A monoclonal antibody detecting a new human T cell antigen, HuLy-m2

HuLy-m2 is a new T cell antigen detected by a monoclonal antibody. The antibody (anti-HuLy-m2) reacts with 85% of human T cells, 20% B cells, and 50% null cells in peripheral blood. The antibody is cytotoxic, binds protein A, and is of the IgG2a subclass. HuLy-m2 antigen consists of a glycoprotein dimer Mr 37,000 subunit structure, in which sialic acid and carbohydrate play some role in antigenicity. Capping studies showed the HuLy-m2 antigen to be distinct from the previously described OKT-3, 4, 8, HuLy-m1 (OKT11), and HuLy-m3 antigens.     

Anti-tumor x anti-CD3 heteroconjugates direct human peripheral blood lymphocytes to lyse colon tumor cells

T lymphocytes normally recognize antigens through the antigen receptor complex (TCR/CD3) but can be redirected to bind and lyse unrecognized tumor cells by anti-tumor X anti-CD3 heteroconjugates. Chemical coupling of an antibody directed against the T cell receptor complex and an antibody directed against tumor antigen produces a conjugated antibody that activates the T cell lytic mechanism and bridges the T cell and tumor cell. We tested the lytic activity of heteroconjugate-treated cultured human peripheral blood lymphocytes (PBLs) in 4-h radioactive chromium release assays with human colon tumor cell targets. PBLs were enriched for T cells by the depletion of Leu11a+ and Leu19+ cells, prior to culture in rIL-2 and anti-CD3. Cultured human PBLs depleted of Leu11a+ and Leu19+ cells produced low levels of tumor cell lysis in the absence of antibodies. Anti-tumor X anti-CD3 heteroconjugates significantly enhanced tumor cell lysis by cultured PBLs when tested against four different colon tumor cell lines (P less than 0.005), but, heteroconjugates in the absence of PBLs did not augment tumor cell lysis. Cultured PBLs treated with monoclonal anti-tumor antibody, with monoclonal anti-CD3 antibody, or with irrelevant heteroconjugate did not enhance tumor cell lysis. We conclude that heteroconjugate-directed lysis is mediated by PBLs and that both the anti-tumor antibody and the anti-CD3 antibody are essential for heteroconjugate function. In addition, heteroconjugate-enhanced tumor cell lysis is mediated through a mechanism other than antibody-dependent cellular cytotoxicity or nonspecific T cell receptor crosslinking.(ABSTRACT TRUNCATED AT 250 WORDS)     

Cell-mediated cytotoxicity to porcine aortic endothelial cells is not dependent on galactosyl residues when baboon peripheral blood lymphocytes are previously primed with pig xenoantigens

BACKGROUND: In the pig-to-baboon model, acute vascular rejection remains the main hurdle for successful long-term xenograft survival. The production of galactosyl knockout pigs could solve concomitantly the problem of hyperacute and acute vascular rejection. This work studies in vitro the cell-mediated cytotoxicity of natural killer (NK) and T cells after priming of baboon peripheral blood lymphocytes (PBLs) with pig antigens to evaluate whether cytotoxicity is galactosyl-dependent. MATERIAL AND METHODS: PBLs from naive and primed baboons were used as effectors on primary porcine aortic endothelial cells (PAECs) to assess cytotoxicity. Untreated or galactosidase-digested PAECs were used to evidence the role of galactosyl residues on cell-mediated cytotoxicity. Two rat-anti baboon monoclonal antibodies were tested to inhibit either T+NK cells (LO-CD2b) or NK cells alone (LO-CD94). RESULTS: When using PBLs from naive animals, spontaneous lysis occurred and was inhibited by both LOCD-2b and LO-CD94. In comparison, lysis of PAECs was significantly higher when baboon PBLs were first primed in vivo with pig xenoantigens. In this case, cytotoxicity was completely inhibited by LO-CD2b but only partially by LO-CD94. Reduction of galactosyl residues by galactosidase digestion showed that PAEC lysis almost completely disappeared with naive baboon PBLs but not with primed baboon PBLs, thereby indicating that anti-pig T-cell response is not dependent on galactosyl residues. CONCLUSION: Galactosyl knockout pigs could solve hyperacute rejection and also prevent the activation of NK cells even after xenogeneic priming. T cells will then be the next hurdle for the success of xenografting.     

