Thymus tissues were obtained from pediatric patients during heart surgery for congenital heart diseases. Mononuclear cells were purified by centrifugation through an ionic density gradient medium (Lymphoprep Medium, Nycomed Pharma AS, Oslo, Norway).

Cell staining and cell sorting
T cells were purified by incubating with biotinylated antibodies specific to human CD19, CD40 and Ig, followed by streptavidin-coupled magnetic microbeads (Miltenyi Biotec, Gladbach, Germany). Cells were passed over a magnetized separation column to deplete B cells. For FACS, mAbs used in this study were FITC-conjugated anti-CD57, PE-conjugated anti-CD45RO, biotinylated anti-CD4 with streptavidin–allophycocyanin, PE-conjugated anti-CD3 and FITC-conjugated anti-CD8 (all purchased from BD/Pharmingen). The stained cells were analyzed on a FACScaliber with CellQuest software. Cells were sorted using MoFlo® (Cytomation, Ft. Collins, CO) into Trizol or PBS with 10% FCS. The purity of each isolated T cell subset was routinely above 90%.

RNA preparation and reverse transcriptase–PCR
Total RNA was prepared and reverse transcribed into cDNA with Oligo dT using the SuperScript kit (GIBCO). Thirty-five cycles of amplification were carried out using the following specific primer sets: Fas sense 5'-CTG CAT CAT GA TGG CCA ATT CTG C-3', antisense 5'-GCC TTT AAC TTG ACT TAG TGT CAT-3'; Fas ligand (FasL) sense 5'-CAG CTC TTC CAC CTG CAG AAG-3', antisense 5'-AGA TTC CTC AAA ATT GAT CAG AGA GAG-3'; GAPDH sense 5'-CAT GTG AGG TCG GAG TCA ACG GAT TTG GT-3', antisense 5'-CAT GTG GGC CAT GAG GTC CAC CAC-3'. The PCR products were analyzed on agarose gels and the net intensity of individual bands was analyzed using Kodak 1D image analysis software.

cDNA arrays
Human Apoptosis 4/Bcl-2 family and regulators microarray kits were obtained from SuperArray (Bethesda, MD). The procedure was carried out as recommended by the manufacturer. The filters were exposed on PhosphorImager and the specific array signal spots were analyzed with GEArray Analyzer software provided by SuperArray.

In vitro culture of T cell subsets and apoptosis induction
For measuring ex vivo apoptosis, purified CD4+ T cells (3 x 106 per well) are cultured in 24-well plates for 48 h in the presence of 100 ng ml–1 plate-bound anti-human Fas mAb (clone CH11, UPSTATE) or FasL (UPSTATE). The apoptotic cells are enumerated by AnnexinV, CD4, CD45RO and CD57 staining and analyzed by flow cytometry gated on GC (CD4+, CD45RO+, CD57+), memory (CD4+, CD45RO+, CD57–) and naive (CD4+, CD45RO–, CD57–) T cells.

For in vitro apoptosis induction, purified T cell subsets (2 x 106 per well) and equal number of irradiated (3000 rads) T cell-depleted tonsillar cells were cultured in 24-well plates for 5 days in the presence of anti-CD3 (5 µg ml–1)/anti-CD28 (2.5 µg ml–1) or ConA (5 µg ml–1)/IL-2 (5 µg ml–1). Viable T cells were purified by centrifugation through an ionic density gradient medium and cultured in triplicate for 48 h at 4 x 105 per well in 96-well plates in the presence or absence of coated anti-CD3 or anti-Fas mAbs (10 µg ml–1). The percentage of apoptotic cells was calculated as above.

For apoptosis induction in the presence of caspase inhibitors, human thymocytes or purified tonsil T cell subsets were cultured with 1 µM dexamethasone, immobilized anti-CD3 or anti-Fas (5 µg ml–1) mAbs in the presence of various concentrations of peptide-fluoromethyl ketone (fmk) (all purchased from Enzyme Systems Products, CA). Cell death was assessed 24 h later by trypan blue exclusion and calculated as a percentage of live cells/total cells.

