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.     

Immunomodulation of T-cell responses with Vibrio cholerae O135 capsular polysaccharide and its protein conjugate, novel cholera vaccine study models

We studied T-cell immune responses to surface capsular polysaccharide (CPS) of Vibrio cholerae O135 and its protein conjugate. CPS and CPS-bovine serum albumin (BSA) activation and presentation are characterized with induced alterations in expression and upregulation of membrane antigens CD25, CD11b, CD16/32, MHCII and CD45 on blood- and spleen-derived T cells. Expression of the early activation marker CD25 revealed efficient CPS-BSA conjugate activation especially of CD4(+) CD3(+) and CD8(+) CD3(+) cells. Specific CPS-BSA-induced CD25(+) T-cell subsets in blood were observed after the first application, i.e. a 4.2-fold increase of CD4(+) CD25(+) and 7.6-fold increase of CD8(+) CD25(+) vs. preimmune levels was determined. The upregulation of surface antigens MHCII and CD45 involved in antigen presentation and cell activation of CD3(+) cells and their significant reciprocal correlation (R(2) =?0.92) observed only with CPS-BSA conjugate suggested efficient T-cell dependency and presentation. The pattern of accelerated T-cell activation and engagement of T cells as antigen-presenting cells throughout CPS-BSA immunization contrary to CPS alone was also confirmed in CD4(+) /CD8(+) /CD3(+) splenic cells. The results revealed different T-cell antigen presentation and activation following administration of CPS and CPS-BSA conjugates, as supported also by evaluation of CD45, MHCII and CD25 expression on CD19(+) B cells.     

Suppression of allograft rejection by Tim-1-Fc through cross-linking with a novel Tim-1 binding partner on T cells

Engagement of T-cell immunoglobulin mucin (Tim)-1 on T cells with its ligand, Tim-4, on antigen presenting cells delivers positive costimulatory signals to T cells. However, the molecular mechanisms for Tim-1-mediated regulation of T-cell activation and differentiation are relatively poorly understood. Here we investigated the role of Tim-1 in T-cell responses and allograft rejection using recombinant human Tim-1 extracellular domain and IgG1-Fc fusion proteins (Tim-1-Fc). In vitro assays confirmed that Tim-1-Fc selectively binds to CD4(+) effector T cells, but not dendritic cells or natural regulatory T cells (nTregs). Tim-1-Fc was able to inhibit the responses of purified CD4(+) T cells that do not express Tim-4 to stimulation by anti-CD3/CD28 mAbs, and this inhibition was associated with reduced AKT and ERK1/2 phosphorylation, but it had no influence on nTregs. Moreover, Tim-1-Fc inhibited the proliferation of CD4(+) T cells stimulated by allogeneic dendritic cells. Treatment of recipient mice with Tim-1-Fc significantly prolonged cardiac allograft survival in a fully MHC-mismatched strain combination, which was associated with impaired Th1 response and preserved Th2 and nTregs function. Importantly, the frequency of Foxp3(+) cells in splenic CD4(+) T cells was increased, thus shifting the balance toward regulators, even though Tim-1-Fc did not induce Foxp3 expression in CD4(+)CD25(-) T cells directly. These results indicate that Tim-1-Fc can inhibit T-cell responses through an unknown Tim-1 binding partner on T cells, and it is a promising immunosuppressive agent for preventing allograft rejection.     

Preferential infection shortens the life span of human immunodeficiency virus-specific CD4+ T cells in vivo

CD4(+) T-cell help is essential for effective immune responses to viruses. In human immunodeficiency virus (HIV) infection, CD4(+) T cells specific for HIV are infected by the virus at higher frequencies than other memory CD4(+) T cells. Here, we demonstrate that HIV-specific CD4(+) T cells are barely detectable in most infected individuals and that the corresponding CD4(+) T cells exhibit an immature phenotype compared to both cytomegalovirus (CMV)-specific CD4(+) T cells and other memory CD4(+) T cells. However, in two individuals, we observed a rare and diametrically opposed pattern in which HIV-specific CD4(+) T-cell populations of large magnitude exhibited a terminally differentiated immunophenotype; these cells were not preferentially infected in vivo. Clonotypic analysis revealed that the HIV-specific CD4(+) T cells from these individuals were cross-reactive with CMV. Thus, preferential infection can be circumvented in the presence of cross-reactive CD4(+) T cells driven to maturity by coinfecting viral antigens, and this physical proximity rather than activation status per se is an important determinant of preferential infection based on antigen specificity. These data demonstrate that preferential infection reduces the life span of HIV-specific CD4(+) T cells in vivo and thereby compromises the generation of effective immune responses to the virus itself; further, this central feature in the pathophysiology of HIV infection can be influenced by the cross-reactivity of responding CD4(+) T cells.     

