Naive and memory T cells induce different types of graft-versus-host disease

The goal of this study was to compare the ability of donor naive and alloantigen-primed effector memory T cells to induce graft-vs-host disease after bone marrow transplantation in MHC-mismatched irradiated host mice. Purified CD4(+) naive (CD62L(high)CD44(low)) T cells and CD4(+) effector memory (CD62L(low)CD44(high)) T cells obtained from unprimed donors and donors primed to host alloantigens, respectively, were injected into host mice, and the rapidity, severity, and pattern of tissue injury of graft-vs-host disease was assessed. Unexpectedly, the naive T cells induced a more acute and severe colitis than the primed memory cells. Whereas the naive T cells expressing CD62L and CCR7 lymph node homing receptors vigorously expanded in mesenteric lymph nodes and colon by day 6 after transplantation, the primed memory T cells without these receptors had 20- to 100-fold lower accumulation at this early time point. These differences were reflected in the significantly more rapid decline in survival and weight loss induced by naive T cells. The primed memory T cells had a greater capacity to induce chronic colitis and liver injury and secrete IL-2 and IFN-gamma in response to alloantigenic stimulation compared with memory T cells from unprimed donors. Nevertheless, the expected increase in potency as compared with naive T cells was not observed due to differences in the pattern and kinetics of tissue injury.     

Protracted protection to Plasmodium berghei malaria is linked to functionally and phenotypically heterogeneous liver memory CD8+ T cells

We previously demonstrated that protection induced by radiation-attenuated (gamma) Plasmodium berghei sporozoites is linked to MHC class I-restricted CD8(+) T cells specific for exoerythrocytic-stage Ags, and that activated intrahepatic memory CD8(+) T cells are associated with protracted protection. In this study, we further investigated intrahepatic memory CD8(+) T cells to elucidate mechanisms required for their maintenance. Using phenotypic markers indicative of activation (CD44, CD45RB), migration (CD62L), and IFN-gamma production, we identified two subsets of intrahepatic memory CD8(+) T cells: the CD44(high)CD45RB(low)CD62L(low)CD122(low) phenotype, representing the dominant effector memory set, and the CD44(high)CD45RB(high)CD62L(low/high)CD122(high) phenotype, representing the central memory set. Only the effector memory CD8(+) T cells responded swiftly to sporozoite challenge by producing sustained IFN-gamma; the central memory T cells responded with delay, and the IFN-gamma reactivity was short-lived. In addition, the subsets of liver memory CD8(+) T cells segregated according to the expression of CD122 (IL-15R) in that only the central memory CD8(+) T cells were CD122(high), whereas the effector memory CD8(+) T cells were CD122(low). Moreover, the effector memory CD8(+) T cells declined as protection waned in mice treated with primaquine, a drug that interferes with the formation of liver-stage Ags. We propose that protracted protection induced by P. berghei radiation-attenuated sporozoites depends in part on a network of interactive liver memory CD8(+) T cell subsets, each representing a different phase of activation or differentiation, and the balance of which is profoundly affected by the repository of liver-stage Ag and IL-15.     

A pool of central memory-like CD4 T cells contains effector memory precursors

The L51S mutation in the D10.G4.1 TCR alpha-chain reduces the affinity of the TCR to its ligand by affecting the interactions among the TCR, the beta-chain of I-A(k), and the bound peptide. We show that this mutation drives the generation of a pool of memory CD44(high)CD62L(neg)CD45RB(neg) CD4 TCR transgenic T cells. Their activation threshold is low, such that they proliferate in response to lower concentrations of agonist peptides than naive L51S CD4 T cells. Unlike effector memory CD4 T cells, however, they lack immediate effector function in response to TCR stimulation. These cells express IL-2R alpha only after culture with specific peptide. Although they can be recovered from lymph nodes, the majority lack the expression of the lymph node homing receptor CCR7. When these cells receive a second TCR stimulation in vitro, they differentiate into potent Th2-like effector cells, producing high levels of IL-4 at doses of agonist peptide too low to stimulate cytokine release from similarly differentiated naive L51S CD4 T cells. Having these properties, the L51S TCR transgenic memory CD4 T cells cannot be classified as either strict central memory or effector memory, but, rather, as a pool of memory T cells containing effector memory precursors.     

