Evidence that the leukocyte-common antigen is required for antigen-induced T lymphocyte proliferation

The leukocyte-common antigen (L-CA) is a family of large molecular weight glycoproteins uniquely expressed on the surface of all nucleated cells of hematopoietic origin. The glycoprotein consists of a heavily glycosylated exterior domain, a single membrane spanning region, and a large cytoplasmic domain that contains tyrosine phosphatase activity. To investigate the function of this family, we generated T cell clones that lacked L-CA (L-CA-). The expression of the alpha beta T cell receptor, CD3, CD4, IL-2 receptor (p55), LFA-1, Thy-1, and Pgp-1 (CD44) was normal. The L-CA- T cell clones failed to proliferate in response to antigen or cross-linked CD3; however, they could still proliferate in response to IL-2. An L-CA+ revertant was obtained and the ability to proliferate in response to antigen and cross-linked CD3 was restored. These data indicate that L-CA is required for T cells to enter into cell cycle in response to antigen.     

Molecular interactions required for triggering alloantigen-specific cytolytic T lymphocytes

Affinity-purified class I proteins in detergent solution can be directly immobilized on plastic by a simple dilution procedure. The immobilized Ag retains its native structure and will trigger specific, TCR-mediated degranulation of cloned CTL. Stimulation of the response is dependent on the surface density of Ag, and displays a critical threshold density below which response does not occur. Individual clones differ with respect to the threshold density required for activation, but these differences are not large. With one exception, the cloned lines examined respond to Ag densities comparable to that found on normal allogeneic cells, and critical threshold densities varied over about a fourfold range. Coimmobilization of alloantigen and nonantigenic class I protein of a different specificity has the effect of decreasing the threshold density of alloantigen required for response to occur. This augmentation is specific for class I, coimmobilized class II protein does not affect responses, and is very likely mediated by Lyt-2 (CD8) interaction with nonpolymorphic determinants on the class I protein. Thus, class I alloantigen is the necessary and sufficient ligand for activation of most allogeneic CTL clones, and both TCR and Lyt-2 interactions contribute to the response. The results described here for effector CTL are compared with those previously found in examining the ligand requirements for activation of precursor CTL.     

Protein kinase C required for cytotoxic T lymphocyte triggering

The role of protein kinase C (PK-C) in triggering the lytic response of cytotoxic T lymphocytes (CTL) has been examined. Both target cell lysis and the release of CTL-associated serine esterase (SE), a marker for cytotoxic granules, were used as indicators of the CTL lytic response. We found triggering of the CTL lytic response occurred when both a PK-C activator, phorbol 12-myristate 13-acetate (PMA), and a calcium ionophore, ionomycin, were added to CTL. The previously described inactivation of the CTL lytic response by long term treatment (24 hr) with PMA was also investigated. CTL cultured with PMA for 24 hr were unable to mediate target cell lysis or release SE; this inability to respond correlated with an absence of PK-C activity. Incubation of the PMA-treated CTL in the absence of PMA for an additional 24 hr resulted in recovery of PK-C activity, SE release, and the lytic response. These experiments strongly suggest that PK-C is involved with the transmembrane signaling required for SE release which is a necessary event in CTL-mediated target cell lysis.     

CD152 ligation by CD80 on T cells is required for the induction of unresponsiveness by costimulation-deficient antigen presentation

Two apparently contradictory observations have been made concerning peripheral T cell tolerance; costimulation-deficient Ag presentation leads to unresponsiveness, and CTLA4 (CD152) ligation is required for unresponsiveness to be induced. This issue was addressed using a CD80- CD86low B cell line to present Ag to DO.11.10 naive CD4+ T cells. Proliferation was substantially enhanced by anti-CD80 or anti-CD152, but was inhibited by anti-CD86. Furthermore, anti-CD80 partially, and anti-CD152 totally protected cloned DO.11.10 T cells from the induction of unresponsiveness following culture with peptide and Chinese hamster ovary H2-Ad+ CD80- CD86- cells. Fab of anti-CD80 caused similar enhancement, and coimmobilized anti-CD80 failed to costimulate the anti-CD3 response of purified T cells, indicating that direct signaling by anti-CD80 was not responsible for these effects. The possibility that anti-CD80 liberated CD28 molecules that were sequestered by the T cell-expressed CD80, enabling them to coaggregate with TCR:CD3 complexes was excluded by finding that anti-CD80 and anti-CD152 individually caused maximal enhancement, rather than having additive effects. These data suggest that T cell-expressed CD80 has a regulatory function and plays a key role in the induction of unresponsiveness due to costimulation-deficient Ag presentation by the ligation of CD152 on neighboring, or even the same, T cell.     