Concurrent allorecognition has a limited impact on posttransplant vaccination

Transplantation of allogeneic hematopoietic stem cells with or without immunocompetent lymphocytes has proved a successful strategy in the treatment of hematological malignancies. We have recently shown that this approach can also cure mouse prostate cancer, provided that it is combined with tumor-specific vaccination. Whether the response to alloantigens acts by providing helper function to enhance vaccine-specific responses or in other ways impinges on vaccine immunogenicity remains to be clarified, and this question is of clinical relevance. In this study, we have addressed this issue by comparing the immunogenicity of dendritic cells pulsed with a peptide derived from a tumor/viral model Ag in recipients of donor cells either syngeneic to the host or differing for either Y-encoded or multiple minor H antigens. We report that vaccination elicits comparable proliferation and differentiation of peptide-specific CD8(+) T cells despite concurrent expansion and differentiation of minor H antigen-specific IFN-γ effector T cells. Depletion of alloreactive CD4(+) T cells reduced alloreactivity but not vaccine-induced CD8(+) T cell priming, suggesting that alloresponses do not provide helper functions in peripheral lymphoid tissues. Vaccine-mediated T cell priming was also preserved in the case of multiple minor H antigen disparities, prone to graft-versus-host disease. Thus, in the context of nonmyeloablative allotransplantation aimed at restoring an effective tumor-specific T cell repertoire, minor H antigen-specific T cells do not interfere with vaccine-induced T cell priming, supporting the notion that posttransplant vaccination is a valuable strategy to boost tumor and pathogen-specific protective immunity.     

The NK cell response to mouse cytomegalovirus infection affects the level and kinetics of the early CD8(+) T-cell response

Natural killer (NK) cells and CD8(+) T cells play a prominent role in the clearance of mouse cytomegalovirus (MCMV) infection. The role of NK cells in modulating the CD8(+) T-cell response to MCMV infection is still the subject of intensive research. For analyzing the impact of NK cells on mounting of a CD8(+) T-cell response and the contribution of these cells to virus control during the first days postinfection (p.i.), we used C57BL/6 mice in which NK cells are specifically activated through the Ly49H receptor engaged by the MCMV-encoded ligand m157. Our results indicate that the requirement for CD8(+) T cells in early MCMV control inversely correlates with the engagement of Ly49H. While depletion of CD8(+) T cells has only a minor effect on the early control of wild-type MCMV, CD8(+) T cells are essential in the control of Δm157 virus. The frequencies of virus epitope-specific CD8(+) T cells and their activation status were higher in mice infected with Δm157 virus. In addition, these mice showed elevated levels of alpha interferon (IFN-α) and several other proinflammatory cytokines as early as 1.5 days p.i. Although the numbers of conventional dendritic cells (cDCs) were reduced later during infection, particularly in Δm157-infected mice, they were not significantly affected at the peak of the cytokine response. Altogether, we concluded that increased antigen load, preservation of early cDCs' function, and higher levels of innate cytokines collectively account for an enhanced CD8(+) T-cell response in C57BL/6 mice infected with a virus unable to activate NK cells via the Ly49H-m157 interaction.     

