Ia Molecules Research Articles

The Structure and Stability of the Monomorphic HLA-G Are Influenced by the Nature of the Bound Peptide

The highly polymorphic major histocompatibility complex class Ia (MHC-Ia) molecules present a broad array of peptides to the clonotypically diverse αβ T-cell receptors. In contrast, MHC-Ib molecules exhibit limited polymorphism and bind a more restricted peptide repertoire, in keeping with their major role in innate immunity. Nevertheless, some MHC-Ib molecules do play a role in adaptive immunity. While human leukocyte antigen E (HLA-E), the MHC-Ib molecule, binds a very restricted repertoire of peptides, the peptide binding preferences of HLA-G, the class Ib molecule, are less stringent, although the basis by which HLA-G can bind various peptides is unclear. To investigate how HLA-G can accommodate different peptides, we compared the structure of HLA-G bound to three naturally abundant self-peptides (RIIPRHLQL, KGPPAALTL and KLPQAFYIL) and their thermal stabilities. The conformation of HLA-GKGPPAALTL was very similar to that of the HLA-GRIIPRHLQL structure. However, the structure of HLA-GKLPQAFYIL not only differed in the conformation of the bound peptide but also caused a small shift in the α2 helix of HLA-G. Furthermore, the relative stability of HLA-G was observed to be dependent on the nature of the bound peptide. These peptide-dependent effects on the substructure of the monomorphic HLA-G are likely to impact on its recognition by receptors of both innate and adaptive immune systems.

HLA-E monoclonal antibodies recognize shared peptide sequences on classical HLA class Ia: Relevance to human natural HLA antibodies

The non-classical HLA-Ib molecule, HLA-E share several peptide sequence similarities with the heavy chains of classical HLA class Ia (-B and -C) molecules. Therefore, the antibodies to HLA-E, that recognize shared sequences, may bind to HLA-Ia alleles. This hypothesis is tested by examining the affinity of HLA-E monoclonal antibodies (HLA-E-MAbs) to HLA-Ia molecules and by inhibiting the antibody binding to both HLA-E and HLA-Ia with the shared peptide sequence(s). Single recombinant HLA molecule-coated beads are used for antibody binding. The antibody binding is evaluated by measuring mean fluorescence index [MFI] with Luminex multiplex flow-cytometric technology. The peptide-inhibition experiments are carried out with synthetic shared peptides, most prevalent to HLA-E and HLA-Ia alleles. The number of HLA-Ia alleles recognized by the HLA-E-MAbs varies with the density of the antigen (quantity of antigen-coated beads) and dilution of MAb. Binding of HLA-E-MAbs to β2 microglobulin (β2m)-free HLA-Ia antigens confirms the location of the epitopes on the heavy chain (HC) of the antigens. Strikingly, the nature of alleles of HLA-Ia recognized by different HLA-E-MAbs is identical. The binding of HLA-E-MAbs to the HLA-Ia is inhibited dosimetrically by the adjacent peptides, 115QFAYDGKDY123 and 137DTAAQI142, but not by 126LNEDLRSWTA135, another closer shared peptide sequence. The inhibitory peptide sequences in HLA-E are at the α2-helix terminal facing β2m. The HLA-Ia alleles recognized by HLA-E-MAb (e.g., MEM-E/02) are similar to those recognized by the natural anti-HLA antibodies found in the sera of healthy non-alloimmunized males. This study postulates that some, if not all, of the natural HLA-Ia antibodies seen in healthy males could be anti-HLA-E antibodies cross-reacting with HLA-Ia alleles.

