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The Journal of Immunology, 2001, 166: 7200-7207.
Copyright © 2001 by The American Association of Immunologists

Differential Survival of Transferred CD8 T Cells and Host Reconstitution Depending on TCR Avidity for Host-Expressed Alloantigen1

Nathalie Auphan-Anezin2 and Anne-Marie Schmitt-Verhulst

Centre d’Immunologie de Marseille-Luminy, Centre National de la Recherche Scientifique-Institut National de la Santé et de la Recherche Médicale-Université de la Méditerranée, Campus de Luminy, Marseille, France


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
We transferred naive alloreactive CD8 T cells from TCR transgenic mice to irradiated recipients expressing a partial (H-2Kbm8) or a full (H-2Kb) agonist alloantigen (alloAg). The consequences were strikingly distinct, resulting in acceleration of host lymphopoiesis in the former group, but in strong graft-vs-host reaction, preventing host lymphocyte reconstitution in the latter group. This was correlated, respectively, with long-term persistence and with rapid disappearance of the transferred CD8 T cells. Analysis of transferred T cells showed that initial T cell expansion and modulation of expression of activation markers CD44 and CD62L, as well as induction of cytotoxic function, were similar in both groups. However, IL-2 production and subsequent up-regulation of CD25, early perforin-independent cytolysis, and early down-regulation of Bcl-2 expression were detected only in T cells transferred in hosts expressing full agonist alloAg. Expansion of transferred CD8 T cells was not dependent on either IL-2 or CD25 expression. This expansion could lead to either accelerated host reconstitution or to strong graft-vs-host, depending on the nature of the alloAg. Thus, the extent of Ag stimulation may be a crucial parameter in protocols of alloreactive T cell immunotherapy.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Following TCR engagement by specific peptide/MHC complexes on APCs, T cell responses occur in three distinct phases. The first phase is Ag specific and leads to the differentiation of naive T cells into effector cells. The second phase is characterized by a massive apoptosis of activated T cells (named activation-induced cell death, AICD)3 that allows the contraction of the immune response and the maintenance of homeostasis. The third step is the establishment of a stable pool of memory T cells (1). Quantitative changes in any of these phases can determine the outcome in terms of effector mechanisms and duration of T cell immunity. It is not clear to what extent changes in the initial Ag stimulation influence these different phases. To date, short-term in vitro models have shown that the nature of the peptide ligand can influence the effector functions elicited from T cell clones (2, 3) or naive T cells (4, 5).

T cell immunotherapy is being considered in various pathological situations such as, for instance, in allogeneic bone marrow transplantation, in which removal of mature CD8 T cells from the injected marrow has been shown to result in an increased rate of marrow rejection (reviewed in Ref. 6). CD8 T cells sensitized to endogenous peptides presented on cells from HLA-mismatched patients are also considered as reagents for tumor immunotherapy (7). In this context, it is important to understand the parameters controlling the development of T cell effector functions upon their transfer in immunosuppressed allogeneic hosts.

In a previous study, we determined the basis for partial reactivity of naive CD8 T cells expressing an alloreactive TCR in their response to a mutant alloantigen (alloAg) that behaved as a partial agonist (5). Having this model in which both full and partial agonists are endogenously expressed alloAg in mice (8), it was interesting, in adoptive transfer experiments, to evaluate the fate and effector program acquisition of transferred CD8 T cells as well as their consequences on the host in remission from sublethal irradiation.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals

Mice transgenic (tg) for the BM3.3 TCR (9) on the CBA/Ca background (tgTCR), C57BL/6 (B6), and C57BL/6.C-H-2bm8 (bm8) mice were bred in the Centre d’Immunologie de Marseille-Luminy animal facility, as well as (CBA/J x C57BL/6)F1 (abbreviated as CBA x B6) and (CBA/J x bm8)F1 (abbreviated as CBA x bm8) mice. We obtained IL-2°/° mice (10) from J. Theze (Institut Pasteur, Paris, France) and crossed them with tgTCR mice.

