Analysis of the response of B cells from CBA/N-defective mice to nonspecific T cell help.

We have investigated the induction of antibody responses to erythrocyte (RBC)-bound antigens in the (CBA/N x B10)F1 mouse. Male B cells, which express the CBA/N defect, were shown to be unresponsive to RBC antigens when the delivered T cell helper activity was solely nonspecific. Thus we demonstrated that defective B cells did not respond to concanavalin A supernatants or bystander helper activity, in spite of the fact that CBA/N-defective mice could produce these T cell activities. The defective B cell did not respond to RBC-bound antigen in the presence of RBC-primed T cells, although the magnitude of this response was usually twofold less than normal controls. The insensitivity of CBA/N defective B cells to nonspecific T cell helper activities seemed to involve at least the inability of RBC antigens to activate defective B cells in the absence of antigen-specific T cell help.

Idiotype profile of an immune response. II. Reversal of the relative dominance of major and minor cross-reactive idiotypes in arsonate-specific T-independent responses.

Two different cross-reactive idiotype (CRI) groups are distinguishable in the Ab response of A/J mice to the p-azobenzenearsonate (ABA) hapten: CRIA and CRIm. These two groups showed distinct patterns of relative dominance in the ensuing response depending on whether the inducing Ag was a T cell-dependent (TD) form of ABA, such as ABA-KLH or ABA-CGG, or a T-independent type 1 (TI-1) form, such as ABA-Brucella abortus or ABA-lipopolysaccharide (LPS), and on whether the response was elicited in vivo or in vitro. The CRI+ component of primary in vivo plaque-forming cell (PFC) responses to TD ABA Ags was largely (greater than 90%) CRIA+ as was, to a slightly lesser extent (greater than 75%) the CRI+ portion of secondary or hyperimmune serum Ab or PFC responses to the same Ags. In contrast, in vivo primary and hyperimmune PFC responses to ABA-Bru or ABA-LPS showed a significantly lower CRIA/CRI ratio, averaging 0.5-0.6, with some individual mice giving figures as low as 0.2, indicating predominance of CRIm over CRIA. Serological analysis of hyperimmune anti-ABA Abs from a group of 5 A/J mice immunized with ABA-Bru gave a figure of less than 0.5 for the CRIA/CRI ratio. The most striking disparity from the TD pattern was seen in primary in vitro PFC responses to the TI ABA Ags; here ratios of less than 0.2 were generally seen. Since T cell removal did not alter the Id pattern in the TI responses, CRIA-specific Ts cells do not account for the weak expression of CRIA in such responses. We propose a model that explains these results on the basis of differential expression of IdX dominance by two distinct B cell subpopulations--equatable to the Lyb-5+ and Lyb-5- B cell subsets--along with differential relative activation of these subsets in different types of responses. Examination of anti-ABA PFC responses of F1 progeny of CBA/N and A/J mice to ABA-Bru lends support to this hypothesis since CRIA expression was significantly lower in mice with the xid defect.

Unbalanced X chromosome mosaicism in B cells of mice with X-linked immunodeficiency.

The immunodeficiency in CBA/N mice is reflected by abnormal development of a subset of B lymphocytes. However, it is not clear how xid, the mutant gene in CBA/N mice, affects the development of this subset. Specifically, it is not known if the xid gene influences the development of the B cell subset directly or indirectly by providing the improper developmental milieu through effects on other cells. We investigated this question using female mice heterozygous for two x chromosomal genes, xid and Pgk-1 (phosphoglycerate kinase-1). Since females are mosaic because of x chromosome inactivation, their lymphocytes can be studied for the choice of the x chromosome, using the two PGK-1 isoenzymes as the cytological marker. We find that B lymphocytes in the spleen prefer the x chromosome without xid while the remaining splenocytes and cells from other tissues do not. This suggests that xid affects B lymphocytes directly and not through their developmental milieu. Furthermore, our data suggest that the precursors for IgG1- and IgG3-producing cells may be both few and different.

