Physical and functional association of the major histocompatibility complex class I heavy chain alpha3 domain with the transporter associated with antigen processing.

CD8+ T lymphocytes recognize antigens as short, MHC class I-associated peptides derived by processing of cytoplasmic proteins. The transporter associated with antigen processing translocates peptides from the cytosol into the ER lumen, where they bind to the nascent class I molecules. To date, the precise location of the class I-TAP interaction site remains unclear. We provide evidence that this site is contained within the heavy chain alpha3 domain. Substitution of a 15 amino acid portion of the H-2Db alpha3 domain (aa 219-233) with the analogous MHC class II (H-2IAd) beta2 domain region (aa 133-147) results in loss of surface expression which can be partially restored upon incubation at 26 degrees C in the presence of excess peptide and beta2-microglobulin. Mutant H-2Db (Db219-233) associates poorly with the TAP complex, and cannot present endogenously-derived antigenic peptides requiring TAP-dependent translocation to the ER. However, this presentation defect can be overcome through use of an ER targeting sequence which bypasses TAP-dependent peptide translocation. Thus, the alpha3 domain serves as an important site of interaction (directly or indirectly) with the TAP complex and is necessary for TAP-dependent peptide loading and class I surface expression.

Paradoxical arrest in lupus activity in BXSB mice with highly autoreactive T cells.

T cells with high avidity for antigens are thought to mediate more effective immunity against foreign antigens and cause more severe autoimmunity. The impact of T cell receptor (TCR) avidity on the development of lupus has not been investigated. We took advantage of a transgenic mouse strain (designated MTB) that has a diverse T cell population and a globally stronger reactivity to self. [MTBxBXSB]F1 mice displayed accelerated lupus relative to the [WTxBXSB]F1 controls. The severity of lupus and the activation of T cells subsided with aging, when elevated IL-10 production by Tr1 cells was observed. Thus, chronic high avidity interactions of T cells with self-antigens can lead to an age associated increase in IL-10 production. This could explain the age-associated reduction of the incidence of lupus, as well as other autoimmune diseases. Furthermore, the principle of Tr1 differentiation based on diverse T cells with high avidity for self may potentially be used as a therapeutic strategy in the treatment of lupus.

Evidence for target tissue regulation of resistance to the induction of experimental allergic encephalomyelitis in AO rats.

A myelin basic protein (MBP)-specific T cell line derived from F1 hybrids between experimental allergic encephalomyelitis (EAE)-susceptible DA (RT1avl) strain and EAE-resistant AO (RT1u) strain was capable of inducing clinical EAE in F1 hybrids and DA, but not in AO rats. In vitro restimulation with MBP presented by AO antigen-presenting cells (APC) resulted in the generation of a MBP-specific subline restricted by RT1u MHC products which induced clinical EAE in F1 hybrids but not in the AO parental strain. Deletion of hosts' leukocytes using sublethal irradiation and cytotoxic drugs did not abrogate the resistance of AO rats, which argues against the involvement of hosts' lymphoid cells in the regulation of autoagression. Thus, mechanism(s) regulating the activity of autoagressive T cells on functional elements in the target tissue might be responsible for differences in susceptibility to EAE.

Analysis of T cell subsets after induction of experimental autoimmune encephalomyelitis in susceptible and resistant strains of rats.

T cell subsets in the peripheral blood, draining lymph node (DLN) and spinal cord lesions were analysed after the induction of experimental autoimmune encephalomyelitis (EAE) in susceptible (DA) and relatively resistant (AO) rats. In DA rats, a significantly higher number of CD4+ cells were generated in the DLN, in response to both nervous tissue antigens and complete Freund's adjuvant (CFA), compared to AO rats. In the peripheral blood of DA rats, the percentage as well as absolute number of CD4+ cells increased in the preclinical phase of EAE, but declined as the disease developed. The percentage of CD8+ cells decreased in both these phases of EAE. In resistant AO rats, however, there were no significant changes in the T lymphocyte subset percentages after EAE induction, although the absolute number of peripheral blood CD4+ cells again increased in the preclinical stage of EAE. In the CFA-treated control DA rats, the absolute number of CD4+ cells was increased in the preclinical phase. However, no decline comparable to that seen in diseased animals followed. It is concluded that the generation of CD4+ cells in response to this antigen is strain specific and, since the cells are released into the circulation, will affect the balance between the T cell subsets in the peripheral blood during the development of EAE.

