Enhanced major histocompatibility complex class I-dependent presentation of antigens modified with cationic and fusogenic peptides

Soluble extracellular protein antigens are notoriously poor stimulators of CD8+ cytotoxic T-lymphocyte (CTL) responses, largely because these antigens have inefficient access to an endogenous cytosolic pathway of the major histocompatibility complex (MHC) class I-dependent antigen presentation. Here, we present a strategy that facilitates antigen penetration into the cytosol of antigen-presenting cells (APC) by addition to the antigen of charge-modifying peptide sequences. As a result of this intervention, the charge modification enhances antigen uptake into APC by counteracting the repulsive cell surface charge, and then endosomal membranes are disrupted with a subsequent release of antigen into the cytosol. This technology significantly improves MHC class I-dependent antigen presentation to CTL, enabling a more efficient generation of specific CTL immunity in vivo. The strategy described here has potential for use in developing efficient vaccines for antigen-specific immunotherapy of human malignancies.     

Antigen presentation of a modified tumor-derived peptide by tumor infiltrating lymphocytes

CD8+ T-lymphocytes recognize peptides in the context of major histocompatibility complex (MHC) class I antigens. Upon activation, these cells differentiate into effector cytotoxic T lymphocytes (CTL) and no longer require formal antigen presentation by professional antigen presenting cells (APC). Subsequently, any cell expressing MHC class I/cognate peptide can stimulate CTL. Using TIL specific for a melanoma antigen-derived peptide, IMDQVPFSV (g209 2M), we sought to determine whether these CTL could present peptide to each other. Our findings demonstrate that peptide presentation of the g209 2M peptide epitope by TIL is comparable to conventional methods of using T2 cells as APC. We report here that CTL are capable of self-presentation of antigenic peptide to neighboring CTL resulting in IFN-gamma secretion, proliferation, and lysis of peptide-loaded CTL. These results demonstrate that human TIL possess both APC functions as well as cytotoxic functions and that this phenomenon could influence CTL activity elicited by immunotherapy.     

Cross-presentation of Listeria monocytogenes-derived CD4 T cell epitopes

Listeriolysin O (LLO) mediates the evasion of Listeria monocytogenes from the phagolysosome into the cytoplasm of the host cell. The recognition of infected cells by CD4 T cells is thought to be limited by the evasion of bacteria from the phagolysosome and also by the direct LLO-mediated inhibition of CD4 T cell activation. To analyze the influence of these immunoevasive mechanisms on the antilisterial CD4 T cell response, the expansion of L. monocytogenes-specific CD4 and CD8 T cells was monitored in infected mice. It was found that expansion of L. monocytogenes-specific CD4 T cells occurred synchronously with CD8 T cell expansion. The analysis of Ag presentation by macrophages and dendritic cells isolated from spleens of infected mice revealed efficient presentation of L. monocytogenes-derived CD4 T cell epitopes that was not dependent on the actA-mediated intercellular spread of bacteria. The further in vitro Ag presentation analysis revealed that although L. monocytogenes-infected macrophages and dendritic cells were poor presenters of CD4 T cell epitopes, more efficient presentation occurred after cocultivation of noninfected dendritic cells or macrophages with infected cells. These data indicate that the suppressive effect of LLO on the antilisterial CD4 T cell response is maintained only in infected APC and support the hypothesis that cross-priming plays a role in the induction of the strong CD4 T cell response in Listeria-infected mice.     

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.     

MHC class II presentation of endogenous tumor antigen by cellular vaccines depends on the endocytic pathway but not H2-M

