Immunochemical and functional analysis of Ia-like antigens on human monocyte-macrophages with monoclonal antibodies

The distribution, structural profile and functional properties of Ia-like antigens synthesized by human monocyte-macrophages have been analyzed using monoclonal antibodies to common determinants of these antigens. Up to 45 and 70%- of monocyte-macrophages isolated from the fluid of blisters induced with cantharidin and from peripheral blood, respectively, react with monoclonal antibodies to human Ia-like antigens. The level of Ia-like antigens on monocytes-macrophages appears to be similar to that on cultured B lymphoid cells. Monoclonal antibodies to common determinants of Ia-like antigens specifically block antigen presentation by monocyte-macrophages to T lymphocytes as well as proliferative response of T lymphocytes to autologous and allogeneic monocytes-macrophages. These results indicate that common determinants of Ia-like antigens play a role in the interaction of monocytes-macrophages with T lymphocytes.     

Purification of immunologically functional subsets of human Ia-like antigens on a monoclonal antibody (Q5/13) immunoadsorbent

Serologic and immunochemical asays have shown that the monoclonal antibody Q5/13 recognizes an antigenic determinant expressed on a subset of human Ia-like antigens. Testing with a panel of HLA typed B lymphoid cells has shown that this determinant is different from those defining the serologic polymorphism of HLA-DR antigens. The monoclonal antibody Q5/13 has been used to purify subsets of human Ia-like antigens, which are immunologically functional. These reagents should facilitate the characterization of structural and functional properties of human Ia-like antigens.     

Monoclonal antibodies to HLA-A,B, and Ia-like antigens inhibit colony formation by human myeloid progenitor cells

Hybridomas derived from the fusion of murine myeloma cells with splenocytes from mice immunized with human cultured lymphoid cells secreted monoclonal antibodies to human cell surface antigens. Serologic and immunochemical assays showed that 4 monoclonal antibodies (Ab Q2/47, Q2/61, Q2/70, Q2/80) recognize framework determinants of Ia-like antigens and 1 monoclonal antibody (Ab Q1/28) reacts with determinants expressed on the heavy chain of HLA-A,B antigens. Both anti-HLA-A,B and anti-Ia-like antigen monoclonal antibodies caused complement-dependent inhibition of granulocyte-macrophage colony formation by human bone marrow grown in soft agar. Mixing experiments excluded the possibility of an indirect effect on progenitor cells by lysis of auxiliary cells. These results indicate that human myeloid progenitor cells express HLA-A,B and Ia-like antigens.     

MLC-derived human helper factor(s) that promote B cell differentiation: induction, functional characterization, and role of Ia antigens

Utilizing a PFC assay to quantitate the polyclonal activation of human peripheral blood B lymphocytes, we have investigated the induction and functional activity of MLC-derived human helper factor(s). Our data demonstrate that highly purified responder T cells, but not B or null cells, are required for the elaboration of MLC helper factor(s) that trigger the in vitro differentiation of B lymphocytes into PFC. Helper factor can trigger B cell maturation in the absence of helper T cells, since complement- (C) mediated lysis of the small (less than 5%) fraction of T cells present in anti-F(ab)2 immunoabsorbent column purified B cell population eliminates the PWM induced, but not the helper factor-induced PFC response. Responder T cells required for helper factor production do not bear surface membrane Ia, since alpha p23,30 + C treatment of this population does not affect helper factor generation. In contrast, alpha p23,30 + C treatment of the allogeneic stimulator cell population eliminates helper factor production. Taken together, these results demonstrate that interaction between Ia-bearing stimulator cells and Ia- responder T cells is required for the production of MLC-derived helper factor. In additional experiments, we determined that alpha p23,30, in the absence of C, totally abrogates the PFC response triggered by MLC helper factors. This result suggests an important role for Ia antigens in the functional activity of preformed helper factor molecules.     