Flow cytometric analyses

Reagents used for immunofluorescence staining were: biotin mAb Ti98, an anticlonotypic mAb specific for the BM3.3 TCR (11) conjugated in the laboratory; FITC anti-CD44; FITC anti-IFN-{gamma}; PE anti-IL-2; and APC anti-CD8{alpha} (BD Biosciences, Mountain View, CA). For IFN-{gamma} and IL-2 intracellular staining, cells were restimulated in vitro for 4 h with 200 ng/ml ionomycin + 10 ng/ml PMA in the presence of 10 µg/ml brefeldin A. Cells were then fixed in 2% paraformaldehyde and permeabilized with 5 µg/ml saponin. The same protocol was used for intracellular staining of Bcl-2 using a FITC-conjugated hamster anti-mouse Bcl-2 mAb as compared with an isotypic control (PharMingen). For detection of early stage apoptosis, cells were labeled with annexin that was coupled to Cy5 using the fluorolink-Ab Cy5-labeling kit (Amersham Life Science, Arlington Heights, IL). Briefly, cells were incubated for 15 min with an appropriate dilution of annexin-Cy5 (provided by D. Marguet, Centre d’Immunologie de Marseille-Luminy) in 10 mM HEPES (pH 7.4), 150 mM NaCl, 5 mM KCl, 1 mM MgCl2, and 1.8 mM CaCl2. After staining, 2.104 viable cells in each sample were analyzed using a FACSCalibur cytofluorometer (BD Biosciences).

Cell purification

CD8+ cells were purified from lymph nodes of tgTCR mice by negative selection using rat anti-CD4 mAb supernatant (H129.19.6) (12) and a mix of both anti-mouse and anti-rat IgGs Dynabeads (Dynal, Oslo, Norway). In all experiments, CD8 T cells represented 90 to 98% of the enriched population.

Adoptive transfer

A total of 107 tgTCR+ CD8+ cells were resuspended in 0.1 ml of PBS and injected i.v. in recipients that had been exposed to a 5 Gy irradiation the day before. When indicated, mice were injected i.p. with 20 µg rIL-2 diluted in PBS (Proleukin; Chiron B.V., Amsterdam, The Netherlands).

CFSE staining

Determination of number of T cell divisions was done by flow cytometry using the fluorescent dye CFSE, which was shown to exhibit sequential halving of intracellular fluorescence intensity at each division step (13). Purified tgTCR+ CD8+ cells were incubated for 10 min at 37°C with 5 µM CFSE (Molecular Probes, Eugene, OR). After two washes, labeled cells were adoptively transferred, as described above.

Cytotoxic assays

In cytolytic assays, target cells were either RMA (H-2b) lymphoma cells or the TAP-2-negative variant RMA-S as a negative control (the percentage of lysis on the latter target never exceeded 5%; data not shown). After labeling with 51Cr (New England Nuclear, Boston, MA), targets (104) were incubated with effector cells for 4 h at 37°C, in absence or presence of 1 mM EGTA and 3 mM MgCl2.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Transferred tgTCR CD8 T cells can be long-lived and protective or short-lived graft-vs-host (GVH) inducers, depending on the alloAg expressed by the irradiated host

