Demonstration of non-idiotypic variable heavy chain (VH) antigens on human peripheral blood lymphocytes

In this study we have obtained evidence for the expression of Vh-like determinants on unstimulated human T lymphocytes as well as T lymphoblasts. These determinants were detected with antisera raised against isolated VH fragments of a human IgG3 cryoglobulin (KUP). The antisera detect idiotypic, VH subgroup and HV framework determinants and behave as anti-immunoglobulin antibodies when tested against peripheral blood mononuclear cells in immunofluorescence experiments. However, sensitive radiolabelling and immunoprecipitation techniques revealed a certain reactivity against highly purified T lymphocytes. The specificity of these T-cell-reactive antibodies has not been fully established, but the results suggest that the antisera contain antibodies directed at VH fragment-specific antigens or antigens not exposed on native immunoglobulins or isolated heavy chains.     

The Still Elusive T Cell Receptor

The contention that VH constitutes a part of T-cell receptors for antigens was probed by purifying rabbit T cells and analysing these cells for non-immunoglobulin VH, i.e. VH not associated with L chain. A number of anti-VH antisera were employed for this purpose, the most important being goat antiserum, reacting with common a1 allotype determinants (allotype determinants expressed on free VH and H chain as well as on intact immunoglobulins), rat antibody against common non-allotype VH determinants (VH framework determinants expressed on VH and H chain as well as on intact immunoglobulins) and chicken antibody against unmasked non-allotype determinants (VH determinants accessible only in the absence of L chain). VH and L chain was quantified by radioimmunoassays on extracts and supernatants from unstimulated T cells as well as from T cells stimulated by concanavalin A and by allogeneic cells. Absolute depletion of Ig-containing and -producing cells was not achieved but in no case was an excess of VH over L chain observed. This indicated that all detected VH originated from cells of the B lineage. The cells were also cultured in the presence of labelled amino acids followed by analysis of detergent extracts and supernatants by immunoadsorption and sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) fluorography. Again, no evidence for T-cell VH could be found. Affinity purified anit-VH antibody was used to label viable rabbit T cells through the use of secondary fluorescence-labelled anti-immunoglobulin antibody. No VH-specific labelling of T cells could be observed. Mixed lymphocyte cultures were carried out in the presence of affinity-purified anti-VH antibodies. No inhibition of the reaction could be discerned. The failure to detect T-cell VH is in agreement with the recent finding that the VH-genome in T cells is not rearranged in a functional manner similar to that in B cells.     

Inhibition of Lymphocyte Activation by Antisera to Embryonic Antigens Shared With Human Placental Trophoblast*

ABSTRACT: In order to further examine the inhibition of lymphocyte activation reported with conventional antisera to human trophoblast membranes, we studied their effects on cells stimulated by antigen in mixed lymphocyte culture (MLC) and by the mitogen phytohemagglutinin. The results were compared with the effects of antisera known to recognize intrinsic membrane determinants on activated T cells (DR antigens and placental alkaline phosphatase) and with those of antisera to normal human serum components such as transferrin which may bind to the activated lymphocyte membrane. The results indicated that antibodies to placental alkaline phosphatase, which constitutes the predominant specificity of conventional trophoblast membrane antisera, caused inhibition both of MLC response and, to a lesser extent, of activation induced by mitogen. Similar inhibition was obtained with antisera to human DR antigens, while antisera to normal human serum and transferrin were not suppressive. These findings, together with time course studies and immunocytochemical studies of the homologous antigens, suggest that these antisera mediate their inhibitory effects in part through binding to antigens which appear on cells as they undergo activation.     

Sheep, rabbit and chicken antisera against a human VH fragment: reactivity with immunoglobulins and lymphocytes

Antisera against a human VH fragment obtained from an IgG3, VH II, kappa protein (KUP) were raised in rabbits, sheep and chicken. The three types of anti-VH antisera reacted equally well with both intact immunoglobulin molecules and isolated heavy chains. The antisera did not detect any free heavy chain specific antigens by sensitive enzyme-linked immunosorbent assay (ELISA) tests although they reacted with some antigens which were more or less hidden on intact immunoglobulin molecules but well expressed on isolated heavy chains. The antisera reacted with more than 90% of IgG, IgA and IgM present in normal pooled serum. Experiments with T cells from normal peripheral blood indicated that the sheep and rabbit anti-VH antisera reacted with a 70,000 mol.wt T-cell surface antigen.     

