Regulatory role of a monomorphic determinant of HLA Class I antigens in T cell proliferation

The role of HLA Class I antigens in T cell proliferation was investigated by using the anti-HLA Class I monoclonal antibodies (MoAb) CR10-215, CR10-325, and CR11-115. MoAb CR10-215 and CR11-115 recognize the same (or spatially close) monomorphic determinant, which is distinct and spatially distant from that reacting with MoAb CR10-325. Addition of MoAb CR10-215 and CR11-115 to cultures of peripheral blood mononuclear cells stimulated with MoAb OKT3, MoAb Pan T2, PHA, or PPD inhibited cell proliferation. The blocking is specific in that the anti-HLA Class I MoAb CR10-325 and the Pan T MoAb Pan T1 had no effect on the proliferation. The inhibitory activity of MoAb CR10-215 and CR11-115 does not reflect i) toxic effects, ii) induction of suppressor cells and factors, iii) blocking of the binding of mitogens to lymphocytes, iv) inhibition of the production of interleukin 1 (IL 1) and interleukin 2 (IL 2), or v) function of IL 2 receptor. Anti-HLA Class I MoAb were able to inhibit the proliferation of purified, Tac-, T cells. The inhibited cells did not express Tac antigen, as assayed by direct immunofluorescence, with MoAb anti-Tac, but released a normal amount of IL 2 in culture medium. These results indicate that monomorphic determinants of the HLA Class I complex are involved in the regulation of T cell proliferation. The effect appears to occur at the level of IL 2 receptor expression.     

Syngeneic anti-idiotypic antisera to murine monoclonal antibodies to monomorphic and polymorphic determinants of HLA class I antigens

BALB/c mice were immunized with syngeneic anti-HLA class I monoclonal antibodies. The latter included the anti-HLA-A2, A28 monoclonal antibody (MoAb) CR11-351, the MoAb Q6/64 to a determinant restricted to HLA-B antigens and the MoAb CR10-215 and CR11-115 to the same (or spatially close) monomorphic determinant. Anti-idiotypic antibodies could be detected in bleedings obtained 3 days after the first booster, increased in titer in bleedings obtained after the second booster, and persisted at high levels in subsequent bleedings. The four anti-HLA class I MoAb did not differ in their ability to elicit syngeneic anti-idiotypic antibodies. Cross-blocking studies with a panel of anti-HLA class I, anti-HLA class II, and anti-human melanoma-associated antigen (MAA) MoAb showed that the anti-MoAb CR10-215 and anti-MoAb CR11-115 antisera contain only antibodies to private idiotopes, whereas the anti-HLA MoAb CR11-351 and anti-MoAb Q6/64 antisera also contain antibodies to public idiotopes. The latter are expressed by the anti-HLA class I MoAb CR11-351, Q1/28, Q6/64, and 6/31, and by the anti-HLA class II MoAb Q5/6, Q5/13, 127, and 441. Public idiotopes were not detected on the nine anti-MAA MoAb tested. Public idiotopes do not interfere with the binding of anti-HLA MoAb with the corresponding antigenic determinants. On the other hand private idiotopes are located within the antigen-combining site, because anti-idiotypic antisera specifically inhibit the binding of the corresponding immunizing anti-HLA class I MoAb to cultured human lymphoid cells in a dose-dependent manner. Analysis by isoelectric focusing of the anti-HLA class I MoAb antisera showed that the spectrotype of the anti-MoAb CR11-351 antiserum comprises four components that focus in the pH 6.9 to 6.2 range, the spectrotype of anti-MoAb Q6/64 antiserum comprises three components that focus in the pH 6.5 to 6.1 range, the spectrotype of the anti-MoAb CR10-215 antiserum comprises three components that focus in the pH 6.4 to 6.1 range, and the spectrotype of the anti-MoAb CR11-115 antiserum comprises three components that focus in the pH 6.6 to 6.4 range.     

