Activation of human CD8+ alpha beta TCR+ cells by Mycobacterium tuberculosis via an alternate class I MHC antigen-processing pathway

Human immune responses to M. tuberculosis are characterized by activation of multiple T cell subsets including CD4+, CD8+, and gammadelta T cells, and the role of CD8+ alphabeta TCR+ T cells in this response is poorly understood. Stimulation of T cells from healthy tuberculin skin test-positive persons with live M. tuberculosis-H37Ra or soluble M. tuberculosis Ags readily up-regulated IL-2Ralpha (CD25) expression on CD8+ T cells. Purified resting and activated CD8+ T cells produced IFN-gamma and proliferated to both M. tuberculosis bacilli and soluble mycobacterial Ags with monocytes as APC. Precursor frequency of mycobacterial Ag-specific CD8+ T cells by IFN-gamma enzyme-linked immunospot was 5-10-fold lower than the precursor frequency of CD4+ T cells, and IFN-gamma secretion by CD8+ T cells was 50-100-fold lower. CD8+ T cells secreted approximately 10-fold less IFN-gamma per cell than CD4+ T cells in response to mycobacterial Ags. CD8+ T cell responses to M. tuberculosis bacilli were blocked by anti-MHC class I antibody and required Ag processing. Processing of M. tuberculosis bacilli by monocytes for presentation to MHC class I-restricted CD8+ T cells was insensitive to brefeldin A treatment, which blocks the conventional MHC class I Ag-processing pathway. These results represent the first demonstration that human cells can process pathogen Ags via an alternate Ag-processing pathway for MHC class I and suggest a mechanism for participation of IFN-gamma-secreting CD8+ T cells in the human immune responses to M. tuberculosis.     

Perforin-deficient CD8+ T cells: in vivo priming and antigen-specific immunity against Listeria monocytogenes

CD8+ T cells require perforin to mediate immunity against some, but not all, intracellular pathogens. Previous studies with H-2b MHC perforin gene knockout (PO) mice revealed both perforin-dependent and perforin-independent pathways of CD8+ T cell-mediated immunity to Listeria monocytogenes (LM). In this study, we address two previously unresolved issues regarding the requirement for perforin in antilisterial immunity: 1) Is CD8+ T cell-mediated, perforin-independent immunity specific for a single Ag or generalizable to multiple Ags? 2) Is there a deficiency in the priming of the CD8+ T cell compartment of PO mice following an immunizing challenge with LM? We used H-2d MHC PO mice to generate CD8+ T cell lines individually specific for three known Ags expressed by a recombinant strain of virulent LM. Adoptive transfer experiments into BALB/c host mice revealed that immunity can be mediated by PO CD8+ T cells specific for all Ags examined, indicating that perforin-independent immunity is not limited to CD8+ T cells that recognize listeriolysin O. Analysis of epitope-specific CD8+ T cell expansion by MHC class I tetramer staining and ELISPOT revealed no deficiency in either the primary or secondary response to LM infection in PO mice. These results demonstrate that the perforin-independent pathway of antilisterial resistance mediated by CD8+ T cells is generalizable to multiple epitopes. Furthermore, the results show that reduced antilisterial resistance observed with polyclonal PO CD8+ T cells is a consequence of a deficiency in effector function and not a result of suboptimal CD8+ T cell priming.     

Dual role of dendritic cells in the induction and down-regulation of antigen-specific cutaneous inflammation

We have previously reported that contact sensitivity (CS) to dinitrofluorobenzene (DNFB) in C57BL/6 mice was mediated by MHC class I-restricted CD8+ T cells and down-regulated by MHC class II-restricted CD4+ T cells. In this study, we analyzed the contribution of dendritic cells (DC) in the induction of these two T cell subsets endowed with opposite functions. Hapten-pulsed skin- and bone marrow-derived DC, obtained from either normal C57BL/6 mice or from MHC class II (I+ II-) and MHC class I (I- II+)-deficient mice, were tested for their ability to prime normal mice for CS to dinitrofluorobenzene. Expression of MHC class I molecules by transferred DC was mandatory both for the induction of CS and for the generation of hapten-specific CD8+ T cells in lymphoid organs. I+ II- DC were as potent as I+ II+ DC in priming for CS, demonstrating that activation of effector CD8+ T cells can occur independently of CD4+ T cell help. I- II+ DC could not immunize for CS, although they could sensitize for a delayed-type hypersensitivity reaction to protein Ags. Moreover, I- II+ DC injected simultaneously with cutaneous sensitization down-regulated the inflammatory response, suggesting that hapten presentation by MHC class II molecules could prime regulatory CD4+ T cells. These results indicate that DC can present haptenated peptides by both MHC class I and class II molecules and activate Ag-specific CD8+ effector and CD4+ regulatory T cell subsets, concurrently and independently.     

