The major histocompatibility complex-restricted antigen receptor on T cells: the genetics of expression of an allotype

The monoclonal antibody KJ16-133 binds an allelic determinant expressed on the antigen-specific, major histocompatibility complex (MHC)-restricted receptors on approximately 20% of T cells in most mouse strains. The locus controlling the presence or absence of the determinant mapped 9.8 +/- 2.2 centimorgans from the Igk/Ly-2 locus on chromosome 6 in mice, and may be the beta-chain locus. Other genetic loci were identified that controlled the frequency of cells that expressed the allele in positive mice. One of these was the MHC itself, which may control expression of the beta-chain allele by controlling T cell repertoire. The identity of the other, as yet unmapped locus is unknown. KJ16-133 was used to show that T cell receptor gene products are expressed in a manner consistent with allelic exclusion.     

Chromosome‐level assembly of wild Bactrian camel genome reveals organization of immune gene loci

Camelids are characterized by their unique adaptive immune system that exhibits the generation of homodimeric heavy‐chain immunoglobulins, somatic hypermutation of T‐cell receptors, and low genetic diversity of major histocompatibility complex (MHC) genes. However, short‐read assemblies are typically highly fragmented in these gene loci owing to their repetitive and polymorphic nature. Here, we constructed a chromosome‐level assembly of wild Bactrian camel genome based on high‐coverage long‐read sequencing and chromatin interaction mapping. The assembly with a contig N50 of 5.37 Mb and a scaffold N50 of 76.03 Mb, represents the most contiguous camelid genome to date. The genomic organization of immunoglobulin heavy‐chain locus was similar between the wild Bactrian camel and alpaca, and genes encoding for conventional and heavy‐chain antibodies were intermixed. The organizations of two immunoglobulin light‐chain loci and four T cell receptor loci were also fully deciphered using the new assembly. Additionally, the complete classical MHC region was resolved into a single contig. The high‐quality assembly presented here provides an essential reference for future investigations examining the camelid immune system.     

Biological implication for loss of function at major histocompatibility complex loci

Despite relatively frequent gene or segment duplications, the number of functional loci in the major histocompatibility complex (MHC) is relatively small. The dual function of MHC molecules (triggering the immune system and limiting T-cell receptor repertoires) is likely to balance the number of functional loci. The effect of this dual function on the number of functional MHC loci has been argued mainly in the theoretical and computer simulation studies, but the evidence from empirical data has not been fully examined. Here, we attempt to evaluate this effect based on the analysis of nucleotide sequence data. We hypothesized that due to the dual function, even becoming a pseudogene (pseudogenization) of MHC is advantageous for the organisms. To evaluate this hypothesis, we compared the distribution of the waiting time (T (W)) till pseudogenization for HLA (human MHC) with that of the human olfactory receptor (OR) and bitter taste receptor (T2R) genes. The result shows that T (W) in HLA has a tendency to be relatively shorter as the emergence time (T) of the gene becomes older, while in OR T (W) becomes proportionally longer as T becomes older and in T2R it is almost null irrespective of T. Furthermore, T (W) in HLA is strongly influenced by the extent of functional differentiation in the peptide-binding region. Taken together, these results show that MHC molecules have optimal numbers of functional loci, and these numbers are regulated by the advantageous pseudogenization of duplicated copies.     

Nonequivalence of Classical MHC Class I Loci in Ability to Direct Effective Antiviral Immunity

Structural diversity in the peptide binding sites of the redundant classical MHC antigen presenting molecules is strongly selected in humans and mice. Although the encoded antigen presenting molecules overlap in antigen presenting function, differences in polymorphism at the MHC I A, B and C loci in humans and higher primates indicate these loci are not functionally equivalent. The structural basis of these differences is not known. We hypothesize that classical class I loci differ in their ability to direct effective immunity against intracellular pathogens. Using a picornavirus infection model and chimeric H-2 transgenes, we examined locus specific functional determinants distinguishing the ability of class I sister genes to direct effective anti viral immunity. Whereas, parental FVB and transgenic FVB mice expressing the H-2K^b gene are highly susceptible to persisting Theiler''s virus infection within the CNS and subsequent demyelination, mice expressing the D^b transgene clear the virus and are protected from demyelination. Remarkably, animals expressing a chimeric transgene, comprised primarily of K^b but encoding the peptide binding domain of D^b, develop a robust anti viral CTL response yet fail to clear virus and develop significant demyelination. Differences in expression of the chimeric K^bα1α2D^b gene (low) and D^b (high) in the CNS of infected mice mirror expression levels of their endogenous H-2^q counterparts in FVB mice. These findings demonstrate that locus specific elements other than those specifying peptide binding and T cell receptor interaction can determine ability to clear virus infection. This finding provides a basis for understanding locus-specific differences in MHC polymorphism, characterized best in human populations.     