Reduced L-selectin (CD62LLow) expression identifies tumor-specific type 1 T cells from lymph nodes draining an autologous tumor cell vaccine

Reduced expression of CD62L can identify tumor-specific T cells in lymph nodes draining murine tumors. Here, we examined whether this strategy could isolate tumor-specific T cells from vaccinated patients. Tumor vaccine-draining lymph node (TVDLN) T cells of seven patients were separated into populations with reduced (CD62LLow) or high levels of CD62L (CD62LHigh). Effector T cells generated from CD62LLow cells maintained or enriched the autologous tumor-specific type 1 cytokine response compared to unseparated TVDLN T cells in four of four patients showing tumor-specific cytokine secretion. Interestingly, effector T cells generated from CD62LLow or CD62LHigh TVDLN were polarized towards a dominant type 1 or type 2 cytokine profile, respectively. For CD62LLow T cells the type 1 cytokine profile appeared determined prior to culture. Since a tumor-specific type 1 cytokine profile appears critical for mediating anti-tumor activity in vivo, this approach might be used to isolate T cells for adoptive immunotherapy.     

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.     

CD27low CD4 T lymphocytes that accumulate in the mouse lungs during mycobacterial infection differentiate from CD27high precursors in situ, produce IFN-gamma, and protect the host against tuberculosis infection

The generation of effector, IFN-gamma producing T lymphocytes and their accumulation at sites of infection are critical for host protection against various infectious diseases. The activation and differentiation of naive T lymphocytes into effector memory cells starts in lymphoid tissues, but it is not clear whether the Ag-experienced cells that leave lymph nodes (LN) are mature or if they undergo further changes in the periphery. We have previously shown that CD44(high)CD62L(low) effector CD4 T lymphocytes generated during the course of mycobacterial infection can be segregated into two subsets on the basis of CD27 receptor expression. Only the CD27(low) subset exhibited a high capacity for IFN-gamma secretion, indicating that low CD27 expression is characteristic of fully differentiated effector CD4 T lymphocytes. We demonstrate now that CD27(low) IFN-gamma-producing CD4 T lymphocytes accumulate in the lungs but are rare in LNs. Several factors contribute to their preferential accumulation. First, CD27(low) CD4 T lymphocytes present in the LN are highly susceptible to apoptosis. Second, circulating CD27(low) CD4 T cells do not enter the LN but efficiently migrate to the lungs. Third, CD27(high) effector CD4 T cells that enter the lungs down-regulate CD27 expression in situ. In genetically heterogeneous mice that exhibit varying susceptibility to tuberculosis, the accumulation of mature CD27(low) CD4 T cells in the lungs correlates with the degree of protection against infection. Thus, we propose that terminal maturation of effector CD4 T lymphocytes in the periphery provides the host with efficient local defense and avoids potentially harmful actions of inflammatory cytokines in lymphoid organs.     

A spontaneously arising pancreatic tumor does not promote the differentiation of naive CD8+ T lymphocytes into effector CTL

In this report, we address whether a growing tumor provides sufficient inflammatory signals to promote activation, clonal expansion, and acquisition of effector functions by naive tumor-specific CD8(+) T lymphocytes. CD8(+) T lymphocytes obtained from hemagglutinin (HA)-specific clone 4 TCR-transgenic mice were injected into recipient mice that spontaneously develop pancreatic tumors expressing HA as a tumor-associated Ag (RIP-Tag2-HA mice). When 3 x 10(6) clone 4 CD8(+) T cells were transferred into tumor-bearing mice, the cells became activated in the pancreatic lymph nodes where they proliferated and acquired effector functions such as cytolytic activity and IFN-gamma production. Surprisingly, reducing the number of adoptively transferred CD8(+) T cells led to a parallel reduction in the proportion of the activated cells that exhibited effector functions, suggesting that CTL differentiation was induced by the large numbers of activated CD8(+) T cells and not the tumor environment. Provision of tumor-specific CD4(+) helper cells provided the signals required to promote both the development of CTL effector functions and increased clonal expansion, resulting in tumor eradication. Considering that only small numbers of tumor-specific CD8(+) T cells would be present in a conventional T cell repertoire, these data suggest that tumor growth alone may not provide the inflammatory signals necessary to support the development of CD8(+) T cell effector functions.     

