MHC Class I Molecules Research Articles

P0192 The concentration of soluble mica as prognostic factor in nasopharyngeal carcinoma

Background In Indonesia, nasopharyngeal carcinoma (NPC), a malignancy of the head and neck, has a high mortality rate because patients are usually diagnosed at an advance stage and, in some, metastasis has even occured. When diagnosed at an early stage, 5-year survival rates can reach more than 80%, but at advance stages, 5-year survival rate is only 10–40%. Epstein-Barr virus is associated with the pathogenesis of NPC. These viral-induced carcinoma express the stress surface protein molecules MHC class I-related chain A (MICA), part of an important activation pathway to trigger the immune system to attack tumour cells. However, the virus and cancer cells might downregulate the expression of membrane-bound MICA (mMICA) molecules and increase the soluble MICA (sMICA) concentration, therefore reducing the immunogenicity of the tumour cell and decreasing the prognosis. We aimed to determine the concentration of sMICA as prognostic factor in NPC. Methods We used immunohistochemical analysis to determine mMICA and ELISA to determine sMICA concentration from the sera. We used Kruskal–Wallis and Mann–Whitney test for analysis. Findings mMICA expression was present in all NPC tissue (100%) in various concentrations. Mean sMICA concentrations was 245.8 pg/dL in NPC stage II, 369.9 pg/mL in stage III, and 464.8 pg/mL in NPC stage IV. The higher the sMICA concentration, the higher the NPC stage. There was no significant difference in sMICA concentration between stage III and stage IV NPC (p = 0.344). Interpretation Increased concentration of sMICA protein is a prognostic factor in NPC.

Equid herpesvirus type 4 uses a restricted set of equine major histocompatibility complex class I proteins as entry receptors.

Equid herpesvirus type 1 (EHV-1) was shown to use an unusual receptor for cellular entry - MHC-I molecules. Here, we demonstrated that the closely related EHV, EHV-4, also uses this strategy for cellular invasion, both in equine cells in culture and in the heterologous, non-permissive murine mastocytoma cell line (P815) after stable transfection with horse MHC-I genes. Using a panel of P815 cell lines transfected with individual horse MHC-I genes, we provided support for the hypothesis that EHV-1 and EHV-4 target classical polymorphic MHC-I molecules as viral entry receptors. All known equine MHC-I molecules from the two principal classical polymorphic loci specify alanine at position 173 (A173), whilst other MHC-I loci encoded different amino acids at this position and did not permit viral entry. Site-directed mutagenesis of position 173 diminished or enhanced viral entry, depending upon the initial amino acid. However, there were other, as yet undefined, constraints to this process: MHC-I genes from two non-classical loci carried A173 but did not enable viral entry in P815 transfectants. Our study suggested that the capacity to bind MHC-I molecules arose in the common ancestor of EHV-1 and EHV-4. The widespread occurrence of A173 in classical polymorphic horse MHC-I molecules indicated that horses of most MHC haplotypes should be susceptible to infection via this entry portal.

Haplotypes of Polymorphic Antigen Processing Genes for Low Molecular Mass Polypeptides (LMP2 and LMP7) are Strongly Associated with Type 1 Diabetes in North India

Objective: Type 1 diabetes (T1D) is a multifactorial autoimmune disorder where several genes have been implicated. Aberrant presentation of auto-antigens on the MHC molecules is the hallmark of autoimmune disorders. The antigen is processed into small peptides by low molecular mass polypeptides, LMP2 and LMP7 before they are presented on the MHC class-I molecules. We have studied the associations of the Single nucleotide polymorphisms (SNPs) in these antigen processing genes and their haplotypes which may be detrimental for the processing of the auto-antigens in T1D. Methods: 239 T1D subjects and 752 normal healthy controls from North India were studied for LMP2 codon 60 G/A (R/H), LMP7 codon 49 of C/A (Q/K) and LMP7 Intron 6 G/T polymorphisms using PCR-RFLP. HLA class-II alleles were studied using bead based Luminex assays. Haplotypes of LMP2 and LMP7 were constructed using SHEsis online software. χ2 test was used to study the significance of differences between patients and controls. Results: The G (R) allele (p<0.009) and homozygous GG (RR) genotype (p<0.01) of LMP2 codon 60, C (Q) allele (p<0.0098) and homozygous CC (QQ) (p<0.03) of LMP 7 codon 49 and LMP7 Intron 6 G allele (p<0.01) were significantly increased in T1D subjects compared to controls. Haplotype analysis showed that haplotypes GCG and ACT (LMP2 codon 60- LMP7codon 49- LMP7 intron 6) (p<5.9×10-13, p<1.9×10-8 respectively) were significantly increased and haplotypes GCT and ACG (p<1.9×10-12, p<1.9×10-13 respectively) were significantly reduced in T1D patients irrespective of the gender, age at onset of T1D and the predisposing HLA DRB1*03:01. Conclusion: Association of LMP2 and LMP7 haplotypes GCG and ACT with T1D may have a role in processing of auto-antigens to be presented by MHC class-I molecules to cytotoxic T cells in T1D.