Regulation of oxidative stress responses by ataxia-telangiectasia mutated is required for T cell proliferation

Mutations in the gene encoding ataxia-telangiectasia (A-T) mutated (Atm) cause the disease A-T, characterized by immunodeficiency, the molecular basis of which is not known. Following stimulation through the TCR, Atm-deficient T cells and normal T cells in which Atm is inhibited undergo apoptosis rather than proliferation. Apoptosis is prevented by scavenging reactive oxygen species (ROS) during activation. Atm therefore plays a critical role in T cell proliferation by regulating responses to ROS generated following T cell activation. The inability of Atm-deficient T cells to control responses to ROS is therefore the molecular basis of immunodeficiency associated with A-T.     

Colitis immunoregulation by CD8+ T cell requires T cell cytotoxicity and B cell peptide antigen presentation

Deficient immunoregulation by CD4+ T cells is an important susceptibility trait for inflammatory bowel disease, but the role of other regulatory cell types is less understood. This study addresses the role and mechanistic interaction of B cells and CD8+ T cells in controlling immune-mediated colitis. The genetic requirements for B cells and CD8+ T cells to confer protective immunoregulation were assessed by cotransfer with colitogenic Galphai2-/- T cells into immune-deficient mice. Disease activity in Galphai2-/- T cell recipients was evaluated by CD4+ T intestinal lymphocyte abundance, cytokine production levels, and large intestine histology. B cells deficient in B7.1/B7.2, CD40, major histocompatibility complex (MHC) II (Abb), or native B cell antigen receptor (MD4) were competent for colitis protection. However, transporter-1-deficient B cells failed to protect, indicating a requirement for peptide MHC I presentation to CD8+ T cells. CD8+ T cells deficient in native T cell receptor repertoire (OT-1) or cytolysis (perforin-/-) also were nonprotective. These finding reveal an integrated role for antigen-specific perforin-dependent CD8+ T cell cytotoxicity in colitis immunoregulatory and its efficient induction by a subset of mesenteric B lymphocytes.     

Hyaluronan synthesis is required for IL-2-mediated T cell proliferation

Hyaluronan (HA) is a glycosaminoglycan composed of N-acetylglucosamine and glucuronic acid subunits. Previous studies have suggested that CD44 expressed by T cells bind exogenous HA for their proliferation. However, HA endogenously synthesized by T cells may participate in their autocrine proliferation. In this study, we examined the role of endogenous HA in T cell proliferation using the highly specific HA synthase inhibitor, 4-methylumbelliferone (4-MU). We found that 4-MU inhibited the mitogen-induced synthesis of HA by T cells. Moreover, 4-MU inhibited T cell proliferation in a dose-dependent manner when cells were cultured with different stimuli, including Con A, PMA/ionomycin, and allogeneic spleen cells. Furthermore, 4-MU inhibited mitogen-stimulated IL-2 secretion, suggesting that HA may play a role in the production of this cytokine. Addition of IL-2 to T cells treated with 4-MU and Con A reversed the block in cell proliferation, showing that impaired IL-2 production is a likely mechanism for the inhibited division of T cells. Surprisingly, an anti-CD44 Ab antagonistic for HA binding did not reduce IL-2 secretion or T cell proliferation. Importantly, 4-MU did not alter the surface expression of CD44 or the ability of CD44 to bind to HA. Thus, HA-mediated IL-2 production and T cell proliferation are CD44 independent. Our results strongly suggest that HA synthesized by T cells themselves is critical for their IL-2-mediated proliferation and have revealed a previously unrecognized role for endogenous HA in T cell biology.     

