Inhibition of HLA-DR assembly,transport, and loading by human cytomegalovirus glycoprotein US3: a novel mechanism for evading major histocompatibility complex class II antigen presentation

Human cytomegalovirus (HCMV) establishes persistent lifelong infections and replicates slowly. To withstand robust immunity, HCMV utilizes numerous immune evasion strategies. The HCMV gene cassette encoding US2 to US11 encodes four homologous glycoproteins, US2, US3, US6, and US11, that inhibit the major histocompatibility complex class I (MHC-I) antigen presentation pathway, probably inhibiting recognition by CD8(+) T lymphocytes. US2 also inhibits the MHC-II antigen presentation pathway, causing degradation of human leukocyte antigen (HLA)-DR-alpha and -DM-alpha and preventing recognition by CD4(+) T cells. We investigated the effects of seven of the US2 to US11 glycoproteins on the MHC-II pathway. Each of the glycoproteins was expressed by using replication-defective adenovirus vectors. In addition to US2, US3 inhibited recognition of antigen by CD4(+) T cells by a novel mechanism. US3 bound to class II alpha/beta complexes in the endoplasmic reticulum (ER), reducing their association with Ii. Class II molecules moved normally from the ER to the Golgi apparatus in US3-expressing cells but were not sorted efficiently to the class II loading compartment. As a consequence, formation of peptide-loaded class II complexes was reduced. We concluded that US3 and US2 can collaborate to inhibit class II-mediated presentation of endogenous HCMV antigens to CD4(+) T cells, allowing virus-infected cells to resist recognition by CD4(+) T cells.     

Binding of human cytomegalovirus US2 to major histocompatibility complex class I and II proteins is not sufficient for their degradation

Human cytomegalovirus (HCMV) glycoprotein US2 causes degradation of major histocompatibility complex (MHC) class I heavy-chain (HC), class II DR-alpha and DM-alpha proteins, and HFE, a nonclassical MHC protein. In US2-expressing cells, MHC proteins present in the endoplasmic reticulum (ER) are degraded by cytosolic proteasomes. It appears that US2 binding triggers a normal cellular pathway by which misfolded or aberrant proteins are translocated from the ER to cytoplasmic proteasomes. To better understand how US2 binds MHC proteins and causes their degradation, we constructed a panel of US2 mutants. Mutants truncated from the N terminus as far as residue 40 or from the C terminus to amino acid 140 could bind to class I and class II proteins. Nevertheless, mutants lacking just the cytosolic tail (residues 187 to 199) were unable to cause degradation of both class I and II proteins. Chimeric proteins were constructed in which US2 sequences were replaced with homologous sequences from US3, an HCMV glycoprotein that can also bind to class I and II proteins. One of these US2/US3 chimeras bound to class II but not to class I, and a second bound class I HC better than wild-type US2. Therefore, US2 residues involved in the binding to MHC class I differ subtly from those involved in binding to class II proteins. Moreover, our results demonstrate that the binding of US2 to class I and II proteins is not sufficient to cause degradation of MHC proteins. The cytosolic tail of US2 and certain US2 lumenal sequences, which are not involved in binding to MHC proteins, are required for degradation. Our results are consistent with the hypothesis that US2 couples MHC proteins to components of the ER degradation pathway, enormously increasing the rate of degradation of MHC proteins.     

Reactivation of latent human cytomegalovirus in CD14(+) monocytes is differentiation dependent.