Human anti-pig cell-mediated cytotoxicity in vitro involves non-T as well as T cell components

Abstract: Human anti-pig cell-mediated cytotoxicity was studied in vitro. Unprimed human peripheral blood lymphocytes (PBL) were able to lyse pig targets but not human targets when the assay was performed in human serum (HS). Much less, but still detectable, lysis was obtained using fetal calf serum (FCS). Human PBLs cultured for 6 days in FCS without stimulating cells showed substantial lysis of pig targets. This lysis was increased by the addition of human IL-2. Unseparated human PBLs stimulated for 6 days with pig cells in serum-free media lysed pig targets expressing the same or different SLA antigens as the stimulating cells. This lysis could not be significantly inhibited by anti-CD3 or anti-CD8 blocking antibodies during the effector phase of the assay. T cell-enriched human PBLs treated with anti-CD 16 and anti-CD56 antibodies plus complement also lysed pig targets. These effector cells were significantly inhibited by anti-CD3 and anti-CD8 antibodies but not by anti-CD4 antibodies. Furthermore, these primed T cell effectors could only lyse pig targets that shared the same MHC class I antigens as the sensitizing stimulators.
These results suggest that human anti-pig cell-mediated cytotoxicity in vitro has at least three different components in bulk culture: 1) an ADCC component depending on natural antibodies from human serum, 2) an NK and/or a LAK cell component that is enriched by in vitro culture and interleukin-2 (IL-2), and (3) an allospecific T cell component, involving CD3+, CD8+, class I-specific effector cells.     

Hu Ly-M3--a human leukocyte antigen

A monoclonal antibody, 5-4.8, was produced against human peripheral blood lymphocytes and it appears to be leukocyte-specific in that it reacts with a common determinant (called Hu Ly-m3) present on the peripheral blood T, B and null lymphocytes of 40 individuals. The antibody also reacts with thymocytes, spleen cells, and bone marrow cells (30%) and weakly with granulocytes and platelets--but not with heart, liver, or kidney. Affinity to lentil-lectin and molecular weight analysis demonstrated that Hu Ly-m3 is a glycoprotein consisting of a single chain of 47,000 daltons which is not HLA because it is not present on all cells; because it is present on the surface of the phenotypically HLA- Daudi cell line; and because soluble HLA antigens did not inhibit the binding of the 5-4.8 antibody.     

Suppressive effect of mizoribine on humoral antibody production in DBA/2 mice

The mode of action of mizoribine (MZR) as a B cell inhibitor was studied using DBA/2 mice. Its in vitro administration significantly delayed the primary response in hemagglutinin production against sheep erythrocytes by suppressing the IgM antibody formation. In vitro plaque-forming cell (PFC) response against both T-dependent and T-independent antigens, such as TNP-SRBC and TNP-Brucella abortus, was dose-dependently suppressed by MZR. Since PFC formation by the T-depleted fraction of splenocytes was likewise suppressed, MZR may inhibit humoral antibody response by directly affecting the B cells (and/or macrophages) as well as by modulating the regulatory T cells. MZR may only act on a certain stage of the cell cycle of B lymphocytes following antigenic stimulation. It may not interfere with the initial antigen recognition or with mature B cells.     

Gamma-interferon production capacity and T lymphocyte subpopulation after allogeneic bone marrow transplantation

T cell phenotypes after bone marrow transplantation (BMT) were investigated using monoclonal antibodies (moAbs) reactive to lymphocyte cell surface antigens. Patients' T cells showed decreased percentages of OKT4, 4A and 9.3-positive T cells, and increased percentages of OKT8, human Ia, and Leu-7-positive T cells. These changes in T cell phenotype persisted for a long period after BMT and had no correlation with the occurrence of graft-versus-host disease (GVHD). No lymphocyte activation antigens such as TIA (Tac) or transferrin receptor (5E9) were detected after BMT. The capacity of the patients' lymphocytes to produce gamma-interferon (IFN-gamma) was measured after incubation of lymphocytes with mitogen. Patients' lymphocytes produced significantly lower levels of IFN-gamma than the normal controls. This failure of IFN-gamma production showed no correlation with stimulation index of mitogen blastogenesis or changes of T cell population. Thus, not only T cell phenotype but also measurement of IFN-gamma production of lymphocyte may be useful in detecting immunological abnormalities in patients who receive BMT.     