Effect of rabbit anti-human B-cell antigens on the response of lymphocytes stimulated by blastogenic factor.

It has been shown that specific antisera to B-cell determinants can block stimulation in the human mixed lymphocyte reaction. Therefore, it is of interest to study the effect of anti-human B-cell serum on blastogenic activities of the cell-free culture medium (CFM) derived from cultures of human blood lymphocytes. B-cell antigen was prepared from human B-cell line as a glycoprotein complex of mol. wt 27,000 and 33,000. Rabbit antisera to the B-cell antigen after absorption with human platelets or T-cell line (MOLT 4) was shown to react only against B cells but not T cells. The antisera suppressed human mixed lymphocyte reaction but did not affect the response of lymphocytes to PHA. The proliferative response of T-cell enriched population induced by blastogenic factor from lyphocyte cultures was markedly suppressed by the antisera. The inhibited reactivity irrespective of the source (autologous, allogeneic, mixed, T or B cells) of CFM. This is compatible with an effect caused by their interactions with the responding cell rather than blastogenic factor in the CFM. The results of the kinetic experiments suggest that addition of the antiserum at intervals after initiation of the culture only prevents the CFM stimulation of the responder cells that have not yet become committed to divide.     

Induction of mouse syngeneic MLR by in vivo xenogeneic immunization with HLA-DR antigens

To study in mice the effects of in vivo xenogeneic immunization with human major histocompatibility complex (MHC) class II antigens, the animals were injected with HLA-DR antigens and their proliferative responses tested in vitro. The results showed that small amounts of HLA-DR proteins, acting as nominal antigens, were not only able to prime mice for a secondary in vitro xenogeneic mixed lymphocyte reaction but also induced a syngeneic mixed lymphocyte reaction. In contrast, allogeneic or syngeneic immunization of mice with soluble MHC class II molecules failed to stimulate an autoreactive response. The syngeneic mixed lymphocyte reaction was primarily directed against syngeneic MHC class II molecules since the murine T lymphocytes reacted against MHC class II-positive dendritic spleen cells and MHC class II-transfected mouse fibroblasts. A self-reactive T-cell line induced under these experimental conditions did not react in xenogeneic and allogeneic mixed lymphocyte reactions. However, these T lymphocytes proliferated when human peripheral blood lymphocytes of various haplotypes were presented in the context of syngeneic mouse antigen presenting cells.     

Comparison with monoclonal anti-Ia antibodies of antigenic determinants inducing lymphocyte proliferation and immune interferon production in primary and secondary murine mixed lymphocyte reactions

The nature of Ia antigens inducing lymphocyte proliferation and immune interferon (IFN-γ) production in primary and secondary murine mixed lymphocyte cultures was studied by means of monoclonal anti-Ia antibodies. In primary mixed lymphocyte cultures, both lymphocyte proliferation and IFN-γ production were found to be controlled by Ia determinants coded for by the I-A subregion. E alloantigen-recognizing antibodies displayed consistent stimulation of both lymphocyte functions, instead of inhibition, suggesting a regulatory mechanism of the E molecule on lymphocyte activation. In secondary mixed lymphocyte cultures fewer Ia determinants seemed to be involved in lymphocyte proliferation, since only antibodies directed against specificities Ia.17 and Ia.19 were found capable of blocking lymphocyte proliferation. In contrast, IFN-γ production, although significantly decreased by antibodies against specificities Ia.17 and Ia.19, was not completely abrogated, even by a mixture of all anti-Ia antibodies. The data indicate that (a) in primary mixed lymphocyte reaction lymphocyte proliferation and IFN-γ production are triggered by the same Ia specificities coded by the I-A subregion and (b) in secondary mixed lymphocyte reaction, whereas lymphocyte proliferation is restricted to fewer determinants, IFN-γ production shows a lower threshold of activation, being induced also by determinants ‘silent’ in primary mixed lymphocyte reaction.     

