4-1BB ligand induces cell division, sustains survival, and enhances effector function of CD4 and CD8 T cells with similar efficacy

A costimulatory member of the TNFR family, 4-1BB, is expressed on activated T cells. Although some reports have suggested that 4-1BB is primarily involved in CD8 T cell activation, in this report we demonstrate that both CD4 and CD8 T cells respond to 4-1BB ligand (4-1BBL) with similar efficacy. CD4 and CD8 TCR transgenic T cells up-regulate 4-1BB, OX40, and CD27 and respond to 4-1BBL-mediated costimulation during a primary response to peptide Ag. 4-1BBL enhanced proliferation, cytokine production, and CTL effector function of TCR transgenic T cells. To compare CD4 vs CD8 responses to 4-1BBL under similar conditions of antigenic stimulation, we performed MLRs with purified CD4 or CD8 responders from CD28(+/+) and CD28(-/-) mice. We found that CD8 T cells produced IL-2 and IFN-gamma in a 4-1BBL-dependent manner, whereas under the same conditions the CD4 T cells produced IL-2 and IL-4. 4-1BBL promoted survival of CD4 and CD8 T cells, particularly at late stages of the MLR. CD4 and CD8 T cells both responded to anti-CD3 plus s4-1BBL with a similar cytokine profile as observed in the MLR. CD4 and CD8 T cells exhibited enhanced proliferation and earlier cell division when stimulated with anti-CD3 plus anti-CD28 compared with anti-CD3 plus 4-1BBL, and both subsets responded comparably to anti-CD3 plus 4-1BBL. These data support the idea that CD28 plays a primary role in initial T cell expansion, whereas 4-1BB/4-1BBL sustains both CD4 and CD8 T cell responses, as well as enhances cell division and T cell effector function.     

4-1BB and OX40 dual costimulation synergistically stimulate primary specific CD8 T cells for robust effector function

CD40, 4-1BB, and OX40 are costimulatory molecules belonging to the TNF/nerve growth factor superfamily of receptors. We examined whether simultaneous costimulation affected the responses of T cells using several different in vivo tracking models in mice. We show that enforced dual costimulation through 4-1BB and OX40, but not through CD40, induced profound specific CD8 T cell clonal expansion. In contrast, the response of specific CD4 T cells to dual costimulation was additive rather than synergistic. The synergistic response of the specific CD8 T cells persevered for several weeks, and the expanded effector cells resided throughout lymphoid and nonlymphoid tissue. Dual costimulation through 4-1BB and OX40 did not increase BrdU incorporation nor an increase in the number of rounds of T cell division in comparison to single costimulators, but rather enhanced accumulation in a cell-intrinsic manner. Mechanistically speaking, we show that CD8 T cell clonal expansion and effector function did not require T help, but accumulation in (non)lymphoid tissue was predominantly CD4 T cell dependent. To determine whether this approach would be useful in a physiological setting, we demonstrated that dual costimulation mediated rejection of an established murine sarcoma. Importantly, effector function directed toward established tumors was CD8 T cell dependent while being entirely CD4 T cell independent, and the timing of enforced dual costimulation was exquisitely regulated. Collectively, these data suggest that simultaneous dual costimulation through 4-1BB and OX40 induces a massive burst of CD8 T cell effector function sufficient to therapeutically treat established tumors even under immunocompromising conditions.     

CD8 T cell clonal expansion and development of effector function require prolonged exposure to antigen, costimulation, and signal 3 cytokine

Full activation of naive CD8 T cells requires Ag, costimulation, and a third signal that can be provided by IL-12. Brief exposure (6 h) to Ag and B7-1 is sufficient to stimulate multiple rounds of cell division, but clonal expansion and development of effector function are minimal even when signal 3 is present. Full activation instead requires concerted signaling by Ag, B7-1, and IL-12 for greater than 40 h. Thus, the gene expression program required for cell division can be initiated by brief interaction with Ag and costimulation, but maintaining the expression of the genes needed for survival and effector function requires prolonged signaling by a signal 3 cytokine in concert with Ag and costimulation.     

