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.     

OK-432 develops CTL and LAK activity in mononuclear cells from regional lymph nodes of lung cancer patients

We examined the effect of OK-432 on induction of cytotoxic T lymphocytes (CTL) directed against autologous tumor cells (ATC) and lymphokine-activated killer (LAK) cells from mononuclear cells separated from regional lymph node cells (RLMNCs) of 49 lung cancer patients. We also examined the phenotypic changes of RLMNCs during incubation with or without OK-432. Significant CTL activity and LAK activity against ATC developed from RLMNCs after stimulation with OK-432 or IL-2. Sequential treatment with OK-432 plus IL-2 or IL-2 plus OK-432 also developed significant CTL activity and LAK activity from RLMNCs. The CTL activity produced by OK-432 alone was as high as the CTL activity developed by IL-2 alone, OK-432 plus IL-2, or IL-2 plus OK-432. There was no significant difference in the CTL activities achieved by these four treatments. The proportion of CD25+ cells in RLMNCs after incubation with OK-432 was twice that before incubation. Although OK-432 increased IL-2 receptor expression on RLMNCs, it showed no synergistic effect with IL-2 in developing CTL and LAK activity. After incubation with OK-432, the proportion of HLA-DR + cells was also increased significantly. Moreover, the proportions of HLA-ABC+ and HLA-DR+ (class I and class II major histocompatibility complex antigens) cells in ATC were significantly larger than in Daudi cells. OK-432 alone could develop CTL activity against ATC from the RLMNCs of lung cancer patients that was as high as that developed by IL-2 alone or by sequential treatment with OK-432 plus IL-2 or IL-2 plus OK-432. The CTL developed from the RLMNCs of lung cancer patients may recognize class I and/or II antigens on the surface of ATC. These results indicated that treatment with OK-432 might be therapeutically useful for lung cancer patients as a CTL inducer rather than a LAK inducer.     

Histocompatibility leukocyte antigen-A2-restricted and tumor-specific cytotoxic T lymphocytes from tumor-infiltrating lymphocytes of patient with testicular embryonal cancer

T lymphocytes play an important role in tumor rejection. To understand T cell-mediated specific immunity at the tumor site of testicular embryonal cancer, we investigated whether interleukin-2 (IL-2)-activated tumor-infiltrating lymphocytes (TIL) of a patient with testicular embryonal cancer show histocompatibility leukocyte antigen (HLA)-class I-restricted and tumor-specific cytotoxicity. We established a CD3+CD4-CD8+ cytotoxic T lymphocyte (CTL) line from the IL-2-activated TIL of a 37-year-old patient with testicular embryonal cancer. A 6 h 51Cr-release assay was performed to measure the cytotoxicity of the CTL. The CD3+CD4-CD8+ CTL line showed cytotoxicity against HLA-A2+ tumor cells, including freshly isolated autologous tumor cells, adenocarcinoma cell lines from various organs (lung, breast, pancreas, colon and kidney) and squamous cell carcinomas (esophagus and oral cavity). No other cell lines examined, including an autologous tumor cell line and HLA-A2- tumor cell lines, were lysed by this CTL line. These results suggest the existence of HLA-A2-restricted and tumor-specific CTL at the tumor site of testicular embryonal cancer.     

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.     

Factors determining drug residence in skin during transdermal absorption: studies on beta-blocking agents

A high rate of induction (9 of 10 cases) of human autologous cytotoxic T lymphocytes (CTL) was achieved in vitro from peripheral blood mononuclear cells of renal carcinoma patients by applying an interleukin (IL)-cocktail consisting of IL-1, -2, -4, and -6. The CTL specifically lysed their own target carcinoma cells within 24 h but did not kill neighboring autologous normal kidney cells or allogeneic renal cancer cell lines. In the case of TUHR4TKB, for which autologous CTL were not induced, no expression of MHC class-I molecules was observed on the surface of these carcinoma cells, although they were sensitive to autologous natural killer cells. The results imply that adoptive immunotherapy for metastasized renal carcinoma will be feasible with autologous CTL in combination with natural killer cells.     

