Tumor-specific CD4+ T cells from a patient with renal cell carcinoma recognize diverse shared antigens

Murine models for immune-mediated tumor regression have defined an essential role for CD4+ T helper (Th) cells, but the contribution of these cells to antitumoral immune responses in humans remains poorly defined. Here, we investigated the Th cell response against the autologous tumor in a patient with metastasized renal cell carcinoma (RCC) exhibiting objective clinical response to immunotherapy. Peripheral blood T cells of the patient were repeatedly stimulated in vitro using either autologous IFNgamma-treated whole tumor cells or Epstein-Barr virus-immortalized B cells (EBV-B) pulsed with tumor cell lysate. CD4+ T-cell clones recognizing autologous tumor cells but not EBV-B cells were efficiently reactivated and expanded with both types of stimulator cells, establishing the latter as potentially useful for isolating CD4+ T cells reactive against MHC class II-negative tumors. Two T-cell clones from both stimulation protocol were further characterized. The restricting MHC class II molecules were defined by using allogeneic EBV-B cells pulsed with tumor lysate, and the expression pattern of the antigens was examined by analyzing lysates from normal kidney cells, allogeneic RCCs as well as tumors of different histologic origin. Furthermore, the subcellular localization of the antigens recognized by the T-cell clones was examined by fractionating the tumor lysate, and the Th phenotype was determined by assessing the cytokines released after T cell activation. These experiments show that a dual Th1/Th2, MHC class II-restricted T-helper-cell response against diverse shared tumor antigens has been elicited in this patient.     

Cellular immune response to human renal-cell carcinomas: Definition of a common antigen recognized by HLA-A2-restricted cytotoxic T-Lymphocyte (CTL) clones

Cytotoxic T lymphocyte (CTL) clones directed against autologous renal-cell carcinoma (RCC) cell lines were generated by mixed lymphocyte/tumor-cell culture (MLTC) using peripheral blood lymphocytes (PBL). A CD8+, CD4- CTL clone MZ1257-CTL 5/30 with high cytolytic activity for the autologous tumor cell line MZ1257-RCC was established. No lysis of the autologous EBV-transformed B lymphocytes (EBV-B) or K562 cells was observed. A panel of HLA-A2-matched allogeneic RCC lines was recognized by CTL 5/30. Further specificity analysis showed a cross-reactivity with HLA-A2-matched allogeneic tumor cells of various origins, especially melanoma. CTL 5/30 was also cross-reactive with several HLA-A2-positive allogeneic normal kidney cells in culture. The restriction element identified for CTL 5/30 was HLA-A2, as shown by blocking of cytotoxicity using an anti-HLA-A2 monoclonal antibody (MAb) and by resistance of an HLA-A2-negative melanoma variant SK29-MEL. 1.22 against lysis by CTL 5/30. In this report we demonstrate HLA-A2-restricted recognition of a T-cell-defined antigen on autologous renal-cancer cells. This antigen is also expressed and recognized in association with HLA-A2 on normal kidney cells in culture and other HLA-A2-positive tumor cells. It may therefore be a normal differentiation antigen to which tolerance is incomplete in the renal-cell cancer system investigated.     

CD4(+), HLA class I-restricted, cytolytic T-lymphocyte clone against primary malignant melanoma cells

The involvement of HLA-class I in target cell lysis by CD4(+) cytolytic T cells (CTL) has been a controversial issue. A CTL clone of CD4 phenotype was derived from the peripheral blood lymphocytes of a patient with primary melanoma. The CTL clone stably lysed the autologous primary melanoma cells for approximately 9 months in culture. Both the Valpha2/Vbeta8 T-cell receptor and CD4 were involved in CTL cytotoxicity. Of a large panel of allogeneic primary and metastatic melanoma or colorectal carcinoma cells, autologous and allogeneic Epstein-Barr virus-transformed B cells and autologous fibroblasts, only allogeneic metastatic melanoma cells matched with the autologous tumor cells for HLA-class I (B57[17]) were lysed and induced IFN-gamma secretion by the CTL clone. Lysis of the autologous tumor cells was significantly blocked by monoclonal antibody to HLA-B17. Importantly, allogeneic, HLA-class I- and class II-unmatched melanoma cells were lysed by the CTL only following transfection of the cells with B57[17] cDNA. Our results provide direct evidence for the involvement of both CD4 and HLA-class I in tumor cell lysis by CD4(+) CTL.     