We have developed cell-based cancer vaccines that activate anti-tumor immunity by directly presenting endogenously synthesized tumor antigens to CD4⁺ T helper lymphocytes via MHC class II molecules. The vaccines are non-conventional antigen-presenting cells because they express MHC class II, do not express invariant chain or H-2M, and preferentially present endogenous antigen. To further improve therapeutic efficacy we have studied the intracellular trafficking pathway of MHC class II molecules in the vaccines using endoplasmic reticulum-localized lysozyme as a model antigen. Experiments using endocytic and cytosolic pathway inhibitors (chloroquine, primaquine, and brefeldin A) and protease inhibitors (lactacystin, LLnL, E64, and leupeptin) indicate antigen presentation depends on the endocytic pathway, although antigen degradation is not mediated by endosomal or proteasomal proteases. Because H2-M facilitates presentation of exogenous antigen via the endocytic pathway, we investigated whether transfection of vaccine cells with H-2M could potentiate endogenous antigen presentation. In contrast to its role in conventional antigen presentation, H-2M had no effect on endogenous antigen presentation by vaccine cells or on vaccine efficacy. These results suggest that antigen/MHC class II complexes in the vaccines may follow a novel route for processing and presentation and may produce a repertoire of class II-restricted peptides different from those presented by professional APC. The therapeutic efficacy of the vaccines, therefore, may reside in their ability to present novel tumor peptides, consequently activating tumor-specific CD4⁺ T cells that would not otherwise be activated.     

The intact structural form of LLO in endosomes cannot protect against listeriosis

LLO is the major immuno-dominant antigen in listeriosis and is also required for protective immunity. Two forms of LLO can be observed in endosomal membranes, a LLO intact form and a Ctsd-processed LLO_1-491 form. Endosomes obtained from resting macrophages contained only LLO intact forms, while endosomes obtained from IFN-activated macrophages contained both forms. Both types of endosomes elicited LLO_90-91/CD8⁺ and LLO_189-201/CD4⁺ specific immune responses. However, only endosomes containing the Ctsd-processed LLO_1-491 form showed significant CD4⁺ and CD8⁺ T cell responses similar to LM infected bone marrow derived macrophages and characteristic of protective Listeria immunity. Moreover, endosomes with intact LLO could not confer protection as vaccine carriers against murine listeriosis. While endosomes with Ctsd-processed LLO_1-491 form showed a moderate ability, slightly lower than high efficiency vaccine vectors as MØ infected with LM. These studies argue that all cell-free membrane vesicles might serve as valid vaccine carriers against infectious agents. Exclusively those cell-free vesicles MIIC competent for LLO processing are protective vaccines vectors since they recruit significant numbers of mature dendritic cells to the vaccination sites and contain a LLO_1-491 form that might be accessible for MHC class I and class II antigen presentation.     

H2-M3-restricted T cells participate in the priming of antigen-specific CD4+ T cells

H2-M3-restricted CD8+ T cells provide early protection against bacterial infections. In this study, we demonstrate that activated H2-M3-restricted T cells provide early signals for efficient CD4+ T cell priming. C57BL/6 mice immunized with dendritic cells coated with the MHC class II-restricted listeriolysin O peptide LLO(190-201) (LLO) generated CD4+ T cells capable of responding to Listeria monocytogenes (LM) infection. Inclusion of a H2-M3-restricted formylated peptide fMIGWII (fMIG), but not MHC class Ia-restricted peptides, during immunization with LLO significantly increased IFN-gamma-producing CD4+ T cell numbers, which was associated with increased protection against LM infection. Studies with a CD4+ T cell-depleting mAb indicate that the reduction in bacterial load in fMIG plus LLO immunized mice is likely due to augmented numbers of LLO-specific CD4+ T cells, generated with the help of H2-M3-restricted CD8+ T cells. We also found that augmentation of LLO-specific CD4+ T lymphocytes with H2-M3-restricted T cells requires presentation of LLO and fMIG by the same dendritic cells. Interestingly, the augmented CD4+ T cell response generated with fMIG also increased primary LM-specific responses by MHC class Ia-restricted CD8 T cells. Coimmunization with LLO and fMIG also increases the number of memory Ag-specific CD4+ T cells. We also demonstrate that CD8 T cells restricted to another MHC class Ib molecule, Qa-1, whose human equivalent is HLA-E, are also able to enhance Ag-specific CD4+ T cell responses. These results reveal a novel function for H2-M3- and Qa-1-restricted T cells; provision of help to CD4+ Th cells during the primary response.     