Characterization of CD25‐Positive T Cells During Syngeneic Pregnancy: Production of Stimulatory Class II MHC Molecules

Various studies demonstrate that immunosuppression vis-à-vis paternal alloantigens may play a role for successful pregnancy. However, if this theory is true, the question that remains unanswered is how do syngeneic pregnancies manage to produce viable embryos. In allogeneic murine pregnancies immunosuppression is mediated by regulatory CD25(+)/Foxp3/CTLA-4 T cells. In order to evaluate whether these cells also play a role in syngeneic pregnancies, CD25(+)CD4(+) and CD25(+)CD8(+) were isolated from spleens of pregnant mice and examined as to the expression of specific suppressive and stimulatory markers, cytokine and soluble MHC class II antigen production. Interestingly, the CD25(+) cells and their products displayed an MHC-restricted stimulatory activity on total spleen cell proliferation assays. Although the CD25(+)CD4(+) and CD25(+)CD8(+) cells expressed Foxp3, they lacked CTLA-4, while expressing CD28. Non-specific proliferative effect was shown to be mediated by IL-3 and IL-4. The MHC-restricted proliferative effect; however, could be attributed to IA(d) molecules, which were detected in all culture supernatants of the T cell subpopulations tested and their elimination ablated the observed stimulatory activity. The detection of Ea, Eb1, Eb2 in addition to the Aa and Ab transcripts in these cells indicated the possible involvement of other class II gene combinations in this type of regulation. The results indicate that in the absence of paternal MHC alloantigens, maternal CD25(+) cells are involved in the development of immunostimulation to ensure foetal survival.

Classical Major Histocompatibility Complex Class I Molecules in Motoneurons: New Actors at the Neuromuscular Junction

Major histocompatibility complex (MHC) class I molecules have fundamental functions in the immune system. Recent studies have suggested that these molecules may also have non-immune functions in the nervous system, in particular related to synaptic function and plasticity. Because adult motoneurons express mRNAs for MHC class I molecules, we have examined their subcellular expression pattern in vivo and their role for the synaptic connectivity of these neurons. We observed immunoreactivity for classical MHC class I (Ia) protein in motoneuron somata, but the predominant expression was found in axons and presynaptically at neuromuscular junctions (NMJs). Peripheral nerve lesion induced a strong increase of motoneuron MHC class Ia (H2-K(b)/D(b)) mRNA, indicating a role for MHC class Ia molecules during regeneration. Accordingly, there was an accumulation of MHC class Ia proteins at the cut ends and in growth cones of motor axons after lesion. In K(b-/-)D(b-/-) mice (lacking MHC class Ia molecules), the time course for recovery of grip ability in reinnervated muscles was significantly delayed. Muscles from K(b-/-)D(b-/-) mice displayed an increased density and a disturbed distribution of NMJs and fewer terminal Schwann cells/NMJ compared with wild-type mice. A population of Schwann cells in sciatic nerves expressed the paired Ig receptor B, which binds to MHC class I molecules. These results provide the first evidence that neuronal MHC class Ia molecules are present in motor axons, that they are important for organization of NMJs and motor recovery after nerve lesion, and that their actions may be mediated via Schwann cells.

Oligomeric Structure of Colicin Ia Channel in Lipid Bilayer Membranes

Colicin Ia is a soluble, harpoon-shaped bacteriocin which translocates across the periplasmic space of sensitive Escherichia coli cell by parasitizing an outer membrane receptor and forms voltage-gated ion channels in the inner membrane. This process leads to cell death, which has been thought to be caused by a single colicin Ia molecule. To directly visualize the three-dimensional structure of the channel, we generated two-dimensional crystals of colicin Ia inserted in lipid-bilayer membranes and determined a approximately 17 three-dimensional model by electron crystallography. Supported by velocity sedimentation, chemical cross-linking and single-particle image analysis, the three-dimensional structure is a crown-shaped oligomer enclosing a approximately 35 A-wide extrabilayer vestibule. Our study suggests that lipid insertion instigates a global conformational change in colicin Ia and that more than one molecule participates in the channel architecture with the vestibule, possibly facilitating the known large scale peptide translocation upon channel opening.