Serological response of melanoma patients to vaccines prepared from VSV lysates of autologous and allogeneic cultured melanoma cells

Melanoma patients were vaccinated with cell-free lysates prepared from vesicular stomatitis virus (VSV)-infected cultured autologous and allogeneic melanoma cells. Eleven patients received vaccines produced from the melanoma cell line SK-MEL-13. This cell line, derived from the melanoma of Patient AH, expresses a differentiation antigen (initially defined by autologous antibody) that is restricted to melanomas and other cells of neural crest origin (an example of a Class 2 melanoma antigen). Thirteen patients received vaccines prepared from autologous melanoma cells, the only known source of autologous unique (Class 1) melanoma antigens. VSV lysates were used for vaccination because VSV infection of tumor cells has been shown to augment the immunogenicity of tumor antigens. All patients but one vaccinated with VSV lysates of autologous melanoma cells developed antibodies against VSV, and all patients vaccinated with VSV lysates of SK-MEL-13 developed antibodies against HLA-related antigens. Antibodies against a Class 1 (unique) melanoma antigen were detected in only one case, and antibodies against Class 2 (shared) melanoma antigens were not found in any of the patients. The authors conclude that VSV lysates of melanoma cells are not effective in increasing the serologic response of melanoma patients to Class 1 or 2 melanoma antigens.     

Melanoma-specific CD4+ T lymphocytes recognize human melanoma antigens processed and presented by epstein-barr virus-transformed B cells

While much emphasis has been placed on the role of MHC class I-restricted CDS⁺ T cells in the recognition of tumor-specific antigens (Ag), evidence has accumulated that CD4⁺ T cells also play a critical role in the anti-tumor immune response. However, little information exists on the nature of MHC class II-restricted human tumor Ag. In an attempt to develop in vitro systems to characterize such Ag, we examined the ability of Epstein-Barr virus(EBV) transformed B cells to present melanoma-associated Ag to melanoma-specific CD4⁺ cells. CD4⁺ T cells cultured from lymphocytes infiltrating a s.c. melanoma metastasis secreted TNF-α and GM-CSF specifically in response to autologous cultured melanoma cells expressing MHC class II molecules. These CD4⁺ cells also recognized MHC class II-compatible EBV-B cells pulsed with extracts of autologous melanoma cells, but failed to recognize EBV-B cells pulsed with autologous non-transformed cells or a variety of allogeneic tumors or normal cells. B cells pre-fixed with paraformaldehyde were incapable of Ag presentation, suggesting that intracellular processing events were occurring. Antibody-blocking studies defined HLA-DR as the dominant if not exclusive restriction locus in this T-B interaction, and HLA-DR genocyping revealed DRBI 0404 to be the probable restriction element. In a second patient, a CD4⁺ T-cell clone cultured from a melanoma lesion recognized autologous tumor Ag presented by autologous EBV-B; no cross-reactivity was observed with the other tumor system investigated, nor with autologous CD4⁺ T cells specific for tetanus toxoid. These findings demonstrate that tumor Ag can be processed and presented by EBV-transformed B cells to MHC class II-restricted tumor-specific CD4⁺ T cells. They also provide a model system for direct identification of these tumor-derived antigens. © 1994 Wiley-Liss, Inc.     

Peptidome from renal cell carcinoma contains antigens recognized by CD4+ T cells and shared among tumors of different histology.

