Activation of thymic T cells by MHC alloantigen requires syngeneic, activated CD4+ T cells and B cells as APC

We examine here the in vitro requirements to activate immunocompetent T cells, present among thymocytes, to give rise to CTL, CD4+ T cells producing IL-2 and CD8+ T cells producing IFN-gamma. These thymocytes are naive in not having received antigen-dependent signals characteristic of the periphery. Their activation, upon stimulation with allogeneic spleen cells depleted of T cells, referred to here as allogeneic antigen-presenting cells (APCs), to produce allo-MHC-specific effector T cells, requires activated (radiation resistant) CD4+ T cells, syngeneic with the responding thymocytes. We refer here to these T cells as 'help'. Furthermore, optimal T cell activation requires an Ig+ B220+ cell in the allogeneic APC population, most probably a B cell. The allogeneic APCs cannot be replaced by conventional bone marrow (BM)-derived dendritic cells (DCs) activated by CD40 ligation or exposure to LPS. The requirements for both help and allogeneic B cells in the activation of thymocytes contrast with the requirements to generate substantial responses from splenic T cell populations. Activated, BM-derived DCs stimulate substantial splenic responses without help. These different requirements for activation could reflect the fact that thymocytes have not received an exit-thymus signal and/or that splenic T cells are heterogeneous, containing naive, memory and partially-activated T cells.     

Kinetics of Class II MHC expression on cytotoxic T cells generated by skin allograft

In the present work the kinetics of class II MHC expression on OX8+ lymphocytes generated by skin allograft and its relationship to the lytic activity were studied. Mononuclear cells from the spleen of LEW (RT1(1) rats bearing BN (RT1n) skin graft for 3, 5 or 7 days were sorted out by sequential immune affinity using columns of Degalan-V26 beads treated with anti-rat or anti-mouse Ig. After depletion of B cells, T cells were precoated with W3/25 MoAb (anti-CD4 equivalent) and sorted out using an anti-mouse Ig column. The W3/25-/OX8+ cells (CD8 equivalent) were then coated with OX4 MoAb (anti-RT1.B) or murine A.TH anti-A.TL alloantiserum (anti I-E, cross-reacts with RT1.D) and were passed through a new anti-mouse Ig column in order to obtain the four subpopulations, RT1.B+, .B-, .D+ and .D-. Their specific lytic activity against BN Con A-stimulated cells increased from the 3rd to the 7th d after the skin graft. The lytic activity observed on the 3rd and 5th d was associated with all four subpopulations analyzed. In contrast, on the 7th d, the lytic activity was concentrated in the RT1.B+ subpopulation. These results, associated with the increase in the number of OX8+/RT1.B+ cells along with days after graft, suggest that RT1.B expression is not essential but is associated with the effectiveness of the cytotoxic activity. It is also possible that RT1.B expression is a marker of cytotoxic T-cell differentiation.     

Cytokines and suppressor macrophages cause tumor-bearing host CD8+ T cells to suppress recognition of allogeneic and syngeneic MHC class II molecules.

Quantitative and qualitative tumor-associated changes in T cell phenotype and function were identified in CD8+ T cells. Tumor growth changed splenic CD4+/CD8+ T cell ratios and induced the appearance of more cells with the CD8+ phenotype. In comparison to equal concentrations of normal host (NH) counterparts, tumor-bearing host (TBH) CD8+ T cells were highly suppressive to allorecognition and autorecognition. Suppression was not due to quantitative reductions in CD4+ T cells, although minor qualitative differences were observed. Suppression appeared to be mediated partly by prostaglandin E2 (PGE2). Interferon-gamma (IFN-gamma) and interleukin-4 (IL-4) contributed to TBH CD8+ T cell-mediated suppression. Blocking studies using monoclonal antibodies (mAb) in conjunction with indomethacin suggested that cytokine networks involving IFN-gamma, IL-4, and PGE2 were disrupted during tumor growth and promoted TBH CD8+ T cell suppression. Alloresponses and autoresponses were significantly suppressed when TBH CD8+ T cells mediated these reactions simultaneously with TBH Ia- macrophages. Inhibition of PGE2 production was unable to reverse the additive suppression caused by these two cell types. These results collectively suggest that tumor-induced changes in CD8+ T cells lead to suppressed allo-recognition and autorecognition through both soluble mediator molecules and cellular interactions.     

