Platelet activation by cross-linking HLA class I molecules and Fc receptor.

The effect on platelet activation of monoclonal antibodies directed against common determinants of the HLA class I heavy chain molecule was studied. Cross-linking W6/32, an anti-HLA class I of IgG2a subclass, led to platelet activation. Two other antibodies of the same subclass did not have this effect on platelets. The lack of activity of the F(ab')2 fragments suggests that the activation signal is mediated by the platelet Fc receptor (Fc gamma RII). Indeed, except for a higher sensitivity of W6/32 to aspirin and apyrase, activations by cross-linking IV-3 (an anti-Fc gamma RII) and W6/32 are similar at the level of InsP3 formation, calcium mobilization, pH modifications, and activation of protein kinase C and myosin kinase. When HLA class I molecules and Fc gamma RII are cross-linked together, platelet activation occurs. This is not observed when a control IgG2a is substituted for W6/32 or when CD9 and Fc receptor are cross-linked together. This suggests that HLA class I molecules and Fc gamma RII synergize to activate platelets.     

HLA class I molecules consistently present internal influenza epitopes

Cytotoxic T lymphocytes (CTL) limit influenza virus replication and prevent morbidity and mortality upon recognition of HLA class I presented epitopes on the surface of virus infected cells, yet the number and origin of the viral epitopes that decorate the infected cell are unknown. To understand the presentation of influenza virus ligands by human MHC class I molecules, HLA-B*0702-presented viral peptides were directly identified following influenza infection. After transfection with soluble class I molecules, peptide ligands unique to infected cells were eluted from isolated MHC molecules and identified by comparative mass spectrometry (MS). Then CTL were gathered following infection with influenza and viral peptides were tested for immune recognition. We found that the class I molecule B*0702 presents 3–6 viral ligands following infection with different strains of influenza. Peptide ligands derived from the internal viral nucleoprotein (NP_418–426 and NP_473–481) and from the internal viral polymerase subunit PB1 (PB1_329–337) were presented by B*0702 following infection with each of 3 different influenza strains; ligands NP_418–426, NP_473–481, and PB1_329–337 derived from internal viral proteins were consistently revealed by class I HLA. In contrast, ligands derived from hemagglutinin (HA) and matrix protein (M1) were presented intermittently on a strain-by-strain basis. When tested for immune recognition, HLA-B*0702 transgenic mice responded to NP_418–426 and PB1_329–337 consistently and NP_473–481 intermittently while ligands from HA and M1 were not recognized. These data demonstrate an emerging pattern whereby class I HLA reveal a handful of internal viral ligands and whereby CTL recognize consistently presented influenza ligands.     

Human T-cell clones used to define functional epitopes on HLA class II molecules

Polyclonal reagents have been used to define HLA class II molecules in conventional serologic and cellular typing. We generated human alloreactive T-cell clones to analyze the functional fine specificities of HLA class II molecules that might be important for the phenomenon of HLA and disease association. We chose to examine HLA-Dw14, an HLA-D specificity that has been associated with juvenile rheumatoid arthritis. In this paper we have presented data that suggest that conventional cellular typing does not reflect the distribution of T-cell epitopes on major histocompatibility complex class II molecules. We describe three alloreactive T-cell clones that have defined three separate Dw14-associated T-cell epitopes. Two of these epitopes were on a DR-region molecule; the third was located on a DQ-region product. In a panel of unrelated DR4-positive donors, these three DW14-associated determinants were present in a high frequency but were not linked to each other. Within the tested panel of DR4-positive cells, all possible combinatorial arrangements of these three allodeterminants were seen. The concurrent expression of any two of the three allodeterminants was equivalent to a positive typing response for Dw14. Our finding that HLA-Dw14 is not characterized by a unique allodeterminant but by the combinatorial recognition of independently distributed T-cell interaction sites suggests that analysis of HLA and disease association may be more clearly demonstrated through the use of human T-cell clones.     