The Role of CD2 Family Members in NK-Cell Regulation of B-Cell Antibody Production

Natural Killer (NK) cells, an important component of the innate immune system, can mount much more rapid responses upon activation than adaptive antigen specific responses. Among the various functions attributed to NK cells their effect on antibody production merits special attention. The modification of IgG subclasses distribution as well as the amplification of the B cell response can be functionally relevant both for mediation of antibody-dependent cellular cytotoxicity (ADCC) and for control of dysregulated autoantibody production. In this review recent experimental evidence for the mechanistic basis of the effect of NK cells on B cell-responses will be covered. Thus, it will be shown that these effects are mediated not only via activation of cytokine and Toll-like receptors (TLR), but also by direct receptor-ligand interactions. Importantly, the function of these receptor/ligands, CD48 and CD244, do not require recognition of class I-MHC molecules but are more dependent on inflammatory conditions brought about by infection or oncogenesis.

The p36 Isoform of Murine Cytomegalovirus m152 Protein Suffices for Mediating Innate and Adaptive Immune Evasion

The MHC-class I (MHC-I)-like viral (MHC-Iv) m152 gene product of murine cytomegalovirus (mCMV) was the first immune evasion molecule described for a member of the β-subfamily of herpesviruses as a paradigm for analogous functions of human cytomegalovirus proteins. Notably, by interacting with classical MHC-I molecules and with MHC-I-like RAE1 family ligands of the activatory natural killer (NK) cell receptor NKG2D, it inhibits presentation of antigenic peptides to CD8 T cells and the NKG2D-dependent activation of NK cells, respectively, thus simultaneously interfering with adaptive and innate immune recognition of infected cells. Although the m152 gene product exists in differentially glycosylated isoforms whose individual contributions to immune evasion are unknown, it has entered the scientific literature as m152/gp40, based on the quantitatively most prominent isoform but with no functional justification. By construction of a recombinant mCMV in which all three N-glycosylation sites are mutated (N61Q, N208Q, and N241Q), we show here that N-linked glycosylation is not essential for functional interaction of the m152 immune evasion protein with either MHC-I or RAE1. These data add an important functional detail to recent structural analysis of the m152/RAE1γ complex that has revealed N-glycosylations at positions Asn61 and Asn208 of m152 distant from the m152/RAE1γ interface.

Optimized Tetramer Analysis Reveals Ly49 Promiscuity for MHC Ligands

Murine Ly49 receptors, which are expressed mainly on NK and NKT cells, interact with MHC class I (MHC-I) molecules with varying specificity. Differing reports of Ly49/MHC binding affinities may be affected by multiple factors, including cis versus trans competition and species origin of the MHC-I L chain (β2-microglobulin). To determine the contribution of each of these factors, Ly49G, Ly49I, Ly49O, Ly49V, and Ly49Q receptors from the 129 mouse strain were expressed individually on human 293T cells or the mouse cell lines MHC-I-deficient C1498, H-2(b)-expressing MC57G, and H-2(k)-expressing L929. The capacity to bind to H-2D(b)- and H-2K(b)-soluble MHC-I tetramers containing either human or murine β2-microglobulin L chains was tested for all five Ly49 receptors in all four cell lines. We found that most of these five inhibitory Ly49 receptors show binding for one or both self-MHC-I molecules in soluble tetramer binding assays when three conditions are fulfilled: 1) lack of competing cis interactions, 2) tetramer L chain is of mouse origin, and 3) Ly49 is expressed in mouse and not human cell lines. Furthermore, Ly49Q, the single known MHC-I receptor on plasmacytoid dendritic cells, was shown to bind H-2D(b) in addition to H-2K(b) when the above conditions were met, suggesting that Ly49Q functions as a pan-MHC-Ia receptor on plasmacytoid dendritic cells. In this study, we have optimized the parameters for soluble tetramer binding analyses to enhance future Ly49 ligand identification and to better evaluate specific contributions by different Ly49/MHC-I pairs to NK cell education and function.

