Ultrastructure of IL2-stimulated tumor-infiltrating lymphocytes showing cytolytic activity against tumor cells.

Tumor-infiltrating lymphocytes (TIL) obtained from tumor tissue and pleural effusion of breast carcinoma were cultured with interleukin-2 (IL2) and thus activated. The ultrastructure of TIL stimulated by IL2 to kill various breast carcinoma cells was then investigated. Freshly isolated TIL cultured with autologous tumor cells for 48 h without IL2 were small, round and showed neither binding to nor killing of tumor cells. TIL stimulated to proliferate by IL2 became effector cells and showed cytotoxicity against tumor cells. Ultrastructurally, the effector TIL resembled large granular lymphocytes, and adhered to tumor cells through interdigitation or close apposition of the two plasma membranes accompanied by spot-like close membrane contacts. At the site of each spot-like contact, there was a 5-nm intercellular space. The morphology of the TIL processes did not differ from those of LAK and other CTL or NK cell processes during contact, invagination or the killing of target cells. The granules in TIL were considered to participate in the cytotoxic effect. Phenotypically heterogeneous TIL, CD8+/CD57- and CD8+/CD57+, adhered to autologous tumor cells and MCF7 (human breast carcinoma cell line). However, it was unclear which cell or cells acted as the effector for tumor-cell killing.     

Characterization of T-Cell Receptor Vβ Repertoire in Ovarian Tumour-Reacting CD3+ CD8+ CD4− CTL Lines

T cells from tumour infiltrating lymphocytes (TIL) cultured in media containing IL-2 were shown to mediate in vitro and in vivo antitumour responses. To characterize the T-cell antigen receptor (TCR) Vβ expression in autologous cytotoxic effectors we isolated CD3⁺ CD8⁺ CD4⁻ cells from cultures of TIL and tumour-associated lymphocytes (TAL) and analysed the TCR Vβ repertoire of CD3⁺ CD8⁺ CD4⁻ lines of known HLA-A, -B and -C phenotype, using polymerase chain reaction (PCR). These lines showed preferential lysis of autologous tumours and lysed, to a much lesser extent, NK and LAK cellsensitive targets. Tumour lysis was inhibited by antibodies to CD3 and MHC class I antigens indicating that they are cytotoxic T lymphocytes (CTL). These CD8⁺ CTL lines expressed a broad distribution of TCR Vβ repertoire which was dominated by particular groups of Vβ families in each CTL line. However, no predominant expression of one or the same Vβ segment in all CTL lines was observed although statistical correlations between Vβ family usage and magnitude of the antitumour cytolytic response were found. These results suggest that certain TCR Vβ families may be selected by antigen in ovarian tumour-reactive T cells and this selection may be affected by Ag expression, and/or host factors. To our knowledge, this is the first documentation of TCR Vβ repertoire of human ovarian tumour-reactive CD3⁺ CD8⁺ CD4⁻ CTL from different individuals of known HLA types.     

Interleukin-2 and Serum Thymic Factor Enable Autologous Rosette-forming T Lymphocytes to Generate Helper and Cytotoxic Functions

The autologous rosette-forming T cells (Tar cells) isolated by means of their ability to form rosettes with autologous erythrocytes were characterized by the use of OKT monoclonal anti-human T-cell subset antibodies and a monoclonal anti-HLA-DR antibody. We found that the phenotype of Tar cells was OKT 3⁺4⁺8⁺Dr⁻ as determined by both indirect imnrunofluorescence microscopy and complement-mediated killing of ⁵¹Cr-labelled Tar cells. In addition, we found that Tar lymphocytes were able to develop cytotoxicity against allogeneic and trinitrophenol (TNP)-conjugated autologous target cells in the presence of interleukin-2 (IL-2) or serum thymic factor. However, these cells showed little or no cytotoxicity in the absence of interleukin-2 or serum thymic factor. Tar lymphocytes generated helper function for B lymphocytes in the presence of interleukin-2 in both pokeweed mitogen (PWM)- and purified protein derivative (PPD)-stimulated cultures. Nevertheless, non-IL-2-treated Tar cells did not exhibit any helper activity on B cells. Finally, pretreatment of Tar cells with 1000–1500 rad of X ray made these cells unable to develop helper function for B lymphocytes. It is concluded that: (1) OKT 3⁺4⁺8⁺Dr⁻ Tar cells are able to generate cytotoxicity against alloantigens and TNP-labelled self structures provided they are stimulated by IL-2 or serum thymic factor; (2) these cells need both to proliferate and to receive help from IL-2 to develop helper cells capable of assisting B-lymphocyte differentiation into plasma cells in both PWM- and PPD-stimulated cultures.     

