Inhibition of cytolytic T lymphocyte proliferation by autologous CD4+/CD25+ regulatory T cells in a colorectal carcinoma patient is mediated by transforming growth factor-beta

Cancer patients often develop CTLs that lyse autologous tumor cells in culture. However, tumors can progress in vivo despite the presence of CTLs. Various mechanisms have been reported to down-modulate CTL functions. In this study, the role of CD4+/CD25+ regulatory T cells in CTL induction and proliferation of established CTLs was investigated in a patient with CRC. CD4+ cytotoxic and regulatory T-cell lines were derived from the peripheral blood mononuclear cells of the same patient in mixed-lymphocyte tumor culture. The cytotoxic T-cell line and a clonal derivative specifically lysed the autologous tumor cells but not the B lymphocytes. Only HLA-A1-matched allogeneic CRC cells were lysed by the CTL clone. The clone produced IFN-gamma and TNF-alpha. The regulatory CD4+/CD25+ T-cell line was tumor cell-dependent in its growth but did not lyse autologous tumor cells. This T-cell line suppressed pokeweed mitogen responses of allogeneic lymphocytes, proliferative activity of the established, autologous CTLs, and induction of CTLs in autologous, freshly isolated peripheral blood mononuclear cells. The immunosuppressive effect of the CD4+/CD25+ regulatory T cells was mediated by transforming growth factor-beta and did not require cell-to-cell contact. Thus, although CRC patients can develop specific CTLs against their tumors, the development of regulatory T cells may allow the escape of tumor cells from immune surveillance by the CTLs in vivo.     

Induction of multiple myeloma-specific cytotoxic T lymphocyte stimulation by dendritic cell pulsing with purified and optimized myeloma cell lysates

We investigated the possibility of immunotherapy for multiple myeloma (MM) using myeloma-specific cytotoxic T lymphocytes (CTLs) that were stimulated in vitro by dendritic cells (DCs) pulsing with purified and optimized myeloma lysates. CD14(+) cells were cultured in the presence of GM-CSF and IL-4. On day 6, the immature DCs were pulsed with the purified myeloma cell lysates, and then maturation of the DCs was induced by the addition of a cytokine cocktail. There were no differences in the phenotypic expressions of mature DCs that were generated by pulsing with CD138(+) cell lysates or total cell lysates. In optimization of the concentration of myeloma lysates, DCs pulsed with 10 microg/mL of myeloma lysate had greater allogeneic T-cell stimulatory capacities than those pulsed with higher concentrations of myeloma lysates. The CTL lines generated by DCs pulsed with myeloma lysates demonstrated potent cytotoxic activities against autologous target cells, but not against HLA-A2(-) cell lines or K562 cell lines. The DCs pulsed with myeloma lysates demonstrated a higher stimulatory capacity for autologous CTL compared with mature nonpulsed DCs. These results suggested that the DCs pulsed with purified and optimized myeloma cell lysates could generate potent myeloma-specific CTLs for approaches in MM.     

Generation of NY-ESO-1-specific CD4+ and CD8+ T cells by a single peptide with dual MHC class I and class II specificities: a new strategy for vaccine design

The existence of overlapping CD8+ and CD4+ T-cell epitopes within certain tumor antigens provides an opportunity to test the hypothesis that relatively short peptides could be used to generate both CD8+ and CD4+ T cells against tumor. In this report, T-cell responses to a fragment of the tumor antigen NY-ESO-1 that contained an HLA-DP4-restricted helper T cell epitope as well as an HLA-A2-restricted cytotoxic T cell epitope were analyzed. One peptide, ESO:157-170 (SLLMWITQCFLPVF) was recognized by both NY-ESO-1-reactive CD8+ and CD4+ T-cell clones. Both CD4+ and CD8+ T cells were efficiently generated from the peripheral blood of multiple melanoma patients after in vitro stimulations using ESO:157-170. Dual-specific peptides containing both cytotoxic T-cell and helper T-cell epitopes may represent an attractive strategy of vaccine design aimed at generating tumor-reactive CD4+ and CD8+ T cells.     

