Association with HLA DQ of early onset myasthenia gravis in Southeast Texas region of the United States.

Forty-four Caucasian American myasthenia gravis (MG) patients from Southeast Texas underwent high resolution HLA DQ analysis. For the majority of patients who were late onset or male, no significant associations with DQ were observed. However, associations with DQ increased in female patients and early onset patients. At the allele level, DQB1 *0503, *0604, *0502 and *0402 collectively contributed to a positive association of the DQ locus with early onset MG (EOMG), while individually failing to show significant association. At DQ level, the novel haplotype DQA1*0401:DQB1*0201 was the primary factor in the association of combined DQ loci with early onset. In addition, *0104:*0503, *0102:*0604, *0102:*0502 and *0303:*0402 collectively contributed to the positive association of the haplotype loci. DR3-DQ2.5cis, a well known risk factor for MG in Western Eurasia, was not found associated with disease in any group. For typical EOMG [early onset, no thymoma, anti-acetylcholine receptor (AChR) antibody (Ab) positive] no association with DQA1 locus was found, however DQB1*0604 demonstrated an 'uncorrected' positive association. A few DQ haplotype (DQA1:DQB1) were positively associated with typical EOMG; a positive individual association for *0401:*0201 was complimented by the contributions of *0102:*0604 and *0303:*0402 haplotypes. A small minority of patients that were atypical and EOMG had a strong genetic association with DQA1*0104:DQB1*0503, the group included an anti-MuSK Ab positive and an anti-AChR negative patient. This report finds common ground with European studies regarding MuSK association; however similarities in association for typical early onset disease resembled HLA risk factors in East Asia and Southern Europe.

Cell interactions in the primary immune response in vitro: a requirement for specific cell clusters.

Mouse spleen cells were found to associate in cell clusters during the primary immune response to sheep erythrocytes in vitro. About 10% of the cell clusters had the following unique properties; (a) they contained most, if not all, antibody-forming cells, (b) they contained only cells forming antibody to one antigen when cell cultures were immunized with two antigens, (c) the cells in clusters reaggregated specifically after dispersion, and (d) the specific reaggregation of clusters appeared to be blocked by antibody to the antigen. The integrity of cell clusters was required for the proliferation of antibody-forming cells, and prevention of clustering by mechanical means or by excess antibody blocked the immune response. Antibody and antigenic determinants on the surfaces of cells probably provide the basis for interaction. The unique microenvironment of cell clusters was essential for the primary immune response in vitro.

Activation of B lymphocytes by monovalent anti-Lyb-2 antibodies.

Although activation of B lymphocytes by antigen or anti-Ig antibody has been shown to require cross-linking of surface Ig molecules, cross-linking is not necessary for B cell activation by anti-Lyb-2 monoclonal antibody (MAb). Monovalent Fab' fragments of anti-Lyb-2 MAb are as effective as the intact antibody in inducing blast cell transformation of small B cells and B cell proliferation in the apparent absence of T cells and adherent cells. In the presence of factors from T cells, B cells activated by Fab' fragments of anti-Lyb-2 MAb were induced to mature into Ig-secreting cells. Since monovalent Fab' fragments probably cannot induce receptor aggregation, it appears that receptor occupancy is sufficient to induce B cell activation with anti-Lyb-2 MAb.

Mice whose B cells cannot produce the T15 idiotype also lack an antigen-specific helper T cell required for T15 expression.

The X-linked CBA/N defect in B cell function precludes an antibody response to phosphorylcholine (PC). Accordingly, (CBA/N X BALB/c)F1 male mice are unresponsive to PC and lack circulating immunoglobulin bearing the T15 idiotype characteristic of BALB/C anti-PC antibody. In contrast, (CBA/N X BALB/c)F1 female mice respond to PC and greater than 80% of the anti-PC antibody is T15+. No T-cell abnormalities are known to be associated with the CBA/N mutation. These experiments compared the ability of helper T cells from either (CBA/N X BALB/c)F1 male (T15-) or F1 female (T15+) mice to help F1 female B cells respond to PC and to influence the level of T15 expression. The results indicate that although F1 male T cells collaborated with F1 female B cells just as efficiently as F1 female T cells for the total anti-PC response, the percentage of T15 expression induced by F1 male T cells fell dramatically. The (CBA/N X BALB/c)F1 male thus appear to lack a helper T-cell subset required for dominant idiotype production. This helper T cell defect could be repaired by adding F1 female T cells primed to a second carrier to F1 male T cells and restimulating the cell mixture with PC coupled to the antigen used to prime the F1 male cells plus free second carrier. This result implies that conventional helper T cells derived from the F1 male donor can collaborate with a distinct helper T-cell subset from the F1 female donor which recognizes both carrier and idiotype to induce an anti-PC antibody response dominated by the T15 clonotype.