The molecular mechanism of apoptosis induced by xenogeneic cytotoxicity

Abstract: In order to clarify the role of natural killer (NK) cells in delayed xenograft rejection (DXR) of discordant xenotransplantation, we used in vitro xenogeneic combination of human NK cells and pig kidney target cells (PK13, and investigated the mechanism of xenogeneic cytotoxicity caused by human NK cells. In the presence of decomplemented human serum or human IgG, freshly isolated human peripheral blood lymphocytes (PBLs) caused both membrane (⁵¹Cr release) and DNA (³H release) damage on PK15. In contrast, only membrane damage was detected in the presence of normal human serum. To clarify the participation of perforin/granzymes-cell mediated cytotoxicity (P/G-CMC), when EGTA or concanamycin B (CMB) was added to the cytotoxicity assays, both cytotoxicities were completely inhibited by these drugs in a dose-dependent manner. In terms of the involvement of Fas/FasL-based cytotoxicity (F-CMC), while the cytotoxicity assays were performed in the presence of antagonistic anti-human FasL mAb, this antibody was not able to block the cytotoxicity. From these results, it is concluded that xenogeneic cytotoxicity is due to NK cell dependent ADCC (antibody-dependent cell-mediated cytotoxicity), and their effector mechanism can cause apoptosis on target cells via P/G-CMC.     

What is the role of T-lymphocyte surveillance in neoplastic disease?

Dramatic advances have recently been made in our comprehension of how thymus-derived (T) lymphocytes function. Principles of both antigen-specific and nonspecific modes of their action in cell-mediated immunity are summarized as follows. First, different functions are carried out by distinct, separable populations of T lymphocytes. Second, all T-cell responses depend on interaction between cells of different functional subclasses. Thus, target cell killing of cytotoxic T lymphocytes depends on separate, coincident recognition of antigen by pre-cytotoxic T lymphocytes and by amplifier T cells that supply a nonspecific but critical lymphokine. Immunosuppressive drugs such as cyclosporin A and dexamethasone block this interaction at different points.Antigen-specific amplifier T cells also secrete various polypeptide factors that augment the impact of macrophage cytotoxicity, and immune interferon, which stimulates natural killer (NK) cells. Thus, even in cases where T cells are not the effectors, activation of T cells can potentiate host defenses. However, immune paralysis can result from stimulation of antigen-specific suppressor T cells. These act on effector cells directly or on their amplifier cells. The route of immunization often influences the balance between suppression and activation. Suppressor and amplifier T cells may be killed differentially by drugs like cyclophosphamide.Other cases of nonresponsiveness result from the way T cells actually recognize antigen. T cells only react with antigen in cell-surface complexes with self-histocompatibility antigen. Some otherwise-stimulatory antigens cannot form immunogenic complexes with the products of certain histocompatibility alleles, so that nonresponsiveness to those antigens is genetically predetermined. Overall, the centrality of T-lymphocyte surveillance in controlling spontaneous neoplasms is challenged by the low incidence of malignancy in T cell-deficient animals. This controversy will be examined both with reference to the lesions in T-cell development in these cases and with reference to the auxiliary roles played by T cells in amplifying the responses of non-T effector cells.     

Analysis of immune response to tumor specific antigen restricted to HLA class II on renal cell carcinoma and its recognition mechanism

PURPOSE: In this study, we studied the immune response against to human renal cell carcinoma and its antigensity. METHODS: Mixed lymphocyte tumor culture test was performed using tumor cells as stimulator cells, peripheral blood lymphocytes from tumor patient (autologous) or healthy volunteer (allogeneic) as responder cells, and tumor cells or peripheral blood lymphocytes from tumor patient as target cells. The cytotoxic activity of mixed lymphocyte tumor culture test was assayed by 51Cr-relase test, and cell surface antigens presented on tumor cells or peripheral blood lymphocytes were assayed by antibody block test. RESULTS: The cytotoxic activity against to tumor cells was induced from allogeneic peripheral blood lymphocytes by mixed lymphocyte tumor culture test. Its cytotoxic activity was inhibited by anti-CD8 antibody treatment of peripheral blood lymphocytes and anti-HLA class II antibody treatment of tumor cells. Furthermore, allogeneic peripheral blood lymphocytes induced to tumor cells did not damage peripheral blood lymphocyte of the tumor patient derivation. CONCLUSION: Renal cell carcinoma may express tumor specific antigen restricted to HLA class II antigens that could be recognized by allogeneic CD8 positive T lymphocytes.     

