Thymocyte antigens do not induce tolerance in the CD4+ T cell compartment.

Thymocytes fail to tolerize the developing T cell repertoire to self MHC class I (MHC I) Ags because transgenic (CD2Kb) mice expressing H-2Kb solely in lymphoid cell lineages reject skin grafts mismatched only for H-2Kb. In this study, we examined why thymocytes fail to tolerize the T cell repertoire to self MHC I Ags. The ability of CD2Kb mice to reject H-2Kb skin grafts was age dependent because CD2Kb mice older than 20 wk accepted skin grafts. T cells from younger CD2Kb mice proliferated, but did not develop cytotoxic functions in vitro in response to H-2Kb. Proliferative responses were dominated by H-2Kb-specific, CD4+ T cells rather than CD8+ T cells. Representative CD4+ T cell clones from CD2Kb mice were MHC II restricted and recognized processed H-2Kb. TCR transgenic mice were generated from one CD4+ T cell clone (361) to monitor development of H-2Kb-specific immature thymocytes when all thymic cells or lymphoid cell lineages only expressed H-2Kb. Thymocyte precursors were not eliminated and mice were not tolerant to H-2Kb when Tg361 TCR transgenic mice were intercrossed with CD2Kb mice. In contrast, all thymocyte precursors were eliminated efficiently in thymic microenvironments in which all cells expressed H-2Kb. We conclude that self MHC I Ags expressed exclusively in thymocytes do not induce T cell tolerance because presentation of processed self MHC I Ags on self MHC II molecules fails to induce negative selection of CD4+ T cell precursors. This suggests that some self Ags are effectively compartmentalized and cannot induce self-tolerance in the T cell repertoire.

The role of protein kinase C in CD8+ T lymphocyte effector responses.

Rare CD8+ T cells present in beta2microglobulin-deficient (beta2m-/-) mice reject allogeneic tumors but not syngeneic wild-type tumors. The lack of syngeneic tumor rejection in vivo is correlated with a partial response of beta2m-/- CD8+ cell lines to syngeneic tumor cells in vitro. This partial response is characterized by perforin/granzyme-mediated cytolytic activity in the absence of cytokine secretion or proliferation. Allogeneic tumors induce cytolysis, cytokine secretion, and proliferation. Cytokine secretion may therefore be an important effector mechanism for tumor rejection by CD8+ T cells. To determine the missing signaling events needed for cytokine secretion as well as the events inducing the isolated cytotoxic response, we attempted to restore cytokine secretion of beta2m-/- CD8+ cells to syngeneic MHC class I. Phorbol ester and syngeneic tumor cells acted synergistically to induce full responsiveness of beta2m-/- CD8+ cells. However, this synergistic induction of cytokine secretion used a different pathway than that induced by alloantigen. Protein kinase C (PKC) inhibitor prevented the syngeneic class I plus PMA-induced cytokine secretion, but not allo-class I-induced cytokine secretion. In contrast to the PKC independent alloantigen-induced cytokine secretion, cytolysis of both allogeneic and syngeneic targets was PKC dependent. The differential dependence of effector functions on PKC activation was also found in beta2m+/+ CD8+ T cells. Thus, two distinct signaling pathways (PKC dependent and PKC independent) may ultimately converge to induce cytokine secretion in CD8+ cells. The TCR engagement-initiated pathway is PKC independent, whereas the phorbol ester-activated pathway is PKC dependent.

Apparent split tolerance of CD8+ T cells from beta 2-microglobulin-deficient (beta 2m-/-) mice to syngeneic beta 2m+/+ cells.

beta 2-microglobulin-deficient (beta 2m-/-) mice express reduced levels of MHC class I molecules and, consequently, have impaired positive selection of CD8+ T lymphocytes in the thymus. However, small numbers of CD8+ CTLs can be found in beta 2m-/- mice after immunization with allogeneic as well as syngeneic beta 2m+/+ tumor or spleen cells. It has been proposed, therefore, that because of the low ligand density in beta 2m-/- mice, negative selection does not remove cells capable of recognizing syngeneic MHC class I expressed at normal levels. We report here that beta 2m-/- CD8+ T cells are partially tolerant to syngeneic beta 2m+/+ cells. Despite the ability of beta 2m-/- mice to raise CD8+ CTLs against syngeneic beta 2m+/+ cells, these CD8+ cells do not proliferate and do not secrete IFN-gamma or IL-3/granulocyte-macrophage-CSF upon in vitro stimulation with syngeneic beta 2m+/+ cells. In contrast, all of these cellular responses are displayed by the beta 2m-/- CD8+ cells upon recognition of the allogeneic MHC class I. These in vitro findings of partial responsiveness to syngeneic and of full responsiveness to allogeneic MHC class I correlate well with the ability of beta 2m-/- mice to reject allogeneic, but not syngeneic, tumors in vivo. It appears, thus, that the significantly reduced levels of MHC class I molecules found in beta 2m-/- mice, although not capable of inducing deletion of all reactive clones, can induce deletion of high affinity clones and, therefore, maintain tolerance to self-MHC class I, even when expressed at much higher (beta 2m+/+) levels.

Characterization of lymphokine-activated killer cells from peripheral blood and lymph nodes of non-Hodgkin's lymphoma patients.

Peripheral blood lymphocytes (PBL) and lymph node lymphocytes (LNL) from non-Hodgkin's lymphoma patients were tested for LAK cell cytotoxicity using appropriate targets in a short-term 51chromium-release assay. The results showed a significant depression in LNL-LAK activity suggesting the reduced capacity of LNL to generate LAK cells. LNL-LAK cells demonstrated significantly low percentages of cells expressing CD16, CD56 and CD25 as compared to PBL-LAK of patients and healthy donors. The reduced capacity to generate LAK cells in lymph nodes could be due to the presence of low numbers of NK cells which are thought to be the main precursors of LAK cells. The IL-2 producing ability of lymph node mononuclear cells was found to be significantly higher than that of peripheral blood mononuclear cells from both healthy donors and NHL patients.

Lymphokine activated killer cells and interleukin-2 production in patients with cervical carcinoma.

Lymphokine activated killer cells were generated from peripheral blood lymphocytes (PBL) in patients with cervical carcinoma. The cytotoxic potential of these cells was determined against three tumor cell lines (K-562, RAJI and MCF-7) using a short-term 51Cr-release assay. Both normals and patients showed comparable levels of LAK activity. Phenotypic analysis of cells having LAK activity showed a heterogeneous population. A significant increase in the expression of CD25 marker (IL-2R, Tac) of PBL population was observed after IL-2 culture. In addition, the indigenous ability of peripheral blood mononuclear cells to produce IL-2 in response to mitogen PHA was almost the same as observed in case of normal individuals.

Generation and characterization of lymphokine-activated killer cells from non-Hodgkin's lymphoma patients.

Lymphokine-activated killer cells generated from peripheral blood of Non-Hodgkin's lymphoma patients showed comparable levels of cytotoxicity. The patients with advanced disseminated disease displayed better augmentation with recombinant interleukin-2 (rIL-2) than the patients with localized disease. Thus, the defective cytotoxic potential of patients could be corrected in a culture of effector cells with IL-2. The indigenous ability to produce IL-2 in response to activation of peripheral blood mononuclear cells with phytohemagglutin showed slight depression and a positive correlation with the stage of the disease. Phenotypic analysis revealed a heterogenous population of cells involved in cytotoxic activity. Thus, IL-2 merits further evaluation in malignant lymphomas, particularly in relation to other variable therapies in conjunction with chemotherapy.