Lack of shared lymphocyte-stimulating determinants between H-2I and HLA-D/DR using mouse primed lymphocyte typing cells

A number of anti-H-2 alloantisera containing antibody reactive with I region gene products (Ia) of the major histocompatibility complex cross-react with determinants expressed by human peripheral blood B lymphocytes. Such data have led to the conclusion that Ia and DR antigens share cross-reacting determinants. We have attempted to generate mouse primed T lymphocyte populations specific for defined I region gene product determinants which concomitantly recognize DR determinants on human peripheral blood leukocytes in primed lymphocyte typing (PLT) analysis. Mouse PLT cells were generated in primary MLC using strain combinations identical to those in which positive mouse/human cross-reacting antisera have been obtained. The resulting PLT cells exhibited strong, yet specific, secondary MLC responses against mouse cells expressing the Ia determinants used as the primary stimulus. In contrast, when examined on panels of human peripheral blood leukocytes, no reactivity was detected. This lack of cross-reactivity suggests that mouse T cells primed toward Ia determinants do not regularly recognize cross-reacting determinants of DR or D-associated antigens expressed on human PBLs. Consequently, mPLT cells are not a useful reagent in defining HLA-D region polymorphism.     

Cellular Distribution, Purification, and Molecular Nature of Human la Antigens

Human Ia antigens were extensively purified (1390-fold increase in specific activity) in 32% yield from BRI 8 cells, a lymphoblastoid B-cell line. Purification was monitored by using allogeneic antisera arising by foetal-maternal stimulation. The product, a glycoprotein fraction, contained the Ia antigens, the HLA-A and -B antigens, and a glycoprotein of unknown function. The glycoprotein fraction was composed of four glycosylated polypeptides with molecular weights of 43,000, 39,000, 33,000, and 28,000, and beta2-microglobulin; no polypeptide was linked to another by disulphide bridges. The A and B antigens only were absorbed by antibody against beta2-microglobulin. The Ia antigens comprised one each of the 33,000 and 28,000 molecular weight glycosylated polypeptides noncovalently linked together. Thus, only these chains were absorbed by xenogeneic anti-Ia antisera and were cross-linked by dimethyl-3-3'-dithiobispropionimidate dihydrochloride. The dimeric molecule bound deoxycholate (0.26 g/g of protein) and, when solubilized in deoxycholate, has a molecular weight of 77,000. The Ia allo- and xeno-antigenic activities were labile to heating and proteolysis and are probably determined by the polypeptide structure. Xenogeneic specific anti-Ia antisera were raised in rabbits and mice by immunizing with the glycoprotein fraction. These antisera reacted with B lymphocytes and monocytes but not T lymphocytes and fibroblasts. Their Fab fragments blocked the cytotoxicity of the allogeneic antisera for B lymphocytes and were potent inhibitors of the mixed lymphocyte reaction.     

Rabbit anti-EL4 serum. A reagent which discriminates between murine cytotoxic and suppressor cells.

Antisera against the C57B1 (H-2b) mouse lymphoma, EL4, were prepared in rabbits. After absorption with mouse liver, red cells and thymocytes, the antisera appeared to be cytotoxic for a subpopulation of peripheral T cells. The absorbed antisera blocked the immunosuppressive function observed when spleen cells from mice undergoing a graft versus host reaction were added to cells responding in vitro to sheep erythrocytes. This antiserum was unreactive against the cytotoxic cells also induced in this system. These results substantiate our previous finding that these antisera are specific for T-suppressor as opposed to T-helper or cytotoxic lymphocyte subpopulations.     

Correlated expression of VH framework and VH idiotypic determinants on T helper cells and on functionally undefined T cells binding group A streptococcal carbohydrate

Antibodies to framework determinants of the VH and V lambda fragments of MOPC 315 and antisera to the VH idiotype determinants of the A 5 A antibody were used to analyze the antigen receptors of mouse T (and B) cells. This was done by using the antibodies as inhibitors in (a) an assay in which the binding of radiolabeled streptococcal carbohydrate (A-CHO) antigen by primed and unprimed T and B cells is determined and (b) an assay in which the helper activity of group A streptococcal vaccine-primed T cells is determined. The results suggest that the major proportion of primed and unprimed T cells binding A-CHO (70-90%) exhibit VH framework and VH idiotypic determinants. This population appears to include the helper T cells. A minor proportion of T cells (10-30%) express V lambda-related framework determinants and lack VH framework and VH idiotypic determinants. This population does not include T helper cells. Taken together, the data suggest that a subpopulation of T cells, including the helper cells, uses entire Ig VH regions as part of their antigen receptor system.     