Mapping the locus of the H-Y gene on the human Y chromosome.

The H-Y locus is on the short arm of the human Y chromosome in most individuals but on the long arm in at least one of 17 individuals with structural abnormalities of the Y.

H-Y antigen and the genetics of sex determination.

Widespread phylogenetic conservation of H-Y antigen indicates persistence of a vital function. It has been proposed that this function is the primary determination of mammalian sex. According to this proposal, the indifferent embryonic gonad is induced to differentiate as a testis in the presence of H-Y antigen, and as an ovary in the absence of H-Y antigen. But presence of H-Y antigen does not guarantee testicular differentiation. Other factors may be required: a gene that activates the H-Y structural locus, and another gene that codes for specific H-Y antigen receptors.

Absence of IgD-CD27(+) memory B cell population in X-linked hyper-IgM syndrome.

The present study analyzed peripheral blood B cell populations separated by IgD and CD27 expression in six males with X-linked hyper-IgM syndrome (XHIM). Costimulation of mononuclear cells from most of the patients induced no to low levels of class switching from IgM to IgG and IgA with Staphylococcus aureus Cowan strain (SAC) plus IL-2 or anti-CD40 mAb (anti-CD40) plus IL-10. Measurable levels of IgE were secreted in some of the patients after stimulation with anti-CD40 plus IL-4. Costimulation with SAC plus IL-2 plus anti-CD40 plus IL-10 yielded secretion of significant levels of IgG in addition to IgM, but not IgA. The most striking finding was that peripheral blood B cells from all of the six patients were composed of only IgD+ CD27(-) and IgD+ CD27(+) B cells; IgD- CD27(+) memory B cells were greatly decreased. IgD+ CD27(+) B cells from an XHIM patient produced IgM predominantly. Our data indicate that the low response of IgG production in XHIM patients is due to reduced numbers of IgD- CD27(+) memory B cells. However, the IgG production can be induced by stimulation of immunoglobulin receptors and CD40 in cooperation with such cytokines as IL-2 and IL-10 in vitro.

Chronic ECHO type 5 virus meningoencephalitis in X-linked hypogammaglobulinemia: treatment with immune plasma.

A patient with X-linked hypogammaglobulinemia developed chronic meningoencephalitis. ECHO virus type 5 was repeatedly cultured from cerebrospinal fluid (CSF). Infusions of high-titer, specific plasma resulted in clinical improvement, but failed to eradicate the virus. After more intensive plasma infusions, the virus could not be cultured from the CSF. The patient died 8 months after institution of intensive therapy. The cause of death was unknown. Autopsy showed persistence of perivascular and meningeal inflammation. Specific anti-ECHO-virus-5 plasma was shown to be more effective in lowering CSF ECHO-virus titers than was plasma without anti-ECHO-virus antibody.

Serological detection of H-Y antigen in humans with a cellular radioimmunobinding assay and monoclonal antibody.

A sensitive and reliable serological assay for the detection of H-Y antigen is described which uses monoclonal H-Y antibody and a cellular radioimmunobinding assay on cultured human fibroblasts. Cell lines from 2 normal human females, 2 normal human males and one XX human male were tested in 3 separate assays. Cells from XY and XX males were found to contain H-Y antigen; however, the reaction against the XX male cells was found to be intermediate between the XY male and normal XX female cells.

Study of H-Y antigen in abnormal sex determination with monoclonal antibody and an ELISA.