Antigen-specific helper T cells required for dominant idiotype expression are not H-2 restricted.

Two synergizing antigen-specific helper T (Th) cell populations are required for an optimal TEPC15 (T15)-dominated antiphosphorylcholine (PC) plaque- forming cell response . In these studies, the two Th cell sets are shown to differ in their requirements for recognition of self-major histocompatibility complex (MHC)-encoded determinants by testing the ability of Th cells from F(1) {arrow} parent bone marrow chimeras to collaborate with PC-specific B cells bearing MHC-encoded determinants of either parental haplotypes. Previous studies have shown that one antigen-specific Th cell population is required for T-dependent anti-PC responses and activates PC-specific B cells only if the hapten, PC, is physically linked to the priming antigen. This Th cell, referred to as ThMHC, induces anti-PC responses that are mainly non-T15 in character, and it appears to be identical to the conventional antigen- specific Th cell. In these experiments, using T cells from (A X B)F(1) {arrow} parent A chimeras, ThMHC cells requiring hapten-carrier association provide help for F(1) and parent A B cells but not for B cells from parent B, thus confirming that the activity of the conventional Th cell is H-2 restricted . The second antigen-specific Th cell population, whose function is measured in the presence of the ThMHC cell set, preferentially activates T15-bearing B cells. This Th cell set (ThId) is missing in mice expressing low levels of T15-bearing antibody and can be restored by the addition of antigen-specific T cells from donors expressing high levels of circulating T15 Id. These studies demonstrate that T cells from F(1) {arrow} parent chimeras that express substantial levels of T15-bearing anti-PC antibody could provide ThId cell activity for the selective activation of T15-bearing B cells of F(1) and both parental H-2 types. These results imply that whereas the activity of conventional, ThMHC, cells is clearly H-2 restricted, ThId cells from the same chimeric donors are not required to recognize antigen in association with self-MHC-encoded determinants for successful T-B collaboration .

Interaction of helper T cells and Lyb5+ B cells responding to phosphorylcholine is MHC-restricted.

The requirements for T cell/B cell interaction for the induction of primary in vitro antibody responses to phosphorylcholine (PC)-keyhole limpet hemocyanin (KLH) were examined. Long-term helper T cell lines derived from KLH-primed (CBA/N X BALB/c) F1 female mice (H-2k/d) were able to support a T15-idiotype dominant, IgM anti-PC response of BALB/c (H-2d) B cells and macrophages, but could not activate PC-specific responses by BALB.B (H-2b) B cells, even in the presence of irradiated H-2k/d antigen-presenting cells. Polyclonal IgM secretion in the same cultures did not appear to depend upon a major histocompatibility complex (MHC)-restricted T-B interaction. Since IgM anti-PC responses seem to be entirely derived from the Lyb5+ B cell subpopulation, we conclude that at least some Lyb5+ B cells can only be activated by MHC-restricted T-B interactions. We also found that xid B cells from (CBA/N X BALB/c) F1 male mice could be polyclonally activated by helper T cell lines by an apparently MHC-unrestricted interaction. Our data thus suggests that residence in the Lyb5- or Lyb5+ B cell subset does not determine the T:B interaction requirements for antibody synthesis.

Ontogeny of mouse lymphocyte function. II. Development of the ability to produce antibody is modulated by T lymphocytes.

The relative functional maturity of neonatal mouse spleen T- and B-cell populations was assessed by comparing the ability to respond to the thymic-independent antigen, DNP-Ficoll, or thymic-dependent SRBC by producing antibody in vitro. Although mouse spleen cells responded to DNP-Ficoll at an earlier age than they responded to SRBC or TNP-SRBC, the reason for the lag in the T-dependent response was confounded by the finding of high numbers of suppressor T lymphocytes in the neonatal spleen. Thus, small numbers of neonatal spleen T cells or thymocytes significantly decreased the in vitro antibody response of adult spleen cells. Although B lymphocytes appear to be functionally mature soon after birth, their acitivity may be modulated by an excess of suppressor T cells; e.g., the reconstitution of helper cell function in the neonatal spleen required anti-theta treatment before addition of adult helper cells. Suppressive activity attributable to T cells seems to play a dominant role in determining the ability of the neonatal animal to react positively or negatively to antigenic stimulation.

