Monomorphic Determinants Research Articles

Expression of monomorphic HLA-determinants, transferrin receptor 1 (TfR1) in molecular subtypes of breast cancer

Background. Immunotropic drugs are widely used in the modern strategy of cancer treatment. Importance is given to immunological markers of the tumor, which determine the prognosis of the disease, the effectiveness of treatment. Therefore, the study of their expression is one of the leading scientific directions. Of particular interest is the study of monomorphic HLA determinants, transferrin receptor 1 (TfR1), depending on its biological subtype of breast cancer.Aim. To evaluate the frequency of expression of HLA class I, II, TfR1 molecules by breast cancer cells and determine their relationship with the molecular biological subtype of the tumor.Materials and methods. This study included 120 patients with breast cancer who received treatment at the N.N. Blokhin National Medical Research Center of Oncology, Ministry of Health of Russia. Tumor stages II and III prevailed: 56.7 % and 33.4 %, respectively. A moderate degree of differentiation (G2) was more often noted. The luminal subtype was 58.3 % (n = 70), non-luminal – in 41.7 % (n = 50). Immunophenotyping of the primary tumor was performed by immunofluorescence on cryostat sections. The reaction was evaluated using a ZEISS Axioscope 5 luminescent microscope (Zeiss AG, Germany). The frequency of expression of HLA class I and II molecules were studied depending on the clinical and morphological characteristics of breast cancer. The frequency of expression of HLA class I, HLA-DR, TfR1, molecules, toumor infiltration of СD45+, CD38+, depending on the molecular subtype of breast cancer was studied.Results. It was found that the frequency of expression of monomorphic determinants of the HLA class I in luminal and non-luminal subtypes of breast cancer was comparable; HLA-DR was expressed significantly more often in the luminal subtype of breast cancer: 37.3 % and 18.0 %, respectively, p = 0.022. The frequency of TfR1 expression was significantly higher in the luminal subtype of cancer compared to non-luminal, p = 0.014. Predominantly monomorphic type of reaction was observed: in 76.5 % (n = 39) of cases. The mosaic type of the TfR1 reaction was noted in 7.8 % of the samples. TfR1 monomorphic expression was detected in 50.0 % (n = 30) of cases in non-luminal cancer, the mosaic expression – in 20.0 % (n = 12) of cases. A pronounced degree of lymphoid infiltration, in particular plasmacytic, was established in non-luminal subtype of breast cancer: 70.7 % (n = 29) and 35.0 % (n = 14), respectively, p = 0.001. An association was noted between the expression of HLA I class molecules and the severity of general leukocyte infiltration, p = 0.007.Conclusion. The frequency of expression of HLA class I monomorphic determinants did not differ in molecular subtypes of breast cancer. The expression of the HLA class II molecule was significantly more frequently observed in the luminal subtype of breast cancer. The expression of HLA class I monomorphic determinants is associated with the degree of lymphoid infiltration of the tumor. In the non-luminal subtype, plasmacytic infiltration is more pronounced. The expression of transferrin receptors is significantly more pronounced in the luminal subtype.

SPECIFIC FEATURES OF THE IMMUNE STATUS IN RHEUMATOID ARTHRITIS IN RELATION TO THE CLIMATIC, GEOGRAPHICAL, AND ENVIRONMENTAL ZONES OF UZBEKISTAN

The feature of rheumatoid arthritis (RA) is the predominant clinical picture of chronic synovial joint inflammation accompanied by progressive cartilage and bone destruction, the pathogenesis of which is greatly due to autoimmune mechanisms. Some investigations demonstrate that a set of poor climatic and geographical factors may influence the development of immune disorders that are a trigger in the development of RA. Objective: to comparative analyze immunological parameters in RA in dwellers from different regions of Uzbekistan. Subjects and methods. The study enrolled RA patients from three Uzbekistan zones: 1) Tashkent (n = 25), 2) Xorazm Region (n = 25); 3) Namangan Region (n = 28). Results and discussion. Comparative analysis of the immune status indicated that the patients living in Zone 2 exhibited a significantly reduced T cell immune response (p < 0.05), a considerably higher count of B lymphocytes (p < 0.001), and significantly lower levels of CD11b+, CD18+, CD25+, and CD95+ cells (p < 0.05) than did those from the two other zones. Examination of the frequency of the monomorphic determinant of histocompatibility Class I (HLA-I) antigens revealed its significant decrease in the patients residing in Zones 1 and 3 (p < 0.05) whereas the RA patients from Zone 2 had a significantly higher frequency of HLA-DR (p < 0.05). Conclusion. The comparative analysis shows that the immunological parameters differ in the patients from the three zones of Uzbekistan, which may suggest that the climatic and geographical factors of the environment may influence the course of the disease.