Human Ia-Like Antigens Associated with HLA-D

Antisera raised with HLA-A- and -B-compatible, HLA-D-disparate combinations were cytotoxic to B lymphocytes from the immunizing donor's HLA-D phenotype. Four antisera recognized structures closely associated with the HLA-D determinants Dw2, Dw3, Dw4, and LD 108. One antiserum had a broad reactivity pattern, including Dw3, Dw6, and some unknown specificity(ies). In population and family studies these B-lymphocyte antigens behaved as if they were governed by one genetic locus in the B-D region of the HLA complex. Furthermore, the antisera were cytotoxic to a minor concavalin-A-reactive T-cell subpopulation. The antisera had previously been shown to inhibit the stimulating cells in mixed lymphocyte culture and to be capable of inhibiting the Fc receptor in the EA rosette assay. We conclude that the antisera produced by this method recognize Ia-like antigens closely associated with the HLA-D determinants.     

Serological identification of five HLA-D associated (Ia-like) determinants.

Five antisera raised within HLA-A and -B compatible, HLA-D disparate combinations were reactive in a modified NIH microcytotoxicity test with B lymphocytes, but not with T lymphocytes from the immunizing donor, as well as with B lymphocytes from most or all donors sharing his immunizing HLA-D phenotype. Four antisera recognized structures closely associated with the HLA-D determinants Dw2, Dw3, Dw4 and LD 108. One serum had a broad reactivity pattern including Dw3, Dw6 and some unknown specificity(ies). In population and family studies, these B lymphocyte antigens behaved as if they were governed by one genetic locus in the B-D region of the HLA complex. We conclude that the antisera produced by this method recognize Ia-like antigens identical to or very closely associated with the HLA-D determinants.     

Stimulation of lymphocytes by allogeneic lymphocytes and lymphoblasts in the presence of anti-HLA antisera.

Anti-HLA alloantisera inhibit mixed lymphocyte responses in which normal lymphocytes are used as stimulator cells. These same antisera are unable to inhibit lymphocyte proliferative responses stimulated by lymphoblastoid cells from cultured lymphoid cell lines. They also fail to inhibit either the generation of cytotoxic effector cells by lymphoblastoid cells or lymphocyte-mediated cytotoxicity against the lymphoblasts. Although the number of HLA antigens on the surface of lymphoblasts is reported to be greater than on normal lymphocytes, the failure of alloantisera to inhibit lymphoblast-induced responses in vitro does not appear to be due to insufficient amounts of antiserum to react with the antigenic sites. Rather, the data are interpreted to suggest that antigens which are not HLA and are not closely associated with HLA on the lymphocyte membrane are responsible for the stimulation of allogeneic lymphocytes by lymphoblastoid cells. Although lymphoid cell lines are known to contain the genome of the Epstein-Barr virus, antisera against products of the viral genome fail to inhibit proliferative responses to lymphoblastoid cells, suggesting that these antigens do not directly participate in lymphocyte activation.     

GENETIC STUDIES IN INBRED RATS. IV. XENOANTISERA AGAINST RAT ERYTHROCYTE (Ag-C) ANTIGENS*

Antibodies to the erythrocyte Ag-C antigens were raised by immunizing New Zealand white rabbits with rat red cells. Evidence that Ag-C antigens are not on lymphocytes includes: (1) Ag-C could not be detected on rat peripheral, splenic or thymic lymphocytes by lymphocytotoxicity using anti-Ag-C antisera, (2) xeno-antisera prepared against rat lymphocytes from which Ag-B antibodies were absorbed did not agglutinate rat red cells, and (3) absorption of reagent Ag-C antisera with lymphocytes did not remove the anti-Ag-C activity. Platelets also failed to absorb Ag-C antibodies from reagent anti-Ag-C antisera. The mixed lymphocyte reaction between strains within the same major histocompatibility (Ag-B) group but differing in Ag-C type showed no stimulation. This finding corroborates the studies which failed to demonstrate Ag-C on lymphocytes. Isoagglutinins for red cells were not detected in any of the strains of rats tested, but rabbit xenoagglutinins to rat and sheep red cells were found. Reagent Ag-C antisera were used to type some previously unreported strains of inbred rats.     