A single autoimmune T cell receptor recognizes more than a million different peptides

The T cell receptor (TCR) orchestrates immune responses by binding to foreign peptides presented at the cell surface in the context of major histocompatibility complex (MHC) molecules. Effective immunity requires that all possible foreign peptide-MHC molecules are recognized or risks leaving holes in immune coverage that pathogens could quickly evolve to exploit. It is unclear how a limited pool of <10(8) human TCRs can successfully provide immunity to the vast array of possible different peptides that could be produced from 20 proteogenic amino acids and presented by self-MHC molecules (>10(15) distinct peptide-MHCs). One possibility is that T cell immunity incorporates an extremely high level of receptor degeneracy, enabling each TCR to recognize multiple peptides. However, the extent of such TCR degeneracy has never been fully quantified. Here, we perform a comprehensive experimental and mathematical analysis to reveal that a single patient-derived autoimmune CD8(+) T cell clone of pathogenic relevance in human type I diabetes recognizes >one million distinct decamer peptides in the context of a single MHC class I molecule. A large number of peptides that acted as substantially better agonists than the wild-type "index" preproinsulin-derived peptide (ALWGPDPAAA) were identified. The RQFGPDFPTI peptide (sampled from >10(8) peptides) was >100-fold more potent than the index peptide despite differing from this sequence at 7 of 10 positions. Quantification of this previously unappreciated high level of CD8(+) T cell cross-reactivity represents an important step toward understanding the system requirements for adaptive immunity and highlights the enormous potential of TCR degeneracy to be the causative factor in autoimmune disease.     

The major histocompatibility complex of primates

The major histocompatibility complex (MHC) encodes cell surface glycoproteins that function in self-nonself recognition and in allograft rejection. Among primates, the MHC has been well defined only in the human; in the chimpanzee and in two species of macaque monkeys the MHC is less well characterized. Serologic, biochemical and genetic evidence indicates that the basic organization of the MHC linkage group has been phylogenetically conserved. However, the number of genes and their linear relationship on the chromosomes differ between species. Class I MHC loci encode molecules that are the most polymorphic genes known. These molecules are ubiquitous in their tissue distribution and typically are recognized together with nominal antigens by cytotoxic lymphocytes. Class II MHC loci constitute a smaller family of serotypes serving as restricting elements for regulatory T lymphocytes. The distribution of class II antigens is limited mainly to cell types serving immune functions, and their expression is subject to up and down modulation. Class III loci code for components C2, C4 and Factor B (Bf) of the complement system.Interspecies differences in the extent of polymorphism occur, but the significance of this finding in relation to fitness and natural selection is unclear. Detailed information on the structure and regulation of MHC gene expression will be required to understand fully the biologic role of the MHC and the evolutionary relationships between species. Meanwhile, MHC testing has numerous applications to biomedical research, especially in preclinical tissue and organ transplantation studies, the study of disease mechanisms, parentage determination and breeding colony management. In this review, the current status of MHC definition in nonhuman primates will be summarized. Special emphasis is placed on the CyLA system of M. fascicularis which is a major focus in our laboratory. A highly polymorphic cynomolgus MHC has been partially characterized and consists of at least 14 A locus, 11 B locus, 7 C locus class I allelic specificities, 9 Ia-like class II antigens and 6 Bf (class III) variants.     