Ligation of HLA class ii molecules on endothelial cells induces p-selectin cell surface expression and myeloid cell recruitment

Aim A hallmark of chronic antibody-mediated rejection is the presence of leukocytes infiltrates within the allograft. We tested the hypothesis that ligation of HLA class II on EC mediates myeloid cell recruitment by inducing p-selectin presentation through Weibel Palade body exocytosis. Methods Class II expression was induced by adenoviral transfection of EC with CIITA. Western blots were used to quantitate protein phosphorylation. Monocyte adherence was measured by counting CFSE-labeled monocytes (MonoMac 6) recruited to HLA-class II Ab stimulated EC in the presence/absence of Fc receptor blockade with human IgG. Transendothelial cell migration was determined using a transwell assay. P-selectin expression was detected by Cell ElISA. Results Class II ligation by antibody (Ab) against a monomorphic epitope determinant on class II antigens significantly increased monocyte transendothelial migration in a Fc-independent manner. Class II Ab-mediated myeloid cell transendothelial migration was attenuated by blockading with recombinant PSGL-1 (rPSGL-1). Class II Ab caused a 1.54 ±± 0.11 fold increases in monocyte adherence that was inhibited by pretreatment with rPSGL-1. In addition, pretreatment of EC with Src inhibitor PP2 and ERK inhibitor U0126 resulted in decreased myeloid cell recruitment (Fig. 1). Furthermore, PP2 and U0126 diminished class II induced p-selectin cell surface expression (Fig. 2). Conclusions We provide evidence that class II DSA promotes a MAPK-mediated monocyte adherence by upregulating EC p-selectin expression, stimulating monocyte adherence and infiltration into allografts, leading to transplant vasculopathy. Figure options Download full-size image Download as PowerPoint slide Figure options Download full-size image Download as PowerPoint slide

OR20 HLA Class II ligation by antibody induces endothelial cell permeability by promoting endocytosis of Ve-cadherin

Aim Increased microvascular dilatation and permeability occurs during allograft rejection and contributes to fibrotic remodeling and organ dysfunction in transplant recipients. Endothelial cells (EC) control vascular permeability by regulating cell-to-cell junctions. We hypothesize that HLA class II ligation on EC transduces signals that disrupt cell–cell tight junctions resulting in increased cell permeability. Methods Class II expression was achieved by transducing EC with adenoviral recombinant CIITA. EC were stimulated with mAb against monomorphic determinant present on all HLA class II antigens (F26C6G1). Western blots were used to characterize protein phosphorylation. Cell permeability was measured by FITC-dextran transwell permeability assay and Ve-Cadherin (Ve-Cad) surface expression was determined by flow cytometry. Cell junctions and Ve-cad internalization were quantified by immunofluorescence microscopy. Results Treatment of EC with anti-class II Ab activated Src, stimulated Ve-Cad phosphorylation at tyr685 and increased Ve-Cad internalization (Fig). Class II ligation decreased Ve-cad surface expression by 42 ± 5%, which was blocked by PP2. In the presence of chloroquine, class II ligation resulted in a 27 ± 4% increase in Ve-cad internalization. Immunofluorescent studies showed that Ve-Cad in the vesicles was phosphorylated and class II ligation increased pY685-Ve-Cad expression. Preincubation of EC with PP2 decreased Ve-cad phosphorylation and internalization. Class II Ab increased FITC-dextran permeability confirming that class II signaling resulting in increased vascular permeability. Our data show that class II ligation on EC mediates the disassembly of intercellular junctions by stimulating Ve-Cad phosphorylation and internalization in a Src dependent manner. Conclusions We provide evidence that HLA class II DSA promote endocytosis of Ve-Cad via Src activation, disrupting endothelial barrier function and contributing to fibrotic remodeling and organ dysfunction. Download : Download full-size image

P017 : HLA EXPRESSION DIFFERENCES ON B CELL SUBSETS ISOLATED FROM DECEASED DONOR TISSUE

We previously reported that differences in HLA class II expression on B cells isolated from different deceased donor tissues can impact flow cytometric crossmatch (FCXM) reactivity. In this current study, we examined HLA surface expression on different B cell subsets found in secondary lymphoid tissue. HLA expression was measured using FITC conjugated antibodies specific for monomorphic determinants on HLA class I and II molecules. Lymphocyte subsets were analyzed using antibodies specific for CD3 (PE), CD19 (APC-Cy7), CD38 (PerCP), and CD27 (APC). Data was acquired on a BD FACSCanto II using FACSDiva software and comparisons of median fluorescence values (MF) were performed using Student t tests. The majority of B cells isolated from spleen ( n = 9, mean = 78%) and lymph nodes ( n = 10, mean = 79%) expressed CD38, consistent with an enrichment of activated B cells in these tissues. CD38 is transiently expressed on activated T cells and was found on approximately half of all T cells residing in spleen ( n = 9, mean = 40%) and lymph node ( n = 10, mean = 58%). Memory lymphocytes, defined by CD27 expression, were also found in spleen ( n = 14, mean = 37% CD19+CD27+ and 22% CD3+CD27+) and lymph node ( n = 17, mean = 38% CD19+CD27+ and 43% CD3+CD27+). Mean HLA class I expression was higher on CD27+ B cells isolated from spleen (36,789 MF) and lymph nodes (34,170 MF) compared to CD27− B cells (mean = 17,617 MF, p = 7.1 × 10 −7 and 15,287 MF, p = 8.1 × 10 −6 , respectively). HLA class I expression on T cells, however, was not impacted by CD27 expression. Mean HLA class I expression on CD3+CD27+ cells isolated from spleen (17,450 MF) and node (14,083 MF) were similar to that found on CD3+CD27− cells (spleen: 19,259 MF and node: 14,040 MF). Interestingly, HLA class II expression was higher on CD27+ B cells isolated from spleen (CD19+CD27+: 3902 MF vs CD19+CD27−: 2097 MF, p = 0.003), but not lymph nodes (CD19+CD27+: 2553 MF vs CD19+CD27−: 2245 MF, p = 0.51). Significant variability in HLA expression was observed on CD27 B cell subsets isolated from secondary lymphoid tissues. Higher levels of HLA class II on splenic, but not lymph node derived CD27+ B cells, identifies spleen as a preferred B cells source for FCXM testing. Moreover, additional gating parameters that include CD27, may allow for enhanced FCXM sensitivity with B lymphocytes.