Peptide loading of nascent MHC class I molecules.

A critical molecular interaction during assembly of the major histocompatibility complex (MHC) class I molecules takes place between the heavy chain and the transporter-associated with antigen-processing (TAP) complex. The recent mapping of regions of the heavy chain involved in the binding to TAP suggests a complex molecular interaction essential for the cell surface expression of the MHC class I. The advances made in understanding the TAP-MHC class I interaction are reviewed and discussed here.

Anti-CD3-induced cell death in T cell hybridomas: mitochondrial failure and DNA fragmentation are distinct events.

Triggering of the T cell receptor of T cell hybridomas leads to interleukin (IL) 2 secretion, inhibition of spontaneous growth, degradation of genomic DNA and cell death. We have investigated the relationship between the ability of mitochondria to convert 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), DNA fragmentation and growth arrest in hybridomas stimulated with anti-CD3/T cell receptor antibodies. We describe a variant T hybridoma whose mitochondrial function remains unaffected upon stimulation with anti-CD3 antibody, although it does undergo DNA fragmentation. By contrast, treatment of another anti-CD3-stimulated T hybridoma with endonuclease inhibitor completely inhibits the DNA fragmentation response but not mitochondrial failure induced by anti-CD3 antibody. Thus, we have been able to dissociate anti-CD3-induced mitochondrial failure and DNA fragmentation, suggesting that they are separate events. Although both undoubtedly contribute to cell death induced by activation the primary cause of death may be mitochondrial failure rather than DNA fragmentation.

Dual MHC class I and class II restriction of a single T cell receptor: distinct modes of tolerance induction by two classes of autoantigens.

In the final stages of thymic development, immature T cells undergo three distinct processes (positive selection, negative selection, and lineage commitment) that all depend on interactions of thymocyte TCRs with MHC molecules. It is currently thought that TCRs are preferentially restricted by either MHC class I or class II molecules. In this report, we present direct evidence that the TCR previously described as H-Y/H-2Db specific cross-reacts with H-2IAb if expressed in CD4+ cells. We also demonstrate an increase in thymocyte numbers in H-Y TCR-trangenic mice deficient in MHC class II, suggesting a relatively discrete form of negative selection by MHC class II compared with that induced by H-Y/H-2Db. We propose that inability to generate CD4+ T cells expressing H-Y TCR in different experimental settings may be due to tolerance to self-MHC class II. These results, therefore, support an intriguing possibility that tolerance to self may influence and/or interfere with the outcome of the lineage commitment.

MHC class I is required for peripheral accumulation of CD8+ thymic emigrants.

MHC molecules influence the fate of T lymphocytes at two important stages of their differentiation. Recognition of self peptide/MHC complexes in the thymus determines whether immature T cells should live and mature into immunocompetent T cells or whether they should die. In the periphery, recognition of Ags presented by MHC molecules induces T cell activation, proliferation, and differentiation into effector/memory T cells. We describe in this work a third role that MHC molecules play in T cell physiology. CD8+ thymic emigrants require presence of MHC class I molecules in the periphery to seed the peripheral lymphoid organs. Numbers of CD8+ T cells are reduced severely in both the thymus and the periphery of beta2-microglobulin-deficient (beta2m[-/-]) mice. When grafted with wild-type (beta2m[+/+]) thymic epithelium, immature beta2m(-/-) T cells that populate the graft develop into functional mature CD8+ cells. However, significant numbers of peripheral CD8+ cells in grafted beta2m(-/-) mice can be observed only after injection of MHC class I-expressing cells in the periphery. Thus, naive T cells in the periphery do not passively await antigenic stimulation, but actively engage in interactions with self MHC molecules that may promote their survival.

Altered effector responses of H-Y transgenic CD8+ cells.