Education of Murine NK Cells Requires Both cis and trans Recognition of MHC Class I Molecules

Although NK cells use invariant receptors to identify diseased cells, they nevertheless adapt to their environment, including the presence of certain MHC class I (MHC-I) molecules. This NK cell education, which is mediated by inhibitory receptors specific for MHC-I molecules, changes the responsiveness of activating NK cell receptors (licensing) and modifies the repertoire of MHC-I receptors used by NK cells. The fact that certain MHC-I receptors have the unusual capacity to recognize MHC-I molecules expressed by other cells (trans) and by the NK cell itself (cis) has raised the question regarding possible contributions of the two types of interactions to NK cell education. Although the analysis of an MHC-I receptor variant suggested a role for cis interaction for NK cell licensing, adoptive NK cell transfer experiments supported a key role for trans recognition. To reconcile some of these findings, we have analyzed the impact of cell type-specific deletion of an MHC-I molecule and of a novel MHC-I receptor variant on the education of murine NK cells when these mature under steady-state conditions in vivo. We find that MHC-I expression by NK cells (cis) and by T cells (trans), and MHC-I recognition in cis and in trans, are both needed for NK cell licensing. Unexpectedly, modifications of the MHC-I receptor repertoire are chiefly dependent on cis binding, which provides additional support for an essential role for this unconventional type of interaction for NK cell education. These data suggest that two separate functions of MHC-I receptors are needed to adapt NK cells to self-MHC-I.

Extensive diversification of MHC in Chinese giant salamanders Andrias davidianus (Anda-MHC) reveals novel splice variants

A series of MHC alleles (including 26 class IA, 27 class IIA, and 17 class IIB) were identified from Chinese giant salamander Andrias davidianus (Anda-MHC). These genes are similar to classical MHC molecules in terms of characteristic domains, functional residues, deduced tertiary structures and genetic diversity. The majority of variation between alleles is found in the putative peptide-binding region (PBR), which is driven by positive Darwinian selection. The coexistence of two isoforms in MHC IA, IIA, and IIB alleles are shown: one full-length transcript and one novel splice variant. Despite lake of the external domains, these variants exhibit similar subcellular localization with the full-length transcripts. Moreover, the expression of MHC isoforms are up-regulated upon in vivo and in vitro stimulation with Andrias davidianus ranavirus (ADRV), suggesting their potential roles in the immune response. The results provide insights into understanding MHC variation and function in this ancient and endangered urodele amphibian.

Expressions of immunogenic molecules in low-dose radiotherapy-treated human renal clear cell carcinoma 786-0 cells

To explore the effects of low-dose radiation on the expression of immunogenic membrane molecules calreticulin (CRT) and MHC-I/II on the surface of human renal clear cell carcinoma 786-0 cells. The inhibitory activity of low-dose radiation on cell line 786-0 was examined by CCK-8 assay. And the post-radiation membrane expressions of CRT, MHC-I and MHC-II were measured by flow cytometry while CRT was visualized by immunofluorescence photography. The inhibition rates on the proliferative capacities of four 786-0 cell lines rose with the incremental radiation doses of 0, 6, 12 and 24 Gy. And the CRT expression levels of each experimental group was significantly higher than that of the control group (P < 0.001). Along with incremental doses of irradiation, the average calreticulin fluorescence intensities increased gradually initially and then there was a downward trend. The membrane expressions of MHC-I and MHC-II of each experimental group was significantly higher than those of the control group (P < 0.05). As the irradiation dose increased, the average MHC-I fluorescence intensities increased gradually in a dose-dependent manner. The low-dose radiotherapy may up-regulate CRT and MHC class I/II related with the immunogenicity of tumor cells to induce immune response against tumors.

