Iwona Stroynowski

ANTIGEN PRESENTATION BY NON-CLASSICAL CLASS I MOLECULES

Non-classical class I genes are no longer clearly distinguished from classical ones in mammals, and they are found also in fishes, frogs and chickens. They contribute to immune responses against pathogens. Given the number and diversity of class Ib products, their various tissue distribution patterns, and the wide range of peptides they bind, new functions are to be expected.

The Nonclassical Major Histocompatibility Complex Molecule Qa-2 Protects Tumor Cells from NK Cell- and Lymphokine-Activated Killer Cell-Mediated Cytolysis

The cytotoxic activity of NK cells is regulated by class I MHC proteins. Although much has been learned about NK recognition of class I autologous targets, the mechanisms of NK self-tolerance are poorly understood. To examine the role of a nonpolymorphic, ubiquitously expressed class Ib Ag, Q9, we expressed it on class I-deficient and NK-sensitive B78H1 melanoma. Presence of this Qa-2 family member on tumor cells partially protected targets from lysis by bulk lymphokine-activated killer (LAK) cells. H-2Kb-expressing B78H1 targets also reduced LAK cell activity, while H-2Db offered no protection. Importantly, blocking with F(ab′)2 specific for Q9 or removal of this GPI-attached molecule by phospholipase C cleavage restored killing to the level of vector-transfected cells. Experiments with LAK cells derived from H2b SCID and B6 mice established that NK1.1+TCR− NK and NK1.1+TCR+ LAK cells were the prevalent cytolytic populations inhibitable by Q9. Treatment of mice with poly(I:C) also resulted in generation of Q9-regulated splenic cytotoxicity. LAK cells from different mouse strains responded to Q9, suggesting that the protective effect of this molecule is not detectably influenced by Ly49 polymorphisms or the presence/absence of Q9 in NK-harboring hosts. We propose that Q9 expressed on melanoma cells serves as a ligand for yet unidentified NK inhibitory receptor(s) expressed on NK1.1+ NK/T cells.

Expression of a nonclassical MHC class Ib molecule in the eye.

BACKGROUND MHC class Ia molecules are absent, or expressed at low levels, on cells lining the anterior chamber of the eye, an immune-privileged site. Although the scarcity of class Ia MHC antigens may protect cells from T cell-mediated tissue injury, it may also render them vulnerable to natural killer cell-mediated cytolysis. There is growing evidence that MHC class Ib molecules share similar functions to class Ia. In this study, we examine the expression and distribution of Qa-2, one of the best-characterized murine MHC class Ib molecules in the eye. METHODS The transcription of Qa-2 mRNA in whole eye and eye-derived cells was analyzed by sensitive and specific RNase protection and reverse transcription-polymerase chain reaction assays. Immunohistochemistry, flow cytometry, and ELISA were used to determine whether Qa-2 was expressed as cell surface proteins. Expression levels of Qa-2 were monitored in resting cells and cells stimulated with interferon-gamma. RESULTS Expression of membrane-bound and soluble Qa-2 isoforms was detected in various tissues of the eye, including cell subsets lining the anterior chamber. Immunohistological staining revealed Qa-2 expression on corneal epithelium as well as endothelium, iris ciliary bodies, and retina. These observations were confirmed by analysis of cultured, eye-derived cells. Qa-2 expression was inducible by interferon-gamma. Qa-2 was not detected in lens cells. CONCLUSIONS The results demonstrate that membrane-bound and soluble MHC class Ib molecules are expressed by eye cells. Expression of Qa-2 in the corneal endothelium and other substructures lining the anterior chamber suggests that this class Ib protein may contribute to the immune-privileged status of the anterior chamber.

Hepatocytes Express Abundant Surface Class I MHC and Efficiently Use Transporter Associated with Antigen Processing, Tapasin, and Low Molecular Weight Polypeptide Proteasome Subunit Components of Antigen Processing and Presentation Pathway

Hepatic expression levels of class I MHC Ags are generally regarded as very low. Because the status of these Ags and their ability to present peptides are important for the understanding of pathogen clearance and tolerogenic properties of the liver, we set out to identify the factors contributing to the reported phenotype. Unexpectedly, we found that the surface densities of Kb and Db on C57BL/6 mouse hepatocytes are nearly as high as on splenocytes, as are the lysate concentrations of mRNA encoding H chain and β2-microglobulin (β2m). In contrast, the components of the peptide-loading pathway are reduced in hepatocytes. Despite the difference in the stoichiometric ratios of H chain/β2m/peptide-loading machineries, both cell types express predominantly thermostable class I and are critically dependent on TAP and tapasin for display of surface Ags. Minor differences in the expression patterns in tapasin−/− background suggest cell specificity in class I assembly. Under immunostimulatory conditions, such as exposure to IFN-γ or Listeria monocytogenes, hepatocytes respond with a vigorous mRNA synthesis of the components of the Ag presentation pathway (up to 10-fold enhancement) but up-regulate H chain and β2m to a lesser degree (<2-fold). This type of response should promote rapid influx of newly generated peptides into the endoplasmic reticulum and preferential presentation of foreign/induced Ag by hepatic class I.