Differential expression of natural killer cell markers: human versus baboon

The use of baboons as a model for the study of allo- and xenotransplantation has become increasingly important, but there are few studies on the basic immunological responses in baboons that might be relevant for a rejection reaction. In present study, the cell-surface phenotype, cytokine-induced activation and growth, and cytotoxicity of baboon and human natural killer (NK) and lymphokine-activated killer (LAK) cells were compared. A panel of murine monoclonal antibodies specific for human cell-surface markers expressed on lymphocytes was used to compare relevant baboon and human peripheral blood lymphocytes (PBL). Baboon PBL were 52.1+/-2.9% CD8+, 18.5+/-2.2% CD16+, 3.0+/-0.5% CD25+, and 5.5+/-1.8% CD69+. The corresponding proportions in humans were 23.8+/-7.1%, 12.8+/-3.2%, 4.5+/-1.0%, and 2.3+/-1.1%. In contrast to human PBL, less than 1% of baboon lymphocytes expressed CD56, CD57, and CD122 (interleukin [IL]-2Rbeta). Baboon lymphocytes showed NK cytotoxic activity against the human K562 and CEM cell lines, which was comparable to human NK activity. Depletion of baboon CD16+ or CD8+ cells led to dramatic decreases in NK cytotoxicity, and removal of both subsets completely abrogated NK activity. Incubation of baboon lymphocytes with human recombinant IL-2 for 1 week led to the appearance of CD56+ cells (11.3+/-2.8%). Most of the baboon CD56+ cells induced in culture were in S and G2 phases of cell cycle. Both baboon and human IL-2-activated lymphocytes were highly cytotoxic against the human LAK-sensitive cell line Daudi. Depletion of baboon CD8+ but not CD56+ cells significantly decreased LAK activity. These studies revealed differences in the NK system of humans and baboons that should be taken into consideration when analyzing immune responses to allo- and xenotransplantation in baboons.     

Clonal and functional analysis for the augmentation of tumour-infiltrating lymphocytes by interleukin 4

In the adoptive immunotherapy for cancer, the amounts of induced effector cells play a major role in improving therapeutic efficacy. We have already demonstrated that interleukin 4 (IL-4) augments proliferation of tumour-infiltrating lymphocytes (TILs) without altering the cytotoxic activity against autologous tumour cells. The present study is designed to investigate how IL-4 augments TILs by using established TIL clones in terms of IL-2/IL-2 receptor system. CD4+, CD8+ and CD4+ CD8+ (double positive) TIL clones were established from cancer patients. At clonal level, IL-4 augmented the proliferation of IL-2-activated TIL clones irrespective of phenotypes. In order to clarify the mechanism of IL-4 at clonal level, the blocking assay by anti-IL-2 receptor alpha and beta chain and binding assay of IL-2 on the cell surface and the measurement of the internalisation of IL-2 in the cell were performed. It was clarified that IL-4 up-regulated the IL-2 receptor and then augmented the action of IL-2 molecule on the cell surface stimulated by IL-4. Furthermore, binding IL-2 internalised rapidly into the cells. Thus, it is suggested that signal transduction is augmented and proliferation of TILs is enhanced by IL-4 via the action of IL-2/IL-2 receptor system.     

Cryopreservation of teeth. Organizational aspects of a tissue bank for tooth tissues

Squamous cell carcinoma (SCC) is one of the most common cancers in human. SCC, particularly, esophageal and lung SCC are relatively resistant to currently available regimens of chemotherapy or radiation therapy. Therefore, development of a specific immunotherapy using tumor specific cytotoxic T lymphocytes (CTL) would be important to offer other treatment modalities. However, generation of HLA class I-restricted CTL recognizing SCC has been rarely reported. We established the HLA A2601-restricted CTL cell line recognizing a peptide antigen expressed on SCC. This CD4- CD8+ cytotoxic T lymphocyte (KE-4 CTL) cell line was established in a patient with esophageal cancer. The KE-4 CTL recognized a peptide antigen on esohageal and lung SCC in an HLA A2601-restricted manner as evaluated by cytotoxity against a panel of tumor cells, transfection experiments with HLA A2601 cDNA, and reconstitution with eleted peptides. None of normal cells tested was lysed by this CTL. These results suggest the exstence of HLA A2601-restricted CTL precursors recognizing a peptide antigen on SCC in a patient with esophageal cancer.     

