Positive selection of a Qa-1-restricted T cell receptor with specificity for insulin

The phenotype and development of T cells from transgenic mice expressing a T cell receptor with specificity for insulin presented by the MHC class Ib molecule Qa-1(b) was investigated. Peripheral T cells from the transgenic mice express CD8 and, after activation, kill Qa-1(b)-positive lymphoid target cells in the presence of soluble insulin. Thymic selection requires expression of Qa-1(b) but not the dominant Qa-1-associated peptide, Qdm. In contrast to conventional T cells, selection is at least as efficient when the selecting ligand is expressed only on hematopoietic lineage cells as compared to expression on epithelial cells in the thymus. Our findings suggest that there is a dedicated population of Qa-1-restricted T cells that are selected by interaction with Qa-1 and that the cellular requirements for selection may differ from conventional T cells.     

Regulation of activated CD4+ T cells by NK cells via the Qa-1-NKG2A inhibitory pathway

The ability of natural-killer cells to regulate adaptive immunity is not well understood. Here we define an interaction between the class Ib major histocompatibility complex (MHC) molecule Qa-1-Qdm on activated T cells responsible for adaptive immunity and CD94-NKG2A inhibitory receptors expressed by natural-killer cells by using Qa-1-deficient and Qa-1 knockin mice containing a point mutation that selectively abolishes Qa-1-Qdm binding to CD94-NKG2A receptors. The Qa-1-NKG2A interaction protected activated CD4+ T cells from lysis by a subset of NKG2A+ NK cells and was essential for T cell expansion and development of immunologic memory. Antibody-dependent blockade of this Qa-1-NKG2A interaction resulted in potent NK-dependent elimination of activated autoreactive T cells and amelioration of experimental autoimmune encephalomyelitis. These findings extend the functional reach of the NK system to include regulation of adaptive T cell responses and suggest a new clinical strategy for elimination of antigen-activated T cells in the context of autoimmune disease and transplantation.     

Qa-1/Tla region alloantigen-specific CTL with alpha beta receptor

Recent studies involving T cells that express gamma delta T-cell receptor (gamma delta TcR) have raised the possibility that Qa-1/Tla region class I major histocompatibility complex (MHC)-like molecules are antigen-presenting molecules for gamma delta TcR. In this report, cytotoxic T lymphocyte (CTL) clones specific for a Qa-1/Tla region gene product were isolated from a bulk B10. QBR (Kb, Ib, Dq Qa-1/Tlab) anti-B10.MBR (Kb, Ik, Dq, Qa/Tlaa) CTL line. These CTL lysed blasts from all Qa-1a strains regardless of the H-2 haplotype, indicating that the recognition of the Qa-1 antigen by these CTL is not restricted by other class I molecules. In bulk populations, CTL activity of this specificity was found only in the CD8+CD4- subpopulation. Accordingly, all established CTL clones were phenotyped as Thy-1+, CD8+CD4-. Furthermore, these clones were shown to express alpha beta TcR rather than gamma delta TcR. Thus, the results indicate that Qa-1 antigen can be recognized by alpha beta TcR T cells in a manner similar to recognition of classical class I molecules.     

Qa-1, a nonclassical class I histocompatibility molecule with roles in innate and adaptive immunity

Qa-1, a nonclassical class I histocompatibility molecule expressed in mice, predominantly assembles with a single nonameric peptide, Qdm, derived from the signal sequence of certain class Ia molecules. The Qa-1/Qdm complex is the primary ligand for CD94/NKG2A inhibitory receptors expressed on a major fraction of natural killer (NK) cells. Cells become susceptible to killing by NK cells under conditions where surface expression of the Qa-1/Qdm inhibitory ligand is reduced. The CD94/NKG2 "missing-self" recognition system serves as mechanism for removing cells that have abnormalities in the intracellular machinery required for assembly and expression of class I-peptides complexes, as a consequence of viral infection, for example. Despite its highly focused peptide-binding specificity, Qa-1 also has a capacity to act as an antigen-presentation molecule for CD8+ T cells. It appears that a small subpopulation of these T cells undergoes positive selection by interaction with Qa-1 in the thymus, and they maintain their specificity for Qa-1 after maturation. The role of these unusual T cells in adaptive immune responses remains to be defined.     

CD94 is essential for NK cell-mediated resistance to a lethal viral disease

It is well established that natural killer (NK) cells confer resistance to many viral diseases, but in only a few instances the molecular mechanisms whereby NK cells recognize virus-infected cells are known. Here we show that CD94, a molecule preferentially expressed by NK cells, is essential for the resistance of C57BL/6 mice to mousepox, a disease caused by the Orthopoxvirus ectromelia virus. Ectromelia virus-infected cells expressing the major histocompatibility complex (MHC) class Ib molecule Qa-1(b) are specifically recognized by the activating receptor formed by CD94 and NKG2E. Because CD94-NKG2 receptors and their ligands are highly conserved in rodents and humans, a similar mechanism may exist during human infections with the smallpox and monkeypox viruses, which are highly homologous to ectromelia virus.     