Standardized and highly efficient expansion of Epstein-Barr virus-specific CD4+ T cells by using virus-like particles

Epstein-Barr virus (EBV)-specific T-cell lines generated by repeated stimulation with EBV-immortalized lymphoblastoid B-cell lines (LCL) have been successfully used to treat EBV-associated posttransplant lymphoproliferative disease (PTLD) in hematopoietic stem cell transplant recipients. However, PTLD in solid-organ transplant recipients and other EBV-associated malignancies respond less efficiently to this adoptive T-cell therapy. LCL-stimulated T-cell preparations are polyclonal and contain CD4(+) and CD8(+) T cells, but the composition varies greatly between lines. Because T-cell lines with higher CD4(+) T-cell proportions show improved clinical efficacy, we assessed which factors might compromise the expansion of this T-cell population. Here we show that spontaneous virus production by LCL and, hence, the presentation of viral antigens varies intra- and interindividually and is further impaired by acyclovir treatment of LCL. Moreover, the stimulation of T cells with LCL grown in medium supplemented with fetal calf serum (FCS) caused the expansion of FCS-reactive CD4(+) T cells, whereas human serum from EBV-seropositive donors diminished viral antigen presentation. To overcome these limitations, we used peripheral blood mononuclear cells pulsed with nontransforming virus-like particles as antigen-presenting cells. This strategy facilitated the specific and rapid expansion of EBV-specific CD4(+) T cells and, thus, might contribute to the development of standardized protocols for the generation of T-cell lines with improved clinical efficacy.     

T cells targeted against a single minor histocompatibility antigen can cure solid tumors

T cells responsive to minor histocompatibility (H) antigens are extremely effective in curing leukemia but it remains unknown whether they can eradicate solid tumors. We report that injection of CD8(+) T cells primed against the immunodominant H7(a) minor H antigen can cure established melanomas in mice. Tumor rejection was initiated by preferential extravasation at the tumor site of interferon (IFN)-gamma-producing H7(a)-specific T cells. Intratumoral release of IFN-gamma had two crucial effects: inhibition of tumor angiogenesis and upregulation of major histocompatibility complex (MHC) class I expression on tumor cells. Despite ubiquitous expression of H7(a), dissemination of a few H7(a)-specific T cells in extralymphoid organs caused neither graft-versus-host disease (GVHD) nor vitiligo because host nonhematopoietic cells were protected by their low expression of MHC class I. Our preclinical model yields unique insights into how minor H antigen-based immunotherapy could be used to treat human solid tumors.     

Human immunodeficiency virus type 1- and cytomegalovirus-specific cytotoxic T lymphocytes can persist at high frequency for prolonged periods in the absence of circulating peripheral CD4(+) T cells

CD4(+) T cells are thought to be critical in the maintenance of virus-specific CD8(+) cytotoxic T-cell (CTL) responses. In human immunodeficiency virus type 1 (HIV-1) infection, a selective decline in HIV-1-specific CTL as the CD4(+) T-cell count decreases has been reported. Using HLA-peptide tetrameric complexes, we show the presence at high frequency of HIV-1- and cytomegalovirus-specific CD8(+) T cells when the peripheral CD4(+) T-cell count was low or zero in three HIV-1-infected patients. No direct virus-specific CD8(+)-mediated effector activity was seen in these subjects, suggesting antigen unresponsiveness, although tetramer-sorted cells could be expanded in vitro in the presence of interleukin-2 into responsive effector cells. Thus, virus-specific CD8(+) T cells can be maintained in the peripheral circulation at high frequency in the absence of circulating peripheral CD4(+) T cells, but these cells may lack direct effector activity. Strategies designed to overcome this antigen unresponsiveness may be of value in therapies for the treatment of AIDS.     

Virus-specific CD4+ and CD8+ cytotoxic T-cell responses and long-term T-cell memory in individuals vaccinated against polio

The presence of poliovirus (PV)-specific CD4(+) T cells in individuals vaccinated against polio has been shown, but CD8(+) T-cell responses have not been described. Here, we functionally characterize the CD4(+) T-cell response and show for the first time that dendritic cells and macrophages can stimulate PV-specific CD8(+) T-cell responses in vitro from vaccinees. Both CD4(+) T and CD8(+) T cells secrete gamma interferon in response to PV antigens and are cytotoxic via the perforin/granzyme B-mediated pathway. Furthermore, the T cells also recognize and kill Sabin 1 vaccine-infected targets. The macrophage-stimulated CD4(+) T and CD8(+) T cells most likely represent memory T cells that persist for long periods in vaccinated individuals. Thus, immunity to PV vaccination involves not only an effective neutralizing antibody titer but also long-term CD4(+) and CD8(+) cytotoxic T-cell responses.     