We have previously demonstrated reactivation of latent human cytomegalovirus (HCMV) in myeloid lineage cells obtained from healthy donors. Virus was obtained from allogenically stimulated monocyte-derived macrophages (Allo-MDM), but not from macrophages differentiated by mitogenic stimulation (ConA-MDM). In the present study, the cellular and cytokine components essential for HCMV replication and reactivation were examined in Allo-MDM. The importance of both CD4(+) and CD8(+) T cells in the generation of HCMV-permissive Allo-MDM was demonstrated by negative selection or blocking experiments using antibodies directed against both HLA class I and HLA class II molecules. Interestingly, contact of monocytes with CD4 or CD8 T cells was not essential for reactivation of HCMV, since virus was observed in macrophages derived from CD14(+) monocytes stimulated by supernatants produced by allogeneic stimulation of peripheral blood mononuclear cells. Examination of the cytokines produced in Allo-MDM and ConA-MDM cultures indicated a significant difference in the kinetics of production and quantity of these factors. Further examination of the cytokines essential for the generation of HCMV-permissive Allo-MDM identified gamma interferon (IFN-gamma) but not interleukin-1 or -2, tumor necrosis factor alpha, or granulocyte-macrophage colony-stimulating factor as critical components in the generation of these macrophages. In addition, although IFN-gamma was crucial for reactivation of latent HCMV, addition of IFN-gamma to unstimulated macrophage cultures was insufficient to reactivate virus. Thus, this study characterizes two distinct monocyte-derived cell types which can be distinguished by their ability to reactivate and support HCMV replication and identifies the critical importance of IFN-gamma in the reactivation of HCMV.     

Immune surveillance via self digestion

The adaptive immune system is orchestrated by CD4+ T cells. These cells detect peptides presented on Major Histocompatibility Complex (MHC) class II molecules, which are loaded in late endosomes with products of lysosomal proteolysis. One pathway by which proteins gain access to degradation in lysosomes is macroautophagy. We recently showed that constitutive macroautophagy can be detected in cells relevant for the immune system, including dendritic cells. In these antigen presenting cells, autophagosomes frequently fused with MHC class II antigen loading compartments and targeting of Influenza matrix protein 1 (MP1) for macroautophagy enhanced MHC class II presentation to MP1-specific CD4+ T cell clones up to 20 fold. Our findings indicate that macroautophagy is a constitutive and efficient pathway of antigen delivery for MHC class II presentation. We suggest that this pathway samples intracellular proteins for immune surveillance and induction of tolerance in CD4+ T cells, and could be targeted for improved MHC class II presentation of vaccine antigens.     

Human cytomegalovirus US7, US8, US9, and US10 are cytoplasmic glycoproteins, not found at cell surfaces, and US9 does not mediate cell-to-cell spread

Human cytomegalovirus (HCMV) expresses a large number of membrane proteins with unknown functions. One class of these membrane proteins apparently acts to allow HCMV to escape detection by the immune system. The best characterized of these are the glycoproteins encoded within the US2 to US11 region of the HCMV genome that mediate resistance to CD8(+) and CD4(+) T cells. US2, US3, US6, and US11 block various aspects of the major histocompatibility complex (MHC) class I and class II antigen presentation pathways, functioning in cytoplasmic membranes to cause retention, degradation, or mislocalization of MHC proteins. Distantly homologous genes in this region, US7, US8, US9, and US10, are not well characterized. Here, we report expression of the glycoproteins encoded by US7 to US10 by using replication-defective adenovirus (Ad) vectors. US7, US9, and US10 remained sensitive to endoglycosidase H and were exclusively or largely present in the endoplasmic reticulum (ER) as determined by confocal microscopy. US8 reached the Golgi apparatus and trans-Golgi network and was more quickly degraded. Previous studies suggested that US9 could localize to cell junctions and mediate cell-to-cell spread in ARPE-19 retinal epithelial cells. We found no evidence of US9 at cell junctions of HEC-1A epithelial cells. HCMV recombinants lacking US9 produced smaller plaques on ARPE-19 cell monolayers but also exhibited defects in virus replication compared with wild-type HCMV in these cells. Other HCMV recombinants constructed in a similar fashion that were able to express US9 also produced small plaques and some of these exhibited defects in production of infectious progeny in ARPE-19 cells. Thus, there was no correlation between defects in cell-to-cell spread (plaque size) and loss of expression of US9, and it is possible that US9(-) mutants produce smaller plaques because they produce fewer progeny. Together, our results do not support the hypothesis that US9 plays a direct role in HCMV cell-to-cell spread.     