4-1BB costimulation enhances HSV-1-specific CD8+ T cell responses by the induction of CD11c+CD8+ T cells

Since 4-1BB plays a predominant role in CD8+ T cell responses, we investigated the effects of 4-1BB triggering on the primary and memory CD8+ T responses to HSV-1 infection. 4-1BB was detected on 10-15% of CD4+ and CD8+ T cells following the infection. 4-1BB-positive T cells were in the proliferative mode and showed the enhanced expression of anti-apoptotic proteins. Agonistic anti-4-1BB treatment exerted preferential expansion of CD8+ T cells and gB/H-2Kb-positive CD8+ T cells, and enhanced cytotoxicity against HSV-1 that was mainly mediated by CD11c+CD8+ T cells. CD11c+CD8+ T cells were re-expanded following re-challenge with HSV-1 at post-infection day 50, indicating that CD11c+CD8+ phenotype was maintained in memory CD8+ T cell pool. Our studies demonstrated that 4-1BB stimulation enhanced both primary and memory anti-HSV-1 CD8+ T cell responses, which was mediated by a massive expansion of antigen-specific CD11c+CD8+ T cells.     

4-1BB costimulation promotes human T cell adhesion to fibronectin

CD28 and 4-1BB (CD137) are costimulatory molecules for T cells. In this study we investigated the role of 4-1BB in T cell adhesion to fibronectin (FN). Unlike CD28, 4-1BB is present in only a small subset of T cells prepared from fresh human peripheral blood mononuclear cells, but was induced after prolonged TCR/CD28 activation in vitro. 4-1BB-expressing T cells were characteristically unique in their strong responsiveness to FN. Anti-4-1BB cross-linking synergized CD28 costimulation by lowering the threshold of CD3 signal required for CD28-mediated maximal proliferative response. In addition to increasing proliferative responses, 4-1BB promoted T cell adhesion to FN in the presence of CD28 costimulation. 4-1BB-mediated cell adhesion to FN was blocked by anti-beta1 integrin, suggesting that 4-1BB mediates beta1 integrin activation. The role of 4-1BB in inducing CD4(+) T cell adhesion to FN was confirmed by showing that the human leukemic CD4(+) T cell line, Jurkat, when transfected with cDNA encoding 4-1BB, became adherent to FN with anti-4-1BB stimulation. Taken together, our results suggest that 4-1BB-promoted T cell adhesion to extracellular matrix proteins is an important postactivation process for T cell migration.     

Potent costimulation of human CD8 T cells by anti-4-1BB and anti-CD28 on synthetic artificial antigen presenting cells

The in vitro generation of cytotoxic T lymphocytes (CTLs) for anticancer immunotherapy is a promising approach to take patient-specific therapy from the bench to the bedside. Two criteria must be met by protocols for the expansion of CTLs: high yield of functional cells and suitability for good manufacturing practice (GMP). The antigen presenting cells (APCs) used to expand the CTLs are the key to achieving both targets but they pose a challenge: Unspecific stimulation is not feasible because only memory T cells are expanded and not rare naïve CTL precursors; in addition, antigen-specific stimulation by cell-based APCs is cumbersome and problematic in a clinical setting. However, synthetic artificial APCs which can be loaded reproducibly with MHC-peptide monomers and antibodies specific for costimulatory molecules could resolve these problems. The purpose of this study was to investigate the potential of complex synthetic artificial APCs in triggering the costimulatory molecules CD28 and 4-1BB on the T cell. Anti-4-1BB antibodies were added to an established system of microbeads coated with MHC-peptide monomers and anti-CD28. Triggering via CD28 and 4-1BB resulted in strong costimulatory synergy. The quantitative ratio between these signals determined the outcome of the stimulation with optimal results when anti-4-1BB and anti-CD28 were applied in a 3:1 ratio. Functional CTLs of an effector memory subtype (CD45RA^? CCR7^?) were generated in high numbers. We present a highly defined APC platform using off-the-shelf reagents for the convenient generation of large numbers of antigen-specific CTLs.     

The 4-1BB costimulation augments the proliferation of CD4+CD25+ regulatory T cells

The thymus-derived CD4(+)CD25(+) T cells belong to a subset of regulatory T cells potentially capable of suppressing the proliferation of pathogenic effector T cells. Intriguingly, these suppressor cells are themselves anergic, proliferating poorly to mitogenic stimulation in culture. In this study, we find that the 4-1BB costimulator receptor, best known for promoting the proliferation and survival of CD8(+) T cells, also induces the proliferation of the CD4(+)CD25(+) regulatory T cells both in culture and in vivo. The proliferating CD4(+)CD25(+) T cells produce no detectable IL-2, suggesting that 4-1BB costimulation of these cells does not involve IL-2 production. The 4-1BB-expanded CD4(+)CD25(+) T cells are functional, as they remain suppressive to other T cells in coculture. These results support the notion that the peripheral expansion of the CD4(+)CD25(+) T cells is controlled in part by costimulation.     