Apoptosis analysis by flow cytometry
The level of apoptosis was assessed using AnnexinV staining (BD/Pharmingen). AnnexinV–FITC was used in combination with anti-human CD4–allophycoerythrin, CD45RO–PE and CD57–biotin, followed by incubation with streptavidin–PerCP (Pharmingen). The cells were labeled with AnnexinV–FITC for 30 min in binding buffer. Cells within the live lymphocyte gate were analyzed using FACScaliber.

Immunohistology
Tonsil sections were stained to detect apoptotic cells using the in situ cell death detection kit (Roche Diagnostics GmbH, Germany). CD57+ GC T cells were labeled by biotinylated anti-CD57 mAb (Pharmingen) followed by SA-AP (Southern Biotechnology, Birmingham, AL). Bound HRP and AP activities were visualized with 3-aminoethylcarbazole and naphthol-AS-MX phosphate/Fast Blue BB (Sigma), respectively.


   Results
Top
 Abstract
 Introduction
 Methods
 Results
Discussion
 References
 
In vivo apoptosis of GC Th cells
In human tonsil, the vast majority of GC T cells are phenotypically distinct Th cells that are CD4+, CD45RO+ and CD57+ (9, 12, 13). It has also been reported that the expression of receptor for chemokine CXCL13, CXCR5, may define T cells with B cell helper function (10–12). To study the functional property of GC T cells, we separated human tonsil CD4+ T cells into three subsets, GC, memory/effector and naive T cells, according to the surface expression of CD45RO and CD57. Among tonsil CD4+ T cells, the proportion of GC, memory/effector or naive T cells is ~5–10%, 35–45% or 45–55%, respectively (Fig. 1A). All the CXCR5+ CD4+ T cells are antigen experienced (CD45RO+), ~15% of them are GC T cells and the rest of them are memory/effector T cells (Fig. 1B). Among CXCR5– CD4+ T cells, ~50–60% of them are naive T cells, 40–50% exhibit memory/effector T cell phenotype and very few CXCR5– T cells belong to the GC T cell subset (Fig. 1B). Thus, both CXCR5+ and CXCR5– T cell populations are heterogeneous in terms of cellular differentiation and helper function. These results suggest that although the majority of T cells up-regulate CXCR5 expression after activation, only a small subset of these activated T cells can gain access to the GC area and become effective GC Th cells. Throughout this study, we have used CD4+, CD45RO+ and CD57+ to define and isolate GC T cell subset.


Figure 1
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  		Fig. 1 Sub-populations of tonsillar CD4+ T cells. (A) According to the expression of CD45RO and CD57, tonsil CD4+ T cells were divided into three subsets, CD45RO+CD57+ GC T cells, CD45RO+CD57– memory/effector T cells and CD45RO–CD57– naive T cells. The percentages of each sub-population in CD4+ T cells are shown in the right panel. (B) Composition of CXCR5+ or CXCR5– T cells. CXCR5+ or CXCR5– CD4+ T cells were gated and further analyzed for their CD45RO and CD57 expression.

 
To confirm whether CD4+CD45RO+CD57+ GC T cell fraction contains the most effective Th cells, we tested the efficacy of various T cell subsets in helping B cell antibody production in vitro. Purified CD19+ B cells were co-cultured with sorted T cell subsets for 10 days and antibody production in the supernatant was measured by ELISA. Our data show that the ability of GC T cells to promote antibody production is much greater than that of T cells with memory/effector or naive phenotypes, whose co-cultures with tonsil B cells only produces minimal levels of IgM or IgG (data not shown). Thus, these functional assays further confirm that CD4+CD45RO+CD57+ GC T cells are the most effective helper cells for antibody response, while CXCR5 expression is an activation marker and not truly associated with Th function. These data are consistent with previous work showing that GC T cells effectively promote tonsil B cells to secret antibodies (12, 16, 17).

