Pretreatment of rat pancreatic islets with MHC I-A monoclonal antibody: in-vitro and in-vivo effects on islet antigenicity.

Pancreatic islet allografts are rejected very rapidly due to their high immunogenicity. Several attempts have been made to reduce the immunogenicity prior to transplantation. We treated islets with MHC Ia (class II) antigen monoclonal antibody and complement in order to lyse Ia antigen bearing cells within the islets. This treatment caused a distinct reduction of Ia antigen positive cells, which was demonstrated by indirect immunofluorescence tests. A total depletion of the Ia antigens, however, could not be achieved. As a second in-vitro test to evaluate the effect of the antibody treatment a mixed lymphocyte islet culture (MLIC) was performed. The partial Ia antigen reduction provoked a significantly reduced but not completely suppressed allogenic T-cell response. Antibody treatment did neither impair the morphological integrity nor the function of the islets as could be demonstrated by syngenic transplantation of islets pretreated by antibodies in the same way. After allotransplantation of antibody-pretreated islets across a major histocompatibility barrier into non immunosuppressed rats, however, there was no significant prolongation of the graft survival. Thus, a partial reduction of the number of the Ia antigen positive cells within the islets is not sufficient for the prevention of allograft rejection.     

Studies on the synergy between collagen and adjuvant arthritis in rats

Intravenous administration of subarthritogenic doses of anticollagen IgG and adjuvant-sensitized spleen cells to syngeneic naive rats induces an erosive arthritis in recipients. The onset of the clinical disease in recipients is rapid and the disease is severe when compared to those recipients receiving cells alone. Immunocytochemical analysis of the knee synovium indicates the accumulation in the adipose tissue of Ia+ (ED1+)macrophages, OX-19+ T lymphocytes, and neutrophils. A large proportion of the lining cells of the proliferative synovium are Ia+. The knee synovium is extremely edematous and contains fibrin. If recipient rats are decomplemented, clinical disease is delayed and the number of mononuclear and polymorphonuclear cells accumulating in the synovium is decreased. Similar results are observed if recipient rats are treated with anti-Ia+ antibody. However, anti-Ia+ treatment does not induce depletion of serum complement.     

Accessory cell function in a Con A response: role of Ia and interleukin 1

Accessory cell (A-cell) function in a Con A response was analyzed. Irradiated P388D1 cells efficiently induced a proliferative response to Con A of T cells purified from spleen cells, whereas paraformaldehyde-fixed P388D1 cells failed to serve as A cells. Although IL-1 containing culture supernatant (SN) of a macrophage hybridoma induced the Con A response of the T-cell preparations, the depletion of Ia+ cells by the treatment with anti-Ia antibody and complement abrogated the response in the presence of IL-1. Fixed P388D1 cells and the hybridoma SN synergized in the reconstitution of the response. A 15,000-Da fraction of the hybridoma SN or human recombinant IL-1 alpha was able to substitute the hybridoma SN for the response. The reconstitution of the response by IL-1 and fixed P388D1 cells was inhibited by the addition of monoclonal anti-Ia antibody. These results indicate that IL-1 or fixed P388D1 cell does not exert a sufficient signal by itself and both of them are required for the reconstitution of a Con A response of highly purified T cells, and that Ia on fixed P388D1 cells play an important role.     

Lactoferrin acts on I-A and I-E/C antigen+ subpopulations of mouse peritoneal macrophages in the absence of T lymphocytes and other cell types to inhibit production of granulocyte-macrophage colony stimulatory factors in vitro

