Alloantigen affinity and CD4 help determine severity of graft-versus-host disease mediated by CD8 donor T cells

TCR affinity dictates T cell selection in the thymus and also has a high impact on the fate of peripheral T cells. Graft-vs-host disease (GVHD) is a pathological process initiated by activation of donor T cells after adoptive transfer into an allogeneic recipient. How TCR affinity affects the potential of alloreactive T cells to induce GVHD is unclear. Using alloreactive CD4+ and CD8+ TCR transgenic (Tg) T cells, GVHD models are presented that allow for the visualization of how CD8+ alloreactive T cells behave in response to alloantigens with different TCR affinity in the absence or presence of CD4 help. In a nonmyeloablative transplant model where GVHD lethality is due to marrow aplasia, alloreactive CD8+ TCR Tg T cells induced significantly more severe GVHD in the recipients that express an intermediate-affinity alloantigen than in the recipients that express a high-affinity alloantigen. In a myeloablative transplant model where GVHD lethality is due to epithelium injury, CD8+ TCR Tg cells were also more pathogenic in the recipients with an intermediate-affinity alloantigen than in those with a high-affinity alloantigen. The presence of alloreactive CD4+ TCR Tg cells enhanced the potential of CD8+ TCR Tg cells to cause GVHD in recipients with an intermediate-, but not with a high-, affinity alloantigen. These findings underscore that alloantigen affinity and CD4 help control the fate and pathogenicity of alloreactive CD8+ T cells in vivo.     

Opposing effects of ICOS on graft-versus-host disease mediated by CD4 and CD8 T cells

ICOS, a CD28 family member expressed on activated CD4(+) and CD8(+) T cells, plays important roles in T cell activation and effector function. Here we studied the role of ICOS in graft-vs-host disease (GVHD) mediated by CD4(+) or CD8(+) T cells in allogeneic bone marrow transplantation. In comparison of wild-type and ICOS-deficient T cells, we found that recipients of ICOS(-/-) CD4(+) T cells exhibited significantly less GVHD morbidity and delayed mortality. ICOS(-/-) CD4(+) T cells had no defect in expansion, but expressed significantly less Fas ligand and produced significantly lower levels of IFN-gamma and TNF-alpha. Thus, ICOS(-/-) CD4(+) T cells were impaired in effector functions that lead to GVHD. In contrast, recipients of ICOS(-/-) CD8(+) T cells exhibited significantly enhanced GVHD morbidity and accelerated mortality. In the absence of ICOS signaling, either using ICOS-deficient donors or ICOS ligand-deficient recipients, the levels of expansion and Tc1 cytokine production of CD8(+) T cells were significantly increased. The level of expansion was inversely correlated with the level of apoptosis, suggesting that increased ability of ICOS(-/-) CD8(+) T cells to induce GVHD resulted from the enhanced survival and expansion of those cells. Our findings indicate that ICOS has paradoxical effects on the regulation of alloreactive CD4(+) and CD8(+) T cells in GVHD.     

Alloreactive memory T cells are responsible for the persistence of graft-versus-host disease

Graft-vs-host disease (GVHD) is caused by a donor T cell anti-host reaction that evolves over several weeks to months, suggesting a requirement for persistent alloreactive T cells. Using the C3H.SW anti-C57BL/6 (B6) mouse model of human GVHD directed against minor histocompatibility Ags, we found that donor CD8(+) T cells secreting high levels of IFN-gamma in GVHD B6 mice receiving C3H.SW naive CD8(+) T cells peaked by day 14, declined by day 28 after transplantation, and persisted thereafter, corresponding to the kinetics of a memory T cell response. Donor CD8(+) T cells recovered on day 42 after allogeneic bone marrow transplantation expressed the phenotype of CD44(high)CD122(high)CD25(low), were able to homeostatically survive in response to IL-2, IL-7, and IL-15 and rapidly proliferated upon restimulation with host dendritic cells. Both allogeneic effector memory (CD44(high)CD62L(low)) and central memory (CD44(high)CD62L(high)) CD8(+) T cells were identified in B6 mice with ongoing GVHD, with effector memory CD8(+) T cells as the dominant (>80%) population. Administration of these allogeneic memory CD8(+) T cells into secondary B6 recipients caused virulent GVHD. A similar allogeneic memory CD4(+) T cell population with the ability to mediate persistent GVHD was also identified in BALB/b mice receiving minor histocompatibility Ag-mismatched B6 T cell-replete bone marrow transplantation. These results indicate that allogeneic memory T cells are generated in vivo during GVH reactions and are able to cause GVHD, resulting in persistent host tissue injury. Thus, in vivo blockade of both alloreactive effector and memory T cell-mediated host tissue injury may prove to be valuable for GVHD prevention and treatment.     