Role of HLA class I and class II antigens in activation and differentiation of B cells

The monoclonal antibodies (MoAb) CR10-214, CR11-115, and Q1/28 to distinct monomorphic determinants of HLA class I antigens, the MoAb CL413 and PTF29.12 recognizing monomorphic determinants of HLA-DR antigens, the anti-HLA-DQw1 MoAb KS11, the anti-HLA-DPw1 MoAb B7/21, and the anti-HLA-DR,DP MoAb CR11-462 were tested for their ability to modulate human B-lymphocyte proliferation and maturation to IgM-forming cells. Purified tonsillar B cells were stimulated with Staphylococcus aureus bacteria of the Cowan first strain (SAC) or anti-human mu-chain xenoantibodies, as well as in growth factor- or T-cell-dependent activation cultures. The B-cell proliferative responses induced by SAC or by mitogenic concentrations of anti-mu-chain xenoantibodies were inhibited by some of the anti-HLA class I and anti-HLA class II monoclonal antibodies tested. The same antibodies were effective inhibitors of the proliferation of B cells stimulated with interferon-gamma (IFN-gamma) or interleukin-2 (IL-2) and with submitogenic concentrations of anti-mu-chain xenoantibodies. The proliferation induced by IL-2 of SAC-preactivated B cells was inhibited by some of the anti-HLA class II monoclonal antibodies, but not by the anti-HLA class I monoclonal antibodies tested. This inhibition appeared to reflect at least in part a direct effect on later events of the B-cell activation cascade, since some anti-HLA class II monoclonal antibodies still exerted considerable inhibitory activity when added together with IL-2 to SAC-preactivated B cells after the third day of culture. Anti HLA-DR, DQ, and DP monoclonal antibodies consistently inhibited the IgM production induced in B cells by T cells alone, T cells plus pokeweed mitogen (PWM), SAC plus IL-2, or IL-2 alone. In contrast, two of the three anti-HLA class I monoclonal antibodies tested inhibited the IgM production in cultures stimulated with SAC plus IL-2 and one the IgM production induced by IL-2 alone, but none of them had inhibitory effects on T-cell dependent IgM production. The results reported herein indicate that HLA class II molecules directly participate in different phases of the B-cell activation cascade. In addition, our data also suggest that HLA class I molecules can be involved in the events leading to B-cell proliferation and differentiation into immunoglobulin-secreting cells.     

Lack of a role of monocytes in the inhibition by monoclonal antibodies to monomorphic and polymorphic determinants of HLA class I antigens of PHA-P-induced peripheral blood mononuclear cell proliferation

This study aimed at characterizing the mechanism(s) underlying the regulatory role of distinct determinants of HLA Class I antigens in PHA-P-induced T cell proliferation and the involvement of monocytes in this phenomenon. The anti-HLA-A2,A28 monoclonal antibodies (MoAb) CR11-351, the MoAb Q6/64 to a determinant restricted to the gene products of the I antigens HLA-B locus, and the MoAb CR10-215 and W6/32 to distinct monomorphic determinants of HLA Class I antigens were found to inhibit PHA-P-induced peripheral blood mononuclear cell (PBMC) proliferation in a dose-dependent fashion. The inhibition is specific and reflects neither inhibition of PHA-P binding to cells nor a toxic effect of the anti-HLA Class I MoAb. The latter differed in the concentration required to induce inhibition, in the influence of the concentration of PHA-P used as mitogen, in the differential effect on the donors used as a source of PBMC, and/or in the requirement of the Fc portion to induce inhibition. At variance with the information in the literature, the inhibitory effect of anti-HLA Class I MoAb on PHA-P-induced PBMC proliferation neither reflected their interaction with accessory cells nor was mediated by suppressor factors released by monocytes stimulated with PHA-P in the presence of anti-HLA Class I MoAb. Therefore, the regulatory role of HLA Class I antigens in T cell proliferation is not likely to be mediated by monocytes and/or factors released from them, but may reflect an involvement of these molecules in T cell activation pathways.     