A peptide derived from hepatitis C virus (HCV) core protein inducing cellular responses in patients with HCV with various HLA class IA alleles

C35-44 peptide is a well known HLA-A2-restricted CTL epitope originating from hepatitis C virus (HCV) core protein. It was reported that the majority of HCV positive patients had significant levels of serum IgG specific to this peptide. This study addressed whether C35-44 peptide could induce CTL activity restricted to various HLA class IA alleles or could not. This peptide demonstrated binding activity to HLA-A*2402, -A*2601, -A*3101, and -A*3303 molecules, but not to HLA-A*1101 by means of stabilization assay. This peptide also induced CTL activity restricted to each of them, except HLA-A11(+) peripheral blood mononuclear cells from HCV 1b(+) patients by means of (51)Cr-release assay. With regard to HLA-A2 subtypes, this peptide demonstrated binding activity to HLA-A*0201 and -A*0206, but not to -A*0207 molecules. Furthermore, this peptide induced CTL activity from both the patients and healthy donors with all the HLA class IA molecules mentioned above by means of interferon-gamma production assay. These results may provide new insights for the development of a novel peptide vaccine against HCV compatible with various HLA class IA types.

Serum-derived MHC class II molecules: Potent regulators of the cellular and humoral immune response

Soluble MHC antigens are detected in body fluids but their role and origin are still unclear. This study examined whether serum IA antigens, isolated from BALB/c mice (sIA d), could modulate the immune response. Specific purification procedures isolated intact IA molecules, which were thereafter applied to functional assays. Thus, sIA d were shown to stimulate spleen cell proliferation and the major target was identified to be the CD4 + cell population. Inhibition of the CD4 co-receptor using specific neutralizing antibodies destroyed the sIA d-mediated proliferative activity, while sIA d successfully antagonized surface IA d antigens for binding to anti-IA d antibody. Serum-IA d stimulated BALB/c versus C3H/HeN but not C3H/HeN versus BALB/c mixed lymphocyte reactions, while increasing responsiveness to Legionella pneumophila. However, sIA d displayed an inhibitory activity during the effector phase of the humoral response, since they inhibited the anti-DNP-specific IgM production to a DNP–HSA hapten–carrier system. Furthermore, sIA d molecules increased Th1/Th2 cytokines during the L. pneumophila stimulus, while decreasing IL-2, GM-CSF and increasing IL-4, IL-15 during the DNP–HSA stimulus. These results suggest that sIA d, following steps similar to surface class II antigen binding mechanisms, stimulate the initiation of a humoral or cellular immune response but rather inhibit the effector phase of the reactions, attributing thus soluble class II MHC antigens important immunomodulatory roles.