56-OR

The development of anti-donor HLA class II antibodies (Ab) is associated with chronic allograft rejection. The endothelial cell (EC) barrier is controlled by adhesive structures called cadherins which regulate permeability, migration and proliferation. We hypothesize that Ab ligation of HLA class II molecules on EC contributes to chronic rejection by transducing intracellular signaling cascades leading to remodeling of the actin cytoskeleton, redistribution of VE-cadherin and changes in EC barrier function. Class II expression on primary cultures of EC was achieved by adenoviral infection with recombinant Class II Transactivator (CIITA) sub-cloned with pAd/PL-DEST. Mouse monoclonal Ab that reacts with a monomorphic determinant of class II antigens (F26C6G1) was used to treat EC. Stress fiber formation was assessed using Texas Red-phalloidin staining. Protein phosphorylation was assessed by Western blot. VE-cadherin localization was measured by immunofluoresence. Treatment of class II-positive EC with class II Ab significantly increased Akt, ERK and p70 S6 Kinase phosphorylation. Class II-augmented ERK phosphorylation was accompanied by a substantial increase in stress fiber formation that was abrogated by pretreatment with the ERK inhibitor UO126 (Fig. 1A). Treatment of EC with class II Ab markedly reduced VE-cadherin localization in EC tight junctions which could be reversed by pretreatment with U0126 (Fig. 1B). Ligation of HLA class II molecules with Ab stimulates intracellular signaling cascades leading to activation of the ERK signaling pathway, actin cytoskeleton remodeling, relocalization of VE cadherin and changes in EC barrier function.

6-P

Flow cytometric crossmatches (FCXM) are used to detect low-level donor-specific antibody and determine transplantation eligibility. We measured HLA class I and II surface expression using different lymphocyte sources (peripheral blood (PBL), spleen, or nodes) to investigate the impact of HLA expression on detecting low-level donor specific antibody. HLA expression was measured on a BD FACSCanto II using fluorescently conjugated antibodies specific for monomorphic determinants on HLA class I and II; measurements were converted to Molecules of Soluble Fluorescence (MESF) units. FCXM reactivity was determined using the ratio of test serum to negative control serum and MESF units. Compared to Live Donor (LD) PBLs (n=249), mean HLA class I expression on lymphocytes isolated from Deceased Donor (DD) (n=25) spleen and lymph nodes was lower (5.8% and 10.4%, respectively). The mean HLA class II expression was significantly lower on DD splenic (90.4%, p < 0.05) and lymph node (125.6%, p < 0.05) lymphocytes. We then compared lymphocytes isolated from the same DD (n=4) to better assess the variability of HLA expression on cells derived from different tissues. Compared to DD PBL, HLA class I expression was higher on splenic (54%) and lymph node lymphocytes (34%). HLA class II expression on DD PBLs was lower than that from spleen (63%), but higher than the lymph node-derived lymphocytes (99%). Variation in HLA class II expression on cells from different tissues also impacted crossmatch reactivity. HLA class II donor specific sera, expected to yield a positive FCXM, consistently showed the highest reactivity with splenic lymphocytes; intermediate reactivity with PBL; and the lowest reactivity with lymph node cells. HLA expression may not be routinely analyzed; however, it can substantially impact crossmatch reactivity. Laboratories using a single positive threshold for all FCXMs should consider that splenic lymphocytes may yield reactivity most comparable to that of PBLs from LDs.

