Complexes of soluble HLA antigens and anti-HLA autoantibodies in human sera: Possible role in maintenance of self-tolerance

The MHC plays an essential role in regulating the process of self-nonself discrimination. T cells recognize nonself antigens only in the context of self-MHC gene products. It is not clear, however, whether B cells are also endowed with immune receptors for self-MHC antigens. We have explored the existence of antibodies against self-MHC antigens (HLA) in the human by analyzing the specificity of anti-HLA antibodies developed by a population of 727 dialysis patients who had been monitored monthly over a period of 3-78 months. Anti-HLA autoantibodies were identified by serum screening in 119 patients. Twenty-five of these 119 patients had not been exposed to alloantigens before, indicating that the production of anti-HLA autoantibodies is not necessarily stimulated by allogeneic HLA antigens. Cross-matching of sera with autologous lymphocytes, confirmed the autoreactive nature of these anti-HLA antibodies which were of IgM or of IgG isotype in approximately equal numbers of patients. The fine specificities of anti-HLA autoantibodies was ascertained in studies which showed that antibodies can be adsorbed, and the eluted, from the membranes of target cells carrying then relevant HLA antigen. Since the antibodies characterized in our studies may be functionally active in vivo we examined the possibility that their level is controlled by anti-idiotypic antibodies or by soluble HLA antigens. We found that the titer of anti-HLA autoantibodies increased significantly if soluble HLA antigens were depleted from the serum. Our data suggest that circulating HLA antigens form immune complexes with anti-HLA autoantibodies and contribute to the maintenance of self-tolerance by inhibiting antibody binding to membrane HLA antigens. The finding that the immune repertoire includes B cells with receptors for self-MHC opens new perspectives for the study of network perturbations in autoimmune diseases.     

Isolation of pure villous cytotrophoblast from term human placenta using immunomagnetic microspheres

A procedure has been developed which yields a pure population of villous cytotrophoblast from term human placenta. As a first step, a cell preparation highly enriched for cytotrophoblast (identified by positive cytokeratin staining) was obtained using a modification of the method of Kliman et al. (1986). The remaining contaminating cells (identified by positive vimentin staining) were then removed by treatment with mouse monoclonal antibodies against class I and class II major histocompatibility antigens followed by magnetic microspheres coated with goat anti-mouse IgG. The rationale for this step was based on the fact that villous trophoblast fails to express HLA antigens whereas cells from the villous mesenchyme do express these surface antigens. Rosetted cells were immobilized using a magnet allowing the non-rosetted cells to be easily withdrawn by pipette. When the non-rosetted cells were placed in primary culture, no HLA-positive or vimentin-positive cells could be detected using immunofluorescence microscopy, indicating complete removal of these components by the immunomagnetic separation procedure. The cells were positive for cytokeratin and, after 24 h, showed positive staining for pregnancy-specific beta 1-glycoprotein (SP1) and human chorionic gonadotropin. Recovery of cytotrophoblast was greater than 92% with only a slight loss of viability.     

Cell culture density modulates the incorporation of HLA antigens by enveloped viruses

Uninfected, as well as feline leukemia virus (FeLV) infected human cells cultured under high cell density conditions undergo changes in the expression of major histocompatibility complex (MHC) antigens, as determined by indirect trace binding radio immunoassay (RIA) using monoclonal anti-HLA antibodies and by decreased sensitivity to complement mediated cytotoxicity by anti-HLA alloantibodies. FeLV particles produced by the viral infected cells are also sensitive to neutralization by anti-HLA antibodies, suggesting that enveloped viral particles incorporated MHC antigens in the viral envelope. The amount of HLA antigens expressed in the viral enveloped, closely reflects the expression of HLA antigens by the virus-producer lymphoid cells. FeLV-infected HsB-2 (T) and SB (B) lymphoid cells cultured under high cell concentration condition show decreased expression of some HLA antigens (A2, B12, B17), and the viral particles produced by those cells also incorporate lower amounts of such antigens. Our results, based on the findings that human lymphoid cells (uninfected, as well as FeLV infected) show decreased expression of some HLA membrane determinants when growth under high cell density conditions, indicate that no viral selective mechanism operates in the incorporation of HLA determinants by enveloped viruses. Instead, our results suggest that viruses pick up MHC antigens from the host cell membrane according to the concentration of those antigens on the surface of the cells at the time of virus budding.     

