The specificity and significance of the inhibition of Fc receptor binding by anti H-2 sera

The possibility that Ia antigens are unique among H-2 antigens in their relationship to the Fc receptor was investigated in an EA rosette assay. Antibody specific for antigens in various regions of theH-2 complex was incubated with mouse cells, and the ability of the cells to form rosettes with antibody-coated chicken erythrocytes was tested. Antibody raised against the H-2 antigens of Ia-negative tumor cells was highly effective in inhibiting rosette formation. A variety of antisera againstK-, I-, andD-region antigens tested in recombinant mice inhibited EA rosette formation, suggesting that antigens in each of these regions could be detected in rosette inhibition. The F(ab′)₂ fragments of all antisera tested also produced specific EA rosette inhibition. Finally, antibody against Ia antigens failed to inhibit bone marrow RFCs, although antibody against H-2K and H-2D antigens did inhibit. Although H-2 serology is in a state of rapid change at present, it must be concluded that in this assay, antibody against antigens in theK andD regions as well as theI region can inhibit EA rosette formation. Inhibition of these rosettes by anti H-2 sera is therefore not due to a special association of Ia antigens with Fc receptors.     

A novel receptor on allograft (H-2d)-induced macrophage (H-2b) toward an allogeneic major histocompatibility complex class I molecule, H-2Dd, in mice

The generation of knockout mice demonstrated that noncytotoxic CD4(+), but not cytotoxic CD8(+), T cells were essential for the rejection of skin or organ allografts. Earlier we reported that allograftinduced macrophages (AIM) in mice lysed allografts with H-2 haplotype specificity, implying screening of grafts by AIM. Here, we isolated a cDNA clone encoding a novel receptor on AIM (H-2D(b)) for an allogeneic major histocompatibility complex (MHC) class I molecule, H-2D(d), by using H-2D(d) tetramer and a monoclonal antibody (mAb; R15) specific for AIM. The cDNA (1,181-bp) encoded a 342-amino acid polypeptide with a calculated molecular mass of 45 kDa and was found to be expressed on AIM, but not on resident macrophages or other cells, infiltrating into the rejection site. HEK293T cells transfected with this cDNA reacted with R15 mAb and H-2D(d), but not H-2L(d), H-2K(d), H-2D(b), H-2K(b), H-2D(k), or H-2K(k), molecules; and the H-2D(d) binding was suppressed by the addition of R15 or anti-H-2D(d) mAb. AIM yielded a specific saturation isotherm in the presence of increasing concentrations of H-2D(d), but not H-2D(b) or H-2D(k), molecules. The dissociation constant of AIM toward H-2D(d) tetramers was 1.9 x 10(-9) M ; and the binding was completely inhibited by the addition of R15 or anti-H-2D(d) mAb. These results reveal that a novel receptor for an allogeneic H-2D(d) molecule was induced on effector macrophages responsible for allograft (H-2(d)) rejection in H-2(b) mice.     

Intra-H-2 recombination in the mouse. III. I-Region characterization of haplotypes H-2at2, H-2at3 and H-2a2

Serological characterization of three K-S interval recombinant strains, TBR2 (H-2at2), TBR3 (H-2at3) and AIR 1 (H-2a2) was performed using anti-H-2, Ia, Ss and Slp antisera. The data presented here reveal that the crossover events in both TBR2 and TBR3 occurred between the I-A and I-E subregions. In both cases, the H-2K and I-A subregions were derived fron the H-2t1 of chromosome, while the I-E, S and H-2D regions were derived from the H-2b chromosome (KsAkEbSbDb). The H-2a2 chromosome resulted from a crossover event between the H-2a1 and H-2i9 chromosomes. Ia and Ss typing of AIR 1 suggested that the K to I-E regions originated from H-2a1 and the S and D regions originated from H-2i9 (KkAkEkSbDd).     

Properties of H-2L locus products in allogeneic and H-2 restricted, trinitrophenyl-specific cytotoxic responses.

The role of the recently defined L antigen (a second D region product) in allogeneic and TNP-specific syngeneic primary CML responses has been investigated. The lysis by anti-L specific cytotoxic effector cells was not inhibited when the target cells were pretreated with an antiserum directed against K and D, whereas an antiserum against L completely abrogated this response. Therefore, H-2L products are recognized on the target cell independently of H-2K and H-2D locus products. Both A.SW cells as well as B10 cells were found to respond to Ld alloantigens, in addition to Dd alloantigens when stimulated by cells differing only in the D region. The results of cold target blocking and antiserum inhibition experiments failed to detect cytotoxic cells with specificity of L antigens in association with TNP, under conditions in which TNP-specific effectors to K and D antigens were demonstrable. These findings suggest that there is a more limited involvement of H-2L locus products than the H-2K or H-2D locus products in the induction and specificity of these responses.     