44-OR

Aim The presence of HLA Ab in the allograft is associated with transplant vasculopathy (TV). Ligation of HLA class I elicits signaling events and induces the remodeling of actin cytoskeleton leading to cell survival, migration and proliferation in EC. However, the mechanism by which anti-HLA class II Ab contribute to TV development is not clear. We hypothesize that HLA II ligation induces signaling cascades mediating cytoskeleton reorganization. Methods To express HLA II, human aortic EC were infected with adenoviruses encoding Class II transactivator (Ad-CIITA). Expression of HLA II was determined by flow cytometric analysis using Ab against a monomorphic epitope on HLA II or locus specific Ab against HLA-DR, DQ and DP. Ad-CIITA infected EC were stimulated with anti-HLA II Ab and protein phosphorylation was examined by Western blot. Stress fiber formation was evaluated by fluorescence microscopy in EC stained with Texas Red-phalloidin. The FAK siRNA was utilized to deplete FAK expression. Results Infection with Ad-CIITA for 2d induced expression of HLA II in 80% of EC. Staining with locus specific Ab showed HLA-DR was expressed at the highest level with comparable levels of DP and DQ. Treatment of EC with HLA II Ab significantly increased the phosphorylation of FAK, Src, ERK and p70 S6 kinase (S6K). Ligation of HLA II also significantly augmented formation of stress fibers in EC compared to isotype control IgG [Fig. 1] Knockdown of FAK attenuated phosphorylation of Src, ERK and S6K and also abolished class II-mediated stress fiber formation. Download : Download full-size image Conclusions Ab ligation of HLA II molecules stimulates signal transduction and induces actin cytoskeleton remodeling in EC in a FAK dependent manner.

Liver-infiltrating T helper cells in autoimmune chronic active hepatitis stimulate the production of autoantibodies against the human asialoglycoprotein receptor in vitro.

Autoantibodies against the human asialoglycoprotein receptor (ASGPR) occur in the sera of patients with autoimmune liver disorders. Liver-infiltrating T cell clones that specifically recognize the ASGPR have been described in patients with autoimmune chronic active hepatitis (AI-CAH) and primary biliary cirrhosis (PBC). Recently, we have shown that peripheral blood mononuclear cells (PBMC) from patients with AI-CAH or PBC but not chronic viral hepatitis secreted anti-ASGPR antibodies in vitro. In this study we characterized the influence of liver-infiltrating T cells on the secretion of ASGPR-specific autoantibodies by autologous B cells in cell culture supernatants. T cell clones from liver biopsies of three patients with chronic autoimmune liver disorders (one with AI-CAH, two with PBC) were isolated and investigated for their proliferative response to soluble ASGPR and their helper function provided to autoantibody-secreting B lymphocytes. PBMC from these patients secreted autoantibodies spontaneously in their cell culture supernatants and showed a proliferative response to ASGPR. T cell-depleted PBMC, however, lacked spontaneous antibody secretion. Four CD4+CD8- liver-infiltrating T cell clones showed a proliferative response to ASGPR and also induced spontaneous anti-ASGPR antibody production in cell culture supernatants when added to autologous T cell depleted PBMC. Activated supernatants of these T cell clones failed to induce antibody production. None of seven CD4+CD8- and two CD4-CD8+ T cell clones non-responding to ASGPR provided this help for antibody secretion. Anti-ASGPR secretion in vitro could not be inhibited by the addition of MoAbs raised against monomorphic determinants on HLA class II molecules. The addition of purified ASGPR or polyclonal-activating pokeweed mitogen showed no influence on the production of autoantibodies in these cultures. These data show that B lymphocytes require T cell help for the production of ASGPR-specific antibodies. This help can be provided by ASGPR-responsive T helper cells via cellular interactions.

Loss of expression of antigen-presenting molecules in human pancreatic cancer and pancreatic cancer cell lines

Tumours evade immune recognition and destruction through loss or down-regulation of expression of antigen processing and antigen-presenting molecules such as the human leucocyte antigen (HLA class I) and transporter for antigen presentation (TAP). This study examined the expression of HLA class I, class II and TAP in human pancreatic carcinoma tissue and 19 immortalized pancreatic cancer lines using a panel of antibodies directed against allele-specific as well as monomorphic determinants of these molecules. In tissue samples, reduction or loss of HLA class I and TAP was observed in 76% of cases, loss or down-regulation of TAP expression in 53%. In pancreatic cell lines down-regulation or loss of class I and TAP expression was also observed frequently. However, reductions in class I and TAP expression were reversible upon exposure to interferon-gamma in vitro, suggesting a regulatory rather than structural defect in these genes. De novo class II expression was observed in 26% of tumours and 42% of cell lines and may reflect the differentiation status of the cells. The high rate of class I and TAP loss has implications for immunotherapy strategies for pancreatic cancer, as such changes could facilitate a selective growth advantage for malignant cells. However, the reinduction of expression of these molecules with cytokines such as interferon-gamma may ultimately allow their cytotoxic T cell-mediated destruction.

Defective Human Leukocyte Antigen Class I-associated Antigen Presentation Caused by a Novel β2-Microglobulin Loss-of-function in Melanoma Cells

The major histocompatibility complex class I molecules consist of three subunits, the 45-kDa heavy chain, the 12-kDa beta(2)-microglobulin (beta(2)m), and an approximately 8-9-residue antigenic peptide. Without beta(2)m, the major histocompatibility complex class I molecules cannot assemble, thereby abolishing their transport to the cell membrane and the subsequent recognition by antigen-specific T cells. Here we report a case of defective antigen presentation caused by the expression of a beta(2)m with a Cys-to-Trp substitution at position 25 (beta(2)m(C25W)). This substitution causes misfolding and degradation of beta(2)m(C25W) but does not result in complete lack of human leukocyte antigen (HLA) class I molecule expression on the surface of melanoma VMM5B cells. Despite HLA class I expression, VMM5B cells are not recognized by HLA class I-restricted, melanoma antigen-specific cytotoxic T lymphocytes even following loading with exogenous peptides or transduction with melanoma antigen-expressing viruses. Lysis of VMM5B cells is restored only following reconstitution with exogenous or endogenous wild-type beta(2)m protein. Together, our results indicate impairment of antigenic peptide presentation because of a dysfunctional beta(2)m and provide a mechanism for the lack of close association between HLA class I expression and susceptibility of tumor cells to cytotoxic T lymphocytes-mediated lysis in malignant diseases.