Control of T-cell activation by CD4+ CD25+ suppressor T cells

Summary: Depletion of the minor (∼10%) subpopulation of CD4⁺ T cells that co-expresses CD25 (interleukin (IL)-2 receptor α-chain) by thymectomy of neonates on the third day of life or by treatment of adult CD4⁺ T cells with anti-CD25 and complement results in the development of organ-specific autoimmunity. Autoimmune disease can be prevented by reconstitution of the animals with CD4⁺ CD25⁺ cells. CD4⁺ CD25⁺-mediated protection of autoimmune gastritis does not require the suppressor cytokines IL-4, IL-10, or transforming growth factor (TGF)-β. Mice that express a transgenic T-cell receptor (TCR) derived from a thymectomized newborn that recognizes the gastric parietal cell antigen H/K ATPase all develop severe autoimmune gastritis very early in life. CD4⁺ CD25⁺ T cells are also powerful suppressors of the activation of both CD4⁺ and CD8⁺ T cells in vitro . Suppression is mediated by a cell contact-dependent, cytokine-independent T–T interaction. Activation of CD4⁺ CD25⁺ via their TCR generates suppressor effector cells that are capable of non-specifically suppressing the activation of any CD4⁺ or CD8⁺ T cell. Activation of suppressor effector function is independent of co-stimulation mediated by CD28/CTLA-4 interactions with CD80/CD86. We propose that CD4⁺ CD25⁺ T cells recognize organ-specific antigens, are recruited to sites of autoimmune damage where they are activated by their target antigen, and then physically interact with autoreactive CD4⁺ or CD8⁺ effector cells to suppress the development of autoimmune disease.     

Participation of the CD69 Antigen in the T-Cell Activation Process of Patients with Systemic Lupus Erythematosus

Accumulating evidence has implicated T cells in the pathogenesis of systemic lupus erythematosus (SLE). The CD69 antigen is an integral membrane protein rapidly induced on the surface of activated lymphocytes. We obtained CD4⁺ and CD8⁺ T cells from normal subjects and patients with SLE. The percentage of CD69 expression in freshly isolated cells and after in-vitro incubation with mitogens was quantified by three-colour immunofluorescent staining. Expression of this protein was increased in both CD4⁺ and CD8⁺ T-cell subsets from SLE patients when compared with normal cells, although the difference was significant only in the CD8⁺ T-cell subset ( P  = 0.05). Cellular activation increased CD69 expression. When stimulated with anti-CD2/CD2R or phytohaemagglutinin (PHA), the percentage and absolute numbers of CD69⁺ cells were lower in patients than in controls. Addition of anti-interleukin (IL)-10 monoclonal antibody (MoAb) increased the percentage of in-vitro CD69 expression in SLE cells. These results suggest that the peripheral blood lymphocytes from patients with SLE have an intrinsic defect that alters their activation process, including the expression of CD69, and might explain some of the T immunoregulatory abnormalities observed in these patients.     