We have previously shown (5, 14) that for alloreactive tgTCR BM3.3, specific for H-2Kb (Kb), the natural mutant H-2Kbm8 (Kbm8) behaved as a partial agonist in vitro, being efficient for induction of cytotoxic effectors and IFN-{gamma} secretion, but inefficient to drive tgTCR cell proliferation due to a defect in IL-2 production. This triggering of distinct transcriptional programs relied on the stimulation of a particular set of transcription factors (5). In the present study, we addressed the question of the in vivo consequences of exposure of the same tgTCR+ CD8+ T cells to either full or partial agonist alloAg. For this purpose, 107 tgTCR+ CD8+ T cells were transferred into irradiated (CBA x B6)F1 or (CBA x bm8)F1 mice. At intervals, recipients were sacrificed, and their splenocytes were counted and phenotyped to evaluate the representation of injected tgTCR+ CD8+ cells (Fig. 1GoA). Spleens of noninjected mice or of injected syngeneic (CBA) controls contained very few cells that survived the irradiation. By day 10, partial host lymphoid reconstitution had occurred in both types of control mice (Fig. 1GoB). In syngeneic recipients, the injected tgTCR+ CD8+ T cells not only did not expand, but most of them never reached the spleen, as fewer than 106 cells had a tgTCR+ CD8+ phenotype (Fig. 1GoA). In mice expressing full agonist Kb, splenic host cells present at day 3 after transfer declined in number until day 7 (Fig. 1GoB). By day 4, the injected cell number was multiplied by a factor of three in the spleen (Fig. 1GoA). These cells were also present in the bone marrow and thymus (data not shown). At day 7, >90% of the (CBA x B6)F1 mice died from a strong GVH reaction. In the 10% of mice of that group that survived, spleens were totally devoid of cells, even up to 4 wk later (results not shown). In recipients expressing partial agonist Kbm8, the number of injected tgTCR+ CD8+ T cells increased from day 2 to 3 (Fig. 1GoA). From day 4 to day 5, this population dropped by 50%. However, a second wave of increase in tgTCR+ CD8+ T cells was detected in the spleen between day 5 and 8 (Fig. 1GoA), which correlated with host lymphoid reconstitution at an accelerated rate as compared with that of control mice (Fig. 1GoB). After day 10, the number of tgTCR+ CD8+ T cells slowly decreased (Fig. 1GoA), but 3 x 106 were still detectable at day 30 after the transfer (data not shown). In this situation, recipient mice did not show any signs of GVH and survived for at least 60 days. Thus, depending on the nature of the expressed alloAg, the fate of the host was drastically distinct, as a strong GVH reaction ending with death was observed when tgTCR+ CD8+ T cells were activated in vivo by a full agonist, whereas a partial agonist failed to induce a GVH reaction. Moreover, this discrepancy was maintained when a smaller number (106) of tgTCR+ CD8+ T cells was transferred (data not shown), suggesting that the response of the injected tgTCR+ CD8+ T cells was qualitatively different in both types of hosts.



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  		FIGURE 1. Expansion of transferred tgTCR+ CD8+ T cells and host reconstitution differ depending on whether the irradiated host expressed full or partial agonist alloAg. A total of 107 tgTCR+ CD8+ T cells were i.v. injected in either (CBA x bm8)F1, (CBA x B6)F1, or CBA-irradiated recipients. A noninjected control group that represents the mean of three mice of each haplotype was also included in this experiment. From day 2 to day 13 after transfer, tgTCR+ CD8+ cell number was evaluated from immunofluorescence analysis and shown in A, whereas host splenocyte number was reported in B. Results are expressed as the mean of six independent experiments, with two mice of each haplotype tested at each time point. In C, tgTCR+ CD8+ T cells were labeled with CFSE before injection. From day 3 to day 10 after the transfer, splenocytes were recovered and analyzed by triple immunofluorescence for surface expression of CD8 and tgTCR, as well as for the CFSE fluorescent dye. Histograms of CFSE profiles on gated tgTCR+ CD8+ cells are shown, and thin line represents CFSE fluorescence level before injection. CFSE MRFI is indicated in brackets, with the corresponding number of cell divisions underneath. Data are representative of three independent experiments, with two mice of each haplotype tested at each time point.

 
We next examined whether the differential outcome for tgTCR+ CD8+ T cells when activated in vivo by a full or a partial agonist could be explained by distinct proliferation rates, as we had observed in vitro (5). To this end, tgTCR+ CD8+ T cells were labeled with the fluorescent dye CFSE before transfer into irradiated recipients. A homogeneous CFSEhigh tgTCR+ CD8+ population was observed in spleens of syngeneic controls at day 5 (Fig. 1GoC). By day 10, a fraction of these cells went through a first round of division, as assessed by the appearance of a tgTCR-expressing CD8+ subset harboring half of the initial level of CFSE staining. In (CBA x B6)F1 mice, tgTCR+ CD8+ T cells had already achieved four cell divisions by day 3, and seven by day 5, showing that one cell division was accomplished every 18 h, in a highly synchronous way. In (CBA x bm8)F1 recipients, the majority of tgTCR+ CD8+ T cells had divided four times by day 4 and six times by day 5, with a generation time around 20 h. It also appeared that a few cells at day 5 had retained an intermediate CFSE fluorescence intensity, corresponding to one to four divisions. In addition, by day 7, a second wave of tgTCR+ CD8+ T cell expansion was observed in (CBA x bm8)F1 recipient spleens (Fig. 1GoA). Several possible explanations may be considered for this secondary expansion phase: either the small pool of CFSEhigh tgTCR+ CD8+ cells underwent cell division by day 7, or different tgTCR+ CD8+ cell routing may occur within the animal, leading to the sequestration of the injected T cell population in particular organs and to a delayed arrival in the spleen, where it was analyzed.