To examine the dynamics of CD4+CD45RO+CD57+ GC T cells in vivo, we have estimated the proportion of GC T cells that are undergoing apoptosis by in situ or ex vivo approaches. By AnnexinV staining, we found that among freshly isolated tonsil T cells, the proportion of CD4+CD45RO+CD57+ GC T cells that are undergoing apoptosis was significantly higher than that of naive T cells or memory/effector T cells (Fig. 2A). 22.7 ± 3.5% (mean ± SE) of GC T cells were undergoing apoptosis, whereas in naive and memory T cells, the percentages of apoptotic cells were 7.7 ± 1.0 and 7.4 ± 2.6%, respectively. These results were substantiated by in situ labeling of apoptotic cells (Fig. 2B), which showed that a significant proportion of CD4+CD45RO+CD57+ GC T cells were labeled by the TUNEL assay. Thus, our findings demonstrate that, compared with other CD4+ T cells in the periphery, GC T cells exhibit highest rate of apoptosis in vivo.


Figure 2
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  		Fig. 2 In vivo apoptosis of GC T cells. (A) Ex vivo labeling of tonsillar T cell subsets by AnnexinV. Red line, GC T cells; blue line, memory/effector T cells; green line, naive T cells. (B) In situ labeling of apoptotic cells by TUNEL assay. Tonsil sections were labeled by TUNEL assay for apoptotic nuclei (red) and anti-CD57 mAb for GC T cells (blue). Arrows indicate apoptotic GC T cells. Original magnification, x200.

 
GC T cells exhibit elevated propensity to both TCR- and Fas-mediated apoptosis
To investigate the mechanisms that control the homeostasis of Th cells during GC response, we first examined the sensitivity of T cell subsets to apoptosis induced by TCR- and Fas-mediated signaling. When freshly isolated T cell sub-populations were tested for their sensitivity to apoptosis induction, we found that apoptosis induced by immobilized anti-CD3 or anti-Fas antibody was significantly higher in CD4+CD45RO+CD57+ GC T cells than in naive or memory T cells (Fig. 3A and B). Similar results were obtained when freshly isolated T cells were signaled by FasL (data not shown).


Figure 3
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  		Fig. 3 Activation-induced apoptotic death in GC T cells. (A and B) Ex vivo apoptosis induction by anti-Fas or anti-CD3. Freshly isolated GC (black bars), naive (gray bars) and memory (open bars) T cells were cultured in the presence of immobilized anti-Fas or anti-CD3 mAb for 48 h and percentages of apoptotic cells were enumerated. Data (mean ± SE) are representative of three independent experiments. (C and D) Apoptosis induced following in vitro activation. Purified GC (black bars), naive (gray bars) and memory (open bars) T cells were cultured in the presence of anti-CD3 and anti-CD28 mAbs for 5 days. Live cells were then purified and further cultured in the presence of immobilized anti-Fas or anti-CD3 mAbs for 48 h. Data (mean ± SE) are representative of five independent experiments. (E) Apoptosis induction in the absence or presence of exogenous IL-2. Cells were stimulated with anti-CD3/anti-CD28 mAbs in the absence (open bars) or presence (black bars) of IL-2 for 5 days and apoptotic cells were enumerated by AnnexinV staining. Data (mean ± SE) are representative of three independent experiments.

 
We further examined the sensitivity to apoptosis induction in T cell subsets after activation in vitro. T cell subsets were cultured with anti-CD3 and anti-CD28 antibodies for 5 days. Then, live cells were purified and further cultured with anti-Fas or anti-CD3 antibodies for 48 h. The results show that all the T cell subsets had increased sensitivity to apoptosis induction by immobilized anti-Fas (Fig. 3C) or anti-CD3 (Fig. 3D) antibody compared with apoptosis induced in freshly isolated T cell subsets. However, CD4+CD45RO+CD57+ GC T cell population still contained a significantly higher proportion of apoptotic cells than naive or memory T cells. Interestingly, memory T cell subset contained the lowest level of apoptotic cells following in vitro activation, suggesting that memory T cells are the most resistant subset to activation-induced apoptosis, which may indicate a distinct property of memory T cells for sustained memory responses including clonal expansion.