Human cytomegalovirus (HCMV) US2 protein interacts with human CD1d (hCD1d) and down-regulates invariant NKT (iNKT) cell activity

To avoid host immune surveillance, human cytomegalovirus (HCMV) encoded endoplasmic reticulum (ER)-membrane glycoprotein US2, which interferes with antigen presenting mechanism of major histocompatibility complex (MHC) class Ia and class II molecules. However, not many attempts have been made to study the effect of HCMV US2 on the expression of MHC class Ib molecules. In this study, we examined the effect of HCMV US2 on the expression and function of human CD1d (hCD1d), which presents glycolipid antigens to invariant NKT (iNKT) cells. Our results clearly showed that the physiological interaction between ER lumenal domain of HCMV US2 and α3 domain of hCD1d was observed within ER. Compared with mature form of hCD1d, immature form of hCD1d is more susceptible to ubiquitin-dependent proteasomal degradation mediated by HCMV US2. Moreover, the ectopic expression of HCMV US2 leads to the down-modulation of iNKT cell activity without significant change of hCD1d expression. These results will advance our understanding of the function of HCMV US2 in immune evasive mechanisms against anti-viral immunity of iNKT cells.     

Human cytomegalovirus disrupts constitutive MHC class II expression

CD8(+) and CD4(+) T lymphocytes are important in controlling human CMV (HCMV) infection, but the virus has evolved protean mechanisms to inhibit MHC-based Ag presentation and escape T lymphocyte immunosurveillance. Herein, the interaction of HCMV with the MHC class II Ag presentation pathway was investigated in cells stably transfected with class II transactivator. Flow cytometry experiments demonstrate that HCMV infection decreases cell-surface MHC class II expression. HCMV down-regulates MHC class II surface expression without a significant effect on class II RNA or steady-state protein levels. SDS-stability and confocal microscopy experiments demonstrate normal levels of steady-state peptide-loaded class II molecules in infected cells and that class II molecules reach late endosomal and HLA-DM positive peptide-loading compartments. However, MHC class II positive vesicles are retained in an abnormal perinuclear distribution. Finally, experiments with a mutant HCMV strain demonstrate that this novel mechanism of decreased MHC class II expression is not mediated by one of the known HCMV immunomodulatory genes. These defects in MHC class II expression combined with previously identified CMV strategies for decreasing MHC class I expression enables infected cells to evade T lymphocyte immunosurveillance.     

Human cytomegalovirus gene products US2 and US11 differ in their ability to attack major histocompatibility class I heavy chains in dendritic cells

Human cytomegalovirus (HCMV) encodes several proteins that inhibit major histocompatibility complex (MHC) class I-dependent antigen presentation. The HCMV products US2 and US11 are each sufficient for causing the dislocation of human and murine MHC class I heavy chains from the lumen of the endoplasmic reticulum to the cytosol, where the heavy chains are readily degraded. The apparent redundancy of US2 and US11 has been probed predominantly in cultured cell lines, where differences in their specificities were shown for murine and human MHC class I locus products. Here, we expressed US11 and US2 via adenovirus vectors and show that US11 exhibits a superior ability to degrade MHC class I molecules in primary human dendritic cells. MHC class II complexes are unaffected by US2- and US11-mediated attack. We suggest that multiple HCMV-encoded immunoevasions have evolved complementary functions in response to diverse host cell types and tissues.     

Antigens and autophagy: the path less travelled?

The Epstein-Barr virus (EBV)-coded nuclear antigen (EBNA) 1, a latent cycle protein endogenously expressed in EBV-transformed B lymphoblastoid cell lines (LCLs), is reported to be processed for CD4(+) T cell recognition by an intracellular route involving antigen delivery to the endosome/lyosome (MHC class II loading) compartment via macroautophagy. In contrast we find that, in the same cell type, two other virus-coded nuclear proteins of the latent cycle, EBNA2 and EBNA3C, are processed by a different route that is unaffected by autophagy inhibition. This involves the intercellular transfer of an antigenic moiety, detectable in cell-free culture supernatants, and its uptake and processing as exogenous antigen by neighboring cells. The process is cumulative and leads over several days of LCL culture to high levels of CD4+ T cell epitope display. The presentation of certain EBV lytic cycle proteins to CD4+ T cells has also recently been found to involve a similar intercellular antigen transfer. It becomes important to know why, even in the same cell type, some antigens but not others appear to access the MHC class II presentation pathway by autophagy.     