Spontaneous Borna disease in sheep and horses: immunophenotyping of inflammatory cells and detection of MHC-I and MHC-II antigen expression in Borna encephalitis lesions

Borna disease (BD) has been recognized as a virally induced T-cell dependent immunopathological disorder of the central nervous system (CNS), as shown by experimental infection of rats with Borna disease virus (BDV). In contrast to the rat model, little is known about the pathogenesis of spontaneous BD in sheep and horses. The present study describes the brain lesions of 12 ovine and 11 equine cases of naturally occurring BD. A set of monoclonal and polyclonal antibodies was used in order to determine the cells operative in encephalitic lesions and to detect expression of MHC-I and MHC-II products in the brains of affected animals. In all cases investigated, a reaction pattern similar to that reported for the acute phase of BD in experimentally infected rats was noted. In brief, the majority of inflammatory cells in perivascular infiltrates (PVI) as well as parenchymal and meningeal infiltrates were CD3 +. CD4 + cells outnumbered CD8 + cells in PVI as well as in the parenchyma. Macrophages (defined by lysozyme immunoreactivity) were seen less often and B-cells or plasma cells (cells positive for lambda or kappa light chains) were demonstrated at lower numbers. TCR-1 + cells were found on very rare occasions in PVI of some sheep. MHC-I and MHC-II products were constantly expressed on inflammatory cells but inconsistently on astrocytes and neurons. Neuronal degeneration was not a major feature.     

Respiratory syncytial virus infection of human respiratory epithelial cells up-regulates class I MHC expression through the induction of IFN-beta and IL-1 alpha

CD8+ T cells mediate some of the damage to the lung epithelium following respiratory syncytial virus (RSV) infection. Since CD8+ T cells recognize antigen-laden class I MHC molecules on the target cells, we examined in this study the expression of class I MHC by RSV-infected respiratory epithelial cells. Respiratory epithelial cell lines and bronchial epithelial cells from normal human tissue responded to RSV infection with an increased expression of class I MHC as determined by flow cytometry and immunoprecipitation of class I MHC from metabolically radiolabeled cells. The increase in class I MHC expression was dependent on infectious, replicating virus. UV-irradiated culture supernatants from RSV-infected A549 cells, when added to fresh A549 cell cultures, induced an increase in class I MHC expression by those cells. The class I MHC increasing activity within supernatants from A549 cells was due, in large part, to IFN-beta, and to a lesser extent to IL-1 alpha. The addition of neutralizing Abs to both cytokines completely blocked the increase in class I MHC expression by cells treated with the above-mentioned supernatants. These results demonstrate that RSV infection elicits IFN-beta production by respiratory epithelial cells, which in turn leads to an increase in their synthesis of class I MHC, which would facilitate their recognition and lysis by RSV-specific CD8+ T cells.     

Adoptively transferred CD4+ lymphocytes from CD8 -/- mice are sufficient to mediate the rejection of MHC class II or class I disparate skin grafts