Syngeneic anti-idiotypic antisera to murine anti-HLA class II monoclonal antibodies

BALB/c mice have been immunized with six anti-HLA Class II monoclonal antibodies (MoAb); the latter included MoAb CR11-462, Q5/6, and Q5/13 to monomorphic determinants, the anti-HLA-DR1,4,w6,w8,w9 MoAb AC1.59, the anti-HLA-DRw9 MoAb AB7ae9, and the anti-HLA-DQw3 MoAb AC6G. The six monoclonal antibodies markedly differ in their ability to induce anti-idiotypic antibodies, because the latter were not detected in the sera from the mice immunized with the MoAb AB7ae9 and with the MoAb AC6G. The MoAb AC1.59 and CR11-462 elicited antibodies to private idiotypes, and the MoAb Q5/6 and Q5/13 elicited antibodies to private and public idiotypes. The titer of the latter in the anti-MoAb Q5/6 antiserum appears to be markedly lower than that of the former ones; no marked difference was detected in the titer of the two types of antibodies in the anti-MoAb Q5/13 antiserum. Blocking experiments with a panel of monoclonal antibodies showed that the MoAb Q5/13 shares idiotypes with the anti-HLA Class I MoAb Q5/6, 127, and 441 and with the anti-HLA Class I MoAb CR11-351, Q1/28, Q6/64, and 6/31, but does not share idiotypes with any of the nine anti-human melanoma-associated antigen MoAb tested. The spectrotypes of the anti-MoAb CR11-462 and anti-MoAb Q5/13 antisera comprise two major components in the pH 6.2 to 6.7 range, that of the anti-MoAb Q5/6 antiserum comprises two major components in the pH 6.5 to 6.8 range, and that of the anti-MoAb AC1.59 antiserum comprises a number of components in the pH 5.6 to 7.2 range.     

Reactivity patterns with HLA-A2 variants indicate lack of identity between determinants defined by monoclonal antibodies and cytotoxic-T-cell clones

The anti-HLA-A2 monoclonal antibodies (MoAb) CR11-351 and 4B inhibit the binding of each other to HLA-A2 lymphoid cells and block the cytotoxicity of the anti-HLA-A2 cytotoxic-T-cell clone R32. The blocking does not reflect reactivity of the MoAb CR11-351 and 4B and of the cytotoxic-T-cell clone R32 with the same determinant, since they display differential reactivity with four HLA-A2 variants which carry amino acid substitutions at different positions. These results show for the first time in the human system that Class I HLA variants represent useful reagents to compare the fine specificities of monoclonal antibodies and T-cell clones. Furthermore our data suggest that T-cell recognition depends upon the tertiary structure of the antigen.     

Monoclonal antibody OKT3-induced T cell proliferation: differential role of HLA class II determinants expressed by T cells and monocytes.

Monoclonal antibodies (MAb) to monomorphic determinants of HLA Class II antigens inhibit monocyte-dependent T cell proliferation induced by MAb OKT3 to a different extent, suggesting a differential regulatory role of the corresponding determinants in T cell proliferation. To elucidate the mechanism(s) underlying this pattern, the MAb CR10-343 and Q5/6 with high inhibitory effect and MAb CR11-462 and CR12-356 with low inhibitory effect were characterized. Cross-inhibition studies showed that the four MAb recognize distinct determinants. The determinants recognized by MAb CR10-343 and CR12-462 are spatially close. The determinants recognized by the four MAb appear to be functionally independent in MAb OKT3-induced T cell proliferation, since the inhibitory effect of the combination of MAb CR10-343 and Q5/6 and of the MAb CR11-462 and CR12-356 was additional but not synergistic. To compare the functional activity of HLA Class II determinants expressed by monocytes and by activated T cells in MAb OKT3-induced T cell proliferation, the effect of the four MAb on MAb OKT3-induced T cell proliferation in a monocyte-dependent and in a monocyte-free system was studied. Dose-response and proliferation kinetics studies showed that the four MAb display a similar inhibitory effect on MAb OKT3-induced T cell proliferation in a monocyte-free system. These results suggest fine differences in the role played by monocyte- and T cell-bound HLA Class II determinants in the regulation of MAb OKT3-induced T cell proliferation. This functional heterogeneity may enhance the flexibility of HLA Class II antigens to mediate cell-cell interactions involved in the proliferative response to a variety of mitogenic stimuli.     