PURPOSE: Renal cell carcinoma (RCC) is considered immunogenic; nonetheless, rare tumor-associated antigens have been identified or are expressed in RCC. Peptidome (i.e., the total content of natural peptides of whole cells) from other tumors, such as melanoma, has proved to be immunogenic. The aims of this study were to determine whether peptidome from RCC is immunogenic and whether it contains tumor peptides shared among allogenic RCCs. EXPERIMENTAL DESIGN: Autologous dendritic cells pulsed with RCC peptidome were used to activate in vitro CD4(+) T cells from healthy donors and a metastatic RCC patient. CD4(+) T-cell polyclonal lines and clones were characterized for tumor cell recognition by proliferation assay, killing activity, and cytokine secretion. RESULTS: CD4(+) T-cell lines and clones recognized HLA-DR-matched allogenic RCC and, for the patient, the autologous tumor. RCC-reactive CD4(+) T cells showed a heterogeneous Th1 or Th0/Th2 pattern of cytokine secretion. Moreover, RCC-reactive CD4(+) T cells recognized also melanoma, colon carcinoma, cervical carcinoma, pancreas carcinoma, lung carcinoma, gastric carcinoma, and lymphoma cells but not autologous T-cell blasts. CONCLUSIONS: Our results show that (a) the RCC peptidome contain antigens recognized by CD4(+) T cells and (b) shared among tumors of different histology and (c) it induces both Th1-type and Th2/Th0-type immune responses. These data support the use of the peptidome from allogenic RCC for specific immunotherapy in RCC and possibly in other neoplastic diseases. Moreover, the CD4(+) T-cell clones generated here are useful tools for tumor antigen identification.     

Dendritic cells fused with allogeneic colorectal cancer cell line present multiple colorectal cancer-specific antigens and induce antitumor immunity against autologous tumor cells.

The aim of antitumor immunotherapy is to induce CTL responses against autologous tumors. Previous work has shown that fusion of human dendritic cells and autologous tumor cells induce CTL responses against autologous tumor cells in vitro. However, in the clinical setting of patients with colorectal carcinoma, a major difficulty is the preparation of sufficient amounts of autologous tumor cells. In the present study, autologous dendritic cells from patients with colorectal carcinoma were fused to allogeneic colorectal tumor cell line, COLM-6 (HLA-A2(-)/HLA-24(-)), carcinoembryonic antigen (CEA)(+), and MUC1(+) as an alternative strategy to deliver shared colorectal carcinoma antigens to dendritic cells. Stimulation of autologous T cells by the fusion cells generated with autologous dendritic cells (HLA-A2(+) and/or HLA-A24(+)) and allogeneic COLM-6 resulted in MHC class I- and MHC class II-restricted proliferation of CD4(+) and CD8(+) T cells, high levels of IFN-gamma production in both CD4(+) and CD8(+) T cells, and the simultaneous induction of CEA- and MUC1-specific CTL responses restricted by HLA-A2 and/or HLA-A24. Finally, CTL induced by dendritic cell/allogeneic COLM-6 fusion cells were able to kill autologous colorectal carcinoma by HLA-A2- and/or HLA-A24-restricted mechanisms. The demonstration of CTL activity against shared tumor-associated antigens using an allogeneic tumor cell line, COLM-6, provides that the presence of alloantigens does not prevent the development of CTL with activity against autologous colorectal carcinoma cells. The fusion of allogeneic colorectal carcinoma cell line and autologous dendritic cells could have potential applicability to the field of antitumor immunotherapy through the cross-priming against shared tumor antigens and provides a platform for adoptive immunotherapy.     

Production of stable cytolytic T-cell clones directed against autologous human melanoma

We have attempted to optimize the production of stable human cytolytic T lymphocyte clones directed against autologous melanoma cell lines. MLTC were restimulated every week with irradiated melanoma cells in medium containing human serum and IL-2. After 21 to 35 days, in 5 out of 6 patients, these cultures expressed a preferential cytolytic activity against the autologous melanoma cells, as compared to autologous EBV-B cells or NK target K562. Limiting dilution of MLTC responder cells was performed at times varying from days 7 to 28, in medium containing IL-2 and allogeneic EBV-B cells as feeders. Approximately 1% of these responder cells gave rise to CTL clones that lysed the autologous melanoma cells, but did not lyse K562 or autologous B cells. It was possible to maintain in culture for several months a large number of CTL clones that retained this specificity with high activity, and multiplied more than 5-fold every week. Some of these CTL clones were dependent on the presence of the autologous melanoma cells for their growth. With one melanoma, the use of autologous CTL clones made it possible to identify 3 different antigens on the tumor cells.     