Human monoclonal HLA antibodies reveal interspecies crossreactive swine MHC class I epitopes relevant for xenotransplantation

Crossreactivity of anti-HLA antibodies with SLA alleles may limit the use of pig xenografts in some highly sensitized patients. An understanding of the molecular basis for this crossreactivity may allow better selection of xenograft donors. We have tested 68 human monoclonal HLA class I antibodies (mAbs) for reactivity with pig lymphocytes from SLA defined pigs and found nine to be crossreactive. Eight of nine were broadly HLA reactive IgM-mAbs. The putative HLA epitopes for seven mAbs. were conserved in the aminoacid sequence of the SLA alleles studied. The lack of reactivity of a large number of mAbs largely correlated with the absence of the putative epitopes in the SLA alleles studied. We conclude that most patients with anti-HLA class I antibodies should be able to find pig donors lacking SLA antigens that cross react with their antibodies and that many of the crossreacting epitopes can be defined by analysis of shared epitopes in the aminoacid sequence of human and pig MHC antigens.     

Signal transduction in lymphocyte activation through crosslinking of HLA class I molecules

The inhibitory effect of anti-HLA class I monoclonal antibodies on lymphocyte proliferation has been well documented. However, recent data suggest that anti-HLA class I monoclonal antibodies can enhance lymphocyte proliferation via both anti-CD3-induced {1,2} and anti-CD2-induced {3} activation pathways. Here we demonstrate that both inhibition and activation can be regulated by the degree of aggregation of HLA class I antigens. Crosslinking of monoclonal antibodies specific for HLA-A, HLA-B, or monomorphic determinants (using anti-IgG2 and/or anti-Ig “second step” monoclonal antibodies) increased the capacity of the anti-HLA class I monoclonal antibodies to inhibit phytohemagglutinin-induced proliferation. However, the cytosolic free calcium concentration was increased in CD4+ cells, CD8+ cells, B cells, and CD16+ cells when anti-HLA class I monoclonal antibodies were crosslinked, suggesting that an activation signal was generated by aggregation of the corresponding antigens. Indeed, inositol 1,4,5-trisphosphate could be detected in peripheral blood lymphocytes following crosslinking of anti-HLA class I monoclonal antibodies. Class I aggregation also induced proliferation of peripheral blood mononuclear cells in the presence of submitogenic doses of phorbol 12-myristate 13-acetate. Strong conditions of crosslinking (monomorphic monoclonal antibody plus both anti-IgG2 and anti-Ig ) induced CD25 expression and responsiveness to recombinant interleukin 2. Our results suggest that aggregation of HLA class I antigens primed cells to become activated in the presence of progression signals including phorbol 12-myristate 13-acetate, recombinant interleukin 2, or anti-CD5 plus anti-CD28 monoclonal antibodies.     

Nonrandom association of cellular antigens with HTLV-III virions

Retroviruses are known to incorporate cellular antigens as they bud from infected cells. To identify the cellular antigens that associate with the AIDS-retrovirus, we evaluated a preparation of HTLV-III antigens with a panel of monoclonal antibodies reactive with a variety of antigens expressed on the H9 T-cell line used to produce the virus. Only monoclonal antibodies that identified HLA class-II antigens, beta-2 microglobulin, and a single anti-HLA class-I antibody were reactive in an ELISA of solubilized HTLV-III virus. No reactivity was seen with 11 monoclonal antibodies to T-cell antigens or with five antibodies to determinants on HLA class-I A or B molecules. These data suggest that on H9 cells the association of budding HTLV-III virions with cellular antigens may be a nonrandom process in which some HLA antigens, particularly class-II antigens, are selectively incorporated into the viral envelope. It is possible that a selective association of HLA class II antigens with budding HTLV-III virions may also occur for T cells infected in vivo, and could have relevance for the pathogenesis of this virus.     

Class I Antigens of the Major Histocompatibility Complex on Cytotrophoblast of the Human Placental Basal Plate

ABSTRACT: The lack of class I antigens of the major histocompatibility complex (MHC) on syncytiotropho-blast has been proposed as an explanation for the survival of the allogeneic fetus. These antigens, however, have recently been detected on nonvillous trophoblastic columns of the early human placenta. By using a combination of immunofluorescence techniques to identify trophoblast, we have studied transplantation antigens of the cytotrophoblastic shell present in the basal plate of normal full-term human placentae. With the use of two different monoclonal antibodies to a common determinant of HLA (clones W6/32 and 61D2), it was shown that this subset of trophoblast does express class 1 MHC antigens. However, while these cells reacted uniformly with W6/32, only rare reactivity with 61D2 was found. Reactivity of polyclonal antisera to β-mi croglobulin correlated with that seen using W6/32. These results are similar to those recently observed in a subset of trophoblast of the amniochorion to which the term “metatrophoblast” has been given.     