It has been shown that GC Th cells are anergic to TCR stimulation and rely on external IL-2 to proliferate (13). We have also observed similar results showing that GC T cells proliferate poorly after further TCR signaling in vitro and require exogenous IL-2 to proliferate (data not shown). Thus, it would be interesting to determine whether GC Th cells still exhibit higher rate of apoptosis in the presence of exogenous IL-2. We have analyzed in vitro TCR-mediated apoptosis induction in GC Th cells in the absence or presence of exogenous IL-2 and compared with that of memory/effector or naive T cells. Our findings have demonstrated that CD4+CD45RO+CD57+ GC T cells exhibit elevated apoptosis induction even in the presence of exogenous IL-2 (Fig. 3E). Therefore, these results suggest that the increased apoptosis induction in GC Th cells is not due to their inability to proliferate.

The propensity of different T cell subsets to apoptosis elicited by Fas ligation was well reflected by their levels of Fas and FasL expression. Both freshly isolated CD4+CD45RO+CD57+ GC T cells and T cells with memory/effector phenotypes express high level of Fas, but only CD4+CD45RO+CD57+ GC T cells express significant level of FasL on their surface (Fig. 4A and C). After in vitro activation, all T cell subsets increased their Fas expression; however, there was significant difference between their ability to up-regulate FasL expression, with the highest FasL expression on CD4+CD45RO+CD57+ GC T cells (Fig. 4B and C). About 20% of CD4+CD45RO+CD57+ GC T cells express high levels of both Fas and FasL, whereas ~5% of naive T cells after activation express intermediate levels of Fas and FasL, and very few T cells with the memory/effector phenotype increased their FasL expression after further stimulation in vitro. Given the fact that GC T cells are mostly concentrated in the light zone of the GCs (1), these results suggest that Fas-FasL-mediated suicidal apoptosis may play an important role in the decline of GC T cells.


Figure 4
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  		Fig. 4 Fas and FasL expression in tonsillar T cell subsets. (A) Fas and FasL expression on freshly isolated tonsil T cell sub-populations. Percentages of Fas+/FasL+ cells in each T cell subset are shown. (B) Fas and FasL expression on tonsil T cell sub-populations 3 days after stimulation with anti-CD3 and anti-CD28 mAbs. Percentages of Fas+/FasL+ cells are shown. (C) Mean fluorescence intensity (MFI) of Fas or FasL expression on T cell subsets before (open bars) or after (black bars) stimulation with anti-CD3 and anti-CD28 mAbs. (D) Representative reverse transcriptase–PCR analysis of Fas and FasL expression in T cell subsets. (E) The expression levels of Fas and FasL in GC (black bars), naive (gray bars) or memory/effector (open bars) T cells were normalized by the expression level of the control gene, GAPDH. Data are representative of three independent experiments.

 
The expression levels of Fas and FasL on different T cell subsets were further examined by semi-quantitative reverse transcriptase–PCR (Fig. 4D and E). Consistent with the results of Fas/FasL expression determined by flow cytometric analyses, only CD4+CD45RO+CD57+ GC T cells contained high message levels for both Fas and FasL, whereas FasL expression in naive or memory/effector T cells was minimal.

Therefore, our findings that GC T cells express both Fas and FasL suggest that GC T cells may play a role in controlling the clone size of both T and B cells in the GC. Importantly, these findings demonstrate that, although both GC and memory/effector T cell subsets have encountered and been activated by antigens, they respond to further stimulation quite differently. Our results suggest that CD4+CD45RO+CD57+ GC T cells may respond to activation signals by committing suicidal apoptosis, resulting in limited clone size of this unique T helper subset. On the other hand, other activated T cells including memory/effector T cells respond to further TCR signaling by continuing cellular proliferation, leading to sustained clonal expansion.

GC T cells show similar sensitivity to caspase inhibitors as developing thymocytes
It has been demonstrated that T cell maturation is accompanied by a markedly diminished sensitivity to the broad-spectrum cell permeable caspase inhibitor, benzyloxycarbonyl (Cbz)-Val-Ala-Asp (Ome)-fluoromethyl ketone (zVAD-fmk), in that zVAD-fmk blocks apoptosis induced by various stimuli in developing thymocytes but not in more mature peripheral T cells (18). To understand the mechanisms that regulate apoptosis induction in different T cell subsets, we wanted to determine whether CD4+CD45RO+CD57+ GC Th cells exhibit distinctive sensitivity to caspase inhibitors. Here, we investigate the effects of zVAD-fmk and its truncated analog Boc-Asp (Ome)-fmk (BD-fmk) on blocking apoptosis induction in thymocytes and different tonsil T cell subsets in order to explore the possibility that different caspases are recruited in different T cell subsets during apoptosis induction.