Salmonella infection of bone marrow-derived macrophages and dendritic cells: influence on antigen presentation and initiating an immune response

Traditionally macrophages (MPhi) have been considered to be the key type of antigen presenting cells (APC) to combat bacterial infections by phagocytosing and destroying bacteria and presenting bacteria-derived antigens to T cells. However, data in recent years have demonstrated that dendritic cells (DC), at their immature stage of differentiation, are capable of phagocytosing particulate antigens including bacteria. Thus, DC may also be important APC for initiating an immune response to bacterial infections. Our studies focus on studying how DC and MPhi process antigens derived from bacteria with no known mechanism of phagosomal escape (i.e. Salmonella typhimurium) for T cell stimulation as well as what role these APC types have in Salmonella infection in vivo. Using an in vitro antigen processing and presentation assay with bone marrow-derived (BM) APC showed that, in addition to peritoneal elicited MPhi and BMMPhi, BMDC can phagocytose and process Escherichia coli and S. typhimurium for peptide presentation on major histocompatibility complex (MHC) class I (MHC-I) and class II MHC-II. These studies showed that both elicited peritoneal MPhi and BMMPhi use an alternate MHC-I presentation pathway that does not require the transporter associated with antigen processing (TAP) or the proteasome and involves peptide loading onto a preformed pool of post-Golgi MHC-I molecules. In contrast, DC process E. coli and S. typhimurium for peptide presentation on MHC-I using the cytosolic MHC-I presentation pathway that requires TAP, the proteasome and uses newly synthesized MHC-I molecules. We further investigated the interaction of Salmonella with BMDC and BMMPhi by analyzing surface molecule expression and cytokine secretion following S. typhimurium infection of BMDC and BMMPhi. These data reveal that Salmonella co-incubation with BMDC as well as BMMPhi results in upregulation of MHC-I and MHC-II as well as several co-stimulatory molecules including CD80 and CD86. Salmonella infection of BMDC or BMMPhi also results in secretion of cytokines including IL-6 and IL-12. Finally, injecting mice with BMDC that have been loaded in vitro with S. typhimurium primes na?ve CD4(+) and CD8(+) T cells to Salmonella-encoded antigens. Taken together, our data suggest that DC may be an important type of APC that contributes to the immune response to Salmonella.     

Listeriolysin O expressed in a bacterial vaccine suppresses CD4+CD25high regulatory T cell function in vivo

CD4(+)CD25(high) regulatory T cells (Treg) protect the host from autoimmune diseases but are also obstacles against cancer therapies. An ideal cancer vaccine would stimulate specific cytotoxic responses and reduce/suppress Treg function. In this study, we showed that Escherichia coli expressing listeriolysin O and OVA (E. coli LLO/OVA) demonstrated remarkable levels of protection against OVA-expressing tumor cells. By contrast, E. coli expressing OVA only (E. coli OVA) showed poor protection. High-avidity OVA-specific CTL were induced in E. coli LLO/OVA-vaccinated mice, and CD8(+) depletion--but not NK cell depletion, abolished the antitumor activity of the E. coli LLO/OVA vaccine. Phenotypic analysis of T cells following vaccination with either vaccine revealed preferential generation of CD44(high)CD62L(low) CD8(+) effector memory T cells over CD44(high)CD62L(high) central memory T cells. Unexpectedly, CD4(+) depletion turned E. coli OVA into a vaccine as effective as E. coli LLO/OVA suggesting that a subset of CD4(+) cells suppressed the CD8(+) T cell-mediated antitumor response. Further depletion experiments demonstrated that these suppressive cells consisted of CD4(+)CD25(high) regulatory T cells. We therefore assessed these vaccines for Treg function and found that although CD4(+)CD25(high) expansion and Foxp3 expression within this population was similar in all groups of mice, Treg cells from E. coli LLO/OVA-vaccinated animals were unable to suppress conventional T cells proliferation. These findings provide the first evidence that LLO expression affects Treg cell function and may have important implications for enhancing antitumor vaccination strategies in humans.     