Memory T cells are enriched in lymph nodes of selectin-ligand-deficient mice

Fucosyltransferase-IV and -VII double knockout (FtDKO) mice reveal profound impairment in T cell trafficking to lymph nodes (LNs) due to an inability to synthesize selectin ligands. We observed an increase in the proportion of memory/effector (CD44(high)) T cells in LNs of FtDKO mice. We infected FtDKO mice with lymphocytic choriomeningitis virus to generate and track Ag-specific CD44(high)CD8 T cells in secondary lymphoid organs. Although frequencies were similar, total Ag-specific effector CD44(high)CD8 T cells were significantly reduced in LNs, but not blood, of FtDKO mice at day 8. In contrast, frequencies of Ag-specific memory CD44(high)CD8 T cells were up to 8-fold higher in LNs of FtDKO mice at day 60. Because wild-type mice treated with anti-CD62L treatment also showed increased frequencies of CD44(high) T cells in LNs, we hypothesized that memory T cells were preferentially retained in, or preferentially migrated to, FtDKO LNs. We analyzed T cell entry and egress in LNs using adoptive transfer of bone fide naive or memory T cells. Memory T cells were not retained longer in LNs compared with naive T cells; however, T cell exit slowed significantly as T cell numbers declined. Memory T cells were profoundly impaired in entering LNs of FtDKO mice; however, memory T cells exhibited greater homeostatic proliferation in FtDKO mice. These results suggest that memory T cells are enriched in LNs with T cell deficits by several mechanisms, including longer T cell retention and increased homeostatic proliferation.     

Tumors hamper the immunogenic competence of CD4+ T cell-directed dendritic cell vaccination

Dendritic cells loaded with tumor-derived peptides induce protective CTL responses and are under evaluation in clinical trails. We report in this study that prophylactic administration of dendritic cells loaded with a MHC class II-restricted peptide derived from a model tumor Ag (Leishmania receptor for activated C kinase (LACK)) confers protection against LACK-expressing TS/A tumors, whereas therapeutic vaccination fails to cure tumor-bearing mice. Although CD4+ T cell-directed dendritic cell vaccination primed effector-like (CD44(high)CD62L(low), IL-2(+), IFN-gamma(+)) and central memory-like lymphocytes (CD44(high)CD62L(high), only IL-2(+)) in tumor-free mice, this was not the case in tumor-bearing animals in which both priming and persistence of CD4+ T cell memory were suppressed. Suppression was specific for the tumor-associated Ag LACK, and did not depend on CD25+ T cells. Because T cell help is needed for protective immunity, we speculate that the ability of tumors to limit vaccine-induced CD4+ T cell memory could provide a partial explanation for the limited efficacy of current strategies.     

Persistence and function of central and effector memory CD4+ T cells following infection with a gastrointestinal helminth

Immunity in the gastrointestinal tract is important for resistance to many pathogens, but the memory T cells that mediate such immunity are poorly characterized. In this study, we show that following sterile cure of a primary infection with the gastrointestinal parasite Trichuris muris, memory CD4+ T cells persist in the draining mesenteric lymph node and protect mice against reinfection. The memory CD4+ T cells that developed were a heterogeneous population, consisting of both CD62L(high) central memory T cells (T(CM)) and CD62L(low) effector memory T cells (T(EM)) that were competent to produce the Th type 2 effector cytokine, IL-4. Unlike memory T cells that develop following exposure to several other pathogens, both CD4+ T(CM) and T(EM) populations persisted in the absence of chronic infection, and, critically, both populations were able to transfer protective immunity to naive recipients. CD62L(high)CD4+ T(CM) were not apparent early after infection, but emerged following clearance of primary infection, suggesting that they may be derived from CD4+ T(EM). Consistent with this theory, transfer of CD62L(low)CD4+ T(EM) into naive recipients resulted in the development of a population of protective CD62L(high)CD4+ T(CM). Taken together, these studies show that distinct subsets of memory CD4+ T cells develop after infection with Trichuris, persist in the GALT, and mediate protective immunity to rechallenge.     