The Qa-1b molecule binds to a large subpopulation of murine NK cells

Recent studies on human NK cells have demonstrated that the NK cell CD94/NKG2 receptors bind to the nonclassical MHC class I molecule HLA-E. A functional CD94/NKG2 complex has not yet been identified in rodents, but cDNA encoding rat and mouse CD94 and NKG2 have recently been cloned, suggesting that CD94/NKG2 receptors may exist in species other than man. The mouse nonclassical MHC class I molecule Qa-1 shares several features with HLA-E. This suggests that Qa-1 may be similarly recognized by murine NK cells. To study the ability of Qa-1 to bind to murine NK cells, we have produced a soluble tetrameric form of Qa-1^b . In the present study, we demonstrate that Qa-1^b tetramers distinctly bind to a large subset of fresh or IL-2-activated NK1.1⁺ /CD3⁻ splenocytes independently of the expression of Ly49 inhibitory receptors. Binding occurs whether NK cells have evolved in an MHC class I-expressing or in an MHC class I-deficient environment. Our data suggest the existence of a Qa-1-recognizing structure on a large subpopulation of murine NK cells that may be similar to the human CD94/NKG2 heterodimeric complex.     

An important role of Tyk2 in APC function of dendritic cells for priming CD8+ T cells producing IFN-gamma

Tyrosine kinase 2 (Tyk2) contributes to the signals triggered by IL-12 for IFN-gamma production by NK cells and T cells. We found in this study that Tyk2-deficient (-/-) mice showed increased susceptibility at the early stage after an i.p. infection with Listeria monocytogenes, accompanied by impaired IFN-gamma production. The numbers of both MHC class Ib (H2-M3)- or MHC class Ia (Kb)-restricted CD8+ T cells producing IFN-gamma and exhibiting cytotoxicity were significantly decreased in Tyk2-/- mice after infection with L. monocytogenes. Using an adoptive transfer system of OT-I cells expressing OVA(257-264)/Kb-specific TCR into Tyk2-/- mice followed by challenge with recombinant L. monocytogenes expressing OVA, we found that the defective Tyk2 signaling in the host environment was at least partially responsible for the impaired CD8+ T cytotoxic-1 (Tc1) cell responses in Tyk2-/- mice following the infection. Adoptive transfer with MHC class Ib- or MHC class Ia-binding peptide-pulsed BM-derived DC from Tyk2-/- mice induced lower levels of the Ag-specific CD8+ Tc1 cells producing IFN-gamma. These results suggest that Tyk2 signaling is also important for DC function in the induction of MHC class Ia- and class Ib-restricted CD8+ Tc1 cells following L. monocytogenes infection.     

Nonclassical MHC class Ib-restricted cytotoxic T cells monitor antigen processing in the endoplasmic reticulum

The aminopeptidase ERAAP is essential for trimming peptides presented by major histocompatibility complex (MHC) class I molecules. Inhibition of ERAAP by cytomegalovirus results in evasion of the immune response by this virus, and polymorphisms in ERAAP are associated with autoimmune disorders. How normal ERAAP function is monitored is unknown. We found that inhibition of ERAAP rapidly induced presentation of the peptide FYAEATPML (FL9) by the MHC class Ib molecule Qa-1(b). Antigen-experienced T cells specific for the Qa-1(b)-FL9 complex were frequent in naive mice. Wild-type mice immunized with ERAAP-deficient cells mounted a potent CD8(+) T cell response specific for Qa-1(b)-FL9. MHC class Ib-restricted cytolytic effector cells specifically eliminated ERAAP-deficient cells in vitro and in vivo. Thus, nonclassical Qa-1(b)-peptide complexes direct cytotoxic T cells to targets with defective antigen processing in the endoplasmic reticulum.     