Generation of human autologous melanoma-specific cytotoxic T-cells using HLA-A2-matched allogeneic melanomas

Autologous tumor-specific cytotoxic T-lymphocytes (CTLs), generated by repeated stimulation with autologous melanoma and expanded in interleukin 2, are major histocompatibility complex restricted. These CTLs recognize a common tumor-associated antigen in the presence of HLA class I determinants, suggesting that allogeneic melanomas which express the restricting HLA-A region antigen could substitute for the autologous tumor in the generation of CTLs. This was investigated in the HLA-A2 system. Four T-cell lines were established by stimulation of lymphocytes with either autologous tumor or an HLA-A2-matched allogeneic melanoma. Allogeneic stimulated CTLs specifically lysed the autologous tumor and demonstrated an identical pattern of HLA-A2 restriction, when compared to the autologous stimulated CTLs. Lysis by the allogeneic stimulated CTLs was blocked by a monoclonal antibody to HLA class I antigens; lysis was also inhibited by both autologous tumor or HLA-A2 allogeneic melanomas when evaluated in cold target competition studies. The allogeneic stimulated CTLs proliferated in response to both autologous tumor and HLA-A2 melanomas, but not in response to HLA-A2 nonmelanomas. By phenotypic analysis these CTLs were CD3+ and predominantly CD8+ cells. We conclude that autologous tumor-specific CTLs can be generated using HLA-A region-matched allogeneic melanomas for stimulation. Since established, HLA-typed melanoma tumor lines can be used in the absence of autologous tumor; this procedure can be applied clinically to a broad patient population and may prove useful in the adoptive immunotherapy of melanoma.     

Shared MHC class II-dependent melanoma ribosomal protein L8 identified by phage display

Antigens recognized by T helper (Th) cells in the context of MHC class II molecules have vaccine potential against cancer and infectious agents. We have described previously a melanoma patient's HLA-DR7-restricted Th cell clone recognizing an antigen, which is shared among melanoma and glioma cells derived from various patients. Here, this antigen was cloned using a novel antigen phage display approach. The antigen was identified as the ribosomal protein L8 (RPL8). A peptide of RPL8 significantly stimulated proliferation and/or cytokine expression of the Th cell clone and lymphocytes in four of nine HLA-DR7(+) melanoma patients but not in healthy volunteers. The RPL8 antigen may represent a relevant vaccine target for patients with melanoma, glioma, and breast carcinoma whose tumors express this protein.     

CD40-stimulated B lymphocytes pulsed with tumor antigens are effective antigen-presenting cells that can generate specific T cells

Although they are considered as antigen-presenting cells, the role of antigen-unspecific B lymphocytes in antigen presentation and T-lymphocyte stimulation remains controversial. In this paper, we tested the capacity of normal human peripheral activated B cells to stimulate T cells using melanoma antigens or melanoma cell lysates. B lymphocytes activated through CD40 ligation and then pulsed with tumor antigens efficiently processed and presented MHC class II-restricted peptides to specific CD4(+) T-cell clones. This suggests that CD40-activated B cells have the functional and molecular competence to present MHC class II epitopes when pulsed with exogenous antigens, thereby making them a relevant source of antigen-presenting cells to generate T cells. To test this hypothesis, CD40-activated B cells were pulsed with a lysate prepared from melanoma cells and used to stimulate peripheral autologous T cells. Interestingly, T cells specific to melanoma antigens were generated. Additional analysis of these T-cell clones revealed that they recognized MHC class II-restricted epitopes from tyrosinase, a known melanoma tumor antigen. The efficient antigen presentation by antigen-unspecific activated B cells was correlated with a down-regulation in the expression of HLA-DO, a B cell-specific protein known to interfere with HLA-DM function. Because HLA-DM is important in MHC class II peptide loading, the observed decrease in HLA-DO may partially explain the enhanced antigen presentation after B-cell activation. Results globally suggest that when they are properly activated, antigen-unspecific B-lymphocytes can present exogenous antigens by MHC class II molecules and stimulate peripheral antigen-specific T cells. Antigen presentation by activated B cells could be exploited for immunotherapy by allowing the in vitro generation of T cells specific against antigens expressed by tumors or viruses.     

Major histocompatibility complex class I restricted cytotoxic T cells specific for natural melanoma peptides recognize unidentified shared melanoma antigen(s).