Altogether, we report in this study that in vivo proliferation of CD8 T cells was induced by expression of either full or partial agonist alloAg. This was in contrast to our previous in vitro work (5), in which partial agonist Kbm8 was unable to drive tgTCR+ CD8+ cell proliferation due to a lack of IL-2 production. Thus, it was interesting to determine whether in vivo T cell expansion was or not sustained by IL-2 (see above). T cell proliferation that occurs following TCR engagement is usually accompanied by the acquisition of an activated phenotype. We next examined phenotypical changes associated with in vivo activation by Kb or Kbm8. This was also of particular interest because in vitro Kbm8 stimulation failed to induce modulation of such surface markers (5).

Distinct kinetics and magnitude of activation marker and tgTCR modulation induced in vivo by full or partial agonist alloAg

T cell activation can be followed by changes in surface expression of markers such as CD44 and CD62L. Immunofluorescence analysis showed (Fig. 2Go) that ex vivo tgTCR+ CD8+ cells have a naive phenotype, being all CD44 negative (thin line in Fig. 2GoA). These tgTCR+ CD8+ cells kept a naive phenotype when injected in syngeneic controls. In contrast, nearly 100% of tgTCR+ CD8+ present in (CBA x B6)F1 recipients at day 3 had up-regulated CD44 at a high level (Fig. 2GoA), as well as CD69 (data not shown). On tgTCR+ CD8+ T cells recovered from (CBA x bm8)F1 mice, CD44 expression was also efficiently increased at day 3 (Fig. 2GoA). The same was observed for CD69 (data not shown),



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  		FIGURE 2. Kinetics of changes in expression of CD44, CD62L, and tgTCR induced in vivo by full or partial agonist alloAg. From day 3 to day 7 after transfer, splenocytes were recovered from the recipients and analyzed by triple immunofluorescence for surface expression of CD8, tgTCR, and either CD44 (A) or CD62L (B). In A and B, histograms relative to activation marker profiles on gated tgTCR+ CD8+ cells are shown, and thin lines represent, respectively, CD44 and CD62L on naive tgTCR+ CD8+ cells before injection. In C, tgTCR expression is shown after normalization to the MRFI detected on naive tgTCR+ CD8+ cells before injection (day 0 = 100%); data are representative of at least three independent experiments, with two mice of each haplotype tested at each time point.

 
Following T cell activation, CD62L is down-regulated. Again, this cell surface marker confirmed that ex vivo CD8+ tgTCR+ cells have a naive phenotype, being CD62Lhigh (thin line in Fig. 2GoB). In contrast, in vivo activation by the full agonist Kb rapidly induced the complete down-regulation of CD62L (Fig. 2GoB). In comparison, the in vivo response to the partial agonist appeared heterogeneous and delayed, as revealed by the altered CD62L expression. This observation may reflect the asynchronous stimulation of tgTCR+ CD8+ T cells by the partial agonist. In our previous characterization of the in vitro response of tgTCR+ CD8+ cells to partial agonist Kbm8, we also observed a heterogeneous response, which could be influenced by the level of costimulating molecules on the APCs (5). In vivo, different factors can influence the outcome of tgTCR+ cell stimulation by a partial agonist: 1) the expression of specific Kbm8/peptide complexes could be weak and heterogeneous in the different organs; 2) the type of APCs encountered, which may express different degrees of costimulating components. Altogether, each encounter with a partial agonist might not be productive for T cell stimulation. One possible way to address this issue was to measure TCR down-modulation that is believed to reflect the effectiveness of TCR engagement. In this study, in syngeneic CBA controls, tgTCR+ CD8+ T cells maintained a very high and homogenous surface expression of the tgTCR (Fig. 2GoC). In (CBA x B6)F1 mice, at day 3, the tgTCR was already massively down-modulated, whereas tgTCR+ CD8+ T cells recovered from (CBA x bm8)F1 showed only a mild decrease in tgTCR level between days 3 and 7, tgTCR down-regulation being more significant at day 10, even if heterogeneous (Fig. 2GoC and data not shown). Therefore, it seemed that tgTCR engagement by Kbm8 was more efficient during the second, than the first wave of tgTCR cell expansion.