Microfluidic biofunctionalisation protocols to form multi‐valent interactions for cell rolling and phenotype modification investigations

In this study, we propose a fast, simple method to biofunctionalise microfluidic systems for cellomic investigations based on micro-fluidic protocols. Many available processes either require expensive and time-consuming protocols or are incompatible with the fabrication of microfluidic systems. Our method differs from the existing since it is applicable to an assembled system, uses few microlitres of reagents and it is based on the use of microbeads. The microbeads have specific surface moieties to link the biomolecules and couple cell receptors. Furthermore, the microbeads serve as arm spacer and offer the benefit of the multi-valent interaction. Microfluidics was adapted together with topology and biochemistry surface modifications to offer the microenvironment for cellomic studies. Based on this principle, we exploit the streptavidin-biotin interaction to couple antibodies to the biofunctionalised microfluidic environment within 5 h using 200 μL of reagents and biomolecules. We selected the antibodies able to form complexes with the MHC class I (MHC-I) molecules present on the cell membrane and involved in the immune surveillance. To test the microfluidic system, tumour cell lines (RMA) were rolled across the coupled antibodies to recognise and strip MHC-I molecules. As result, we show that cell rolling performed inside a microfluidic chamber functionalised with beads and the opportune antibody facilitate the removal of MHC class I molecules. We showed that the level of median fluorescent intensity of the MHC-I molecules is 300 for cells treated in a not biofunctionalised surface. It decreased to 275 for cells treated in a flat biofunctionalised surface and to 250 for cells treated on a surface where biofunctionalised microbeads were immobilised. The cells with reduced expression of MHC-I molecules showed, after cytotoxicity tests, susceptibility 3.5 times higher than normal cells.

Antigen-activated dendritic cells ameliorate influenza A infections.

Influenza A viruses cause significant morbidity and mortality worldwide. There is a need for alternative or adjunct therapies, as resistance to currently used antiviral drugs is emerging rapidly. We tested ligand epitope antigen presentation system (LEAPS) technology as a new immune-based treatment for influenza virus infection in a mouse model. Influenza-J-LEAPS peptides were synthesized by conjugating the binding ligand derived from the β2-microglobulin chain of the human MHC class I molecule (J-LEAPS) with 15 to 30 amino acid-long peptides derived from influenza virus NP, M, or HA proteins. DCs were stimulated with influenza-J-LEAPS peptides (influenza-J-LEAPS) and injected intravenously into infected mice. Antigen-specific LEAPS-stimulated DCs were effective in reducing influenza virus replication in the lungs and enhancing survival of infected animals. Additionally, they augmented influenza-specific T cell responses in the lungs and reduced the severity of disease by limiting excessive cytokine responses, which are known to contribute to morbidity and mortality following influenza virus infection. Our data demonstrate that influenza-J-LEAPS-pulsed DCs reduce virus replication in the lungs, enhance survival, and modulate the protective immune responses that eliminate the virus while preventing excessive cytokines that could injure the host. This approach shows promise as an adjunct to antiviral treatment of influenza virus infections.

Human antigen-specific invariant NKT cells generated by the TIL 1383I T cell receptor gene transfer (P2098)

Abstract Human invariant NKT cells (iNKT cells) are a novel lymphocyte population characterized by an invariant T-cell receptor (Vα24/Vβ11). iNKT cells activated with αGalCer and IL-2 have been shown to produce large amounts of cytokines such as IFN-γ and also have potent killing activity against various tumor cell lines. One major hurdle to studying the anti-tumor activity of iNKT cells is they don’t recognize antigens presented by classical MHC molecules and their tumor antigen specificity is unknown. Therefore, we expressed the MHC class I restricted TIL 1383I TCR into iNKT cells to better study their biology and anti-tumor activity. The 1383I TCR is a high affinity TCR that recognizes the melanoma antigen tyrosinase presented by HLA-A2. In this study, we report the production and function of the TIL 1383I TCR transduced human iNKT cells. iNKT cells were activated and expanded from normal PBMCs by stimulating them with αGalCer in the presence of IL-2 and IL-15. The iNKT cells were isolated using iNKT magnetic beads and transduced using retroviral vectors encoding the TIL 1383I TCR. The TIL 1383I TCR transduced iNKT expression was measured by flow cytometry (FACS) and their anti-tumor activity by intracellular cytokine staining. The TIL 1383I TCR transduced iNKT cells specifically produced IFN-γ in response to tyrosinase peptide loaded T2 cells and HLA-A2+/tyrosinase+ human melanomas. These results indicate that iNKT cells can be engineered to recognize normal tumor antigens.

