Immunological properties of Fc receptor on lymphocytes. 3. Stimulating activities of Fc receptor-bearing and -nonbearing cells in mixed-lymphocyte reaction.

By separating FcR⁺ and FcR^? cells from stimulator spleens using an EA rosetting procedure, it was found that EA-rosetting (FcR⁺) cells stimulate mixed-lymphocyte culture reaction (MLR) far more effectively than do non-EA-rosetting (FcR^?) cells. The difference in stimulatory activity is observed in MLR of both H-2 and M-locus different combinations and cannot be explained by the proportion of B cells and macrophages contained in each population. The finding that FcR⁺ cells can stimulate allogeneic responding T cells more effectively than FcR^? cells suggests a close association of FcR with Ia and Mls antigens on the cell surface.     

Immunological properties of Fc receptor on lymphocytes. 1. Functional differences between Fc receptor-positive and negative lymphocytes in humoral immune responses.

Using an EA rosetting system, it was observed that Fc receptors (FcR) were present on the surface of T cells as well as B cells, and that functional differences existed between FcR-positive (FcR⁺) and FcR-negative (FcR^?) cells in both T and B cells in in vivo humoral immune responses. Approximately 15% of splenic T cells obtained by nylon wool passage are FcR⁺. The number of surface immunoglobulinbearing cells as detected by immunofluorescent staining accounted for less than 10% of these FcR⁺ cells. FcR⁺ and FcR^? T+B-cell populations obtained from spleens contain 60 and 20% of surface immunoglobulin-positive cells, respectively. In the adoptive primary response in which horse RBC and dinitrophenyl-conjugated dextran (DNP-DE) were used as T-dependent and T-independent antigens, respectively, the majority of precursor B cells were FcR^?. In the secondary response using hapten-primed B cells and carrier-primed T cells, the majority of memory B cells for a haptenic determinant were also FcR^?. Furthermore, the majority of functional cells exerting helper activity in the same hapten-carrier system are FcR^? cells, and FcR⁺ T cells collaborate much less effectively with either memory B cells or helper FcR^? T cells.     

Immunological properties of Fc receptor on lymphocytes. 5. Suppressive regulation of humoral immune response by Fc receptor bearing B lymphocytes.

High anti-DNP PFC responses to DNP-DE or DNP-KLH were obtained by transferring normal or primed FcR^? B cell fractions into irradiated syngeneic recipients. On the other hand, the FcR⁺ B cell fraction showed a low precursor activity. Trypsinization of the FcR⁺ B cells, to eliminate remaining antigen-antibody complexes on the surface, failed to augment the response in comparison with that of trypsin-untreated FcR⁺ B cells. Therefore, the weak precursor activity of FcR⁺ B cells seemed to be inherent. No synergistic interaction between the FcR⁺ B and precursor FcR^? B cells, to give rise to the maximum PFC response, was observed. On the contrary, the FcR⁺ B cells significantly suppressed the PFC responses of FcR^? B cells. This kind of suppression could be mediated by a factor released from the FcR⁺ B cell, but not from the FcR^? B or original-unrosetted spleen cell fraction. The factor was not attributable to macrophages, because the FcR⁺ B cells isolated from normal spleen cells, of which macrophages were depleted by Sephadex G-10 columns, could produce the factor with the same activity. Stimulation by specific antigen is not necessary for the induction of the factor(s) as well as of the suppressing FcR⁺ B cells. It seems to be necessary to stimulate FcR by antigen-antibody complexes to produce or release this factor.     