Differential regulatory role of monomorphic and polymorphic determinants of histocompatibility leukocyte antigen class I antigens in monoclonal antibody OKT3-induced T cell proliferation

Monoclonal antibodies (mAb) to monomorphic and polymorphic determinants on the heavy chain of histocompatibility leukocyte antigen (HLA) class I antigens inhibit mAb OKT3-induced T cell proliferation, whereas the anti-beta 2-microglobulin mAb NAMB-1 does not affect it. The inhibitory effect of anti-HLA class I mAb is specific, is not an Fc-mediated phenomenon, does not require accessory cells, and does not involve early stages of T cell activation. Distinct determinants of HLA class I antigens regulate T cell proliferation by different mechanisms, because the anti-HLA-A2, A28 mAb CR11-351, and the mAb W6/32 to a framework determinant of HLA class I antigens block interleukin 2 (IL-2) secretion and IL-2 receptor expression, whereas the mAb CR10-215 to a monomorphic determinant blocks only IL-2 receptor expression. The mAb CR10-215 and W6/32 induced a 50% of maximal inhibition of T cell proliferation, when added after 27 and 12 hr, respectively, of incubation of peripheral blood mononuclear cells with mAb OKT3. On the other hand, the mAb CR11-351 inhibited T cell proliferation even when added after 38 hr of incubation of peripheral blood mononuclear cells with mAb OKT3 and was the only one to inhibit proliferation of cycling T lymphocytes. It is suggested that HLA class I antigens regulate T cell proliferation by interacting with cell-surface molecules involved in T cell activation. The differential inhibitory activity of the anti-HLA class I monoclonal antibodies tested may reflect the different ability of the corresponding determinants to interact with activation molecules.     

Enhancing effect of anti-HLA class I monoclonal antibodies on T cell proliferation induced via CD2 molecule

The mAb 131 to a determinant preferentially expressed on the gene products of the HLA-A locus, the mAb Q6/64 and 4E to determinants preferentially expressed on the gene products of the HLA-B locus, the anti-HLA-A2,A28 mAb CR11-351, HO-2, HO-3, HO-4, and KS1, and the anti-HLA-B7 cross-reacting group mAb KS4 enhanced proliferation of T cells in most, if not all, the PBMC preparations stimulated with the anti-CD2 mAb 9-1 + 9.6. The mAb CR10-215, W6/32, and 6/31 to distinct monomorphic determinants of HLA class I antigens enhanced CD2-induced T cell proliferation in at most 30% of the PBMC preparations tested. The anti human beta 2-microglobulin (beta 2-mu) mAb NAMB-1 displayed no detectable effect on the proliferation of T cells stimulated with the mAb 9-1 + 9.6. The enhancing effect of anti-HLA class I mAb is specific, is dose dependent, is not abrogated by the addition of exogenous IL-1 and IL-2 to the cultures, and reflects the interaction of anti-HLA class I mAb with T cells. Enhancement of CD2 mediated proliferation of T cells is not a unique property of anti-HLA class I mAb, since the anti-HLA class II mAb Q5/6 and Q5/13 also had a similar effect. Analysis of the kinetics of the enhancing effect of anti-HLA class I mAb suggests that they modulate an early event of T cell activation and may affect the interaction of T cells with mAb 9-1. Phenotyping of T lymphocytes activated by mAb 9-1 + 9.6 in the presence of anti-HLA class I mAb suggests that the enhancing effect of anti-HLA class I mAb may reflect the recruitment of a higher percentage of T cells. The present study has shown for the first time that certain, but not all, the determinants of the HLA class I molecular complex are involved in the proliferation of T cells stimulated with the anti-CD2 mAb 9-1 + 9.6. Furthermore, the inhibitory effect of mAb CR11-351, KS1, Q6/64, and W6/32 on the proliferation of T cells stimulated with mAb OKT3 or with mAb BMA 031 indicates that the same determinants of HLA class I antigens play a differential regulatory role in T cell proliferation induced via the CD2 and CD3 pathway.     