Swedish gynecologists'' and general practitioners'' views on the climacteric period: knowledge, attitudes and management strategies

The cytolytic T lymphocyte (CTL) response has often been used to assess the reconstitution of T cell function after allogeneic or autologous bone marrow transplantation (BMT). Less is known, however, about the reconstitution of the CTL response after peripheral blood stem cell transplantation (PBSCT). Therefore, we investigated the CTL response against Epstein-Barr virus (EBV) of patients undergoing autologous PBSCT. CTLs of six patients with relapsed non-Hodgkin's lymphoma and multiple myeloma were established before and at different times after PBSCT by in vitro stimulation of peripheral blood lymphocytes with autologous EBV-transformed lymphoblastoid cell lines (LCLs). The efficiency of T cell priming by LCLs was assessed at the time of initiation of CTL lines; the proliferative response was strongly reduced during the first 4 months and increased 5 months or more following PBSCT. Cytolytic activity was measured after three or four restimulations of CTLs. All patients investigated had a detectable EBV-specific CTL response which was poor during the first weeks after transplantation, accompanied by a strong non-MHC-restricted cytotoxic activity and a high proportion of CD56-positive T cells. Five or more months after PBSCT, a specific CTL response against EBV was seen which was similar to the situation prior to PBSCT, while the unspecific cytotoxic response decreased. Blocking experiments with monoclonal anti-CD3, anti-CD8 or anti-MHC I antibodies resulted in substantial inhibition of autologous LCL lysis, whereas anti-CD4 or anti-MHC II antibodies had no effect. Finally, autologous PHA blasts of a patient with the HLA haplotype A1/9+, B5/8+, Cw4/7+, were loaded with various EBNA-derived nonapeptides known to be presented by HLA B8 or A11, and exposed to autologous, EBV-directed CTLs. Specific lysis by CTLs only occurred with HLA B8-, but not with HLA A11-restricted nonapeptides. This demonstrated the existence of an MHC I-restricted anti-EBV CTL response after PBSCT. Taken together, the results show that the anlaysis of the EBV-directed CTL activity may serve as a surrogate marker to assess the reconstitution of the cellular immune response in patients undergoing autologous PBSCT.     

Potentiation of lysis of leukaemia cells by a bispecific antibody to CD33 and CD16 (Fc gamma RIII) expressed by human natural killer (NK) cells

Bispecific antibodies recognizing tumour-associated antigens and trigger molecules expressed on immune effector cells have been shown to redirect cytotoxicity of several types of peripheral blood cells against relevant tumour targets. Among various effector cells, natural killer (NK) cells appear to play a role in defence against leukaemia. Here we report the successful chemical conjugation of monoclonal antibodies to CD33 and CD16 to create a bispecific antibody (BsAb 251 x 3G8). This bispecific antibody is capable of augmenting the killing of otherwise resistant leukaemia cells by peripheral blood lymphocytes (PBL), purified resting NK (R-NK) cells, and activated NK (A-NK) cells. BsAb 251 x 3G8 may play a role in the therapy of acute myeloid leukaemia (AML) through redirecting the cytotoxic activity of endogenous or adoptively transferred NK cells.     

Progressive loss of IL-2-expandable HIV-1-specific cytotoxic T lymphocytes during asymptomatic HIV infection

In HIV-1 infection, circulating HIV-1-specific cytotoxic T lymphocytes (CTL) exist in different states of activation, including activated cytotoxic cells and memory cells. We report that a subpopulation of HIV-1-specific CTL is capable of clonal expansion upon culture with IL-2 without exogenous antigen. The IL-2-expandable HIV-1-specific CTL precursor frequency was reduced in patients with advancing infection, although HIV-1-specific memory CTL could still be detected by stimulation in vitro with allele-specific HIV-1 peptide. Longitudinal analysis during advancing infection showed a progressive decline in the IL-2-expandable HIV-1-specific CTL precursor (CTLp) frequency without a decline in Epstein-Barr virus (EBV)-specific or allo-specific CTLp frequencies. To address mechanisms that may contribute to the decline in the IL-2-expandable HIV-specific CTL response, the requirements for in vitro generation of HIV-1-specific and EBV-specific effector CTL were examined. In the absence of exogenous IL-2 in limiting dilution, generation of EBV-specific CD8+ effector CTL was dependent upon help from CD4+ cells. CD4+ help for EBV-specific CD8+ CTL was observed in asymptomatic HIV infection but not in advanced infection. In the presence of exogenous IL-2, CD4+ cells could also provide help for the optimal generation of HIV-1 peptide-specific CD8+ CTL, because in vitro depletion of CD4+ cells prior to culture using stimulation with an MHC class I-restricted HIV-1 peptide reduced the peptide-specific CD8+ CTL response. We conclude that there is a decline in the IL-2-expandable HIV-1-specific CTL response during advancing infection. There are a number of possible mechanisms for this decline, including a reduction in CD4+ T cell help for in vivo antigen-activated CD8+ T cells.     