Global expression profiling of peripheral Qa-1-restricted CD8αα+TCRαβ+ regulatory T cells reveals innate-like features: implications for immune-regulatory repertoire

Among peripheral regulatory T cells, CD8(+) T cells also play an important role in the maintenance of immune homeostasis. A subset of CD8(+) Treg that express αβ T cell receptor (TCR) and CD8αα homodimers can recognize TCR-derived peptides in the context of the class Ib MHC molecule Qa-1. To gain a better understanding of the nature and phenotype of CD8αα(+)TCRαβ+ Treg, a global gene expression profiling using microarray, real-time quantitative polymerase chain reaction, and flow-cytometric analysis was performed using functional Treg clones and lines. The study findings show that CD8(+) Treg shared gene profile expressed by innate-like lymphocytes, including murine intraepithelial lymphocytes and thymic CD8αα(+)TCRαβ+ T-cell populations. In addition, this subset displays differential expression of several key regulatory molecules, including CD200. CD8αα(+) Treg expressed higher levels of a number of natural killer cell-related receptors and molecules belonging to the TNF superfamily. Collectively, peripheral class Ib-reactive CD8αα(+)TCRαβ+ T cells represent a unique regulatory population different from class Ia major histocompatibility complex-restricted conventional T cells. These studies have important implications for the regulatory mechanisms mediated by the CD8(+) Treg population in general.     

Contribution of NK, NK T, gamma delta T, and alpha beta T cells to the gamma interferon response required for liver protection against Trypanosoma cruzi

In the present work, we show that intracellular Trypanosoma cruzi is rarely found in the livers of acutely infected mice, but inflammation is commonly observed. The presence of numerous intrahepatic amastigotes in infected gamma interferon (IFN-gamma)-deficient mice corroborates the notion that the liver is protected by an efficient local immunity. The contribution of different cell populations was suggested by data showing that CD4- and CD8-deficient mice were able to restrain liver parasite growth. Therefore, we have characterized the liver-infiltrating lymphocytes and determined the sources of IFN-gamma during acute T. cruzi infection. We observed that natural killer (NK) cells increased by day 7, while T and B cells increased by day 14. Among CD3+ cells, CD4+, CD8+, and CD4- CD8- cell populations were greatly expanded. A large fraction of CD3+ cells were positive for PanNK, a beta1 integrin expressed by NK and NK T cells. However, these lymphocytes were not classic NK T cells because they did not express NK1.1 and showed no preferential usage of Vbeta8. Otherwise, liver NK T (CD3+ NK1.1+) cells were not increased in acutely infected mice. The majority of PanNK+ CD4+ and PanNK+ CD8+ cells expressed T-cell receptor alphabeta (TCRalphabeta), whereas PanNK+ CD4- CD8- cells were positive for TCRgammadelta. In fact, gammadelta T cells showed the most remarkable increase (40- to 100-fold) among liver lymphocytes. Most importantly, intracellular analysis revealed high levels of IFN-gamma production at day 7 by NK cells and at day 14 by CD4+, CD8+, and CD4- CD8- TCRgammadelta+ cells. We concluded that NK cells are a precocious source of IFN-gamma in the livers of acutely infected mice, and, as the disease progresses, conventional CD4+ and CD8+ T cells and gammadelta T cells, but not classic NK-T cells, may provide the IFN-gamma required for liver protection against T. cruzi.     

Regulation of self-tolerance by Qa-1-restricted CD8(+) regulatory T cells

Mounting an efficient immune response to pathogens while avoiding damage to host tissues is the central task of the immune system. Emerging evidence has highlighted the contribution of the CD8⁺ lineage of regulatory T cells to the maintenance of self-tolerance. Specific recognition of the MHC class Ib molecule Qa-1 complexed to peptides expressed by activated CD4⁺ T cells by regulatory CD8⁺ T cells triggers an inhibitory interaction that prevents autoimmune responses. Conversely, defective Qa-1-restricted CD8⁺ regulatory activity can result in development of systemic autoimmune disease. Here, we review recent research into the cellular and molecular basis of these regulatory T cells, their mechanism of suppressive activity and the potential application of these insights into new treatments for autoimmune disease and cancer.     

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.     

Role of Qa-1(b)-binding receptors in the specificity of developing NK cells

NK cells acquire the ability to recognize MHC class I molecules during development. Studies with Qa-1(b) tetramers (Qa-1 tetramers) showed that nearly all NK1.1(+) cells from newborn C57BL/6 mice express Qa-1-binding receptors. Cytotoxic activity of these cells is fully inhibited by Qa-1 ligands on target cells. In contrast, neither receptors for H-2K(b) nor H-2D(b) were observed on NK1.1(+) cells from newborn mice. After birth, frequencies of Qa-1 tetramer(+)/ NK1.1(+) cells gradually decrease as the number of Ly49(+) /NK1.1(+) cells increases. Cell transfer studies showed that Qa-1 tetramer(+) cells from newborn mice do not lose expression of Qa-1 receptors, but that they further acquire expression of Ly49 molecules. Acquisition of Qa-1-binding receptors appears largely independent of host MHC class I molecules, as observed in studies using beta2-microglobulin-deficient (beta2m(-/-)) mice as well as K(b)/ D(b-/-) and K(b)/D(b)/beta2m(-/-) mice. The present results suggest that Qa-1-binding receptors play an important role in the specificity of developing NK cells, and suggest that these cells rely mainly on inhibitory receptors specific for non-classical MHC class I molecules to maintain self tolerance during the first weeks of life.     