The primary role of CD8+ T cells is to destroy virus-infected or tumor cells expressing cognate antigens in the form of peptide-MHC class I complexes. This destruction is primarily achieved by the actions of lytic mediators and/or lymphokines. In this report, we show that mature, H-Y/Db-specific CD8+ T cells from H-Y TCR transgenic mice were unable to efficiently release lytic mediators after antigenic stimulation. However, anti-TCR antibody induced granule exocytosis and target cell lysis, arguing against signaling and/or cytolytic machinery defects in CD8+ cells, and demonstrating that male antigen induced differentiation of 'naive' into effector CD8+ cells. Stimulation of H-Y-specific effector CD8+ T cells with male stimulators, although insufficient to induce lytic granule release, was sufficient for H-Y-specific IFN-gamma production. Unexpectedly, this effector-phase IFN-gamma production was dependent on B7-2 engagement. We hypothesize that altered effector functions in H-Y-specific CD8+ cells are due to the low affinity of TCR-antigen-MHC interaction and/or the elevated threshold of CD8+ T cell activation.

Phosphorylation of murine CD8 alpha is not essential for responses of T cell hybridomas to antigen.

CD8 T cell differentiation antigens, expressed on class I-restricted T cells, have a key role in the control of recognition and response of these cells to antigen. It has been suggested that these molecules function as co-receptors together with antigen-specific T cell receptors to regulate T cell responses. We have addressed the question of whether cytoplasmic serine phosphorylation, which occurs on CD8 molecules after activation by antigen or phorbol esters, is relevant to its co-receptor function. By mutagenesis, we show that phorbol ester-induced phosphorylation occurs exclusively on CD8 alpha serine residue 216. However, inhibition of CD8 polypeptide phosphorylation does not appear to have a detrimental effect on several responses of CD8-dependent transfectants to antigen. This is in contrast to results reported with CD4 (N.Glaichenhaus, N.Shastri, D.R. Littmann and J.M.Turner. 1991. Cell, 64:511), suggesting that CD4 and CD8 molecules may play somewhat different roles in the control of T cell activation.

Prevention of antigen-induced microtubule organizing center reorientation in cytotoxic T cells by modulation of protein kinase C activity.

Lysis of target cells (TC) by cytotoxic T lymphocytes (CTL) is achieved by directional exocytosis of cytolytic molecules-perforin and granzymes. They are stored within lytic granules which can be readily released following antigenic stimulation. Secretion of lytic molecules appears to be controlled by protein kinase C (PKC) activity, since specific modulators of PKC activity abolish the lysis of TC. We have examined the effect of PKC modulation on some of the earliest events in the perforin/granzyme-mediated cytotoxicity. De novo synthesis of perforin mRNA, required for the refilling of granules and sustained cytotoxicity, seems to be unaltered in the presence of PKC modulators. Immunofluorescent studies of CTL-TC conjugates revealed that PKC modulation impairs reorientation of the microtubule organizing center toward the contact point with the TC, which accounts for the specific direction of lytic granules exocytosis. Thus, it appears that PKC regulates exocytosis of lytic granules by governing microtubule reorganization, one of the initial steps in perforin/granzyme-mediated cytotoxicity.

A thymic epithelial cell line induces both positive and negative selection in the thymus.

TCR engagement in the thymus results in both survival and elimination signals for developing thymocytes. To examine whether both signals can be provided by the same cell type, we investigated the ability of a thymic epithelial cell (TEC) line 427.1, previously shown to allow positive selection in the thymus, to induce clonal deletion of immature thymocytes. [H-2b/s-->H-2s] bone marrow chimeras are non-responsive to antigens in the context of H-2b. However, chimeras that underwent intrathymic injection of H-2b/s 427.1 cells expressing vesicular stomatitis virus (VSV) nucleocapsid antigen acquired the ability to raise influenza, but not VSV specific H-2b restricted cytotoxic T lymphocyte (CTL) responses. The ability of 427.1 cells to delete CD4+CD8+ thymocytes was determined using mice transgenic for the TCR specific for ovalbumin (OVA) in the context of H-2Kb. OVA transfected, but not mock transfected 427.1 TECs, induced in vitro deletion of CD4+CD8+ TCR transgenic thymocytes manifested as a down-modulation of CD4 and CD8 molecules, a shift in the side versus forward scatter characteristics of thymocytes, and appearance of thymocytes with subdiploid content of DNA indicated the ongoing process of DNA fragmentation. The finding that the same TEC line is capable of inducing both positive and negative selection in the thymus suggests that thymocytes bearing TCRs specific for self peptides expressed by positively selecting thymic epithelium can be deleted. Therefore the expression of a unique set of MHC associated peptides by TECs does not appear to be the basis for the positive outcome of the TCR ligation on immature thymocytes.