Accessory cell stimulation of T cell proliferation requires active antigen processing, Ia-restricted antigen presentation, and a separate nonspecific 2nd signal

The roles of Ia+ accessory cells in H-2-restricted stimulation of antigen-specific T cell proliferation were explored in an in vitro model. L-glutamic acid60-L-alanine30-L-tyrosine10-(GAT) primed BALB/c nylon wool-passed T cells were depleted of Ia+ antigen-presenting cells (APC) by treatment with monoclonal anti-Ia antibody plus complement. Such cells failed to respond to soluble GAT, or to soluble GAT in the presence of phorbol myristic acetate (PMA), which is known to stimulate production of, or replace, IL-1 in vitro. Addition of gamma-irradiated syngeneic spleen cells reconstituted the response to soluble GAT, but addition of ultraviolet (UV) light-irradiated spleen cells did not, even in the presence of PMA. Preincubation of cells with GAT for 24 hr, followed by washing, then gamma irradiation, generated a cell population able to stimulate GAT-primed T cells to proliferate. The same pulsed cells exposed to UV irradiation failed to stimulate T cell responses unless PMA was added to the cultures. The relevant cells in this UV-irradiated population are Ia+. It is concluded that a finite period of time for interaction of metabolically intact APC with antigen is required before creation of an appropriate (Ia + antigen) signal recognized by the T cell. In addition to such Ia-restricted antigen presentation, however, a 2nd nonspecific signal, again requiring metabolically active APC for elaboration, is necessary for detectable T cell activation. These studies thus define 3 separable activities of APC during the process of H-2 restricted T cell activation.     

Metabolically active antigen presenting cells are required for human T cell proliferation in response to the superantigen streptococcal M protein

Abstract M protein from type 5 group A streptococci has been identified as a member of the family of polyclonal T cell activators termed superantigens because it preferentially stimulates T cells bearing specific Vβ elements of the T cell receptor (TCR). In this study the molecular and cellular requirements for presentation of this protein to T cells were investigated. Only accessory cells (AC) expressing class II major histocompatibility complex (MHC) molecules were capable of supporting T cell activation in response to a 22 kDa fragment of M protein (pep M). Despite the need for class II elements, processing of pep M5 by the antigen-presenting cells (APC) was not required for T cell proliferation induced by pep M5. Fixation of APC by paraformaldehyde (PF) treatment impaired their ability to induce optimal T cell proliferation in response to pep M5 withouth significantly affecting interleukin (IL-2) production. In contrast, PF-fixation of cells from the B cell lymphoma line, Raji, did not affect their ability to present pep M5 to human T cells. Addition of rIL-1 and IL-6 to PF-treated APC restored pep M5-induced blastogenesis. Our data suggest that pep M5 directly associates with HLA class II molecules forming a complex that can induce IL-2 production but not optimal proliferation by T cells. Additional signals provided by the AC are required to trigger optimal T cell proliferation in response to this superantigen.     

The CD154-CD40 T cell costimulation pathway is required for host sensitization of CD8(+) T cells by skin grafts via direct antigen presentation

Although the CD154-CD40 T cell costimulation pathway has been shown to mediate alloimmune responses in normal recipients, little is known about its role in sensitized hosts. In this work, by using novel models of cardiac allograft rejection in skin-sensitized CD154- and CD40-deficient mice, we reaffirm the key role of CD154-CD40 signaling in host sensitization to alloantigen in vivo. First, we identified CD8(+) T cells as principal effectors in executing accelerated rejection in our model. Disruption of CD154-CD40 signaling in recipients at the T cell side (CD154-deficient) but not at the APC side (CD40-deficient) abrogated accelerated (<2 days) rejection and resulted in long-term (>100 days) graft survival. This suggests that the CD154-dependent mechanism in host CD8(+) T cell sensitization operates via the direct Ag presentation. Then, in comparative studies of alloimmune responses in CD154-deficient and wild-type recipients, we showed that, although alloreactive B cell responses were inhibited, alloreactive T cell responses were down-regulated selectively in the CD8(+) T cell compartment, leaving CD4(+) T cells largely unaffected. This unique alteration in host alloreactivity, seen not only in peripheral lymphocytes but also in allograft infiltrate, may represent the key mechanism by which disruption of CD154-CD40 signaling prevents sensitization to alloantigen in vivo and leads to long-term allograft survival.     