IL-2-independent in vivo expansion of tgTCR+ CD8+ T cells

We next analyzed the cytokine production elicited in vivo by full or partial agonist alloAg. For this purpose, tgTCR+ CD8+ T cells were harvested from recipient spleens and assayed for intracellular cytokines either directly (not shown) or after activation by calcium ionophore and phorbol ester in a 4-h in vitro culture. The latter treatment did not induce cytokines in either naive or CBA-transferred tgTCR+ CD8+ T cells (Fig. 3Go). A high level of intracellular staining for IL-2 was found in ex vivo tgTCR+ CD8+ T cells retrieved from (CBA x B6)F1 mice after 3 days, this production being slightly enhanced after an in vitro reactivation (Fig. 3Go and data not shown). This IL-2 production was not detectable thereafter. Activation of tgTCR+ CD8+ cells in (CBA x bm8)F1 recipients did not induce the capacity to produce IL-2, even after 4-h in vitro restimulation, whether the tgTCR+ CD8+ cells were taken in the first (days 3–5), or second (day 7) wave of T cell expansion (Fig. 3Go and data not shown).



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  		FIGURE 3. Differential IL-2 secretion induced in vivo by full or partial agonist alloAg. After transfer, splenocytes were recovered from the recipients and tested by intracellular staining for IL-2 after a 4-h culture in medium supplemented with ionomycin + PMA (bold line) in the presence of brefeldin A. Cells cultured in medium alone showed the same IL-2 staining as the one observed after ionomycin + PMA stimulation (data not shown). Histograms relative to IL-2 profiles on gated tgTCR+ CD8+ cells are shown on days 0, 3, 4, and 5. Thin lines represent on each cell type a negative fluorescent control. Data are representative of three independent experiments, with two mice of each haplotype tested at each time point.

 
The effects of IL-2 on peripheral T cells are dependent upon the induction of CD25, the IL-2R{alpha}-chain contributing high affinity for IL-2. TgTCR+ CD8+ T cells activated in (CBA x B6)F1 mice had up-regulated CD25 expression, such phenotypic change being absent on cells recovered from either (CBA x bm8)F1 or syngeneic recipients (Fig. 4GoA). To assess whether the absence of CD25 up-regulation in (CBA x bm8)F1 hosts resulted from poor TCR engagement or from lack of IL-2, we analyzed the effect 1) of IL-2 deprivation in transfers in (CBA x B6)F1 mice, and 2) of exogenous addition of IL-2 in transfers in (CBA x bm8)F1 mice. When tgTCR+ cells from IL-2°/° mice were transferred in (CBA x B6)F1 hosts, no more CD25 up-regulation was detected (Fig. 4GoC), indicating that in addition to a tgTCR-mediated signal, an IL-2-dependent signal was also required to induce the increased expression of its own receptor. Furthermore, injections of rIL-2 (twice 20 µg at days 1 and 2 after the transfer of tgTCR+ CD8+ cells) in (CBA x bm8)F1 hosts led to efficient up-regulation of CD25 that was absent in the same recipients injected with PBS (Fig. 4GoB). Thus, IL-2 secretion by tgTCR+ CD8+ T cells appears to be the limiting factor for CD25 expression in the transfer protocol. IL-2 production was also defective in response to partial agonist in vitro (5), in which it limited T cell proliferation. However, in contrast to the in vitro situation, lack of IL-2 production did not prevent T cell proliferation in vivo. Indeed, we clearly observed that when tgTCR+ CD8+ T cells from IL-2°/° mice were transferred in (CBA x B6)F1 hosts, T cell division also occurred (Fig. 4GoD). Thus, in agreement with our previous in vitro work (5), stimulation of tgTCR+ CD8+ by a partial agonist failed to induce IL-2 secretion, and therefore prevented up-regulation of CD25. However, a discrepancy between in vitro and in vivo stimulation by such altered ligand appeared, as in vivo proliferation was not prevented by lack of IL-2.