Functional dichotomy between OX40 and 4-1BB in modulating effector CD8 T cell responses

Members of the TNFR family are thought to deliver costimulatory signals to T cells and modulate their function and survival. In this study, we compare the role of two closely related TNFR family molecules, OX40 and 4-1BB, in generating effector CD8 T cells to Ag delivered by adenovirus. OX40 and 4-1BB were both induced on responding naive CD8 T cells, but 4-1BB exhibited faster and more sustained kinetics than OX40. OX40-deficient CD8 T cells initially expanded normally; however, their accumulation and survival at late times in the primary response was significantly impaired. In contrast, 4-1BB-deficient CD8 T cells displayed hyperresponsiveness, expanding more than wild-type cells. The 4-1BB-deficient CD8 T cells also showed enhanced maturation attributes, whereas OX40-deficient CD8 T cells had multiple defects in the expression of effector cell surface markers, the synthesis of cytokines, and in cytotoxic activity. These results suggest that, in contrast to current ideas, OX40 and 4-1BB can have a clear functional dichotomy in modulating effector CD8 T cell responses. OX40 can positively regulate effector function and late accumulation/survival, whereas 4-1BB can initially operate in a negative manner to limit primary CD8 responses.     

Disparate primary and secondary allospecific CD8+ T cell cytolytic effector function in the presence or absence of host CD4+ T cells

The role of CD4+ T cells in promoting CD8+ T cell effector activity in response to transplant Ags in vivo has not been reported. We used a hepatocellular allograft model known to initiate both CD4-dependent and CD4-independent rejection responses to investigate the contribution of CD4+ T cells to the development, function, and persistence of allospecific CD8+ T cell effectors in vivo. Complete MHC-mismatched hepatocellular allografts were transplanted into C57BL/6 (CD4-sufficient) or CD4 knockout (CD4-deficient) hosts. The development of in vivo allospecific cytotoxicity was determined by clearance of CFSE-labeled target cells. CD8+ T cell cytotoxic effector activity was enhanced in response to allogeneic hepatocellular grafts with a greater magnitude of allocytotoxicity and a prolonged persistence of CTL effector activity in CD4-sufficient hosts compared with CD4-deficient hosts. Cytolytic activity was mediated by CD8+ T cells in both recipient groups. In response to a second hepatocyte transplant, rejection kinetics were enhanced in both CD4-sufficient and CD4-deficient hepatocyte recipients. However, only CD4-sufficient hosts developed recall CTL responses with an augmented magnitude and persistence of allocytotoxicity in comparison with primary CTL responses. These studies show important functional differences between alloreactive CD8+ T cell cytolytic effectors that mature in vivo in the presence or absence of CD4+ T cells.     

Antigen-specific CD4 effector T cells: Analysis of factors regulating clonal expansion and cytokine production: Clonal expansion and cytokine production by CD4 effector T cells

In order to fully understand T cell-mediated immunity, the mechanisms that regulate clonal expansion and cytokine production by CD4⁺ antigen-specific effector T cells in response to a wide range of antigenic stimulation needs clarification. For this purpose, panels of antigen-specific CD4⁺ T cell clones with different thresholds for antigen-induced proliferation were generated by repeated stimulation with high- or low-dose antigen. Differences in antigen sensitivities did not correlate with expression of TCR, CD4, adhesion or costimulatory molecules. There was no significant difference in antigen-dependent cytokine production by TG40 cells transfected with TCR obtained from either high- or low-dose-responding T cell clones, suggesting that the affinity of TCRs for their ligands is not primary determinant of T cell antigen reactivity. The proliferative responses of all T cell clones to both peptide stimulation and to TCRβ crosslinking revealed parallel dose-response curves. These results suggest that the TCR signal strength of effector T cells and threshold of antigen reactivity is determined by an intrinsic property, such as the TCR signalosome and/or intracellular signaling machinery. Finally, the antigen responses of high- and low-peptide-responding T cell clones reveal that clonal expansion and cytokine production of effector T cells occur independently of antigen concentration. Based on these results, the mechanisms underlying selection of high “avidity” effector and memory T cells in response to pathogen are discussed.     