Effector memory Th1 CD4 T cells are maintained in a mouse model of chronic malaria

Protection against malaria often decays in the absence of infection, suggesting that protective immunological memory depends on stimulation. Here we have used CD4(+) T cells from a transgenic mouse carrying a T cell receptor specific for a malaria protein, Merozoite Surface Protein-1, to investigate memory in a Plasmodium chabaudi infection. CD4(+) memory T cells (CD44(hi)IL-7Rα(+)) developed during the chronic infection, and were readily distinguishable from effector (CD62L(lo)IL-7Rα(-)) cells in acute infection. On the basis of cell surface phenotype, we classified memory CD4(+) T cells into three subsets: central memory, and early and late effector memory cells, and found that early effector memory cells (CD62L(lo)CD27(+)) dominated the chronic infection. We demonstrate a linear pathway of differentiation from central memory to early and then late effector memory cells. In adoptive transfer, CD44(hi) memory cells from chronically infected mice were more effective at delaying and reducing parasitemia and pathology than memory cells from drug-treated mice without chronic infection, and contained a greater proportion of effector cells producing IFN-γ and TNFα, which may have contributed to the enhanced protection. These findings may explain the observation that in humans with chronic malaria, activated effector memory cells are best maintained in conditions of repeated exposure.     

Long-term engraftment and expansion of tumor-derived memory T cells following the implantation of non-disrupted pieces of human lung tumor into NOD-scid IL2Rgamma(null) mice

Non-disrupted pieces of primary human lung tumor implanted into NOD-scid IL2Rgamma(null) mice consistently result in successful xenografts in which tissue architecture, including tumor-associated leukocytes, stromal fibroblasts, and tumor cells are preserved for prolonged periods with limited host-vs-graft interference. Human CD45(+) tumor-associated leukocytes within the xenograft are predominantly CD3(+) T cells with fewer CD138(+) plasma cells. The effector memory T cells that had been shown to be quiescent in human lung tumor microenvironments can be activated in situ as determined by the production of human IFN-gamma in response to exogenous IL-12. Plasma cells remain functional as evidenced by production of human Ig. Significant levels of human IFN-gamma and Ig were detected in sera from xenograft-bearing mice for up to 9 wk postengraftment. Tumor-associated T cells were found to migrate from the microenvironment of the xenograft to the lung, liver, and primarily the spleen. At 8 wk postengraftment, a significant portion of cells isolated from the mouse spleens were found to be human CD45(+) cells. The majority of CD45(+) cells were CD3(+) and expressed a phenotype consistent with an effector memory T cell, consisting of CD4(+) or CD8(+) T cells that were CD45RO(+), CD44(+), CD62L(-), and CD25(-). Following adoptive transfer into non-tumor bearing NOD-scid IL2Rgamma(null) mice, these human T cells were found to expand in the spleen, produce IFN-gamma, and maintain an effector memory phenotype. We conclude that the NOD-scid IL2Rgamma(null) tumor xenograft model provides an opportunity to study tumor and tumor-stromal cell interactions in situ for prolonged periods.     

Lymphatic tracing and T cell responses following oral vaccination with live Mycobacterium bovis (BCG)

Oral vaccination of mice with lipid-encapsulated Mycobacterium bovis bacille Calmette-Guérin (BCG) expands a subset of interferon-gamma (IFN-gamma)-secreting T cells and mediates protection against aerosol mycobacterial challenge. We have traced the movement of the live vaccine through the regional lymphatics of mice and monitored the resultant immune response. Six hours after oral vaccination BCG was detected in low numbers systemically and in draining lymphatic tissue. However, after 48 h, BCG was predominantly associated with alimentary tract lymphatic tissues, such as the cervical and mesenteric lymph nodes and Peyer's patches. Lymphocytes that produced IFN-gamma in response to PPD-B or BCG-pulsed dendritic cells predominated in the spleen and were almost exclusively CD4(+), CD44(+) and CD62L(-), thus resembling an effector memory T cell population. Despite the fact that an oral route was used for immunization, splenic IFN-gamma-secreting T cells in vaccinated mice did not express the mucosal homing antigens alpha(4)beta(7) integrin or alphaIEL (CD103). However, a proportion of BCG-specific CD4(+) T cells expressed the CD29 integrin (beta(1)) chain, potentially involved in lung homing function. Thus, oral priming with M. bovis BCG appears to induce a subset of spleen-resident CD4(+) T cells with the potential to provide protective immunity in the lung.     