TAP1-deficiency does not alter atherosclerosis development in Apoe-/- mice

Antigen presenting cells (APC) have the ability to present both extra-cellular and intra-cellular antigens via MHC class I molecules to CD8(+) T cells. The cross presentation of extra-cellular antigens is reduced in mice with deficient Antigen Peptide Transporter 1 (TAP1)-dependent MHC class I antigen presentation, and these mice are characterized by a diminished CD8(+) T cell population. We have recently reported an increased activation of CD8(+) T cells in hypercholesterolemic Apoe(-/-) mice. Therefore, this study included TAP1-deficient Apoe(-/-) mice (Apoe(-/-)Tap1(-/-)) to test the atherogenicity of CD8(+) T cells and TAP1-dependent cross presentation in a hypercholesterolemic environment. As expected the CD8(+) T cell numbers were low in Apoe(-/-)Tap1(-/-) mice in comparison to Apoe(-/-) mice, constituting ~1% of the lymphocyte population. In spite of this there were no differences in the extent of atherosclerosis as assessed by en face Oil Red O staining of the aorta and cross-sections of the aortic root between Apoe(-/-)Tap1(-/-) and Apoe(-/-) mice. Moreover, no differences were detected in lesion infiltration of macrophages or CD3(+) T cells in Apoe(-/-)Tap1(-/-) compared to Apoe(-/-) mice. The CD3(+)CD4(+) T cell fraction was increased in Apoe(-/-)Tap1(-/-) mice, suggesting a compensation for the decreased CD8(+) T cell population. Interestingly, the fraction of CD8(+) effector memory T cells was increased but this appeared to have little impact on the atherosclerosis development.In conclusion, Apoe(-/-)Tap1(-/-) mice develop atherosclerosis equal to Apoe(-/-) mice, indicating a minor role for CD8(+) T cells and TAP1-dependent antigen presentation in the disease process.     

Antigen presentation by Toxoplasma gondii-infected cells to CD4+ proliferative T cells and CD8+ cytotoxic cells

Cytotoxic cells specific for Toxoplasma gondii-infected cells were detected in the peripheral blood leukocytes from a patient with acute toxoplasmosis. The cytotoxicity was mediated by CD5+, CD4-, CD8+ cells. The cytotoxic T cells lysed Toxoplasma-infected target cells with HLA class I restriction. Two types of T cell clones were established from peripheral blood leukocytes of a patient with chronic toxoplasmosis; one was a CD5+, CD4-, CD8+ cytotoxic cell specific for Toxoplasma-infected cells, and the other was a CD5+, CD4+, CD8- proliferative cell that responded to Toxoplasma antigen. Toxoplasma-infected cell-specific cytotoxic cloned T cells recognize the infected target cells in the context of the HLA class I molecules, and the CD8 molecule was involved in the cytotoxicity. Toxoplasma antigen-specific proliferative cloned T cells were stimulated by Toxoplasma antigen-pulsed or Toxoplasma-infected cells in conjunction with HLA-DR molecule on the target cells. Thus, antigen presentation by Toxoplasma-infected cells for activation of both cytotoxic and proliferative T cells has been demonstrated.     

Efficient presentation of exogenous antigen by liver endothelial cells to CD8+ T cells results in antigen-specific T-cell tolerance

Myeloid antigen-presenting cells (APC) are known to cross-present exogenous antigen on major histocompatibility class I molecules to CD8+ T cells and thereby induce protective immunity against infecting microorganisms. Here we report that liver sinusoidal endothelial cells (LSEC) are organ-resident, non-myeloid APC capable of cross-presenting soluble exogenous antigen to CD8+ T cells. Though LSEC employ similar molecular mechanisms for cross-presentation as dendritic cells, the outcome of cross-presentation by LSEC is CD8+ T cell tolerance rather than immunity. As uptake of circulating antigens into LSEC occurs efficiently in vivo, it is likely that cross-presentation by LSEC contributes to CD8+ T cell tolerance observed in situations where soluble antigen is present in the circulation.     

Hepatitis B virus (HBV)-specific cytotoxic T-cell (CTL) response in humans: characterization of HLA class II-restricted CTLs that recognize endogenously synthesized HBV envelope antigens.

