Immune checkpoints in viral latency.

The dynamics of the relationship between the immune system and latent viruses are highly complex. Latent viruses not only avoid elimination by the host's primary immune response, they also remain with the host for life in the presence of strong acquired immunity, often exhibiting periodic reactivation and recurrence from the latent state. The continual battle between reemergent infectious virus and immunological memory cells provides an essential virus-host regulatory loop in latency. In this review, we speculate on the critical importance of immune interference mechanisms by viruses contributing to the regulatory loop in viral homeostasis of latency. Central to the notion of viral homeostasis, we further invoke the concept of threshold limits in naive and memory states of immunity to account for the failure of the host to completely eradicate these intracellular parasites.

The impact of duration versus extent of TCR occupancy on T cell activation: a revision of the kinetic proofreading model.

The widely accepted kinetic proofreading theory proposes that rapid TCR dissociation from a peptide/MHC ligand allows for stimulation of early but not late T cell activation events, explaining why low-affinity TCR ligands are poor agonists. We identified a low-affinity TCR ligand which stimulated late T cell responses but, contrary to predictions from kinetic proofreading, inefficiently induced early activation events. Furthermore, responses induced by this ligand were kinetically delayed compared to its high-affinity counterpart. Using peptide/MHC tetramers, we showed that activation characteristics could be dissociated from TCR occupancy by the peptide/MHC ligands. Our data argue that T cell responses are triggered by a cumulative signal which is reached at different time points for different TCR ligands.

Hijacking and exploitation of IL-10 by intracellular pathogens.

Macrophages play a central role in infections, as a target for pathogens and in activation of the immune system. Interleukin-10 (IL-10), a cytokine produced by macrophages, is a potent immunosuppressive factor. Some intracellular pathogens specifically target macrophages for infection and use IL-10 to dampen the host immune response and stall their elimination from the host. Certain viruses induce production of cellular IL-10 by macrophages, whereas other viruses encode their own viral IL-10 homologs. Additionally, specific bacteria, including several Mycobacteria spp. and Listeria monocytogenes, can survive and replicate in macrophages while inducing cellular IL-10, highlighting a potential role for IL-10 of macrophage origin in the immunosuppressive etiology of these pathogens. Thus, the exploitation of IL-10 appears to be a common mechanism of immunosuppression by a diverse group of intracellular pathogens that can infect macrophages.

T cell receptor binding kinetics and special role of Valpha in T cell development and activation.

The kinetics of the interaction between T cell receptor (TCR) and major histocompatibility complex (MHC) has an important role in determining thymocyte-positive and -negative selection in the thymus, as well as in T cell activation. The alpha chain of the TCR is the major player in determining how the TCR fits onto the MHC ligand, and thus has a major role in determining whether a T cell develops as class I or class II restricted. In this article, we summarize recent data from our laboratory and others on the role of polymorphism in the Valpha combining site in determining MHC class restriction, and on kinetic parameters in thymocyte selection.

Allelic exclusion of the T cell receptor alpha-chain: developmental regulation of a post-translational event.

Allelic exclusion of the alpha and beta chains of the T cell receptor is maintained by different mechanisms. Exclusion of the beta-chain is primarily by allowing the successful rearrangement of only one of the two beta-chain loci. In the case of the alpha-chain, rearrangement on both chromosomes is very common, as is expression of alpha-chain mRNA and protein encoded by both loci. For the most part, however, functional alpha-chain allelic exclusion is maintained at the cell surface after positive selection in the thymus. The mechanism by which this is accomplished is not yet known, but recent evidence indicates that it is an active process coupled to signalling through the T cell receptor.

Cutting edge: trimolecular interaction of TCR with MHC class II and bacterial superantigen shows a similar affinity to MHC:peptide ligands.

Bacterial superantigens such as Staphylococcus aureus enterotoxin A (SEA) are very potent stimulators of T cells. They bind to the Vbeta region of the TCR and to MHC class II, stimulating T cells at nanomolar concentrations. Using surface plasmon resonance measurements, we find that binding between the individual components of the complex (TCR-class II, TCR-SEA, SEA-class II) is very weak, but that the stability of the trimolecular complex is considerably enhanced, reaching an affinity similar to that found for TCR interactions with MHC:peptide ligand. Thus, the potency of SEA in stimulation of T cells is not due to particularly strong affinities between the proteins, but to a cooperative effect of interactions in the TCR-SEA-MHC class II trimolecular complex that brings the kinetics into a similar range to binding of conventional Ags. This range may be the optimum for T cell activation.

Murine cytomegalovirus infection down-regulates MHC class II expression on macrophages by induction of IL-10.