Regulatory role of monomorphic and polymorphic determinants of HLA class I antigens in the proliferation of T cells stimulated by autologous and allogeneic B and T lymphoid cells

Monoclonal antibodies (mAb's) to monomorphic and polymorphic determinants of HLA Class I antigens were shown to inhibit proliferation of T cells stimulated with autologous and allogeneic B and T lymphocytes. Inhibition of proliferative responses was lower when T cells were used as stimulators than when B cells were used. The inhibitory activity was similar for mAb's to monomorphic and polymorphic determinants of HLA Class I antigens, suggesting that the density of antigen-antibody complexes on the cell membrane does not play a major role in the phenomenon. The anti-HLA Class I mAb's exerted their inhibitory effect at the level of both the responding and the stimulating cells. Addition of exogenous interleukin 2 to the mixed cultures did not affect the mAb-mediated inhibition.     

Effect of monoclonal antibodies (MoAb) to class I and class II HLA antigens on lectin- and MoAb OKT3-induced lymphocyte proliferation

We have examined the effect of several monoclonal antibodies (MoAb) to monomorphic determinants of class II HLA antigens, and MoAb to monomorphic determinants of class I HLA antigens and to beta-2-microglobulin (beta 2-mu) on lectin- and MoAb OKT3-induced proliferation of human peripheral blood mononuclear cells (PBMNC) and cultured T cells (CTC). Some, but not all, anti-class II HLA MoAb inhibited the proliferative response of PBMNC to MoAb OKT3 and pokeweed mitogen (PWM). The degree of inhibitory effect varied considerably. This effect was not limited to anti-class II HLA MoAb since anti-class I HLA MoAb and anti-beta 2-mu MoAb also inhibited MoAb OKT3- or PWM-induced proliferative responses. In contrast, the response of PBMNC to phytohemagglutinin (PHA) and concanavalin A (Con A) was not blocked by any anti-class II HLA MoAb. However, some anti-class II HLA MoAb also inhibited the proliferative response of CTC plus allogeneic peripheral blood adherent accessory cells (AC) to PHA or Con A as well as to MoAb OKT3 or PWM. This may be attributable to the substantially greater class II HLA antigen expression by CTC than by fresh lymphocytes. Pretreatment of either CTC or AC with anti-class II HLA MoAb inhibited OKT3-induced proliferation. In contrast, pretreatment of CTC, but not AC, with anti-class I HLA MoAb inhibited the proliferative response of CTC to OKT3. Pretreatment of CTC with anti-class I HLA MoAb inhibited PHA-, Con A and PWM-induced proliferation, to a greater degree than the anti-class II HLA MoAb. It appears as if lymphocyte activation by different mitogens exhibits variable requirements for the presence of cells expressing major histocompatibility determinants. Binding of Ab to membrane markers may interfere with lymphocyte-AC cooperation, perhaps by inhibiting binding of mitogens to their receptors or by interfering with lymphocyte and AC function. We also have examined the role of class II HLA antigens on CTC by depleting class II HLA-positive cells. As expected, elimination of class II HLA-positive AC with anti-class II HLA MoAb plus complement caused a decrease in proliferation of CTC in response to all the mitogens tested. In contrast, elimination of class II HLA-positive CTC was shown to clearly increase proliferation of CTC, perhaps because this may deplete class II HLA-positive suppressor cells.     

The monoclonal antibody CR11-351 discriminates HLA-A2 variants identified by T cells

Serologic and immunochemical assays have shown that the monoclonal antibody (MoAb) CR11-351 recognizes a determinant expressed by HLA-A2 and A28 alloantigens. The MoAb CR11-351 blocks the cytotoxicity of some, but not all, anti-HLA-A2 and anti-HLA-A28 alloantisera tested. These findings suggest that each allospecificity consists of several determinants, only some of which are spatially close to the determinant defined by the MoAb CR11-351. The binding of the MoAb CR11-351 to HLA-A2 lymphoid cells is not effected by their precoating with the HLA-A, B-specific MoAb CR10-214, Q6/64, and 6/31 but is enhanced by at least 20% by the MoAb CR10-131, CR10-402 and by the beta 2-m-specific MoAb NAMB-1. The MoAb CR11-351 did not react with one of four HLA-A2 variants which are indistinguishable with conventional anti-HLA-A2 sera, but are not recognized by "normal" HLA-A2-restricted cytotoxic T cells and possess structurally distinct HLA-A2 heavy chains. Therefore the MoAb CR11-351 provides the first evidence of a serologically detectable difference between the four HLA-A2 variants and "normal" HLA-A2 antigens.     