Lack of expansion of major histocompatibility complex class Ib-restricted effector cells following recovery from secondary infection with the intracellular pathogen Listeria monocytogenes

Sublethal infection of BALB/c mice with the intracellular bacterial pathogen Listeria monocytogenes leads to the development of antilisterial immunity with concurrent stimulation of major histocompatibility complex (MHC) class Ia- and Ib-restricted CD8+ effector T cells. Secondary L. monocytogenes infection is followed by an accelerated increase in the number of Listeria-specific CD8+ cells and rapid clearance of the bacterium from the murine host. Recovery from secondary infection is associated with increased levels of effector cell function, as measured by gamma interferon secretion following coculture of immune cells with L. monocytogenes infected APCs in vitro, as well as antilisterial cytotoxicity, as measured by effector cell recognition of L. monocytogenes-infected target cells. We assessed the frequency of L. monocytogenes-specific MHC class I-restricted cells following secondary infection by ELISPOT assays utilizing coculture of immune cells with L. monocytogenes-infected antigen-presenting cells that express MHC class Ia and/or Ib molecules. We found that the antilisterial Qa-1b (MHC class Ib)-restricted effector subset is not detected as an expanded population following secondary infection compared to the frequency of this effector population as measured following recovery from primary infection. This is in contrast to the frequency of antilisterial H2-Kd (MHC class Ia)-restricted effector cells, which following recovery from secondary infection are detected as an expanded population, and appears to undergo a substantial expansion event 3 to 4 days post-secondary infection. These results are consistent with the conclusion that although Listeria-specific MHC class Ib-restricted effector cells are present following recovery from secondary infection, this subset does not appear to undergo the expansion phase that is detected for the MHC class Ia-restricted effector cell response.     

Cellular sources and targets of IFN-gamma-mediated protection against viral demyelination and neurological deficits

IFN-gamma is an anti-viral and immunomodulatory cytokine critical for resistance to multiple pathogens. Using mice with targeted disruption of the gene for IFN-gamma, we previously demonstrated that this cytokine is critical for resistance to viral persistence and demyelination in the Theiler's virus model of multiple sclerosis. During viral infections, IFN-gamma is produced by natural killer (NK) cells, CD4(+) and CD8(+) T cells; however, the proportions of lymphocyte subsets responding to virus infection influences the contributions to IFN-gamma-mediated protection. To determine the lymphocyte subsets that produce IFN-gamma to maintain resistance, we used adoptive transfer strategies to generate mice with lymphocyte-specific deficiencies in IFN-gamma-production. We demonstrate that IFN-gamma production by both CD4(+) and CD8(+) T cell subsets is critical for resistance to Theiler's murine encephalomyelitis virus (TMEV)-induced demyelination and neurological disease, and that CD4(+) T cells make a greater contribution to IFN-gamma-mediated protection. To determine the cellular targets of IFN-gamma-mediated responses, we used adoptive transfer studies and bone marrow chimerism to generate mice in which either hematopoietic or somatic cells lacked the ability to express IFN-gamma receptor. We demonstrate that IFN-gamma receptor must be present on central nervous system glia, but not bone marrow-derived lymphocytes, in order to maintain resistance to TMEV-induced demyelination.     

CD160: a unique activating NK cell receptor

Here we discuss CD160 an essential NK cell activating receptor that remains poorly understood. CD160 receptor exhibits a number of unique structural and functional characteristics that are not common to other killer immunoglobulin-like receptors that recognize major histocompatibility complex (MHC) class I molecules: (1) In addition to humans and mice, the cd160 gene is conserved in several other mammal species; (2) cd160 is located outside the NK gene complex and the Leukocyte Receptor Complex in humans; (3) CD160 expression is associated to the CD56(dim) CD16+ cytotoxic NK cell phenotype; (4) both human and mouse CD160 recognize MHC class Ia and Ib molecules; (5) unlike the other MHC class I-dependent activating NK receptors, CD160 is a glycosylphosphatidylinositol-anchored molecule with a single immunoglobulin-like domain, and does not bear immunoreceptor tyrosine-based activation motifs. Consequently, CD160 cannot signal by itself, requiring the recruitment of adaptor proteins. CD160 recruits phosphoinositide-3 kinase to trigger cytotoxicity and cytokine secretion; (6) specific engagement of NK CD160 receptor expressed by circulating NK cells produces proinflammatory cytokines IFN-γ, TNF-α, and, most notably, IL-6 and IL-8 as well as MIP1-β chemokine. The level of CD160-mediated IFN-γ production is always higher than the one observed after engagement of the CD16 receptor.