Herpesviruses utilize many strategies for weakening the host immune response. For CMV, this includes avoidance of NK clearance and inhibition of MHC class I and class II presentation pathways. In this study, we report that mouse CMV (MCMV) specifically causes a premature and transient activation of host IL-10 very early in the course of infection, resulting in a dramatic and selective reduction in MHC class II surface expression. The expression of IL-10 is normally late in the immune response to a pathogen, serving to dampen the response by suppression of the production of inflammatory cytokines. In infection of macrophages, we show that MCMV induces the production of IL-10, leading to an early and selective reduction in the expression of MHC class II on the surface of the cells. Inhibition of MHC class II expression was not observed in the presence of neutralizing Abs to IL-10 or in macrophages from IL-10-deficient mice. Moreover, MCMV-infected IL-10-deficient mice developed an early and significantly more robust macrophage MHC class II induction than normal mice. Altogether, our results demonstrate that viral induction of an IL-10 autocrine pathway plays an essential early role in selectively reducing MHC class II expression on the surface of APC prior to stimulation by IFN-gamma.

Qualitative and quantitative differences in T cell receptor binding of agonist and antagonist ligands.

The kinetics of interaction between TCR and MHC-peptide show a general relationship between affinity and the biological response, but the reported kinetic differences between antigenic and antagonistic peptides are very small. Here, we show a remarkable difference in the kinetics of TCR interactions with strong agonist ligands at 37 degrees C compared to 25 degrees C. This difference is not seen with antagonist/positive selecting ligands. The interaction at 37 degrees C shows biphasic binding kinetics best described by a model of TCR dimerization. The altered kinetics greatly increase the stability of complexes with agonist ligands, accounting for the large differences in biological response compared to other ligands. Thus, there may be an allosteric, as well as a kinetic, component to the discrimination between agonists and antagonists.

Reciprocal expression in CD4 or CD8 subsets of different members of the V alpha 11 gene family correlates with sequence polymorphism.

Previous staining studies with TCR V alpha 11-specific mAbs showed that V alpha 11.1/11.2 (AV11S1 and S2) expression was selectively favored in the CD4+ peripheral T cell population. As this phenomenon was essentially independent of the MHC haplotype, it was suggested that AV11S1 and S2 TCRs exert a preference for recognition of class II MHC molecules. The V alpha segment of the TCR alpha-chain is suggested to have a primary role in shaping the T cell repertoire due to selection for class I or II molecules acting through the complementarity determining regions (CDR) 1 alpha and CDR2 alpha residues. We have analyzed the repertoire of V alpha 11 family members expressed in C57BL/6 mice and have identified a new member of this family; AV11S8. We show that, whereas AV11S1 and S2 are more frequent in CD4+ cells, AV11S3 and S8 are more frequent in CD8+ cells. The sequences in the CDR1 alpha and CDR2 alpha correlate with differential expression in CD4+ or CD8+ cells, a phenomenon that is also observed in BALB/c mice. With no apparent restriction in TCR J alpha usage or CDR3 alpha length in C57BL/6, these findings support the idea of V alpha-dependent T cell repertoire selection through preferential recognition of MHC class I or class II molecules.

Thymic skewing of the CD4/CD8 ratio maps with the T-cell receptor alpha-chain locus.

The thymic preference for CD4+ T cells over CD8+ T cells is often attributed to a default pathway favouring CD4+ T cells or to homeostatic mechanisms. It is also clear, however, that T-cell receptor (TCR) preferences for major histocompatibility complex (MHC) class I versus class II binding will strongly influence an individual clone's skewing to the CD4 or CD8 subset. The variable region of each TCR alpha chain (V alpha) studied to date is found to be overrepresented in either CD4+ or CD8+ cells, suggesting that each V alpha element can interact more favourably with either MHC class I or class II molecules. Indeed, TCRs appear to have an intrinsic ability to interact with MHC molecules, and single amino acid residues present in germline-encoded complementarity determining region 1 (CDR1) and CDR2 of the V alpha element can be responsible for determining MHC specificity. Interestingly, the degree of CD4/CD8 skewing is variable among different mouse strains and in human populations. Here, we have shown that polymorphism in CD4/CD8 skewing between B6 and BALB/c mice is determined by the stem cell genotype and not by environmental effects, and that it maps in or near the TCR alpha-chain complex, Tcra. This was confirmed by comparing Tcra(b) with Tcra(a) or Tcra(c) haplotypes in congenic mice. We propose that the array of V alpha genes in various Tcra haplotypes exerts influence over the proportion of CD4 and CD8 subsets generated and may account in part for the observed thymic skewing. Thus, while it has been suggested that the TCR genes have been selected by evolution for MHC binding, our results further indicate selection for class II MHC preference.