Selective deletion of antigen-specific, activated T cells by a humanized MAB to CD2 (MEDI-507) is mediated by NK cells

CD2 is a 50-kDa transmembrane glycoprotein that plays an important role in T and natural killer (NT) lymphocyte functions. CD2 serves as both an adhesion molecule and as a costimulatory molecule through interactions with its ligand, CD58, on antigen presenting or target cells. Consistent with earlier studies using a rat anti-CD2 mAb, we have shown that treatment of alloantigen stimulated T lymphocytes with a humanized mAb, MEDI-507 (IgG1, kappa), induced hyporesponsiveness to subsequent stimulation with alloantigen but not to mitogen (phytohemagglutinin). Fluorescence-activated cell sorting analysis of cells from mixed lymphocyte reaction (MLR) treated with MEDI-507 revealed pronounced deletion of T and NK cells, consistent with lack of proliferation in the MLR. MEDI-507 F(ab')2 fragments did not have inhibitory activity or induce deletion of lymphocytes in the MLR. Removal of the NK cell subset by magnetic bead depletion using anti-CD16 and anti-CD56 mAbs eliminated both the T cell deletion and the inhibitory effect. Reconstitution of NK depleted responder populations using autologous NK cells restored the MEDI-507-mediated deletion activity to levels measured in the original MLR. Formaldehyde-fixed NK cells failed to mediate the MEDI-507-induced deletion effect. Altogether, our studies indicate that activated T cells with MEDI-507 bound to CD2 are preferential targets for autologous NK cells through a nonapoptotic cytotoxic mechanism.     

The distinct effects of FK506 on the activation, proliferation, and differentiation of human B lymphocytes.

We examined the effect of FK506 on the activation, proliferation and differentiation of human B lymphocytes in vitro. FK506 inhibited the proliferative response of resting B cells induced by Staphylococcus aureus Cowan strain I (SAC) and phorbol myristate acetate (PMA) in a dose-dependent manner. Inhibition of cell proliferation by FK506 was caused by a selective block of G0 to G1 phase transition leading to cell arrest. In addition, the proliferative response of in vivo-activated B cells and lymphokine-driven B cell proliferation were also found to be sensitive to FK506. Interestingly, FK506 did not affect the expression of activation antigens such as CD23, IL-2 receptor (CD25), and transferrin receptor (CD71). Finally, FK506 had little effect on B cell antibody generation in a T cell-independent system. Conversely, FK506 suppressed neither proliferation nor immunoglobulin secretion in a human B lymphoblastoid cell line. These results indicate that FK506 has discrete effects on the different stages of the B cell maturation.     

The effect of a chimeric mouse-human CD7 antibody on human T, natural killer, and lymphokine-activated killer cell activity in vitro.

A chimeric CD7 antibody has been constructed with mouse variable and human constant regions and is currently being assessed in the prophylaxis of renal graft rejection. In this study we have investigated if this antibody or its murine parental form inhibits the function of a number of immune effector mechanisms involved in host defense against infection and/or malignancy. Most memory T cells and all natural killer cells express the CD7 antigen and could therefore be affected by CD7 antibody. Murine and chimeric CD7 antibodies significantly inhibit the alloproliferation of naive (65 +/- 4% and 66 +/- 8%, respectively) but not memory T cells (86 +/- 2% and 98 +/- 4%, respectively) in a primary mixed lymphocyte reaction relative to the negative control CD10 antibody (P less than 0.001). The memory T cell proliferative response to recall antigen is also largely unaffected by murine and chimeric CD7 antibodies relative to the negative control antibody (91 +/- 12% and 103 +/- 10%, respectively). The CD7 antigen is almost completely modulated from the surface of NK cells after incubation for 24 hr with either the murine or chimeric CD7, but not the CD10, negative control. The modulation of CD7 antigen by antibody, however, does not affect the cytotoxic function of either the NK or lymphokine-activated killer cells significantly. Preincubation with the chimeric antibody however, consistently showed a small inhibition relative to the negative control of 75-80% in NK assays and to 80-90% in LAK assays. These data suggest that both murine and chimeric CD7 antibodies may have a selective effect on alloproliferation but may largely spare a major component of the host's innate immunity as well as memory T cell proliferation to previously encountered antigens.     