Protein kinase C delta stimulates antigen presentation by Class II MHC in murine dendritic cells

Maturation of dendritic cells (DCs) regulates protein sorting in endosomal compartments to promote the surface expression of molecules involved in T cell activation. MHC Class II complexes are mobilized to the surface via intracellular effector molecules that remain largely unknown. We here show that protein kinase C (PKC) stimulates Class II antigen surface expression, using knock-in mice that express a Class II-green fluorescent protein fusion protein as a read out. Selective inhibition of PKCdelta counteracts the ability of DCs to stimulate Class II MHC-restricted antigen-specific T cells. Activation of PKC does not affect antigen uptake, peptide loading and surface display of Class I MHC and transferrin receptor in DCs. We show that activation-induced Class II MHC surface expression is dependent on activation of PKCdelta and conclude that this event is pivotal for optimal CD4 T cell activation.     

Different repertoires of mouse T cells for bovine insulin presented by syngeneic and allogeneic cells

Splenic T cells were primed, after removal of alloreactive cells, to beef insulin on allogeneic antigen-presenting cells (APC). The fine specificity of in vitro secondary response was tested in combinations H-2b (responder) T cell-H-2k (nonresponder) APC, and vice versa, using separated chains of beef and pork insulin. The response in both combinations exhibited identical specificity patterns demonstrating that both responder and nonresponder APC could present the same array of insulin epitopes to allogeneic T cells. The determinants presented to allogeneic T cells include the A-chain loop epitope and the B-chain determinant(s) that were found to be immunogenic for H-2b and H-2d T cells, respectively, in the context of syngeneic major histocompatibility complex (HC) molecules. In addition, minor determinants were detected in the A chain outside the loop that are not immunogenic in syngeneic T cell-APC combinations. Inhibition of T cell proliferation with monoclonal antibodies has shown that class II MHC molecules of the nonresponder (Ak alpha Ak beta, Ek alpha Ek beta) as well as those of the responder APC (Ab alpha Ab beta) are equally capable of presenting virtually all insulin epitopes recognizable by T cells. The data, therefore, demonstrate that the selective recognition of different insulin epitopes observed in syngeneic or semisyngeneic T cell-APC combinations does not result from determinant selection at the level of APC.     

MHC Class II tetramers and the pursuit of antigen-specific T cells: define, deviate, delete

Selective expansion and activation of a very small number of antigen-specific CD4(+) T cells is a remarkable and essential property of the adaptive immune response. Antigen-specific T cells were until recently identified only indirectly by functional assays, such as antigen-induced cytokine secretion and proliferation. The advent of MHC Class II tetramers has added a pivotal tool to our research armamentarium, allowing the definition of allo- and autoimmune responses in deeper detail. Rare antigen-specific CD4(+) cells can now be selectively identified, isolated and characterized. The same tetramer reagents also provide a new mean of stimulating T cells, more closely reproducing the MHC-peptide/TCR interaction. This property allows the use of tetramers to direct T cells toward the more desirable outcome, that is, activation (in malignancies and infectious diseases) or Th2/T regulatory cell deviation, anergy and deletion (in autoimmune diseases). These experimental approaches hold promise for diagnostic, prognostic and therapeutic applications.     

CIITA反义RNA抑制MHCII类分子表达

为探索利用CIITA (MHCclassIItransactivator)基因反义RNA抑制MHCII类分子表达的可能性 ,为CIITA的应用研究奠定基础。利用RT PCR扩增能与CIITAcDNA第 95至 5 0 0bp互补的片段 ,并构建成pcDNA3/CIITAa 重组质粒 ,脂质体转染法将其转入人HeLa/Raji细胞和猪PIEC/L2 3细胞 ,流式细胞术动态检测反义RNA对人 /猪MHCII类分子的抑制率和抑制程度。结果显示HeLa、Raji、PIEC和L2 3四种细胞MHCII类分子受抑制率分别为 4 6 7% ( 7/ 15 ) ,4 0 % ( 6 / 15 ) ,4 6 7% ( 7/ 15 )和33 3% ( 5 / 15 )。典型受抑克隆细胞MHCII类分子受抑程度高达 85 % ,反义RNA对MHCII类分子的抑制时间持续 35～ 5 0d。CIITA反义RNA能有效抑制人 /猪MHCII类分子的表达 ,它在自身免疫和移植免疫中有一定的应用前景     

Targeting antigen to MHC Class I and Class II antigen presentation pathways for malaria DNA vaccines