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  		FIGURE 4. Kinetics of changes in expression of CD25 induced in vivo by full or partial agonist alloAg. From day 3 to day 7 after transfer, splenocytes were recovered from the recipients and analyzed by triple immunofluorescence for surface expression of CD8, tgTCR, and CD25. In A, B, and C, histograms relative to CD25 profiles on gated tgTCR+ CD8+ cells are shown, and thin lines represent CD25 on naive tgTCR+ CD8+ cells before injection. In A, transfer was done as in Figs. 1Go and 2Go. In B, host mice were i.v. injected at days 1 and 2 with 20 µg of rIL-2. In C and D, the injected tgTCR+ CD8+ cells were from tgTCR x IL-2+/+ or tgTCR x IL-2°/° mice. At day 4 after the transfer, CD25 expression (C) and CFSE labeling (D) are shown, with the thin lines representing the corresponding fluorescence level before injection. In D, CFSE MRFI is indicated in brackets, with the corresponding number of cell divisions underneath. Data are representative of three (A), two (B), and one (C and D) experiments, with two mice of each haplotype tested at each time point.

 
Differential cytotoxic activities induced in vivo by full or partial agonist alloAg

We further asked whether differential cytotoxic effector functions of transferred tgTCR cells influenced the fate of the hosts. Therefore, total splenocytes recovered from irradiated recipients at different intervals after the transfer were directly tested for their ability to kill a Kb-expressing target cell (Fig. 5GoA). Ex vivo splenocytes from syngeneic CBA control mice did not exhibit any Kb-specific cytotoxic activity. In contrast, T cells from either transferred (CBA x B6)F1 or (CBA x bm8)F1 mice displayed efficient cytolysis of Kb target cells. The kinetics of induction of the CTL function seemed to be delayed by 1 day in (CBA x bm8)F1 as compared with (CBA x B6)F1 mice, a time lag reminiscent of that observed for CD62L down-regulation (see above). The same assay was conducted in the presence of EGTA (Fig. 5GoB) to discriminate between the calcium-sensitive perforin component and the calcium-independent cytotoxicity exerted by other mechanisms, including Fas ligand-expressing cells. Under such conditions, a peak of perforin-independent cytotoxic activity was detected at day 4 for effectors isolated from (CBA x B6)F1 mice, a time point in which perforin-mediated killing was also maximal. However, significant perforin-independent lysis was observed for (CBA x bm8)F1 recipient splenocytes at a later time point (day 9), and in consequence was desynchronized from the earlier perforin cytotoxicity. Hence, whereas both perforin-dependent and perforin-independent killing were stimulated in vivo by both kinds of alloAg, these results suggest a difference in the time required for the induction of the perforin-independent pathways.



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  		FIGURE 5. Differential kinetics of cytotoxic effector functions induced in vivo by full or partial agonist alloAg. In A, Kb-specific cytotoxic activity was directly measured in a 4-h 51Cr release assay on RMA target cells, and in B, the assay in the presence of EGTA reveals perforin-independent cytotoxicity. Results are expressed as lytic units calculated as the (target/tgTCR+ CD8+) ratio corresponding to a same level of cytotoxic activity. Results are expressed as the mean of two mice of each haplotype tested at each time point of one representative experiment among four.

 
Altogether, tgTCR+ CD8+ T cells were fully activated in (CBA x B6)F1 hosts and rapidly disappeared (Fig. 1GoA), suggesting that they may be susceptible to an AICD process. This possibility was addressed next.

