Expression of both I-A and I-E/C subregion antigens on accessory cells required for in vitro generation of cytotoxic T lymphocytes against alloantigens or TNBS-modified syngeneic cells

We have previously reported that radioresistant, Thy 1-negative accessory cells (SAC) are required for the in vitro generation of cytotoxic T-effector cells to allogeneic or trinitrophenyl-modified syngeneic cells. These SAC were found to provide accessory functions irrespective of whether they were syngeneic, semi-syngeneic, or allogeneic to the responding cells. To further characterize the accessory cells active in CML, the expression of Ia antigens on this functional population was assessed by pretreated SAC with anti-Ia reagents and complement and by testing the accessory cell function of these treated populations. The results of these studies demonstrated that the relevant accessory cells for allogeneic and TNP-self CTL express Ia determinants encoded by genes mapping in the I-A and I-E/C subregions. For the TNP-self CTL the accessory function of both SAC syngeneic or allogeneic to the responding and stimulating cells was specifically abrogated by treatment with anti-Ia reagents and complement.     

Cloned murine Ia+ BK-BI-2.6.C6 T cells function as accessory cells presenting protein antigens to long-term-cultured antigen-specific T cell lines

A variant clone, BK-BI-2.6.C6, was derived from the murine bovine insulin-reactive T cell line BK-BI-2.6 with helper/amplifier phenotype. Variant cells have lost reactivity to insulin, but have acquired constitutive IL 2 receptor expression, growing in IL 2-containing medium without feeder cells. In contrast to their ancestor line, variant cells synthesize and express I-A and I-E region-dependent class II molecules as indicated by metabolic radiolabeling, immunoprecipitation with subregion-specific monoclonal antibodies and two-dimensional (2D) gel electrophoresis (1D isoelectric focusing, 2D SDS-PAGE). BK-BI-2.6.C6 cells can act as accessory cells, presenting the protein antigens bovine insulin and ovalbumin to antigen-dependent long-term cultured T cell lines BK-BI-1.2 and BK-OVA-1 in the context of I-A restriction elements. Antigen recognition on presenting BK-BI-2.6.C6 accessory cells resulted in highly efficient IL 2 production. However, in contrast to splenic antigen-presenting cells, BK-BI-2.6.C6 cells did not initiate antigen-specific [3H]thymidine incorporation by the T cell lines tested. Further study of accessory function of Ia+ T cell clones might provide insight into processes regulating T cell responses to antigen.     

Expression of I-A and I-E,C region-coded Ia antigens on functional B cell subpopulations.

Ia antigens from specific subregions have been examined on functional B cell populations. Expression of both I-A and I-E,C region antigens was demonstrated on cells required for both lipopolysaccharide mitogenesis and polyclonal activation. Similar I-A and I-E,C subregion expression was found on cells required for response to the T-independent antigen, polyvinylpyrrolidone. TNP-specific IgM and hen egg lysozyme-specific IgG plaque-forming cells also express I-A and I-E,C region antigens. No evidence was found for an Ia- population responsive in the systems tested. Further, no evidence of preferential expression of I-A or I-E,C region antigens was observed in any system examined. Therefore, it appears that B cells express both I-A and I-E,C region-coded Ia antigens.     

T lymphocyte-enriched murine peritoneal exudate cells. I. A reliable assay for antigen-induced T lymphocyte proliferation.