CTLs were generated in vitro from two healthy donors and one melanoma patient by stimulation of CD8+ T cells with autologous dendritic cells pulsed with natural melanoma peptides (NMPs), obtained by acid treatment of HLA-matched melanoma cells. CTLs showed MHC class I-restricted melanoma-specific cytolytic activity. Importantly, CTLs from the patient, induced with NMPs obtained from an allogeneic HLA-A-matched melanoma, killed the autologous tumor. COS-7 cells cotransfected with the cDNA of 13 melanoma antigens and the HLA-A1-restricting allele did not induce cytokines release from NMP-specific CTLs, suggesting that they recognize unidentified shared melanoma antigens and that they may be valuable for identification of new tumor antigens. These results strongly support the use of autologous and/or allogeneic NMP-pulsed dendritic cells as cancer vaccines in patients whose neoplasms do not express or have lost expression of known tumor antigens.     

Interleukin-2-induced, melanoma-specific T cells recognize CAMEL, an unexpected translation product of LAGE-1.

Melanoma-specific cytotoxic T lymphocytes (CTLs) were induced by in vitro stimulation of peripheral blood mononuclear cells of a melanoma patient with autologous IL-2-producing melanoma 518/IL2.14 cells. CTL clone 1/29 recognized, in addition to autologous melanoma cell lines, a panel of HLA-A*0201-expressing allogeneic melanoma cell lines but was not reactive with normal melanocytes. Here, we report the full molecular characterization of the target structure for CTL 1/29, which was identified by cDNA expression cloning. The recognized antigen was named CAMEL (CTL-recognized antigen on melanoma). The CAMEL cDNA turned out to be derived from the LAGE-1 gene, a recently described tumor antigen that is strongly homologous to NY-ESO-1. CAMEL, however, is not encoded by the putative open reading frame (ORF) of LAGE-1 but by an alternative frame starting from the second ATG of the mRNA. The first 11 amino acids of the CAMEL protein, MLMAQEALAFL, constitute the epitope of CTL 1/29. This epitope is also encoded by a similar alternative ORF in NY-ESO-1. In summary, CTL induction with IL-2-transfected melanoma cells has revealed a new tumor antigen that may serve as a target for immunotherapy.     

A phase I trial of tumor lysate-pulsed dendritic cells in the treatment of advanced cancer.

PURPOSE: The objectives of this study were to assess the toxicity and immunological response induced by the intradermal (i.d) administration of tumor lysate-pulsed dendritic cells (DCs). EXPERIMENTAL DESIGN: Patients with stage IV solid malignancies were treated in cohorts that received 10(6), 10(7), and 10(8) DCs i.d. every 2 weeks for three vaccines. Each vaccine was composed of a mixture of half DCs pulsed with autologous tumor lysate and the other half with keyhole limpet hemocyanin (KLH). Peripheral blood mononuclear cells (PBMCs) harvested 1 month after the last immunization was compared with pretreatment PBMCs for immunological response. Delayed-type hypersensitivity reactivity to tumor antigen and KLH was also assessed. RESULTS: Fourteen patients received all three vaccines and were evaluable for toxicity and/or immunological monitoring. There were no grade 3 or 4 toxicities associated with the vaccines or major evidence of autoimmunity. Local accumulation of CD4(+) and CD8(+) T cells were found at the vaccination sites. There was a significant proliferative response of PBMCs to KLH induced by the vaccine. In 5 of 6 patients, the vaccine resulted in increased IFN-gamma production by PBMCs to KLH in an ELISPOT assay. Using the same assay, 3 of 7 patients' PBMCs displayed increased IFN-gamma production in response to autologous tumor lysate. One patient with melanoma also was observed to have an increased frequency of MART-1- and gp100-reactive CD8(+) T cells after vaccination. By delayed-type hypersensitivity testing, 8 of 9 and 4 of 10 patients demonstrated reactivity to KLH and autologous tumor, respectively. Two patients with melanoma experienced a partial and a minor response, respectively. CONCLUSION: The administration of tumor lysate-pulsed DCs is nontoxic and capable of inducing immunological response to tumor antigen. Additional studies are necessary to improve tumor rejection responses.     