Feedback regulation of immune responses by immune complexes; possible involvement of a suppressive lymphokine by FcR gamma-bearing B cell

Inoculations of antigen-antibody complexes (immune complexes) with the intact Fc portion generates suppressor cells in vivo by binding to FcR gamma on B cells via Fc portions. The cell type responsible for the suppression appears to be B cells bearing FcR gamma. Neither T cells nor macrophages participate in both the inductive and effective phases of this type of regulation. The suppression caused by splenic B cells, previously stimulated with immune complexes in vivo, is mediated by humoral factor(s) released from them. The suppressive factor(s) have H-2 gene product(s) coded by the right-hand side of the H-2 gene complex, but not for FcR gamma themselves or immunoglobulins. It has shared component(s) with suppressive B cell factor (SBF) released from FcR gamma + B cells stimulated with immune complexes in vitro, and it resembles SBF in its mode of action. These findings indicate that immune complexes, the final products of antibody responses, control the immune responses by stimulating surface FcR gamma on B cells. It is of interest that this type of regulation functions in vivo.     

Cytolytic T cells activated by H-2-controlled E molecules cross-react with A molecules

Cell-mediated lymphocytotoxicity was generated in four strain combinations differing only by the cell-surface expression of the class II E molecule controlled by the H-2 complex. The four combinations were: B10.D2(R107) anti-B10.A(3R), B10.A(4R) anti-B10.A(2R), B10.GD anti-B10.D2(R101), and B10.S(7R) anti-B10.S(9R). In all four of these combinations, the stimulator expresses E molecules on the cell surface, while the responder does not. The cytolytic T lymphocytes generated in the B10.D2(R107) anti-B10.A(3R) and B10.A(4R) anti-B10.A(2R) combinations reacted not only with the stimulator but also with strains that do not express cell-surface E molecules, in particular, strains carrying the H-2 ^f and H-2 ^q haplotypes. The cross-reactivity with E-negative strains could be blocked by monoclonal antibodies specific for the A^f or A^q molecules but not by antibodies recognizing determinants on E or class I (K) molecules. The anti-H-2^f cross-reactivity could be inhibited by H-2 ^q cold targets and, reciprocally, the anti-H-2^q reactivity could be blocked by H-2 ^f cold targets. These findings are interpreted as indicating that the cytolytic T lymphocytes stimulated by E molecules can recognize and lyse cells lacking E molecules but expressing A molecules. The observed E-A cross-reactivity supports the notion of structural and functional relatedness between the A and E molecules and suggests a common evolutionary origin of the A- and E-encoding loci.     

Further studies on non-H-2 histocompatibility alloantibodies in the mouse

Immunofluorescence tests indicate that alloantibodies specific for mouse histocompatibility antigens H-1^a, H-3^a, and H-13^a have been produced, using four different immunizations. Furthermore, an immunization employing donors and recipients which were H-2^k at the MHC produced stronger anti-H-3^a and anti-H-13^a than did immunizations where donors and recipients were H-2^b at the MHC.     

The spatial relationship of the viral and H-2 antigens recognized by anti-viral CTLs

With the use of monospecific rabbit anti-G protein and mouse monoclonal anti-H-2K^k, we have analyzed the spatial relationship of the serologically defined H-2K^k antigens and the major surface glycoprotein (G protein) of vesicular stomatitis virus (VSV) to those antigens recognized by B10.A (k, d) anti-VSV cytotoxic T lymphocytes (CTLs). The ability of monoclonal anti-H-2K^k or rabbit anti-G protein to inhibit specifically the cytolytic activity of B10.A anti-VSV CTLs indicates that the G protein and the H-2K^k molecules are in close proximity to the viral and H-2K^k antigens recognized by the anti-VSV (CTLs. By the method of sequential immunoprecipitation, we also demonstrated that only 10–30 percent of the serologically defined G and H-2K^k molecules are in theG-H-2K ^k complexes.Abbreviations used in this paper Con A Concanavalin A - cpm counts per minure - CTLs cytotoxic T lymphocytes - E: T ratio effector: target ratio - G major surface glycoprotein of VSV - MHC major histocompatibility complex - MOI multiplicity of infection - NP40 Nonidet-P40 - PAGE polyacrylamide gel electrophoresis - PBS phosphate-buffered saline - SaCI Staphylococcus aureus, Cowan I strain - SDS sodium dodecyl sulfate - UV ultraviolet light     

Immunological properties of Fc receptor on lymphocytes. 2. Differentiation from FcR- to FcR+ cells and their functional differences in in vitro antibody response.