Polymorphism within a TCRAV family influences the repertoire through class I/II restriction.

Antibody-staining experiments have shown that closely related members of the TCRAV3 family are reciprocally selected into the CD4 or CD8 peripheral T cell subsets. This has been attributed to the individual AV3 members interacting preferentially with either MHC class I or MHC class II molecules. Single amino acid residues present in the complementarity-determining regions (CDR) CDR1alpha and CDR2alpha are important in determining MHC class specificity. We have now extended these observations to survey the expressed repertoire of the AV3 family in C57BL/6 mice. Three of the four expressed AV3 members are preferentially selected into the CD4+ subset of T cells. These share the same amino acid residue in both CDR1alpha and CDR2alpha that differ from the only CD8-skewed member. Preferential expression of an individual AV3 is not caused by other endogenous alpha- or beta-chains, by any conserved CDR3 sequence, or by the usage of TCRAJ regions. This study shows that residues in the CDR1 and CDR2 regions are primary determinants for MHC class discrimination and suggests that polymorphism found within a TCRAV family has an important effect on the overall shaping of the T cell repertoire.

Posttranslational regulation of TCR Valpha allelic exclusion during T cell differentiation.

We have previously shown that phenotypic allelic exclusion of TCR alpha-chain is functional only in mature thymocytes. A significant proportion of immature thymocytes (TCRlow) express more than one cell surface alpha-chain, but mature thymocytes (TCRhigh) show phenotypic allelic exclusion and express only a single alpha-chain. We have analyzed thymocytes for both surface and intracellular alpha-chain expression and find that the majority of mature thymocytes express a second alpha-chain intracellularly. This result is predicted by a model in which the developmentally regulated allelic exclusion of the TCR alpha-chain is caused by competition between alpha-chains for the beta-chain rather than by models in which one alpha-chain is down-regulated or in which selection favors cells with only a single alpha-chain species. Changes in the relative amounts of alpha- and beta-chains available for pairing may therefore allow competition between the two alpha-chains for the beta-chain. Peripheral T cells also frequently express second alpha-chains in the cytoplasm (18-27%), despite a rather low frequency of dual alpha-chain expression on the cell surface (2-4%). The frequency of nonsurface expressed alpha-chains is reduced somewhat compared with thymocytes, indicating that an additional level of control of allelic exclusion operates during the maturation of peripheral T cells.

Natural killer cells: influence of the home environment.

Recent results show that the repertoire of anti-MHC class 1-specific inhibitory receptors expressed by natural killer cells is influenced by self class 1 molecules in such a way as to minimize the number of cells with multiple self-reactive inhibitory receptors.

V alpha 3.2 selection in MHC class I mutant mice: evidence for an alternate orientation of TCR-MHC class I interaction.

TCR V alpha elements are expressed preferentially in CD4 or CD8 subsets in a manner that is largely independent of MHC haplotype. It is likely that the V alphas interact preferentially with conserved regions of class I or class II molecules. To investigate the topology of binding of TCR to MHC-peptide complexes, we screened a panel of H-2Kbm mutants for differential V alpha expression. One strain, bm23, showed a consistent alteration in V alpha expression, with increased V alpha 3.2 expression in CD8 peripheral T cells. This overselection is manifest in CD8 single-positive thymocytes and appears to be due to enhanced positive selection on Kbm23. There is no apparent effect of V beta elements. The Kbm23 molecule is unique compared with Kb and the other Kbm molecules at residue 75 on the helix of the alpha 1 domain, suggesting an interaction between V alpha 3.2 and the alpha 1 helix at this point. Such an interaction is inconsistent with the orientation of TCR and MHC defined in two crystal structures, but is consistent with an orientation where the TCR is rotated by 180 degrees relative to MHC.

Selection of phage-displayed superantigen by binding to cell-surface MHC class II.

We have expressed the superantigen staphylococcal enterotoxin A (SEA) on the surface of bacteriophage as a fusion with the gene VIII protein (gVIIIp). This phage-displayed superantigen retains the properties inherent in the natural protein. It binds to MHC class II and activates T-cells bearing appropriate V beta regions. A flexible 5-amino acid linker sequence between the SEA molecule and the phage coat protein improved the production of functional phage-displayed SEA. Binding to MHC class II-expressing cells effectively selected SEA-phage from non-SEA-phage background. This indicates that this will be an effective method for selecting new specificities of superantigen from libraries of SEA mutants and for cloning of novel superantigens.