Analysis of the underlying cellular mechanisms of anti-CD154-induced graft tolerance: the interplay of clonal anergy and immune regulation

Although it has been shown that CD4(+)CD25(+) regulatory T cells (T(reg)) contribute to long-term graft acceptance, their impact on the effector compartment and the mechanism by which they exert suppression in vivo remain unresolved. Using a CD4(+) TCR transgenic model for graft tolerance, we have unveiled the independent contributions of anergy and active suppression to the fate of immune and tolerant alloreactive T cells in vivo. First, it is shown that anti-CD154-induced tolerance resulted in the abortive expansion of the alloreactive, effector T cell pool. Second, commensurate with reduced expansion, there was a loss of cytokine production, activation marker expression, and absence of memory T cell markers. All these parameters defined the tolerant alloreactive T cells and correlated with the inability to mediate graft rejection. Third, the tolerant alloreactive T cell phenotype that is induced by CD154 was reversed by the in vivo depletion of T(reg). Reversal of the tolerant phenotype was followed by rapid rejection of the allograft. Fourth, in addition to T(reg) depletion, costimulation of the tolerant alloreactive T cells or activation of the APC compartment also reverted alloreactive T cell tolerance and restored an activated phenotype. Finally, it is shown that the suppression is long-lived, and in the absence of anti-CD154 and donor-specific transfusion, these T(reg) can chronically suppress effector cell responses, allowing long-lived graft acceptance.     

Host-reactive CD8+ memory stem cells in graft-versus-host disease

Graft-versus-host disease (GVHD) is caused by alloreactive donor T cells that trigger host tissue injury. GVHD develops over weeks or months, but how this immune response is maintained over time is unknown. In mouse models of human GVHD, we identify a new subset of postmitotic CD44(lo)CD62L(hi)CD8(+) T cells that generate and sustain all allogeneic T-cell subsets in GVHD reactions, including central memory, effector memory and effector CD8(+) T cells, while self-renewing. These cells express Sca-1, CD122 and Bcl-2, and induce GVHD upon transfer into secondary recipients. The postmitotic CD44(lo)CD62L(hi)CD8(+) T cells persist throughout the course of GVHD, are generated in the initial phase in response to alloantigens and dendritic cells and require interleukin-15. Thus, their long life, ability to self-renew and multipotentiality define these cells as candidate memory stem cells. Memory stem cells will be important targets for understanding and influencing diverse chronic immune reactions, including GVHD.     

Accelerated onset and increased severity of acute graft-versus-host disease following adoptive transfer of DR6-deficient T cells

DR6 is a recently identified member of the TNFR family. In a previous study, we have shown that DR6 KO mice have enhanced CD4(+) T cell proliferation and Th2 cytokine production. Acute graft-vs-host disease (GVHD) results from the activation and expansion of alloreactive donor T cells following bone marrow transplantation. In this article, we demonstrate that the transfer of donor T cells from DR6 KO mice into allogeneic recipient mice in a parent into an F(1) model of acute GVHD results in a more rapid onset of GVHD with increased severity. Recipients of DR6 KO T cells exhibit earlier systemic symptoms of GVHD, more rapid weight loss, earlier histopathological organ damage in the thymus, spleen, and intestines, and earlier mortality. The rapid onset of GVHD in these mice may be attributable to the enhanced activation and expansion of DR6 KO CD4(+) and CD8(+) T cells. Our findings support the hypothesis that DR6 serves as an important regulatory molecule in T cell immune responses. The identification and use of DR6 ligands and/or agonistic Abs to DR6 may represent useful therapeutics in the treatment of T cell-mediated diseases such as GVHD.     