CD8 regulatory T cells: What's old is now new

CD8 regulatory T cells have had a long history in immunology. They were the first described subset of cells capable of inducing immune suppression as shown initially by Gershon and Kondo in the 1970s.1, 2 However, difficulties in characterization of these CD8 T cells and potential markers limited this area considerably. In the current issue, Cone et al.3 describe CD8 Tregs specific for antigen delivered to an immune privileged site—the eye—and their ability to limit effector T-cell responses in an ‘unconventional’ major histocompatibility complex (MHC) class Ib-restricted manner. Besides the initial difficulties in identifying CD8 Tregs, these regulatory cells were also outshone by the emergence of Tregs in the CD4 T-cell compartment. Hall et al.4 and Sakaguchi et al.5 showed that a subset of CD4 T cells expressing CD25 (the interleukin-2 receptor) was capable of inducing immune suppression. Initially described as CD4+ CD25+ T cells, the recent identification of the transcription factor foxp3 has defined a lineage of T cells that are generated in the thymus or induced by peripheral conversion, and can inhibit the autoimmune disease by suppression of autoreactive T cells. The importance of CD8 Tregs has been recently revived by observations in both T-cell vaccination and in knockout mice.6, 7 Cone et al.3 add to this re-emerging field the description of CD8 Tregs that can limit the delayed type hypersensitivity T-cell responses against antigen delivered to the eye, and show that this is restricted not by MHC Class Ia molecules but rather by Qa-1, an MHC Class Ib molecule expressed on effector T cells. Activated T cells can be used to vaccinate mice, and this induces a regulatory response that limits disease in a number of models including experimental autoimmune encephalomyelitis (EAE), the mouse model of multiple sclerosis. This has been shown to be due to the induction of CD8 T cells that interact with effector T cells limiting immune responses (CD8 Tregs).6, 7 Further, this response occurs not through the classical Class Ia MHC molecules, but through a Class Ib molecule Qa-1 (HLA-E in man).6, 7 The importance of this molecule's interaction with CD8 Tregs was shown in Qa-1 knockout mice, which in the absence of CD8 Treg function develop autoimmune disease.6 CD8 Tregs appear to act not only through direct lysis of effector T cells via perforin but also through immunosuppressive cytokines. Qa-1 is a very interesting molecule and is expressed on a range of cells in particular activated T cells, B cells and dendritic cells. It can bind β-2 globulin, such as a classical MHC Class Ia molecule and can present a range of peptides.8 The peptides it presents are derived either from the leader sequence of MHC molecules termed Qdm molecules or from self peptides including preproinsulin and heat-shock protein, and this is a transporter associated with antigen processing (TAP)-dependent process. In addition to acting as a restriction element for CD8 T cells, it is also a ligand for the inhibitory receptor NKG2A on natural killer (NK) cells. Thus, it can both inhibit NK cell lysis of T cells or can lead to CD8 Treg inhibition of T cells. Distinct binding sites on Qa-1 for CD8 Tregs and NK cell, NKG2A, have been defined, and mutations of each lead to opposing effects. The lack of activation of CD8 Tregs leads to worsening disease, whereas loss of NK inhibition leads to less severe disease (Figure 1).9 The numbers of CD8 Tregs are relatively small: <1% in peripheral circulation, though much higher among intestinal epithelial lymphocytes. A number of markers of CD8 Tregs have been studied including CD28low, CD122+ and CD8αα expression compared with the normal CD8αβ heterodimer.10 Additionally, foxp3 expression has been identified in some CD8s though this may reflect activation-induced foxp3 rather than a true regulatory phenotype. The use of Qa-1 tetramers while initially promising is limited by the relatively low binding efficiency of peptides to the molecule and also by the tetramer binding to NKG2A on large numbers of CD8 T cells. The restricted nature of CD8 Tregs has also been shown by T cell receptor (TCR) repertoire analysis. In summary, Qa-1-restricted CD8 Tregs appear to represent a unique regulatory subset that has a role in limiting autoimmune disease. The study by Cone et al.3 adds to the current literature by showing local immune suppression by CD8 Tregs and their restriction by Qa-1 rather than Class Ia molecules. Other data in a parallel study by these authors also show the role of the cytokines transforming growth factor-β and interferon-γ in the function of CD8 Tregs in suppressing the delayed type hypersensitivity.11 Qa-1-restricted CD8+ Tregs limit the effector T cells compared with the NK cells, which are inhibited by Qa-1 through NKG2A.

An MHC class Ib–restricted CD8 T cell response confers antiviral immunity

Although immunity against intracellular pathogens is primarily provided by CD8 T lymphocytes that recognize pathogen-derived peptides presented by major histocompatibility complex (MHC) class Ia molecules, MHC class Ib–restricted CD8 T cells have been implicated in antiviral immunity. Using mouse polyoma virus (PyV), we found that MHC class Ia–deficient (Kb−/−Db−/−) mice efficiently control this persistently infecting mouse pathogen. CD8 T cell depletion mitigates clearance of PyV in Kb−/−Db−/− mice. We identified the ligand for PyV-specific CD8 T cells in Kb−/−Db−/− mice as a nonamer peptide from the VP2 capsid protein presented by Q9, a member of the β2 microglobulin–associated Qa-2 family. Using Q9-VP2 tetramers, we monitored delayed but progressive expansion of these antigen-specific CD8αβ T cells in Kb−/−Db−/− mice. Importantly, we demonstrate that Q9-VP2–specific CD8 T cells more effectively clear wild-type PyV than a VP2 epitopenull mutant PyV. Finally, we show that wild-type mice also generate Q9-restricted VP2 epitope–specific CD8 T cells to PyV infection. To our knowledge, this is the first evidence for a defined MHC class Ib–restricted antiviral CD8 T cell response that contributes to host defense. This study motivates efforts to uncover MHC class Ib–restricted CD8 T cell responses in other viral infections, and given the limited polymorphism of MHC class Ib molecules, it raises the possibility of developing peptide-based viral vaccines having broad coverage across MHC haplotypes.