CD4(-)CD8alphaalpha subset of CD1d-restricted NKT cells controls T cell expansion

Valpha24 invariant (Valpha24i) CD1d-restricted NKT cells are widely regarded to have immune regulatory properties. They are known to have a role in preventing autoimmune diseases and are involved in optimally mounted immune responses to pathogens and tumor cells. We were interested in understanding how these cells provide protection in autoimmune diseases. We first observed, using EBV/MHC I tetrameric complexes, that expansion of Ag-specific cells in human PBMCs was reduced when CD1d-restricted NKT cells were concomitantly activated. This was accompanied by an increase in a CD4(-)CD8alphaalpha(+) subset of Valpha24i NKT cells. To delineate if a specific subset of NKT cells was responsible for this effect, we generated different subsets of human CD4(-) and CD4(+) Valpha24i NKT clones and demonstrate that a CD4(-)CD8alphaalpha(+) subset with highly efficient cytolytic ability was unique among the clones in being able to suppress the proliferation and expansion of activated T cells in vitro. Activated clones were able to kill CD1d-bearing dendritic or target cells. We suggest that one mechanism by which CD1d-restricted NKT cells can exert a regulatory role is by containing the proliferation of activated T cells, possibly through timely lysis of APCs or activated T cells bearing CD1d.     

A switch in costimulation from CD28 to 4-1BB during primary versus secondary CD8 T cell response to influenza in vivo

4-1BBL(-/-) mice exhibit normal primary CD8 T cell responses to influenza virus, but show decreased CD8 T cell numbers late in the primary response as well as decreased secondary responses. In contrast, CD28(-/-) mice are defective in initial CD8 T cell expansion. Using agonistic anti-4-1BB Ab to replace the CD28 or 4-1BB signal, we examined the timing of the required signals for CD28 vs 4-1BB costimulation. A single dose of agonistic anti-4-1BB Ab added only during priming restores the secondary CD8 T cell response in CD28(-/-) mice. Once the T cell numbers in the primary response reach a minimum threshold, a full secondary response is achieved even in the absence of CD28. In contrast, anti-4-1BB added during priming fails to correct the defective secondary response in 4-1BBL(-/-) mice, whereas addition of anti-4-1BB during challenge fully restores this response. Thus, there is a switch in costimulatory requirement from CD28 to 4-1BB during primary vs recall responses. Adoptive transfer studies show that T cells primed in 4-1BBL(-/-) or wild-type mice are equally capable of re-expansion when rechallenged in wild-type mice. These studies rule out a model in which signals delivered through 4-1BB during priming program the T cells to give a full recall response and suggest that 4-1BB-4-1BBL interactions take place at later stages in the immune response. The results indicate that anti-4-1BB or 4-1BBL therapy will be most effective during the boost phase of a prime-boost vaccination strategy.     

Double-negative (CD4- CD8-) T cells with an alpha/beta T cell receptor. Non-MHC-restricted cytolytic activity and lymphokine production

T cell lines with a novel phenotype (CD3+ TCR-alpha/beta+ CD4- CD8-) were developed from the peripheral blood of a patient with a combined immunodeficiency and tissue injury resembling graft-vs-host disease. One of these IL-2-dependent T cell lines demonstrated non-MHC-restricted cytolytic function against tumor targets, syngeneic and allogeneic fibroblasts, and PHA blasts from allogeneic donors. The other cell line only became cytotoxic in the presence of lectin or anti-CD3 antibody. The two cell lines also differed in their expression of the T-200 gene products CD45RO (gp180) and CD45RA (gp220). Both cell lines produced tumor necrosis factor-alpha and -beta and IFN-gamma activity when activated with mitogens or PMA and IL-1. The in vitro functions of these T-cell lines suggest a potential role for alpha/beta double-negative T lymphocytes in tissue injury resembling graft-vs-host disease.     