NLRP12 protein function in the regulation of MHC-I expression in monocytes of patients transplanted with HPCs and their relationship to the reconstitution of CD8 T lymphocytes. (P1333)

Abstract The reconstitution of the T CD8 lymphocyte population in patients subject to hematopoietic cell transplantation (HCT) depends largely of homeostatic peripheral expansion (HPE) of the T CD8 lymphocytes that were not eliminated during the pre-transplant conditioning regimen or the ones that are contained in the transplanted cells. In order to have an efficient HPE, MHC-I molecules, IL-7 and IL-15 are required. Evidences obtained in our lab indicate that the low and/or heterogeneous MHC-I levels of expression correlate with a inefficient reconstitution of T CD8 lymphocytes in patients that received allogeneic HCT. The deficiency in MHC-I expression could be due to a decrease in the expression of the NLRP12 protein, which has been proposed as a positive regulator of classical and non-classical MHC-I molecules expression. Therefore the objective of the following project is to determine the involvement of NALP12 in the MHC-I expression in monocytes from patients subject to HCT. We studied a cohort of 11 patients, 6 of which received autologous transplant and 5 with allogeneic transplant. Most of the patients present normal levels of T CD8 lymphocytes and a homogeneous expression of MHC-I. The patients that received autologous transplantation show a correlation of r=0.56. Our results suggest that NALP12 could act as an enhancer for the expression of the MHC-I molecules and that this expression could be affected in patients subject to allogeneic HPC transplantation.

Nectin and nectin-like molecules: immune regulator, adhesion molecule and virus receptors (P1001)

Abstract Nectin and nectin-like proteins are cell adhesion molecules that mediate the formation of cell adherens junctions by forming homo- or hetero-dimers. Some members of this protein family can also be used by immune receptors to mediate immune recognition. For instance, nectin-like 2 (Necl-2) is used as a ligand for the immune system by interaction with the immune receptor CRTAM (class-I MHC restricted T-cell associated molecule), which is mainly expressed on the surface of cytotoxic lymphocyte cells. However, the Necl-2/CRTAM binding mode and its relationship to cell adhesion are not known. Here, we report a Necl-2/CRTAM complex structure, demonstrating that Necl-2 binding to CRTAM competes with the dimerization of CRTAM and possibly Necl-2. Necl-2 occupies the CRTAM homodimer interface, making homodimerization impossible. Mutational and functional analyses identified key amino acids (double "lock-and-key") responsible for the binding. Our work illustrates how the cell adhesion molecule Necl-2 competitively binds the immune receptor CRTAM. Otherwise, the nectin proteins can also be employed as the virus receptors, such as herpes simplex virus and measles virus.

The molecular basis for Mucosal-Associated Invariant T cell recognition of MR1 proteins

Mucosal-associated invariant T (MAIT) cells are an evolutionarily conserved αβ T-cell lineage that express a semi-invariant T-cell receptor (TCR) restricted to the MHC related-1 (MR1) protein. MAIT cells are dependent upon MR1 expression and exposure to microbes for their development and stimulation, yet these cells can exhibit microbial-independent stimulation when responding to MR1 from different species. We have used this microbial-independent, cross-species reactivity of MAIT cells to define the molecular basis of MAIT-TCR/MR1 engagement and present here a 2.85 Å complex structure of a human MAIT-TCR bound to bovine MR1. The MR1 binding groove is similar in backbone structure to classical peptide-presenting MHC class I molecules (MHCp), yet is partially occluded by large aromatic residues that form cavities suitable for small ligand presentation. The docking of the MAIT-TCR on MR1 is perpendicular to the MR1 surface and straddles the MR1 α1 and α2 helices, similar to classical αβ TCR engagement of MHCp. However, the MAIT-TCR contacts are dominated by the α-chain, focused on the MR1 α2 helix. TCR β-chain contacts are mostly through the variable CDR3β loop that is positioned proximal to the CDR3α loop directly over the MR1 open groove. The elucidation of the MAIT TCR/MR1 complex structure explains how the semi-invariant MAIT-TCR engages the nonpolymorphic MR1 protein, and sheds light onto ligand discrimination by this cell type. Importantly, this structure also provides a critical link in our understanding of the evolution of αβ T-cell recognition of MHC and MHC-like ligands.