Induction and clonal expansion of tumor-specific cytotoxic T lymphocytes from renal cell carcinoma patients after stimulation with autologous dendritic cells loaded with tumor cells

Melanoma and renal cell carcinoma (RCC) are considered to be the most immunogenic tumors in humans. To generate conditions to induce primary T-cell responses against RCC and to allow further expansion of tumor-specific cytotoxic T lymphocytes (CTL) for adoptive transfer, peripheral blood mononuclear cells from RCC patients were stimulated with primary autologous tumor cells or monocyte-derived dendritic cells (DC) loaded with either tumor lysate (TU-LY) or apoptotic tumor cells (TU-AP). Whereas repetitive stimulation (4x) with tumor cells alone induced a predominant population of CD3(-) natural killer cells, 4 weeks of stimulation with tumor-loaded DC favored induction and expansion of CD4+ T cells (>80%). However, 2 weekly stimulation cycles with tumor-loaded DC followed by restimulation with autologous irradiated tumor cells alone were optimal for induction of tumor-specific CTL responses in vitro. Using these culture conditions a marked increase of CD4+ T cells was observed during the first 2 weeks of stimulation with tumor-loaded DC. Subsequent restimulation with autologous tumor cells alone gave rise to 500-fold expansion of CD8+ T cells. These CD8+ T cells were shown to exhibit strong major histocompatibility complex class I-restricted cytotoxic activity against the autologous tumor. Comparison of TU-LY and TU-AP as a source of tumor antigen for loading DC did not show any difference in stimulating tumor-specific CTL. Length pattern analysis of the complementary determining region 3 (CDR3) of the T-cell receptor Vbeta chain revealed expansion of oligoclonal CTL populations with outgrowth of 1 or 2 clones after prolonged stimulation with autologous tumor cells. Our study demonstrated an efficient method for generating tumor-specific CTL in vitro that may be used to identify tumor cell antigens or that can be expanded for adoptive T-cell transfer in tumor immunotherapy.     

Glioblastoma patients exhibit circulating tumor-specific CD8+ T cells.

PURPOSE: There is growing interest in developing cellular immune therapies for glioblastoma multiforme, but little is known about tumor-specific T-cell responses. A glioblastoma multiforme-specific T-cell assay was developed using monocyte-derived dendritic cells to present tumor antigens from the established glioblastoma multiforme cell line U118. EXPERIMENTAL DESIGN: Peripheral blood mononuclear cells (PBMC) and tumor cells were obtained from nine patients with newly diagnosed brain tumors: five glioblastoma multiforme, two oligodendroglioma, one ependymoma, and one astrocytoma. PBMCs were incubated overnight with autologous tumor cells or autologous dendritic cells loaded with a U118 cell lysate, and responses were detected by IFN-gamma ELISPOT and cytokine flow cytometry assays. RESULTS: PBMCs from all glioblastoma multiforme patients exhibited IFN-gamma responses to autologous tumor but not to HLA-mismatched U118 cells. Glioblastoma multiforme-specific IFN-gamma responses were primarily mediated by CD8+ T cells and represented approximately 2% of total CD8+ T cells. Additionally, all glioblastoma multiforme patients responded to autologous dendritic cells loaded with U118 lysate but not with low-grade astrocytoma cell lysates. PBMCs from four patients with other brain tumor types and one normal donor failed to respond to U118 lysate-loaded autologous dendritic cells. These data indicate that the IFN-gamma responses to U118 lysate-loaded autologous dendritic cells are glioblastoma multiforme specific. Moreover, PBMCs stimulated 1 to 2 weeks with U118 lysate-loaded dendritic cells exhibited MHC class I-restricted cytotoxicity against autologous tumor cells. CONCLUSIONS: Glioblastoma multiforme patients exhibit circulating tumor-specific CD8+ T cells that recognize shared tumor antigens from the glioblastoma multiforme cell line U118. These data show that glioblastoma multiformes are immunogenic and support the development of immunotherapy trials.     

An analysis of autologous T-cell anti-tumour responses in colon-carcinoma patients following active specific immunization (ASI).