Analysis of the monocyte Fc receptors and antibody-mediated cellular interactions required for the induction of T cell proliferation by anti-T3 antibodies

The induction of human T cell proliferation by antibodies that cross-link T3 antigens is dependent on functional interactions of anti-T3 antibodies with monocyte Fc receptors. In this report, we used a panel of anti-T3 antibodies of differing heavy chain isotype and a variety of other monoclonal antibodies to analyze several features of the antibody-mediated interactions between T cells and monocytes that are required for mitogenesis. Whereas three IgG2a anti-T3 antibodies were mitogenic for cells from all individuals, IgM and IgG2b anti-T3 antibodies did not induce T cell proliferation in any donor and could block the proliferative responses induced by other mitogenic anti-T3 antibodies. Dose-response analyses with four IgG1 anti-T3 antibodies demonstrated donor heterogeneity as reported by other investigators. However, in contrast to these previous reports, high concentrations of IgG1 anti-T3 antibodies were found to be mitogenic for all donors, indicating that this heterogeneity is based on relative rather than absolute defects in low responder monocytes. Cell mixing experiments in which monocytes from two different low responder donors were co-cultured with T cells and IgG1 anti-T3 antibodies did not identify any complementary defects, suggesting that the low responder phenotype results from a relatively restricted polymorphism. To assess the nature of the signals required for inducing T cell proliferation, nonmitogenic anti-T3 antibodies were co-cultured with other pan-T cell antibodies having the IgG2a isotype. The combination of signals from T3 antigen cross-linkage and those independently generated by other IgG2a antibodies bound to monocyte Fc receptors did not induce T cell proliferation. Hence, it appears that the T3 antigen or closely associated structures must be clustered at the monocyte membrane for mitogenesis. Finally, in competitive inhibition experiments, the isotype specificity of monocyte Fc receptors involved in the induction of T cell proliferation was examined. Two distinct Fc receptor sites, one that binds murine IgG2a and IgG3 antibodies and a second that binds murine IgG1 antibodies, were identified. Murine IgM or IgG2b did not appear to bind either of these receptor sites. Taken together, these data indicate that human monocytes have two distinct Fc receptor sites, which must specifically and directly interact with T cell-bound anti-T3 antibodies for mitogenesis.     

Memory CD8+ T cell responses expand when antigen presentation overcomes T cell self-regulation

Antimicrobial memory CD8+ T cell responses are not readily expanded by either repeated infections or immunizations. This is a major obstacle to the development of T cell vaccines. Prime-boost immunization with heterologous microbes sharing the same CD8+ epitope can induce a large expansion of the CD8+ response; however, different vectors vary greatly in their ability to boost for reasons that are poorly understood. To investigate how efficient memory T cell expansion can occur, we evaluated immune regulatory events and Ag presentation after secondary immunization with strong and weak boosting vectors. We found that dendritic cells were essential for T cell boosting and that Ag presentation by these cells was regulated by cognate memory CD8+ T cells. When weak boosting vectors were used for secondary immunization, pre-established CD8+ T cells were able to effectively curtail Ag presentation, resulting in limited CD8+ T cell expansion. In contrast, a strong boosting vector, vaccinia virus, induced highly efficient Ag presentation that overcame regulation by cognate T cells and induced large numbers of memory CD8+ T cells to expand. Thus, efficient targeting of Ag to dendritic cells in the face of cognate immunity is an important requirement for T cell expansion.     

AKAPs in lipid rafts are required for optimal antigen presentation by dendritic cells

Dendritic cell (DC) maturation and antigen presentation are regulated by activation of protein kinase A (PKA) signaling pathways, through unknown mechanisms. We have recently shown that interfering with PKA signaling through the use of anchoring inhibitor peptides hinders antigen presentation and DC maturation. These experiments provide evidence that DC maturation and antigen presentation are regulated by A-kinase anchoring proteins (AKAPs). Herein, we determine that the presence of AKAPs and PKA in lipid rafts regulates antigen presentation. Using a combination of western blotting and immuno-cytochemistry, we illustrate the presence of AKAP149, AKAP79, Ezrin and the regulatory subunits of PKA in DC lipid rafts. Incubation of DCs with the type II anchoring inhibitor, AKAP-in silico (AKAP-IS), removes Ezrin and RII from the lipid raft without disrupting raft formation. Addition of a lipid raft disruptor, methyl-β-cyclodextrin, blocks the efficacy of AKAP-IS, suggesting that the lipid raft must be intact for AKAP-IS to inhibit antigen presentation. Ezrin and AKAP79 are present in the lipid raft of stimulated KG1 cells, but Ezrin is not present in the lipid raft of unstimulated KG1 cells and AKAP79 levels are greatly diminished, suggesting that Ezrin and AKAP79 may be the key AKAPs responsible for regulating antigen presentation.     