The relationship between Ia antigens on mouse resident peritoneal macrophages and the ability of lactoferrin (LF) to inhibit the production of granulocyte-macrophage colony stimulatory factors (GM-CSF) from these cells was investigated. Detection of the suppressive influence of LF on release of GM-CSF from greater than or equal to 10(5) macrophages/ml/plate required that the conditioned media being assessed for GM-CSF be prepared in the presence of indomethacin and/or be preincubated with anti-ferritin antiserum to respectively stop production of E-type prostaglandins and to remove acidic isoferritin-inhibitory activities that can mask the effects of LF. Treatment of mouse macrophages with monoclonal antibodies to the I-A and I-E/C subregions of Ia antigens in a complement C-dependent cytotoxicity assay killed less than 15% of the cells, but removed all Ia antigen+ macrophages and reduced GM-CSF production by approximately 50%. LF decreased GM-CSF production by untreated macrophages by approximately 50%, but had no effect on macrophages insensitive to treatment with anti-Ia plus C. Macrophages left at 37 degrees C for 5 and 24 hr were not killed by treatment with monoclonal anti-Ia plus C and GM-CSF production by these macrophages was not suppressed by LF. Treatment of macrophages with monoclonal anti-H-2K or anti-Mac-1 plus C reduced GM-CSF production greater than 95%. Anti-I-A, -I-E/C, -H-2K, or -Mac-1, in the absence of C, had no effect on viability of macrophages or on production of GM-CSF, but anti-I-A and -I-E/C each blocked the inhibitory action of LF. Lower concentrations of these antibodies could block the action of LF when anti-I-A and anti-I-E/C were mixed together better than when they were each used separately. The removal of Thy-1.2+ cells from unseparated or adherent peritoneal cells resulted in populations of cells that were up to 100% positive for nonspecific esterase, and did not influence GM-CSF production from these cells, the reduction of GM-CSF from these cells by LF, or the reduction of GM-CSF by the removal of Ia antigen+ cells. The results were similar whether or not T cells were removed from the assay marrow by treatment with antibodies Ly-1.1, Ly-2.2, and Qa4 plus C.(ABSTRACT TRUNCATED AT 400 WORDS)     

Treatment with humanized monoclonal antibody against CD154 prevents acute renal allograft rejection in nonhuman primates.

CD154 is the ligand for the receptor CD40. This ligand-receptor pair mediates endothelial and antigen-presenting cell activation, and facilitates the interaction of these cells with T cells and platelets. We demonstrate here that administration of a CD154-specific monoclonal antibody (hu5C8) allows for renal allotransplantation in outbred, MHC-mismatched rhesus monkeys without acute rejection. The effect persisted for more than 10 months after therapy termination, and no additional drug was required to achieve extended graft survival. Indeed, the use of tacrolimus or chronic steroids seemed to antagonize the anti-rejection effect. Monkeys treated with antibody against CD154 remained healthy during and after therapy. The mechanism of action does not require global depletion of T or B cells. Long-term survivors lost their mixed lymphocyte reactivity in a donor-specific manner, but still formed donor-specific antibody and generated T cells that infiltrated the grafted organ without any obvious effect on graft function. Thus, therapy with antibody against CD154 is a promising agent for clinical use in human allotransplantation.     

Unique T cell Ia antigen expressed on a hybrid cell line producing antigen-specific augmenting T cell factor

Hybrid cell lines were established by fusion between keyhole limpet hemocyanin(KLH) binding T cells of A/J mice and an AKR T cell tumor line, BW5147. Hybrids were selected for the presence of Ia antigen and KLH-specific augmenting activity of their extracts in the secondary antibody response. The detailed phenotypic and functional analysis of 1 of these clones, FL10, is reported here. The hybrid was positive for both Thy1.1 and Thy1.2 antigens and possessed the Lyt-1+,2-,3- phenotype. Both VH and Ia determinants were detected on their cell surface. The IA locus was mapped in the I-A subregion, but the Ia specificities were serologically distinct from those of B cell Ia antigen. This was demonstrated by the fact that anti-Ia antiserum preabsorbed with B cells could react with the hybrid cells, whereas none of the monoclonal anti-Ia specific for private and public determinations of Iak could. The extract from the cell line specifically augmented the in vitro secondary antibody response against dinitrophenylated KLH, and this activity was removed by absorption with antigen and conventional anti-Ia antisera. The results indicate that the cell line, FL10, carries Ia antigen unique to the T cell, which is associated with the antigen-specific augmenting molecule.     