Similarity of the carbohydrate structures of H-2 and Ia glycoproteins

The glycopeptides produced by pronase digestion of two H-2K, two H-2D, and three Ia glycoprotein antigens were examined for size and charge. The glycopeptides derived from all of the antigens examined were found to have m.w. of 3250 +/- 200 daltons with a similar and variable composition of sialic acid residues. These data, when combined with the similarity in monosaccharide incorporation, suggest that the general parameters of the carbohydrate structure of the Ia glycoproteins from different I subregions and H-2 glycoproteins are highly similar if not identical.     

Studies on mouse autoreactive cells: I. Role of H-2 antigens in mouse autologous rosette formation

A minority (1–2%) of normal mouse lymphoid cells bind autologous erythrocytes and form rosettes. In this study we examined the antigenic specificity involved in the formation of such rosettes. A significant difference in the incidence of rosettes formed, respectively, with autologous and allogeneic mouse erythrocytes is found. Moreover, preincubation of lymphoid cells with low concentrations of syngeneic erythrocytic ghosts causes significant competitive inhibition of subsequent rosette formation. Allogeneic ghosts obtained from nonrelated or from congenic resistant strains of mice do not display this inhibitory effect under the same conditions. It is thus suggested that mouse autologous rosette-forming cells bear receptors for syngeneic H-2 antigens that are involved in the binding of autologous erythrocytes. More precisely, compatibility between lymphocyte and erythrocyte restricted to K or D only is sufficient to ensure a level of rosettes similar to that obtained when complete identity occurs for K, I, and D regions.     

Effects of masking H-2 and Ir gene products on the in vitro responses of cells from immunized and naive mice.

Mouse spleen cells from immunized and naive animals were cultured for 3 days with SRBC and antisera to H-2K, H-2D, Ia, or aggregated IgG. Direct and indirect antibody responses were impaired by each of these agents only when the responding cells had been obtained from unimmunized mice. It was found that as lymphoid cells reacted to the antigen and progressed toward a fully matured antibody response, they first became resistant to inhibition by anti-H-2K and H-2D sera and somewhat later they lost their vulnerability to agents that reacted with Ia antigens.     

Characterization of T-cell-soluble factors modulating the expression of Ia and H-2 antigens on BALB/c B lymphoma cell lines

The effect of supernatants of concanavalin A-activated spleen cells (CAS) on the expression of various antigens, especially Ia antigens, on BALB/c B lymphoid cells, was examined. This study demonstrates the following: (i) CAS enhanced the expression of Ia antigens on four out of five BALB/c lymphoid cell lines. (ii) CAS selectively modulates the expression of Ia and H-2D, but not sIgM or viral gp70 expression, on X16C 8.5 tumor cells. The enhanced levels of Ia expression on B lymphoid tumor cells were also detected by using anti-Ia monoclonal antibodies. (iii) The molecular weight of soluble factor(s) affecting Ia and H-2 was approximately 40,000 estimated by gel filtration on a Sephadex G-200 column. (iv) Type 1 interferon but not interleukin 1, interleukin 2, or T-cell-replacing factor enhanced the expressions of Ia and H-2D antigens. (v) The activity of CAS-modulating Ia and H-2 antigens was eliminated by acidic treatment. It was concluded from this study that at least one of the factor(s) in CAS, modulating the antigenic expression of B-lymphoid cells, was interferon-like in nature. From our findings, a possible immunoregulatory mechanism by interferon was suggested: T cells, after stimulation of mitogens or antigens, secrete interferons which modulate the expression of Ia and H-2 on B cells. Then B cells, whose Ia and H-2 were modulated selectively by T-soluble factors(s), might interact with T cells much more efficiently.     

The I-A beta b region (65-85) is a binding site for the superantigen, staphylococcal enterotoxin A

Ia antigen is a receptor for the superantigen staphylococcal enterotoxin A (SEA). Peptides I-A beta b(30-60), I-A beta b(50-70), I-A beta b(65-85), and I-A beta b(80-100) of the MHC class II antigen beta chain on mouse (H-2b) accessory cells were synthesized. Only I-A beta b(65-85) inhibited SEA binding to the mouse B-cell lymphoma line, A20 (H-2d) and the human Burkitt's lymphoma line, Raji (HLA-DR). The I-A beta b(65-85) sequence is a predicted alpha-helix along the hypothetical antigen binding cleft of the Ia molecule. I-A beta b(65-85) also directly and specifically bound both the intact SEA molecule and its Ia binding site, represented by the peptide SEA(1-45). The results suggest that I-A beta b region (65-85) is a necessary site for Ia molecular interaction with the superantigen SEA. Further, the data suggest that the same helical region of other Ia antigens binds SEA irrespective of haplotype and species.     