The in vitro activation of murine thymus-derived (T) lymphocytes by soluble protein and synthetic antigens has been difficult to assess because of the lack of a specific and reliable proliferation assay. The present report describes the development of an assay system which overcomes these problems by making use of a population of nylon wool column-purified T lymphocytes obtained from thioglycollate-induced peritoneal exudates of immunized mice. PETLES (peritoneal exudate, T lymphocyte-enriched cells) were composed mainly of T lymphocytes, eosinophils and small numbers of macrophages. Contamination with bone marrow-derived (B) lymphocytes averaged only 2%. When PETLES from immunized mice were stimulated in microtiter cultures with the immunizing antigen, large degrees of proliferation ensued as measured by incorporation of 3H-methyl-thymidine 5 days after initiation. As few as 1.25 x 10(4) cells and as little as 50 ng/ml of antigen gave significant stimulation. Maximum responses were obtained witn a series of 10 experiments under these optimal conditions, gave a mean incorporation of 70,900 cpm while the controls cultured without antigen showed only 3,600 cpm. PETLES from nonimmunized mice or from mice immunized to other antigens did not respond to DNP5OVA although they did respond to mitogens. The antigen-induced proliferation was shown to require the presence of immune T lymphocytes by two criteria: elimination of the response by treatment with anti-Thy 1.2 serum plus complement and failure to reconstitute the response when the few remaining immune B lymphocytes left after anti-Thy 1.2 treatment were added to nonimmune T lymphocytes. In addition, the system exhibited carrier specificity. Because of the paucity of B lymphocytes in the population, their contribution to the overall magnitude of the proliferative response was negligible as demonstrated by the small response to B cell mitogens. Thus, the assay appears to be a quantitative as well as a qualitative assay for one aspect of T lymphocyte function. This technique should prove useful for the study of murine T lymphocytes in vitro.     

Fanconi anemia complementation group C is required for proliferation of murine primordial germ cells

Fanconi anemia is a polygenic trait hypothesized to be a DNA damage repair disease. We show that all three Fanconi anemia loci that have been cloned are expressed in the embryonic gonad during the period of primordial germ cell proliferation. Mice mutant for the Fanconi anemia complementation group C locus (Fancc) have reduced germ cell numbers as early as embryonic day E12.5, suggesting the Fancc protein functions prior to meiosis in both sexes. Depletion in the mutant occurs at a time when all three loci would be expressed in a wild-type gonad, implying a function in the early germline. Determination of the mitotic index of primordial germ cells by BrdU incorporation shows that germ cells in Fancc(-/-) mice proliferate significantly more slowly than littermate controls. This study demonstrates Fancc is required for mitotic proliferation of primordial germ cells.     

The superantigen Pseudomonas exotoxin A requires additional functions from accessory cells for T lymphocyte proliferation

We have examined the functions required of accessory cells (AC) for murine thymocyte proliferation induced by Pseudomonas exotoxin A (PE) and have compared these functions to those required of a known superantigen, staphylococcal enterotoxin B (SEB). We demonstrate that PE, like SEB, preferentially stimulates PNA+ thymocytes expressing a specific V beta element within the T cell receptor. However, PE requires functions from AC that are distinct from those required by SEB. AC treated with paraformaldehyde (PCHO) prior to stimulation supported thymocyte proliferation induced by SEB but not PE. However, when AC were treated with PCHO subsequent to stimulation with PE, thymocyte proliferation was observed, which suggests that PE requires antigen processing in addition to presentation. Furthermore, treatment of AC with lysosomotropic agents abrogated thymocyte proliferation induced by PE but not SEB. Antibodies to MHC class II molecules inhibited thymocyte proliferation induced by both PE and SEB. In addition, we observed that interleukin 1 alpha (IL-1 alpha) participated in the proliferation of thymocytes induced by PE but not SEB. Thus, our data indicate that PE is a unique microbial superantigen that requires additional AC functions for T lymphocyte proliferation.     

The murine Kupffer cell. I. Characterization of the cell serving accessory function in antigen-specific T cell proliferation.

Murine Kupffer cells, the tissue macrophages of the liver, were isolated by collagenase digestion, differential sedimentation over Metrizamide, and glass adherence. The resultant cell population was more than 86% phagocytic, and 95% of cells stained positively for alpha-naphthyl butyrate esterase activity. The cells also had cell surface receptors for complement (C) and the Fc portion of IgG. In addition, a large proportion of Kupffer cells was shown to bear Ia antigens: about half of the cells bore I-A subregion-encoded antigens and about half bore I-BJE or I-EC subregion-encoded antigens. Kupffer cell populations were capable of reconstituting antigen-stimulated proliferative responses of antigen-primed, macrophage-depleted, lymph node T cells. The ability to reconstitute proliferation was enriched in the adherent population and was resistant to radiation and treatment with an anti-Thy antiserum and C. We conclude that isolated murine Kupffer cells bear the Ia phenotype of accessory cells that function in antigen presentation and that Kupffer cells can participate in the induction of antigen-specific immune responses. These data suggest that Kupffer cells may play a role in modulating responses to enterically derived antigens.     