The expression of Ia antigenic determinants on macrophages required for the in vitro antibody response.

A subpopulation of antigen-presenting macrophages required for an in vitro antibody response to burro erythrocytes was deleted by pretreating the splenic macrophages with anti-Ia serum and complement (C). The in vitro response of the macrophage depleted T-B cell population could not be restored by the addition of macrophages resistant to anti-Ia antibodies and C (Ia-). The response of Ia- macrophages and the macrophage-depleted T-B cells was only reconstituted by the addition of Ia+ macrophages. Macrophages pretreated with anti-Ia antibodies restricted to react with determinants of one I subregion could not support the in vitro antibody response when added to cultures whose macrophages were pretreated with anti-Ia serum and C specific for the I-J subregion. These results confirmed that Ia determinants of the I-A, the I-E, and the I-C subregions were all expressed on the I-J+ macrophage required for an in vitro antibody response.     

Mouse skin graft prolongation with donor strain bone marrow and anti-lymphocyte serum: surface markers of the active bone marrow cells

The survival of C3H/HeJ skin grafts on B6AF1 mice treated with anti-lymphocyte serum (ALS) can be significantly prolonged by the injection of the host with C3H/HeJ bone marrow. Although the prolongation is apparently due at least in part to the ultimate presence in the host of specific suppressor cells of donor origin, little is known about the nature of the cells in the marrow inoculum that are responsible for this effect. The present investigation was undertaken to characterize surface markers of the active bone marrow cells. Marker-positive populations were either depleted and enriched by panning techniques or depleted by killing with specific antibody and complement, and then were assayed for their ability to prolong graft survival. Cells that were adherent to anti-Ia-coated plates extended graft survival only slightly better than did treatment with ALS alone, whereas nonadherent (Ia-depleted) cells, as well as cells treated with anti Ia and complement, retained good prolonging activity. Similarly, panning on anti-immunoglobulin (Ig)-coated plates produced an active, Ig+-depleted population and an inactive adherent population, and killing of Thy-1+ cells with antibody and complement did not compromise the ability of the bone marrow inoculum to prolong graft survival. Complement receptor-positive (EAC+) and Fc gamma receptor-positive cells (EA+) were separated by panning on plates coated with sheep erythrocytes/antibody/complement and erythrocytes/7S antibody respectively. Adherent, EAC+-enriched cells were only slightly active, whereas the nonadherent, EAC-depleted population was fully active in graft prolongation. However, both Fc gamma R+ (EA+)-enriched and depleted populations were active, with the enriched fraction producing significantly better prolongation than the depleted population. Thus, the bone marrow cells that can prolong skin graft survival in ALS-treated mice appear to be Ia-, Thy-1+, largely complement receptor negative, and Ig-, but are largely positive for Fc gamma receptors.     

Subpopulations of the T4+ inducer T cell subset in man: evidence for an amplifier population preferentially expressing Ia antigen upon activation

Prior studies demonstrated that Ia molecules were expressed on a fraction of human peripheral T4+ inducer cells upon stimulation by soluble antigen. In the present study, we utilized a fluorescence-activated cell sorter to separate antigen-activated T4+,Ia+ and T4+,Ia- populations and characterized their function. It was found that the T4+,Ia+ population contained the majority of proliferating T cells as assessed by tritiated thymidine incorporation. This proliferation largely appeared to be nonspecific. Despite macrophage repletion, elimination of the Ia+ subset of T cells with monoclonal anti-Ia antibody and complement treatment equally diminished subsequent proliferation to both the triggering antigen and an unrelated antigen. Moreover, the antigen-induced Ia+ subset of T cells alone produced a nonspecific helper factor, LMF. In contrast, the the T4+,Ia- population showed minimal proliferation to soluble antigen and did not generate LMF. Nevertheless, both T4+,Ia+ and T4+,Ia- inducer T cells were required to generate maximal immunoglobulin production by B cells in an antigen-driven system. We conclude that the human T4+ inducer T cell subset is comprised of at least 2 functionally distinct subpopulations, which are capable of acting in a synergistic fashion to provide help to B cells.     