Binding of bacterial lipopolysaccharide to histocompatibility-2-complex proteins of mouse lymphocytes.

The membrane binding sites for lipopolysaccharide (LPS) were isolated by affinity chromatography of the solubilized membranes prepared from 125I-labeled mouse B-cells and T-cells on an affinity adsorbent prepared by coupling Salmonella minnesota R595 LPS to activated Sepharose 4B. The membrane proteins bound to the affinity adsorbent and eluted with 1.0% Triton X-100 were analyzed according to their mobility on polyacrylamide gel electrophoresis in sodium dodecylsulphate. These membrane proteins were further identified by immunoprecipitation with specific antisera. Immunoglobulins, possibly immunoglobulins M and D, were identified in the eluate from the B-cell membranes. The histocompatibility-2-complex proteins (H-2D, H-2K and Ia antigens) were also found to be binding sites for LPS on both B-cells and T-cells.     

Interferon-gamma induces enhanced expression of Ia and H-2 antigens on B lymphoid, macrophage, and myeloid cell lines

The levels of class II major histocompatibility complex (MHC) antigens (la antigens) on cells of a cultured B lymphoma line (WEHI-279) were significantly increased after 24 hr incubation with medium conditioned by concanavalin A-stimulated mouse or rat spleen cells, or by an azobenzenearsonate- (ABA) specific T cell clone that had been stimulated with ABA-coupled spleen cells or concanavalin A. The levels and properties of the la-inducing activity correlated with those of interferon-gamma (IFN-gamma) measured by inhibition of virus plaque formation. Both the la-inducing activity and the IFN-gamma from the T cell clone had an apparent m.w. of 40,000 determined by gel filtration, were sensitive to treatment with trypsin or exposure to pH 2, but were stable to heat (56 degrees C, 1 hr). The induction of la antigens on WEHI-279 cells was dose-dependent, and the maximum response occurred at a concentration corresponding to 1 to 2 U/ml of antiviral activity. This T cell-derived IFN-gamma-like molecule also increased the expression of cell surface la antigens on another B cell line (WEHI-231), and cell lines of macrophage (J774) and myeloid (WEHI-3B and WEHI-265) origin. Furthermore, in all cases the levels of class I MHC (H-2K or H-2D) antigens were also increased. Similar patterns of induction of Ia and H-2 antigens were obtained with supernatants containing IFN-gamma produced by a monkey cell line (COS) that had been transfected with a plasmid bearing the cloned murine IFN-gamma gene. This activity was sensitive to pH 2 and was not present in the supernatant from COS cells that were not transfected with the murine IFN-gamma gene. These results established that IFN-gamma is the T cell-derived molecule that induces the enhanced expression of Ia and H-2 antigens on B cells and macrophages. A major physiologic role of IFN-gamma may be to regulate immune function through the enhanced expression of MHC antigens.     

Functional changes in murine macrophages infected with cytomegalovirus relate to H-2-determined sensitivity to infection

Peritoneal macrophages were infected with murine cytomegalovirus in vitro, and indices of infection and macrophage function were monitored over 4 days. When the cells were assessed for the expression of viral antigen or for cytopathic effects, infection was found to be solely determined by the H-2 phenotype. Less than 10% of the macrophages from resistant H-2k strains were affected, whereas 90% of H-2d cells and approximately 80% of H-2b and H-2a cells became infected. Similar trends were demonstrable by the measurement of viral DNA. In H-2a cells (B10.A), Dd conferred sensitivity despite the resistant K and class II phenotype. The findings suggest a critical association between the class I antigens and an early stage in the infectious process. Indices of infection were paralleled by a loss of Fc receptor expression and optimal colloidal gold uptake, whereas most cells remained trypan blue negative, retained dehydrogenase and acid phosphatase activities, and did not release infectious virus during the period of study. This is consistent with a role for macrophages in the persistence of cytomegalovirus in the host.     

Characterization of a spontaneous murine B cell leukemia (BCL1). I. Cell surface expression of IgM, IgD, Ia, and FcR.