An effective malaria vaccine which protects against all stages of Plasmodium infection may need to elicit robust CD8(+) and CD4(+) T cell and antibody responses. To achieve this, we have investigated strategies designed to improve the immunogenicity of DNA vaccines encoding the Plasmodium yoelii pre-erythrocytic stage antigens PyCSP and PyHEP17, by targeting the encoded proteins to the MHC Classes I and II processing and presentation pathways. For enhancement of CD8(+) T cell responses, we targeted the antigens for degradation by the ubiquitin (Ub)/proteosome pathway following the N-terminal rule. We constructed plasmids containing PyCSP or PyHEP17 genes fused to the Ub gene: plasmids where the N-terminal antigen residues were mutated from the stabilizing amino acid methionine to destabilizing arginine, plasmids where the C-terminal residues of Ub were mutated from glycine to alanine, and plasmids in which the potential hydrophobic leader sequences of the antigens were deleted. For enhancement of CD4(+) T cell and antibody responses, we targeted the antigens for degradation by the endosomal/lysosomal pathway by linking the antigen to the lysosome-associated membrane protein (LAMP). We found that immunization with DNA vaccine encoding PyHEP17 fused to Ub and bearing arginine induced higher IFN-gamma, cytotoxic and proliferative T cell responses than unmodified vaccines. However, no effect was seen for PyCSP using the same targeting strategies. Regarding Class II antigen targeting, fusion to LAMP did not enhance antibody responses to either PyHEP17 or PyCSP, and resulted in a marginal increase in lymphoproliferative CD4(+) T cell responses. Our data highlight the antigen dependence of immune enhancement strategies that target antigen to the MHC Class I and II pathways for vaccine development.     

T cells recognize antigen alone and not MHC molecules

It is a central dogma of contemporary immunology that T cells engaged in immune responses to foreign antigens or cells recognize determinants on major histocompatibility complex (MHC) molecules. Here Ole Werdelin argues that this dogma is false. Taking the case of T-cell responses which are controlled by MHC class II molecules, he suggests that la molecules serve to bind antigen fragments and stabilize them in the membrane of presenting cells, shielding them from proteolytic degradation and permitting T cells to bind the epitopes so displayed.     

Comparative immunogenicity of irradiated and nonirradiated syngeneic and xenogeneic tumor cells containing a common specific transplantation antigen

The immunogenic activity of native and irradiated syngeneic and xenogeneic tumor cells, carrying a common transplantation antigen specific for SV40 on their surface, was compared in Syrian hamsters. The results showed that syngeneic tumor cells were more immunogenic for the recipient than xenogeneic tumor cells containing antigen of the same specificity. Irradiation makes tumor cells, including xenogeneic cells, more immunogenic, possibly on account of the ability of the unirradiated cells to escape recognition on account of their ability to divide.Laboratory of Immunology of Tumors, Oncologic Scientific Center, Academy of Medical Sciences of the USSR, Moscow. (Presented by Academician of the Academy of Medical Sciences of the USSR L. M. Shabad.) Translated from Byulleten' Éksperimental'noi Biologii i Meditsiny, Vol. 86, No. 8, pp. 225–228, August, 1978.     

Method for studying immunologic cell-mediated response to transplanted syngeneic, allogeneic and xenogeneic fibroblasts in mice

A method is described for studying the types of host cells which react with transplanted xenogeneic, allogeneic and syngenetic fibroblasts. Fibroblasts were placed in an 0.15 mm glass fissure, thus limiting the wound reaction and making possible study of host cells infiltrating into the chamber. Under the conditions of the method, in early stages of the reaction to xenogeneic and allogeneic fibroblasts, mostly granulocytes migrated into the transplanted fissures, whereas syngeneic fibroblasts caused infiltration of only a small number of granulocytes.     

Class II MHC antigen expression by human keratinocytes results from lympho-epidermal interactions and gamma-interferon production

Human peripheral blood lymphocytes were co-cultured with either allogeneic, autologous or purified protein derivative of tuberculin (PPD) pulsed autologous epidermal cells. In these mixed skin cell-lymphocyte culture reactions (MSLR), lymphocytes are stimulated to proliferate by epidermal cells. The supernatants of MSLR were examined for their capacity to induce class II MHC antigen expression on separately cultured epidermal cells. It is shown that supernatants from allogeneic and PPD pulsed autologous MSLR contained the factor(s) which stimulated HLA-DR antigen synthesis and expression by 30-40% of cultured epidermal cells. Kinetic analysis revealed a production rate maximum between 72 and 96 h of lympho-epidermal co-cultures. The factor mediating the induction of HLA-DR antigen expression on epidermal cells is thought to be gamma-interferon, because it was sensitive to pH 2 as well as heat incubation. Furthermore, anti-gamma-interferon monoclonal antibody abolished its activity. It is proposed, that HLA-DR antigen expression by keratinocytes observed in vivo in different dermatological inflammatory disorders originates from lympho-epidermal interactions and local gamma-interferon production as documented here in experiments in vitro.     