Selective expansion of a specific anti-tumor CD8+ cytotoxic T lymphocyte clone in the bulk culture of tumor-infiltrating lymphocytes from a melanoma patient: cytotoxic activity and T cell receptor gene rearrangements

In vitro culture of metastatic melanoma fragments with 150 units of recombinant interleukin 2 resulted in the successive expansion of CD4+ and then CD8+ tumor-infiltrating lymphocytes (TIL) throughout a 2-month period. TIL cultured for 43 days and consisting of 95% CD8+ and 10% CD4+ T lymphocytes were cloned by limiting dilution (LD). Thirteen CD8+ and thirty-one CD4+ clones were obtained, indicating that the frequency of clonogenic CD8+ proliferative T lymphocytes was much lower than that of their CD4+ homologues. When LD was performed in the presence of autologous melanoma cells the frequency of CD8+ clones was increased by factor 4. The DNA from TIL of day 43 bulk culture and of six CD8+ clones was hybridized with T cell receptor (TcR) beta and gamma probes. Identical configuration of the nonfunctional gamma and functional beta TcR genes was found in "bulk culture" and cloned TIL. The CD8+ clones therefore derived from a clonal population of CD8+ cells which had expanded in vitro before the LD. All the CD8+ clones tested were strongly cytotoxic for autologous melanoma cells but did not kill autologous fibroblasts or concanavalin A blasts, or any of the 10 allogeneic tumor targets tested, including 5 melanomas, 2 breast cell lines, 1 neuroblastoma, K-562 and the Epstein-Barr virus-transformed cell line used as a feeder. Furthermore, specific killing was inhibited by monoclonal antibodies against CD3, CD8, TcR alpha/beta and against class I major histocompatibility complex antigens indicating that these cytotoxic T lymphocyte clones recognized autologous tumor cells through the TcR, in an HLA class I-restricted manner. These data show that it is feasible to obtain tumor-specific cytotoxic T lymphocytes from melanoma TIL with a simple culture technique and that a single clone could be expanded to more than 10(10) cells which should allow testing of immunotherapeutic potential of these cells by adoptive transfer into melanoma patients.     

CD8+ Cells are the Main Producers of IL10 and IFNγ after Superantigen Stimulation

Purified CD8⁺ T cells were recently shown to produce TH1 as well as TH2 types of cytokines upon restimulation, indicating an important role for these cells in regulation of immune responses. However, it is not known if the CD8⁺ cells would contribute to cytokine production in the presence of cytokine secreting CD4⁺ cells. In the present study the authors have investigated the proportion of cytokine-producing CD4⁺ and CD8⁺ cells in the spleen after in vitro or in vivo stimulation. They found that stimulation of spleen cells with the superantigen Staphylococcal Enterotoxin B (SEB) in the presence of IL4 promoted production of IL10 and IFNγ predominately by CD8⁺ cells. In contrast, the production of IL4 was almost exclusively confined to the CD4⁺ subset. When priming with SEB in vivo before subsequent restimulation in vitro , a protocol previously shown to induce anergy, up to 80% of the IL10 and IFNγ positive cell expressed the CD8 marker. Taken together, these results emphasize the important role of cytokine-producing CD8⁺ cells and indicate that CD4⁺ and CD8⁺ T cells may, in a given situation, produce distinct cytokines.     

Primary Stimulation of CD4+ Cells in the Presence of IL-4 or IFN-γ Alters the Frequencies of Cytokine-Producing Cells at Restimulation

The induction of specific effector functions in naive T cells may be directed by accessory signals during activation. These could be elicited through binding to cell surface molecules or through factors secreted by antigen-presenting cells or other simultaneously activated cells. We have investigated the influence of CD8⁺ cells and of exogenousiy added cytokines (interleukin (IL)-2, IL-4 and interferon (IFN)-γ) on the cylokine production in splenic CD4⁺ T cells. IL-2, IL-4, IL-5 and IFN-γ production in CD4⁺ cells was measured at the single cell level during primary mitogen stimulation in vitro in the presence or absence of factors or CD8⁺ cells. On day 5 the cells were restimulated with mitogen alone and analysed to evaluate the short-term development of cytokine-producing cells in such cultures. Preactivation in the presence of either exogenous IL-4 or IFN-γ led to an increased production of IL-4 and IFN-γ respectively at restimtilation, and the effects of both IL-4 and IFN-γ were augmented by IL-2. After preactivation in the presence of IL-2 and IL-4, every third CD4⁺ cell could be induced to produce IL-4. Exogenous IL-4 or IFN-γ further decreased each other's production. Depletion of CD8⁺ cells before activation resulted in a slight increase of IL-4-producing cells, indicating that simultaneous activation of CD8⁺ cells will influence lymphokine production in CD4⁺ cells. The results suggest that the pattern of lymphokines induced in naive cells may be influenced by factors secreted by preactivated CD4⁺ and CD8⁺ cells, and that naive cells are preferentially 'recruited' to produce similar cytokines.     