Recent studies revealed that CD4+ cells initiate allograft rejection through direct recognition of allogeneic MHC class II Ags and indirect recognition of MHC peptides processed by self APCs. Both pathways were shown to help CD8+ cells that eventually lysed allogeneic MHC class I-presenting targets. There was little evidence, however, that CD4+ cells are sufficient for graft rejection. We studied skin graft rejection by CD8-deficient (CD8 -/-) mice. We showed that BALB/cJ(H-2d) CD8 -/- mice could reject allogeneic skin from C57BL/6J(H-2b) mice deficient in MHC class I or in MHC class II Ags. To understand the role of CD4+ cells in this process, we isolated them from CD8 -/- mice and transferred them to BALB/cJ nude mice that had been grafted with allogeneic skin (H-2b) from animals deficient in MHC class I or MHC class II. Nude mice injected with CD4+ cells rejected MHC class II and, albeit more slowly, MHC class I disparate skins. We showed in vitro evidence that CD4+ cells were not cytotoxic toward MHC class I or MHC class II disparate targets and that they recognized MHC class I allogeneic targets through indirect recognition. CD4+ cells produced Th1 cytokines, but not IL-4, following stimulation with allogeneic cells. Furthermore, intragraft TNF-alpha was elevated in skin grafted onto nude mice reconstituted with CD4+ cells compared with nonreconstituted mice. This suggests that MHC class II- or MHC class I-guided CD4+ cells alone are sufficient to induce rejection by the generation of cytokine-induced lesions.     

Distribution and temporal regulation of the immune response in the rat brain to intracerebroventricular injection of interferon-gamma

The response to intracerebroventricular administration of interferon (IFN)-gamma was examined in the adult Wistar rat brain: major histocompatibility complex (MHC) antigens class I and II, CD8 and CD4 antigens, and the macrophage/microglia antigen OX42 were analyzed in respect to saline-injected cases over 1 week. The glial cell type expressing MHC antigens was characterized with double labeling. IFN-gamma was thus found to induce MHC class I and II expression in microglia, identified by tomato lectin histochemistry, and not in GFAP-immunostained astrocytes. MHC antigen-expressing microglia was detected in the periventricular parenchyma, several fields of the cerebral cortex, cerebellum, major fiber tracts, and brainstem superficial parenchyma. Different gradients of density and staining intensity of the MHC-immunopositive elements were observed in these regions, in which MHC class I antigens persisted up to 1 week, when MHC class II induction had declined. Quantitative analysis pointed out the proliferation of OX42-immunoreactive cells in periventricular and basal brain regions. CD8+ T cells were observed in periventricular regions, basal forebrain, and fiber tracts 3 days after IFN-gamma injection and their density markedly increased by 7 days. CD4+ T cells were also seen and they were fewer than CD8+ ones. However, numerous CD4+ microglial cells were observed in periventricular and subpial regions, especially 1 week after IFN-gamma injection. Our data indicate that this proinflammatory cytokine mediates in vivo microglia activation and T cell infiltration in the brain and that the cells involved in this immune response display a regional selectivity and a different temporal regulation of antigen expression.     

Bacterial surface proteins recognized by CD4+ T cells during murine infection with Listeria monocytogenes

Optimal immunity to the Gram-positive pathogen Listeria monocytogenes (LM) requires both CD8+ and CD4+ antigen-specific T cell responses. Understanding how CD4+ T cells function in an immune response to LM and how bacterial proteins are processed to peptide/MHC class II complexes in infected cells requires identification of these proteins. Using LacZ-inducible, LM-specific CD4+ T cells as probes, we identified two immunogenic LM proteins by a novel expression cloning strategy. The antigenic peptides contained within these proteins were defined by deletion analysis of the genes, and their antigenicity was confirmed with synthetic peptides. The nucleotide sequences of the genes showed that they encode previously unknown LM proteins that are homologous to surface proteins in other bacterial species. Consistent with their surface topology, mild trypsin treatment of LM protoplasts ablated T cell recognition of these Ags. These findings establish a general strategy for identifying unknown CD4+ T cell Ags and demonstrate that LM surface proteins can provide the peptides for presentation by MHC class II molecules that are specific targets for CD4+ T cells during murine LM infection.     