Analysis of Ir gene function using monoclonal antibodies: independent regulation of GAT and GLPhe T cell responses by I-A and I-E subregion products on a single accessory cell population

T cell proliferative responses to the synthetic polypeptides GAT and GLPhe are under Ir gene control. GAT responses are regulated by gene(s) in the I-A subregion, and GLPhe responses are controlled by a pair of complementing genes mapping to the I-A and I-E subregions. We demonstrate that monoclonal antibody to the I-A gene product inhibits GAT proliferation but not the GLPhe response, whereas a monoclonal antibody to the I-E associated Ia-7 determinant inhibits GLPhe but not GAT proliferation, which indicates independent involvement of each Ia determinant in antigen presentation for the T cell response to these antigens. Use of the same subregion-specific monoclonal antibodies in complement-dependent lysis demonstrates that the antigen-presenting cells for GAT and GLPhe express both I-A and I-E products. The possibility that an Ia subregion-specific "self-receptor" functions on the reactive T cells as a regulatory element is discussed.     

Inhibition of proliferation of MIs- and Ia-reactive cloned T cells by a monoclonal antibody against a determinant shared by I-A and I-E

The interaction between cloned amplifier T cells and stimulating cells has been probed with the use of a monoclonal antibody selected for its ability to inhibit stimulation of Mls- and la-reactive T cells. The antibody recognizes a public determinant on I-A- and I-E-coded molecules of a variety of murine H-2 haplotypes. The antibody inhibits the proliferation of the I-Ab-reactive T cell clone 48.1 and of the Mls-reactive T cell clones L2 and Fa 13. The antibody may be added as late as 3 hr after initiation of culture to cause inhibition of L2 proliferation and release of interleukin 2. A profound inhibition of primary and secondary mixed leukocyte responses also is observed. The results indicate that a large fraction of Mls reactive T cells require interaction with la antigens.     

Interleukin-4-mediated downregulation of cytotoxic T lymphocyte activity is associated with reduced proliferation of antigen-specific CD8+ T cells

During virus infection, exogenous IL-4 strongly downregulates expression of antiviral cytokines and cytotoxic T lymphocyte (CTL) responses. In this study, we have employed a T cell receptor (TCR) transgenic system to more closely investigate the effect of IL-4 on CTL activity. This system involves mice transgenic for an H2-Kb restricted TCR recognising an ovalbumin (OVA)-specific peptide (OT-I mice), and recombinant vaccinia viruses expressing the gene for OVA (VV-OVA), or OVA together with IL-4 (VV-OVA-IL-4). Spleen cells from OT-I mice were adoptively transferred to irradiated C57BL/6 mice infected with VV-OVA or VV-OVA-IL-4. Five days following transfer, markedly stronger CTL activity was detected in VV-OVA- than in VV-OVA-IL-4-infected recipients. The reduction in CTL activity was associated with a reduction in the number of OVA-specific CD8+ T cells. Proliferation of cells from VV-OVA-IL-4-infected recipients was dramatically reduced, and this is a likely explanation for the IL-4-mediated reduction in the total number of OVA-specific cells and the reduced cytotoxic activity. On a per cell basis, the production of IFNgamma and cytotoxic activity of OVA-specific CD8+ cells was not influenced by IL-4. Taken together, our results indicate that the reduction in CTL activity by exogenous IL-4 is due to a reduced number of antigen-specific effectors, and does not involve a downregulation of effector function of these cells.     