A newly developed enzyme-linked immunosorbent assay (ELISA) has been applied to the study of H-Y antigen in cases of XY, XYY, and X,dicY gonadal dysgenesis, testicular feminization syndrome, XXXXY syndrome, and XX true hermaphroditism. Monoclonal H-Y antibody was absorbed with cells from each of eight patients and from normal male and female controls, and then reacted with a plated antigen source in a system subsuming the addition of biotinylated secondary antibody, avidin-biotin-enzyme complex and substrate, and thereby the generation of a color. Positive absorption decreased the reaction, and this allowed sensitive measurement of H-Y phenotype in an electronic optical density reader. The ELISA obviates many of the technical difficulties encountered in complement-mediated cytotoxicity systems and can be used in the study of clinical cases of aberrant sex determination and in the evaluation of current models of the genetics of sex determination.

[Y-chromosome polysomy and expression of the H-Y antigen]. / Polysomie du chromosome Y et expression de l'antigène H-Y.

The sex-differentiation H-Y antigen was studied in 5 males subjects with Y-chromosome polysomy. All had gonadal deficiency and significant decrease of H-Y antigen as compared with 77 normal 46, XY male controls. Men with additional Y-gonosomes may be hypogonadic or, more often, fertile, as was probably the case with the 3 patients with very high H-Y antigen titers previously described by other research workers. The phenotypic polymorphism of these subjects therefore seems to be parallel with their H-Y antigen polymorphism and there may be, in our 5 subjects, a relationship between abnormal spermatogenesis and H-Y antigen deficiency.

Immunity to Plasmodium yoelii and Babesia microti: modulation by the CBA/N X-chromosome.

CBA/N mice carry an X-linked recessive defect expressed in cells of the B cell lineage. The major deficiency in these mice is an almost complete inability to respond to certain thymus-independent antigens, such as pneumococcal polysaccharide type III (S III). We have examined the responses of mice carrying the CBA/N X-chromosome to the malaria parasite Plasmodium yoelii and the piroplasm Babesia microti. We have found that the duration and severity of these infections is increased in mice carrying the CBA/N X-chromosome and that this is associated with a markedly defective IgM antibody response to the parasitized red cell and a failure to produce autoantibodies to bromelain-treated mouse RBCs. An autoimmune response directed at modified determinants on the red cell membrane may be one of the factors involved in the control of these infections.

Clinical, pathologic, and genetic findings in a case of 46,XY pure gonadal dysgenesis (Swyer's syndrome). II. Presence of H-Y antigen.

Evidence that expression of histocompatibility-Y (H-Y) antigen on human cells is determined by a Y-linked gene is provided by data demonstrating that male subjects with two Y chromosomes have higher antigen levels than male subjects with one Y chromosome. The widespread evolutionary conservation of H-Y antigen and its association with the Y chromosome suggest that the antigen has a specific, crucial function. We surmise that this function is the differentiation of the embryonic gonad into whichever mature gonad, testis or ovary, typifies the heterogametic sex of each species. Of particular interest are individuals whose gonadal sex does not correspond to their somatic genotype. In the present article, we report positive results in the first case of 46,XY pure gonadal dysgenesis (Swyer's syndrome) to be typed for H-Y antigen. This case suggests that the presence of H-Y antigen may not be sufficient to complete masculinization of the embryonic mammalian gonad. Alternatively, a mutant gene may govern expression of a cell surface component which cross reacts with H-Y antigen but which lacks the ability to function in the virilization of the gonad.

H-Y antigen: localization of the H-Y gene.

Testicular development in a patient with deletion of the distal (fluorescent) segment of the Y chromosome is described. The presence of a normal dose of H-Y antigen was demonstrated by Goldberg's cytotoxicity test. It is concluded that the distal fluorescent segment of the Y chromosome is void of genes regulating H-Y antigen activity.

The recognition of abnormal sex chromosome constitution by HLA-restricted anti-H-Y cytotoxic T cells and antibody.