Antigen processing for presentation to CD4+ T cells.

The immune system surveys the organism for the presence of foreign or abnormal structures. An important role in the immune response is assumed by T lymphocytes that recognize foreign antigen while tolerating self-proteins. T lymphocytes can recognize only peptide fragments that are presented to them by molecules of the major histocompatibility complex (MHC). Antigen processing for presentation to T cells involves distinct cellular compartments where peptides and MHC molecules interact. Whereas class I MHC molecules (recognized by CD8+ cytotoxic T cells) acquire peptides in an early biosynthetic compartment, class II molecules (recognized by CD4+ helper T cells) acquire peptides most efficiently in an endocytic compartment. It has emerged recently that the class II processing compartment can be fed not only from the outside with exogenous antigen but also from endogenous sources, including membrane-associated and cytosolic proteins. The potential sources of proteins that can trigger a helper T cell response during viral infections and that can induce self-tolerance are thus much wider than previously anticipated.     

Human cytomegalovirus US2 causes similar effects on both major histocompatibility complex class I and II proteins in epithelial and glial cells

The human cytomegalovirus (HCMV) glycoprotein US2 specifically binds to major histocompatibility complex (MHC) class I heavy chain (HC) and class II proteins DRalpha and DMalpha, triggering their degradation by proteasomes. Effects of US2 on class II proteins were originally characterized in HCMV- or adenovirus vector-infected U373 astroglioma cells. Here, we have extended characterization of US2-mediated degradation of class II DRalpha to two other cell lines, including biologically relevant epithelial cells. Comparison of the effects of US2 in cells expressing both class I and II proteins demonstrated only a slight preference for class I HC. Moreover, US2 caused degradation of DRalpha and DMalpha when these proteins were expressed by transfection without DRbeta, invariant chain (Ii), or DMbeta. Therefore, US2 binds to alpha chains of DR and DM and triggers endoplasmic reticulum degradation without formation of class II DR alphabeta/Ii or DM alphabeta complexes. Similar levels of degradation of class II alpha were observed in cells expressing vastly different amounts of class II, suggesting that cellular factors, other than class II, were limiting. We concluded that US2 has broad effects in a variety of cells that express both class I and II proteins and is relevant to HCMV infection in vivo.     

Immune cell populations in the equine corpus luteum throughout the oestrous cycle and early pregnancy: an immunohistochemical and flow cytometric study

Recent evidence indicates that the cells of the immune system and their large network of secretory products, or cytokines, play an active role in the ovary throughout the oestrous cycle. In the present study, immune cell populations (T and B lymphocytes, macrophages, granulocytes and eosinophils) and expression of major histocompatibility complex (MHC) class II were investigated in corpora lutea from mares in early (days 2-4), mid- (days 7-10) and late (days 12-14) dioestrus, the post-luteolytic phase (days 16-17) and early pregnancy. The number of T lymphocytes within the corpus luteum increased in the late luteal phase. CD4+ cells did not increase until day 16, whereas the number of CD8+ cells increased before functional luteolysis; an apparently selective luteal infiltration of CD8+ cells was observed. MHC class II expression by non-steroidogenic cells was increased in samples from days 16-17, as was the number of infiltrating macrophages. Flow cytometry revealed very low expression of MHC class II by large luteal cells at all stages of the oestrous cycle. In early pregnancy, the number of CD4+ and CD8+ cells and macrophages decreased, as did MHC class II expression, compared with mid-dioestrous samples. B cells were present in very small numbers in all samples examined. Eosinophils were similarly sparsely distributed and numbers decreased further in pregnancy. After exogenous PGF2 alpha administration, populations of CD4+ cells and non-specific esterase staining cells were significantly smaller than after natural luteolysis, whereas eosinophil numbers were increased compared with samples from days 16-17. However, the number of CD8+ and CD5+ cells and MHC class II expression were not significantly different from those observed after natural luteolysis. These findings indicate that populations of immune cells in the equine corpus luteum vary during its lifespan. The selective increase in CD8+ cells before functional luteolysis indicates that they have a physiological role in the regression of the corpus luteum.     