As part of a phase-II clinical trial of post-operative active specific immunization (ASI) with virus-modified autologous tumour cells (AuTu) in colorectal carcinoma patients, we have analyzed in vitro anti-AuTu immune responses with lymphocytes isolated from the peripheral blood (PBL) of 5 treated patients. The PBL of 3 "responder patients", those who developed a positive DTH reaction to AuTu, when stimulated in standard in vitro autologous lymphocyte tumour-cell cultures (ALTC), showed cytotoxic anti-AuTu reactivity only in association with natural-killer-cell(NK)-like activity. We removed nonspecific cytotoxic cells (CD56-positive) from PBL of colon carcinoma or melanoma patients and positively selected T cells with strong CD8 staining (CD8hi) using FACS. Following in vitro stimulation, specific cytotoxic T cells (CTL) directed against either autologous EBV-transformed B cells (AuEBV-B) or autologous melanoma cells were identified in the CD8hi T-cell population. However, even using this novel technique, no specific CTL against autologous colon carcinoma cell lines were detected in PBL from ASI-treated patients (2 DTH responders and 2 DTH non-responders). If AuTu-specific CTL precursors existed in these blood samples, their frequency must have been very low (less than 1 in 8 x 10(4) CD8 positive T cells). Sorted CD4 T cells from these patients, in the presence of autologous antigen-presenting cells, showed no specific anti-tumour proliferative response, and in one instance we observed inhibition of proliferation in the presence of tumour cells.     

Tumor cytolysis by lymphocytes infiltrating ovarian malignant ascites

Tumor-associated lymphocytes (TAL) were isolated from the ascitic fluid of patients with adenocarcinoma of the ovary. These cells proliferated and expanded by 100-600-fold as either CD3+ CD4+ or CD3+ CD8+ cultures in the presence of moderate concentrations (50-200 cetus units/ml) of recombinant interleukin 2 and reached high numbers (5 x 10(8)-1 x 10(9)). After expansion of 16 TAL samples from 15 patients, 5 of the 7 isolated ovarian cytotoxic T-lymphocyte cell lines of T-cell receptor (TCR) (alpha beta)+ CD3+ CD8+ CD4- phenotype exhibited preferential cytolytic activity against autologous tumor targets and significantly lower cytolytic activity against allogeneic tumor targets and the natural killer-sensitive cell line K562. The cytolytic activity of the CD8+ TAL was inhibited by operationally anti-TCR (alpha beta) monoclonal antibody and monoclonal antibody specific for the CD3 differentiation antigen, indicating that the TCR and CD3 are involved in the cytolytic process. The other TAL cultures demonstrated similar cytolytic activity against both autologous and allogeneic tumors. The phenotype of these TAL was predominantly TCR (alpha beta)+ CD3+ CD4+ CD8-. Certain CD3+ CD8+ T-cell clones isolated from representative TAL exhibited preferential autologous tumor-specific cytotoxicity that may be major histocompatibility complex restricted. Other CD3+ CD8+ and CD3+ CD4+ clones exhibited nonmajor histocompatibility complex restricted cytotoxicity. These results demonstrate that CD3+ CD4+ and CD3+ CD8+ T-cells present in the ovarian malignant ascites can be propagated in large numbers and for long time intervals as T-cell lines in vitro. This finding may be significant for further investigation of ovarian tumor-specific cytotoxic T-lymphocytes and future adoptive specific immunotherapy studies.     

Direct Activation of Human CD8+ Cytotoxic T Lymphocytes by Interleukin-18

Direct activation of human cytotoxic T lymphocytes (CTL) by interleukin (IL)-18 was observed in a system in which CTL effective against autologous tumor cells were generated. Peripheral blood mononuclear cells (PBMC) from tumor-bearing patients, after removal of natural killer (NK) cells, were cultured in a medium containing IL-1, -2, -4, and -6, with or without IL-18, and stimulated with autologous tumor cells. IL-18 increased the activity of the CTL and the proportion of autologous CD8⁺ T cells present after 28 days in the induction culture. When purified CD8⁺ T cells were cultured in the presence of IL-18 and IL-2 for 7 days, the CTL showed enhanced cytotoxic activity against autologous tumor cells. Moreover, a purified CD8⁺ T cell population, which did not exhibit any apparent cytotoxic activity against autologous tumor cells, displayed cytotoxic activity after 7-day incubation with IL-18. These results suggest that IL-18 may be useful to generate autologous CTL in humans and may thereby contribute to adoptive immunotherapy for tumors.     