Expression of MHC class I determinants on erythrocytes of SLE patients

Strong expression of MHC Class I determinants had been observed on the erythrocytes of three genetically C4 deficient patients who all had SLE. In a study of 35 other SLE patients who were not C4 deficient, 30 showed a marked increase in the expression of MHC Class I on their erythrocytes. There was a correlation between the expression of erythrocyte Class I and disease activity. The polymorphic HLA determinants were detected by haemagglutination with human cytotoxic antisera from untransfused pregnant women. A shared monomorphic epitope of HLA-A, -B and -C, and beta 2-microglobulin were detected by haemagglutination with monoclonal antibodies. A monoclonal antibody for a monomorphic epitope on MHC Class II alpha and beta chains did not react. Erythrocytes from a group of RA patients and a group of normal controls had moderate and low expression respectively. We suggest that MHC Class I may be induced on erythrocytes maturing in a milieu containing mediators derived from activated cells of the immune system. Aberrant tissue expression of MHC antigens may be more widespread than has been previously recognized in diseases mediated by immune mechanisms.     

Unusual expression of HLA molecules at the surface of murine cells transfected with HLA-B7 or HLA-A11 genes

The HLA-B7 and HLA-A11 molecules expressed on murine transfectants have been analysed by one- and two-dimensional polyacrylamide gel electrophoresis (PAGE). Two different murine cells, L and P815-HTR have been compared, because it has been previously established that P815 transfectants were much more sensitive to human cytolytic cells than L transfectants. Three kinds of HLA molecules were present on these cells: (1) normal HLA molecules with 2D-PAGE profiles identical to those of the molecules isolated from human cells; (2) HLA molecules of usual size but with more various charges than HLA molecules detected on human cells. This heterogeneity was constantly found with cells expressing HLA-B7 or -A11 antigens, both in L and in P815 transfectants, including several clones. These forms were detected by anti-HLA monoclonal antibodies and by antipeptide (from HLA-B7) antibodies; (3) other unusual products corresponding to shorter heavy chains: molecules of various mol. wts and charges were detected in HLA-B7 but not in HLA-A11 transfectants. They were observed using antipeptide sera but were not seen with anti-HLA monoclonal antibodies. These products were possibly related to the DNA used for transfection and it cannot be excluded that such abnormalities only detectable by antipeptide sera would exist in other transfectants. The functional discrepancies between P815 and L transfectants cannot be clearly explained by these biochemical results.     

Immune recognition at the maternal-fetal interface: overview.

Trophoblast antigens at the maternal-fetal interface that are capable of stimulating maternal immune responses have been studied. Candidates are blood group I and P, HLA, Fc gamma-receptors, TLX, and phospholipids. Antigens I and P on trophoblast have been implicated in pregnancy loss but incompatible i,p mothers are rare. HLA-G is expressed on cytotrophoblast; however, no evidence for HLA-G allotypy or maternal responses to these molecules exists, although HLA-G has been implicated in recruitment of suppressor T cells. Receptors for IgG (Fc gamma-RI, Fc gamma-RII and Fc gamma-III) are present on trophoblast but allotypy is limited to the NA1-NA2 antigen system associated with Fc gamma-RIII on neutrophils. Maternal Fc-gamma R blocking antibodies have been linked to pregnancy success. The TLX alloantigen system was described by using xenogeneic antisera. Idiotype-antiidiotype regulated maternal responses to TLX are proposed as necessary for successful pregnancy. Several putative TLX monoclonal antibodies (Mab) recognize a regulator of complement activation called MCP (membrane cofactor protein, or CD46). Mab to MCP do not exhibit allotypy. Syncytial and cytotrophoblastic membranes are rich sources of MCP. Preliminary data suggest that a conformational site induced by C3b (iC3) binding to MCP may be responsible for TLX allotypy. Certain pregnancy loss patients produce antiphospholipid antibodies (aPA). Some investigators believe that aPA recognize a plasma protein cofactor, beta 2 GPI and not phospholipid per se. We produced three Mab specific for beta 2 GPI, one of which fails to recognize beta 2 GPI bound to phospholipid [corrected].(ABSTRACT TRUNCATED AT 250 WORDS)     

Evidence for a novel HLA antigen found on human extravillous trophoblast and a choriocarcinoma cell line

We describe the characterization of a novel HLA Class I molecule, which we have isolated from chorionic cytotrophoblast cell membranes, and from a trophoblast-derived choriocarcinoma cell line, BeWo; classical HLA Class I antigens are not expressed on these cells. This antigen is an electrophoretically non-polymorphic glycoprotein of approximately 40,000 molecular weight, which is found in association with beta 2 microglobulin, and which is detected by monoclonal antibodies recognizing monomorphic determinants of HLA Class I. Elucidation of the nature and origin of this molecule may provide valuable information regarding the immune barrier that exists between mother and fetus.     