MHC class II presentation of endogenous tumor antigen by cellular vaccines depends on the endocytic pathway but not H2-M

We have developed cell-based cancer vaccines that activate anti-tumor immunity by directly presenting endogenously synthesized tumor antigens to CD4⁺ T helper lymphocytes via MHC class II molecules. The vaccines are non-conventional antigen-presenting cells because they express MHC class II, do not express invariant chain or H-2M, and preferentially present endogenous antigen. To further improve therapeutic efficacy we have studied the intracellular trafficking pathway of MHC class II molecules in the vaccines using endoplasmic reticulum-localized lysozyme as a model antigen. Experiments using endocytic and cytosolic pathway inhibitors (chloroquine, primaquine, and brefeldin A) and protease inhibitors (lactacystin, LLnL, E64, and leupeptin) indicate antigen presentation depends on the endocytic pathway, although antigen degradation is not mediated by endosomal or proteasomal proteases. Because H2-M facilitates presentation of exogenous antigen via the endocytic pathway, we investigated whether transfection of vaccine cells with H-2M could potentiate endogenous antigen presentation. In contrast to its role in conventional antigen presentation, H-2M had no effect on endogenous antigen presentation by vaccine cells or on vaccine efficacy. These results suggest that antigen/MHC class II complexes in the vaccines may follow a novel route for processing and presentation and may produce a repertoire of class II-restricted peptides different from those presented by professional APC. The therapeutic efficacy of the vaccines, therefore, may reside in their ability to present novel tumor peptides, consequently activating tumor-specific CD4⁺ T cells that would not otherwise be activated.     

Human cytomegalovirus US3 chimeras containing US2 cytosolic residues acquire major histocompatibility class I and II protein degradation properties

Human cytomegalovirus (HCMV) glycoprotein US2 increases the proteasome-mediated degradation of major histocompatibility complex (MHC) class I heavy chain (HC), class II DR-alpha and DM-alpha proteins, and HFE, a nonclassical MHC protein. US2-initiated degradation of MHC proteins apparently involves the recruitment of cellular proteins that participate in a process known as endoplasmic reticulum (ER)-associated degradation. ER-associated degradation is a normal process by which misfolded proteins are recognized and translocated into the cytoplasm for degradation by proteasomes. It has been demonstrated that truncated forms of US2, especially those lacking the cytoplasmic domain (CT), can bind MHC proteins but do not cause their degradation. To further assess how the US2 CT domain interacts with the cellular components of the ER-associated degradation pathway, we constructed chimeric proteins in which the US2 CT domain or the CT and transmembrane (TM) domains replaced those of the HCMV glycoprotein US3. US3 also binds both class I and II proteins but does not cause their degradation. Remarkably, chimeras containing the US2 CT domain caused the degradation of both MHC class I and II proteins although this degradation was less than that by wild-type US2. Therefore, the US2 CT and TM domains can confer on US3 the capacity to degrade MHC proteins. We also analyzed complexes containing MHC proteins and US2, US3, US11, or US3/US2 chimeras for the presence of cdc48/p97 ATPase, a protein that binds polyubiquitinated proteins and likely functions in the extraction of substrates from the ER membrane before the substrates meet proteasomes. p97 ATPase was present in immunoprecipitates containing US2, US11, and two chimeras that included the US2 CT domain, but not in US3 complexes. Therefore, it appears that the CT domain of US2 participates in recruiting p97 ATPase into ER-associated degradation complexes.     