Anti-idiotype induced regulation of helper cell function for the response to phosphorylcholine in adult BALB/c mice.

An adoptive secondary antibody response to phosphorylcholine (PC) can be generated by the transfer of keyhole limpet hemocyanin (KLH)-primed T cells, PC-bovine gamma globulin-primed B cells, and PC-KLH into irradiated syngeneic BALB/c mice. If the KLH-primed T-cell donors were pretreated with anti-idiotype antibodies directed against the BALB/c PC-binding myeloma TEPC 15, their T cells were unable to collaborate effectively with PC-primed B cells; moreover, they could suppress the helper activity of T cells from normal mice for the PC-KLH response. The Ly phenotype of these T cells was found to be Ly 1-, 2+. The specificity of the suppressor T-cell population induced by anti-T15 treatment appears to be both for idiotype (hapten) and carrier, since the suppressor T cells fail to interfere with the antibody response to PC on a heterologous carrier, nor do they suppress the response to trinitrophenol-KLH.

Functional maturation of thymic lymphocyte populations in vitro.

Mouse thymocytes were cultured for short periods of time either alone or with one of two supporting cell populations, splenic adherent cells or thymic epithelial cells. The thymus-derived (T) cell activity of thymocytes cultured on supporting cell populations increased dramatically during 2 days of culture, as assayed in the mixed lymphocyte interaction (MLI), response to phytomitogens, and helper cell activity in the in vitro antibody response. The level of activity attained was equal to that of spleen and lymph node lymphocytes and greater than that of steroid-resistant thymocytes. The cultured thymocytes had surface antigens characteristic of mature T lymphocytes with regard to theta and H-2. The appearance of functionally active lymphocytes in vitro depended upon cell division. Most of the active cultured cells arose from cells already undergoing maturation, i.e., from cells with reduced theta determinants and increased H-2 determinants. We therefore have generated a population of thymocytes indistinguishable from peripheral T lymphocytes using simple in vitro techniques. The extent to which the production of these active lymphocytes depends upon in vitro differentiation is discussed.

Negative selection in vivo reveals expression of strong Mls determinants in mice with X-linked immunodeficiency.

Evidence is presented that mice with X-linked immunodeficiency (xid) express strong Mlsa,d determinants, a putative marker of the mature subset of B cells. Although young (3-5 wk) (CBA/N X DBA/2)F1 male (xid+) mice stimulated only very weak mixed lymphocyte reactions (MLR) to Mlsa,d determinants, older mice (greater than 7 wk) regularly elicited conspicuous responses, despite being totally unresponsive to TNP-Ficoll. Expression of Mlsa,d determinants by xid+ mice was also detected by the procedure of negative selection in vivo. Thus BALB/c T cells were totally depleted of Mlsa,d reactivity after blood to lymph recirculation through 10-wk old irradiated xid+ (CBA/N X DBA/2)1 male mice. Significantly, a marked (90%) reduction in the anti-Mlsa,d response also occurred with T cell filtration through 3-wk xid+ mice, i.e., mice that elicit only minimal primary MLR; filtration through 3-wk xid- normal female mice led to near-complete (99%) negative selection. Collectively these data indicate either, (a) that xid+ mice contain appreciable numbers of cells with at least some of the properties of mature B cells, or (b) that the expression of Mlsa,d determinants is not restricted to mature B cells. In either case, B cells from xid mice cannot be viewed as a simple model for immature normal B cells.

Retroviral induction of acute lymphoproliferative disease and profound immunosuppression in adult C57BL/6 mice.