Effective adoptive therapy of tap-deficient lymphoma using diverse high avidity alloreactive T cells

High avidity for antigen and diversity of T cell receptor (TCR) repertoire are essential for effective immunity against cancer. We have previously created a transgenic mouse strain with increased TCR avidity in a diverse T cell population. In this report, we show that strong alloreactive responses of transgenic T cells against targets with low MHC class I expression can be used for effective adoptive transfer of tumor immunity in vivo. Alloreactive transgenic T cells could be an effective therapeutic approach counteracting tumor evasion of the immune system.     

Innate immunity to cytomegalovirus: the Cmv1 locus and its role in natural killer cell function

The identification and characterization of genetic loci that contribute to patterns of susceptibility/resistance to infection provide important insights into the mechanisms of innate and adaptive immunity. Genetic heterogeneity across the population makes the characterization of such traits in humans technically difficult; however, inbred animal models represent an ideal tool for such analyses. This review illustrates the power of mouse genetics as utilized for the identification and characterization of the locus conferring early resistance to murine cytomegalovirus infection, Cmv1. This locus encodes an activating C-type lectin receptor of the Ly49 family that promotes natural killer (NK) cell cytolysis of infected cells. Although NK cells are usually able to detect and destroy virally infected cells via recognition of the downregulation of MHC class I molecules, the Cmv1 locus provides the first example of an NK receptor that is able to mediate clearance of viral infection via direct recognition of a virally encoded protein.     

Major-histocompatibility-complex-class-II-positive cells and interleukin-2-dependent proliferation of immune T cells are required to reject carcinoma cells in the guinea pig

Summary Tumor immunity induced by bacillus Calmette-Guérin was studied in the line 10 hepatocellular carcinoma (line 10) in the strain-2 guinea pig. Line 10 immunity was investigatedin vitro with a lymphocyte proliferation assay using line 10 tumor protein extracted with 3 M KCl andin vivo by adoptive transfer of line-10-immune spleen cells. Monoclonal antibodies against guinea pig leucocyte markers were used to block functional properties of the immune cells in order to determine which cell types or cell markers are involved in the immune response to the line 10 tumor.In vitro cells from the spleen, peripheral blood and regional lymph node of immune animals reacted with a proliferative response to line 10 protein. This antigen-specific response was caused by T cells and was regulated by major histocompatibility complex (MHC) class II molecules. In blocking experiments it was found that CT5 (anti-PanT), or MSgp4 [anti-(MHC class I antigen)] monoclonal antibodies did not block but some-times stimulated the proliferative response. The effect of H159 (anti-PanT) was irregular, while H155 [anti-(T helper)], and 5C3 [anti-(IL-2 receptor)] monoclonal antibodies blocked the response almost completely. We studied the relevance of the resultsin vitro obtained and found that mAb 5C3 [anti-(IL-2 receptor)] inhibited the adoptive transfer of line 10 immunity, suggesting that the rejection of line 10 cells is caused by a mechanism that is interleukin-2 (IL-2)-dependent. Moreover, complement lysis of MHC-class-II-antigen-positive immune spleen cells inhibited completely the rejection of the line 10 tumor cell challenge in the adoptive-transfer experiments. In conclusion, our data show that MHC class II molecules or cells possessing these molecules are involved in immunity against line 10 tumor cells, as (a) monoclonal antibodies against MHC class II antigens inhibited thein vitro proliferative response of T cells to tumor antigens and (b) removal of MHC-class-II-positive immune spleen cells abrogated the antitumor effect in the adoptive-transfer experiments. Interleukin-2-dependent proliferation of immune T cells is required for the rejection of line 10 tumor cells.     

Specificity on a knife-edge: the alphabeta T cell receptor

The interaction between the alphabeta T cell receptor (TCR) and the peptide bound to the major histocompatibility complex class I molecule (pMHC-I) constitutes a central interaction in adaptive immunity. How these receptors interact with such low affinity while maintaining exquisite specificity for peptide antigen and host MHC (MHC-I restriction) remains a challenge to be explained by structural immunologists. Moreover, how this extracellular interaction is transmitted as an intracellular signal via the CD3 complex remains unresolved. Nevertheless, several structures of TCRs, non-liganded and ligated to a defined pMHC-I, combined with detailed biophysical analyses, have provided insight of the structural basis of MHC-I restriction. In addition, structures of isolated CD3 components have enabled T cell signalling mechanisms to be postulated. Recent findings in this area, which include seven distinct TCR/pMHC-I complexes, have fundamental implications in adaptive immunity as well as therapeutic applications to modulate the adaptive immune response.     