Clinical relevance of soluble HLA class I molecules in Waldenstrom Macroglobulinemia

Objectives: Waldenstrom Macroglobulinemia (WM) is a B‐cell neoplasm characterised by secretion of IgM by lymphoplasmacytic bone marrow cells and by cytopenias and hypogammaglobulinemia in a subset of patients. Beta‐2 microglobulin (b2m) is a major prognostic factor in WM and the heavy chain of HLA class I molecules, which are known to have immunosuppressive properties and have been implicated in the pathogeny of several malignancies. Methods: We assessed the serum levels of the total soluble HLA‐I molecules and the HLA‐Gs molecules in 105 patients with IgM‐related disorders [WM (n = 42) and IgM MGUS (n = 63)], and compared the results to 41 healthy subjects. Results: We found higher levels of HLA‐Is in WM, compared to IgM MGUS and healthy donors. HLA‐Gs levels were similar in WM and in IgM MGUS, but higher than in healthy donors. The association between HLA‐Is at the cut‐off of 1.8 μg/mL and known markers of poor prognosis was then evaluated among WM patients using univariate and multivariate methods. Based on this, high HLA‐Is level was strongly associated with high serum β2M level >3 mg/L [OR = 2, (CI 95% 1.1–5.7); P = 0.04], age > 65 yrs [OR = 1.5, (CI 95% 0.5–4.1), P = 0.06] and haemoglobin ≤11.5 g/dL [OR = 3.3, (CI 95% 1.2–9.7); P = 0.03]. High levels of serum HLA‐Is were also found in patients with cryoglobulinemia, however irrespectively of WM or IgM‐MGUS status. Conclusion: Together our results suggest a possible role for soluble MHC class I molecules in WM disease. Further investigations are necessary to further demonstrate the prognostic impact of soluble MHC class I molecules in Waldenstrom Macroglobulinemia.     

Fluorine-18 fluorodeoxyglucose positron emission tomography in mature T-cell and natural killer cell malignancies

Fluorine-18 fluorodeoxyglucose (FDG)-positron emission tomography (PET) is useful in Hodgkin and B-cell lymphomas. Few data exist on T-cell and natural killer (NK)-cell lymphomas. Thirty consecutive T-cell and NK-cell lymphomas were investigated with PET-computerized tomography (CT). In 12 NK-cell lymphomas, all nasal/extranasal lesions were FDG-avid. In nasal/maxillary areas, FDG-avid tumours were consistently more localised than on CT, suggesting that soft tissue masses on CT were partly due to inflammation. These findings have important implications in radiotherapy planning. In two NK-cell lymphomas, PET did not detect morphologically occult marrow infiltration uncovered by in situ hybridisation for Epstein-Barr-virus-encoded small RNA. In angioimmunoblastic lymphoma (n = 7), peripheral T-cell lymphoma, unspecified (PTCL-U, n = 4) and anaplastic large cell lymphoma (ALCL, n = 3), involved nodal/extranodal sites shown on CT and/or biopsy were concordantly PET-positive. In one PTCL-U, PET detected FDG-avid marrow infiltrations not shown on biopsies. In contrast, cutaneous ALCL (n = 1) and mycosis fungoides (n = 2) showed minimal FDG uptake. In one case of T-cell large granular lymphocyte leukaemia, marrow, nodal and bowel infiltrations were not FDG-avid. PET maximum standardised uptake value did not correlate with clinicopathological features and prognosis. These observations defined the pre-treatment value of PET-CT in T-cell and NK-cell lymphomas. The post-treatment role requires further studies.     

Two abundant proteasome subtypes that uniquely process some antigens presented by HLA class I molecules

Most antigenic peptides presented by MHC class I molecules result from the degradation of intracellular proteins by the proteasome. In lymphoid tissues and cells exposed to IFNγ, the standard proteasome is replaced by the immunoproteasome, in which all of the standard catalytic subunits β1, β2, and β5 are replaced by their inducible counterparts β1i, β2i, and β5i, which have different cleavage specificities. The immunoproteasome thereby shapes the repertoire of antigenic peptides. The existence of additional forms of proteasomes bearing a mixed assortment of standard and inducible catalytic subunits has been suggested. Using a new set of unique subunit-specific antibodies, we have now isolated, quantified, and characterized human proteasomes that are intermediate between the standard proteasome and the immunoproteasome. They contain only one (β5i) or two (β1i and β5i) of the three inducible catalytic subunits of the immunoproteasome. These intermediate proteasomes represent between one-third and one-half of the proteasome content of human liver, colon, small intestine, and kidney. They are also present in human tumor cells and dendritic cells. We identified two tumor antigens of clinical interest that are processed exclusively either by intermediate proteasomes β5i (MAGE-A3(271-279)) or by intermediate proteasomes β1i-β5i (MAGE-A10(254-262)). The existence of these intermediate proteasomes broadens the repertoire of antigens presented to CD8 T cells and implies that the antigens presented by a given cell depend on their proteasome content.     