We have shown that a mixture of murine leukemia viruses (MuLV) causes the acute onset of lymphoproliferation and immunosuppression when injected into adult C57BL/6 mice. The ecotropic/MCF (mink cell focus-inducing) mixture of MuLV stimulates polyclonal B lymphocyte proliferation and differentiation to antibody-secreting cells. Serum Ig levels are elevated for all isotypes except IgA. The viral infection leads to a rapid decline in T lymphocyte responses to mitogens and alloantigens, as well as a decrease in helper cell activity. Specific antibody responses to both T-dependent and T-independent antigens are impaired, and the response of B lymphocytes to mitogens is abolished. The profound immunosuppression seems to be due to the MuLV-induced polyclonal activation of lymphocytes. No active suppression of normal lymphocyte responses by cells from virus-infected mice was observed. The disease induced by the LP-BM5 MuLV isolate thus seems a promising model for the study of lymphocyte activation and the mechanisms of retrovirus-induced immunosuppression.

Adoptive transfer of neonatal T lymphocytes rescues immunoglobulin production in mice with severe combined immune deficiency.

Mice with the autosomal recessive severe combined immune deficiency (scid) mutation lack mature lymphocytes because of defective joining of T cell receptor and immunoglobulin (Ig) gene segments. Penetrance of this mutation is incomplete since 10-25% of SCID mice produce some T or B lymphocytes. This "leaky" phenotype could be due to a reversion of the mutation in some mice or to a constant, low frequency of functional lymphocytes generated in all SCID mice with variable survival of such cells. We report here that all SCID mice can be stimulated to produce functional B cells by the transfer of normal neonatal, but not adult, T cells. T cell-induced rescue of C.B-17scid B cells results in high levels of Ig expressing the Ighb allotype of the SCID recipient. These results show that all SCID mice generate some functional B cells, the majority of which do not survive in the absence of a subset of T cells present in high frequency in the neonate.

The immunoglobulin allotype contributed by peritoneal cavity B cells dominates in SCID mice reconstituted with allotype-disparate mixtures of splenic and peritoneal cavity B cells.

We have studied potential regulatory interactions between mature B lymphocyte populations by analysis of C.B-17 severe combined immunodeficient (SCID) mice reconstituted simultaneously with immunoglobulin allotype-congenic mixtures of spleen (SP) and peritoneal cavity (PerC) B cells. We have previously shown that the independent transfer of B cells from these sources leads to the long-term survival of donor B cells and reconstitution of immunoglobulin levels in SCID mice (Riggs, J.E., D.L. Robertson, R.S. Stowers, and D.E. Mosier, manuscript submitted for publication). SP and PerC B cells differ in numerous respects, with the PerC having higher proportions of large, activated B cells that express the IgM greater than IgD phenotype and greater numbers of CD5 B cells. The injection of equal numbers of B cells from SP and PerC into SCID recipients (e.g., BALB/c SP + C.B-17 Per C----SCID) has led to the following observations: (a) serum IgM allotypes in B cell chimeras revealed strict dominance by the allotype contributed by the PerC B cells; (b) this dominance was not due to regulatory T cells; (c) B cells of the unexpressed (i.e., SP) allotype were present in the chimera in the spleen but not the peritoneal cavity; and (d) immunization with TI and TD antigens failed to elicit the SP IgM allotype, whereas secondary TD antigen immunization elicited low levels of the SP IgG2a allotype. Additional experiments demonstrated concurrent expression of IgM allotypes derived from both SP and PerC B cells in recipients that: (a) received a 10-fold excess of SP B cells; (b) received SP B cells before PerC B cell transfer; or (c) received SP B cells intravenously and PerC B cells intraperitoneally. We conclude that the establishment of IgM synthesis by PerC B cells leads to a feedback inhibition of subsequent IgM synthesis by SP B cells, and that the frequency of B cells that can lead to this effect is substantially higher in peritoneal cavity than in spleen. These data provide further confirmation of regulatory interactions between B cells in the absence of T lymphocytes, but confound the interpretation of experiments supporting the existence of a separate CD5+ B cell lineage.

Ly phenotype and mechanism of action of mouse neonatal suppressor T cells.

Neonatal suppressor T cells were isolated from the thymuses of 10- to 14-day old BDF mice infected at birth with mouse thymic virus. Such cells were enriched for suppressive activity directed against antibody formation by adult B cells and represented a relatively homogenous population of outer cortical cells. Their surface antigen phenotype was found to be: Ly 1+, Ly 2+, TL+, Thy 1+, and H-2+. The cells were larger and contained more DNA than thymocytes from age-matched controls. These findings identify neonatal suppressor T cells as a unique subpopulation separate from most inducible suppressor cells in the adult mouse. The mechanism of action of neonatal suppressor T cells seems to be a reduction in the number of B cells initially triggered by antigen.