Are extensive T cell epitope polymorphisms in the Plasmodium falciparum circumsporozoite antigen, a leading sporozoite vaccine candidate, selected by immune pressure?

Protective cellular immune responses depend on MHC presentation of pathogen-derived Ag fragments. MHC diversity renders this process sensitive to point mutations coding for altered amino acid sequence of the short target Ag-derived peptides epitopes. Thus, in a given host, a pathogen with an altered epitope sequence will be more likely to escape detection and elimination by the immune system. At a population level, selection by immune pressure will increase the likelihood of polymorphism in important pathogen antigenic epitopes. This mechanism of immune evasion is found in viruses and other pathogens. The detection of polymorphic hot spots in an Ag is often taken as a strong indication of its role in protective immunity. We provide evidence that polymorphisms in the T cell epitopes of a malaria vaccine candidate are unlikely to have been selected by immune pressure in the human host.     

High variability in the MHC class II DA beta chain of the brushtail possum (<Emphasis Type="Italic">Trichosurus vulpecula</Emphasis>)

The diversity of class II major histocompatibility complex (MHC) loci was investigated in the brushtail possum, an important marsupial pest species in New Zealand. Immunocontraception, a form of fertility control that generates an autoimmune response, is being developed as a population control method for the possum. Because the immune response is partly under genetic control, an understanding of immunogenetics in possum will be crucial to the development of immunocontraceptive vaccines. MHC molecules are critical in the vertebrate immune response. Class II MHC molecules bind and present exogenously derived peptides to T lymphocytes and may be important in the presentation of immunocontraceptives. We used polymerase chain reaction primers designed to amplify the peptide binding region of possum class II MHC genes to isolate sequences from 49 animals. We have previously described 19 novel alleles from the DAB locus in the possum (Holland et al., Immunogenetics 60:449–460, 2008). Here, we report on another 11 novel alleles isolated from possum DAB, making this the most diverse marsupial locus described so far. This high level of diversity indicates that DAB is an important MHC locus in the possum and will need to be taken into account in the design of immunocontraceptive vaccines.     

Peptide Modulation of Class I Major Histocompatibility Complex Protein Molecular Flexibility and the Implications for Immune Recognition

T cells use the αβ T cell receptor (TCR) to recognize antigenic peptides presented by class I major histocompatibility complex proteins (pMHCs) on the surfaces of antigen-presenting cells. Flexibility in both TCRs and peptides plays an important role in antigen recognition and discrimination. Less clear is the role of flexibility in the MHC protein; although recent observations have indicated that mobility in the MHC can impact TCR recognition in a peptide-dependent fashion, the extent of this behavior is unknown. Here, using hydrogen/deuterium exchange, fluorescence anisotropy, and structural analyses, we show that the flexibility of the peptide binding groove of the class I MHC protein HLA-A*0201 varies significantly with different peptides. The variations extend throughout the binding groove, impacting regions contacted by TCRs as well as other activating and inhibitory receptors of the immune system. Our results are consistent with statistical mechanical models of protein structure and dynamics, in which the binding of different peptides alters the populations and exchange kinetics of substates in the MHC conformational ensemble. Altered MHC flexibility will influence receptor engagement, impacting conformational adaptations, entropic penalties associated with receptor recognition, and the populations of binding-competent states. Our results highlight a previously unrecognized aspect of the “altered self” mechanism of immune recognition and have implications for specificity, cross-reactivity, and antigenicity in cellular immunity.     