Induction of cytotoxicity from human lymphocytes coated with bispecific antibody against human glioma cells

A bifunctional hetero-F (ab') 2 fragment containing the Fab portions from anti-CD3 and anti-glioma monoclonal antibodies was prepared. The antibody simultaneously recognized two different molecules, the CD3 complex on effector T cells and human glioma-associated antigens on target glioma cells. This bispecific F (ab') 2 fragment induced peripheral blood lymphocytes (PBLs) from healthy donors to lyse cells of the human glioma cell line, U251MG, that is resistant to natural killer cell-mediated cytolysis. Compared with lymphokine-activated killer (LAK) activity which is obtained by exposure to interleukin (IL)-2 for more than 3 days, the maximum bispecific antibody-dependent cytotoxicity can be generated only after 24 hour exposure to IL-2. And cytotoxicity of lymphocytes triggered by the bispecific antibody was dependent upon the concentration of IL-2 in the culture medium. The effect of the bispecific antibody on LAK cells was tested in patients suffering from malignant glioma. One patient who received specific targeting therapy (LAK plus bispecific antibody) showed the disappearance of high density tumor mass from CT scan. But the patient who received only LAK therapy showed the recurrence of tumor one year after LAK treatment. These are preliminary data, but may be a promising approach in cancer immunotherapy.     

Liver Transplantation in a Patient With CD40 Ligand Deficiency and Hyper‐IgM Syndrome: Clinical and Immunological Assessments

Monoclonal antibodies that disrupt CD40–CD40 ligand (CD40L) interactions are likely to have use in human transplantation. However, the extent of the immunosuppressive effects of CD40–CD40L blockade in humans is unknown. Hyper‐IgM syndrome (HIGM) is a rare primary immunodeficiency syndrome characterized by defects in the CD40–CD40L pathway, severe immune deficiency (IgG), and high or normal IgM levels. However, the effects of CD40L deficiency on T‐ and natural killer (NK)‐cell function is not established. Here, we present a patient with HIGM syndrome who underwent liver transplantation for hepatitis C virus infection. Posttransplantation, NK‐cell antibody‐dependent cytokine release (γ‐interferon) to alloantigens and T cell responses to viral antigens and mitogens were assessed and showed normal CD4⁺, CD8⁺, and NK‐cell responses. We also examined antibody‐dependent cellular cytotoxicity against a CD40⁺ and HLA‐expressing cell line. These experiments confirmed that the patient's NK cells were equivalent to those of normal subjects in mediating antibody‐dependent cellular cytotoxicity despite the absence of CD40–CD40L interactions. Mitogenic stimulation of the patient's peripheral blood mononuclear cells showed no expression of CD40L on T and NK cells compared with increased expression in normal subjects. Taken together, these data suggest that absence of CD40L expression is responsible for aberrant B cell immunity but had little impact on NK‐ and T cell immune responses in vitro.     

Mesenchymal stem cells inhibit the formation of cytotoxic T lymphocytes, but not activated cytotoxic T lymphocytes or natural killer cells

BACKGROUND: Mesenchymal stem cells (MSCs) can reduce the incidence of graft-versus-host disease because of their ability to inhibit T-lymphocyte proliferation. There are no publications on the effect that MSCs have on cytotoxic T lymphocytes (CTLs) and natural killer (NK) cells, effector cells vital for the graft-versus-leukemia effect. METHODS: Cytotoxic T cells were primed in mixed lymphocyte culture (MLC) against irradiated stimulator lymphocytes, and irradiated third-party MSCs were added at different time points. The CTLs were collected, and their cytotoxic potential was analyzed in a chromium-release assay against the same stimulator cells as in the MLC. Purified NK cells were mixed with irradiated MSCs, and the lysis was measured in chromium-release assay against K562 target cells. RESULTS: We found that MSCs inhibited CTL-mediated lysis by 70% if added at the beginning of the 6-day MLC. The lysis was not affected on day 3 or in the cytotoxic phase. Furthermore, MSCs inhibited the formation of cytotoxic lymphocytes when the cells were separated in a transwell system, which indicates that the effect is mediated by a soluble factor. NK cell-mediated lysis of K562 cells was not inhibited by MSCs. MSCs did not induce proliferation of allogeneic lymphocytes, and they were not lysed by allogeneic CTLs or NK cells. CONCLUSION: Our findings indicate that MSCs escape recognition by CTLs and alloreactive NK cells, and inhibit the formation of cytotoxic T cells by secreting a soluble factor, but that they do not interfere with CTLs and NK cell lysis.     