Common gamma chain cytokines promote rapid in vitro expansion of allo-specific human CD8+ suppressor T cells

Human CD8(+) regulatory T cells, particularly the CD8(+)CD28(-) T suppressor cells, have emerged as an important modulator of alloimmunity. Understanding the conditions under which these cells are induced and/or expanded would greatly facilitate their application in future clinical trials. In the current study, we develop a novel strategy that combines common gamma chain (γc) cytokines IL-2, IL-7 and IL-15 and donor antigen presenting cells (APCs) to stimulate full HLA-mismatched allogeneic human CD8(+) T cells which results in significant expansions of donor-specific CD8(+)CD28(-) T suppressor cells in vitro. The expanded CD8(+)CD28(-) T cells exhibit increased expressions of CTLA-4, FoxP3, and CD25, while down-regulate expressions of CD56, CD57, CD127, and perforin. Furthermore, these cells suppress proliferation of CD4(+) T cells in a contact-dependent and cytokine-independent manner. Interestingly, the specificity of suppression is restricted by the donor HLA class I antigens but promiscuous to HLA class II antigens, providing a potential mechanism for linked suppression. Taken together, our results demonstrate a novel role for common γc cytokines in combination with donor APCs in the expansion of donor-specific CD8(+)CD28(-) T suppressor cells, and represent a robust strategy for in vitro generation of such cells for adoptive cellular immunotherapy in transplantation.     

Subcellular antigen location influences T-cell activation during acute infection with Toxoplasma gondii

Effective control of the intracellular protozoan parasite Toxoplasma gondii depends on the activation of antigen-specific CD8(+) T-cells that manage acute disease and prevent recrudescence during chronic infection. T-cell activation in turn, requires presentation of parasite antigens by MHC-I molecules on the surface of antigen presenting cells. CD8(+) T-cell epitopes have been defined for several T. gondii proteins, but it is unclear how these antigens enter into the presentation pathway. We have exploited the well-characterized model antigen ovalbumin (OVA) to investigate the ability of parasite proteins to enter the MHC-I presentation pathway, by engineering recombinant expression in various organelles. CD8(+) T-cell activation was assayed using 'B3Z' reporter cells in vitro, or adoptively-transferred OVA-specific 'OT-I' CD8(+) T-cells in vivo. As expected, OVA secreted into the parasitophorous vacuole strongly stimulated antigen-presenting cells. Lower levels of activation were observed using glycophosphatidyl inositol (GPI) anchored OVA associated with (or shed from) the parasite surface. Little CD8(+) T-cell activation was detected using parasites expressing intracellular OVA in the cytosol, mitochondrion, or inner membrane complex (IMC). These results indicate that effective presentation of parasite proteins to CD8(+) T-cells is a consequence of active protein secretion by T. gondii and escape from the parasitophorous vacuole, rather than degradation of phagocytosed parasites or parasite products.     

Dendritic cells crosspresent antigens from live B16 cells more efficiently than from apoptotic cells and protect from melanoma in a therapeutic model

Dendritic cells (DC) are able to elicit anti-tumoral CD8(+) T cell responses by cross-presenting exogenous antigens in association with major histocompatibility complex (MHC) class I molecules. Therefore they are crucial actors in cell-based cancer immunotherapy. Although apoptotic cells are usually considered to be the best source of antigens, live cells are also able to provide antigens for cross-presentation by DC. We have recently shown that prophylactic immunotherapy by DC after capture of antigens from live B16 melanoma cells induced strong CD8(+) T-cell responses and protection against a lethal tumor challenge in vivo in C57Bl/6 mice. Here, we showed that DC cross-presenting antigens from live B16 cells can also inhibit melanoma lung dissemination in a therapeutic protocol in mice. DC were first incubated with live tumor cells for antigen uptake and processing, then purified and irradiated for safety prior to injection. This treatment induced stronger tumor-specific CD8(+) T-cell responses than treatment by DC cross-presenting antigens from apoptotic cells. Apoptotic B16 cells induced more IL-10 secretion by DC than live B16 cells. They underwent strong native antigen degradation and led to the expression of fewer MHC class I/epitope complexes on the surface of DC than live cells. Therefore, the possibility to use live cells as sources of tumor antigens must be taken into account to improve the efficiency of cancer immunotherapy.     