CD4+ T helper responses in squamous cell carcinoma of the head and neck

Anti-tumor immunity plays an important role in the development of and protection from malignancy. However, there is a lack of information regarding induction of CD4+ T helper responses in patients with squamous cell carcinoma (SCCHN). To explore anti-tumor immune responses against SCCHN, a permanent cell line, Gun-1 was established from a squamous cell carcinoma of the hypopharynx. In addition to its characterization, we performed mixed lymphocyte-tumor cell cultures (MLTC) using peripheral blood lymphocytes and autologous tumor cells. Furthermore, T cell responses to wild type (wt) p53-derived peptides were assessed. Gun-1 cells overexpressed p53 and were negative for HLA-A2 expression. No tumor-specific or wt p53-specific CD8+ CTL lines could be established from peripheral blood mononuclear cells (PBMCs) of this patient. Autologous tumor-specific HLA-DR-restricted CD4+ T helper clone was obtained by limiting dilutions using bulk populations from MLTC. This clone produced IFN-gamma but not IL-5 in response to autologous tumor cells. In addition, CD4+ T cells were generated from the patient's PBMCs which responded to two HLA-DP5-restricted wt p53-derived peptides. Our results suggest that the immune cells specific for autologous tumor as well as wt p53-derived epitopes are present in the peripheral circulation of this cancer patient. However, helper-type CD4+ T lymphocytes represent the predominant anti-tumor response.     

Identification of known and novel immunogenic T-cell epitopes from tumor antigens recognized by peripheral blood T cells from patients responding to IL-2-based treatment

In previous studies CD8+ T cells specific for melanocyte antigens have been frequently found in melanoma patients responding to interleukin-2 (IL-2)-based therapies. In our study we analyzed the suitability of using circulating T cells from melanoma patients with clinical response after IL-2-based therapy to identify novel T-cell epitopes from defined tumor antigens. Using unstimulated peripheral blood mononuclear cells and the interferon-gamma (IFN-gamma) ELISPOT assay, we studied CD8(+) T-cell responses against 5 peptides from the tumor antigen tyrosinase (Tyr) selected by epitope prediction using an HLA-A1-binding computer algorithm. T cells specifically secreting IFN-gamma in response to 3 of these 5 peptides, namely, Tyr (454-463), Tyr (146-156) and Tyr (243-251), could be detected in 4 of 4 HLA-A1-positive patients with clinical response. In contrast, no T-cell responses against these peptides were seen in 6 HLA-A1-positive melanoma patients with progressive disease and in 8 healthy subjects. We could generate specific cytotoxic T lymphocytes (CTL) against Tyr (454-463) using peptide-pulsed autologous dendritic cells as antigen-presenting cells. The induced CTLs efficiently killed melanoma cells that express HLA-A1 and tyrosinase. The peptides Tyr (146-156) and Tyr (243-251) had recently been identified as CTL epitopes by other groups. Further ex vivo characterization of the T cells reactive against the novel epitope Tyr (454-463) in 1 patient by multicolor flow cytometry showed specific CD3+/CD8+/IFN-gamma+ T cells with frequencies of up to 0.41% of the CD3+/CD8+ T-cell population. Most of this T-cell population also expressed granzyme B. Our data confirm that in patients with tumor regressions induced by immunotherapy or chemoimmunotherapy circulating T cells reactive with tyrosinase epitopes can frequently be detected. Peripheral blood T cells from such patients are a valuable source for screening peptides selected by epitope prediction This strategy facilitates the rapid identification of immunogenic T-cell epitopes that are probable targets of immune-mediated tumor rejection.     

Interleukin 1 beta synergises with interleukin 2 in the outgrowth of autologous tumour-reactive CD8+ effectors.