Immune Surveillance via Self Digestion

The adaptive immune system is orchestrated by CD4⁺ T cells. These cells detect peptides presented on Major Histocompatiblity Complex (MHC) class II molecules, which are loaded in late endosomes with products of lysosomal proteolysis. One pathway by which proteins gain access to degradation in lysosomes is macroautophagy. We recently showed that constitutive macroautophagy can be detected in cells relevant for the immune system, including dendritic cells. In these antigen presenting cells, autophagosomes frequently fused with MHC class II antigen loading compartments and targeting of Influenza matrix protein 1 (MP1) for macroautophagy enhanced MHC class II presentation to MP1-specific CD4⁺ T cell clones up to 20 fold. Our findings indicate that macroautophagy is a constitutive and efficient pathway of antigen delivery for MHC class II presentation. We suggest that this pathway samples intracellular proteins for immune surveillance and induction of tolerance in CD4⁺ T cells, and could be targeted for improved MHC class II presentation of vaccine antigens.

Addendum to:

MHC Class II Antigen Loading Compartments Continuously Receive Input from Autophagosomes

Dorothee Schmid, Marc Pypaert and Christian Münz

Immunity 2006; In press     

Expression of herpes simplex virus ICP47 and human cytomegalovirus US11 prevents recognition of transgene products by CD8(+) cytotoxic T lymphocytes

The in vivo persistence of gene-modified cells may be limited by the development of a host immune response to vector-encoded proteins. Herpesviruses evade cytotoxic T-lymphocyte (CTL) recognition by expressing genes which interfere selectively with presentation of viral antigens by class I major histocompatibility complex (MHC) molecules. Here, we studied the use of retroviral vectors encoding herpes simplex virus ICP47, human cytomegalovirus (HCMV) US3, or HCMV US11 to decrease presentation of viral proteins and transgene products to CD8(+) CTL. Human fibroblasts and T cells transduced to express the ICP47, US3, or US11 genes alone exhibited a decrease in cell surface class I MHC expression. The combination of ICP47 and US11 rendered fibroblasts negative for surface class I MHC and allowed a class I MHC-low population of T cells to be sorted by flow cytometry. Fibroblasts and T cells expressing both ICP47 and US11 were protected from CTL-mediated lysis and failed to stimulate specific memory T-cell responses to transgene products in vitro. Our findings suggest that expression of immunoregulatory viral gene products could be a potential strategy to prolong transgene expression in vivo.     

Placental extravillous cytotrophoblasts persistently express class I major histocompatibility complex molecules after human cytomegalovirus infection

Human cytomegalovirus (HCMV) downregulates the class I major histocompatibility complexes (MHCs), HLA-A and -B, in infected fibroblasts to escape from antigen-specific cytotoxic T lymphocytes. The HCMV genes responsible for the downregulation of MHCs are US2, US3, US6, and US11, which encode type I membrane proteins working at the endoplasmic reticulum (ER). However, it is largely unknown whether HCMV downregulates the class I MHC molecules in placental extravillous cytotrophoblasts (EVT), which express HLA-C, -E, and -G to protect a semiallogenic fetus from maternal natural killer (NK) cells at the fetomaternal interface. Here, we report that differentiated EVT prepared from human first-trimester chorionic villi persistently express class I MHC molecules upon HCMV infection. When these US proteins were expressed in uninfected EVT, they were localized at the ER in the entire cytoplasm. However, subsequent HCMV infection resulted in dissociation of these US proteins from the ER, which relocated toward the cell membrane. In fibroblasts, these US proteins were localized at the ER before and after HCMV infection. These results suggest that the US gene products are not integrated into ER of HCMV-infected EVT and fail to downregulate class I MHC molecules.     

Some cloned murine CD4+ T cells recognize H-2Ld class I MHC determinants directly. Other cloned CD4+ T cells recognize H-2Ld class I MHC determinants in the context of class II MHC molecules.