Expression of class II, but not class I, major histocompatibility complex molecules is required for granuloma formation in infection with Schistosoma mansoni

Previous studies have suggested that granulomatous inflammation in schistosomiasis is mediated by CD4+ T helper lymphocytes sensitized to parasite egg antigens. However, CD8+ T cells have also frequently been associated with the immune response to schistosome eggs. To examine more precisely the role of CD4+ and CD8+ T cells in the pathology of the schistosomal infection, we used mice with targeted mutations in major histocompatibility complex (MHC) class II or class I molecules. These mutations lead, respectively, to the virtual absence of CD4+ and CD8+ T cells. The results clearly show that schistosome-infected MHC class II mutant mice failed to form granulomas around parasite eggs. In contrast, infected MHC class I mutant mice displayed characteristic granulomatous lesions that were comparable to those in wild-type control mice. Moreover, lymphoid cells from MHC class II mutant mice were unable to react to egg antigens with either proliferative or cytokine [interferon-gamma, interleukin (IL)-4, IL-10] responses; nor were they able to present egg antigens to specifically sensitized CD4+ T helper cells from infected syngeneic control mice. By comparison, cells from MHC class I mutant mice exercised all these functions in a manner comparable with those from wild-type controls. These observations clearly demonstrate that schistosomal egg granulomas are mediated by MHC class II-restricted CD4+ T helper cells. They also suggest that CD8+ T cells do not become sensitized to egg antigens and play little role, if any, in the pathogenesis of schistosomiasis.     

Specific suppression of human CD4+ Th cell responses to pig MHC antigens by CD8+CD28- regulatory T cells

Evidence that T cells can down-regulate the immune response by producing or consuming certain cytokines or by lysing APCs or Th cells has been provided in various systems. However, the generation and characterization of suppressor T cell lines have met with limited success. Here we show that xenospecific suppressor T cells can be generated by in vitro stimulation of human T cells with pig APCs. Similar to allospecific suppressors, these xenospecific suppressor T cells carry the CD8+CD28- phenotype and react to MHC class I Ags expressed by the APCs used for priming. TCR spectratyping of T suppressor cells showed oligoclonal usage of TCR-Vbeta families, indicating that xenostimulation of CD8+CD28- T cells results in Ag-driven selection of a limited Vbeta repertoire. Xenospecific T suppressor cells prevent the up-regulation of CD154 molecules on the membrane of Th cells, inhibiting their ability to react against the immunizing MHC class II xenoantigens. The mechanism of this suppression, therefore, appears to be blockade of CD154/CD40 interaction required for efficient costimulation of activated T cells.     

Helicobacter pylori infection in immunized mice lacking major histocompatibility complex class I and class II functions

The role of major histocompatibility complex (MHC) class I- and class II-restricted functions in Helicobacter pylori infection and immunity upon oral immunization was examined in vivo. Experimental challenge with H. pylori SS1 resulted in significantly greater (P 相似文献   

Development and antigen specificity of CD8+ cytotoxic T lymphocytes in beta 2-microglobulin-negative, MHC class I-deficient mice in response to immunization with tumor cells

beta 2-Microglobulin knockout mice (beta 2-m-/-) with MHC class I expression deficiency are able to develop functional TCR(+)-alpha beta, CD8+ CTLs in response to tumor cell injection. The i.p. injection of beta 2-m-/- mice with tumor results in the massive accumulation of highly lytic CD8+ CTLs in the peritoneum and causes the local recruitment of CD8+ T cells into lymph nodes and spleens of immune animals. The accumulation of CD8+ CTLs in peritoneum is accompanied by the rejection of tumor cells and the survival of animals. The deficiency in MHC class I expression in beta 2-m/- mice is reflected in the delayed tumor rejection and CD8+ cell accumulation during the primary anti-tumor response in comparison with normal mice. The secondary response, however, is identical in normal and MHC class I-deficient mice. The rejection of tumor cells appears to be MHC class I directed because no rejection of tumors, no accumulation of CD8+ CTLs, and no survival of animals were observed when syngeneic tumor cells were used for injection with the notable exception of anti-minor Ag response. The Ag specificity of CD8+ CTLs in beta 2-m-/- mice is demonstrated using a panel of tumor target cells and class I transfectants. Although no substantial differences were found in the number and specificity of peritoneal CD8+ CTLs in beta 2-m-/- and normal mice using tumor rejection studies, the analysis of TCR-V beta phenotype using the panel of mAbs revealed the reduction in proportion of TCR-V beta 5 and TCR-V beta 6 used by CD8+ cell population from beta 2-m-/- mice. Development of lytic and H-2-directed CD8+ cells in regional lymph nodes was also observed after footpad immunization of beta 2-m-/- mice with TNP-labeled C57BL/6 splenocytes, suggesting anti-minor Ag reaction.     