Using peritoneal fluid or pleural effusion obtained from 20 patients with lung, ovarian or metastatic breast cancer, we separated tumour cells from malignant effusion-associated mononuclear cells (MEMNCs) using discontinuous Ficoll-Hypaque density gradients. CD3+ T lymphocytes represented the main population of MEMNCs. The mean +/- s.d. CD4/CD8 ratio of MEMNC suspensions was 1.18 +/- 0.40. MEMNCs proliferated and expanded in vitro with human interleukin 2 (IL-2) either as CD3+ CD8+ cells or as CD3+ CD4+ cells or as mixed populations of CD8+ and CD4+ cells. Preferential cytolytic activity against autologous tumour cells was demonstrated in IL-2-activated MEMNC cultures with excess CD3+ CD8+ cells. In contrast, effectors derived from IL-2-activated cultures with excess CD3+ CD4+ cells lysed both autologous and allogeneic tumour target cells. The addition on day 0 of interleukin 1 beta (IL-1 beta) to MEMNCs cultured in the presence of IL-2 was effective in promoting the growth of CD3+ CD8+ cells and augmenting the cytotoxicity against autologous tumour. Simultaneously, the production of gamma-interferon (IFN-gamma) was increased in these cultures. This is the first report suggesting that IL-1 beta synergises with IL-2 to induce autologous tumour-specific CD8+ cytotoxic T lymphocytes (CTLs) within the MEMNC population. Selective enrichment in T-cell subsets by IL-1 beta may be useful in cellular adoptive immunotherapy using cells isolated from malignant effusions.     

High frequencies of circulating melanoma-reactive CD8+ T cells in patients with advanced melanoma

To determine whether circulating tumor-reactive T cells are present in melanoma patients, unstimulated T cells from peripheral blood were tested for recognition of HLA-A2- or HLA-A1-matched melanoma cell lines using the ELISPOT assay. Eleven out of 19 patients with metastatic melanoma had a T-cell response with up to 0.81%, 0.78%, 0. 53%, 0.12%, 0.10%, 0.09%, 0.07%, 0.06%, 0.06%, 0.04%, and 0.04% of peripheral blood mononuclear cells (PBMC) secreting IFNgamma upon exposure to various HLA-A2- or HLA-A1-matched melanoma cell lines. These T-cell responses were mediated by CD8+ T cells and could specifically be blocked by an anti-HLA-A2 antibody in HLA-A2-positive patients. Separation experiments performed in one melanoma patient showed tumor-reactive T cells in both the CD8+ effector T cell (CD45RA+/IFNgamma+) as well as the CD8+ memory T-cell compartment (CD45RO+/IFNgamma+). In 3 out of 5 patients, in whom autologous cell lines were available, similar frequencies of T cells in response to HLA-A1- or HLA-A2-matched allogeneic and autologous tumor cells were observed, while 2 patients had a T-cell response restricted to either the autologous or the allogeneic cell lines. These results give evidence for the presence of tumor-reactive CD8+ T cells in more than half of melanoma patients tested. Although some of these patients have clinical evidence for an immunological-mediated tumor control, several patients have growing tumors suggesting presence of escape mechanisms.     

Tumor lysate and IL-18 loaded dendritic cells elicits Th1 response,tumor-specific CD8+ cytotoxic T cells in patients with malignant glioma

In this study, we demonstrate that tumor lysate-loaded dendritic cells can elicit a specific CD8+ cytotoxic T lymphocyte response against autologous tumor cells in patients with malignant glioma. CTL from three of five patients expressed strong cytolytic activity against autologous glioma cells, did not lyse autologous lymphoblasts and were variably cytotoxic against the LAK-sensitive cell line Daudi. Also, DCs pulsed normal brain lysate failed to induce cytolytic activity against autologous glioma cells, suggesting the lack of autoimmune response. Two of five patients CD8+ T cells expressed a modest cytotoxicity against autologous glioma cells. CD8+ T cells isolated during these ineffective primings secreted large amounts of IL-10, less amounts of IFN-γ as detected by ELISA, Type 2 bias in the CD8+ T cell response accounts for the lack of cytotoxic effector function from these patients. Cytotoxicity against autologous glioma cells could be significantly inhibited by anti-HLA class I antibody. These data demonstrate that tumor lysate-loaded DC can be an effective tool in inducing glioma-specific CD8+ CTL able to kill autologous glioma cells in vitro. However, high levels of tumor specific tolerance in some patients may account for a significant barrier to therapeutic vaccination. Moreover, cytotoxic responses were augmented by transfecting DC with the gene for IL-18. For all five patients, CD8+T cells treated with IL18 transfected DC produced Th1 response. These results may have important implications for the treatment of malignant glioma patients with immunotherapy. DCs loaded with total tumor lysate and IL-18 may represent a method for inducing Th1 immunoresponses against the entire repertoire of glioma antigens.     