Natural CD4+ CD25+ regulatory T cells. Their role in the control of superantigen responses

Summary: CD4 regulatory T cells have a major role in controlling the immune response to self and foreign antigens. Natural CD4⁺ CD25⁺ T cells are a major component of the regulatory subset. Their absence is associated with the development of autoimmune and inflammatory diseases and with abnormal peripheral T-cell homeostasis. Two main characteristics discriminate natural CD4⁺ CD25⁺ T cells from their CD4⁺ CD25⁻ counterparts, namely their cytokine production profile and their behavior during tolerance induction. Natural CD4⁺ CD25⁺ T cells produce interleukin (IL)-10, a cytokine that contributes to their regulatory role. They do not produce IL-2 and are dependent on exogenous IL-2 for proliferation in vitro and in vivo . Studies of their response to superantigen administration in vivo show that they are resistant to clonal deletion but can be tolerized by anergy. Their resistance to apoptosis may contribute to their continuous regulatory function, as it allows them to maintain permanent control over effector T cells.     

Rapid expression of the CD69 antigen on T cells and natural killer cells upon antigenic stimulation of peripheral blood mononuclear cell suspensions

The CD69 antigen has been identified as the earliest activation marker on the surface of cytokine- or mitogen-activated lymphocytes. The expression of this molecule may be a useful early marker of antigen- or allergen-specific activation of lymphocytes in vitro. We evaluated the expression of the CD69 and CD25 antigens on antigen- or allergen-stimulated lymphocytes and the proliferative responses as detected by thymidine incorporation. Peripheral blood mononuclear cells (PBMC) of allergic patients sensitized to Dermatophagoides pteronyssinus , bovine casein, or nickel sulfate were cultured in the absence or presence of clinically relevant allergens, tetanus toxoid, or recombinant interleukin (IL)-2. The respective binding of CD69 or CD25 antibodies to PBMC and thymidine incorporation were measured. An early expression of CD69, but not of CD25, antigen was detectable after 24-72 h of stimulation on up to 80% of natural killer (NK) cells and up to 10% of CD4⁺ T cells in PBMC cultures. Anti-IL-2 antibodies inhibited these increases of CD69 on NK cells and T cells by up to 60%. After 6 days of antigenic stimulation, the rates of both CD25⁺ and CD69⁺ lymphocytes were higher. Seventy-four percent of the CD25⁺ PBMC but only 55% of the CD69⁺ cells were CD3⁺ T lymphocytes at this time. No qualitative differences were detectable in allergen- or tetanus-toxoid-stimulated PBMC from allergic patients. The high expression of CD69 on NK cells in antigen-stimulated cultures suggests that these cells are easily activated by cytokines from antigen-stimulated T cells. CD69⁺ NK cells may serve as early-indicator cells in cultures with antigen- or allergen-stimulated mononuclear cells.     

Anti CD5 Sustains the Proliferative Response of IgM-Activated Human CD5+B Cells

The CD5 molecule is expressed by most T cells but it is present on a minor B cell subset. Whilst several studies have provided information on the physiological role of T cell CD5, the functional role of CD5 on B lymphocytes remains unclear. To address this question, tonsillar CD5⁺ B cells were sorted by dual-colour fluorescence and FACS. Sorted cells were stimulated with polyclonal anti-IgM antibodies (Ab), and monoclonal (MoAb) F(ab')₂ fragments of anti-CD5. Proliferative responses were evaluated by enumeration of Ki-67 positive cells using quantitative flow cytometry. Co-stimulation with anti-CD5 MoAb for 3 days did not affect the anti-IgM and IL2-induced proliferation of CD5⁺ B cells. This was seen under conditions where the anti-CD5 was soluble, adsorbed to the microwells or cross-linked by anti-mouse antibodies. Fewer CD25⁺ cells were detected, however, in the presence of anti-CD5. In contrast, the proliferative response of CD5⁺ B cells prestimulated for 3 days with IL-2 and anti-IgM, was sustained in a further 3-day culture period when anti-CD5 was added. It is concluded that CD5 occupancy might provide an additional signal to activated CD5⁺ B cells favouring their proliferation and differentiation into autoantibody secreting cells.     