In in vitro plaque-forming cell (PFC) response to particulate as well as to soluble antigen, the functional difference between Fc receptor-bearing (FcR⁺) and nonbearing (FcR^?) murine splenic lymphocytes was analyzed using the EA rosetting method. In the secondary anti-horse red blood cell (HRBC) response of C3H mice, FcR^? cells showed higher IgM and IgG responses than did FcR⁺ cells. When nylon wool (NW)-purified T cells primed with keyhole limpet hemocyanin (KLH) were fractionated into FcR^? and FcR⁺ T cells, helper activity was proven in the former subset in the cooperation with syngeneic spleen cells primed with dinitrophenylated ascaris extract (DNP-Asc). FcR⁺ T cells showed essentially no helper activity. When FcR^? cells were cultured, neogenesis of FcR⁺ cells was observed on Days 3 to 5. The conversion from FcR^? to FcR⁺ cells was prominent in B cells (40 to 50%), whereas NW-purified nonadherent FcR^? T cells converted poorly (15 to 20%). The converting process was accelerated slightly by mitogens, but was least affected by antigens. To examine the possible contribution of neogeneic FcR⁺ T cells in the helper activity, KLH-primed FcR^? T cells were precultured for 7 days with homologous antigen. The specific helper activity of the cultured T cells proved to be unaffected by the depletion of neogeneic FcR⁺ T cells by EA rosetting. The neogeneic FcR⁺ T cells had no helper activity. It was thus suggested that helper T cells remain in the FcR^? cell fraction and do not convert to the FcR⁺ state during the cooperating process.     

Comparative studies on FcR (FcRII, FcRIII, and FcR alpha) functions of murine B cells

Distribution of FcR II, FcRIII, and FcR alpha on murine splenic B cells was examined by using FITC-labeled heat-aggregated IgG of each subclass and IgA. Almost 60 to 80% of B cells expressed both FcRII and FcRIII. However, FcR alpha was expressed on only a small proportion (6%) of B cells that co-expressed FcRII. By inhibition assays with the use of cold IgG of each subclass and IgA in addition to anti-FcRII mAb (2.4G2), it was found that IgG1, IgG2a, and IgG2b utilized the same receptor (FcRII), whereas IgG3 and IgA bound only to their unique receptors, FcRIII and FcR alpha, respectively. Immune complexes IC prepared by IgG1, IgG2a, IgG2b, and IgA anti-TNP mAb with TNP-coupled SRBC inhibited the polyclonal Ig secretion and proliferative responses of B cells stimulated with either IL-4 or LPS. The inhibition of B cell activation was associated with the blockade of the membrane depolarization. Moreover, IC prepared by these antibodies caused production of suppressive B cell factor (SBF) as is the case with rabbit IgG antibody to SRBC, and SBF thus prepared regulated antibody responses in an isotype-nonspecific manner. In contrast, no inhibition for these responses or production of SBF was attained by the IC of IgG3 antibody. We concluded that FcRII and FcR alpha mediates a suppressive signal for B cells by acting on the initial step of activation, whereas FcRIII lacks this activity.     

A study of the ability of H-2Kk-Iak containing subcellular fractions to elicit primary anti-H-2 cytotoxic T lymphocytes

In order to identify an antigen required for elicitation of anti-H-2 cytotoxic T lymphocytes (CTLs), we have purified the H-2-K^k glycoprotein, incorporated it into a lipid vesicle, and tested it for its ability to elicit anti-H-2K^k CTLs. The results indicate that a lipid vesicle containing purified H-2K^k can elicit specific secondary anti-H-2K^k CTLs. In addition it was shown that in association with a partially purified Ia^k fraction the primary anti-H-2K^k CTL response was enhanced. It was also shown that Ia^k antigens alone could elicit an anti-H-2^k CTL response. Although a low primary response was found with purified H-2K^k, it was observed that lipid vesicles containing both H-2K^k and Ia^k glycoproteins could elicit a significantly enhanced primary anti-H-2K^k CTL response. Lipid vesicles containing H-2K^k-Ia^k were tested for their enhanced capacity to elicit anti-H-2 CTLs as well as for their ability to elicit anti-H-2K^k CTLs in the presence of supernatants from concanavalin A-stimulated spleen cells.     