Preliminary study on the changes of NKG2D expression and its role in children with infectious mononucleosis

Objective To investigate the mechanism of disturbed immunological function induced by Epstein-Barr virus(EBV) infection through evaluating NKG2D expressions in NK cells and CD8+ T lymphocytes from children with infectious mononucleosis.Methods Twenty-nine children with infectious mononucleosis (IM) and twenty-five age-matched healthy children were enrolled in the study.NKG2D/NKG2A expressions in NK cells and CD8+T lymphocytes,and NKG2D ligand MHC Ⅰ chain-related molecules A(MICA) and UL-16binding proteins (ULBP-1) expressions on CD14+ mononuclear cells (MC) were analyzed by flow cytometry.The concentrations of cytokines such as IL-7,IL-12,IL-15,IFN-γ,TGF-β and soluble MICA(sMICA) in plasma were determined by enzyme-linked immunosorbent assay (ELISA).Results (1) Compared with the control group,NKG2D expression was significantly decreased in both NK cells and CD8+T lymphocytes from children with IM (P＜0.05),especially in the three cases of suspected EVB-HLH children.(2)There was no difference in the expression of MICA and ULBP-1 in CD14+ MC between the children with IM and the normal control(P＞0.05).(3)The concentrations of IL-15 and TGF-β in plasma were lower than those of the control group (P＜0.05),while the levels of IL-7,IL-12,IFN-γ and sMICA were up-regulated(P＜0.05) in children with IM.(4)NKG2A expression in NKcells in children with IM was significantly increased as compared with healthy controls(P＜0.05),however,there was no differences in the expression of NKG2A in CD8+ T lymphocytes between the two groups (P＞0.05).Conclusion Remarkable down-regulated expressions of NKG2D in NK cells and CD8+T lymphocytes might be one of the factors causing disturbed immunological function in children with EBV infection,which might have a close relationship with abnormal cytokine milieu of IL-15 and IL-12or high concentration of sMICA. Key words: Epstein-Barr virus; Infectious mononucleosis; NKG2D

Increased thymic development of regulatory T cells in NOD mice is functionally dissociated from type I diabetes susceptibility

Regulatory T (Treg) lymphocytes play a central role in the control of autoimmune pathology. Any alteration in Treg-cell biology in mouse strains used for the study of these disorders therefore raises the question of its direct link with disease susceptibility. Paradoxically, in non-obese diabetic (NOD) mice increased numbers of Treg cells develop in the thymus. In this report we identify a locus of <7 Mbp that quantitatively controls Treg-cell development in the thymus of the NOD mouse. This 'Trd1' region is located centromeric to the H2 complex on chromosome 17 and does not include genes encoding classical MHC molecules. The genomic region identified here contains the Idd16 diabetes susceptibility locus and the use of congenic mouse strains allowed us to investigate the potential link between quantitatively altered thymic Treg cells and diabetes susceptibility. Hybrid mice present similar levels of thymic Treg cells as B6 animals but they developed diabetes with the same kinetics as NOD mice. Therefore, the increased Treg-cell development in NOD mice controlled by Trd1 is functionally dissociated from the susceptibility of NOD to diabetes.