Distinct kinetics of decreased Bcl-2 expression and apoptosis after in vivo activation by full or partial agonist alloAg

The antiapoptotic function of Bcl-2 is well established (15), and it has been reported that constitutive expression of Bcl-2 may prevent AICD of CD8+ T cells (16). We thus measured the expression of Bcl-2 protein in ex vivo tgTCR+ CD8+ T cells, and found that at day 5 cells recovered from (CBA x B6)F1 recipients had down-modulated Bcl-2, whereas Bcl-2 levels were unchanged in cells from (CBA x bm8)F1 or syngeneic controls (Fig. 6Go). However, by day 10, a slightly decreased Bcl-2 level was observed in cells activated in (CBA x bm8)F1 recipients (data not shown). This decreased Bcl-2 expression may thus be correlated with the kinetics of AICD susceptibility of tgTCR+ CD8+ T cells stimulated by full vs partial agonist. This hypothesis was further tested by a direct measure of cell death performed by annexin labeling of ex vivo T cells. As shown in Table IGo, the number of tgTCR+ CD8+ cells undergoing apoptosis was already high at day 5 in (CBA x B6)F1 recipients, whereas this number was significantly lower in (CBA x bm8)F1 mice. However, in this latter case, an increase in the number of apoptotic tgTCR+ CD8+ was observed by day 10, at a time in which the splenic tgTCR+ CD8+ cell number started to decrease (Fig. 1GoA). Thus, there was a strict correlation between the Bcl-2 down-regulation and cell death, as measured by phosphatidylserine exposure.



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  		FIGURE 6. Modulated Bcl-2 expression induced in vivo by full or partial agonist alloAg. At day 5 after transfer, splenocytes were recovered from the recipients and analyzed by triple immunofluorescence for surface expression of CD8, tgTCR, and intracellular staining of Bcl-2. Histograms relative to Bcl-2 (bold line) vs negative isotypic control (thin line) profiles on gated tgTCR+ CD8+ cells are shown. Data are representative of three independent experiments, with two mice of each haplotype tested.

 

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  		Table I. Number of annexin-positive tgTCR+CD8+ cells recovered in the different recipients

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Several reports have established that a degree of TCR triggering by self peptide/MHC complexes was required for peripheral T cell survival or homeostatic proliferation in lymphopenic hosts (1, 17, 18, 19). For antigenic T cell stimulation, some correlation between the range of TCR avidity for MHC/peptide ligands and the level of T cell activation has been reported, with full and partial agonist ligands displaying, respectively, strong and intermediate TCR affinity (20, 21). The initial strength of TCR stimulation can be determined either by the avidity of MHC/peptide complexes toward the TCR or by the abundance of specific antigenic complexes. In this latter case, the extent of TCR triggering was shown to determine not only the development of T cell effector functions, but also the fate and the duration of the T cell response that can be shortened by the induction of Ag-driven T cell apoptosis (22).

Altogether, the situation of T cell transfer in irradiated hosts has two major components: the fate of the transferred T cells and the fate of host cell reconstitution. These two components are interdependent as 1) T cell expansion, acquisition of effector function, and survival will depend upon the strength of T cell stimulation and the maintenance of APCs; and 2) the nature of the T cell effector functions and their maintenance will bear on host cell reconstitution.