Characterization of a two-signal-dependent, Ia+ mononuclear phagocyte progenitor subpopulation that is sensitive to inhibition by ferritin

Evidence is presented that the ferritin-inhibitable, Ia+ monocyte progenitor in murine marrow requires two signals for stimulation of clonal proliferation. Escherichia coli K235 lipopolysaccharide (LPS) at 0.1 ng/ml enhanced macrophage colony formation by 25 to 70% in murine marrow cultures stimulated with colony-stimulating factor (CSF-1). The progenitors which responded to LPS and CSF-1 represented a distinct subpopulation. Pretreatment of marrow cells with complement plus anti-Ia, anti-H2, anti-asialo GM1, and anti-Mac-1 antibodies specifically depleted the two-signal-requiring progenitors. In addition, the same progenitors were depleted by preincubation with hydroxyurea, indicating that these cells were in cell cycle when removed from the marrow. When compared with the quiescent progenitors, the Ia+, cycling cells were more sensitive to the antiproliferative effects of interferon alpha/beta but were more resistant to inhibition by E prostaglandins. Pretreatment with T cell-specific antibodies and complement specifically enhanced cloning of quiescent progenitors without affecting cloning of the Ia+, cycling subpopulation. Moreover, rat liver ferritin at 10(-8) to 10(-10) M specifically inhibited clonal proliferation of the Ia+ progenitors. Finally, the requirement for LPS as the additional stimulant could be replaced by the addition of haplotype-specific anti-Ia antibody to CSF-stimulated cultures. In contrast to LPS, anti-IA was competitive with inhibitory ferritin in clonal proliferation of the Ia+ progenitors. The significance of these observations in regulation of monocytopoiesis is discussed.     

Accessory cell stimulation of T cell proliferation requires active antigen processing, Ia-restricted antigen presentation, and a separate nonspecific 2nd signal

The roles of Ia+ accessory cells in H-2-restricted stimulation of antigen-specific T cell proliferation were explored in an in vitro model. L-glutamic acid60-L-alanine30-L-tyrosine10-(GAT) primed BALB/c nylon wool-passed T cells were depleted of Ia+ antigen-presenting cells (APC) by treatment with monoclonal anti-Ia antibody plus complement. Such cells failed to respond to soluble GAT, or to soluble GAT in the presence of phorbol myristic acetate (PMA), which is known to stimulate production of, or replace, IL-1 in vitro. Addition of gamma-irradiated syngeneic spleen cells reconstituted the response to soluble GAT, but addition of ultraviolet (UV) light-irradiated spleen cells did not, even in the presence of PMA. Preincubation of cells with GAT for 24 hr, followed by washing, then gamma irradiation, generated a cell population able to stimulate GAT-primed T cells to proliferate. The same pulsed cells exposed to UV irradiation failed to stimulate T cell responses unless PMA was added to the cultures. The relevant cells in this UV-irradiated population are Ia+. It is concluded that a finite period of time for interaction of metabolically intact APC with antigen is required before creation of an appropriate (Ia + antigen) signal recognized by the T cell. In addition to such Ia-restricted antigen presentation, however, a 2nd nonspecific signal, again requiring metabolically active APC for elaboration, is necessary for detectable T cell activation. These studies thus define 3 separable activities of APC during the process of H-2 restricted T cell activation.     

Cellular stimuli for rejection of sarcoma I tumor allografts by BALB/c recipient mice

We have evaluated the role of passenger leukocytes in Sarcoma I (SaI) tumor allograft rejection by BALB/c recipient mice. SaI tumor cells grown in tissue culture expressed low levels of class I MHC antigens as determined by flow cytometry. Ascites-derived tumor cells contained additional cell populations of A/J host origin which expressed high levels of class I and class II alloantigens. Tissue culture-derived SaI grafts grew progressively or were rejected in delayed fashion by some BALB/c hosts. In contrast, ascites-derived tumor allografts were rejected rapidly by 100% of recipient mice. Passenger cells appear to be responsible for these striking differences in immunogenicity because the addition of allogeneic A/J splenocytes to the tissue culture form of SaI caused rapid rejection. When tissue culture- and ascites-derived tumors simultaneously were grafted on opposite shoulders of recipient mice, both tumors were rejected. These data indicate that passenger cells provide the primary stimulus for SaI tumor allograft rejection by BALB/c mice. The mechanism of SaI enhancement by anti-Ia antibodies may be analogous to the prolonged survival of endocrine grafts after depletion of Ia+ passenger leukocytes.     