CD4+CD25+ regulatory T cells preserve graft-versus-tumor activity while inhibiting graft-versus-host disease after bone marrow transplantation

Mature donor T cells cause graft-versus-host disease (GVHD), but they are also the main mediators of the beneficial graft-versus-tumor (GVT) activity of allogeneic bone marrow transplantation. Suppression of GVHD with maintenance of GVT activity is a desirable outcome for clinical transplantation. We have previously shown that donor-derived CD4+CD25+ regulatory T cells inhibit lethal GVHD after allogeneic bone marrow transplantation across major histocompatibility complex (MHC) class I and II barriers in mice. Here we demonstrate that in host mice with leukemia and lymphoma, CD4+CD25+ regulatory T cells suppress the early expansion of alloreactive donor T cells, their interleukin-2-receptor (IL-2R) alpha-chain expression and their capacity to induce GVHD without abrogating their GVT effector function, mediated primarily by the perforin lysis pathway. Thus, CD4+CD25+ T cells are potent regulatory cells that can separate GVHD from GVT activity mediated by conventional donor T cells.     

Virotherapy Using Myxoma Virus Prevents Lethal Graft-versus-Host Disease following Xeno-Transplantation with Primary Human Hematopoietic Stem Cells

Graft-versus-host disease (GVHD) is a potentially lethal clinical complication arising from the transfer of alloreactive T lymphocytes into immunocompromised recipients. Despite conventional methods of T cell depletion, GVHD remains a major challenge in allogeneic hematopoietic cell transplant. Here, we demonstrate a novel method of preventing GVHD by ex vivo treatment of primary human hematopoietic cell sources with myxoma virus, a rabbit specific poxvirus currently under development for oncolytic virotherapy. This pretreatment dramatically increases post-transplant survival of immunocompromised mice injected with primary human bone marrow or peripheral blood cells and prevents the expansion of human CD3(+) lymphocytes in major recipient organs. Similar viral treatment also prevents human-human mixed alloreactive T lymphocyte reactions in vitro. Our data suggest that ex vivo virotherapy with myxoma virus can be a simple and effective method for preventing GVHD following infusion of hematopoietic products containing alloreactive T lymphocytes such as: allogeneic hematopoietic stem and progenitor cells, donor leukocyte infusions and blood transfusions.     

Cutting edge: sustained expansion of CD8+ T cells requires CD154 expression by Th cells in acute graft versus host disease

Brief treatment with alphaCD154 Ab has been shown to prevent acute graft versus host disease (aGvHD). We extend these data to show that in the absence of CD154 function, donor T cells are unable to expand or generate high level anti-host CTL activity. Using transgenic (Tg) alloreactive CD8+ T cells adoptively transferred into allogeneic recipients, we show that short-term expansion of the CD8+ Tg T cells occurred in the absence of Th cells, and this short-term expansion could be facilitated with an agonistic alphaCD40. While CD40 agonism could enhance short-term expansion, sustained expansion of CD8+ Tg T cells required bona fide CD154-expressing CD4+ alloreactive Th cells. While CD154 was necessary for CD8+ Tg T cell sustained expansion, IL-2 was also implicated as essential. These observations suggest alphaCD154 therapy in GvHD is effective because the treatment causes an abortive CD8 alloresponse leading to the exhaustion or deletion of alloreactive CD8+ clones preventing the development of disease.     

Host conditioning is a primary determinant in modulating the effect of IL-7 on murine graft-versus-host disease

Interleukin-7 has been shown to enhance T cell reconstitution after allogeneic bone marrow transplantation, in part, by expansion of mature donor T cells, but whether IL-7 also exacerbates graft-vs-host disease (GVHD) remains unresolved. To address this issue, we examined the effect of IL-7 on GVHD induction using a well-defined murine GVHD model (B6-->B6AF1/J). Administration of IL-7 to nonirradiated B6AF1/J recipients of B6 T cells resulted in expansion of splenic donor CD4(+) and CD8(+) T cells and increased GVHD mortality. In contrast, administration of IL-7 on the same schedule failed to increase GVHD mortality in either sublethally or lethally irradiated animals that received graded doses of T cells designed to induce varying degrees of GVHD severity. Moreover, IL-7 failed to increase the number of alloreactive T cells when examined in a murine model (B6-->BALB.B) that allowed for direct quantitation of graft-vs-host-reactive T cells. The combination of irradiation and transplantation of alloreactive donor T cells resulted in significantly increased levels of endogenous splenic IL-7 mRNA when compared with nonirradiated transplanted animals, providing a potential explanation for why exogenous IL-7 did not increase GVHD severity in these mice. We conclude that host conditioning modulates the ability of exogenous IL-7 to exacerbate GVHD and that this occurs through induction of endogenous IL-7 production.     