Evaluation of human immunodeficiency virus type 1 (HIV-1)-specific cytotoxic T-lymphocyte responses utilizing B-lymphoblastoid cell lines transduced with the CD4 gene and infected with HIV-1

Analysis of major histocompatibility complex-restricted cytotoxic T lymphocytes (CTL) capable of killing human immunodeficiency virus type 1 (HIV-1)-infected targets is essential for elucidating the basis for HIV-1 disease progression and the potential efficacy of candidate vaccines. The use of primary CD4+ T cells with variable infectivity as targets for such studies has significant limitations, and immortal autologous cells with high levels of CD4 expression that can be consistently infected with HIV-1 would be of much greater utility. Therefore, we transduced Epstein-Barr-virus-transformed B-lymphoblastoid cell lines (LCL) with a retroviral vector, LT4SN, containing the human CD4 gene. Stable LCL in which more than 95% of cells expressed membrane CD4 were obtained. Aliquots were infected with HIV-1, and, after 4 to 7 days, nearly all of the cells contained cytoplasmic gag and produced high levels of p24 antigen. The ability of major histocompatibility complex-restricted CD8+ CTL to lyse such HIV-1-infected CD4-transduced LCL (LCL-CD4HIV-1) was evaluated. These autologous targets were lysed by CTL generated from an HIV-1-uninfected vaccinee over a broad range of effector-to-target ratios. Similarly, the LCL-CD4HIV-1 were efficiently lysed by fresh circulating CTL from HIV-1-infected individuals, as well as by CTL activated by in vitro stimulation. Both HIV-1 env- and gag-specific CTL effectors lysed LCL-CD4HIV-1, consistent with the cellular expression of both HIV-1 genes. The LCL-CD4HIV also functioned as stimulator cells, and thus are capable of amplifying CTL against multiple HIV-1 gene products in HIV-1-infected individuals. The ability to produce HIV-1-susceptible autologous immortalized cell lines that can be employed as target cells should enable a more detailed evaluation of vaccine-induced CTL against both homologous and disparate HIV-1 strains. Furthermore, the use of LCL-CD4HIV-1 should facilitate the analysis of the range of HIV-1 gene products recognized by CTL in seropositive persons.     

Interleukins 4 and 13 upregulate expression of cd44 in human colonic epithelial cell lines

Interleukin 4 (IL-4) inhibits carcinoma cell growth and promotes expression of differentiation-associated products by normal and malignant epithelial cells. The effects of IL-4 and IL-13 on expression of the CD44 transmembrane adhesion receptor were examined in human epithelial cell lines of colonic (HT-29, CaCo-2, DLD-1, T84), breast (MCF-7, ZR75-1) and liver (Hep-G2, PLC/PRF/5) origins as well as mitogen-activated and resting peripheral blood lymphocytes (PBL) and T cell lines (Jurkat, HUT78). Liver and Jurkat cells were negative for CD44. Colonic, breast and HUT78 cells expressed CD44 constitutively and all except DLD-1 and HUT78 also expressed CD44 splice variant (CD44v) epitopes. All cell lines expressed IL-4 receptors, but IL-4 and IL-13 induced upregulation of CD44 only in the colonic cell lines. CD44v was also upregulated, but there was no de novo induction of CD44v in variant-negative cells and no de novo expression of CD44 in the CD44(-) lines. CD44 upregulation in mitogen-activated PBL was not increased by IL-4 and IL-13 and was not inhibited by neutralizing antibodies. Other cytokines tested [interferon gamma (IFN-gamma, tumour necrosis factor alpha (TNF-alpha), transforming growth factor beta1 (TGF-beta1) and IL-6] did not affect CD44 core epitope expression in the cell lines tested.     