Murine T lymphocytes recognize nominal Ag presented by class I or class II MHC molecules. Most CD8+ T cells recognize Ag presented in the context of class I molecules, whereas most CD4+ cells recognize Ag associated with class II molecules. However, it has been shown that a proportion of T cells recognizing class I alloantigens express CD4 surface molecules. Furthermore, CD4+ T cells are sufficient for the rejection of H-2Kbm10 and H-2Kbm11 class I disparate skin grafts. It has been suggested that the CD4 component of an anti-class I response can be ascribed to T cells recognizing class I determinants in the context of class II MHC products. To examine the specificity and effector functions of class I-specific HTL, CD4+ T cells were stimulated with APC that differed from them at a class I locus. Specifically, a MLC was prepared involving an allogeneic difference only at the Ld region. CD4+ clones were derived by limiting dilution of bulk MLC cells. Two clones have been studied in detail. The CD4+ clone 46.2 produced IL-2, IL-3, and IFN-gamma when stimulated with anti-CD3 mAb, whereas the CD4+ clone 93.1 secreted IL-4 in addition to IL-2, IL-3, and IFN-gamma. Cloned 46.2 cells recognized H-2Ld directly, whereas recognition of Ld by 93.1 apparently was restricted by class II MHC molecules. Furthermore, cytolysis by both clones 46.2 and 93.1 was inhibited by the anti-CD4 mAb GK1.5. These results demonstrate that CD4+ T cells can respond to a class I difference and that a proportion of CD4+ T cells can recognize class I MHC determinants directly as well as in the context of class II MHC molecules.     

Function of the CD4 and CD8 molecules on human cytotoxic T lymphocytes: regulation of T cell triggering

The function of the T cell differentiation antigens CD4 (Leu-3/T4) and CD8 (Leu-2/T8) on human cytotoxic T lymphocytes (CTL) is presently seen only in conjugate formation between CTL and target cell via class II or class I MHC antigens rather than in the later killing steps. In this study, human CD4+ and CD8+ CTL clones were used to investigate the effects of monoclonal antibodies against these differentiation antigens on nonspecific triggering of cytotoxicity. Cytotoxicity was induced either by antibodies against the CD3 (T3) antigen or by the lectins Con A and PHA. Anti-CD4 or anti-CD8 antibodies specifically inhibited all types of cytotoxicity of CD4+ or CD8+ CTL, respectively, regardless of the specificity of the CTL for class I or class II HLA antigens and regardless of whether target cells expressed class I or class II antigens. These results are incompatible with an exclusive role of the CD4 and CD8 molecules in MHC class recognition and are discussed with respect to a function as negative signal receptors for these molecules on CTL.     

Requirement for efficient interactions between CD4 and MHC class II molecules for survival of resting CD4+ T lymphocytes in vivo and for activation-induced cell death

Regulation of homeostasis in the immune system includes mechanisms that promote survival of resting T lymphocytes, and others that control activation-induced cell death (AICD). In this study, we report on the use of a transgenic mouse model to test the role of CD4-MHC class II interactions for the susceptibility of CD4+ T lymphocytes to AICD, and for the survival of resting CD4+ T cells in peripheral lymphoid organs. The only I-Abeta gene expressed in these mice is an Abetak transgene with a mutation that prevents MHC class II molecules from interacting with CD4. We show increased apoptosis in CD4+ T lymphocytes derived from wild-type, but not from mutant Abetak transgenic mice following stimulation with staphylococcal enterotoxin A. Therefore, AICD may be impaired in CD4+ T cells derived from mutant Abetak transgenic mice. Importantly, we observed much higher apoptosis in resting CD4+ T cells from mutant Abetak transgenic mice than from wild-type mice. Furthermore, resting CD4+ T cells from mutant Abetak transgenic mice expressed higher levels of cell surface CD95 (Fas, APO-1). Ab-mediated cross-linking of CD95 further increased apoptosis in CD4+ T cells from mutant Abetak transgenic mice, but not from wild-type mice, suggesting apoptosis involved CD95 signaling. When cocultured with APC-expressing wild-type MHC class II molecules, apoptosis in resting CD4+ T lymphocytes from mutant Abetak transgenic mice was reduced. Our results show for the first time that interactions between CD4 and MHC class II molecules are required for the survival of resting CD4+ T cells in peripheral lymphoid organs.