CD8+ Foxp3+ regulatory T cells are induced during graft-versus-host disease and mitigate disease severity

Regulatory T cells (Tregs), in particular CD4(+) Foxp3(+) T cells, have been shown to play an important role in the maintenance of tolerance after allogeneic stem cell transplantation. In the current study, we have identified a population of CD8(+) Foxp3(+) T cells that are induced early during graft-versus-host disease (GVHD), constitute a significant percentage of the entire Treg population, and are present in all major GVHD target organs. These cells expressed many of the same cell surface molecules as found on CD4(+) Tregs and potently suppressed in vitro alloreactive T cell responses. Induction of these cells correlated positively with the degree of MHC disparity between donor and recipient and was significantly greater than that observed for CD4(+)-induced Tregs (iTregs) in nearly all tissue sites. Mice that lacked the ability to make both CD8(+) and CD4(+) iTregs had accelerated GVHD mortality compared with animals that were competent to make both iTreg populations. The absence of both iTreg populations was associated with significantly greater expansion of activated donor T cells and increased numbers of CD4(+) and CD8(+) T cells that secreted IFN-γ and IL-17. The presence of CD8(+) iTregs, however, was sufficient to prevent increased GVHD mortality in the complete absence of CD4(+) Tregs, indicating at least one functional iTreg population was sufficient to prevent an exacerbation in GVHD severity, and that CD8(+) iTregs could compensate for CD4(+) iTregs. These studies define a novel population of CD8(+) Tregs that play a role in mitigating the severity of GVHD after allogeneic stem cell transplantation.     

T cell subsets and in vitro immune regulation in "infectious" transplantation tolerance.

CD4-targeted mAb therapy results in permanent acceptance of cardiac allografts in rat recipients, in conjunction with features of the infectious tolerance pathway. Although CD4(+) T cells play a central role, the actual cellular and molecular tolerogenic mechanisms remain elusive. This study was designed to analyze in vitro alloimmune responses of T lymphocytes from CD4 mAb-treated engrafted hosts. Spleen, but not lymph node, cells lost proliferative response against donor alloantigen in MLR and suppressed test allograft rejection in adoptive transfer studies, suggesting compartmentalization of tolerogenic T cells in transplant recipients. A high dose of exogenous IL-2 restored the allogeneic response of tolerogenic T cells, indicating anergy as a putative mechanism. Vigorous proliferation of the tolerogenic T cells in in vivo MLR supports the existence of alloreactive lymphocytes in tolerogenic T cell repertoire and implies an active operational suppression mechanism. The tolerogenic splenocytes suppressed proliferation of naive splenocytes in vitro, consistent with their in vivo property of dominant immune regulation. Finally, CD45RC(+) but not CD45RC(-) T cells from tolerant hosts were hyporesponsive to alloantigen and suppressed the proliferation of normal T cells in the coculture assay. Thus, nondeletional, anergy-like regulatory mechanisms may operate via CD4(+)CD45RC(+) T cells in the infectious tolerance pathway in transplant recipients.     

"Pruning" of alloreactive CD4+ T cells using 5- (and 6-)carboxyfluorescein diacetate succinimidyl ester prolongs skin allograft survival

Removal of alloreactive cells by either thymic deletion or deletion/anergy in the periphery is regarded as crucial to the development of tolerance. Dyes, such as CFSE, that allow monitoring of cell division suggest that in vitro proliferation could be a used as a way of "pruning" alloreactive cells while retaining a normal immune repertoire with retention of memory to previously encountered pathogens. This would overcome the problems occurring as a result of therapies that use massive depletion of T cells to allow acceptance of organ transplants or bone marrow grafts. We therefore used a skin graft model of CD4-mediated T cell rejection across a major H-2 mismatch (C57BL/6 (H-2(b)) to BALB/c (H-2(d)) mice) to evaluate whether nondividing CD4(+) T cells derived from a mixed lymphocyte culture would exhibit tolerance to a skin graft from the initial stimulator strain. We demonstrate that selective removal of dividing alloreactive CD4(+) T cells resulted in marked specific prolongation of allogeneic skin graft survival, and that the nondividing CD4(+) T cells retained a broad TCR repertoire and the ability to maintain memory. This novel way of depleting alloreactive T cells may serve as a useful strategy in combination with other mechanisms to achieve transplant tolerance.     