28 EXPRESSION OF MHC CLASS I IN THE PLACENTA AND THE LYMPHOCYTES OF BOVINE SOMATIC CLONES

A major limitation for the development of somatic cloning in cattle is the low efficiency for producing live offspring because only 5 to 25% of the reconstructed embryos reach full-term development, with variation between the genotypes. Hill et al. (2002 Biol. Reprod. 67, 55–63) reported that early fetal losses were due to inappropriate expression of trophoblast major histocompatibility complex Class 1 (MHC1) antigens on the placenta of bovine somatic clones. This abnormal expression was present in all clones, with more cells expressing MHC1 in those with growth retardation. These clones, however, originated from only one genotype, and only one antibody recognizing a monomorphic determinant of bovine MHCI was used. In the present study, MHC1 expression was examined throughout gestation in bovine clone placentas originating from 3 different genotypes. Placentas were collected at Days 32, 62, 180, 260, and 280 (term) in clones (n = 8) and in controls obtained by artificial insemination (n = 6). Four different monoclonal antibodies that recognize different determinants of MHC1 molecules (ILA-A88, IL-A19, H58A, and the β2-microglobulin, i.e. the MHC1 light chain) were used. Results showed MHC1 expression in the maternal tissue but not on the fetal side, regardless of group, gestational age, or fetal growth, although patchy expression of β2-microglobulin could be found in both clones and controls in the fetal mesenchyme in some but not all samples. In conclusion, MHC1 protein expression appears normally down-regulated in the placenta of clones, but the antibodies used do not discriminate between classical and non-classical expression. The mRNA expression of classical and non-classical MHC1 genes is currently being explored. These results cast doubt on the hypothesis that the observed fetal losses would be the result of an immunologic rejection due to inappropriate expression of some placental MHC1 genes. Although early observations suggest that immune functions appear normal (Lanza et al. 2001 Science 294, 1893–1894; Chavatte-Palmer et al. 2006 Reprod. Fert. Dev. 18, 121 abst), immunological deficiencies have been reported in some bovine somatic clones after birth (Renard et al. 1999 The Lancet 353, 1489–1491), and they may be related to an abnormal expression of MHC1 genes. We therefore determined the MHC1 haplotype of cell donors from 4 genotypes and 3 of their healthy clones using the Reference Strand Conformation Analysis method with DNA extracted from lymphocytes. Results showed that the MHC1 type was identical, as could be expected in clones. Animals that died of immune deficiencies, however, may have had abnormal MHC1 expression. Further work is therefore being performed on tissues obtained postmortem from cloned animals having been diagnosed with thymic atrophy.

Selective monomorphic and polymorphic HLA class I antigenic determinant loss in surgically removed melanoma lesions

The analysis of human leukocyte antigen (HLA) class I allospecificity expression in malignant lesions has been hampered by the limited availability of HLA class I allospecificity-specific monoclonal antibodies (mAbs) which stain tissues in immunohistochemical (IHC) reactions. During the 12th International Histocompatibility Workshop, the HLA and cancer component made available a panel of mAbs capable of detecting monomorphic, locus- and allo-specific HLA class I antigenic determinants in surgically removed frozen tissue sections by IHC staining. In the present study, we have utilized this panel of mAbs to analyze the expression of HLA class I allospecificities in 33 primary and in 11 metastatic lesions surgically removed from HLA-typed patients with malignant melanoma, as this information contributes to determine the extent of HLA class I antigen abnormalities in melanoma lesions. HLA class I antigens were downregulated in six (18.2%) of the primary lesions and in six (54.5%) of the metastatic lesions. Selective loss of HLA-A and HLA-B antigens was detected in two (6.1%) and in one (3.0%), respectively, of the primary lesions, but in none of the metastases. HLA-A and HLA-B antigens were downregulated in three (9.1%) and four (36.4%) of the primary and metastatic lesions, respectively. Selective loss of one or more HLA class I allospecificities was found in 10 (33.0%) and two (18.0%) of the 33 primary and 11 metastatic melanoma lesions analyzed, respectively. HLA class I antigen abnormalities were present in 16 (48.5%) of the 33 primary lesions analyzed (i.e. six lesions demonstrating abnormal reactivity with HLA class I monomorphic-specific mAb, two lesions demonstrating selective abnormal reactivity with HLA-B locus-specific mAb, one lesion demonstrating selective abnormal reactivity with HLA-A and HLA-B locus-specific mAbs, and seven lesions demonstrating selective abnormal reactivity with HLA class I allele-specific mAb). Furthermore, HLA class I antigen abnormalities were present in nine (81.8%) of the 11 metastatic lesions analyzed (i.e. six lesions demonstrating abnormal reactivity with HLA class I monomorphic-specific mAb, one lesion demonstrating selective abnormal reactivity with HLA-A locus-specific mAb, and two lesions demonstrating selective abnormal reactivity with HLA class I allele-specific mAb). It cannot be ruled out that the frequency of HLA class I allospecificity abnormalities is higher, as the expression of several HLA class I allospecificities could not be investigated because of the lack of appropriate probes. The frequency of HLA class I antigen defects in primary lesions was significantly correlated with primary lesion thickness, an important prognostic marker in melanoma, arguing for a potential clinical significance of HLA class I antigen abnormalities in melanoma. In conclusion, the results of the present study (i) demonstrate that the frequency of HLA class I allospecificity abnormalities in primary melanoma lesions is markedly higher than that of total HLA class I antigen downregulation described in the literature; (ii) corroborate our previous findings that staining of melanoma lesions with mAb to monomorphic determinants of HLA class I antigens does not detect selective HLA class I allospecificity loss; and (iii) demonstrate for the first time selective loss of antigenic determinants expressed on HLA class I molecules in melanoma lesions. The latter finding indicates that at least two mAbs recognizing distinct antigenic determinants on the HLA molecule being investigated should be used for IHC staining of tissue sections in order to prove that lack of immunostaining reflects actual loss of the corresponding HLA molecule and not selective loss of antigenic determinants.