Culture of Regulatory T-Cell Lines from Bronchial Mucosa

T lymphocytes play a major role in many immune responses. In the last decade, special focus has been on the function of Th1 and Th2 effector cells. Now the importance of regulatory CD4⁺CD25⁺ T cells in maintenance of the immunological homeostasis emerges. Sarcoidosis is a multisystem granulomatous disorder often affecting the lungs. The typical sarcoid granulomas consists of epitheloid cells, macrophages and lymphocytes, mainly CD4⁺ T cells of Th1 phenotype. We have cultured T cells from bronchial biopsies of patients with sarcoidosis as well as from controls in high levels of interleukin 2 (IL-2) and IL-4 and demonstrate spontaneously arising CD4⁺ CD25⁺ populations and high concentrations of IL-10 in these cultures. The main difference between cultures of sarcoid origin compared to controls is a very much higher concentration of the inflammatory cytokines IL-6 and TNF-α in cultures of sarcoid origin.     

Subpopulations of T Lymphocytes in the Peripheral Blood, Dermal Lesions and Lymph Nodes of Post Kala-azar Dermal Leishmaniasis Patients

Distribution of different subpopulations of T cells in the dermal lesions, lymph nodes and peripheral blood of post kala-azar dermal leishmaniasis (PKADL) patients was studied by using appropriate phenotypic markers for CD2⁺, CD4⁺ and CD8⁺ cells. Histopathological studies of skin lesions showed marginal to massive infiltration of mononuclear cells depending upon the duration of illness and type of lesions. Thus, while the hypopigmented patches were represented by small focal collections of lymphocytes with scanty parasites in the dermis, these were replaced at the nodular stage with massive granulomas consisting of lymphocytes, plasma cells and histiocytes with numerous amastigotes. The involvement of CD4⁺ and CD8⁺ cell types in these lesions also showed a gradual change from the appearance of a few cells of both the phenotypes in early hypopigmented type to massive accumulation of cells, primarily of CD8⁺ phenotype, in the granuloma of nodular type. However, the observed preponderance of CD8⁺ cells at the lesion site of chronic PKADL patients is in contrast to their peripheral blood CD4⁺/CD8⁺ cell ratio (1.9:1) which remained within the normal limits. Similar studies of lymph nodes from PKADL patients with lymphadenopathy revealed infiltration of the cortical areas by T cells which were more of CD8⁺ than CD4⁺ phenotypes. All these results document the involvement of CD8⁺ cells in leishmanoid lesions. Thus, it is likely that these cells, in association with appropriate subpopulations of CD4⁺ cells, play a profound role in the evolution of dermal pathology in PKADL.     

Isolation of broadly reactive, tumor-specific, HLA Class-I restricted CTL from blood lymphocytes of a breast cancer patient

Blood lymphocytes of a HLA-A2 positive breast cancer patient were stimulated with either MCF-7 or MDA-MB-231, i.e., HLA-A2-matched allogeneic breast carcinoma cell lines. Several CD8⁺ CTL clones with reactivity against the stimulator cells but not against K562 were generated. Reactivity could be blocked with monoclonal antibody (mAb) W6/32, MA2.1, and/or BB7.2, indicating that the clones are HLA-class I and HLA-A2 restricted. The CTL clones generated following stimulation with MCF-7, recognized various other allogeneic HLA-A2⁺ tumor cell lines, including breast carcinoma, renal cell carcinoma, and melanoma cell lines, but not HLA-A2⁻ tumor cell lines. The CTL clones did not recognize normal HLA-A2⁺ cells including breast epithelial cells, renal proximal tubular epithelial cells (PTEC), or EBV-transformed B cells including the autologous EBV cell line. In contrast to the CTL clones induced with MCF-7, the reactivity of the clones stimulated with MDA-MB-231, was limited to the stimulator cell MDA-MB-231. Cytotoxicity assays utilizing T2 cells loaded with peptides as target cells indicated that none of the examined CTL-epitopes derived from HER-2/neu, Muc-1, Ep-CAM-1, and p53 were recognized by the CTL clones generated. Our findings underscore that breast cancer is an immunogenic tumor and that HLA-class I-matched allogeneic tumor cells can be used as stimulator cells to generate tumor-specific CTL from peripheral blood of a breast cancer patient with specificity for an antigenic determinant that is broadly expressed on tumor cells from various origins or with specificity limited to the breast cancer stimulator cell.     