Genetic diversity in cytokines associated with immune variation and resistance to multiple pathogens in a natural rodent population

Pathogens are believed to drive genetic diversity at host loci involved in immunity to infectious disease. To date, studies exploring the genetic basis of pathogen resistance in the wild have focussed almost exclusively on genes of the Major Histocompatibility Complex (MHC); the role of genetic variation elsewhere in the genome as a basis for variation in pathogen resistance has rarely been explored in natural populations. Cytokines are signalling molecules with a role in many immunological and physiological processes. Here we use a natural population of field voles (Microtus agrestis) to examine how genetic diversity at a suite of cytokine and other immune loci impacts the immune response phenotype and resistance to several endemic pathogen species. By using linear models to first control for a range of non-genetic factors, we demonstrate strong effects of genetic variation at cytokine loci both on host immunological parameters and on resistance to multiple pathogens. These effects were primarily localized to three cytokine genes (Interleukin 1 beta (Il1b), Il2, and Il12b), rather than to other cytokines tested, or to membrane-bound, non-cytokine immune loci. The observed genetic effects were as great as for other intrinsic factors such as sex and body weight. Our results demonstrate that genetic diversity at cytokine loci is a novel and important source of individual variation in immune function and pathogen resistance in natural populations. The products of these loci are therefore likely to affect interactions between pathogens and help determine survival and reproductive success in natural populations. Our study also highlights the utility of wild rodents as a model of ecological immunology, to better understand the causes and consequences of variation in immune function in natural populations including humans.     

Birth-and-death evolution in primate MHC class I genes: divergence time estimates

The major histocompatibility complex (MHC) is a multigene family that mediates the host immune response by helping T lymphocytes to recognize and respond to foreign antigens. The high degree of polymorphism and a quick turnover of the genetic loci make the evolution of MHC genes an intriguing subject of study. To understand the evolutionary pattern of this multigene family, we studied the phylogeny and divergence times of six functional MHC class I loci from primate species. On the phylogenetic trees, locus F occupies the most basal position among these loci. Our results suggest that the F locus diverged from the other MHC class I loci about 46-66 MYA. The major diversification of the other class I loci was estimated to have occurred at about 35-49 MYA, which is before the time of separation of Old World-New World monkeys. The gene duplication leading to the classical C locus in great apes appears to have occurred about 21-28 MYA. At approximately the same time the duplication of the B locus occurred in macaques. The oldest allelic lineages of A, B, and C loci in humans seem to have appeared at least 14-19, 10-15, and 13-17 MYA, respectively. Our phylogenetic analysis supports the hypothesis that the nonclassical locus F has diverged from the rest of class I loci very early in primate evolution. The overall phylogenetic pattern observed among class I genes is consistent with the model of birth-and-death evolution.     

MHC Class II Molecules Enhance Toll-Like Receptor Mediated Innate Immune Responses

Background

Major histocompatibility complex (MHC) class II molecules play crucial roles in immune activation by presenting foreign peptides to antigen-specific T helper cells and thereby inducing adaptive immune responses. Although adaptive immunity is a highly effective defense system, it takes several days to become fully operational and needs to be triggered by danger-signals generated during the preceding innate immune response. Here we show that MHC class II molecules synergize with Toll-like receptor (TLR) 2 and TLR4 in inducing an innate immune response.

Methodology/Principal Findings

We found that co-expression of MHC class II molecules and TLR2 or TLR4 in human embryonic kidney (HEK) cells 293 leads to enhanced production of the anti-microbial peptide human-β-defensin (hBD) 2 after treatment with TLR2 stimulus bacterial lipoprotein (BLP) or TLR4 ligand lipopolysaccharide (LPS), respectively. Furthermore, we found that peritoneal macrophages of MHC class II knock-out mice show a decreased responsiveness to TLR2 and TLR4 stimuli compared to macrophages of wild-type mice. Finally, we show that MHC class II molecules are physically and functionally associated with TLR2 in lipid raft domains of the cell membrane.

Conclusions/Significance

These results demonstrate that MHC class II molecules are, in addition to their central role in adaptive immunity, also implicated in generating optimal innate immune responses.     