Soluble HLA class I induces NK cell apoptosis upon the engagement of killer-activating HLA class I receptors through FasL-Fas interaction

The engagement of the activating isoforms of C-type lectin inhibitory receptor (CLIR) or killer Ig-like receptor (KIR) by their natural ligands, represented by soluble HLA-I (sHLA-I) molecules, induced programmed cell death of natural killer (NK) cells. Indeed, NK cell apoptosis elicited by either putative HLA-E and HLA-F (sHLA-I non-A, -B, -C, and -G) or sHLA-I-Cw4 or -Cw3 from untransfected or -Cw4 or -Cw3 alleles transfected HLA-A(-), B(-), C(-), G(-), E(+), F(+) 721.221 lymphoblastoid cell line, respectively, was blocked by covering the corresponding activating receptor with either anti-CLIR- or anti-KIR-specific monoclonal antibodies (mAbs). After sHLA-I-activating receptor interaction, NK cells produced and released Fas ligand (FasL), which in turn led to NK cell apoptosis by interacting with Fas at the NK cell surface. Blocking anti-Fas mAb, or anti-FasL mAb, inhibited sHLA-I-mediated apoptosis via activating receptor in NK cell clones. This apoptosis was inhibited by NK cell treatment with cyclosporin A, whereas this drug had no effect on activating receptor-mediated activation of cytolysis. Conversely, concanamycin A, an inhibitor of vacuolar type H(+)-adenosine triphosphatase (H(+)-ATPase) of granules, inhibited activating receptor-induced NK cell cytolysis, suggesting that activating receptor-mediated apoptosis and cytolysis can use different intracellular pathways. Furthermore, a large amount of interferon-gamma (IFN-gamma) was detectable in culture supernatant of activating receptor(+) NK cells incubated with the appropriate sHLA-I ligand. Again, cyclosporin A, but not concanamycin A, strongly reduced activating receptor-mediated IFN-gamma production. This suggests that activating receptor-induced apoptosis of NK cells could play a role in eliminating potentially harmful NK cell clones and, at the same time, it leads to production of IFN-gamma, an antiviral cytokine able to amplify immune responses.     

Role in T-cell activation for HLA class I molecules from accessory cells: further distinction between activation signals delivered to T cells via CD2 and CD3 molecules

The immunological function of major histocompatibility complex molecules, including HLA class I molecules, is to present antigens and/or their processed peptides to various lymphocyte subpopulations. Thus, they play a pivotal role in regulatory interactions between cells of the immune system, which can result in the activation and function of T cells. We looked for a role of major histocompatibility complex molecules during T-cell activation induced by monoclonal antibody (mAb) or combinations of mAb recognizing the two well-characterized T-cell surface molecules CD3 and CD2. To activate T-cell peripheral blood lymphocytes, we used a CD3 mAb or two different pairs of CD2 mAb, CD2 "GT2 + T11(1)" and CD2 "D66 + T11(1)," which, as we have previously shown, deliver different signals of activation to T cells. Anti-HLA class I mAb blocked the activation induced by CD3 mAb or by CD2 GT2 + T11(1), but it did not block activation induced by CD2 D66 + T11(1). We observed this pattern of inhibition according to the stimulus used to activate T cells both when the anti-HLA class I mAbs were added to cultures of whole peripheral blood mononuclear cells and when they were fixed to monocytes only. In the latter case, purified monocytes were first incubated with the anti-HLA mAb (whether whole immunoglobulin or Fab fragment) and then fixed with paraformaldehyde before culture with autologous purified T cells. Anti-HLA class I fixed on monocytes prevented both interleukin 2 (IL-2) receptor expression and IL-2 synthesis on T cells. The inhibitory effects of anti-class I mAb bound to monocytes were not reversed by adding large amounts of recombinant IL-2 or recombinant IL-1, a finding consistent with the observations that accessory cells surface components can fully complement the signals directly delivered to T cells by CD2 or CD3 mAb. We conclude that HLA class I from accessory cells plays an important role in the early phase of T-cell activation when direct contacts between accessory cells and T cells are required.     