Generation and characterization of gp100 peptide-specific NK-T cell clones

MHC-restricted cytotoxic T lymphocytes (CTLs) specific for antigens expressed by malignant cells are important components of immune responses against human cancer. Peripheral blood monocytes of HLA-A2+ healthy donors were used to induce dendritic cells (DCs) by granulocyte-macrophage colony-stimulating factor and interleukin-4 and loaded with a gp100 peptide (YLEPGPVTA). By applying these peptide-loaded DCs, a CTL line that displayed high cytotoxic reactivity with peptide-loaded target cells was generated. A total of 11 gp100 peptide-specific CTL clones were generated from this cell line. Several of these CTL clones were studied in detail. Of particular interest was clone CTL-45, which, contrary to the parental cell line, displayed strong NK activity and, by flow-cytometric analysis, revealed a CD3+, TCR BV17, CD8+ and CD56+ phenotype. This clone was strictly peptide-specific and effectively killed a panel of melanoma cells expressing HLA-A2 and gp100. Tumor-specific T cells with this kind of dual function are potentially of great clinical importance as they have a backup mechanism that may go into action when tumor cells escape specific killing by losing their HLA-class I molecules.     

The HER2/neu-derived peptide p654-662 is a tumor-associated antigen in human pancreatic cancer recognized by cytotoxic T lymphocytes

The protooncogene HER2/neu encodes a 185-kDa transmembrane protein with extensive homology to the epidermal growth factor receptor. It is overexpressed in several human cancers of epithelial origin, such as pancreatic cancer. Previously, we demonstrated that cytotoxic T lymphocytes (CTL) derived from breast, ovarian, and non-small cell lung cancer recognized a peptide derived from HER2/neu. To evaluate whether this HLA-A2-binding peptide is a tumor-associated antigen (TAA) in pancreatic cancer, the ability of HER2/neu-reactive CTL to lyse human pancreatic carcinoma cells was tested. CTL were generated from tumor-associated T lymphocytes from HLA-A2+ HER2/neu+ breast and ovarian cancer patients. All CTL recognized autologous and allogeneic HER2/ neu+ tumor cells in an HLA-A2-restricted fashion. Furthermore, all CTL recognized p654-662 (GP2) derived from HER2/neu. These CTL also recognized HER2/neu+ pancreatic cancer cells in an HLA-A2-restricted fashion. HER2/neu+ HLA-A2- pancreatic cancer were not or only poorly lysed. Repeated stimulation of HLA-A2+ PBL from pancreatic cancer patients using the HER2/neu-derived peptide resulted in specific recognition of this peptide and, more importantly, HER2/neu+ pancreatic tumors in an HLA-A2-restricted fashion. Autologous HLA-A2+ fibroblasts or HLA-A2+ malignant melanoma cells were not recognized. HLA-A2- peptide-stimulated T lymphocytes showed no significant cytotoxicity. These results demonstrate that this HER2/neu-derived peptide is a shared TAA among several adenocarcinomas including pancreatic carcinoma, suggesting a common mechanism of recognition of these human tumors by T lymphocytes. The identification of the HER2/neu-derived peptide GP2 as a TAA in pancreatic cancer provides an opportunity for the design of novel immunotherapy and vaccine strategies.     

Protection by organotellurium compounds against peroxynitrite-mediated oxidation and nitration reactions

Sixteen metastatic breast cancer patients were immunized with a low dose (5 micrograms) of a 16 amino acid MUC1 peptide (GVTSAPDTRPAPGSTA) conjugated to KLH (BP16-KLH) plus DETOX adjuvant and evaluated for antibody titers against MUC1 peptide and KLH and for cytotoxic lymphocyte (CTL) activity using class 1 HLA-matched MUC1-positive tumor targets. All patients generated strong anti-KLH IgG responses. Only 3 patients developed an anti-MUC1 IgG response, which was weak in magnitude. As it is controversial whether human cancer patients generate class-1-restricted CTL against MUC1, we examined anti-MUC1 CTL activity of PBLs following 4 immunizations with BP16-KLH. The generation of MUC1-specific CTLs required only a 6-day in vitro stimulation of patients' T-cells with synthetic MUC1-peptide-pulsed autologous APCs. The assay for CTL activity was a 4 hour 51Cr release from labeled adenocarcinoma target cells. Eleven of the 16 immunized patients were tested for CTL activity using class-1-matched adenocarcinoma target cell lines. Evidence for class-1-restricted killing of MUC1-expressing tumor cell lines was obtained in 7 of these 11 patients.     