Transgenic expression of Ly49A on T cells impairs a specific antitumor response

Inhibitory MHC receptors determine the reactivity and specificity of NK cells. These receptors can also regulate T cells by modulating TCR-induced effector functions such as cytotoxicity, cytokine production, and proliferation. Here we have assessed the capacity of mouse T cells expressing the inhibitory MHC class I receptor Ly49A to respond to a well-defined tumor Ag in vivo using Ly49A transgenic mice. We find that the presence of Ly49A on the vast majority of lymphocytes prevents the development of a significant Ag-specific CD8+ T cell response and, consequently, the rejection of the tumor. Despite minor alterations in the TCR repertoire of CD8+ T cells in the transgenic lines, precursors of functional tumor-specific CD8+ T cells exist but could not be activated most likely due to a lack of appropriate CD4+ T cell help. Surprisingly, all of these effects are observed in the absence of a known ligand for the Ly49A receptor as defined by its ability to regulate NK cell function. Indeed, we found that the above effects on T cells may be based on a weak interaction of Ly49A with Kb or Db class I molecules. Thus, our data demonstrate that enforced expression of a Ly49A receptor on conventional T cells prevents a specific immune response in vivo and suggest that the functions of T and NK cells are differentially sensitive to the presence of inhibitory MHC class I receptors.     

Lack of genetic diversity across diverse immune genes in an endangered mammal,the Tasmanian devil (Sarcophilus harrisii)

The Tasmanian devil (Sarcophilus harrisii) is threatened with extinction due to the spread of devil facial tumour disease. Polymorphisms in immune genes can provide adaptive potential to resist diseases. Previous studies in diversity at immune loci in wild species have almost exclusively focused on genes of the major histocompatibility complex (MHC); however, these genes only account for a fraction of immune gene diversity. Devils lack diversity at functionally important immunity loci, including MHC and Toll‐like receptor genes. Whether there are polymorphisms at devil immune genes outside these two families is unknown. Here, we identify polymorphisms in a wide range of key immune genes, and develop assays to type single nucleotide polymorphisms (SNPs) within a subset of these genes. A total of 167 immune genes were examined, including cytokines, chemokines and natural killer cell receptors. Using genome‐level data from ten devils, SNPs within coding regions, introns and 10 kb flanking genes of interest were identified. We found low polymorphism across 167 immune genes examined bioinformatically using whole‐genome data. From this data, we developed long amplicon assays to target nine genes. These amplicons were sequenced in 29–220 devils and found to contain 78 SNPs, including eight SNPS within exons. Despite the extreme paucity of genetic diversity within these genes, signatures of balancing selection were exhibited by one chemokine gene, suggesting that remaining diversity may hold adaptive potential. The low functional diversity may leave devils highly vulnerable to infectious disease, and therefore, monitoring and preserving remaining diversity will be critical for the long‐term management of this species. Examining genetic variation in diverse immune genes should be a priority for threatened wildlife species. This study can act as a model for broad‐scale immunogenetic diversity analysis in threatened species.     

MHC class I bound to an immunodominant Theileria parva epitope demonstrates unconventional presentation to T cell receptors

T cell receptor (TCR) recognition of peptide-MHC class I (pMHC) complexes is a crucial event in the adaptive immune response to pathogens. Peptide epitopes often display a strong dominance hierarchy, resulting in focusing of the response on a limited number of the most dominant epitopes. Such T cell responses may be additionally restricted by particular MHC alleles in preference to others. We have studied this poorly understood phenomenon using Theileria parva, a protozoan parasite that causes an often fatal lymphoproliferative disease in cattle. Despite its antigenic complexity, CD8+ T cell responses induced by infection with the parasite show profound immunodominance, as exemplified by the Tp1(214-224) epitope presented by the common and functionally important MHC class I allele N*01301. We present a high-resolution crystal structure of this pMHC complex, demonstrating that the peptide is presented in a distinctive raised conformation. Functional studies using CD8+ T cell clones show that this impacts significantly on TCR recognition. The unconventional structure is generated by a hydrophobic ridge within the MHC peptide binding groove, found in a set of cattle MHC alleles. Extremely rare in all other species, this feature is seen in a small group of mouse MHC class I molecules. The data generated in this analysis contribute to our understanding of the structural basis for T cell-dependent immune responses, providing insight into what determines a highly immunogenic p-MHC complex, and hence can be of value in prediction of antigenic epitopes and vaccine design.