Mumps virus infects Beta cells in human fetal islet cell cultures upregulating the expression of HLA class I molecules

Summary The ability of mumps virus to infect pancreatic Beta cells and cause alterations in their HLA expression was evaluated in cultured human fetal islet cell clusters. Mumps virus could be isolated during the whole culture period (6–8 days) and 60 % of cells, including Beta cells, contained viral nucleocapsid protein at the end of the culturing. A minor decrease in insulin secretion was observed in some of the infected cultures. The infection was invariably associated with an increase in the expression of HLA class I molecules. This enhancement was mediated by soluble factors secreted by infected cells. The infection could not induce the expression of HLA-DR molecules. However, external interferon-gamma was able to cause a clear rise in DR-expression which was observed only on non-Beta-cells. Rubella and coxsackie B4 viruses were also able to enhance the expression of class I molecules while herpes simplex virus type 2 was not. The results suggest that certain viruses are able to infect Beta cells and cause alterations in their immunological appearance. Increased HLA class I expression in infected islets may exaggerate the autoimmune process in pre-diabetic individuals by increasing the activity of autoreactive cytotoxic T cells.     

Loss of surface HLA class I molecules in leukemic myeloblasts is correlated with an increased leukocyte concentration at onset.

BACKGROUND. Reduced expression of HLA class I molecules has been demonstrated for many human neoplasias and is correlated with increased malignancy. METHODS. We investigated the correlation between HLA expression on blasts and the number of peripheral blood leukocytes (WBC) at onset in 34 patients with acute non lymphoblastic (ANLL) leukemia. Leukemic blasts at onset were serologically typed for HLA-A,B specificities, and the patients were classified in three groups according to the number of detectable surface HLA antigens. RESULTS. The amount of WBC at onset in patients lacking HLA antigens was significantly higher than in patients with no antigenic loss. CONCLUSIONS. A decrease in HLA class I molecules on the cell surface is correlated with a greater expansion of the leukemic clone.     

Functional role of HLA class I cell-surface molecules in human T-lymphocyte activation and proliferation

This investigation addressed the role of major histocompatibility complex-encoded class I molecules in the activation and proliferation of human lymphocytes. We studied the effect of antibodies specific for HLA-A and HLA-B locus gene products on mitogen-stimulated peripheral blood mononuclear cell (PBMC) subpopulations. Three individually derived, well-characterized anti-HLA class I monoclonal antibodies were demonstrated to inhibit the proliferation of human PBMC stimulated by either OKT3 or the calcium ionophore ionomycin. The antibody directed against HLA-A, -B, and -C locus gene products (W6/32) and the antibody directed against HLA-B locus gene products (4E) inhibited proliferation induced by either mitogen by 70-90%. The HLA-A locus-specific antibody (131), though inhibiting ionomycin-induced proliferation by 80-90%, was much less effective when OKT3 was the stimulus. The inhibition affected T4+ and T8+ cells and was not mediated by DR+ accessory cells. The inhibitory effect of these antibodies was associated with a decrease in the level of interleukin 2 activity present in culture supernatants, decreased interleukin 2 receptor expression, and decreased transferrin receptor expression and was not overcome by the addition of exogenous interleukin 2. Our results suggest that HLA class I molecules are directly involved in the early critical events of human lymphocyte activation and proliferation.     

HLA class I molecules are associated with CD1a heavy chains on normal human thymus cells

The molecules encoded by the major histocompatibility complex play a pivotal role in regulatory interactions between cells of the immune system, which can result in the activation and function of T cells. The function of the CD1 molecules, which are homologous to the major histocompatibility complex-encoded molecules but are encoded on human chromosome 1, is not known. HLA class I molecules and CD1a heavy chains share the ability to associate with several different cell-surface molecules. We show here, by several technical approaches, that HLA class I molecules are associated with CD1a heavy chains on the surface of normal thymus cells. The functional significance of this association during T-cell differentiation is discussed.     