Cutting edge: cytolytic effector function in human circulating CD8+ T cells closely correlates with CD56 surface expression

Recent data suggest that human effector CD8+ T cells express a distinct CD27-CD45RAhigh (CD57+CD28-CD11ahigh) phenotype. Here, we propose that CTL effector function correlates better with CD56 (neuronal cell adhesion molecule (NCAM)) surface expression. CD56 was absent on cord blood CD8+ T cells, but was expressed by 4-30% of freshly isolated circulating CD8+ T cells from 15 adults. Dramatic oligoclonal expansions in 3/3 individuals were confined to the CD56+ subset of CD8+ T cells. The CD56+ subset generally contained high amounts of intracellular perforin and granzyme B. Finally, direct cytolytic capacity was closely restricted to the CD56+(CD45RAhigh) cells, better than to CD27-CD45RAhigh cells in 5/5 individuals analyzed. Thus, the phenotype corresponding to the circulating effector CD8+ T cell pool may be simplified and more precisely defined by the use of just two surface markers: CD8 and CD56.     

CD28 costimulation is essential for human T regulatory expansion and function

The costimulatory requirements required for peripheral blood T regulatory cells (Tregs) are unclear. Using cell-based artificial APCs we found that CD28 but not ICOS, OX40, 4-1BB, CD27, or CD40 ligand costimulation maintained high levels of Foxp3 expression and in vitro suppressive function. Only CD28 costimulation in the presence of rapamycin consistently generated Tregs that consistently suppressed xenogeneic graft-vs-host disease in immunodeficient mice. Restimulation of Tregs after 8-12 days of culture with CD28 costimulation in the presence of rapamycin resulted in >1000-fold expansion of Tregs in <3 wk. Next, we determined whether other costimulatory pathways could augment the replicative potential of CD28-costimulated Tregs. We observed that while OX40 costimulation augmented the proliferative capacity of CD28-costimulated Tregs, Foxp3 expression and suppressive function were diminished. These studies indicate that the costimulatory requirements for expanding Tregs differ from those for T effector cells and, furthermore, they extend findings from mouse Tregs to demonstrate that human postthymic Tregs require CD28 costimulation to expand and maintain potent suppressive function in vivo.     

Ex vivo expansion of human CD8+ T cells using autologous CD4+ T cell help

Background

Using in vivo mouse models, the mechanisms of CD4⁺ T cell help have been intensively investigated. However, a mechanistic analysis of human CD4⁺ T cell help is largely lacking. Our goal was to elucidate the mechanisms of human CD4⁺ T cell help of CD8⁺ T cell proliferation using a novel in vitro model.

Methods/Principal Findings

We developed a genetically engineered novel human cell-based artificial APC, aAPC/mOKT3, which expresses a membranous form of the anti-CD3 monoclonal antibody OKT3 as well as other immune accessory molecules. Without requiring the addition of allogeneic feeder cells, aAPC/mOKT3 enabled the expansion of both peripheral and tumor-infiltrating T cells, regardless of HLA-restriction. Stimulation with aAPC/mOKT3 did not expand Foxp3⁺ regulatory T cells, and expanded tumor infiltrating lymphocytes predominantly secreted Th1-type cytokines, interferon-γ and IL-2. In this aAPC-based system, the presence of autologous CD4⁺ T cells was associated with significantly improved CD8⁺ T cell expansion in vitro. The CD4⁺ T cell derived cytokines IL-2 and IL-21 were necessary but not sufficient for this effect. However, CD4⁺ T cell help of CD8⁺ T cell proliferation was partially recapitulated by both adding IL-2/IL-21 and by upregulation of IL-21 receptor on CD8⁺ T cells.

Conclusions

We have developed an in vitro model that advances our understanding of the immunobiology of human CD4⁺ T cell help of CD8⁺ T cells. Our data suggests that human CD4⁺ T cell help can be leveraged to expand CD8⁺ T cells in vitro.     

Three memory subsets of human CD8+ T cells differently expressing three cytolytic effector molecules