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.

Modulation of CD8+ T cell response to antigen by the levels of self MHC class I.

The response of H-Y-specific TCR-transgenic CD8(+) T cells to Ag is characterized by poor proliferation, cytolytic activity, and IFN-gamma secretion. IFN-gamma secretion, but not cytotoxic function, can be rescued by the B7.1 molecule, suggesting that costimulation can selectively enhance some, but not all, effector CD8(+) T cell responses. Although the H-Y epitope binds H-2D(b) relatively less well than some other epitopes, it can induce potent CTL responses in nontransgenic mice, suggesting that the observed poor responsiveness of transgenic CD8(+) T cells cannot be ascribed to the epitope itself. Previously reported reactivity of this TCR to H-2A(b) is also not the cause of the poor responsiveness of the H-Y-specific CD8(+) T cells, as H-Y-specific CD8(+) T cells obtained from genetic backgrounds lacking H-2A(b) also responded poorly. Rather, reducing the levels of H-2(b) class I molecules by breeding the mice to (C57BL/6 x B10.D2)F(1) or TAP1(+/-) backgrounds partially restored cytotoxic activity and enhanced proliferative responses. These findings demonstrate that the self MHC class I gene dosage may regulate the extent of CD8(+) T cell responsiveness to Ag.

Experimental autoimmune encephalomyelitis in "low" and "high" interleukin 2 producer rats. I. Cellular basis of induction.

Albino Oxford (AO) rats in comparison to the Dark August (DA) strain exhibit lower susceptibility to the induction of experimental autoimmune encephalomyelitis (EAE), and interleukin 2 (IL-2) production by their spleen and lymph node cells is significantly lower. The cellular analysis of these differences in the outcome of the EAE induction, possibly related to the differences in the IL-2 production, revealed different changes in the T cell subsets in the draining lymph node (DLN) and different cellular composition of the mononuclear infiltrates in the central nervous system (CNS). After the encephalitogenic challenge, the frequency of CD8+ T cells was much higher and the expansion of CD4+ T cells was much lower in the DLN of "low" IL-2 producer rats. AO rats have not shown any clinical sign of EAE, although histological lesions in the early phases of EAE (Day 7-9) were similar to those seen in diseased DA rats. CD4/CD8 T cell ratios and the number of cells bearing receptor for IL-2 (IL-2-R+ cells) and cells bearing class II MHC antigens (Ia+) were significantly lower in the mononuclear cell infiltrates of AO rats. These data are compatible with the notion that CD4+ IL-2-R+ encephalitogenic T cells induce clinical signs of EAE in susceptible animals and show that CD8+ T cells are present in a higher percentage in the lesions of the symptom-free AO rats.

Alpha beta TCR+ cells are a minimal fraction of peripheral CD8+ pool in MHC class I-deficient mice.

MHC class I molecules play a role in the maintenance of the naive peripheral CD8+ T cell pool. The mechanisms of the peripheral maintenance and the life span of residual CD8+ cells present in the periphery of beta 2-microglobulin-deficient (beta 2m-/-) mice are unknown. We here show that very few CD8+ cells in beta 2m-/- mice coexpress CD8 beta, a marker of the thymus-derived CD8+ T cells. Most of the CD8 alpha+ cells express CD11c and can be found in beta 2m/RAG-2 double-deficient mice, demonstrating that these cells do not require rearranged Ag receptors for differentiation and survival and may be of dendritic cell lineage. Rare CD8 alpha+CD8 beta+ cells can be detected following in vivo alloantigenic stimulation 2 wk after the adult thymectomy. Selective MHC class I expression by bone marrow-derived cells does not lead to an accumulation of CD8 beta+ cells in beta 2m-/- mice. These findings demonstrate that 1) thymic export of CD8+ T cells in beta 2m-/- mice is reduced more severely than previously thought; 2) non-T cells expressing CD8 alpha become prominent when CD8+ T cells are virtually absent; 3) at least some beta 2m-/- CD8+ T cells have a life span in the periphery comparable to wild-type CD8+ cells; and 4) similar ligands induce positive selection in the thymus and survival of CD8+ T cells in the periphery.