Tumor-specific responses in lymph nodes draining murine sarcomas are concentrated in cells expressing P-selectin binding sites.

Tumor-draining lymph node (TDLN) cells develop substantial antitumor activity after activation on immobilized alphaCD3 and culture in low-dose IL-2. This study found that the minor subset of TDLN T cells expressing binding sites for the adhesion receptor P-selectin (Plig(high) T cells) produced T lymphoblasts with the most tumor-specific IFN-gamma synthesis in vitro and antitumor activity following adoptive transfer in vivo. The Plig(high) T cells constituted <25% of the cells with the phenotype of recently activated cells including high levels of CD69, CD44, or CD25, and low levels of CD62L. The cultured Plig(high) TDLN were 10- to 20-fold more active against established pulmonary micrometastases than cultured unfractionated TDLN, and >30-fold more active than cultured TDLN cells depleted of the Plig(high) fraction before expansion (Plig(low) cells). Tumor-specific IFN-gamma synthesis in vitro paralleled the antitumor activities of the cultured fractions in vivo, implying that increased Tc1 and Th1 effector functions contributed to the tumor suppression. Neither nonspecific interaction with the P-selectin chimera used for sorting nor endogenous costimulatory activity in the Plig(high) fraction accounted for the marked increase in antitumor activities after culture. The cultured Plig(high) fraction contained a variety of potential effector cells; however, the CD8 and CD4 subsets of alphabeta T cells accounted for 95-97% of its antitumor activity. The authors propose that P-selectin sorting increased antitumor activities by concentrating Tc1 and Th1 pre-effector/effector cells before culture.     

Intratumoral interleukin-2/agonist CD40 antibody drives CD4<Superscript>+</Superscript>-independent resolution of treated-tumors and CD4<Superscript>+</Superscript>-dependent systemic and memory responses

Targeting interleukin-2 (IL-2) and/or agonist anti-CD40 antibody (Ab) into tumors represents an effective vaccination strategy that avoids systemic toxicity and resolves treated-site tumors. Here, we examined IL-2 and/or anti-CD40 Ab-driven local versus systemic T cell function and the installation of T cell memory. Single tumor studies showed that IL-2 induced a potent CD4⁺ and CD8⁺ T cell response that was limited to the draining lymph node and treated-site tumor, and lymph node tumor-specific CD8⁺ T cells did not upregulate CD44. A two-tumor model showed that while IL-2-treated-site tumors resolved, distal tumors continued to grow, implying limited systemic immunity. In contrast, anti-CD40 Ab treatment with or without IL-2 expanded the systemic T cell response to non-draining lymph nodes, and distal tumors resolved. Tumor-specific T cells in lymph nodes of anti-CD40 Ab ± IL-2-treated mice upregulated CD44, demonstrating activation and transition to effector/memory migratory cells. While CD40-activated CD4⁺ T cells were not required for eradicating treated-site tumors, they, plus CD8⁺ T cells, were crucial for removing distal tumors. Rechallenge/depletion experiments showed that the effector/memory phase required the presence of previously CD40/IL-2-activated CD4⁺ and CD8⁺ T cells to prevent recurrence. These novel findings show that different T cell effector mechanisms can operate for the eradication of local treated-site tumors versus untreated distal tumors and that signaling through CD40 generates a whole of body, effector/memory CD4⁺ and CD8⁺ T cell response that is amplified and prolonged via IL-2. Thus, successful immunotherapy needs to generate collaborating CD4⁺ and CD8⁺ T cells for a complete long-term protective cure.     