As expected, apoptosis in human immature thymocytes triggered by different stimuli was blocked by both zVAD-fmk and BD-fmk but not by the control peptide, Cbz-Phe-Ala-fmk (zFA-fmk), which lacks the critical Asp (Fig. 5, top panel). In contrast, only BD-fmk, but not zVAD-fmk, was able to block apoptosis induced in naive or memory/effector T cell subset (Fig. 5, second and third panels). Remarkably, apoptotic death induced in CD4+CD45RO+CD57+ GC T cells was blocked by both zVAD-fmk and BD-fmk (Fig. 5, bottom panel), resembling that in developing thymocytes. Therefore, these findings show that CD4+CD45RO+CD57+ GC T cells exhibit thymocyte-like sensitivity to caspase inhibitors, indicating that, during apoptosis induction, CD4+CD45RO+CD57+ GC T cells may recruit the same members of caspases as immature thymocytes but different members of caspases from those recruited by other peripheral T cell subsets.


Figure 5
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  		Fig. 5 Differential effects of caspase inhibitors on apoptotic death in thymocytes and peripheral T cell subsets. Total human thymocytes and purified tonsillar T cell subsets were treated with 1 µM dexamethasone, immobilized anti-CD3 (5 µg ml–1) or anti-Fas (5 µg ml–1) mAb for 24 h in the presence of various concentrations of BD-fmk (squares), zVAD-fmk (diamonds) or control zFA-fmk (triangles). Data (mean ± SE) are representative of three independent experiments.

 
GC T cells express unique profile of apoptotic antagonists and agonists
To further understand the molecular mechanisms contributing to the unique propensity of CD4+CD45RO+CD57+ GC T cells to apoptosis, we analyzed the expression of Bcl-2 protein family members and related genes in CD4+CD45RO+CD57+ GC T cells and other peripheral T cell subsets. Significantly, the expression pattern of Bcl-2 family genes in CD4+CD45RO+CD57+ GC T cells is opposite to that of naive or memory/effector T cells (Fig. 6). Of particular interest are the extremely low expression levels of both Bcl-2 (Fig. 6A) and Bcl-x (Fig. 6B) in CD4+CD45RO+CD57+ GC T cells. Our results are consistent with the recent histologic study showing that Bcl-2 is down-regulated in GC CD4+ T cells (19). On the other hand, among the important pro-apoptotic molecules including Bad, Bak, Bax and Bid, only Bax expression was slightly decreased in CD4+CD45RO+CD57+ GC T cells (Fig. 6C), the rest were all significantly elevated in GC T cells (Fig. 6D–F). It has been suggested that the overall ratio of death agonists versus antagonists determines the susceptibility to a death stimulus (20). The final balance of these pro-/anti-apoptotic genes is likely to be responsible for the higher susceptibility of CD4+CD45RO+CD57+ GC T cells to apoptosis induction. Thus, this distinctive expression pattern of pro- and anti-apoptotic proteins in CD4+CD45RO+CD57+ GC T cells may contribute to the mechanisms that distinguish CD4+CD45RO+CD57+ GC T cells from other mature peripheral T cell subsets in responding to apoptotic stimuli.


Figure 6
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  		Fig. 6 GC T cells exhibit a unique gene profile of apoptosis and cell cycle control. The gene expression profiles associated with GC T cells (black bars), naive T cells (gray bars) or memory/effector T cells (open bars) were analyzed with a gene array kit. The expression levels of each gene were normalized against the expression of ß-actin. Specific array signal spots were analyzed with GEArray Analyzer software. Identical experiments were repeated three times.