Transcriptional profiling of MHC class I genes in rainbow trout infected with infectious hematopoietic necrosis virus

Major histocompatibility complex (MHC) molecules are important mediators of cell-mediated immunity in vertebrates. MHC class IA molecules are important for host anti-viral immunity as they present intracellular antigens and regulate natural killer cell (NK) activity. MHC class Ib molecules on the other hand are less understood and have demonstrated diverse immune and non-immune functions in mammals. Rainbow trout possess a single classical MHC IA locus ( Onmy-UBA) that is believed to function similar to that of mammalian MHC class Ia. Numerous MHC class Ib genes with undetermined functions have also been described in trout. Here we utilize quantitative reverse transcriptase PCR (qRT-PCR) techniques to survey the levels of basal and inducible transcription for selected trout MHC class Ib genes, sIgM and sentinels of IFN induction in response to viral infection. Basal transcription of all the class Ib genes examined in this study was lower than Onmy-UBA in naïve fish. UBA, along with all of the non-classical genes were induced in fish infected with virus but not in control fish. Our results support a non-classical designation for the majority of the class IB genes surveyed in this study based upon expression levels while also indicating that they may play an important role in anti-viral immunity in trout.

The role of tapasin in MHC class I protein trafficking in embryos and T cells

Preimplantation mouse embryos express both classical (class Ia) and nonclassical (class Ib) MHC class I proteins, and yet are not rejected by the maternal immune system. Although the function of the embryonic MHC class Ia proteins is unknown, one MHC class Ib protein, Qa-2, the product of the preimplantation embryo development ( Ped) gene, actually enhances reproductive success. Similar in structure to MHC class Ia proteins, Qa-2 protein is a trimer of the alpha (heavy) chain, β 2 microglobulin and a bound peptide. Studies on the folding, assembly and trafficking of MHC class Ia molecules to the cell surface have revealed this process to be dependent on multiple protein chaperone molecules, but information on the role of chaperone molecules in Qa-2 expression is incomplete. Here, we report the detection of mRNA for four chaperone molecules (TAP1, TAP2, calnexin and tapasin) in preimplantation embryos. We then focused on the role of the MHC-dedicated chaperone, tapasin, on Qa-2 protein expression. First, we demonstrated that tapasin protein is expressed by preimplantation embryos. Then, we used tapasin knockout mice to evaluate the role of tapasin in Qa-2 protein expression on both T cells and preimplantation embryos. We report here that optimal cell surface expression of Qa-2 is dependent on tapasin in both T cells and preimplantation embryos. Identification of the molecules involved in regulation of MHC class I protein expression in early embryos is an important first step in gaining insight into mechanisms of escape of embryos from destruction by the maternal immune system.

The Heterodimeric Assembly of the CD94-NKG2 Receptor Family and Implications for Human Leukocyte Antigen-E Recognition

The CD94-NKG2 receptor family that regulates NK and T cells is unique among the lectin-like receptors encoded within the natural killer cell complex. The function of the CD94-NKG2 receptors is dictated by the pairing of the invariant CD94 polypeptide with specific NKG2 isoforms to form a family of functionally distinct heterodimeric receptors. However, the structural basis for this selective pairing and how they interact with their ligand, HLA-E, is unknown. We describe the 2.5 A resolution crystal structure of CD94-NKG2A in which the mode of dimerization contrasts with that of other homodimeric NK receptors. Despite structural homology between the CD94 and NKG2A subunits, the dimer interface is asymmetric, thereby providing a structural basis for the preferred heterodimeric assembly. Structure-based sequence comparisons of other CD94-NKG2 family members, combined with extensive mutagenesis studies on HLA-E and CD94-NKG2A, allows a model of the interaction between CD94-NKG2A and HLA-E to be established, in which the invariant CD94 chain plays a more dominant role in interacting with HLA-E in comparison to the variable NKG2 chain.