The surface marker expression of a spontaneous B lymphocyte leukemia discovered in a BALB/c mouse (BCL1) was examined and found to include a subset of markers known to occur on normal B lymphocytes. The tumor cells bore surface Ig that included both mu- and delta-chains associated with the lambda light chain. Alloantigens coded for within the murine MHC, including H-2D, H-2K, and I-region products, were identified on the tumor cells. Although normal B lymphocytes are thought to express products coded for within both the I-A and I-E subregions, the BCL1 expressed only normal amounts of I-E subregion products. In addition, the H-2 and Ia antigens revealed by 2-dimensional gel electrophoresis exhibited an abnormal pattern of post-translational modifications. The Fc, but not the complement-receptor, was present on the surface of tumor cells. The presence of IgD, Ia antigens, and the responsiveness to lipopolysaccharide (see subsequent paper) have led us to postulate that the BCL1 tumor represents a later differentiative stage than murine B lymphocyte tumors previously described.     

Murine IgA binding factors produced by Fc alpha R(+) T cells: role of Fc gamma R(+) cells for the induction of Fc alpha R and formation of IgA-binding factor in Con A-activated cells

The relationship between the production of a T cell factor having affinity for IgA (IgA-binding factor(s); IgA BF) and the expression of Fc receptors specific for IgA (Fc alpha R) was studied by using murine spleen cells activated with concanavalin A (Con A blasts). Fc alpha R was detected by the cytophilic binding of anti-TNP murine IgA myeloma protein (MOPC 315 IgA) to Con A blasts as determined by an indirect rosette method with trinitrophenylated sheep red blood cells (TNP-SRBC). After 18 hr preculture with IgA, Fc alpha R was expressed on 15 to 20% of Con A blasts, which released IgA BF suppressing the in vitro IgA synthesis of the spleen cells stimulated with pokeweed mitogen (PWM). Without preculture with IgA, there was neither induction of Fc alpha R nor the production of IgA BF from Con A blasts. Fc alpha R was not induced on Con A blasts by IgA if Fc gamma R(+) cells were depleted from the blasts by rosetting with SRBC sensitized with rabbit IgG antibody (EA gamma). Even after preculture with IgA, the suppressive IgA BF was undetectable in the culture supernatant of Con A blasts depleted of the Fc gamma R(+) cell population. By using a double rosette method with EA gamma and trinitrophenylated quail red blood cells, Fc alpha R proved to be co-expressed on Fc gamma R(+) precursor T cells in the Con A blasts. The results suggested that both Fc gamma R and Fc alpha R could be co-expressed on Con A blasts, as is the case with T2D4 Fc gamma R(+), Fc alpha R(+) T hybridoma cells, which are known to produce IgG-binding factor(s) (IgG BF) and IgA BF. The relationship between Fc gamma R and Fc alpha R on a single cell was studied by using monoclonal anti-Fc gamma R antibody (2. 4G2 ). The reactivity of 2. 4G2 antibody with T cell Fc gamma R was proved by the inhibition of EA gamma rosette formation by Con A blasts or T2D4 cells. The addition of 2. 4G2 monoclonal antibody, however, did not affect the induction of Fc alpha R on Con A blasts by IgA. Furthermore, the binding of IgA to Fc alpha R already expressed on L5178Y T lymphoma cell line cells was not inhibited by the monoclonal antibody. The results confirmed that Fc alpha R are distinct from Fc gamma R co-expressed on the same Con A blasts, and that the expression of Fc alpha R on Fc gamma R(+) T cells and their production of suppressive IgA BF may be induced by the binding of IgA to Fc alpha R.     

Tumor metastases and cell-mediated immunity in a model system in DBA/2 mice. VIII. Expression and shedding of Fcγ receptors on metastatic tumor cell variants

The expression of receptors for the Fc portion of IgG immunoglobin molecules was studied on tumor cell lines with high and low metastatic capacity. Two tumor cell lines from DBA/2 mice that had high metastatic activity, ESb and MDAY-D2, contained a high percentage of Fc receptor positive cells, as detected in a rosette assay with IgG antibody-coated erythrocytes (EA). In contrast, the low metastatic parental line Eb, from which ESb was derived, contained only a low percentage of EA-rosette-forming cells. ESb ascites tumor cells adapted to tissue culture in the presence of 2-mercaptoethanol (2ME) had a high expression of Fc receptors, whereas a cell line adapted to tissue culture in the absence of 2ME had a low expression of Fc receptors. “Soluble” Fc receptors were detectable by their ability to bind to EA and to cause blocking of rosette formation. They were found to be present in fluids from tumor-bearing animals, such as serum and cell-free ascites. Even animals with an ascites tumor of the low-metastatic line Eb contained “soluble” Fc receptors. The results are discussed with regard to their possible significance for tumor metastasis.     