In this study, we show that weak in vivo TCR engagement by a partial agonist alloAg leads to a long-term survival of the tgTCR+ CD8+-injected cells. The same cells did not expand in syngeneic irradiated hosts, as already reported for two other TCR tg lines (18). In contrast, in hosts expressing full agonist alloAg, the rapid expansion of tgTCR+ CD8+-injected cells was accompanied by their apoptosis. In this latter case, a parallel decline in host splenocytes was observed, whereas in hosts expressing partial agonist alloAg, splenocyte reconstitution was parallel to transferred T cell accumulation. However, it is not clear whether a particular effector function is responsible for the strikingly different outcomes for the irradiated host receiving CD8 T cells in a situation of exposure to a partial or full agonist alloAg. For instance, Fas/Fas ligand and TNF/TNFR(p55) have been implicated as effector mechanisms in GVH (23, 24, 25, 26), but also as mediators of AICD of CD8 T cells in vivo (27, 28, 29), and in particular for host-reactive T cells in GVH (30). A functional heterogeneity of in vitro cultured alloreactive CD8 CTL clones has also been reported: some clones endowed with cytotoxic potential and production of IFN-{gamma} and TNF-{alpha}, but not IL-2 or IL-4, did not cause toxic GVH in irradiated hosts expressing the alloAg, and some clones, in addition, could prevent host rejection of allogeneic bone marrow (31). In the study presented in this work, we showed that the full activation of transferred CD8 T cells in full agonist-expressing hosts triggered early perforin-independent cytotoxicity, IL-2 production, and CD25 up-regulation, which were not observed in partial agonist-expressing hosts. This may contribute to the strong GVH observed in full alloAg-expressing hosts, but might also sensitize the tgTCR+ CD8+ cells to AICD, as previously suggested (32, 33), leading to their rapid disappearance. Although activation of tgTCR+ CD8+ cells by a partial agonist induced differentiation from naive to cytolytic effectors, perforin-dependent and perforin-independent cytotoxicities appeared distinct in terms of activation threshold and kinetics. Together with the fact that the lysis of Kbm8-expressing targets was less efficient than that of Kb-positive targets (about one-third; results not shown), this may explain the absence of GVH in bm8 recipients.

Activation of transferred CD8 T cells in partial agonist-expressing hosts failed to induce IL-2 secretion. This absence of IL-2 not only correlated with a protection from AICD, but it also did not prevent in vivo induced proliferation. This IL-2-independent CD8 T cell expansion in vivo contrasts with our in vitro findings, in which lack of IL-2 production in response to partial agonist alloAg appeared to be the limiting factor for T cell proliferation (5). The requirements for mature T cell survival and expansion at the naive, effector, and memory stage are still poorly understood (1). The description that CD4 T cells from mice deficient for the common cytokine receptor {gamma}-chain are able to expand in response to Ag in vivo would exclude IL-2, IL-4, IL-7, IL-9, and IL-15 as CD4 T cell growth factors (34). For CD8 T cells, IL-7 has been shown to be required for CD8 T cell homeostatic proliferation, but not for proliferation in response to viral infection (35). The nature of IL(s) involved in proliferation and triggered by a partial agonist alloAg needs further investigation. Whether the same or a different cytokine is responsible for enhanced host reconstitution also remains to be addressed.

In conclusion, our study shows that an appropriate interaction between TCR and alloAg determines the fate of a given T cell population adoptively transferred to a myeloablated recipient. At an appropriate level of stimulation, both host lymphoid cells and transferred CD8 T cells expand to the benefit of the lymphoid reconstitution of the host. As cellular immunotherapy has been aimed at improving T cell effector functions and cytokines produced, it may be necessary to consider the extent of Ag stimulation as a major parameter.


   Acknowledgments
 
We thank C. Boyer, C. Chabannnon, A. Gross, S. Guerder, A. Guimezanes, and L. Leserman for criticism on the manuscript. We also thank H. Sanchez and P. Gibier for animal care.


   Footnotes
 
1 This work was supported by institutional grants from Institut National de la Santé et de la Recherche Médicale and Centre National de la Recherche Scientifique, and by grants from Association pour la Recherche sur le Cancer, Ligue Nationale contre le Cancer ("axe immunologie des tumeurs"), and Ligue Nationale contre le Cancer-Comité des Bouches du Rhône. Back

2 Address correspondence and reprint requests to Dr. Nathalie Auphan-Anezin, Centre d’Immunologie de Marseille-Luminy, Centre National de Recherche Scientifique-Institute National de la Santé et de la Recherche Médicale-Université de la Méditerranée, Campus de Luminy, Case 906, 13288 Marseille, Cedex 9, France. E-mail address: auphan{at}ciml.univ-mrs.fr Back

3 Abbreviations used in this paper: AICD, activation-induced cell death; alloAg, alloantigen; GVH, graft-vs-host; MRFI, mean of relative fluorescence intensity; tg, transgenic. Back

Received for publication January 8, 2001. Accepted for publication April 12, 2001.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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