Proliferating cell nuclear antigen as the cell cycle sensor for an HLA-derived peptide blocking T cell proliferation

Synthetic peptides corresponding to structural regions of HLA molecules are novel immunosuppressive agents. A peptide corresponding to residues 65-79 of the alpha-chain of HLA-DQA03011 (DQ65-79) blocks cell cycle progression from early G1 to the G1 restriction point, which inhibits cyclin-dependent kinase-2 activity and phosphorylation of the retinoblastoma protein. A yeast two-hybrid screen identified proliferating cell nuclear Ag (PCNA) as a cellular ligand for this peptide, whose interaction with PCNA was further confirmed by in vitro biochemistry. Electron microscopy demonstrates that the DQ65-79 peptide enters the cell and colocalizes with PCNA in the T cell nucleus in vivo. Binding of the DQ65-79 peptide to PCNA did not block polymerase delta (pol delta)-dependent DNA replication in vitro. These findings support a key role for PCNA as a sensor of cell cycle progression and reveal an unanticipated function for conserved regions of HLA molecules.     

Primary T cell expansion and differentiation in vivo requires antigen presentation by B cells

B cells are well documented as APC; however, their role in supporting and programming the T cell response in vivo is still unclear. Studies using B cell-deficient mice have given rise to contradictory results. We have used mixed BM chimeric mice to define the contribution that B cells make as APC. When the B cell compartment is deficient in MHC class II, while other APC are largely normal, T cell clonal expansion is significantly reduced and the differentiation of T cells into cytokine-secreting effector cells is impaired (in particular, Th2 cells). The development of the memory T cell populations is also decreased. Although MHC class II-mediated presentation by B cells was crucial for an optimal T cell response, neither a B cell-specific lack of CD40 (influencing costimulation) nor lymphotoxin alpha (influencing lymphoid tissue architecture) had any effect on the T cell response. We conclude that in vivo B cells provide extra and essential Ag presentation capacity over and above that provided by dendritic cells, optimizing expansion and allowing the generation of memory and effector T cells.     

Role of the CD22 human B cell antigen in B cell triggering by anti-immunoglobulin

As B cells mature during ontogeny the CD22 human differentiation Ag is exported from the cytoplasm onto the membrane. Surface expression is lost in terminal differentiation and after activation. In tonsils, CD22 is expressed on the surface of 60 to 80% of the dense B cells. Some IgM+ dense cells, however, and buoyant in vivo activated B cells are CD22-. This differential expression of CD22 and the finding that an anti-CD22 mAb augmented anti-Ig induced B cell proliferation suggested that CD22 may play a role in B cell activation. In this study we have found that CD22+ but not CD22- B cells could be triggered by anti-IgM or anti-IgD to have increased free intracellular calcium ([Ca2+]i). The presence of CD22 rather than of IgD seems to determine the ability of B cells to respond to anti-Ig with a [Ca2+]i flux. Also the proliferative response to anti-Ig or anti-Ig + B cell growth factor was restricted to the CD22+ population. Anti-CD22 mAb, although not inducing [Ca2+]i on their own after binding to B cells, did augment [Ca2+]i fluxes by anti-Ig when cross-linked. Together these results suggest that CD22 may regulate triggering of B cells through surface Ig perhaps by acting as a "bridge" to transmit an early signal into the cytoplasm.     