Class II MHC cytoplasmic domain-mediated signaling in B cells: A tail of two signals

In addition to their role in antigen presentation, class II MHC molecules also transmit signals to B lymphocytes. Class II MHC-mediated signals initiate a range of events in B cells, including induction of cell surface proteins, initiation of cell-cycle progression/proliferation, activation of or protection from apoptosis, and antigen-dependent plasma cell differentiation. Although various transmembrane signaling proteins associate with class II MHC molecules, the class II MHC cytoplasmic domains are essential for signals leading to increased intracellular cAMP and activation of protein kinase C (PKC). Although truncation and mutagenesis studies have provided considerable information about the cytoplasmic domain sequences required, how class II MHC molecules elicit cAMP and PKC activation is not known. Further, appropriate T-dependent B cell responses require intact cAMP and PKC signaling, but the extent to which class II MHC signals are involved is also unknown. This review details our current knowledge of class II MHC cytoplasmic domain signaling in B cells with an emphasis on the likely importance of class II MHC signals for T-dependent antibody responses.     

Priming of T cells by exogenous antigen cross-presented on MHC class I molecules

Cross-presentation is the process whereby bone-marrow-derived antigen-presenting cells acquire, process and present exogenous antigen as peptides bound to MHC class I molecules to CD8(+) T cells. Professional antigen-presenting cells acquire cell-associated antigen predominantly in the form of protein, then process and present antigenic peptides on their surface MHC class I molecules via several mechanisms and efficiently cross-prime na?ve CD8(+) T cells in vivo. This pathway is of considerable interest because it has an important role in the immune surveillance of tissues for pathogens and cancers.     

Evidence for antigen presentation to sensitized T cells by thyroid peroxidase (TPO)-specific B cells in mice injected with fibroblasts co-expressing TPO and MHC class II

Injection of AKR/N mice with fibroblasts co-expressing MHC class II and TPO in the absence of adjuvant induces IgG-class TPO antibodies that resemble spontaneously arising human thyroid autoantibodies. We have used this model to examine the effect of iodide on TPO antibody induction as well as to analyse the interaction between T and B cells. Despite its importance as a major environmental factor in thyroid autoimmunity, variable iodide intake had no detectable effects on TPO antibody levels, lymphocytic infiltration of the thyroid or thyroid hormone levels. In terms of T cell responsiveness, splenocytes from TPO fibroblast-injected mice, but not from control mice, proliferated in response to TPO. Intriguingly, B cell-depleted splenocytes (mainly T cells without reduction of macrophages) proliferated in response to TPO only when co-cultured with irradiated autologous splenocytes from TPO fibroblast-injected mice but not from control mice. These data suggest that TPO-specific B cells are involved in antigen presentation to sensitized T cells and are supported by the ability of spleen cells from TPO cell-injected (but not control) mice to secrete TPO antibodies spontaneously in culture. In conclusion, we provide the first evidence for the presence of thyroid autoantigen-specific B cells and their ability to present their autoantigen to sensitized T cells in mice induced to develop TPO antibodies resembling autoantibodies in humans.     

Immunosynapse formation coincides with rapid activation of NK cells by syngeneic T cells and correlates with clustering of MHC class I

T cells cultured for 3 h with antigen-presenting cells (APCs) stimulated syngeneic IL-2-activated NK cells as measured via a standard chromium-release assay. Discrete caps containing both TCR and MHC-I had formed on the surface of these activated T cells. When conjugates were formed between NK cells and these activated T cells, >80% of the contact sites were in the MHC-I(dim) region outside the TCR-MHC-I cap. Stimulation with phorbol myristate acetate plus Ionomycin, which bypasses the need for cell surface events during activation, did not induce either cap formation or NK cell activation. Further, the addition of the protein transport inhibitor Brefeldin A did not block activation of NK cells. MHC-I is the major inhibitory ligand recognized by NK cells. One possible mechanism for the activation of NK cells by TCR-MHC-I-capped T cells is that aggregation of MHC-I into one region leaves the remaining T cell surface denuded of ligands for NK-inhibitory receptors. As a test of this hypothesis, we aggregated MHC-I on T cells with plate-bound anti-MHC-I mAb. This treatment conferred upon the T cells the capacity to activate NK cells, suggesting that MHC-I clustering could contribute to the observed phenomenon.