Dissociation ofH-2 recognition by antibody and cytotoxic T cells of a cloned murine leukemia cell line

The differential expression of H-2 specificities recognized by antibody and by cytotoxic T lymphocytes (CTL) has been studied using a clone (FY7) of the C57BL/6 leukemia cell line FBL-3 (H-2 ^b /H-2 ^b ). Unlike C57BL/10 spleen cells, EL-4 lymphoma cells and Y57-2C leukemia cells (allH-2 ^b /H-2 ^b ), FY7 failed to induce the primary in vitro generation of anti-H-2^b CTL by (B10.A x A)F₁ (H-2 ^a /H-2 ^a or (B10.D2 x BALB/c)F₁ (H-2 ^d /H-2 ^d ) responder spleen cells. In addition, FY7 was not lysed by, and did not competitively inhibit anti-H-2^b CTL. Quantitative absorption tests with H-2K^b and H-2D^b antisera revealed that FY7 expressed these antigens in quantitatively similar amounts to EL-4. The H-2K^b product of FY7 appeared to be identical with that of C57BL/10 spleen cells both in apparent molecular weight and isoelectric point. Yet FY7 failed to inhibit anti-H-2K^b CTL competitively in a cold target inhibition assay. Possible mechanisms are discussed for the lack of T-lymphocyte recognition of the H-2K^b-gene product expressed by FY7.Abbreviations used in this paper CTL cytotoxic T lymphocytes - MHC major histocompatibility complex - MLC mixed lymphocyte culture - PAGE polyacrylamide gel electrophoresis     

MHC recognition by clones of Mls specific T-lymphocytes

To test whether M1s determinants, like other non-MHC or nominal antigens, are recognized by T-cells in association with H-2 determinants, the in vitro proliferative responses of T-cell lines and clones were studied. Lines and clones were prepared by soft agar cloning (B10.BR x BALB/c)F₁ (H-2^k/H-2^d, M1s^b/M1s^b) T-cells responding in a primary MLR to AKD2F1 (H-2^k/H-2^d, M1s^a/M1s^a) stimulator cells. All the T-cell clones obtained could respond equally well in a proliferative assay to the Mls^a determinant in association with the H-2 haplotype of either parent, i. e., DBA/2 (H-2^d, M1s^a), and AKR (H-2^k, M1s^a) both stimulated equally well. When the T-cell lines and clones were screened against stimulators from recombinant inbred (RI) strains, it became apparent that strains exhibiting the H-2^b, M1s^a genotype stimulated poorly or not at all. This shows that the T-cell response to M1s^a involves MHC recognition, and raises the possibility that the response to M1s^a can involve recognition of H-2 specificities shared between the H-2 ^k and H-2 ^d haplotypes.Abbreviations used in this paper MHC major histocompatibility complex - MLC mixed lymphocyte culture - IL-2 interleukin 2 - Con A concanavalin A - RI recombinant inbred Howard Hughes Medical Institute     

Interacting genetic factors controlling the antibody response against the H-2.2 specificity

The antibody response against the H-2.2 specificity has been studied in three H-2 ^d strains, B10.D2, DBA/2, and BALB/c, and their hybrids (B10.D2 × DBA/2)F₁ and (B10.D2 × BALB/c)F₁. The genetic control of the response appears to be complex: The three pure strains are responders, whereas both hybrids when immunized with C3H-HTG are nonresponders. Individual analysis of N3 offspring is compatible with the idea that, in this combination, an Ea-4 incompatibility between donor and immunized strain is necessary for the anti-H-2.2 response to occur. H-2 ^d /H-2 ^k hybrids (B10.BR × B10.D2)F₁ or (B10.BR × DBA/2)F₁ are responders when immunized with C57BL/10 (H-2 ^b ) but not with B10.A(2R) (H-2 ^h ), indicating that simultaneously recognized H-2 specificities are necessary for the anti-H-2.2 response.     