CD4 T cell-dependent CD8 T cell maturation

We have investigated the contribution of CD4 T cells to the optimal priming of functionally robust memory CD8 T cell subsets. Intranasal infection of CD4 T cell-deficient (CD4(-/-)) mice with lymphocytic choriomeningitis virus resulted in the elaboration of virus-specific CD8 T cell responses that cleared the infection. However, by comparison with normal mice, the virus-specific CD8 T cells in CD4(-/-) mice were quantitatively and qualitatively different. In normal mice, lymphocytic choriomeningitis virus-specific memory CD8 T cells are CD44(high), many are CD122(high), and a majority of these cells regain expression of CD62L overtime. These cells produce IFN-gamma and TNF-alpha, and a subset also produces IL-2. In the absence of CD4 T cell help, a distinct subset of memory CD8 T cells develops that remains CD62L(low) up to 1 year after infection and exhibits a CD44(int)CD122(low) phenotype. These cells are qualitatively different from their counterparts in normal hosts, as their capacity to produce TNF-alpha and IL-2 is diminished. In addition, although CD4-independent CD8 T cells can contain the infection following secondary viral challenge, their ability to expand is impaired. These findings suggest that CD4 T cell responses not only contribute to the optimal priming of CD8 T cells in chronically infected hosts, but are also critical for the phenotypic and functional maturation of CD8 T cell responses to Ags that are more rapidly cleared. Moreover, these data imply that the development of CD62L(high) central memory CD8 T cells is arrested in the absence of CD4 T cell help.     

Control of memory CD4 T cell recall by the CD28/B7 costimulatory pathway

The CD28/B7 costimulatory pathway is generally considered dispensable for memory T cell responses, largely based on in vitro studies demonstrating memory T cell activation in the absence of CD28 engagement by B7 ligands. However, the susceptibility of memory CD4 T cells, including central (CD62L(high)) and effector memory (T(EM); CD62L(low)) subsets, to inhibition of CD28-derived costimulation has not been closely examined. In this study, we demonstrate that inhibition of CD28/B7 costimulation with the B7-binding fusion molecule CTLA4Ig has profound and specific effects on secondary responses mediated by memory CD4 T cells generated by priming with Ag or infection with influenza virus. In vitro, CTLA4Ig substantially inhibits IL-2, but not IFN-gamma production from heterogeneous memory CD4 T cells specific for influenza hemagglutinin or OVA in response to peptide challenge. Moreover, IL-2 production from polyclonal influenza-specific memory CD4 T cells in response to virus challenge was completely abrogated by CTLA4Ig with IFN-gamma production partially inhibited. When administered in vivo, CTLA4Ig significantly blocks Ag-driven memory CD4 T cell proliferation and expansion, without affecting early recall and activation. Importantly, CTLA4Ig treatment in vivo induced a striking shift in the phenotype of the responding population from predominantly T(EM) in control-treated mice to predominantly central memory T cells in CTLA4Ig-treated mice, suggesting biased effects of CTLA4Ig on T(EM) responses. Our results identify a novel role for CD28/B7 as a regulator of memory T cell responses, and have important clinical implications for using CTLA4Ig to abrogate the pathologic consequences of T(EM) cells in autoimmunity and chronic disease.     

OX40/OX40L costimulation affects induction of Foxp3+ regulatory T cells in part by expanding memory T cells in vivo

OX40 is a member of the TNFR superfamily and has potent T cell costimulatory activities. OX40 also inhibits the induction of Foxp3(+) regulatory T cells (Tregs) from T effector cells, but the precise mechanism of such inhibition remains unknown. In the present study, we found that CD4(+) T effector cells from OX40 ligand-transgenic (OX40Ltg) mice are highly resistant to TGF-beta mediated induction of Foxp3(+) Tregs, whereas wild-type B6 and OX40 knockout CD4(+) T effector cells can be readily converted to Foxp3(+) T cells. We also found that CD4(+) T effector cells from OX40Ltg mice are heterogeneous and contain a large population of CD44(high)CD62L(-) memory T cells. Analysis of purified OX40Ltg naive and memory CD4(+) T effector cells showed that memory CD4(+) T cells not only resist the induction of Foxp3(+) T cells but also actively suppress the conversion of naive CD4(+) T effector cells to Foxp3(+) Tregs. This suppression is mediated by the production of IFN-gamma by memory T cells but not by cell-cell contact and also involves the induction of T-bet. Importantly, memory CD4(+) T cells have a broad impact on the induction of Foxp3(+) Tregs regardless of their origins and Ag specificities. Our data suggest that one of the mechanisms by which OX40 inhibits the induction of Foxp3(+) Tregs is by inducing memory T cells in vivo. This finding may have important clinical implications in tolerance induction to transplanted tissues.