CLL Cell Proliferation and IL-6 Production Are Regulated by CD160 - MHC Class I Interactions Offering New Therapeutic Targets.

We have previously demonstrated the expression of the Natural Killer cell (NK) marker CD160 in CLL, which is not expressed by normal B cells1. CD160 is a cell surface molecule expressed by human and mouse circulating cytotoxic lymphocytes and exhibits a broad specificity for major histocompatibility complex (MHC) class Ia and Ib molecules 2,3,4. Triggering of CD160 on NK cells leads to cell proliferation and IL-6 production5. IL-6 is a pleiotropic cytokine produced by a variety of cell types, including lymphocytes6. IL-6 expression has been associated with the development of lymphomas7, while higher serum IL-6 levels correlated with shorter survival in CLL8. Without stimulation, CLL cells secreted low basal levels of INF-γ (100pg/ml) and even lower levels of IL-2, IL-4, IL-6, IL-10 and TNF-α (< 50pg/ml) (cytokine bead array, BD Bioscience). Incubation with CL1-R2, an anti-CD160 monoclonal antibody, led to an increase in IL-6 alone (up to 1500pg/ml), which was associated with significant cell proliferation (detected by 3H incorporation) - median 100%, range 50 to 400% (n = 13). Different patterns of response were seen, with all cases showing proliferation to CL1-R2 alone and with the positive control pan anti-immunoglobulin Ab (anti-Ig Ab). In some cases, there was marked synergy between CL1-R2 and anti-Ig Ab. We found that the MEC I and MEC II B-cell lines, derived from a patient with prolymphocytic progression of CLL (DSMZ Institut), express CD160 and show CL1-R2 enhanced proliferation (30 to 55% increase). However, MEC I and MEC II only unregulated IL-10 production (13 to 33% increase). Interestingly, proliferation of CD160Neg Sanchez EBV-immortalized B-cells was inhibited when these cells were incubated with CHO-CD160 transfectants. This inhibition was reversed with an anti-CD160 Ab or anti-MHC-class I W6/32 Ab. The CD160-MHC-I interaction plays a role in CLL biology. MAb engagement of CD160 leads to:i.a direct proliferative signal;ii.CD160-triggered production of IL-6 and IL-6 mediated effects;iii.inhibition of a constitutive negative signal mediated via MHC-I molecules by their ligand CD160 (supported by the inhibition of the Sanchez cells by CD160 interaction with their MHC class I).The CD160-MHC class I axis represents a new pathway in CLL biology offering new therapeutic targets. This work also suggests a role for targeting the IL-6/IL-6R system in CLL - for example, with humanized anti-IL6 R (Actemra), already used for Castleman's disease and Rheumatoid Arthritis. [Display omitted]

Involvement of nonclassical MHC class Ib molecules in heat shock protein‐mediated anti‐tumor responses

Nonclassical MHC class Ib (class Ib) genes are found in all jawed vertebrates, and their products are hypothesized to be indicators of intracellular stress and malignancy. They may be involved in immune recognition of classical MHC class Ia (class Ia)-low or -negative tumor cells through their interaction with T cell receptors and/or non-T cell inhibitory or triggering receptors expressed by NK cells and T cells. In the frog Xenopus, the molecular chaperone gp96 mediates a potent immune response involving antigen-specific classical class Ia-unrestricted CD8+ CTL (CCU-CTL) against a transplantable thymic tumor (15/0) that does not express class Ia molecules. We hypothesized that Xenopus nonclassical class Ib gene products (XNC) are involved in gp96-mediated CCU-CTL anti-tumor responses. To investigate the involvement of class Ib gene products in Xenopus anti-tumor responses, we generated, for the first time in ectothermic vertebrates, stable tumor transfectants expressing short hairpin RNA (shRNA) to silence either XNC directly or beta2m to prevent class Ib surface expression. Both types of 15/0 transfectants are more resistant to CCU-CTL killing, more tumorigenic and more susceptible to NK-like cell killing. This study provides in vitro and in vivo evidence of the evolutionary conservation of class Ib involvement in anti-tumor CD8+ T cell responses.