Cytotoxic T-cell clone against rectal carcinoma induced by stimulation of a patient's peripheral blood mononuclear cells with autologous cultured tumor cells

In an effort to establish cytolytic T lymphocytes (CTLs) against colorectal carcinoma (CRC) by stimulating patients' lymphocytes with autologous tumor cells, we used peripheral blood mononuclear cells (PBMC) from a patient with minimal residual rectal carcinoma following removal of the primary lesion and involved regional lymph nodes as a source to generate CTLs in culture. A CTL line and clone were established from the patient's PBMC following stimulation of PBMC with autologous, cultured tumor cells and interleukin-2. The CTL line and the clone consisted predominantly of CD4⁺ lymphocytes. The CTL clone expressed two T-cell receptor variable α chains (Vα11 and Vα22) and one β chain (Vβ14). The cytokine secretion pattern of the CTL line was of the Th1-type. Both the CTL line and the clone lysed the autologous rectal carcinoma cells, but not the allogeneic, partially human lymphocyte antigen (HLA)-matched or nonmatched CRC cells, autologous Epstein-Barr virus-transformed B cells, K562 (natural killer target) cells or Daudi (lymphokine-activated killer target) cells. Lysis of autologous tumor cells most likely was HLA class I-restricted. Our unique success in generating CTLs against this tumor type may rest in the inclusion of a patient with minimal residual, rather than advanced, disease. Int. J. Cancer 71:325-332, 1997. © 1997 Wiley-Liss Inc.     

Generation of mammaglobin-A-specific CD4 T cells and identification of candidate CD4 epitopes for breast cancer vaccine strategies

BACKGROUND: Mammaglobin-A (MGB) is a breast cancer-associated antigen that is an attractive target for immune intervention. MGB has been shown to induce a specific CD8 T cell response in breast cancer patients, but little is known about a possible MGB-specific CD4 T cell response. METHODS: Peripheral blood-derived CD4(+)CD25(-) T cells were stimulated in vitro with MGB-pulsed antigen-presenting cells (APC). The MGB and human leukocyte antigen (HLA) class II specificity of the CD4 T cell lines was confirmed by cytokine release following restimulation with autologous and allogenic APC pulsed with MGB from different sources. Candidate HLA class II-restricted epitopes were identified by computer algorithm and validated in cytokine release assays. RESULTS: MGB-specific CD4 T cells were successfully generated in cultures from six of seven donors. Restimulation of MGB-specific CD4 T cells with MGB-pulsed APC induced significantly higher levels of interferon (IFN)-gamma release than APC pulsed with an irrelevant protein (P = 0.0004). Cultures from five of seven donors showed a pure Th1 type response as evidenced by the absence of interleukin (IL)-4. MGB-specific CD4 T cells recognized both recombinant and naturally processed MGB presented by APC. This recognition was HLA class II-restricted, as HLA-DR mismatched APC were not recognized. MGB-specific CD4 T cells from three of four donors recognized MGB-derived, HLA class II-restricted peptides pulsed onto APC. CONCLUSIONS: We have successfully generated MGB-specific CD4 T cell cultures and identified candidate MGB HLA class II epitopes. These studies should facilitate study of the CD4 T cell response to MGB, and the development and monitoring of vaccine strategies targeting this unique antigen.     

Generation of tumour-specific cytotoxic T-cell clones from histocompatibility leucocyte antigen-identical siblings of patients with melanoma

Lymphodepletion and infusion of autologous expanded tumour-infiltrating lymphocytes is effective therapy for patients with malignant melanoma. Antitumour responses are likely to be mediated by HLA class I- and II-restricted immune responses directed at tumour antigens. We assessed whether the peripheral blood of normal HLA-matched siblings of patients with melanoma could be used to generate lymphocytes with antimelanoma activity for adoptive immunotherapy after allogeneic blood or marrow transplantation. Melanoma cell lines were derived from two donors and were used to stimulate the mononuclear cells of three HLA-identical siblings. CD4(+) clones dominated cultures. Of these, approximately half were directly cytotoxic towards recipient melanoma cells and secreted interferon-gamma in response to tumour stimulation. More than half of the noncytotoxic clones also secreted interferon-gamma after melanoma stimulation. No CD4(+) clones responded to stimulation with recipient haemopoietic cells. The majority of CD8(+) clones directly lysed recipient melanoma, but did not persist in long-term culture in vitro. No crossreactivity with recipient haemopoietic cells was observed. The antigenic target of one CD4(+) clone was determined to be an HLA-DR11-restricted MAGE-3 epitope. Antigenic targets of the remaining clones were not elucidated, but appeared to be restricted through a non-HLA-DR class II molecule. We conclude that the blood of allogeneic HLA-matched sibling donors contains melanoma-reactive lymphocyte precursors directed at tumour-associated antigens. Adoptive immunotherapy with unselected or ex vivo-stimulated donor lymphocytes after allogeneic stem cell transplantation has a rational basis for the treatment of malignant melanoma.     