Human peripheral blood monocyte-derived interleukin-10-induced semi-mature dendritic cells induce anergic CD4(+) and CD8(+) T cells via presentation of the internalized soluble antigen and cross-presentation of the phagocytosed necrotic cellular fragments

We examined the ability of human monocyte-derived interleukin (IL)-10-induced semi-mature dendritic cells (semi-mDCs) that had been pulsed with soluble protein and necrotic cellular fragments to induce an antigen (Ag)-specific anergy in CD4(+) and CD8(+) T cells. IL-10 converted normal immature DCs (iDCs) into semi-mDCs during the maturation. In contrast to normal iDCs and mature DCs, IL-10-induced semi-mDCs as well as IL-10-treated iDCs not only had reduced their allogeneic T cell-stimulatory capacity, but also induced an allogeneic Ag-specific anergy in T cells. Normal mDCs that had been pulsed with tetanus toxin (TT) or allogeneic necrotic cellular fragments caused further activation of TT-specific CD4(+) T cells or allogeneic fibroblast-specific CD8(+) T cells, Ag-pulsed IL-10-induced semi-mDCs induced an anergic state in both cell types. Thus, our results suggest that IL-10-induced semi-mDCs induce an Ag-specific anergy in CD4(+) and CD8(+) T cells via presentation of the internalized protein and cross-presentation of the phagocytosed cellular fragments.     

Generation of melanoma-specific, cytotoxic CD4(+) T helper 2 cells: requirement of both HLA-DR15 and Fas antigens on melanomas for their lysis by Th2 cells

Recognition of melanoma antigens by HLA class-II-restricted CD4(+) T lymphocytes has been investigated. Two cytotoxic CD4(+) T cell lines were established by stimulating PBLs from a melanoma patient with either parental or IFN-gamma-transduced autologous tumor cells. These T cells secreted IL-4, but not IL-2, IFN-gamma, or TNF-beta, in response to the autologous melanoma cells, suggesting that they belong to the Th2 subtype. Their cytotoxicity was directed against the IFN-gamma-transduced melanoma cells and was HLA-DR-restricted. The autologous and two allogeneic IFN-gamma-modified melanoma cell lines shared melanoma antigen(s) presented in the context of HLA-DR15. HLA-DR15(+) nonmelanoma cells were resistant targets indicating that the shared antigen(s) is melanoma associated. Parental autologous and HLA-DR-matched allogeneic melanoma cell lines, displaying low levels of HLA-DR antigens, induced Th2 proliferation and cytokine release, but were insensitive to lysis prior to upregulation of HLA-DR and Fas antigens by IFN-gamma. Cytolysis was inhibited by anti-HLA-DR and by anti-Fas antibodies, suggesting that the cytolysis is mediated via the Fas pathway. While small amounts of HLA-DR15 molecules on melanoma cells are sufficient for Th2 proliferation and cytokine release, higher amounts of HLA-DR15 and the expression of Fas are required for CD4(+)-mediated lysis.     

Human CD4+ CD25+ regulatory T cells control T-cell responses to human immunodeficiency virus and cytomegalovirus antigens

Regulatory T (T(R)) cells maintain tolerance to self-antigens and control immune responses to alloantigens after organ transplantation. Here, we show that CD4(+) CD25(+) human T(R) cells suppress virus-specific T-cell responses. Depletion of T(R) cells from peripheral blood mononuclear cells enhances T-cell responses to cytomegalovirus and human immunodeficiency virus antigens. We propose that chronic viral infections lead to induction of suppressive T(R) cells that inhibit the antiviral immune response.     