The role of Ia molecules in the activation of T lymphocytes. II. Ia-restricted recognition of allo K/D antigens is required for class I MHC-stimulated mixed lymphocyte responses

The role of Ia molecules in the T cell proliferative response to class I (H2K/D) MHC alloantigens was examined. Proliferation in response to allo-K/D antigenic stimulation, but not to allo-Ia, was markedly inhibited by the addition of monoclonal anti-responder Ia antibodies to cultures in the absence of C. This anti-Ia blocking was observed in responses against both allelic and mutant class I antigens. Partial blocking was observed by using an anti-I-A or anti-I-E monoclonal antibody alone, whereas marked inhibition was seen with these two reagents together when the proliferating cells derived from a responder strain expressing both IA and IE gene products. Syngeneic Ia molecules appear to function as restriction elements, because they are required even in the presence of a source of exogenous second signal, phorbol myristic acetate or IL 1. The K/D-specific response required a responding cell that bears both Lyt-1 and -2 antigens, whereas responses generated to alloantigenic differences, including the I region, require only an Ly-1+ cell. The implications of these data with respect to the repertoire of the alloreactive proliferating T cell and the expression of the Lyt-2 antigen by such cells are discussed.     

Characterization of responding cells in oxidative mitogen stimulation. I. Ia+ cells and Ly-1+2+ cells are required for the proliferative response

Addition of anti-Ia sera to cultures of mouse spleen cells stimulated with oxidative mitogens, neuraminidase/galactose oxidase (NaGO) or sodium periodate (NaIO4), inhibits the subsequent proliferative response 30 to 70%. Anti-H-2K or D sera were not specifically inhibitory. Similar inhibition was seen when cells were pretreated with anti-Ia sera and washed before exposure to the mitogenic enzymes. Treatment with anti-Ia serum and complement depletes greater than 89% of the NaGO and the NaIO4 responses but 2% or less of the phytohemagglutinin (PHA) response. The response to NaGO was sensitive to depletion with anti-Thy-1 serum, rabbit anti-mouse brain serum, anti-Ly-1, or anti-Ly-2 serum. Mixtures of Ly-1 and Ly-2-depleted populations did not restore responsiveness. Thus both an Ia+ cell and an Ly-1+2+ T cell are required for [3H]TdR incorporation in response to NaGO treatment.     

Characterization of responding cells in oxidative mitogen stimulation. III. Presence of I-A- and I-J, E, C-subregion gene products on the surface of required cells

Ia antigens coded by genes of the murine major histocompatibility complex are expressed on the surface of a population of cells critical to the proliferative response of murine spleen cells to the oxidative mitogen neuraminidase/galactose oxidase. By selective depletion with antiserum and complement, Ia antigens coded (or determined) by the I-A and I-J, E, C subregions of the Ir region can be detected on the surface of cells required for the response. In addition, I-A-subregion products have a functional significance in cellular activation which can be demonstrated by blocking experiments with anti-Ia serum in the absence of complement.     

Antibody-dependent cellular cytotoxicity effector cells lack Ia antigens.