Human Ia-like DRw lymphocyte antigens stimulating activity in primary mixed lymphocyte reaction.

The intensity of the primary mixed lymphocyte reaction (MLR) seems to be influenced by at least two distinct determinants of the HLA-D region: the HLA-Dw stimulating products and the human Ia-like B lymphocyte DR (D-related) antigens. In three families, the primary MLR is significantly higher in case of HLA haploidentity, when stimulating and responding cells differ with regard to both Dw and DRw determinants, than when only Dw products are different. Thus, an additional effect of DR antigens during primary MLR is observed. The role of DRw antigens in primary MLR and the results obtained by a primed lymphocyte test which can discriminate between Dw3 and DRw3, indicate that DRw and Dw products could be distinct determinants.     

Antibody against rabbit immunoglobulin VH and L chain allotype determinants in heterologous anti-Ig antisera.

Heterologous anti-rabbit IgG antisera have been examined by radioimmunoassay for the occurrence of anti-VH antibody. Most of the sera contained antibody specific for VH allotype determinants (Aal). The antisera contained as much or more anti-al antibody as estimated in conventional alloantisera. It has further been shown that the anti-L chain antibody in all these sera was specific for L chain allotype determinants. Antibody against H chain-dependent L chain conformational determinants or cross-reacting determinants in CHlgamma and CHlalpha could not be demonstrated.     

Specific Suppression of MLC and CML by Anti-Idiotypic Antibodies in the Mouse

Rabbit antisera directed against idiotypic determinants of alloreactive mouse CBA anti-C57BL/6 T blasts were raised in the following manner: first, a rabbit serum directed against nonspecific CBA blasts cells was prepared by injecting CBA concanavalin A blasts three times at monthly intervals into a rabbit. Second, specific CBA anti-C57BL/6 T lymphoblasts were induced in a mixed lymphocyte culture (MLC), were purified by gravity sedimentation through a fetal calf serum gradient, and, finally, were incubated with the anti-blast serum from the first step. During this incubation, presumably all epitopes of the blast cell population were blocked by anti-blast antibodies, except for the greatly amplified set of CBA anti-C57BL/6 alloreactive idiotypes. The mixture was then injected into fresh rabbits, which were boosted with similar mixtures after 3 and 6 weeks. Blood samples were removed 10 days after each injection. Such sera, when used together with complement, inhibited specifically the stimulation of CBA cells by C57BL/6 antigens in MLC and the CBA anti-C57BL/6 killer cells.     

In vitro generation of cytotoxic lymphocytes against radiation and radiation leukaemia virus-induced tumours. II. A radiation-induced thymoma generates cytyotoxic response in syngeneic but not in allogeneic lymphocytes.

A thymoma cell line (PXT), originally induced by X-irradiation in a C57Bl/6 mouse, was found capable of generating cytotoxic T lymphocytes (CTL) in a syngeneic mixed lymphocyte tumour culture (MLTC), but failed to stimulate allogeneic lymphocytes. Serologically defined H-2 antigens could readily be detected on PXT cells, which were also susceptible to lysis by alloreactive CTL generated against C57Bl/6 lymphoblasts or other thymomas of C57Bl/6 origin. These observations suggest that the PXT line lacks lymphocyte-activating determinants (LAD) essential for allosensitization but possesses other determinants enabling stimulation of syngeneic CTL, and that the cellular events leading to generation of anti-H-2 and anti-'modified self' CTL proceed along distinct T-cell differentiation pathways.     

Two-Chain Subunit Structure of HLA-D Antigens

Both F(ab')2 fragments and intact antibody from rabbit anti-B-cell antisera were shown to specifically block the mixed lymphocyte culture (MLC) reaction. The antisera, which were raised to papain-solubilized spleen cell membrane antigens, appeared to block the stimulator cell and not the responder cell. Specificity of the blocking was shown in that antisera to beta2 microglobulin would not block the MLC-stimulating cell. Membrane antigens from acute lymphocytic leukemia cells were labeled with 125I, solubilized with sodium deoxycholate and immunoprecipitated with either human or rabbit anti-B-cell antisera. Both sources of antisera precipitated polypeptide subunits of 27,000 and 35,000 daltons.