Multicolor flow cytometric analysis for the expression of three effector molecules, i.e., perforin (Per), granzyme A (GraA), and granzyme B (GraB), in human CD8(+) T cells demonstrated that they included five subpopulations, implying the following pathway for the differentiation of CD8(+) T cells: Per(-)GraA(-)GraB(-)-->Per(-)GraA(+)GraB(-)-->Per(low)GraA(+)GraB(-)--> Per(low)GraA(+)GraB(+)-->Per(high)GraA(+)GraB(+). The analysis of the expression of these molecules in the subsets classified by the combination of the expression of CCR7 and CD45RA or by that of CD27, CD28, and CD45RA showed that functional CD8(+) T cell subsets could be partially identified by these phenotypic classifications. However, the functional subsets could be precisely identified by the classification using five cell surface markers or three cell surface markers and three cytolytic molecules. Per(-)GraA(-)GraB(-) and Per(-/low)GraA(+)GraB(-) cells were predominantly found in CCR5(-)CCR7(+) and CCR5(high/low)CCR7(-) subsets, respectively, of CD8(+) T cells expressing the CD27(+)CD28(+)CD45RA(-) phenotype, whereas Per(low)GraA(+)GraB(+) cells were found in the CCR5(low)CCR7(-) subset of those expressing this phenotype and in a part of the CCR5(-/low)CCR7(-) subset of those expressing the CD27(-/low)CD28(-)CD45RA(-/+) phenotype. Ex vivo EBV-specific CD8(+) T cells, which were Per(low/-)GraA(+)GraB(-/+) cells, hardly or very weakly killed the target cells, indicating that these were not effector T cells. These findings suggest that the Per(-)GraA(-)GraB(-), Per(-/low)GraA(+)GraB(-), and Per(low)GraA(+)GraB(+) cells were central memory, early effector memory, and late effector memory T cells, respectively. Per(-/low)GraA(+)GraB(-) cells gained GraB expression after TCR stimulation, indicating that early effector memory T cells could differentiate into late effector and effector T cells. The present study showed the existence of three memory subsets and the pathway for their differentiation.     

Enhanced CD4 T cell responsiveness in the absence of 4-1BB

The 4-1BB (CD137) is a member of the TNFR superfamily, and is expressed on several cell types, including activated T cells. Although 4-1BB ligation by agonistic Ab or 4-1BB ligand-expressing APCs can costimulate T cells, the physiological significance of 4-1BB expression in vivo during T cell responses is still being elucidated. In this study, we have addressed the impact on CD4 T cell priming when 4-1BB is absent after gene targeting. Surprisingly, 4-1BB(-/-) mice generated more enhanced effector CD4 T cell responses to OVA protein in adjuvant, even though Ab responses in 4-1BB(-/-) mice were normal. Using an adoptive transfer system with OT-II TCR transgenic CD4 T cells, we found that 4-1BB(-/-) CD4 cells responding in a 4-1BB-sufficient environment had enhanced cell division compared with wild-type cells and displayed augmented clonal expansion during the primary response. This was not due to a developmental defect as 4-1BB-deficient CD4 cells could respond normally to Ag in vitro. These results demonstrate that the absence of 4-1BB can make CD4 T cells hyperresponsive to protein Ag in vivo, suggesting a new unappreciated negative regulatory role of 4-1BB when expressed on a T cell.     

Blockade of CD26-mediated T cell costimulation with soluble caveolin-1-Ig fusion protein induces anergy in CD4T cells

CD26 binds to caveolin-1 in antigen-presenting cells (APC), and that ligation of CD26 by caveolin-1 induces T cell proliferation in a TCR/CD3-dependent manner. We report herein the effects of CD26-caveolin-1 costimulatory blockade by fusion protein caveolin-1-Ig (Cav-Ig). Soluble Cav-Ig inhibits T cell proliferation and cytokine production in response to recall antigen, or allogeneic APC. Our data hence suggest that blocking of CD26-associated signaling by soluble Cav-Ig may be an effective approach as immunosuppressive therapy.     

Activation-induced cell death limits effector function of CD4 tumor-specific T cells

A number of studies have documented a critical role for tumor-specific CD4(+) cells in the augmentation of immunotherapeutic effector mechanisms. However, in the context of an extensive tumor burden, chronic stimulation of such CD4(+) T cells often leads to the up-regulation of both Fas and Fas ligand, and coexpression of these molecules can potentially result in activation-induced cell death and the subsequent loss of effector activity. To evaluate the importance of T cell persistence in an experimental model of immunotherapy, we used DO11 Th1 cells from wild-type, Fas-deficient, and Fas ligand-deficient mice as effector populations specific for a model tumor Ag consisting of an OVA-derived transmembrane fusion protein. We found that the prolonged survival of Fas-deficient DO11 Th1 cells led to a more sustained tumor-specific response both in vitro and in vivo. Importantly, both Fas- and Fas ligand-deficient Th1 cells delayed tumor growth and cause regression of established tumors more effectively than wild-type Th1 cells, indicating that resistance to activation-induced cell death significantly enhances T cell effector activity.