Specific lysis of murine cells expressing HLA molecules by allospecific human and murine H-2-restricted anti-HLA T killer lymphocytes

The lysis by human and murine anti-HLA cytolytic T lymphocytes (CTL) of murine cells expressing class I HLA molecule after gene transfection has been studied using two different murine cells: LMTK- and P815-HTR-TK-. Weak but significant HLA-A11-specific lysis was found occasionally with human CTL on the HLA-A11+ L cells. On the contrary, P815-A11 or P815-A2 cells were lysed strongly and specifically by HLA-A11 or HLA-A2-specific human CTL. The T8+T4- phenotype of the effector cells was confirmed and the reaction was inhibited by anti-HLA class I monoclonal antibodies. Despite their higher sensitivity to human CTL, the P815-HLA+ cells did not express higher levels of HLA antigens than L cells, and the presence or the absence of human beta 2 microglobulin was irrelevant. Anti-human LFA-1 antibodies abrogated the lysis of P815-A11+ cells showing that the LFA-1 receptor which is apparently lacking on the L cell surface was on the contrary expressed on P815 cells. On the other hand, murine anti-HLA CTL have been prepared by immunizing mice against syngeneic HLA-A11+ L cells. They lysed very efficiently and specifically these cells, but appeared completely devoid of activity against human HLA-A11 target cells. This barrier was apparently due to the H-2 restriction of these H-2k anti-HLA murine CTL, as shown by their inability to lyse allogeneic H-2d cells expressing HLA-A11, and by the blocking of their activity by anti H-2k antibodies. By contrast, xenogeneic anti-HLA CTL obtained by immunizing murine lymphocytes against human cells lysed both human and murine HLA+ cells but they reacted with a monomorphic epitope of the HLA molecule in a nonrestricted way. These results show that human cells lyse very efficiently P815 murine cells expressing HLA class I antigens; the higher sensitivity of P815 cells compared to L cells is probably due to the presence of a LFA-1 receptor on these cells; a class I molecule of human origin can be seen as an H-2-restricted minor histocompatibility antigen in another species.     

Equine T lymphocytes express MHC class II antigens

Six anti-HLA class II mouse monoclonal antibodies (mAbs) were used in conjunction with a rat monoclonal antibody raised against horse lymphocytes to define class II major histocompatibility complex (MHC) molecules in the horse. By utilizing an ELISA assay and complement dependent lymphocytotoxicity assay, five out of the six anti-HLA class II antibodies and the rat anti-horse monoclonal antibody were found to react with a high percentage of peripheral blood lymphocytes. Flow cytometry demonstrated a variable antigen density on peripheral blood lymphocytes and clear evidence for expression by lymphocytes that carried no detectable surface immunoglobulin. None of the antibodies reacted with equine platelets. The mAbs immunoprecipitated an antigenic complex of Mr 29,000-33,000 from horse lymphocytes. It appears that the distribution of MHC class II antigens in the horse is different from that in man but is similar to that in the dog, since MHC class II antigens are expressed on resting peripheral blood lymphocytes which lack membrane-bound immunoglobulins. Correlations between the distribution of MHC class II antigens on lymphocyte subpopulations and their role in immunological phenomena may contribute to our understanding of the functional properties of these molecules.     

Localization in Human Term Placental Bed and Amniochorion of Cells Bearing Trophoblast Antigens Identified by Monoclonal Antibodies*

ABSTRACT: The monoclonal antibodies (mAb) H315, H317, and OKT9 have been used in immunofluorescence to investigate the expression of fetal trophoblast membrane antigens by cells within human term amniochorionic membranes and the marginal area of term placental bed tissue. OKT9 reacted only with trophoblast of placental chorionic villi and did not react with any nonvillous cytotrophoblast population: this mAb is known to identify the cell surface receptor for transferrin. H315 identifies a trophoblast-specific cell-surface antigen and strongly stained both placental villous trophoblast and the cytotrophoblastic layer of amniochorion. This mAb also stained some extravillous cytotrophoblast in the term placental bed, notable interstitial cytotrophoblast within maternal decidua. H317, which identifies placental-type alkaline phosphatase, gave the same distribution pattern as H315.     