Cutting edge: the minor histocompatibility antigen H60 peptide interacts with both H-2Kb and NKG2D

Minor histocompatibility Ags elicit cell-mediated immune responses and graft rejection in individuals receiving MHC-matched tissues. H60 represents a dominant Ag that elicits a strong CTL response in C57BL/6 mice immunized against BALB.B. An 8-aa peptide in the H60 protein is presented by H-2K(b) and this is recognized by the TCR as an alloantigen. The intact H60 glycoprotein is a ligand for the costimulatory NKG2D receptor that is expressed by activated CD8(+) T cells. Thus, H60 may provide both an allogeneic peptide and its own costimulation. We show that mutation of an H-2K(b)-binding anchor residue in the H60 peptide completely abrogates binding of H60 glycoprotein to NKG2D and a synthetic H60 peptide partially blocks the binding of NKG2D to its ligand. Ligands of the human NKG2D receptor are remarkably polymorphic, suggesting that these may also serve as minor histocompatibility Ags.     

Abelson virus-transformed lymphocytes: null cells that modulate H-2.

A-MuLV-transformed lymphoid cells from Balb/c mice had the properties of null lymphocytes. They did not secrete Ig and all but one did not have detectable cell-associated Ig; one line synthesized, but did not secrete, the mu chain of IgM. The cells expressed H-2D and H-2K, but not H-21 histocompatibility antigens or theta-antigen; they had Fc receptors. Most cell lines grew to form donor cell tumors after inoculation into (Balb/c X C57B1/6)F1 mice. The tumor cells have more H-2Dd than cells passaged in vitro. Cell lines carried in vitro progressively lost H-2Dd. A line in which 5-30% of the cells were lysable by anti-H-2Dd was cloned; all eleven clones had H-2Dd (13-69% lysable) demonstrating that H-2 modulates in vitro. A clone with little H-2Dd (10-15% lysable) was tumorigenic even after treatment with anti-H-2Dd sera; at least 50% of the tumor cells were lysed by anti-H-2Dd. Thus A-MuLV-transformed lymphocytes modulate H-2 in vivo to higher levels and in vitro to lower levels.     

Isolation and characterization of membrane receptors for pokeweed mitogens from mouse lymphocytes.

The major glycoproteins that bind pokeweek B-cell mitogen (Pa-1) and pokeweed T-cell mitogen (Pa-2) were isolated and identified from bone-marrow-derived lymphocytes (B-cells) and thymus-derived lymphocytes (T-cells) of C3H/He mice. The surfaces of the cells were 125I-labelled by using the enzyme lactoperoxidase, and the plasma membranes were isolated from the 125I-labelled cells. These membranes were solubilized with Triton X-100 and subjected to affinity chromatography on the affinity adsorbent prepared by coupling mitogen Pa-1 or Pa-2 to activated Sepharose 4B. The glycoproteins specifically eluted with di-N-acetylchitobiose from the affinity adsorbents were analysed according to their mobility on polyacrylamide-gel electrophoresis in sodium dodecyl sulphate. These glycoproteins were further identified by immunoprecipitation with specific antisera. Immunoglobulins, possibly immunoglobulins M and D, were identified in the eluate from the B-cell membranes, but they were not detected in the eluate from the T-cell membranes. The histocompatibility-2-complex proteins (H-2D, H-2K and Ia antigens) were found to be major receptor sites for the pokeweed mitogens on both B-cells and T-cells. However, mitogen Pa-1 (B-cell) has a stronger affinity to Ia antigens than does mitogen Pa-2 (T-cell).     

Effects of anti-beta-2 microglobulin antibodies on human lymphocytes.

Anti-β2 microglobulin antisera prepared in rabbits immunized with β2m purified from the urine of a single patient were cytotoxic for human T and B lymphocytes of all donors tested; lymphocytotoxicity could be fully inhibited by all human sera tested, not by serum from other animal species. Anti-β2 microglobulin antibodies and their F(ab′)2 fragments had little effect on E and EAC rosette formation, suggesting that β2m is not closely associated with receptors for sheep erythrocytes on T lymphocytes or receptors for C3 on B cells. Anti-β2m IgG and F(ab′)2 fragments inhibited EA rosette formation though the latter did not impair lysis of antibody-coated xenogeneic erythrocytes by lymphocytes bearing receptors for the Fc portion of IgG. Some of the antisera had a mild mitogenic effect, all of them inhibited mitogen and antigen-induced lymphocyte proliferation at high concentrations whereas they potentiated these responses at low concentrations. In mixed lymphocyte cultures pretreatment of responding cells markedly depressed the response whereas coating of stimulating cells with β2m antibodies had little or no effect.