Accessory cell function of human melanoma cells in mitogen-induced T cell proliferation

Six out of eight human melanoma cell lines were found to be able to function as accessory cells in PHA-induced proliferation of autologous and allogeneic T cells. The accessory cell function of the melanoma cell lines appears to be similar to that of monocytes, requires the presence of viable cells, and does not correlate with the cell surface binding sites for PHA and with the level of expression of HMW-MAA and of HLA Class I antigens. HLA Class II antigens do not appear to play a major role in these phenomena, since there is no relationship between level of expression of HLA Class II antigens and accessory cell function of melanoma cells. Furthermore, addition of anti-HLA Class II monoclonal antibodies does not affect proliferation of T cells stimulated with PHA in the presence of melanoma cells with accessory cell function. Although melanoma cells exert accessory cell function, functional and immunological assays did not detect IL-1 in the spent medium of the melanoma cell lines. Furthermore, Northern blotting analysis with IL-1 alpha and IL-1 beta probes did not detect IL-1-specific mRNA in melanoma cell lines. These results suggest that PHA-induced proliferation of T cells in the presence of melanoma cells can bypass the requirement for IL-1 or utilizes factors other than IL-1.     

Anchored anti-HLA class I monoclonal antibody fails to induce inhibition of PHA-activated lymphocytes proliferation.

It is known that anti-HLA Class I antibodies inhibit the proliferative response of PHA-activated T-lymphocytes. We found that plastic- or sepharose-linked anti-HLA Class I monoclonal antibody 01.65 does not inhibit either [3H]Thymidine incorporation or recruitment in the cell cycle, nor does it reduce the expression of c-myc mRNA and the membrane expression of Interleukin-2 Receptor and Transferrin Receptor. Furthermore, particulate Protein Kinase C is not affected by anchored anti-HLA Class I monoclonal antibody 01.65. We suggest that anti-HLA Class I monoclonal antibody may act through crosslinking or internalization of HLA Class I antigens.     

Differential expression of HLA-DR, -DQ, and -DP antigens on malignant B cells

HLA class II antigens mediate interactions among cells involved in the immune response and play an important role in the process of self recognition. We made use of conventional alloantisera and six well-characterized monoclonal antibodies (MoAb) to study the HLA class II antigens on CALLA-positive malignant B cell populations and autologous normal B cell lines. Forty additional HLA class II-specific MoAb were also tested for their ability to bind to these cells. By using indirect immunofluorescence and immune precipitation assays, we find that malignant B cells often fail to express one or more of the three known types of HLA class II antigens. Cell lines with the following five phenotypes have been identified: HLA-DR+, -DQ+, -DP+; HLA-DR+, -DQ-, -DP+; HLA-DR-, -DQ+, -DP+; HLA-DR-, -DQ-, -DP+; and HLA-DR-, -DQ-, -DP-. These cell lines have been used to characterize the subregion specificity of MoAb that react with HLA class II antigens. This work confirms the existence of complicated patterns of serologic cross-reactivity between the three different types of HLA class II molecules. It also increases our understanding of the specificity of individual MoAb, thereby facilitating future investigation of the distribution and function of individual antigens. Our studies are consistent with the proposal that altered expression of HLA antigens on tumors might impair recognition of these cells by the immune system of the host, thereby contributing to the proliferation of a malignant clone.     

Structural and functional analysis of monomorphic determinants recognized by monoclonal antibodies reacting with the HLA class I alpha 3 domain.