Anti-TCR Antibody Treatment Activates a Novel Population of Nonintestinal CD8αα+TCRαβ+ Regulatory T Cells and Prevents Experimental Autoimmune Encephalomyelitis

CD8alphaalpha+CD4-TCRalphabeta+ T cells are a special lineage of T cells found predominantly within the intestine as intraepithelial lymphocytes and have been shown to be involved in the maintenance of immune homeostasis. Although these cells are independent of classical MHC class I (class Ia) molecules, their origin and function in peripheral lymphoid tissues are unknown. We have recently identified a novel subset of nonintestinal CD8alphaalpha+CD4-TCRalphabeta+ regulatory T cells (CD8alphaalpha Tregs) that recognize a TCR peptide from the conserved CDR2 region of the TCR Vbeta8.2-chain in the context of a class Ib molecule, Qa-1a, and control- activated Vbeta8.2+ T cells mediating experimental autoimmune encephalomyelitis. Using flow cytometry, spectratyping, and real-time PCR analysis of T cell clones and short-term lines, we have determined the TCR repertoire of the CD8alphaalpha regulatory T cells (Tregs) and found that they predominantly use the TCR Vbeta6 gene segment. In vivo injection of anti-TCR Vbeta6 mAb results in activation of the CD8alphaalpha Tregs, inhibition of the Th1-like pathogenic response to the immunizing Ag, and protection from experimental autoimmune encephalomyelitis. These data suggest that activation of the CD8alphaalpha Tregs present in peripheral lymphoid organs other than the gut can be exploited for the control of T cell-mediated autoimmune diseases.

Identification of an MHC class Ib-restricted CD8 T cell response to polyoma virus infection (B136)

Abstract While MHC class Ia-restricted CD8 T cell responses to viral infections have been extensively characterized, little is known about MHC class Ib-restricted CD8 T cells during viral infection. Mouse polyoma virus establishes a persistent low-level infection and induces tumors in MHC class Ia + class Ib deficient (i.e., b2m−/−) mice. Unexpectedly, mice selectively deficient for MHC class Ia molecules (i.e., Kb−/−Db−/−) mice efficiently control polyoma viral replication and are resistant to polyoma virus-induced tumors. Polyoma virus-specific CD8 T cell cloned lines were isolated from immune Kb−/−Db−/− mice. Using these cloned lines, we mapped their MHC restriction to H2-Q9, an MHC Class Ib molecule of the Qa-2 family. We identified the Q9-restricted viral epitope as a 9mer peptide from a viral capsid protein. In preliminary studies, we found that the magnitude of the Q9-restricted antiviral CD8 T cell response progressively inflates over the course of infection. Experiments are underway to visualize this novel virus-specific class Ib-restricted CD8 T cell response in vivo. This work was supported by R01CA71971.

GATA-6 mediates cell-type specific IFN-γresponse of the HLA-E gene (35.39)