Surface immunoglobulin (sIg)-positive and sIg-negative subpopulations of macrophage-depleted murine splenic lymphocytes were obtained by Sephadex anti-Fab immunoabsorbent fractionation. These lymphocyte subpopulations were analyzed for the presence of Thy 1 and Ia alloantigens and also for Fc receptors by fluorescence microscopy. Concurrently, these lymphocyte subpopulations were studied for effector cell activity in antibody-dependent cellular cytotoxicity (ADCC). Effector cells mediating ADCC were contained in the sIg-negative lymphocyte subpopulation and sIg-positive lymphocytes did not mediate cytotoxicity. The majority of sIg-positive lymphocytes were found to bear Ia antigens and Fc receptors, and these cell surface structures were associated in that treatment of these cells with anti-Ia sera inhibited binding of complexed immunoglobulin to Fc receptors. In contrast, most sIg-negative, Thy 1-negative lymphocytes lacked Ia Antigens, and the Fc receptors detected on such cells were not blocked by anti-Ia sera. In addition, a small subpopulation of sIg-negative, Ia antigen-positive, Fc receptor-positive lymphocytes was found. Elimination of this subpopulation of Ia antigen-positive cells from sIg-negative lymphocytes, by treatment with anti-Ia serum and complement, did not diminish ADCC effector cell activity in the resultant cell population when compared with untreated sIg-negative lymphocytes. Thus, in murine spleen, nonphagocytic mononuclear cells that lack both sIg and Ia antigens were shown to mediate ADCC.     

Prolonged local expression of anti-CD4 antibody by adenovirally transduced allografts can promote long-term graft survival

BACKGROUND: Currently, successful transplantation of allografts requires the systemic use of immunosuppressive drugs. These can cause serious morbidity due to toxicity and increased susceptibility to cancer and infections. Local production of immunosuppressive molecules limited to the graft site would reduce the need for conventional, generalized immunosuppressive therapies and thus educe fewer side effects. This is particularly salient in a disease like type 1 diabetes, which is not immediately life-threatening yet islet allografts can effect a cure. METHODS: We studied the efficacy of locally produced anti-CD4 antibody, mediated by adenovirus (Adv-anti-CD4) transduction of islets, to enhance allograft survival. Adenovirus-transduced islets were transplanted under the kidney capsule of diabetic recipients and graft rejection determined by monitoring blood glucose levels. RESULTS: Adv-anti-CD4 transduction of mouse islets afforded protection against allogeneic rejection after transplantation into fully mismatched recipients. In some recipients, the islet allograft survival was prolonged (persisting for at least 15 weeks), corresponding to the prolonged expression of the anti-CD4 antibody. The effect was local, as absence of CD4+ T lymphocytes was observed primarily at the graft site. CONCLUSIONS: Immunosuppressive effects can be restricted locally by our strategy. Local production of a single antibody against one subset of T lymphocytes can protect mouse islets from allograft rejection during transplantation to treat diabetes. Our findings foreshadow that this strategy may be even more effective when a combination of antibodies are used and that similar strategies may prevent xenograft rejection.     

Identification of a cord blood T cell subset of CD3+4-8-45R+ suppressing interleukin 2 production in the autologous mixed lymphocyte reaction and the mode of action of exogenous IL2 in the induction of IL2 production

As previously reported, the inability of cord blood T cells to produce IL2 in the autologous mixed lymphocyte reaction (AMLR) could be recovered by the treatment of stimulator non-T cells with interferon-gamma (IFN-gamma) and of the AMLR with exogenous IL2. In the present study, we showed that addition of untreated autologous cord blood T cells to the above-mentioned AMLR abrogated the IL2 production in a dose-dependent manner, suggesting active suppression by the untreated T cells because untreated cord blood T cells did not consume IL2. Suppressor activity was abrogated by the treatment of cord blood T cells with monoclonal anti-CD3 antibody plus complement or with monoclonal anti-CD45R (Leu 18) antibody, but not by the treatment with monoclonal anti-CD4 antibody and/or anti-CD8 antibody plus complement. These data showed that the cord blood suppressor T cells were CD3+4-8-45R+. This suppressor activity also disappeared by culturing with rIL2 for 8 hr. As the frequency of CD45R+ cord blood T cells was comparable to that of CD45R+ adult T cells and was minimally affected by the IL2 treatment, functional modulation of CD45R+ suppressor T cells by IL2 is suggested. Moreover, in spite of the inhibitory effect of anti-CD45R antibody on the suppressor activity, IL2 production was not induced merely by addition of anti-CD45R antibody directly to the responder cells in AMLR. Taken together, these data suggest the requirement of exogenous IL2 for IL2 production in that IL2-producing-precursor T cells themselves should be stimulated by IL2 in addition to the modulation of CD45R+ suppressor T cells by IL2.     