Abnormal priming of CD4(+) T cells by dendritic cells expressing hepatitis C virus core and E1 proteins

Patients infected with hepatitis C virus (HCV) have an impaired response against HCV antigens while keeping immune competence for other antigens. We hypothesized that expression of HCV proteins in infected dendritic cells (DC) might impair their antigen-presenting function, leading to a defective anti-HCV T-cell immunity. To test this hypothesis, DC from normal donors were transduced with an adenovirus coding for HCV core and E1 proteins and these cells (DC-CE1) were used to stimulate T lymphocytes. DC-CE1 were poor stimulators of allogeneic reactions and of autologous primary and secondary proliferative responses. Autologous T cells stimulated with DC-CE1 exhibited a pattern of incomplete activation characterized by enhanced CD25 expression but reduced interleukin 2 production. The same pattern of incomplete lymphocyte activation was observed in CD4(+) T cells responding to HCV core in patients with chronic HCV infection. However, CD4(+) response to HCV core was normal in patients who cleared HCV after alpha interferon therapy. Moreover, a normal CD4(+) response to tetanus toxoid was found in both chronic HCV carriers and patients who had eliminated the infection. Our results suggest that expression of HCV structural antigens in infected DC disturbs their antigen-presenting function, leading to incomplete activation of anti-HCV-specific T cells and chronicity of infection. However, presentation of unrelated antigens by noninfected DC would allow normal T-cell immunity to other pathogens.     

Vaccination with CD133+ melanoma induces specific Th17 and Th1 cell�Cmediated antitumor reactivity against parental tumor

Accumulating evidence suggests that cancer cells possess a small subpopulation that survives during potentially lethal stresses, including chemotherapy, radiation treatment, and molecular-targeting therapy. CD133 is a putative marker that distinguishes a minor subpopulation from normal differentiated tumor cells in many cancers. Although it is necessary to eradicate all cancer cells to obtain a cure, effective treatment to eliminate the CD133(+) treatment-tolerant cells has not been elucidated. In this study, we demonstrated that a CD133(+) subpopulation in murine melanoma is immunogenic and that effector T cells specific for the CD133(+) melanoma cells mediated potent antitumor reactivity, curing the mice of the parental melanoma. CD133(+) melanoma antigens preferentially induced type 17 T helper (Th17) cells and Th1 cells but not Th2 cells. CD133(+) melanoma cell-specific CD4(+) T-cell treatment eradicated not only CD133(+) tumor cells but also CD133(-) tumor cells while inducing long-lasting accumulation of lymphocytes and dendritic cells with upregulated MHC class II in tumor tissues. Further, the treatment prevented regulatory T-cell induction. These results indicate that T-cell immunotherapy is a promising treatment option to eradicate CD133(+) drug-tolerant cells to obtain a cure for cancer.     

Existence of MHC class I-restricted alloreactive CD4+ T cells reacting with peptide transporter-deficient cells

It is generally accepted that as the result of positive thymic selection, CD8-expressing T cells recognize peptide antigens presented in the context of MHC class I molecules and CD4-expressing T cells interact with peptide antigens presented by MHC class II molecules. Here we report the generation of TCRalpha/beta(+), CD3(+), CD4(+), CD8(-), MHC class I-restricted alloreactive T-cell clones which were induced using peripheral blood mononuclear cells from healthy individuals following in vitro stimulation with transporter associated with antigen processing (TAP)-deficient cell lines T2. The CD4(+) T-cell clones showed an HLA-A2.1-specific proliferative response against T2 cells which was inhibited by anti-CD3 and anti-CD4 monoclonal antibodies. These results suggest that interaction of the TCR with peptide-bound HLA class I molecules contributes to antigen-specific activation of these co-receptor-mismatched T-cell clones. Antigen recognition by alloreactive MHC class I-restricted CD4(+) T cells was inhibited by removing peptides bound to HLA molecules on T2 cells suggesting that the alloreactive CD4(+) T cells recognize peptides that bind in a TAP-independent manner to HLA-A2 molecules. The existence of such MHC class I-restricted CD4(+) T cells which can recognize HLA-A2 molecules in the absence of TAP function may provide a basis for the development of immunotherapy against TAP-deficient tumor variants which would be tolerant to immunosurveillance by conventional MHC class I-restricted cytotoxic lymphocytes.