In this study, we show that CD4+, hepatitis B virus (HBV) envelope-specific T-cell clones produced by stimulation with a particulate antigen preparation are able to recognize and kill not only autologous antigen-presenting cells incubated with exogenous HBV envelope antigens but also autologous HLA class II-positive cells expressing endogenously synthesized HBV envelope antigens following infection with recombinant vaccinia viruses or transfection with recombinant Epstein-Barr virus expression vectors. Experiments with lysosomotropic agents and brefeldin A suggest that the endosomal compartment is likely involved in the processing of endogenously synthesized viral proteins for recognition by CD4+ T cells. Our study indicates that HBV envelope-specific, HLA class II-restricted CD4+ cytotoxic T lymphocytes can potentially participate in the immune clearance of HBV-infected cells and the pathogenesis of hepatocellular injury in hepatitis B.     

Correlation between Direct Binding Ability of SyntheticT. gondiiSAG1 Peptides to HLA-A2 Measured by a Sensor for Surface Plasmon Resonance and Antigenicity of the Peptides forT. gondii-Infected Cell-Specific CTL

Toxoplasma gondii(T. gondii) -infected B lymphoma cells presentT. gondiiantigens in the context of major histocompatibility complex molecules toT. gondii-specific CD8⁺cytotoxic T cells (CTL). HLA-A2 molecules ofT. gondii-infected human cells have been shown to be used in presenting T. gondiiantigens to CD8⁺CTL. SAG1, one of the major antigenic molecules ofT. gondii,is an antigen forT. gondii-specific CTL, and represents a possible basis for vaccines. The direct binding of nonamer SAG1 peptides to HLA-A2 was assayed here using an automated biosensor system with a sensor for surface plasmon resonance detection. The antigenicity of synthetic SAG1 peptides toT. gondii-specific CD8⁺CTL also was assayed. The present study found a high correlation between the binding ability of synthetic SAG1 peptides to HLA-A2 and the antigenicity of peptides toT. gondii-infected cell-specific CD8⁺CTL.     

Impaired tumor antigen processing by immunoproteasome-expressing CD40-activated B cells and dendritic cells

Professional APCs, such as dendritic cells, are routinely used in vitro for the generation of cytotoxic T lymphocytes specific for tumor antigens. In addition to dendritic cells, CD40-activated B cells and variant K562 leukemic cells can be readily transfected with nucleic acids for in vitro and in vivo antigen presentation. However, the expression of immunoproteasome components in dendritic cells may preclude display of tumor antigens such as Mart1/MelanA. Here, we use three target epitopes, two derived from tumor antigens [Mart1_26?C34 (M26) and Cyp1B1_239?C247 (Cyp239)] and one derived from the influenza A viral antigen [FluM1_58?C66 (FluM58)], to demonstrate that CD40-activated B cells, like dendritic cells, have a limited capability to process certain tumor antigens. In contrast, the K562 HLA-A*0201 transfectant efficiently processes and presents M26 and Cyp239 as well as the influenza FluM58 epitopes to T cells. These results demonstrate that the choice of target APC for gene transfer of tumor antigens may be limited by the relative efficacy of proteasome components to process certain tumor epitopes. Importantly, K562 can be exploited as an artificial APC, efficient in processing both M26 and Cyp239 epitopes and presumably, by extension, other relevant tumor antigens.     

Membrane-bound CD95 ligand expressed on human antigen-presenting cells prevents alloantigen-specific T cell response without impairment of viral and third-party T cell immunity

Genetically modified antigen-presenting cells (APC) represent an attractive strategy for in vitro immunomodulation. In the human system, APC expressing HLA-A1 and a membrane-bound form of CD95L (m-CD95L) were used for selective depletion of HLA-A1-specific T cells. In short-term assays, m-CD95L-expressing APC-induced apoptosis in activated T cells and the constitutive presence of m-CD95L and HLA-A1 expressing APC in long-term T cell cultures prevented the expansion of CD4(+) and CD8(+) HLA-A1-specific T cells and the development of HLA-A1-specific cytotoxicity. However, immunity towards third party, viral and bacterial antigens was maintained and T cells spared from depletion could be induced to develop cytotoxicity towards unrelated antigens. Interestingly, inhibition of HLA-A1-specific T cell response absolutely requires the coexpression of m-CD95L and HLA-A1 antigen on the same APC. Thus, m-CD95L expressing APC might be used in clinical settings to obtain tolerance induction in allogeneic transplantation systems or autoimmune diseases.     