Characterization of autologous tumor-specific T-helper 2 cells in tumor-infiltrating lymphocytes from a patient with metastatic melanoma

Human autologous tumor-specific T-helper 2 (Th2) cells were investigated in melanoma tumor-infiltrating lymphocytes (TILs). Both a CD4⁺ T-cell line and its 5 potential T-cell clones established from TILs of a patient with metastatic melanoma produced significant levels of IL-4, IL-6, IL-10 and granulocyte-macrophage colony-stimulating factor (GM-CSF) in response to autologous, but not any of 12 allogeneic, melanoma cell lines. They also produced IL-3 and IL-8 but not IL-2, IFN-γ, TNF-α or TNF-β in response to autologous tumor cells. Furthermore, they showed autologous melanoma-specific cytotoxicity only in an 18-hr ⁵¹Cr-release assay. Specific IL-4, IL-6 or IL-10 production by the CD4⁺ M73 T-cell line and its clone was inhibited by anti-class 11 DR (but not anti-class 1) MAb, whereas their specific cytotoxicity was inhibited by anti-class 1 (but not anti-class 11) MAb. Anti-CD3 and -CD4 MAb (but not anti-CD8) abrogated both IL-4, IL6 and IL-10 production and cytotoxicity, while anti-IL-4 antibody did not inhibit cytotoxicity. CD4⁺ potential T-cell clones, but not CD8⁺ clones, that were established from freshly isolated TILs without in vitro sensitization by autologous tumor cells also produced IL-4, IL-6 and IL-10 but not IFN-γ or tumor necrosis factor (TNF)α in an autologous tumor-specific fashion. These Th2 cells were neither reactive to EBV-B cells nor suppressive against CD8⁺ T-cell clones. PMA and PHA stimulated these potential T-cell clones, regardless of their specific lymphokine production, to produce IL-3, IL-4, IL-6, IL-8, IL-10, GM-CSF, TNFα and IFN-γ. Our results demonstrate the presence of autologous tumor-specific Th2 cells at the melanoma sites.     

Superior antitumor in vitro responses of allogeneic matched sibling compared with autologous patient CD8+ T cells

Allogeneic cell therapy as a means to break immunotolerance to solid tumors is increasingly used for cancer treatment. To investigate cellular alloimmune responses in a human tumor model, primary cultures were established from renal cell carcinoma (RCC) tissues of 56 patients. In three patients with stable RCC line and human leukocyte antigen (HLA)-identical sibling donor available, allogeneic and autologous RCC reactivities were compared using mixed lymphocyte/tumor cell cultures (MLTC). Responding lymphocytes were exclusively CD8(+) T cells, whereas CD4(+) T cells or natural killer cells were never observed. Sibling MLTC populations showed higher proliferative and cytolytic antitumor responses compared with their autologous counterparts. The allo-MLTC responders originated from the CD8(+) CD62L(high)(+) peripheral blood subpopulation containing naive precursor and central memory T cells. Limiting dilution cloning failed to establish CTL clones from autologous MLTCs or tumor-infiltrating lymphocytes. In contrast, a broad panel of RCC-reactive CTL clones was expanded from each allogeneic MLTC. These sibling CTL clones either recognized exclusively the original RCC tumor line or cross-reacted with nonmalignant kidney cells of patient origin. A minority of CTL clones also recognized patient-derived hematopoietic cells or other allogeneic tumor targets. The MHC-restricting alleles for RCC-reactive sibling CTL clones included HLA-A2, HLA-A3, HLA-A11, HLA-A24, and HLA-B7. In one sibling donor-RCC pair, strongly proliferative CD3(+)CD16(+)CD57(+) CTL clones with non-HLA-restricted antitumor reactivity were established. Our results show superior tumor-reactive CD8 responses of matched allogeneic compared with autologous T cells. These data encourage the generation of antitumor T-cell products from HLA-identical siblings and their potential use in adoptive immunotherapy of metastatic RCC patients.