To identify mAb reacting with the HLA class I alpha 3 domain, 14 mAb recognizing monomorphic determinants expressed on HLA-A, B, and C Ag or restricted to HLA-B Ag were screened in indirect immunofluorescence with mouse L cells expressing HLA-B7/H-2Kb chimeric Ag. mAb CR1S63, CR10-215, CR11-115, and W6/32 were found to react with the HLA class I alpha 3 domain in addition to the alpha 2 domain. mAb Q1/28 and TP25.99 were found to react only with the HLA class I alpha 3 domain. The determinants recognized by the six mAb were mapped on the HLA class I alpha 3 domain by indirect immunofluorescence staining of L cells expressing H-2Kb Ag containing different segments of the HLA-B7 alpha 3 domain chimerized with the H-2Kb alpha 3 domain. mAb TP25.99 reacts with chimeric Ag containing the HLA-B7 184 to 199 stretch, mAb CR10-215 and CR11-115 react with chimeric Ag containing the HLA-B7 184 to 246 stretch, mAb CR1S63 and Q1/28 react with chimeric Ag containing the HLA-B7 184 to 256 stretch, and mAb W6/32 reacts with chimeric Ag containing the whole HLA-B7 alpha 3 domain. Functional analysis using human CD8 alpha-bearing mouse H-2Kb-specific T cell hybridoma cells (HTB-Leu2) showed that only mAb TP25.99 inhibited IL-2 production by HTB-Leu2 cells stimulated with L cells expressing KbKbB7 Ag. This inhibition may occur because of the spatial proximity of the determinant defined by mAb TP25.99 to the CD8 alpha binding loop and/or because of change(s) in the conformation of the CD8 alpha binding loop induced by the binding of mAb TP25.99 to the HLA class I molecule. Furthermore, mAb TP25.99 inhibited the cytotoxicity of CD8-dependent and CD8-independent CTL clones. These results indicate that mAb TP25.99 has unique specificity and functional characteristics. Therefore it represents a valuable probe to characterize the role of the HLA class I alpha 3 domain in immunologic phenomena.     

Cross-linking of insulin receptors to MHC antigens in human B lymphocytes: evidence for selective molecular interactions

Molecular interactions between insulin receptors and MHC antigens were investigated in human B cells. Two B lymphoblastoid cell lines, IM-9 and 526, chosen for their high insulin binding capacity, were found to express 15,000 and 25,000 insulin receptors per cell, respectively. Insulin receptors were labeled with a 125I-photoreactive insulin analogue, and all other surface proteins by lactoperoxidase-catalyzed radioiodination. Neighbor proteins were cross-linked with a cleavable homobifunctional reagent dithio-bis-(succinimidyl propionate) (DSP) and solubilized before immunoprecipitation by anti-HLA monoclonal antibodies. Gel analysis of the precipitated proteins showed that 90% of insulin receptors precipitable by anti-insulin receptor antibodies were precipitated by anti-class I antibodies (anti-heavy chain and anti-beta 2-microglobulin) after cross-linking with 2 mM DSP. In neither IM-9- nor 526 cells could HLA antigens be precipitated by anti-insulin receptor antibodies, suggesting that the concentration of class I antigens largely exceeds the concentration of insulin receptors at the cell surface. In 526 lymphocytes, class I MHC antigens were also found to adjoin class II antigens, since both molecules could be coprecipitated with anti-HLA A, B, C and with anti-HLA-DR antibodies after chemical cross-linking. Down-regulation of insulin receptors by chronic exposure of IM-9 cells to insulin did not affect the amount of MHC molecules present on the cell surface, and conversely, class I MHC molecules were internalized in 526 cells irrespective of the presence of insulin. These results thus show that insulin receptors and MHC antigens form multimolecular complexes in the plasma membrane of cultured human B cells. These interactions, which do not appear to influence the regulation of these proteins on the cell surface, may be involved in the mechanism of hormone signaling.     