Signals for activation and proliferation of murine T lymphocyte clones

Biochemical signals required for the growth of T cell clones were studied. Antigen-specific helper T cell clones, 6-1 and KO.6, could enter the state similar to the resting state where the cells expressed only small numbers of interleukin 2 (IL2) receptors and could not respond to IL2 without antigenic stimulation. A combination of a phorbol ester, 12-O-tetradecanoylphorbol-13-acetate (TPA), and a calcium ionophore, A23187, induced the expression of IL2 receptors on resting 6-1 cells and induced DNA synthesis in the presence of IL2. TPA alone did not induce IL2 receptors. A23187 induced the expression of the receptors to some extent but did not induce DNA synthesis even in the presence of IL2. IL2 receptors induced by A23187 alone were mostly low affinity receptors, whereas the combination of TPA and A23187 induced high affinity receptors in addition to low affinity receptors. Resting KO.6 cells produced IL2 in response to a combination of TPA and A23187, whereas either one of the agents did not induce the production of IL2. Dicaprylin, a permeable diacylglycerol and a potent activator of protein kinase C (the Ca2+/phospholipid-dependent enzyme) could replace TPA in both cases when dicaprylin was repeatedly added to the culture. These results suggest that strong and continuous activation of protein kinase C together with calcium mobilization is required for IL2 production and IL2 receptor expression. On the contrary, signals for DNA synthesis generated by binding of IL2 to IL2 receptors are different from those for IL2 production and IL2 receptor expression, as the combination of TPA and A23187 could not induce DNA synthesis without IL2.     

Surface expression and synthesis of I-A and I-E/C encoded molecules by B lymphocytes and Ig-secreting cells.

Splenocytes from recombinant mice were radiolabeled before or after deletion of subpopulations by cytotoxic anterisera (+C) directed against I-A, I-E/C, IgM, or Ig. Examination of the lysates of the surviving cells by immunoprecipitation demonstrated that 1) virtually all I-A and I-E/C molecules are co-expressed and synthesized by Ig+, IgM+ lymphocytes, 2) I-A, I-E/C, and IgM molecules are present on many of the cells secreting IgM and IgG, and 3) populations of Ig-bearing or Ig-secreting cells that lack detectable I-A and I-E/C antigens can be identified in spleen cell populations. The co-expression of I-A and I-E/C on most cells of the B cell lineage is discussed in terms of our present concepts of Ir gene control of immune responses.     

T cell recognition in the mixed lymphocyte response. II. Ia-positive splenic adherent cells are required for non-I region-induced stimulation

Ia-positive splenic adherent cells (SAC) have been shown to be the predominant stimulators of a mixed lymphocyte response (MLR) to whole H-2 differences, in which most of the proliferative response is directed against I region-encoded determinants. The present studies were undertaken to examine the ability of several purified lymphoid subpopulations to activate T cells in response to the non-H-2-linked MIs products or to products of the K or D regions of H-2. The results demonstrated that adherent cell-depleted populations of T and B cells were nonstimulatory, whereas SAC were potent stimulators for responses involving each of these genetic differences. Treatment of these SAC with anti-Ia and C abrogated their MLR-stimulating ability. In contrast, whereas treatment of SAC with anti-Ia and C abrogated their ability to stimulate an MLR directed against K or D region-encoded determinants, this treatment had no effect on their ability to generate a cytotoxic T lymphocyte response against these same determinants. These findings suggest that in addition to presenting allogeneic I region-encoded determinants, Ia-positive SAC also play a unique role in the presentation of non-I region-encoded alloantigens to proliferating T cells.     

Tyrosine protein phosphorylation is required for protein kinase C-mediated proliferation in T cells.

We have studied the role of tyrosine kinase in PMA-stimulated T cells. Protein kinase C (PKC)-mediated D10A cell proliferation is inhibited by the specific inhibitor of tyrosine kinase, tyrphostin. This inhibitor selectively blocks the mRNA expression of the proto-oncogene c-myc in response to the phorbol ester, PMA. On the other hand, the same doses of this inhibitor do not affect the mRNA expression of the proto-oncogene c-fos in PMA-stimulated D10A cells. Phorbol esters induce in this T cell line the tyrosine phosphorylation of a unique protein of 42 kDa and the enzyme PKC is required for this activity.     