A2B5 Cells from Human Glioblastoma have Cancer Stem Cell Properties

Glioblastomas, like other cancers, harbor small cell populations with the capability of sustaining tumor formation. These cells are referred to as cancer stem cells. We isolated cells expressing the surface marker A2B5 from three human glioblastomas (GBM) and showed that after grafting into nude mice, they generated dense and highly infiltrative tumors. Then, we extensively studied A2B5⁺ cells isolated from 11 human GBM. These cells display neurosphere-like, self-renewal, asymmetrical cell division properties and have multipotency capability. Stereotactic xenografts of dissociated A2B5⁺-derived secondary spheres revealed that as few as 1000 cells produced a tumor. Moreover, flow cytometry characterization of A2B5⁺-derived spheres revealed three distinct populations of cells: A2B5⁺/CD133⁺, A2B5⁺/CD133^- and A2B5^-/CD133^-, with striking proportion differences among GBM. Both A2B5⁺/CD133⁺ and A2B5⁺/CD133^- cell fractions displayed a high proliferative index, the potential to generate spheres and produced tumors in nude mice. Finally, we generated two green fluorescent protein-cell lines that display—after serum induction—distinct proliferative and migratory properties, and differ in their CD133 level of expression. Taken together, our results suggest that transformed A2B5⁺ cells are crucial for the initiation and maintenance of GBM, although CD133 expression is more involved in determining the tumor's behavior.     

Suppression of lymphokine-activated killer cell generation by tumor-infiltrating lymphocytes

A functional analysis of tumor-infiltrating lymphocytes (TILs) from renal cell carcinoma (RCC) and malignant melanoma was performed. TILs were expanded in recombinant interleukin-2 (50 U/ml) in Iscoves medium. Phenotypic and functional (cytolytic vs regulatory) analyses were carried out with the fresh and expanded TIL populations after 4 weeks in culture. Only one TIL population from an RCC case (out of six cases studied) was CD8+ and demonstrated MHC class I-restricted tumor-specific cytotoxicity against the autologous RCC target. TIL populations from the other five cases became predominantly CD4+ and they neither killed the respective autologous tumor cells nor killed the NK-sensitive target K-562 cells. When studied for other functions, two CD4+ TIL populations were found to suppress the lymphokine-activated killer cell response by peripheral blood lymphocytes (PBL) in coculture. Of these two, a TIL population from an RCC case (MJ TIL) was used to study the cellular and molecular mechanisms of suppression. The MJ TIL synthesized a supernatant factor that blocked activation of resting PBL as measured by the induction of high-affinity IL-2 receptor (IL-2R) when stimulated by phytohemagglutinin but did not down-regulate the fully expressed IL-2R on activated T cells. The suppression of high-affinity IL-2R induction on T cells did not result from tumor necrosis factor-alpha and beta or from transforming growth factor-beta as these cytokines were not detected in the cell-free supernatant from the MJ TIL culture. The supernatant factor also suppressed IL-2-mediated enhancement of cytotoxicity by natural killer (NK) cells without demonstrating direct toxic effect on the NK cells. Thus, when TIL are used for adoptive immunocytotherapy, it may be useful to fully characterize them functionally, in vitro.     