HLA-typing analysis following allogeneic bone grafting for sinus lifting

According to the Brazilian Association of Organ Transplants, in 2015, 19,408 bone transplants were performed in Brazil, over 90% by Dental Surgeons. The surgical technique itself has a respectable number of reports regarding its clinical efficacy, as measured by long-term survival of dental implants in grafted areas. Uncertainty remains, however, as to whether fresh frozen grafts from human bone donors remain immunologically innocuous in the body of the host. Six male with no previous medical history of note, including systemic diseases, surgery or blood transfusion were selected. These patients underwent reconstructive procedures (sinus lifting) using fresh frozen human bone from a tissue bank. All patients had venous blood samples collected prior to surgery and 6 months after the procedure. Anti-HLA analysis for the detection of HLA (human leukocyte antigen) antibodies was performed using methods such as the LABScreen PRA Class I and Class II, LABScreen Single Antigen Class I and Class II, Luminex Platform. Reactive individuals to the screening tests (LABScreen PRA) were further investigated to determine the specificity of the antibodies detected (LABScreen Single Antigen) with a cutoff value of median fluorescence intensity ≥500. As a result, it was observed that two patients (33%) were positive in screening tests, one presenting with anti-HLA Class I and II sensitization and the other with anti-HLA class II. The specificity analysis showed that the patients sensitized to HLA class II presented 4 specificities, 3 of which immunologically relevant. In the second individual, 23 specificities were identified, 6 of which immunologically important for HLA class I and 4 specificities for HLA class II, 3 of these were immunologically important. All specificities detected had average fluorescence. These findings are suggestive that sinus-lifting procedures with allogeneic bone can induce immunological sensitization.     

The incidence and clinical relevance of anti-HLA and/or MICA antibodies in patients with long-term survival renal allo-grafts

The purpose of this study was to analyze the incidence and clinical relevance of anti-HLA and/or MICA antibodies in renal allo-grafts transplant recipients with long-term renal survival (> 5 years). This retrospective study collected post-transplant serum samples from a total of 110 patients which were used to detect the incidence of anti-HLA and/or MICA antibodies as well as anti-HLA donor specific antibodies. Among these 110 patients, 72 patients had antibodies against HLA and/or MICA at the time of test, 61 had only anti-HLA antibodies, 31 had anti-MICA antibodies, and 38 were antibody negative. There was no difference in the number of patients developing antibodies against non-donor specific antibodies, donor specific antibodies, Class I donor specific antibodies, Class II donor specific antibodies or MICA antibodies between normal function group (serum creatinine level < 2.0 mg/dL) and dysfunction group (serum creatinine level > 2.0 mg/dL). For the serum creatinine level, estimated glomerular filtration rate and blood urea nitrogen level, patients with different antibodies were not statistically different to antibody-negative patients. Cox regression analysis showed that the type of transplantation and HLA mismatch number were significant negative risk factors for the development of anti-HLA (P < 0.05). Our results suggested that the anti-HLA antibody status has little impact on the renal graft function in the long-term survival allo-graft renal recipients.     

Antigenic profile and functional characterization of human peritoneal macrophages

The antigenic profile and the functional properties of human peritoneal macrophages have been analyzed by using a panel of monoclonal antibodies (MoAb) and functional assays. All peritoneal macrophages were stained by the anti-class I HLA MoAb Q6/64. Between 40 and 100% of the cells were stained by the anti-HLA-DR + DP MoAb Q2/80, Q5/6, and Q5/13; approximately 80% of the cells were stained by the anti-HLA-DQ MoAb BT3.4, and about 95% were stained by the anti-macrophage MoAb OKM1. Peritoneal macrophages were not stained by the anti-dendritic cells MoAb Ki-M4 or by MoAb to T cell subsets, although all of the MoAb were reactive with the appropriate substrates. More than 60% of the cells expressed Fc receptors and C3 receptors, and displayed phagocytic activity. Peritoneal macrophages were effective in stimulating autologous and allogeneic lymphocytes and in presenting soluble antigens to T cells. These reactions were blocked by the anti-HLA-DR + DP MoAb Q5/13, but were not affected by the anti-dendritic cells MoAb Ki-M4 or by the anti-class I HLA MoAb Q6/64. These results suggest that human peritoneal macrophage preparations, without detectable contamination with dendritic cells, can induce proliferation of autologous and allogeneic T cells, and that class II HLA antigens play a significant role in these phenomena.