Extracellular signal-regulated protein kinase 2 is required for efficient generation of B cells bearing antigen-specific immunoglobulin G

Activation of extracellular signal-regulated protein kinase (ERK) has been implicated in proliferation as well as differentiation in a wide variety of cell types. Using B-cell-specific gene-targeted mice, we report here that in T-cell-dependent immune responses, ERK2 is required to generate efficient immunoglobulin G (IgG) production. In its absence, the proportion of antigen-specific surface IgG1-bearing cells and the subsequent number of IgG1 antibody-secreting cells were decreased, despite apparently unimpaired class switch recombination. Notably, this defect was countered by overexpression of the antiapoptotic factor Bcl-2. Together, our results suggest that ERK2 plays a key role in efficient generation of antigen-specific IgG-bearing B cells by promoting their survival.     

Inhibition of macrophage-induced antigen-specific T lymphocyte proliferation by poly I:C: strain and antigen independence

We have previously demonstrated that IFN-alpha/beta, poly I:C (an inducer of IFN-alpha/beta), and IFN-gamma can inhibit the ability of KLH-pulsed peritoneal macrophages to induce proliferation of syngeneic, KLH immune T lymphocytes in CBA/J mice. In this study, we show that this IFN-induced immunosuppression is not restricted to CBA/J (H-2k) mice but is also seen in BALB/cJ (H-2d) mice. A similar inhibition of proliferation is observed with the KLH-specific T cell hybridoma BDK, 100, which requires KLH-pulsed macrophages for optimum proliferation and IL-2 production. The immunosuppression produced by IFN was also independent of the antigen employed. Inhibition of T lymphocyte proliferation was observed when casein, instead of KLH, was used to immunize T cells and to pulse peritoneal macrophages in vivo. Utilizing KLH and casein, the antigen specificity of the inhibition was demonstrated. Therefore, the inhibition by the IFN-inducer poly I:C of macrophage-induced, antigen-specific T cell proliferation is not limited by H-2 type of the mice or to one antigen.     

Accessory cell function of human endothelial cells. I. A subpopulation of Ia positive cells is required for antigen presentation

The expression of class I and class II HLA antigens on preparations of human endothelial cells, isolated from umbilical cord veins, was investigated by immunofluorescence. While virtually all endothelial cells expressed class I antigens, less than 1% were positive for class II antigens, as detected with a panel of 10 different monoclonal antibodies. Antigen specific T cell lines proliferated in response to mumps antigen in the presence of endothelial cells or blood monocytes from HLA-DR matched donors. However, these T cell lines failed to respond in the absence of accessory cells or when accessory cells from HLA-D-region mismatched cord donors were used. The ability of both monocytes and endothelial cells to present antigen was abolished by treatment of the cells with monoclonal antibodies specific for either class I or class II HLA antigens plus complement. Similar treatment with monoclonal antibodies specific for monocytes greatly reduced antigen presentation by endothelial cells. These results indicate that preparations of endothelial cells contain a subpopulation of Ia positive cells, distinct from monocytes, which are required for antigen presentation.     

IL-15 is required for sustained lymphopenia-driven proliferation and accumulation of CD8 T cells

Naive T cells undergo slow homeostatic proliferation in response to T cell lymphopenia, which is also called lymphopenia-induced proliferation (LIP). IL-7 is critically required for this process, but previous studies suggested IL-15 was expendable for LIP of naive CD8 T cells. In contrast, we show that IL-15 is important for sustained CD8 T cell proliferation and accumulation in a lymphopenic setting, as revealed by truncated LIP in IL-15(-/-) hosts. At the same time, we find that IL-12 enhances LIP by acting directly on the CD8 T cells and independently of IL-15, suggesting distinct pathways by which cytokines can regulate homeostatic proliferation. Interestingly, the memory-phenotype CD8 T cell generated by LIP in IL-15(-/-) hosts are phenotypically distinct from the rare endogenous memory-phenotype cells found in IL-15(-/-) animals, suggesting these cells are generated by different means. These findings demonstrate that cytokine requirements for LIP change during the process itself, illustrating the need to identify factors that regulate successive stages of lymphopenia-driven proliferation.