Nucleoplasmic calcium is required for cell proliferation

Ca(2+) signals regulate cell proliferation, but the spatial and temporal specificity of these signals is unknown. Here we use selective buffers of nucleoplasmic or cytoplasmic Ca(2+) to determine that cell proliferation depends upon Ca(2+) signals within the nucleus rather than in the cytoplasm. Nuclear Ca(2+) signals stimulate cell growth rather than inhibit apoptosis and specifically permit cells to advance through early prophase. Selective buffering of nuclear but not cytoplasmic Ca(2+) signals also impairs growth of tumors in vivo. These findings reveal a major physiological and potential pathophysiological role for nucleoplasmic Ca(2+) signals and suggest that this information can be used to design novel therapeutic strategies to regulate conditions of abnormal cell growth.     

Analysis of the role of bleomycin hydrolase in antigen presentation and the generation of CD8 T cell responses

Long oligopeptides (>10 residues) are generated during the catabolism of cellular proteins in the cytosol. To be presented to T cells, such peptides must be trimmed by aminopeptidases to the proper size (typically 8-10 residues) to stably bind to MHC class I molecules. Aminopeptidases also destroy epitopes by trimming them to even shorter lengths. Bleomycin hydrolase (BH) is a cytosolic aminopeptidase that has been suggested to play a key role in generating MHC class I-presented peptides. We show that BH-deficient cells from mice are unimpaired in their ability to present epitopes from N-extended precursors or whole Ags and express normal levels of MHC class I molecules. Similarly, BH-deficient mice develop normal CD8(+) T cell responses to eight epitopes from three different viruses in vivo. Therefore, BH by itself is not essential for the generation or destruction of MHC class I peptides. In contrast, when BH(-/-) mice are crossed to mice lacking another cytosolic aminopeptidase, leucine aminopeptidase, the resulting BH(-/-)leucine aminopeptidase(-/-) progeny show a selective increase in CD8(+) T cell responses to the gp276 epitope from lymphocytic choriomeningitis virus, whereas the ability to present and respond to several other epitopes is unchanged. Therefore, BH does influence presentation of some Ags, although its role is largely redundant with other aminopeptidases.     

T cell receptor triggering induces responsiveness to interleukin 1 and interleukin 2 but does not lead to T cell proliferation

The antigen-like activity of monoclonal antibodies directed at the T3-Ti antigen receptor complex of human T lymphocytes was employed to study activation requirements of resting T cells. Efficient antigen recognition (signal 1) by T lymphocytes requires multimeric antigen receptor triggering because under appropriate experimental conditions soluble ligands do not produce this initial signal for T cell activation. The latter leads to receptiveness for both interleukin 1 (IL 1) and interleukin 2 (IL 2). Importantly, induction of proliferation requires an additional signal (signal 2), namely IL 1, which appears to be required to enable optimal secretion of IL 2. In contrast, presensitized T lymphocytes do not require IL 1 for IL 2 production. In this case, antigen receptor oligomerization is in itself sufficient to induce IL 2 receptor expression, and IL 2 secretion as well.     

Inhibition by brefeldin A of the specific B cell antigen presentation to MHC class II-restricted T cells

We have shown previously that specific Ag presentation is prevented by the inhibition of protein synthesis but nonspecific presentation is not. In the present paper, Ag presentation by Ag-specific B cells was examined for sensitivity to brefeldin A (BFA), which blocks protein export from the endoplasmic reticulum. A20-HL B lymphoma expressing surface receptors specific for TNP was used as a B cell, and TNP-OVA was used as a specific Ag. The presence of BFA during pulsing of A20-HL cells with TNP-OVA inhibited the ability of the pulsed cells to stimulate 42-6A T cell clone, specific for OVA323-339 and Iad. The inhibition was not due to nonspecific toxicity of BFA, because the presence of BFA during pulsing of A20-HL cells with OVA323-339 did not affect their APC function. Ag binding to the receptor on A20-HL cells and internalization by the cells were observed in the presence of BFA. Thus, BFA might inhibit intracellular processing of specific Ag or intracellular complex formation of antigenic peptide from specific Ag with MHC class II molecules. Nonspecific Ag presentation by A20-HL cells, however, was resistant to BFA. A20-HL cells pulsed with OVA in the presence of BFA, even after fixation, could stimulate 42-6A cells to produce IL-2, although the IL-2 production was lower than that induced by A20-HL cells pulsed in the absence of BFA. These results suggest that the processing pathways for specific Ag and nonspecific Ag are different from each other, at least partly, in A20-HL cells.