CD3xCD19 bispecific antibodies and CD28 bivalent antibodies enhance T-cell reactivity against autologous leukemic cells in pediatric B-ALL bone marrow

Bispecific CD3xCD19 antibodies and CD28 co-stimulating antibodies were used to activate T cells in bone marrow aspirates (n = 8) of children with B cell-derived acute lymphoblastic leukemia. Bone marrow specimens were incubated for 10 days with CD3xCD19 bispecific and CD28 antibodies. Changes in the numbers of T lymphocytes and tumoral B cells as well as surface expression of T cell-activation markers were determined by flow cytometry, and cytokines (human IFN-gamma, IL-2, IL-4 and IL-12) were measured in the cell culture supernatant. In 7 of 8 bone marrow samples, an increase in the number of CD4- and CD8-positive T lymphocytes was found, which correlated with an up-regulation of T cell-activation markers. Additionally, we demonstrated a decrease of tumoral B cells in 3 samples and enhanced cytotoxic T-cell activity against autologous malignant B cells. ELISpot analyses in an autologous Epstein-Barr virus model showed that bispecific antibodies (CD3xCD19+CD28) were more potent at generating T-cell responses against autologous and allogeneic tumoral targets than a combination of monospecific antibodies (CD3+CD28). Thus, T-cell targeting by CD3xCD19 bispecific and CD28 antibodies may be used to eliminate leukemic B cells ex vivo and reconstitute immunological control of residual malignant disease by the induction of anti-tumoral T-cell responses.     

Naturally occurring human lymphocyte antigen-A2 restricted CD8+ T-cell response to the cancer testis antigen NY-ESO-1 in melanoma patients

Cancer testis (CT) antigens are particularly interesting candidates for cancer vaccines. However, T-cell reactivity to CT antigens has been detected only occasionally in cancer patients, even after vaccination. A new group of CT antigens has been recently identified using the SEREX technique based on immunoscreening of tumor cDNA expression libraries with autologous sera. We have used fluorescent HLA-A2/peptide tetramers containing an optimized antigenic peptide to directly identify HLA-A2-restricted CD8+ T cells specific for the SEREX-defined CT antigen NY-ESO-1 in melanoma patients. High frequencies of NY-ESO-1-specific CD8+ T cells were readily detected in peptide-stimulated peripheral blood mononuclear cells as well as in lymphocytes infiltrating melanoma lesions from patients with measurable antibody responses to NY-ESO-1. NY-ESO-1-specific CD8+ T cells were also detectable in peptide-stimulated peripheral blood mononuclear cells from some seronegative patients. Whereas the frequencies of NY-ESO-1-specific CD8+ T cells in circulating lymphocytes were usually below the limit of detection by tetramer staining, the presence of NY-ESO-1 CD8+ T cells displaying a memory phenotype was clearly detectable ex vivo in blood from a seropositive patient over an extended period of time. These results indicate that sustained CD8+ T-cell responses to CT antigens can naturally occur both locally and systemically in melanoma patients.     

Human cytotoxic T cells specific for autologous melanoma cells: successful generation from lymph node cells in seven consecutive cases