Abstract The human MHC Class Ib gene, HLA-E, has been shown to inhibit both Natural Killer cells and a subset of CD8+ cytotoxic T-cells by engaging the CD94/NKG2A inhibitory receptor. IFN-γ induces the expression of HLA-E as well as Class Ia molecules, which are required for killing of target cells. Since HLA-E has negative effects on immune cell killing of target cells, we have sought to identify locus-specific mechanisms of IFN-γ induction in order to identify molecular targets for selective activation of Class Ia genes, but not HLA-E. We have previously identified a unique upstream IFN-γ response region in the HLA-E promoter and showed that GATA-1 is required for its function in the K562 leukemic cell line. We have now examined the effect of GATA family members on IFN-γ induction of HLA-E in other cell types. HLA-E CAT reporter gene assays demonstrated that only epithelial cells which express GATA factors, as determined by western blot and real-time PCR, mediate a 2-to-4 fold enhanced response to IFN-γ stimulation. Functional analysis of HLA-E CAT reporter gene constructs containing mutations of the core nucleotides in the GATA binding site resulted in a four-fold decreased response to IFN-γ. Tetracycline regulated siRNA knockdown of GATA 6 expression in the SKOV3 ovarian carcinoma cell line revealed a two-to-three fold decrease in IFN-γ response of both HLA-E promoter driven constructs and the endogenous HLA-E gene. We conclude that GATA factors play a tissue specific role in regulation of IFN-γ mediated HLA-E expression. This work was supported by National Institutes of Health Grant CA87496 and Immunology Training Grant 5T32AI007407-15.

Recognition of H-2Kb by Ly49Q suggests a role for class Ia MHC regulation of plasmacytoid dendritic cell function

Ly49Q is a member of the polymorphic Ly49 family of NK cell receptors that displays both a high degree of conservation and a unique expression pattern restricted to myeloid lineage cells, including plasmacytoid dendritic cells (pDC). The function and ligand specificity of Ly49Q are unknown. Here, we use reporter cell analysis to demonstrate that a high-affinity ligand for Ly49Q is present on H-2b, but not H-2d, H-2k, H-2q, or H-2a-derived tumor cells and normal cells ex vivo. The ligand is peptide-dependent and MHC Ia-like, as revealed by its functional absence on cells deficient in TAP-1, β2m, or H-2KbDb expression. Furthermore, Ly49Q is specific for H-2Kb, as the receptor binds peptide-loaded H-2Kb but not H-2Db complexes, and Ly49Q recognition can be blocked using anti-Kb but not anti-Db mAb. Greater soluble H-2Kb binding to ligand-deficient pDC also suggests cis interactions of Ly49Q and H-2Kb. These results demonstrate that Ly49Q efficiently binds H-2Kb ligand, and suggest that pDC function, like that of NK cells, is regulated by classical MHC Ia molecules. MHC recognition capability by pDC has important implications for the role of this cell type during innate immune responses.

From Vanilla to 28 Flavors: Multiple Varieties of T Regulatory Cells

Numerous T cell subpopulations have now been claimed to exhibit regulatory activity. Shevach discusses the current understanding of the different subsets of T regulatory cells and provides a perspective on the current areas of uncertainly and controversy in the field.

Effective Control of Chronic γ-Herpesvirus Infection by Unconventional MHC Class Ia–Independent CD8 T Cells

Control of virus infection is mediated in part by major histocompatibility complex (MHC) Class Ia presentation of viral peptides to conventional CD8 T cells. Although important, the absolute requirement for MHC Class Ia–dependent CD8 T cells for control of chronic virus infection has not been formally demonstrated. We show here that mice lacking MHC Class Ia molecules (Kb−/−xDb−/− mice) effectively control chronic γ-herpesvirus 68 (γHV68) infection via a robust expansion of β2-microglobulin (β2-m)-dependent, but CD1d-independent, unconventional CD8 T cells. These unconventional CD8 T cells expressed: (1) CD8αβ and CD3, (2) cell surface molecules associated with conventional effector/memory CD8 T cells, (3) TCRαβ with a significant Vβ4, Vβ3, and Vβ10 bias, and (4) the key effector cytokine interferon-γ (IFNγ). Unconventional CD8 T cells utilized a diverse TCR repertoire, and CDR3 analysis suggests that some of that repertoire may be utilized even in the presence of conventional CD8 T cells. This is the first demonstration to our knowledge that β2-m–dependent, but Class Ia–independent, unconventional CD8 T cells can efficiently control chronic virus infection, implicating a role for β2-n–dependent non-classical MHC molecules in control of chronic viral infection. We speculate that similar unconventional CD8 T cells may be able to control of other chronic viral infections, especially when viruses evade immunity by inhibiting generation of Class Ia–restricted T cells.