Expression of TAP1 by human trophoblast

Successful placentation in the human is dependent on the trophoblast evading recognition and destruction by the maternal immune system. However, invasive cytotrophoblast express HLA-G which may be able to present peptide to T cells. Transporter proteins are essential for peptide presentation and major histocompatibility complex (MHC) class I assembly. We have determined their expression by trophoblast in relation to HLA-G, using immunohistochemistry. Antitransporter protein antibody (TAP1) labeling closely paralleled that of MHC class I, but the intensity of its expression was much greater on the HLA-G⁺ extravillous cytotrophoblast than any other fetal or maternal tissue in the first trimester and at term. This suggests that the extravillous cytotrophoblast are very actively assembling MHC class I antigens with peptides. However, expression of MHC class I by the cytotrophoblast was not correspondingly elevated. This pattern could result from HLA-G being shed from the surface of the trophoblast, a process which may play a central role in protecting the fetus from maternal immune attack.     

Major histocompatibility complex restriction of tetanus toxoid-specific human T lymphocyte clones

Peripheral blood mononuclear cells (PBMC) from an HLA DRw6/7 individual were stimulated with tetanus toxoid (TT). T cell blasts were cloned by the limiting dilution technique in the presence of TT and irradiated autologous PBMC (iPBMC). Twelve were propagated under interleukin 2 and restimulated weekly with TT and iPBMC. All proliferated specifically in response to TT or either the alpha or beta chain of the toxin molecule. HLA restriction of specific proliferative responses was analyzed using a panel of HLA-typed unrelated donors and selected families, and blocking experiments with anti-HLA class I and class II monoclonal antibodies (mAb). Three types of restriction were observed: (a) autologous restriction; the inhibition observed using anti-HLA DR mAb as well as family studies performed previously with a similarly restricted clone obtained from the same donor suggest an HLA DRw6-linked restriction; (b) an HLA DR7 restriction was found for 2 clones, specific for alpha or beta chain; the identical pattern of inhibition obtained with two different mAb belonging to the same cluster suggests that these clones may be restricted by the same (or a very close) epitope of the HLA DR7 molecule; (c) an unusual restriction pattern was found for one clone; PBMC from more than 80% of donors could present TT whatever their degree of HLA compatibility with the autologous donor. Family studies were unable to disclose any restriction with known class II (or class I) antigens. While no inhibition was observed with anti-DR or -DC reagents, a mAb that recognizes class I antigen when associated with beta 2-microglobulin did inhibit the proliferation of this clone.     

Distribution of Human Leukocyte Antigen-ABC and -D/DR Antigens in the Unfixed Human Testis

ABSTRACT: The distribution of the major histocompatibility complex (MHC) antigens in the unfixed human testicle was studied by indirect immunofluorescence. Three murine monoclonal antibodies to the common determinants of class I MHC antigens (human leukocyte antigen [HLA]-ABC) and three against class II MHC antigens (HLA-D/DR antigens), respectively, were utilized. No class I MHC antigens were identified on developing testicular germ cells including spermatozoa, but interstitial cells between the seminiferous tubules (including Leydig cells) and blood vessel endothelium expressed the antigen. Class II MHC antigens were not found on any cells within the seminiferous tubules. However, the class II antigen was identified on dendritic-like cells between the seminiferous tubules and on vessel endothelium, although its expression was expectedly limited. These findings indicate that human testicular germ cells express minimal or no MHC antigens.     

Expression of normal and tumor-associated antigens in human breast carcinoma

The expression of six cytoplasmic/membrane antigens (beta 2-microglobulin, HLA, HLA-DR, carcinoembryonic antigen, and two breast tumor-associated antigens (TAAs), B6.2 and B72.3) was investigated in serial sections of 28 human breast carcinomas using monoclonal antibodies and the avidin-biotin complex immunoperoxidase technique. The frequency of expression and linkage between these antigens was determined, and antigenic expression was related to patient age, morphologic differentiation, cytologic grade, and estrogen receptor/progesterone receptor content of the tumor. The expression of beta 2-microglobulin and HLA correlated with morphologic differentiation, well-differentiated and moderately well-differentiated tumors expressing these antigens more often than poorly differentiated tumors. Expression of the TAAs, however, was not related to differentiation. There was no linkage between beta 2-microglobulin/HLA and the TAAs. Carcinoembryonic antigen was found to be linked to the TAA, B6.2. Expression of the TAA, B72.3, correlated with patient age. Eighty percent (23 of 28) of the tumors were positive for carcinoembryonic antigen or at least one of the TAAs. The estrogen receptor/progesterone receptor status of the tumor was not statistically related to the expression of any of the antigens studied. Analysis of tumor antigen profiles may provide important information relevant to prognosis, therapy, and early detection of cancer, as well as insights into the nature of the neoplastic process.