Correction of Defects Responsible for Impaired Qa-2 Class Ib MHC Expression on Melanoma Cells Protects Mice from Tumor Growth

One of the principal mechanisms of tumor immune evasion is alteration of class I MHC expression. We have identified defects contributing to down-regulation of class I MHC expression in the widely studied murine B16 melanoma and its variants B16F1, B16F10, BL6-2, BL6-8 and B78H1. Transcription of the nonclassical class I MHC genes Q8 and Q9 (Qa-2 Ags) has been switched off in the entire panel of melanoma lines, suggesting that this event occurred early during tumor progression. B78H1, unlike B16F1 and B16F10 sublines, is also selectively devoid of TAP2 and low molecular weight protein 7 as well as classical class I MHC Kb and Db transcripts. Cotransfection of B78H1 with TAP2 and class I H chain genes is sufficient to reconstitute surface expression of exogenously delivered class I MHC without concomitant re-expression of endogenous β2-microglobulin-associated class I. The serological absence of endogenous class Ia and Ib at the surface of TAP2-negative as well as TAP2-transfected B78H1 makes this system a suitable model for studying the properties of isolated class I proteins in tumors. We used this system to demonstrate that B78H1 cells genetically manipulated to re-express Q9 Ag have reduced tumor potential in syngeneic B6 mice compared with TAP2-transfected parental melanoma. Both NK cells and CTLs appear to collaborate in restraining growth of Q9-positive tumors. The results implicate Qa-2 in antitumor responses and illustrate the utility of the B78H1 system for identifying in vivo interactions between class I MHC molecules of interest and immune cells of innate and/or adaptive immunity.

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.

A Nonclassical MHC Class I Molecule Restricts CTL-Mediated Rejection of a Syngeneic Melanoma Tumor

Although CTL and polymorphic, classical MHC class I molecules have well defined roles in the immune response against tumors, little is currently known regarding the participation of nonpolymorphic, nonclassical MHC class I in antitumor immunity. Using an MHC class I-deficient melanoma as a model tumor, we demonstrate that Q9, a murine MHC class Ib molecule from the Qa-2 family, expressed on the surface of tumor cells, protects syngeneic hosts from melanoma outgrowth. Q9-mediated protective immunity is lost or greatly diminished in mice deficient in CTL, including β2-microglobulin knockout (KO), CD8 KO, and SCID mice. In contrast, the Q9 antitumor effects are not detectably suppressed in CD4 KO mice with decreased Th cell activity. Killing by antitumor CTL in vitro is Q9 specific and can be blocked by anti-Q9 and anti-CD8 Abs. The adaptive Q9-restricted CTL response leads to immunological memory, because mice that resist the initial tumor challenge reject subsequent challenges with less immunogenic tumor variants and show expansion of CD8+ T cell populations with an activated/memory CD44high phenotype. Collectively, these studies demonstrate that a MHC class Ib molecule can serve as a restriction element for antitumor CTL and mediate protective immune responses in a syngeneic setting.

Novel molecules related to MHC antigens

Several types of molecules related to classical class I and II antigens of the MHC have been recently discovered. At the same time we have learnt more about the functions of non-classical (class Ib) antigens. This has shed light on the possible evolutionary origins and the likely roles that these molecules may play in the immune response.

The murine family of gut-restricted class Ib MHC includes alternatively spliced isoforms of the proposed HLA-G homolog, "blastocyst MHC".

The gastrointestinal tract is populated by a multitude of specialized immune cells endowed with receptors for classical (class Ia) and nonclassical (class Ib) MHC proteins. To identify class I products that engage these receptors and impact immunity/tolerance, we studied gut-transcribed class Ib loci and their polymorphism in inbred, outbred, and wild-derived mice. Intestinal tissues enriched in epithelial cells contained abundant transcripts of ubiquitously expressed and preferentially gut-restricted Q and T class I loci. The latter category included the “blastocyst Mhc” gene, H2-Bl, and its putative paralog, Tw5. Expression of H2-Bl was previously detected only at the maternal/fetal interface, where it was proposed to induce immune tolerance via interactions with CD94/NKG2A receptors. Analysis of coding region polymorphism performed here revealed two major alleles of H2-Bl with conserved residues at positions critical for class I protein folding and peptide binding. Both divergent alleles are maintained in outbred and wild mice under selection for fecundity and pathogen resistance. Surprisingly, we found that alternative splicing of H2-Bl mRNA in gut tissues is prevalent and allele-specific. It leads to strain-dependent expression of diverse repertoires of canonical and noncanonical transcripts that may give rise to distinct ligands for intestinal NK cell, T cell, and/or intraepithelial lymphocyte receptors.

Protective Immunity against Disparate Tumors Is Mediated by a Nonpolymorphic MHC Class I Molecule

Current peptide-based immunotherapies for treatment of model cancers target tumor Ags bound by the classical MHC class I (class Ia) molecules. The extensive polymorphism of class Ia loci greatly limits the effectiveness of these approaches. We demonstrate in this study that the murine nonpolymorphic, nonclassical MHC class I (class Ib) molecule Q9 (Qa-2) promotes potent immune responses against multiple syngeneic tumors. We have previously shown that ectopic expression of Q9 on the surface of class Ia-negative B78H1 melanoma led to efficient CTL-mediated rejection of this tumor. In this study, we report that surface-expressed Q9 on 3LLA9F1 Lewis lung carcinoma and RMA T cell lymphoma also induces potent antitumor CTL responses. Importantly, CTL harvested from animals surviving the initial challenge with Q9-positive 3LLA9F1, RMA, or B78H1 tumors recognized and killed their cognate tumors as well as the other cancer lines. Furthermore, immunization with Q9-expressing 3LLA9F1 or RMA tumor cells established immunological memory that enhanced protection against subsequent challenge with a weakly immunogenic, Q9-bearing melanoma variant. Collectively, the generation of cross-reactive CTL capable of eliminating multiple disparate Q9-expressing tumors suggests that this nonpolymorphic MHC class I molecule serves as a restriction element for a shared tumor Ag(s) common to lung carcinoma, T cell lymphoma, and melanoma.