Analysis of the role of MHC class II presentation in the stimulation of cytotoxic T lymphocytes by antigens targeted into the exogenous antigen-MHC class I presentation pathway

By conjugation of proteins to beads, Ags can be selectively targeted into the MHC class I pathway of phagocytes in vivo and can stimulate CTL responses. Because phagocytes also present particulate Ag on MHC class II molecules, we examined whether these Ags stimulated concomitant CD4 T cell immunity. Although the priming of CD4 T cells with soluble OVA required adjuvants, particulate Ag was stimulatory when injected in saline. We next examined whether CD4 T cell responses played a role in the generation of CTL to particulate Ag. At low concentrations of Ag, OVA primed CTLs in wild-type mice but not in MHC class II-deficient animals, indicating that MHC class II presentation of Ag was essential for CTL generation. These data both support a model where CD4 T cells collaborate with CTLs as part of a three-cell interaction and identify a phagocyte as the third cell in this reaction. Interestingly, injection of higher concentrations of the same Ag primed equivalent CTL responses in both wild-type and MHC class II-deficient mice. These results indicate that a key variable in determining whether CTL generation is helper cell dependent or independent is the dose of immunogen. This may explain in part why CTL responses to abundant Ags, such as viruses, tend to be helper independent, while responses to less abundant Ags, such as minor histocompatibility Ags, require T helper cells. In addition, these results also point to the potential of using particulate Ags to prime or boost responses in settings with CD4 immunodeficiency.     

The cytoplasmic domain of CD4 promotes the development of CD4 lineage T cells

Thymocytes must bind major histocompatibility complex (MHC) proteins on thymic epithelial cells in order to mature into either CD8+ cytotoxic T cells or CD4+ helper T cells. Thymic precursors express both CD8 and CD4, and it has been suggested that the intracellular signals generated by CD8 or CD4 binding to class I or II MHC, respectively, might influence the fate of uncommitted cells. Here we test the notion that intracellular signaling by CD4 directs the development of thymocytes to a CD4 lineage. A hybrid protein consisting of the CD8 extracellular and transmembrane domains and the cytoplasmic domain of CD4 (CD884) should bind class I MHC but deliver a CD4 intracellular signal. We find that expression of a hybrid CD884 protein in thymocytes of transgenic mice leads to the development of large numbers of class I MHC-specific, CD4 lineage T cells. We discuss these results in terms of current models for CD4 and CD8 lineage commitment.     

A molecular basis for how a single TCR interfaces multiple ligands

CD8+ T cells respond to Ags when their clonotypic receptor, the TCR, recognizes nonself peptides displayed by MHC class I molecules. The TCR/ligand interactions are degenerate because, in its life time, the TCR interacts with self MHC class I-self peptide complexes during ontogeny and with self class I complexed with nonself peptides to initiate Ag-specific responses. Additionally, the same TCR has the potential to interact with nonself class I complexed with nonself peptides. How a single TCR interfaces multiple ligands remains unclear. Combinatorial synthetic peptide libraries provide a powerful tool to elucidate the rules that dictate how a single TCR engages multiple ligands. Such libraries were used to probe the requirements for TCR recognition by cloned CD8+ T cells directed against Ags presented by H-2Kb class I molecules. When H-2Kb contact residues were examined, position 3 of the peptides proved more critical than the dominant carboxyl-terminal anchor residue. Thus, secondary anchor residues can play a dominant role in determining the antigenicity of the epitope presented by class I molecules. When the four solvent-exposed potential TCR contact residues were examined, only one or two of these positions required structurally similar residues. Considerable structural variability was tolerated at the remaining two or three solvent-exposed residues of the Kb-binding peptides. The TCR, therefore, requires close physico-chemical complementarity with only a few amino acid residues, thus explaining why TCR/MHC interactions are of low affinity and degenerate.     