Beta-2 microglobulin-free HLA class I heavy chain (FHC) A3 and/or A30 soluble products contribute only minimally to serum FHC expression.

No monoclonal antibodies (mAbs) are presently available to measure the total amount of beta2-microglobulin-free HLA class I heavy chain (FHC) in sera. The available ELISA-based double determinant immunoassay (DDIA), established to measure FHC, uses two mAbs (TP25.99 and HC-10) that recognize a monomorphic determinant expressed on all HLA-B/C FHC products and a determinant expressed only on some HLA-A FHC products. This restricted reactivity implies that, in addition to HLA-B/C, HLA-A FHC products are also detected in individuals bearing HLA A3 and/or A30 allotypes. The aim of this study was to establish whether such restriction results in the detection of low FHC levels in individuals lacking HLA A3 and/or A30 allospecificities. The FHC mean concentration (+/- SD) in 294 healthy blood/bone marrow donors (HBDs) was 0.24 (+/- 0.2) mg/l. The grouping of HBDs according to their HLA-A FHC product reactivity with one, both or no mAbs did not result in any statistically significant differences (Mann-Whitney test: P > 0.05) between their median FHC concentrations. Since the absence of differences in their FHC levels was not attributable to a difference in the percentage distribution of HLA allotypes associated with high or low HLA-B/C FHC expression, our results indicate that FHC HLA A3 and/or A30 products detected in DDIA by these two mAbs only minimally contribute to FHC serum expression and that the assay is not limited by the failure to detect HLA-A FHC products in A3- and/or A30- individuals.

Drug-induced and drug-dependent immune thrombocytopenias.

Thrombocytopenia is a frequent comorbid condition in many in hospital patients. In some patients, drugs are the cause of low platelet counts. While cytotoxic effects of anti-tumor therapy are the most frequent cause, immune mechanisms should also be considered. This review addresses thrombocytopenias in four groups. Heparin-dependent thrombocytopenia (HIT), by far the most frequent drug-induced immune-mediated type of thrombocytopenia, has a unique pathogenesis and clinical consequences. HIT is a clinicopathological syndrome in which antibodies mostly directed against a multimolecular complex of platelet factor 4 and heparin cause paradoxical thromboembolic complications. The mechanisms through which heparin can enhance thrombin generation are discussed and treatment alternatives for affected patients are presented in detail. It is of primary importance to recognize these patients as early as possible and to substitute heparin with a compatible anticoagulatory drug, such as hirudin, danaparoid or argatroban. Patients seem to benefit from therapeutic doses of alternative treatment rather than from low-dose prophylactic doses. With the increasing use of glycoprotein (GP) IIb/IIIa inhibitors in patients with acute coronary syndromes, thrombocytopenias are increasingly recognized as an adverse effect of these drugs. Up to 4% of treated patients are affected. Most important, pseudothrombocytopenia, a laboratory artefact, is as frequent as real drug-induced thrombocytopenia and must be excluded before changes in treatment are considered. The pathogenesis of these thrombocytopenias is still debated; an immune mechanism involving preformed antibodies is likely. However, since these antibodies are also detectable in a high percentage of normal controls and of patients not developing thrombocytopenia, their impact is still unclear. Patients with real thrombocytopenia are at an increased risk of bleeding; treatment consists of cessation of the GP IIb/IIIa inhibitor and platelet transfusions in cases of severe hemorrhage. Classic immune thrombocytopenia can be induced by some drugs, e.g. gold, which trigger anti-platelet antibodies indistinguishable from platelet autoantibodies found in autoimmune thrombocytopenia. Drug-induced and drug-dependent immune thrombocytopenia is induced by antibodies recognizing an epitope on platelet GP formed after binding of a drug to a platelet glycoprotein. Still unresolved is whether antibody binding is the consequence of a conformational change of the antigen, the antibody, or both. These antibodies typically react with monomorphic epitopes on platelet GP, but only in the presence of the drug or a metabolite. Although several platelet GP have been identified as antibody target (GPIb/IX, GPV, GP IIb/IIIa), antibodies in an individual patient are highly specific for a single GP. Clinically, these patients present with very low platelet counts and acute, sometimes severe, hemorrhage. Treatment is restricted to withdrawal of the drug and symptomatic treatment of bleeding.