Pretransplant cytotoxic donor T-cell activity specific to patient HLA class I antigens correlating with mortality after unrelated BMT

Unrelated bone marrow transplantation (BMT) is associated with increased post-transplant complication rates, partly because more transplantation antigens are mismatched than in HLA-identical related BMT. We have shown previously that the cytotoxic T-lymphocyte precursor (CTLp) test performed before transplantation specifically detects HLA class I mismatches demonstrating its usefulness for the identification of new HLA class I alleles. In this study we analysed the clinical relevance of the CTLp test in 41 patients who underwent unrelated BMT between 1990 and 1994. All patient–donor pairs were HLA-A, -B, -DR compatible as defined by AB-serology and oligotyping for DR1-14. The host-reactive CTLp test was performed using previously frozen peripheral blood mononuclear cells (PBMC) as stimulators and PHA blasts as target cells. We found 10 CTLp-positive and 31 CTLp-negative patient–donor pairs. Between the two groups there were no significant differences for age, diagnosis, sex, preconditioning and GvHD prophylaxis. The clinical results for the CTLp positive and the CTLp negative transplants were: severe acute GvHD III–IV 67% and 26% ( P  = 0.0315), transplant-related mortality 60% and 26% ( P  = 0.0085), and patient survival at 3.5 years 10% and 54% ( P  = 0.0006).
Seven patient–donor pairs were mismatched for HLA-DR and/or -DQ subtypes. Only one of these seven class II mismatched pairs had a positive CTLp test. In the remaining nine CTLp positive pairs the CTL reactivity was directed against HLA-A, -B or -C antigens, revealing a statistically significant ( P  < 0.005) correlation between the CTLp frequency and HLA class I matching. In conclusion, the CTLp test helped to select optimally matched bone marrow donors and was particularly useful in association with high resolution oligotyping for DR- and DQ-subtypes for precise matching of both classes of HLA antigens.     

The role of class I histocompatibility antigens in the regulation of T-cell activation

Class I major histocompatibility antigens in humans (HLA antigens) were found to participate in the regulation of T-cell activation and proliferation induced by phytohemagglutinin. W6/32, a monomorphic antibody directed against class I HLA-A,B,C antigens, significantly inhibited the phytohemagglutinin-induced cell proliferation of peripheral blood lymphocytes. Almost complete suppression of cell activation was achieved on a subfraction of peripheral blood lymphocytes enriched in Mo1+ monocyte/macrophage cells. This inhibition of cell proliferation takes place at an early stage of activation and was found to be adherent cell dependent. Removal of monocyte/macrophage type cells from peripheral blood lymphocytes completely abrogated the inhibitory influence of anti-HLA-class I antibody, and, upon adding them back, suppression reappeared. Indirect immunofluorescence demonstrated that the expression of receptors for interleukin 2 and transferrin was impaired in the presence of antibody. Although the amount of interleukin 2 synthesized by these cells was also reduced, the addition of exogenous purified interleukin 2 did not restore cell proliferation. Mitogenesis induced by the Ca2+ ionophore A23187 was similarly suppressed, but mitogenesis induced by the phorbol diester phorbol myristate acetate, which activates cells by directly stimulating protein kinase C, was not suppressed. These results are consistent with a hypothesis that HLA class I antigens regulate an early event(s) of the Ca2+-dependent pathway of activation of T lymphocytes and that this event(s) apparently occurs before protein kinase C stimulation.     

HLA class II molecules and autoimmune hepatitis susceptibility in Japanese patients.

To investigate the association between autoimmune hepatitis and HLA alleles in Japanese patients, serological typing and class II genotyping were performed using the polymerase chain reaction-restriction fragment length polymorphisms (PCR-RFLP) method. Serological typing showed that HLA-B54, -DR4, -DR53, and -DQ4 were significantly more frequent in patients with autoimmune hepatitis than in controls. HLA-DR4 was most frequently associated with autoimmune hepatitis (88.7%). In PCR-RFLP typing, the frequency of DRB1*0405 was significantly higher in autoimmune hepatitis than in controls. However, there was no significant difference in the frequency of Dw between the patients and the controls who were DR4-positive. The significant increase observed in DQA1*0301 and DQB1*0401 was explained by a linkage disequilibrium with DR4. Six DR4-negative patients had DR2, but there was no significant difference in the frequency of the DR2-associated Dw-alleles compared with the DR2-positive controls. No DPB1 allele was significantly associated with autoimmune hepatitis. These findings suggest that the basic amino acid at position 13, which is present only on the DR2 and DR4 B1 molecules (Arg on DR2 and His on DR4), contributes to the susceptibility to autoimmune hepatitis among the Japanese.