T-Lymphocyte Functions in Acute Leukaemia Patients with Severe Chemotherapy-Induced Cytopenia: Characterization of Clonogenic T-Cell Proliferation

Intensive chemotherapy for acute leukaemia is followed by a period of severe chemotherapy-induced leukopenia. We used a limiting dilution assay to investigate whether remaining CD4⁺ and CD8⁺ T lymphocytes derived from such leukopenic patients could be activated and undergo clonogenic proliferation. The activation signal in our model was accessory cells (irradiated normal peripheral blood mononuclear cells) + phytohaemagglutinin (PHA) + interleukin-2 (IL-2). During severe leukopenia a majority of circulating lymphocytes were CD4⁺ T cells. Clonogenic proliferating T lymphocytes were detected for all patients. Higher frequencies of clonogenic cells were detected in the CD8⁺ subset as compared to the CD4⁺ subset. However, for both subsets frequencies of proliferating cells were decreased compared with healthy individuals. The CD4⁺ and CD8⁺ lymphocytes were also capable of proliferation in response to alloactivation, and accessory cells mainly containing acute myelogenous leukaemia blast were efficient as accessory cells for activation. For the CD4⁺ cells, increased proliferation was detected in the presence of acute myelogenous leukaemia (AML) blasts compared with normal accessory cells. Based on our results we conclude that: (1) although acute leukaemia patients with therapy-induced leukopenia have both a quantitative and a qualitative T-cell defect, (2) the remaining T-cell population includes a subset capable of clonogenic proliferation. However, (3) proliferation of the clonogenic CD4⁺ cells can be modulated by AML blasts.     

A Human Melanoma Cell Line, Recognized by Both HLA Class I and Class II Restricted T Cells, is Capable of Initiating both Primary and Secondary Immune Responses

We have characterized a melanoma cell line, FM3, established from a metastasis of a 75 year old female patient (HLA–A2, HLA–DQ7) with malignant melanoma. This cell line expresses both HLA class I and class II antigens, as well as several important accessory molecules at high levels. FM3 cells were shown to function as a stimulator of both allogeneic as well as autologous mixed lymphocyte tumour cell culture (MLTC). From these autologous MLTC we were able to generate cytotoxic T cell clones indicating that FM3 is capable of processing and presenting endogenous antigens.
We have used this cell line in a model system to investigate whether these cells were able to initiate and support an immune response with specificity for selected peptide antigens. The FM3 cell line was capable of presenting a HLA–DQ7 restricted ras derived peptide (5–21, 13Gly–Asp) to a previously established T cell clone, RM70. The ability of FM3 to function as an antigen presenting cell (APC) was comparable to that of an autologous Epstein Barr virus (EBV) transformed B cell line. The CD4⁺ T cell clone RM70 showed a peptide–specific anti–proliferative effect on FM3 cells. This growth inhibition was not due to cytotoxicity as measured in a standard 4h chromium release assay. The FM3 cell line also presented a HLA–A2 restricted nonapeptide derived from the influenza matrix protein. M 1 (58–66) to a CD8⁺ T cell line specific for this peptide. This resulted in an effective killing of the melanoma cells.
Together, these data suggest that some melanomas may initiate an immune response by presenting their own specific antigens in an immunogenic context, and subsequently serve as targets for T cells of both the CD4⁺ and CD8⁺ phenotype.     

Effector mechanisms in transplant rejection

Summary:  Antigens, provided by the allograft, trigger the activation and proliferation of allospecific T cells. As a consequence of this response, effector elements are generated that mediate graft injury and are responsible for the clinical manifestations of allograft rejection. Donor-specific CD8⁺ cytotoxic T lymphocytes play a major role in this process. Likewise, CD4⁺ T cells mediate delayed-type hypersensitivity responses via the production of soluble mediators that function to further activate and guide immune cells to the site of injury. In addition, these mediators may directly alter graft function by modulating vascular tone and permeability or by promoting platelet aggregation. Allospecific CD4⁺ T cells also promote B-cell maturation and differentiation into antibody-secreting plasma cells via CD40–CD40 ligand interactions. Alloantibodies that are produced by these B cells exert most of their detrimental effects on the graft by activating the complement cascade. Alternatively, antibodies can bind Fc receptors on natural killer cells or macrophages and cause target cell lysis via antibody-dependent cell-mediated cytotoxicity. In this review, we discuss these major effector pathways, focusing on their role in the pathogenesis of allograft rejection.