Murine antigen H-2.7: Its genetics,tissue expression,and strain distribution

Reinvestigation of alloantisera containing antibodies to murine antigen H-2.7 revealed that the crucial recombinant, A.TFR1 (H-2 ^an1 ), which was reported to separate theH-2G locus from theSs-Slp loci, has ak-like instead off-like H-2.7 antigen. Therefore, the crossover position inH-2 ^an1 and the position of the G locus in theH-2 map are now uncertain. By using the hemagglutination-serum inhibition test, anti-H-2.7 reactive substance was found to be present in normal mouse serum in a strain-specific manner. Tissue distribution study by absorption analysis indicated that H-2.7 antigen is present, in addition to RBCs, on spleen and lymph node cells, but is absent on thymus cells. Thirty B10.W congenic lines were analysed for the presence of the H-2.7 antigen. Two lines (B 10.CHA2 and B 10.KPA44) were found to be H-2.7 positive by both direct hemagglutination and absorption tests.Abbreviations used in this paper ACT Ammonium chloride Tris-buffer - BSA Bovine serum albumin - HA Hemagglutination - HASI Hemagglutination serum inhibition - HBSS Hanks balanced salt solution - PBS Phosphate buffered saline - PVP Polyvinylpyrrolidone - RBCs Red blood cells     

Close Association of Virus-Specific Cell Surface (S) and H-2 Antigens in Ad12-Infected and -Transformed Mouse Cells

A virus-specific cell surface (S) antigen in adenovirus type 12 (Ad12)-transformed mouse cells has been assumed to be a direct target for cytotoxic thymus-derived lymphocytes (CTL). In this study, the spatial proximity between the S and H-2 antigens was determined by three different methods, the proximity and co-capping tests, and the test for blocking of CTL-mediated lysis by anti-H-2 serum. In the proximity test with Ad12-infected thymic and splenic lymphocytes, and an Ad12-transformed line of C3H/He (H-2^k) mouse cells, anti-H-2^k and anti-S sera reciprocally inhibited fluorescent-antibody staining of the opposite antigens. By contrast, anti-Thy-1, 2 serum as well as anti-Ia and anti-Ig sera failed to show any appreciable effect in this test, when paired with anti-S serum. In addition, the S and H-2 antigens co-capped in the infected thymic lymphocytes, and CTL-mediated lysis of the transformed cells was abrogated equally by treatment of cells with anti-S and anti-H-2 sera. These results clearly demonstrate that there is a close proximity between the S and H-2 antigens on the surface of Ad12-infected and -transformed mouse cells.     

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.     

Studies on non-H-2-linked lymphocyte-activating determinants. IV. Ontogeny of the Mls product on murine B cells.

The minor lymphocyte stimulating (Mls) locus codes for lymphocyte activating determinants (LADs) on murine B lymphocytes, but not T lymphocytes. This observation was strengthened by a series of techniques which allow deletion and addition of T and B cells. These included the use of cytotoxic antisera such as anti-Thy 1.2, anti-MTLA, anti-MBLA, and complement, and the use of a goat anti-μ antisera, and finally the use of a fluorescence activated cell sorter (FACS).The studies in this report document the organ distribution and the ontogenetic appearance of the surface LADs on the surface of B lymphocytes from DBA/2N (H-2^d, Mls^a) and CBA/J (H-2^k, Mls^d) mice. Adult-like ability to stimulate H-2 identical BALB/c (H-2^d, Mls^b) and C3H/He (H-2^k, Mls^c) responder cells appeared at about 4–5 weeks of age. Inability of neonatal cells to induce an Mls-defined MLC was found not to be due to a low frequency of B lymphocytes or to the presence of suppressor cells, but due to the absence of the Mls-coded LADs on their surface. These data support the concept that the Mls-coded LADs are present on adult B lymphocytes and are specific markers of B-cell differentiation, which is preceded by membrane IgM and the δ homologue of human IgD, Ia, and the receptor for the third component of complement.     