Human T-cell populations specifically cytotoxic for autologous melanoma cells have been successfully generated from lymph node cells obtained from seven consecutive patients. The lymph node cells were stimulated in vitro with autologous irradiated melanoma cells; stimulation was repeated every 10-15 days at a tumor cell-to-lymphocyte ratio of approximately 1:20. Cytotoxic activity was assessed by a 4-hour 51Cr release assay. Mean lysis of autologous tumor cells was 47% at an effector-to-target cell ratio of 20:1, while mean lyses of the human myeloid leukemia cell line K562, allogeneic melanoma cells, and an osteosarcoma cell were 20%, 13%, and 11%, respectively. There was no lysis of autologous fibroblasts, fresh lymphocytes, or phytohemagglutinin-stimulated blasts. Three grades of specificity developed sequentially. In grade I, lysis of autologous tumor cells exceeded lysis of allogeneic tumor cells but did not exceed lysis of K562 cells. In grade II, lysis of autologous tumor cells exceeded lysis of K562 cells and all allogeneic tumor cells tested. In grade III, potent lysis of autologous tumor cells (greater than 40%) exceeded lysis of K562 cells and of all allogeneic tumor cells tested. All seven lymphocyte populations reached or exceeded grade I. Six reached or exceeded grade II. Two progressed to grade III. The generated cells were T cells, as determined by phenotypic analysis with flow cytometry. CD4+ cells and CD8+ cells accounted for 83%-100% of the cells. CD8+ T cells were separated from CD4+ T cells by panning with OKT8 and OKT4 antibodies. The resulting CD8-enriched and CD4-enriched populations were compared as effectors in cytotoxicity assays. The results suggest that the cell responsible for lysis of autologous tumor cells is CD8+. The methods used in this study have repeatedly resulted in the successful generation of cytotoxic T lymphocytes specifically cytotoxic for autologous melanoma cells; it is suggested that these cells have potential application for adoptive immunotherapy of melanoma.     

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.     

In vitro induction of myeloid leukemia-specific CD4 and CD8 T cells by CD40 ligand-activated B cells gene modified to express primary granule proteins.

The primary granule proteins (PGP) of myeloid cells are a source of multiple antigens with immunotherapeutic potential for myeloid leukemias. Therefore, we developed a method to induce T-cell responses to PGP protein sequences. We found that gene-transfected antigen-presenting cells efficiently expand functionally competent PGP-specific CD4 and CD8 T cells. The system was optimized using T-cell responses to autologous CD40-activated B cells (CD40-B) transfected with a cytomegalovirus pp65-encoding expression vector. To generate leukemia-specific T cells, expression vectors encoding the PGP proteinase 3 (PR3), human neutrophil elastase, and cathepsin-G were transfected into CD40-B cells to stimulate post-allogeneic stem cell transplantation T cells from five patients with myeloid and three with lymphoid leukemias. T-cell responses to PGP proteinase 3 and human neutrophil elastase were observed in CD8+ and CD4+ T cells only in patients with myeloid leukemias. T-cell responses against cathepsin-G occurred in both myeloid and lymphoblastic leukemias. T cells from a patient with chronic myelogenous leukemia (CML) and from a posttransplant CML patient, expanded against PGP, produced IFN-gamma or were cytotoxic to the patient's CML cells, demonstrating specific antileukemic efficacy. This study emphasizes the clinical potential of PGP for expansion and adoptive transfer of polyclonal leukemia antigen-specific T cells to treat leukemia.     

T Cell Response to Embryonal Carcinoma F9 Cells: Induction and Characterization of T Cell Receptor αβ+ Double-negative Cytotoxic T Lymphocytes

We investigated the mechanism of T cell response to marine embryonal carcinoma F9 cells. Thy-1⁺, CD4⁻, CD8⁻ (double-negative) cytotoxic effector cells were induced in spleen cells obtained from immune A.BY mice to F9 cells, and the cytotoxic activity was major histocompatibility complex (MHC)-unrestricted. Furthermore, CD4⁺ T cells were essential for the induction of double-negative cytotoxic T lymphocytes directed to F9 cells. Most of the double-negative cytotoxic T lymphocyte lines obtained by long-term culture of the effector cells had CD3 molecule and T-cell receptor β chain on their cell surface, and the CDS molecule was found to be involved in target cell recognition. The T cell receptor αβ⁺ double-negative cytotoxic T lymphocyte line (2A5) also lysed various tumor cells in a non-MHC-restricted manner, but did not lyse concanavalin A-stimulated blasts of 129 strain, from which F9 cells had originated. These results indicate that T cell receptor αβ⁺ double-negative cytotoxic T lymphocytes induced by F9 cells recognize a common antigen(s) expressed on F9 cells and other tumor cells but not minor histocompatibility antigens.