Differential effect of interferon on the expression of tumor-associated antigens and histocompatibility antigens on human melanoma cells: relationship to susceptibility to immune lysis mediated by monoclonal antibodies

Incubation of cultured human melanoma cells with human leukocyte interferon did not change the expression of melanoma-associated antigens (MAA) recognized by monoclonal antibodies and of Ia-like antigens but significantly increased the expression of HLA-A,B antigens and of beta 2-microglobulin (beta 2-mu). The effect is dependent on the dose of interferon and on the incubation time. Interferon-treated melanoma cells showed an increased susceptibility to lysis mediated by monoclonal antibodies to HLA-A,B antigens and to human beta 2-mu; on the other hand, interferon-treated melanoma cells did not change in their susceptibility to murine natural killer (NK) cell lysis and to immune lysis mediated by monoclonal antibodies to MAA and to Ia-like antigens, and they displayed a reduced susceptibility to human NK cell lysis. Therefore, the increased susceptibility of interferon-treated melanoma cells to lysis mediated by anti HLA-A,B and anti beta 2-mu monoclonal antibodies is likely to reflect the increase in cell surface expression of the corresponding antigens.     

HLA antigens on human hemopoietic progenitors during embryonic-fetal life: expression and in vitro modulation

We have analyzed the time course of the expression of HLA antigens on hemopoietic progenitors (BFU-E, CFU-E and CFU-GM) from human embryonic-fetal livers (FL). HLA ABC and Ia-like antigens are never detected on progenitor cells at 5-week post-conception. Their expression initiates at 6-week and progressively increases thereafter, up to near-adult levels at 9-week. The time course of HLA antigen expression on erythroid precursors, derived from FL at corresponding gestational ages, parallels that observed on hemopoietic progenitors. Incubation of embryonic progenitors or precursors with medium conditioned by PHA-stimulated adult peripheral blood mononucleated cells induces a marked increase of the expression of both HLA-ABC and Ia-like antigens. Conversely, incubation with recombinant gamma-interferon causes a marked increase of HLA-ABC, but not Ia-like antigen expression, thus in contrast with results observed on adult monocytes.     

Immunofluorescence analysis of normal and malignant lymphoid tissues with selected combination of antisera

Tissue sections stained with combinations of antisera labeled with different fluorochromes (i.e., conventional antisera to human immunoglobulin classes, T lymphocyte antigens, and Ia-like p28,33 antigens used in various double combinations with each other or with different mouse monoclonal antibodies) allow the identification of the different areas of lymph nodes in serial sections and provide great flexibility as well as precision in the analysis of the distribution and relationship of normal and malignant cells. Lymphoid microenvironments in the thymus and the paracortical areas of lymph nodes are described. The close association of T lymphocytes and nonlymphoid cells expressing large amounts of Ia-like antigens (such as interdigitating reticular cells and endothelium) may be relevant for the understanding of immunoregulatory disorders such as dermatopathic and angioimmunoblastic lymphadenopathies and some malignancies (e.g., mycosis fungoides) were the expression of Ia-like antigens on non-T cells seems to be abnormally abundant. The analysis of immunoglobulin and membrane marker expression of normal and malignant B cells and their relation to T cells can also be related to the histology of the disease. These studies are clinically useful for the classification of childhood lymphomas, the differential diagnosis of anaplastic carcinomas and lymphomas, and in the study of the early stages of lymphomas.     

Peripheral blood T-lymphocytes of patients with chronic B-cell lympholeukemia express Ia-like antigens

15.7 +/- 3.5% of sheep rosette-forming cells (SRFC) were isolated from the peripheral blood of 11 patients with B-cell chronic lympholeukemia (B-CLL). SRFC did not express surface immunoglobulins, antigens of nondifferentiated blasts and antigens of early T-cell precursors, while NK-cell antigen expression was low. 6 of 11 patients revealed 44.6 +/- 19.6% of Ia-like antigens in SRFC. Ia-like antigen expression was 4.8 +/- 0.6% in SRFC isolated from the peripheral blood of 19 healthy donors. The expression of Ia-like antigens in T cells of patients with B-CLL is suggested to be related to the activation of regulatory T-lymphocyte subpopulation.     

Sharing of Ia antigens between species. II. Molecular localization of shared Ia determinants implies the existence of more than one I sublocus of the rat MHC.