Divergent appearance of complement receptors and Fc receptors on T cells in a murine mammary tumor system.

The emergence of novel T cells during mammary tumorigenesis has been previously described. T cells with surface markers usually associated with B cells, i.e. complement receptors (CR), appear in the spleens from tumor bearing mice. We now report on the appearance of Fc receptor (FcR) positive T cells in the spleens from the same animals. The kinetics of appearance of the two kinds of cells are similar.Based on evidence from double and triple label assays, it was concluded that FcR and CR are not coexpressed on the same T cell and that the two kinds of T cells which emerge do so in an independent fashion. Furthermore, they appear to represent a branch in the differentiation process influenced by tumor growth. The development of CR⁺ T cells represents an irreversible process as evidenced by the lack of change in the cells' representation following surgical procedures. In contrast the development of FcR⁺ T cells appears to be quite flexible in nature since mere surgical trauma as well as tumor mass removal can effect a decrease in the proportion of such cells.     

Activation of lymphocytes alters Fc receptor-beta 2-microglobulin interrelationship on the lymphocyte surface

The effect of anti-beta 2-microglobulin (anti-B2Mi) on the expression of Fc receptors (FcR) of human lymphocytes was compared on resting and activated cells. Previously we reported that anti-B2Mi induces a "co-shedding" of FcR with the beta 2-microglobulin (B2Mi)-anti-B2Mi complexes when used under the conditions where the redistribution of membrane molecules is allowed (Sármay et al., Cell. Immunol. 56, 452, 1980; Sármay et al. Immunology 36, 339, 1979). Furthermore our group also described two types of FcR-bearing cells, one which shed their FcR during a temperature shift from 4 to 37 degrees C (FcRI+ cells) and the other which has an immobile type FcR under the same circumstances (FcRI+ cells) (Sándor et al., Immunology 38, 553, 1979; Sármay et al., Immunology 34, 315, 1978). In this work we have characterized the FcR released from the membrane as a consequence of anti-B2Mi treatment. We have found that they are the mobile, FcRI type. It was proved that the shedding of this FcRI is a consequence of the anti-B2MI-induced transformation of FcRII into the FcRI form on the membrane of the antibody-treated lymphocytes. On the activated T cells, however, anti-B2Mi is incapable of inducing the same phenomenon in the early phase of activation. In contrast, FcR expression is blocked by anti-B2Mi treatment similarly to that on resting lymphocytes, on the surface of activated B cells, or on activated T cells in the later phases of activation.     

Genetic control of the immune response to the H-2Dk private specificity, H-2.32. II. Genetic linkage analysis of a backcross generation.

B10.AKM mice (H-2M) when immunized with H-2k cells showed very low cytotoxic antibody responses to the H-2Dk private specificity H-2.32, whereas AKR.M and (AKR.M X B10.AKM)F1 mice that possess the same H-2m haplotype mounted reasonable anti-H-2.32 antibody responses. The genetic nature of the non-H-2 linked gene(s) controlling the anti-H-2.32 response was analyzed on the backcross progeny raised between (AKR.M X B10.AKM)F1 and B10.AKM mice. The anti-H-2.32 antibody response was found to be predominantly controlled by a single locus. This locus segregated independently of the Ig heavy chain locus, the Ly2 locus, and the Mls locus. Despite the observed difference in antibody production, no significant differences between AKR.M and B10.AKM mice were detected in induction of H-2Dk-specific killer T cells. Thus, the defect in the response of B10.AKM mice to H-2.32 can be detected at the level of B cell function and is controlled by a single non-H-2-linked genetic locus, but is not attributable to genes linked to the major immunoglobulin structural genes nor to the Mls locus.