Type I Interferons as Stimulators of DC-Mediated Cross-Priming: Impact on Anti-Tumor Response

Induction of potent tumor-specific cytotoxic T-cell responses is a fundamental objective in anticancer therapeutic strategies. This event requires that antigen-presenting cells present tumor-associated antigens (Ag) on their MHC class-I molecule, in a process termed cross-presentation. Dendritic cells (DC) are particularly keen on this task and can induce the cross-priming of CD8+ T cells, when exposed to danger or inflammatory signals that stimulate their activation. Type I interferons (IFN-I), a family of long-known immunostimulatory cytokines, have been proven to produce optimal activation signal for DC-induced cross-priming. Recent in vitro and in vivo evidences have suggested that IFN-I-stimulated cross-priming by DC against tumor-associated Ag is a key mechanism for cancer immunosurveillance and may be usefully exploited to boost anti-tumor CD8+ T-cell responses. Here, we will review the cross-presentation properties of different DC subsets, with special focus on cell-associated and tumor Ag, and discuss how IFN-I can modify this function, with the aim of identifying more specific and effective strategies for improving anticancer responses.

Degenerate Recognition of MHC Class I Molecules with Bw4 and Bw6 Motifs by a Killer Cell Ig-like Receptor 3DL Expressed by Macaque NK Cells

The killer cell Ig-like receptors (KIRs) expressed on the surface of NK cells recognize specific MHC class I (MHC-I) molecules and regulate NK cell activities against pathogen-infected cells and neoplasia. In HIV infection, survival is linked to host KIR and MHC-I genotypes. In the SIV macaque model, however, the role of NK cells is unclear due to the lack of information on KIR-MHC interactions. In this study, we describe, to our knowledge, the first in-depth characterization of KIR-MHC interactions in pigtailed macaques (Macaca nemestrina). Initially, we identified three distinct subsets of macaque NK cells that stained ex vivo with macaque MHC-I tetramers loaded with SIV peptides. We then cloned cDNAs corresponding to 15 distinct KIR3D alleles. One of these, KIR049-4, was an inhibitory KIR3DL that bound MHC-I tetramers and prevented activation, degranulation, and cytokine production by macaque NK cells after engagement with specific MHC-I molecules on the surface of target cells. Furthermore, KIR049-4 recognized a broad range of MHC-I molecules carrying not only the Bw4 motif, but also Bw6 and non-Bw4/Bw6 motifs. This degenerate, yet peptide-dependent, MHC reactivity differs markedly from the fine specificity of human KIRs.

The Successful Induction of Systemic Tolerance to a Class-I MHC Disparity through Adoptive Transfer in a Large Animal Model

Background: We have previously reported that 12 days of high dose CyA uniformly leads to tolerance of MHC class I-mismatched renal grafts in MHC inbred miniature swine. We have now attempted to induce tolerance in secondary recipients by the adoptive transfer of a long-term tolerant kidney with or without tolerated cells. Methods: (1) Adoptive Transfer Donors: Three MHC inbred SLAdd miniature swine received life-supporting MHC class I-mismatched kidneys (SLAgg) with 12 days of high dose CyA to induce tolerance. Three to four months after kidney transplant (KTx) these animals received a Donor Specific Transfusion (DST) to expand T regulatory cells. (2) Adoptive Transfer Recipients: Three recipients, MHC-matched to the adoptive transfer donors (SLAdd), received 150 rad whole body irradiation (WBI) one day before receiving class I mismatched kidneys from the donors. Two of these animals also received 4-5 × 10ˆ9 cells/kg cells, obtained by leukapheresis from the adoptive transfer donors, on day -1. Tolerated kidneys (SLAgg) were transplanted (day 0) without further immunosuppression. Second donor matched kidneys from naïve SLAgg animals were transplanted >100 days later to confirm tolerance. (3) Controls: Three SLAdd animals received 150 rad WBI on day -1 followed by a class I MHC mismatched KTx. (4) Graft function was assessed by serum creatinine and histology. Immunologic responses were assessed by Cell Mediated Lysis (CML) assays. Anti-Donor MHC antibody was assessed by FACS. Results: The three controls rejected the naïve Class I mismatched kidney by day 9. All three recipients accepted tolerated kidneys >100 days and did not reject second donor matched naïve kidneys transplanted without immunosuppression, confirming systemic tolerance. All three animals maintained donor specific unresponsiveness by cellular assays and two animals did not develop antibody to donor. Conclusions: To our knowledge, these results provide the first evidence for the successful transfer of systemic tolerance in a large animal model by the adoptive transfer of tolerated kidneys into 150-rad irradiated miniature swine.