Whole recombinant yeast vaccine activates dendritic cells and elicits protective cell-mediated immunity

There is currently a need for vaccines that stimulate cell-mediated immunity-particularly that mediated by CD8+ cytotoxic T lymphocytes (CTLs)-against viral and tumor antigens. The optimal induction of cell-mediated immunity requires the presentation of antigens by specialized cells of the immune system called dendritic cells (DCs). DCs are unique in their ability to process exogenous antigens via the major histocompatibility complex (MHC) class I pathway as well as in their ability to activate naive, antigen-specific CD8+ and CD4+ T cells. Vaccine strategies that target or activate DCs in order to elicit potent CTL-mediated immunity are the subject of intense research. We report here that whole recombinant Saccharomyces cerevisiae yeast expressing tumor or HIV-1 antigens potently induced antigen-specific, CTL responses, including those mediating tumor protection, in vaccinated animals. Interactions between yeast and DCs led to DC maturation, IL-12 production and the efficient priming of MHC class I- and class II-restricted, antigen-specific T-cell responses. Yeast exerted a strong adjuvant effect, augmenting DC presentation of exogenous whole-protein antigen to MHC class I- and class II-restricted T cells. Recombinant yeast represent a novel vaccine strategy for the induction of broad-based cellular immune responses.     

Whole protein and defined CD8(+) and CD4(+) peptides linked to penetratin targets both MHC class I and II antigen presentation pathways

Cytoplasmic delivery and cross-presentation of proteins and peptides is necessary for processing and presentation of antigens for the generation of cytotoxic T cells. We previously described the use of the 16 amino acid peptide penetratin from the Drosophila Antennapedia homeodomain (penetratin, Antp) to transport cytotoxic T lymphocyte epitopes derived from ovalbumin (OVA) or the Mucin-1 tumor-associated antigen into cells. We have now shown that penetratin covalently conjugated to OVA protein and linked in tandem to CD4(+) and/or CD8(+) T-cell epitopes from OVA-stimulated T cells in vitro (B3Z T-cell hybridoma and OT-I and OT-II T cells). The induction of these responses was directly mediated by the penetratin peptide as linking a nonspecific 16-mer peptide to OVA or mixing did not induce CD8(+) or CD4(+) T-cell responses in vitro. Furthermore, interferon (IFN)-γ-secreting CD4(+) and CD8(+) T cells were induced which suppressed B16.OVA tumor growth in C57BL/6 mice. Tumor protection was mediated by a CD8(+) T-cell-dependent mechanism and did not require CD4(+) help to protect mice 7 days after a boost immunization. Alternatively, 40 days after a boost immunization, the presence of CD4(+) help enhanced antigen-specific IFN-γ-secreting CD8(+) T cells and tumor protection in mice challenged with B16.OVA. Long-term CD8 responses were equally enhanced by antigen-specific and universal CD4 help. In addition, immunization with AntpOVA significantly delayed growth of B16.OVA tumors in mice in a tumor therapy model.     

MHC-class I-restricted CD4 T cells: a nanomolar affinity TCR has improved anti-tumor efficacy in vivo compared to the micromolar wild-type TCR

Clinical studies with immunotherapies for cancer, including adoptive cell transfers of T cells, have shown promising results. It is now widely believed that recruitment of CD4⁺ helper T cells to the tumor would be favorable, as CD4⁺ cells play a pivotal role in cytokine secretion as well as promoting the survival, proliferation, and effector functions of tumor-specific CD8⁺ cytotoxic T lymphocytes. Genetically engineered high-affinity T-cell receptors (TCRs) can be introduced into CD4⁺ helper T cells to redirect them to recognize MHC-class I-restricted antigens, but it is not clear what affinity of the TCR will be optimal in this approach. Here, we show that CD4⁺ T cells expressing a high-affinity TCR (nanomolar K _d value) against a class I tumor antigen mediated more effective tumor treatment than the wild-type affinity TCR (micromolar K _d value). High-affinity TCRs in CD4⁺ cells resulted in enhanced survival and long-term persistence of effector memory T cells in a melanoma tumor model. The results suggest that TCRs with nanomolar affinity could be advantageous for tumor targeting when expressed in CD4⁺ T cells.