 
In addition, CD4+CD45RO+CD57+ GC T cells exhibit a unique expression pattern of genes that control cell cycle. The expression of stratifin (14-3-3 {sigma}), which is a negative regulator of the cell cycle progression and contributes to G2 arrest (21), is significantly down-regulated in CD4+CD45RO+CD57+ GC T cells (Fig. 6G). Other factors that promote cell growth and cell cycle progression, such as ciliary neurotrophic factor, insulin-like growth factor-1 and IL-3, are significantly up-regulated in CD4+CD45RO+CD57+ GC T cells (Fig. 6H–J).


   Discussion
Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
References
 
GC reaction is T cell dependent and directs SHM of Ig genes, affinity maturation of antibody response and generation of the B cell memory compartment. One of the fundamental questions regarding GC biology has been the property of Th cells that reside in the GC. The dynamics of the GC T cell population had not been clearly defined. It is not clear how the wax and wane of Th population is regulated during the GC response. Importantly, the responsiveness and outcome of GC T cells to TCR signaling has not been well studied. In the current study, we have defined the in vivo population sizes of CD4+CD45RO+CD57+ GC T cells that are undergoing programmed cell death. In addition, we have investigated the sensitivity of CD4+CD45RO+CD57+ GC T cells to TCR- and Fas-mediated signals and the molecular mechanisms that regulate apoptosis in this unique subset of Th cells.

Using ex vivo or in situ approaches, we found that, unlike naive or memory/effector T cells, which are in a relatively static state, a significantly higher proportion of CD4+CD45RO+CD57+ GC T cells are constantly undergoing apoptosis in vivo. This unique phenotype of CD4+CD45RO+CD57+ GC T cells resembles that of GC B cells (1, 2, 7, 8, 22–24), suggesting that these GC Th cells may also constantly be subject to the clonal selective process in the GC microenvironment. The higher propensity of CD4+CD45RO+CD57+ GC T cells to apoptosis was further revealed by TCR- or Fas-mediated apoptosis. Freshly isolated CD4+CD45RO+CD57+ GC T cells were significantly more sensitive to apoptosis induction by CD3- or Fas-cross-linking than naive or memory T cells. The low sensitivity to apoptosis in naive or memory/effector T cells may be explained by the fact that all naive T cells and most memory/effector T cells were at resting stage. Indeed, after in vitro activation, both naive and memory T cells increased their sensitivity to apoptosis induction. However, compared with CD4+CD45RO+CD57+ GC T cells, naive or memory T cells still exhibited significantly lower sensitivity to apoptosis induction.

One possible mechanism for the higher propensity of CD4+CD45RO+CD57+ GC T cells to apoptosis is that these GC T cells express high levels of both Fas and FasL. Memory T cells only express Fas but not FasL expression; naive T cells do not express either Fas or FasL. After in vitro activation by anti-CD3 and anti-CD28 mAbs, CD4+CD45RO+CD57+ GC T cells further up-regulated their expression of both Fas and FasL. Although naive and memory T cells increased their Fas expression after activation, FasL+ T cells among these two subsets were very small. Thus, our data indicate that Fas expression reflects the activation status of T cells, whereas FasL expression may indicate the sensitivity of T cells to apoptosis induction. These findings suggest that suicidal apoptosis may be responsible for controlling the population size of activated T cells in the GC. In addition, our data suggest that CD4+CD45RO+CD57+ GC T cells are constantly removed by apoptosis whereas memory T cells are resistant to apoptosis induction. These results are consistent with the notion that memory T cells are long lived and will undergo cell division to generate effector cells upon reactivation. Interestingly, it has been reported that human non-GC CD57+ T cells in peripheral blood up-regulate Fas and FasL expression after activation in vitro, and these T cells exhibit higher susceptibility to apoptosis induction (25), providing another example of Fas-mediated suicidal in T cell apoptosis and homeostasis. It should be noted that GC T cells and peripheral blood CD57+ T cells are totally different T cell subsets, since most of peripheral blood CD57+ T cells are CD8+ and have a capacity to produce a larger amount of IFN-{gamma} (26–29). It has been suggested that peripheral blood CD57+ T cells differentiate extra-thymically and increase dramatically in transplantation, autoimmunity, AIDS and aging (26–32). In addition, unlike GC T cells, which have high expression of Fas and FasL in vivo, peripheral blood CD57+ T cells up-regulate Fas and FasL expression after activation in vitro (25).