Using the cell-mediated lympholysis (CML) technique, we studied lymphocytes of six individuals with discrepancies between the karyotypic and phenotypic sex. Two sets of cytotoxic T cells (CTLs) obtained from two multitransfused female aplastic anemia patients were used as typing reagents. These cells were previously shown to kill allogeneic target cells from HLA-A2- or B7-positive male donors. An antiserum obtained from one of the patients likewise killed HLA-A2 male lymphocytes. The six patients studied were selected for the required antigens. Positive reactions were obtained with lymphocytes from a 46,XY woman with pure gonadal dysgenesis and a 45,XO male. Target cells of the mother of the latter patient were also lysed. One individual with a 45,XO/46,X,del(Y)? karyotype was weakly positive, while three 46,XX males were completely negative. The reactivity of the HLA-A2-restricted H-Y-specific antibody showed the same discriminatory patterns. The results obtained by the HLA-restricted CTLs as well as by the antiserum did not correlate with the presence of testes as is the case in a different test system for the serologically detectable male (SDM) antigen in man. On the other hand, there was a correlation with the presence of cytologically detectable Y-chromosome material in five of the six individuals studied. The HLA-restricted CTLs and the antibody might recognize the classical transplantation antigen H-Y.

Editorial: Y-linked genes and male-sex determination.

PIP: An antigen known as the H-Y antigen behaves as if determined by a y-linked gene. The availability of an assay system has provided remarkable new information. On the basis of a finding that males with XXY and XYY/XXYY karytoypes have a greater amount of antigen on leukocytes than normal males, a genetic determinant for the H-Y antigen can be assigned to the human Y chromosome with reasonable confidence. This antigen may serve as a qualitative and quantitative marker for the presence of a Y chromosome or the region containing the H-Y genetic determinant. Persons with 46,XX karyotype but with male development and with testes occur about once in every 15,000 newborn males. When an autosomal dominant condition in the mouse known as sex-reversed (Sxr) occurs, the XX mouse heterozygous for Sxr is said to be H-Y antigen positive which is consistent with the postulate that Sxr is a translocated portion of the Y chromosome. The H-Y antigen in human males might represent the primary male-determining gene. The H-Y antigen adds a new dimension to the understanding of sex determination.^ieng

Variability in serologically detected male antigen titer and some resulting problems: a critical review.

Seroologically detected male antigen" (also called H-Y antigen) was first described in normal male mammals but now appears to occur in normal female mammals as well. "Serologically detected male predominant" (SDMP) antigen is a more appropriate name since the titer in normal males usually exceeds that of normal females. As we show, in each sex there is a considerable inter-individual variability in SDMP antigen titer, and in moderate-to-large size samples the low end of the male range of titers usually coincides with the high end of the female range. Several major problems arise from failure to recognize and/or to deal adequately with this normal variation in SDMP antigen titer. The chief problem is that the "controls" used (often a single individual) may be inadequate and misleading, leading to unjustified designation of samples as "positive", "negative", or even "deviant" ("intermediate", "reduced") for SDMP antigen titer. Other problems include deficiencies in technique and lack of statistical control for test and sample variability. Adequate attention to these problems, especially to the normal variability in SDMP antigen titer, could reduce the contradictions and inconsistencies which have troubled this field.

Acute SLE in F1 hybrids between SB/Le and NZW mice; prominently enhanced formation of gp70 immune complexes by a Y chromosome-associated factor from SB/Le mice.

Both sexes of the F1 hybrids between SB/Le and NZW mice developed a spontaneous lupus-like disease. Their disease is essentially identical in time course and nature with autoimmune responses that are seen in the F1 hybrids between BXSB and NZW mice. The presence of abnormal Y chromosomes in the SB/Le strain was proved by the finding that the accelerated disease occurred in the F1 hybrid males only when the male parent was SB/Le but not NZW. The acceleration of disease in male F1 hybrids with abnormal Y chromosome was significantly associated with the enhanced formation of gp70 IC but not anti-DNA antibodies. These results indicate that all or almost all of the genetic abnormalities expressed in the BXSB strain are contributed by the SB/Le strain, and the Y chromosome-associated factor enhances the autoimmune response to serum gp70 antigen more markedly than to DNA antigens.