Tumour cell vaccines that secrete interleukin-2 (IL-2) and interferon gamma (IFN gamma) are recognised by T cells while resisting destruction by natural killer (NK) cells

The inoculation into mice of genetically engineered tumour cells that secrete IL-2 or IFN gamma results in rejection, while unmodified parental tumour cells grow progressively. In vivo studies demonstrated synergy between IL-2 and IFN gamma leading to the rejection of the transduced tumour cells. IL-2 is required for T cell proliferation and differentiation. IFN gamma induced the upregulation of MHC class I molecules that present peptides to CD8+ T cells. Furthermore, IFN gamma can correct defects in antigen processing. Thus, for T cells, IL-2/IFN gamma-secreting double cytokine tumour cell vaccines might serve as class I+ peptide/antigen presenting depots for developing effector cells. In contrast to T cells, NK cells exert spontaneous killing and kill class I+ targets less well than those that are class I-. For this reason, they may actually have a detrimental effect by destroying a class I+ tumour cell vaccine before adequate T cell stimulation occurs. Based upon this rationale, we tested the hypothesis that an unrecognised benefit of increased class I expression by tumour cells in response to IFN gamma secretion would be to enable cytokine-secreting vaccine cells to resist destruction by NK cells. Our results demonstrated that T cells recognised tumour cells secreting IFN gamma better than those secreting IL-2. NK cells, in contrast, were inhibited by tumour cells that secreted IFN gamma, but not by those that secreted IL-2. The findings suggest that, in addition to upregulating adhesion molecules, MHC molecules, and correcting defects in antigen presentation pathways, IFN gamma secretion may protect tumour cell vaccines from early NK-mediated destruction, keeping them available for T cell priming.     

MHC class I-restricted lysis of human oligodendrocytes by myelin basic protein peptide-specific CD8 T lymphocytes

Multiple sclerosis (MS) is considered to be an autoimmune disease that is directed either at myelin or at its cell of origin, the oligodendrocytes (OL). The inflammatory lesions in the central nervous system contain multiple myelin Ag-restricted and nonrestricted cell populations with the potential to mediate tissue injury. Previous studies indicate that it is possible to generate MHC class I-restricted myelin peptide-specific cytotoxic CD8 T cells, and that human adult OLs express MHC class I molecules in vitro. The purpose of this study was to demonstrate that myelin basic protein peptide-specific CD8 T cells could induce OL injury. We generated CD8 T cell lines from six healthy donors and five MS patients, and all cell lines were HLA-A2 positive. The obtained CD8 cell lines induced lysis of HLA-A2- but not HLA-A3-transfected HMy2.C1R cells in the presence of myelin basic protein peptide 110-118. In the absence of exogenous peptide, the CD8 T cell lines were cytotoxic to HLA-A2 but not to non-HLA-A2 OLs. Cytotoxicity was blocked with anti-MHC class I-blocking Ab. These results support the postulate that autoreactive CD8 cytotoxic T cells can contribute to the tissue injury in MS.     

Cellular mechanisms involved in protection against influenza virus infection in transgenic mice expressing a TCR receptor specific for class II hemagglutinin peptide in CD4+ and CD8+ T cells

Mice transgenic for a TCR that recognizes peptide110-120 of hemagglutinin of PR8 influenza virus in the context of MHC class II I-Ed molecules express the transgenes in both CD4+ and CD8+ T cells. We have found that these TCR-hemagglutinin (TCR-HA) transgenic mice display a significantly increased resistance to the primary infection with PR8 virus compared with the wild-type mice. The TCR-HA transgenic mice mounted significant MHC type II and enhanced MHC type I-restricted cytotoxicity as well as increased cytokine responses in both spleen and lungs after infection with PR8 virus. In contrast, the primary humoral response against PR8 virus was not significantly different from that of the wild-type mice. In vivo depletion and adoptive cell transfer experiments demonstrated that both CD4+ and CD8+ TCR-HA+ T cell subsets were required for the complete clearance of pulmonary virus following infection with a dose that is 100% lethal in wild-type mice. Whereas CD4+ TCR-HA+ T cells were necessary for effective activation and local recruitment of CD8+ T cells, CD8+ TCR-HA+ T cells showed a Th1-biased pattern and MHC type II-restricted cytotoxicity. However, in the absence of in vivo expression of MHC type I molecules on the infected cells, the protection conferred by the TCR-HA+ T cells was impaired, indicating that the enhanced MHC class I-restricted cytotoxicity due to TCR-HA+ CD4+ Th cells was a critical element for clearance of the pulmonary virus by the transgenic mice.