Hemoglobin autooxidation/oxidation mechanisms and methemoglobin prevention or reduction processes in the bloodstream. Literature review and outline of autooxidation reaction

The human carcinoembryonic antigen (CEA), which is expressed in several cancer types is a potential target for antigen-specific immunotherapy. In this study, we show that dendritic cells (DC) pulsed with an HLA class I restricted CEA cytotoxic T lymphocyte (CTL) peptide epitope can stimulate T cells to kill CEA peptide loaded T2 target cells as well as CEA expressing tumor lines in the presence of interleukin-7 (IL-7) in an HLA-restricted manner. This has been demonstrated for carcinoma patients as well as healthy donors. The DC-CEA + IL-7 stimulated cultures contained predominantly CD3+CD8+CD56- cells indicative of MHC class I restricted CTL. In addition, DC-CEA + IL-7 stimulated cells showed higher levels of CD69 expression compared with cells stimulated with IL-7 alone, implying an activated phenotype. When the T-cell receptor (TCR) from CTL cultures stimulated with DC-CEA + IL-7 was analyzed, an oligoclonal pattern of expression was found for certain V beta subfamilies compared with the polyclonal patterns shown by IL-7 or phytohemagglutinin stimulated T cells from the same donors. This TCR restriction appeared to be maintained and enhanced after additional rounds of restimulation with DC-CEA + IL-7. The association between cytotoxicity and TCR restriction suggests that TCR analysis may be useful as an in vitro indicator to monitor alterations in the T-cell population in response to antigen-specific immunotherapies.     

Lysis of CD4+ T cells expressing HIV-1 gag peptides by gag-specific CD8+ cytotoxic T cells

The vast majority of in vitro experiments testing the cytotoxic T lymphocytes (CTL) activity in HIV infection has been performed with target cells consisting of autologous EBV-transformed B lymphoblastoid cell lines (B-LCLs) expressing Human immunodeficiency virus type I (HIV-1) proteins. However data concerning the lysis of primary CD4+ T lymphocytes expressing HIV-1 antigens by CTLs is still lacking. To study the CTL activity against such primary targets, we used a system involving PBMCs of an HIV+ asymptomatic patient (PT) as effector cells and the CD4+ lymphocytes or B-LCLs of his healthy HLA-identical twin brother (HTW) as target cells. These syngeneic targets were either infected with recombinant vaccinia virus containing HIV-1 gag gene (gag-vac), or coated with HIV-1 gag peptides. We demonstrate in this study that PT CTLs (which were CD3+, CD4-, CD8+, TCRalphabeta+, TCRgammadelta-, CD56-) specifically lysed both types of syngeneic target cells expressing gag-vac; however, CD4+ T cells expressing HIV gag proteins were lysed less efficiently than B-LCLs expressing the same HIV epitopes. On the other hand, no specific lysis was detected when the target cells were uninfected or infected by wild-type vaccinia virus.     

A tumor-specific Th2 clone initiating tumor rejection via primed CD8+ cytotoxic T-lymphocyte activation in mice

We established a CD4+ T-cell clone specific for syngeneic methylcholanthrene-induced sarcoma, S1509a raised in an A/J mouse, involved in tumor regression. The phenotype of the T-cell clone was CD3+, TCR-beta+, CD4+, CD45RB+, LFA-1+, ICAM-1+, CD44+, and VLA-4+. The CD4+ T-cell clone specifically proliferated through antigen stimulation with attenuated S1509a in the presence of syngeneic accessory cells, and this antigen-induced proliferation was inhibited with anti-CD4 and anti-I-Ek monoclonal antibodies. The CD4+ T-cell clone designated YS1093 secreted interleukin (IL) 4, IL-5, and IL-6, but not IFN-gamma, tumor necrosis factor alpha, or IL-2, thus indicating that the clone belongs to the Th2 type. YS1093 cells and their culture supernatant after antigen stimulation augmented the primed cytotoxic T lymphocyte killing activity at the effector phase. YS1093 cells having Th2-type characteristics made the homologous growing tumor regress in the tumor-bearing syngeneic mice when YS1093 cells were transferred into the tumor-bearing mice i.v. The in vivo tumor regression initiated by YS1093 cell transfer essentially required the presence of CD8+ T cells in the tumor-bearing hosts, thus suggesting that some specific Th2 cells are positively involved in tumor regression by activating primed CD8+ cytotoxic T lymphocytes against the homologous tumor in situ.