A mouse alloantiserum B10.D2 anti-B10.BR (H-2d anti-H-2k) cross-reacted with rat lymphocyte surface glycoproteins with characteristics of Ia antigens. Sequential precipitation analysis on solubilized radiolabeled LEW rat lymphocyte antigens with this cross-reactive mouse alloantiserum and the rat alloantiserum BN anti-LEW (Ag-B3 anti-Ag-B1) revealed that the Ia-like antigen detected by the mouse alloantiserum also reacted with the rat anti-Ia antibodies. It was further shown that the rat alloantiserum also detected another set of Ia-like antigens that did not cross-react with the mouse alloantibody. Precipitation analysis with congenic rat strains confirmed that all Ia-like antigens precipitated by the rat alloantibody were encoded by Ag-B linked genes. Thus the shared Ia-like antigen must also be the product of Ag-B-linked gene(s) or be physically associated with such products. In addition, molecules bearing shared antigenic determinants were separable from at least some of the Ia-like antigens detected by the rat alloantiserum, possibly suggesting the existence of more than one sublocus coding for Ia antigens within the rat MHC.     

Role of surface IgM and IgD in the functional differentiation of human B lymphocytes: effect of papain treatment.

Short-term treatment of normal human B lymphocytes with low concentrations of papain resulted in selective and reversible removal of sIgD determinants, whereas HLA and Ia-like antigens, sIgM as well as receptors for E, C3, and FcIgG were unaffected. When studied for their capacity to generate antigen-specific direct PFC, papain-treated (delta-) B cells were highly sensitive to inactivation by even low concentrations of antigen. In addition, these cells were impaired in their ability to cooperate normally with T-helper cells or their humoral product(s).     

H-2K/H-2D and Mls and I region-associated antigens stimulate helper factor(s) involved in the generation of cytotoxic T lymphocytes

This study examines the antigen that stimulate production or release of a soluble helper factor(s) involved in development of cytotoxic T lymphocytes (CTL). Antigens associated with the Mls locus, I and K/D regions of the MHC were all capable of stimulating responder cells in MLC to produce helper factor. These supernatant fluids were all capable of providing "help" for the generation of cytotoxic T lymphocytes in MLC in which spleen cells are stimulated by allogeneic heat-treated thymocytes or splenocytes. Previous reports from our laboratory as well as others have shown that heat-treated cells do not stimulate a cytotoxic response. Heat-treatment of Mls, I, and H-2K/H-2D region incompatible stimulatory cells in MLC eliminated their ability to induce responder cells to produce helper factor, suggesting this is the mechanism whereby heat-treatment reduces the ability of cells to stimulate cell-mediated lympholysis (CML). The inability of supernatant fluids, from MLCs in which heat-treated cells were the stimulators, to assist in the generation of cytotoxic T cells did not appear to be the result of any suppressive factor induced by such treatment. Further, the antigens that stimulate pre-killer cells appear functionally distinct from those heat labile antigens (Mls, I, H-2K/H-2D associated) that stimulate helper factor production since heat-treated allogeneic cells served as stimulators of cytotoxicity provided helper activity was added to the MLC.     

Antigenic phenotype of TdT-positive cells in human peripheral blood

Double immunofluorescence studies for terminal deoxynucleotidyl transferase (TdT) and leucocyte surface membrane antigens have been used to characterize the small subpopulation of TdT-positive cells in human peripheral blood. The predominant antigens demonstrated were those coded for by the major histocompatibility complex, namely HLA-A,B and Ia-like antigens. A small proportion of TdT+ cells expressed antigens restricted to B lymphocytes and their precursors (BA-1+ CALLA+). In contrast, antigens associated with T-lymphocyte differentiation were not detected using a panel of T-cell-specific monoclonal antibodies. These results preclude the possibility that circulating TdT+ cells are immature cortical thymocytes that have "leaked" into the bloodstream. Although bone marrow-derived prothymocytes, which have not yet acquired T-cell lineage markers, may be included amongst this subset, the expression of B-cell related antigens by some TdT+ cells indicates the likely existence of lineage heterogeneity amongst this population of lymphoid cells. The relevance of these findings to the monitoring of human acute lymphoblastic leukaemia is discussed.     