G-CSF-treated donor CD4(+) T cells attenuate acute GVHD through a reduction in Th17 cell differentiation

The immunoregulatory effects of granulocyte colony-stimulating factor (G-CSF) on allogeneic peripheral blood cell transplantation (PBCT) have been demonstrated to reduce acute graft-versus-host disease (GVHD). However, the underlying mechanism is still not clear. In this study, we focused on the direct effects of G-CSF on donor CD4(+) T cell responses after transplantation. We observed that lethally irradiated B6D2F1 recipient mice that are transplanted with CD4(+) T cells from G-CSF-treated B6 donors showed mild attenuations in severity and mortality compared with recipients transplanted with PBS-treated CD4(+) T cells. Notably, skin GVHD was significantly reduced, but no such reduction was observed in other organs. Although there was no difference with respect to alloreactive expansion or Foxp3(+) Treg induction, the use of G-CSF-treated CD4(+) T cells significantly reduced the numbers of IL-17-producing and RORγt-expressing cells in the secondary lymphoid organs of allogeneic recipients after transplantation compared with the use of the control cells. Finally, we found that the suppressor of cytokine signaling-3 (SOCS3) expression in G-CSF-treated donor CD4(+) T cells was much higher than that in control CD4(+) T cells. Our results demonstrate that the inhibition of Th17 cell differentiation by SOCS3 induction is associated with the immunoregulatory role of G-CSF in CD4(+) T cell-mediated acute GVHD.     

Differential roles for CCR5 expression on donor T cells during graft-versus-host disease based on pretransplant conditioning

The coordinated expression of chemokines and receptors may be important in the directed migration of alloreactive T cells during graft-vs-host disease (GVHD). Recent work demonstrated in a murine model that transfer of CCR5-deficient (CCR5(-/-)) donor cells to nonconditioned haploidentical recipients resulted in reduced donor cell infiltration in liver and lymphoid tissues compared with transfer of CCR5(+/+) cells. To investigate the function of CCR5 during GVHD in conditioned transplant recipients, we transferred CCR5(-/-) or wild-type C57BL/6 (B6) T cells to lethally irradiated B6D2 recipients. Unexpectedly, we found an earlier time to onset and a worsening of GVHD using CCR5(-/-) T cells, which was associated with significant increases in the accumulation of alloreactive CD4(+) and CD8(+) T cells in liver and lung. Conversely, the transfer of CCR5(-/-) donor cells to nonirradiated recipients led to reduced infiltration of target organs, confirming previous studies and demonstrating that the role of CCR5 on donor T cells is dependent on conditioning of recipients. Expression of proinflammatory chemokines in target tissues was dependent on conditioning of recipients, such that CXCL10 and CXCL11 were most highly expressed in tissues of irradiated recipients during the first week post-transplant. CCR5(-/-) T cells were shown to have enhanced migration to CXCL10, and blocking this ligand in vivo improved survival in irradiated recipients receiving CCR5(-/-) T cells. Our data indicate that the effects of inhibiting CCR5/ligand interaction on donor T cells during GVHD differ depending on conditioning of recipients, a finding with potentially important clinical significance.     

Ex vivo expansion of CD4+CD25+FoxP3+ T regulatory cells based on synergy between IL-2 and 4-1BB signaling

Naturally occurring CD4(+)CD25(+)FoxP3(+) T regulatory (Treg) cells require three distinct signals transduced via TCR, CD28, and IL-2R for their development and maintenance. These requirements served as the basis for several recently developed ex vivo expansion protocols that relied on the use of solid support-bound Abs to CD3 and CD28 in the presence of high dose IL-2. We report in this study that Treg cells up-regulate the expression of inducible costimulatory receptor 4-1BB in response to IL-2, and stimulation using this receptor via a novel form of 4-1BB ligand (4-1BBL) fused to a modified form of core streptavidin (SA-4-1BBL) was effective in expanding these cells up to 110-fold within 3 wk. Expanded cells up-regulated CD25, 4-1BB, and membranous TGF-beta, suppressed T cell proliferation, and prevented the rejection of allogeneic islets upon adoptive transfer into graft recipients. Importantly, SA-4-1BBL rendered CD4(+)CD25(-) T effector cells refractive to suppression by Treg cells. This dual function of signaling via 4-1BB, vis-à-vis Treg cell expansion and licensing T effector cells resistant to Treg cell suppression, as well as the up-regulation of 4-1BB by IL-2 may serve as important regulatory mechanisms for immune homeostasis following antigenic challenge. Stimulation using a soluble form of SA-4-1BBL represents a novel approach to expand Treg cells with potential therapeutic applications in autoimmunity and transplantation.     