Not only the propensity to apoptosis varies in different T cell subsets but also the utilization of caspase members in different T cell subsets may differ when apoptotic death is triggered by the same input pathway. To investigate the possibility that different caspases are recruited during apoptosis induction between CD4+CD45RO+CD57+ GC T cells and other T cell subsets, we employed two caspase inhibitors, BD-fmk and zVAD-fmk. It has been shown that apoptosis induced in immature thymocytes is equally sensitive to the blocking effect of either BD-fmk or zVAD-fmk, whereas apoptosis in mature T cells is less sensitive to the inhibitory effect of zVAD-fmk (18). Our results show that CD4+CD45RO+CD57+ GC T cells display thymocyte-like susceptibility to caspase inhibitors, in that apoptosis induced in CD4+CD45RO+CD57+ GC T cells is blocked by either zVAD-fmk or BD-fmk, whereas apoptosis induced in other mature T cell subsets in the periphery such as naive and memory T cells is resistant to zVAD-fmk. The underlying mechanism for the differential inhibitory effects of zVAD-fmk on apoptosis induced in immature thymocytes, CD4+CD45RO+CD57+ GC T cells or other mature peripheral T cells may be explained by the differential susceptibility of different caspases to zVAD-fmk. Although considered a broad-spectrum caspase inhibitor, zVAD-fmk has very different potencies in inhibiting enzymatic activities of different human caspases (33, 34), with second-order inactivation rates ({kappa}) that range from 2.9 x 102 M–1 S–1 for caspase-2 to 2.6 x 105 M–1 S–1 for caspase-1 and -8 (33). On the other hand, BD-fmk has been shown to be more specific for caspase-3-like protease than zVAD-fmk (18). Therefore, during apoptosis induction, CD4+CD45RO+CD57+ GC T cells may utilize different members of caspases from other mature peripheral T cell subsets, but the same caspases as immature developing thymocytes. Currently, we are investigating which caspases are differentially activated during apoptosis induction between CD4+CD45RO+CD57+ GC T cells and other peripheral T cell subsets.

Although there were two gene expression profiling studies on human tonsil GC T cells in comparison with other T cells (34, 35), the expression of Bcl-2 family genes and genes that regulate cell cycle are not well defined. Our data from analysis of gene expression by microarray demonstrates that CD4+CD45RO+CD57+ GC T cells exhibit an overall expression pattern with down-regulated anti-apoptotic genes and up-regulated pro-apoptotic genes. On the other hand, CD4+CD45RO+CD57+ GC T cells express a gene array profile with increased expression of genes that promote cell growth and cell cycle progression, whereas genes that negatively regulate cell progression are down-regulated. This gene expression pattern may provide the molecular mechanisms that control the dynamics of CD4+CD45RO+CD57+ GC T cells.

In summary, the present study has identified the unique properties of CD4+CD45RO+CD57+ GC T cells in responding to activation or apoptotic stimuli. Our observations suggest that CD4+CD45RO+CD57+ GC T cells utilize the regulatory mechanisms similar to that of developing thymocytes but distinct from that of mature naive T cells or memory/effector T cells. Since T help signals are critical in controlling the initiation and maintenance of GC reaction (36–40), these findings provide additional mechanisms that regulate the clonal expansion of activated B-lymphocytes and control the GC reaction. Our results suggest that CD4+CD45RO+CD57+ GC T cells are hypersensitive to apoptotic signals and are programmed to be short lived, which may restrict GC reaction within an appropriate magnitude and lifespan.


   Abbreviations
 
Cbz, benzyloxycarbonyl
FasL, Fas ligand
fmk, fluoromethyl ketone
GC, germinal center
SHM, somatic hypermutation
zVAD-fmk, benzyloxycarbonyl (Cbz)-Val-Ala-Asp (Ome)-fluoromethyl ketone

   Notes
 
Transmitting editor: D. Tarlinton

Received 3 November 2005, accepted 28 May 2006.


   References
Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 

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