In vitro influenza virus-specific antibody production in man: antigen-specific and HLA-restricted induction of helper activity mediated by cloned human T lymphocytes

Cloned human T lymphocytes induced with influenza A virus (A/Texas/1/77) and maintained in continuous culture with T cell growth factor were assayed for helper function in the in vitro production of anti-influenza antibody. Helper function mediated by both cloned helper T cells and normal peripheral blood lymphocytes was highly antigen dose-dependent, requiring lower concentrations than that necessary to induce blastogenesis. Optimal help was observed with 1 X 10(2) cloned T cells per culture, whereas excess helper cells inhibited the response. After culture with influenza A virus-induced cloned helper T cells, the antibodies formed were directed against influenza A and not B virus. Furthermore, the cloned helper T cells despite being specific for matrix protein collaborated in the production of predominantly anti-hemagglutinin antibody, suggesting associative recognition of the two discrete antigens. Cellular interactions between cloned helper cells from an HLA-Dw1,3 DR1,3 individual and erythrocyte rosette-negative cells required HLA-Dw1; DR1 compatibility for the production of specific antibody. This was confirmed by using subclones. Finally, it was observed that supernatants of the cloned helper T cells contained functional activity capable of replacing the parent cells in the production of anti-influenza A virus antibody.     

Analysis of structural properties and cellular distribution of avian Ia antigen by using monoclonal antibody to monomorphic determinants

In this report, we describe the analysis of Ia-like antigens in the chicken by using a monoclonal antibody (CIa-1) reactive with monomorphic determinants of the Ia-like (B-L) antigens. This antibody reacts with determinants on B cells in all avian species tested, but does not detect antigens on lymphocytes of representative mammals, reptiles, and amphibians. In addition to B cells, this antibody defines a subpopulation of the monocyte-macrophage series and reacts with mitogen-activated T cells. Immunochemical analysis indicates that the CIa-1 reactive antigen is a 65,000-dalton glycoprotein consisting of an alpha-chain of 32,000 daltons noncovalently bound to a beta-chain of 27,000 daltons. Under nonreducing conditions, the beta-chain migrates with slightly faster mobility. Two-dimensional gel analysis indicates that the beta-chain is the more heterogeneous of the two chains. Thus, the antigen detected by CIa-1 antibody is similar in cell distribution and structure to the murine Ia antigens and human DR antigens. During in ovo development, Ia+Ig- cells were not found in the yolk sac but were detected in the spleen, mesonephros, and bursa of 9-day embryos. Two populations of Ia+Ig- cells were identified in the bursa: 40 to 60% of the bursacytes, mostly larger cells, exhibited brighter immunofluorescence reactivity than the smaller bursacytes.     

Serological detection of B-cell ("Ia-like") antigens in serum of normal donor and in some sera of leukaemic patients

Some sera from normal donors (1/18) and from leukaemic patients (2/7 with acute myeloid leukaemia [AML], 1/4 with chronic lymphatic leukaemia [CLL], 0/3 with acute lymphoblastic leukaemia [ALL]; with high numbers of leukaemic cells expressing Ia-like p28,33 antigen on the leukaemic cell surface) inhibited the complement mediated cytotoxic activity of highly specific xenogenous anti Ia-like sera (which were prepared by immunization of rabbits with insoluble membrane fractions of B-type lymphoid lines) at a titre 1:4 or less. This effect was not observed with antisera directed against other membrane marker determinants (e.g. T lymphocyte specific antigens). These results suggest that at least a small proportion of membrane bound Ia-like antigens can be released from cell surfaces and in some patients these Ia-like moieties are detectable (by sensitive inhibition assays) in the serum.     