Shared idiotope on monoclonal anti-Ia.7 antibodies reactive with determinants in a structural domain of the I-E molecules

In previous studies, heterologous anti-idiotypic (anti-Id) antisera against the C3H.SW 14-4-4S or the A.TH 41.A anti-Ia.7 monoclonal antibodies (mAb) were shown to identify an interstrain cross-reactive idiotypic specificity (IdX.Ia.7) expressed on monoclonal or conventional anti-Ia.7 alloantibodies. The objective of the present investigation was to characterize further this IdX at the idiotopic level. To this end, 11 hybridomas producing IgG1, IgG2a, or IgM anti-Id mAb were derived from a rat immunized with a mixture of 10 A.TH or A.BY anti-Ia.7 mAb. The specificity of the latter anti-Id mAb was determined by direct Id binding radioimmunoassay (RIA) with the use of a panel of 52 anti-Ia mAb derived from hybridomas produced in various inbred mouse strains. These rat anti-Id mAb recognized idiotopes expressed on i) all anti-Ia.7 mAb against determinants in the topographic domain I of the I-Ek molecule but not on 18 other anti-I-Ek mAb directed at epitopes in domains II or III; ii) three of 19 anti-I-Ak mAb; and iii) one A.TL-derived anti-I-As mAb. Competitive Id binding assays revealed that among the 14 IdX+ anti-Ia.7 mAb, one (81.B) was bound to a lesser extent by various rat anti-Id mAb, suggesting that heterogeneity probably exists in this antibody family. By contrast, two isologous (B10.S(7R)) anti-Id mAb to the IdX.Ia.7+ mAb 41.A displayed a specificity restricted to 41.A individual idiotopes (IdI). Rat anti-IdX.Ia.7 and mouse anti-41.A IdI mAb inhibited the binding of 125I-labeled mAb 41.A to CBA spleen cells. These two sets of mAb bound in a noncompetitive fashion to mAb 41.A-coated plates, indicating that their corresponding public or private idiotopes were spatially distinct. These data may have implications for in vivo manipulations of anti-Ia immune responses.     

A role for Ia antigens in thymocyte binding by macrophages

To investigate the membrane structures involved in cellular interactions between thymocytes and macrophages, the relative ability of different murine macrophage populations to spontaneously bind thymocytes was compared. Macrophages derived from the spleen or thymus bound three to four times the number of thymocytes than macrophages from peripheral blood, peritoneum, or bone marrow. This reflects differences both in the number of macrophages binding thymocytes and in the number of thymocytes bound per macrophage. The extent of binding seems to positively correlate with the number of Ia-positive macrophages contained in these populations, as based on previously published values. This was confirmed by showing that elimination of splenic Ia-positive macrophages with anti-Ia and complement treatment dramatically reduced thymocyte binding. In addition, mouse peritoneal washout macrophages incubated for several days with supernatant fluid from concanavalin A-stimulated spleen cells, which induce Ia-antigen expression, exhibited a marked increase in the number of macrophages that bound thymocytes and the number of thymocytes bound per macrophage. To determine if Ia antigens were directly involved in binding, spleen, thymus, or Ia-induced peritoneal macrophages were treated with a monoclonal anti-Ia antibody prior to the addition of thymocytes. Treatment with anti-Ia reduced binding by around 50%, whereas treatment with anti-H-2D antibody had no effect. Monoclonal anti-I-A and anti-I-E antibody treatments of macrophages both inhibited thymocyte binding to similar extents, and treatment of macrophages with both reagents together reduced thymocyte binding by 80%. These results indicate that thymocyte binding is in part dependent on macrophage Ia expression.