Trachea allograft class I molecules directly activate and retain CD8+ T cells that cause obliterative airways disease

Human T cells responding against transplanted allogeneic lung tissue have been implicated in late graft failure secondary to obliterative bronchiolitis. This obliterative airways disease (OAD) also develops in heterotopic murine tracheal allografts in association with graft infiltration by both CD8(+) and CD4(+) T cells. To date, there has been little evidence to suggest that directly alloreactive CD8(+) T cells either promote chronic rejection or lead to the development of OAD following airway allotransplantation. Using L(d)-specific TCR-Tg 2C CD8(+) T cells adoptively transferred into wild-type B6 (H-2(b)) mice and the transplantation of BALB/c (H-2(d)) tracheal allografts, we now show that the direct recognition of donor-specific class I MHC molecules by host CD8(+) T cells leads to their activation, clonal expansion within the graft, and differentiation to an effector phenotype with the capacity to induce airway fibrosis. In addition, these experiments demonstrate that ongoing direct alloantigen recognition within the transplanted airway tissue is necessary for the recruitment and retention of these directly alloreactive CD8(+) T cells. Thus, these experiments are the first to definitively show a role for directly alloreactive CD8(+) T cells in the chronic rejection that leads to OAD.     

Rapid and selective expansion of nonclonotypic T cells in regulatory T cell-deficient, foreign antigen-specific TCR-transgenic scurfy mice: antigen-dependent expansion and TCR analysis

Foreign Ag-specific TCR-transgenic (Tg) mice contain a small fraction of T cells bearing the endogenous Vbeta and Valpha chains as well as a population expressing an intermediate level of Tg TCR. Importantly, these minor nonclonotypic populations contain > or = 99% of the CD4(+)Foxp3(+) regulatory T cells (Treg) and, despite low overall Treg expression, peripheral tolerance is maintained. In the OT-II TCR (OVA-specific, Vbeta5(high)Valpha2(high)) Tg scurfy (Sf) mice (OT-II Sf) that lack Treg, nonclonotypic T cells markedly expanded in the periphery but not in the thymus. Expanded T cells expressed memory/effector phenotype and were enriched in blood and inflamed lungs. In contrast, Vbeta5(high)Valpha2(high) clonotypic T cells were not expanded, displayed the naive phenotype, and found mainly in the lymph nodes. Importantly, Vbeta5(neg) T cells were able to transfer multiorgan inflammation in Rag1(-/-) recipients. T cells bearing dual TCR (dual Vbeta or dual Valpha) were demonstrated frequently in the Vbeta5(int) and Valpha2(int) populations. Our study demonstrated that in the absence of Treg, the lack of peripheral expansion of clonotypic T cells is due to the absence of its high-affinity Ag OVA. Thus, the rapid expansion of nonclonotypic T cells in OT-II Sf mice must require Ag (self and foreign) with sufficient affinity. Our study has implications with respect to the roles of Ag and dual TCR in the selection and regulation of Treg and Treg-controlled Ag-dependent T cell expansion in TCR Tg and TCR Tg Sf mice, respectively.     

Preferential blockade of CD8(+) T cell responses by administration of anti-CD137 ligand monoclonal antibody results in differential effect on development of murine acute and chronic graft-versus-host diseases.

We investigated the effect of CD137 costimulatory blockade in the development of murine acute and chronic graft-vs-host diseases (GVHD). The administration of anti-CD137 ligand (anti-CD137L) mAb at the time of GVHD induction ameliorated the lethality of acute GVHD, but enhanced IgE and anti-dsDNA IgG autoantibody production in chronic GVHD. The anti-CD137L mAb treatment efficiently inhibited donor CD8(+) T cell expansion and IFN-gamma expression by CD8(+) T cells in both GVHD models and CD8(+) T cell-mediated cytotoxicity against host-alloantigen in acute GVHD. However, a clear inhibition of donor CD4(+) T cell expansion and activation has not been observed. On the contrary, in chronic GVHD, the number of CD4(+) T cells producing IL-4 was enhanced by anti-CD137L mAb treatment. This suggests that the reduction of CD8(+) T cells producing IFN-gamma promotes Th2 cell differentiation and may result in exacerbation of chronic GVHD. Our results highlight the effective inactivation of CD8(+) T cells and the lesser effect on CD4(+) T cell inactivation by CD137 blockade. Intervention of the CD137 costimulatory pathway may be beneficial for some selected diseases in which CD8(+) T cells are major effector or pathogenic cells. Otherwise, a combinatorial approach will be required for intervention of CD4(+) T cell function.