Serological cross-reactivity of murine A.TH anti-A.TL antibody (anti-l-Ek antibody) with La-like molecules of human antigen-presenting cells

The purified protein derivative (PPD)-specific proliferative responses of T cells from human peripheral blood are shown to be dependent on antigen-presenting cells (APC) which bear HLA-DR antigen detected by the monoclonal anti-HLA-DR antibody. The serological cross-reactivity of murine A.TH anti-A.TL antibody was observed in human APC. By absorption experiments using H-2 congenic mice, the serological cross-reactivity of A.TH anti-A.TL antibody with human APC is mapped in the I-E subregion. Thus, anti-I-Ek antibody reacts with the Ia-like molecule(s) on human APC. Murine allo-anti-I-Ek antibody does not always react with determinants of Ia-like molecule(s) on human APC, since this antibody did not eliminate PPD-specific proliferative responses in one particular case. Thus, anti-I-Ek antibody seems to react some type of the polymorphic determinants but not of the shared determinants of human Ia-like molecule(s) on APC. The relationship between the cross-reactive molecule detected by murine allo-anti-I-Ek antibody and the HLA-DR antigen remains to be analyzed.     

Serologic and immunochemical characterization of the specificity of four monoclonal antibodies to distinct antigenic determinants expressed on subpopulations of human Ia-like antigens

Serologic and immunochemical assays showed that the monoclonal antibodies Q2/70, Q2/80, Q5/6, and Q5/13 react with human Ia-like antigens. Each monoclonal antibody recognizes distinct antigenic determinants that are different from those defining the serologic polymorphism of Ia-like antigens defined by conventional alloantisera and are expressed on subpopulations of Ia-like antigens. The determinants recognized by the MoAb Q2/70 and Q5/13 are expressed on all HLA-DR allospecificities tested, whereas those reacting with the MoAb Q2/80 and Q5/6 are not detectable on HLA-DR5 and HLA-DR7 allospecificities, respectively.     

HLA-A,B antigens Ia-like antigens, and tumor-associated antigens on prostate carcinoma cell lines: serologic and immunochemical analysis with monoclonal antibodies

Serologic and immunochemical analysis of the antigenic profile of the 2 human prostate carcinoma cell lines DU-145 and H494 with a battery of monoclonal antibodies has shown that both cell lines express HLA-A,B alloantigens and the 94,000 m.w. tumor-associated glycoprotein recognized by the monoclonal antibody 376.96S. In addition, the cell line H494 unexpectedly expresses Ia-like antigens, which are similar in their antigenic profile and structure to B lymphoid cell derived Ia-like antigens. Both Ia-like antigens and tumor-associated antigens can function as targets of cell-dependent lysis mediated by the corresponding monoclonal antibodies.     

The role of macrophages in the generation of T helper cells. V. Evidence for differential activation of short-lived T1 and long-lived T2 lymphocytes by the macrophage factors GRF and NMF.

The generation of T helper cells in vitro requires macrophages or macrophage-derived factors such as genetically related macrophage factor (GRF) or nonspecific macrophage factor (NMF). However, there is a basic difference of T helper cell induction when using particulate antigens. The present study demonstrates that this difference is based on the activation of two different T cell subsets. GRF activates short-lived 'T1' cells which amplify the induction of T2 cells, which are the helper cell precursors. Thus, the genetic restriction of T helper cell induction seen with soluble antigen or GRF lies on the level of macrophage or GRF interaction with T1 cells. NMF (or macrophages) and particulate antigens directly activate the helper cell precursor (T2) indicating no requirement for T1-T2 cooperation. The direct activation of the helper cell precursor with particulate antigens does not require histocompatible macrophages or NMF from histocompatible macrophages. The present results may explain some of the discrepancies reported in the literature concerning the genetic requirements and specificity of T cell activation.     

The role of p23,30-bearing human macrophages in antigen-induced T lymphocyte responses.

The effect of anti-p23,30, a rabbit antiserum to the human Ia-like antigen p23,30, on two macrophage-dependent T-cell functions, proliferation in response to soluble antigens, and production of lymphocyte mitogenic factor (LMF) was studied. T cells depleted of macrophages neither proliferate nor secrete LMF, and these functions are restored by addition of as few as 0.5% adherent macrophages. Treatment of macrophages with anti-p23,30 and C, however, abolishes their capacity to reconstitute these T-cell functions. In contrast, treatment of T cells with anti-p23,30 and C did not affect their capacity to respond in the presence of untreated adherent cells. We conclude that the presence of p23,30-bearing macrophages is critical for the expression of these antigen-induced T-cell responses.