Programmed cell death signaling via cell-surface expression of a single-chain antibody transgene.

The monoclonal antibody, 5H7, is specific for a monomorphic determinant on the a3 domain of human class I MHC (A, B, C). Immobilized 5H7 delivers programmed cell death (PCD) signals to human lymphoid tumor cells as well as peripheral blood mononuclear cells. The potential clinical utility of 5H7 was addressed by design of a single-chain variable antibody (scFv), termed 5H7scFv, which was coupled to glycophosphotidylinostitol (GPI), thereby providing membrane expression of the 5H7 idiotype (5H7scFv-GPI). Membrane expression of 5H7scFv-GPI conferred PCD-inducing properties to cells that do not normally have the capability to process and express whole antibody molecules. The initial construction was undertaken in a bacterial expression system, and appropriate protein folding was determined by binding to class I MHC-expressing cells. 5H7scFv-GPI-transfected Chinese hamster ovary cells demonstrated reconstitution of the 5H7 idiotype and binding to soluble HLA-A2. Cross-linking of class I MHC, via membrane expression of the scFv, provided effective PCD signaling in B and T lymphocyte tumor cells. Peripheral blood mononuclear cells were susceptible to 5H7scFv-GPI-induced PCD, and augmentation of PCD signals was noted with anti-CD3 and anti-CD28 preactivation. Responder cells demonstrated typical histologic features of PCD and Annexin V-fluorescein isothiocyanate binding. Cell surface anchorage of scFv thus provides effective delivery of immune modulatory signals, which may be manipulated for various therapeutic strategies.

Is there a role for NK cells in the pathogenesis of multiple sclerosis? A case study

We report on a patient with multiple sclerosis (MS) in which we documented an elevated percentage of activated CD56+ natural killer (NK) cells in peripheral blood lymphocytes. NK cells from the patient lysed preferentially glioblastoma but not neuroblastoma cells. Killing of glial cells was not inhibited by a monoclonal antibody against a monomorphic determinant of MHC class I gene products. Lymphokine activated killer (LAK) cell function in the MS patient was comparable to that of controls. Analysis of cytokine production during resting or activated states demonstrated that this patient had a deficit in the ability to secrete T cell derived cytokines associated with increased production of TNFα, a product of NK cells. Taken together, these data indicate a possible involvement of NK cells in the pathogenesis of MS.

EXPRESSION OF A GPI-ANCHORED, SINGLE-CHAIN ANTIBODY SPECIFIC FOR A CELL DEATH SIGNALING DOMAIN OF HUMAN CLASS I MHC

173 Recent studies have shown that Fas ligand (FasL) expression confers immune privilege by inducing programmed cell death (PCD). However, early attempts to confer immune privilege to islet allografts via FasL gene transfer have not been reliably successful. 5H7 is a monoclonal antibody (mAb) specific for a monomorphic determinant on the α3 portion of human class I MHC and has been shown to induce PCD in primary human B and T lymphocytes. Here, we describe the construction and expression of a 5H7 single-chain antibody (5H7 scFv) with a GPI (glycophosphoinostitol) anchor that allows cell surface expression of the 5H7 epitope. Methods: The 5H6 scFv included the VL and the VH regions of the 5H7 mAb joined by a 25 amino acid flexible linker. Cloning of the GPI anchor downstream of the VL-VH construct was performed utilizing PCR amplification and specific primer encoded restriction sites. GPI was derived from the C-terminal portion of CD58 and engineered to the 5H7 scFv construct via a second flexible linker. The 5H7 scFv-GPI DNA construct was cloned into the eukaryotic expression vector pCDNA 3.1 to generate pCDNA 3.1-5H7scFv-GPI(pC5H7Fv-GPI). Cos cells (Green Monkey) were transiently transfected by DEA dextran with either pC5H7Fv-GPI or pCDNA 3.1 as a control. Transfectants were screened at 48-72 hours post-transfection by FITC filtered microscopic fluorescence after staining with a biotinylated 5H7 ligand (soluble(s)HLA-A2 tetramer) and strep-avidin FITC. Results: PCR sequencing confirmed proper gene construction and orientation. Immunofluorescent microscopy demonstrated cell membrane bound sHLA-A2 tetramer on cells transfected with pC5H7Fv-GPI, but not on cells transfected with control pCDNA 3.1. Of note, Cos class I MHC has been shown to bind to 5H7 whole mAb, however, this binding did not preclude membrane expression of the 5H7 scFv-GPI.Conclusions: A single-chain form of the mAb 5H7 was constructed with a GPI anchor. Cos cell transfections were successful in transferring the 5H7 epitope to the cell surface as confirmed by sHLA-A2 binding. This 5H7 scFv-GPI construct thus provides a cell membrane expressible form of the parental 5H7 antibody. Experiments are underway to determine the lymphocyte PCD and immune privilege potential of pC5H7scFv-GPI gene transfer.