Induction of xenoreactive CD4+ T-cell anergy by suppressor CD8+CD28- T cells

BACKGROUND: The underlying mechanism of immune suppression mediated by regulatory T cells is not completely understood. In previous studies we have shown that antigen-specific human T suppressor cells (Ts) can be generated in vitro by multiple rounds of stimulation with allogeneic, xenogeneic, or antigen-pulsed autologous antigen-presenting cells (APC). Human Ts express the CD8+CD28- phenotype and require specific recognition of MHC class I/peptide complexes on the surface of APC to block proliferation of T helper cells (Th). The aim of the present study was to explore the activation requirements of Ts as well as the nature of Th unresponsiveness to xenogeneic (swine) antigens induced by Ts. METHODS AND RESULTS: We investigated whether specific antigenic stimulation of Ts is required for their ability to inhibit early activation of xenoreactive Th (up-regulation of CD40 ligand). Flow cytometry studies indicated that Ts function required specific recognition of MHC class I on the surface of the stimulating APC. However, neither proliferation nor protein synthesis was required for the ability of Ts to inhibit Th. Ts drastically reduced the capacity of xenoreactive Th cells to produce interleukin (IL)-2 in response to the specific APC, without affecting their surface expression of IL-2 receptor. The suppressor effect that Ts exerted on Th proliferation could not be circumvented by CD40 ligation on the surface of the APC but could be reversed by the addition of exogenous IL-2. CONCLUSION: These data indicate that Ts induce anergy of xenoreactive human Th cells upon specific recognition of MHC class I antigens. Hence, Ts may prevent the activation of T cell-mediated immune responses against xenogeneic transplants.     

Development and characterization of monoclonal antibodies to lymphocyte cell surface antigens of the rhesus monkey.

Three monoclonal antibodies directed against rhesus lymphocyte cell surface antigens are described. A pan-T mAb, T64, and a T suppressor mAb, T35, showed phenotypic and functional specificity for both human and rhesus cells. In contrast, a third mAb, N42, identifying natural killer cells in rhesus peripheral blood leukocytes, was not crossreactive with the corresponding homologous human cells. N42 reacted with the same cells identified by Leu 11a and Leu 11b in rhesus PBL and in a functional assay decreased NK activity by 80%. N42 precipitated a 50KD protein from rhesus PBL lysates and was reactive with the Fc receptor domain of the NK cell. The T-S functional activity of cells reactive with mAb T35 was demonstrated in a pokeweed-mitogen-driven system for Ig synthesis: removal of the T35 positive cells by complement-mediated lysis led to an enhanced production of Ig by rhesus PBL, and the addition of T35 positive cells to a culture of T helper and B cells resulted in a reduction of this response. T35 was determined to be an IgG2a immunoglobulin and precipitated a 34KD protein from rhesus cell lysates. An IgM immunoglobulin, mAb T64 delineated all T lymphocytes, inhibited E-rosette formation, interfered with the proliferation of cells stimulated with mitogens, and precipitated a 52KD membrane protein. The potential utilization of these mAbs in vivo for organ or tissue transplantation in the rhesus monkey is discussed.     

Identification of target antigens in specific immunotherapy for renal cell carcinoma

PURPOSE: Effective immunotherapy against renal cell carcinoma has not yet been established despite recent advances in specific immunotherapy for various malignancies. A plausible reason is limited information about target antigens of renal cell carcinoma. We searched for useful cancer antigens applicable to immunotherapy for renal cell carcinoma by examining antigen expression in renal cell carcinoma cell lines and testing the ability to induce renal cell carcinoma reactive cytotoxic T lymphocytes. MATERIALS AND METHODS: mRNA expression of a panel of cancer associated antigens was examined using 5 renal cell carcinoma cell lines. Thereafter antigen derived peptides reported to induce cancer reactive cytotoxic T lymphocytes from human leukocyte antigen-A24+ patients with cancer were examined for their potential to induce cytotoxic T lymphocytes from peripheral blood mononuclear cells of human leukocyte antigen-A24+ patients with renal cell carcinoma. RESULTS: Three candidate antigens, including multidrug resistance-associated protein 3, polycomb group protein enhancer of zeste homologue 2 and Her2/neu, were expressed in all 5 renal cell carcinoma cell lines. Six peptides derived from these antigens, including multidrug resistance-associated protein 3(503-511), multidrug resistance-associated protein 3(1293-1302), polycomb group protein enhancer of zeste homologue 2(291-299), polycomb group protein enhancer of zeste homologue 2(735-743), Her2/neu342-350 and Her2/neu485-493, efficiently induced peptide specific and renal cell carcinoma reactive cytotoxic T lymphocytes from human leukocyte antigen-A24+ patients with renal cell carcinoma. Blocking and cold inhibition assays revealed that cytotoxicity against renal cell carcinoma depended on human leukocyte antigen class I restricted and peptide specific CD8+ T cells. CONCLUSIONS: This information could facilitate the development of effective immunotherapy against renal cell carcinoma.