EXPRESSION OF A GPI-ANCHORED, SINGLE-CHAIN ANTIBODY SPECIFIC FOR A CELL DEATH SIGNALING DOMAIN OF HUMAN CLASS I MHC

173 Recent studies have shown that Fas ligand (FasL) expression confers immune privilege by inducing programmed cell death (PCD). However, early attempts to confer immune privilege to islet allografts via FasL gene transfer have not been reliably successful. 5H7 is a monoclonal antibody (mAb) specific for a monomorphic determinant on the α3 portion of human class I MHC and has been shown to induce PCD in primary human B and T lymphocytes. Here, we describe the construction and expression of a 5H7 single-chain antibody (5H7 scFv) with a GPI (glycophosphoinostitol) anchor that allows cell surface expression of the 5H7 epitope. Methods: The 5H7 scFv included the VL and the VH regions of the 5H7 mAb joined by a 25 amino acid flexible linker. Cloning of the GPI anchor downstream of the VL-VH construct was performed utilizing PCR amplification and specific primer encoded restriction sites. GPI was derived from the C-terminal portion of CD58 and engineered to the 5H7 scFv construct via a second flexible linker. The 5H7 scFv-GPI DNA construct was cloned into the eukaryotic expression vector pCDNA 3.1 to generate pCDNA 3.1-5H7scFv-GPI (pC5H7Fv-GPI). Cos cells (Green Monkey) were transiently transfected by DEA dextran with either pC5H7Fv-GPI or pCDNA 3.1 as a control. Transfectants were screened at 48-72 hours post-transfection by FITC filtered microscopic fluorescence after staining with a biotinylated 5H7 ligand (soluble(s)HLA-A2 tetramer) and strep-avidin FITC. Results: PCR sequencing confirmed proper gene construction and orientation. Immunofluorescent microscopy demonstrated cell membrane bound sHLA-A2 tetramer on cells transfected with pC5H7Fv-GPI, but not on cells transfected with control pCDNA 3.1. Of note, Cos cell class I MHC has been shown to bind to 5H7 whole mAb, however, this binding did not preclude membrane expression of the 5H7 scFv-GPI. Conclusions: A single-chain form of the mAb 5H7 was constructed with a GPI anchor. Cos cell transfections were successful in transferring the 5H7 epitope to the cell surface as confirmed by sHLA-A2 binding. This 5H7 scFv-GPI construct thus provides a cell membrane expressible form of the parental 5H7 antibody. Experiments are underway to determine the lymphocyte PCD and immune privilege potential of pC5H7scFv-GPI gene transfer.

A MURINE TRANSGENIC MODEL OF HUMAN CLASS I MHC-MEDIATED PROGRAMMED CELL DEATH

376 Human class I MHC molecules have recently been shown to mediate programmed cell death (PCD) signals via a Fas-independent pathway, suggesting that this pathway may, like Fas/Fas ligand, be useful for conferring immune privilege. The present study was conducted to develop a murine model for PCD mediated via human class I MHC molecules. Methods: B6 mice and B6 mice expressing both HLA B27 and human β2-microglobulin transgenes (Tg++ mice) were used. PCD was evaluated using viable cell recovery, ethidium bromide/acridine orange staining/fluorescent microscopy, and propidium iodide staining/FACS. Anti-human class I MHC mAbs used included W6/32 and 5H7(specific for an α3 domain monomorphic determinant). Results: Initial studies in mice expressing a single human class I MHC transgene did not demonstrate PCD induction, however, human class I MHC transgene expression was relatively low. Mice expressing HLA B27 and human β2 microglobulin transgenes, however, demonstrated higher human class I MHC expression and underwent PCD following treatment with 5H7 mAb. Splenocytes undergoing 5H7-induced PCD demonstrated classic features of PCD: cell shrinkage, cytoplasmic vacuolization, DNA condensation, and apoptotic body formation. Similar to the human studies of 5H7-induced PCD, studies in murine Tg++ cells demonstrated a greater sensitivity of B cells than T cells to PCD induction. The murine model was also similar to the human model in that class I MHC-mediated PCD induction was profoundly influenced by MHC receptor crosslinking and also dependent on the epitope specificity of the inducing anti-class I MHC mAb. Anti-CD3 mAb enhanced PCD induction by 5H7 mAb in both murine and human cells, however, IL-2 reversed 5H7-induced PCD only in murine cells. Conclusions: These studies indicate that 1) high level expression of human class I MHC in murine cells renders them susceptible to PCD induction via anti-human class I MHC mAb, 2) similar to human lymphoid cells, crosslinking and epitope specificity of the anti-class I MHC mAb markedly influence PCD induction, 3) anti-CD3 mAb enhances human class I MHC-mediated PCD in both human and murine cells, and 4) Tg++ mice provide a useful model for study of human class I MHC-mediated PCD.