The role of surface IgD in the response to thymic-independent antigens.

An alloanti-delta antibody was prepared by immunizing C57BL/Ka mice with BALB/c spleen cells. Its specificity for delta-chain was demonstrated by immunoprecipitation and SDS-PAGE of 125I-labeled membrane proteins from BC8 spleen cells. BC8 mice possess C57BL/Ka "background" genes and BALB/c IgH genes. The anti-delta reagent without complement inhibited the primary in vitro anti-TNP antibody response to TNP-AECM-Ficoll by BC8 spleen cells, although it had no effect of the anti-TNP response of congenic C57BL/Ka spleen cells, which lack the delta-allotype identified by this antibody. On the other hand, the anti-delta antibody had no effect on the anti-TNP response of BC8 spleen cells to TNP-BA, except at limiting antigen concentrations. Both TNP-AECM-Ficoll and TNP-BA are T-I antigens, but they differ in that TNP-AECM-Ficoll fails to stimulate in vitro responses by immunologically defective CBA/N and neonatal spleen cells whereas TNP-BA can cause responses from both these animals. These results suggest that the IgD receptor is critical to T-I antibody responses initiated by TNP-AECM-Ficoll but that it is not required for T-I responses stimulated by TNP-BA.

Functional T lymphocytes are required for a murine retrovirus-induced immunodeficiency disease (MAIDS).

Athymic nu/nu mice were found to be resistant to the immunodeficiency disease and lethality induced in normal mice by the injection of the LP-BM5 mixture of murine retroviruses (LP-BM5 MuLV). Susceptibility to disease induction was reconstituted by injection of nu/nu mice with purified, mature T lymphocytes. The extent of viral replication of both the ecotropic and mink cell focus forming (MCF) components of LP-BM5 MuLV was equivalent in both nu/nu and normal animals. Retrovirally-induced immunodeficiency disease in mice (MAIDS) is thus dependent upon the presence of functional T lymphocytes, and high virus titers in athymic mice have little or no effect on the immune system.

Role of a nonimmunoglobulin cell surface determinant in the activation of B lymphocytes by thymus-independent antigens.

Lyb 5 is a B-cell alloantigen which is expressed on 50-60% of B cells. It was defined originally on the basis of cytotoxicity. We have described a new reactivity within the anti-Lyb 5 serum on the basis of selective inhibition of antibody responses in vitro by this antiserum in the absence of complement. This inhibitory activity of anti-Lyb 5.1 serum appears to be due to recognition of antigenic determinants different from the prototype antigens detected in the cytotoxicity assay. Anti-Lyb 5 serum incorporated into spleen cell cultures selectively inhibits antibody responses to a class of thymus-independent antigens (TI-2) previously characterized by their failure to elicit antibody formation in immature mice or in the defective CBA/N strain. Responses to optimal concentrations of TI-1 antigens, which can induce antibody synthesis in these mice, are unaffected by the addition of anti-Lyb 5.1 serum. The B-cell alloantigen defined by this functional assay is designated tentatively Lyb 7 and it is shown to be distinct from cell surface immunoglobulins. Lyb 7 appears to have a role in the activation of B lymphocytes by the TI-2 class of thymus-independent antigens.

Homozygous scid/scid;beige/beige mice have low levels of spontaneous or neonatal T cell-induced B cell generation.

The autosomal recessive scid mutation results in defective immunoglobulin and T cell receptor gene rearrangement. The scid mutation occurred in the allotype congenic C.B-17 line, and up to 25% of C.B-17 scid mice spontaneously produce both T cells and immunoglobulin, a phenotype known as "leaky." Moreover, introduction of neonatal T cells into C.B-17 scid mice leads to immunoglobulin production by 100% of animals. We have produced mice homozygous for both the scid and beige mutations. By contrast with C.B-17 scid mice, BALB/c scid.beige mice have a < 2% incidence of "leakiness." This percentage does not increase with age, and introduction of neonatal T cells fails to rescue immunoglobulin production. This suggests that a gene (or genes) closely linked to the beige locus regulates B and/or T cell development.

Inability of mice with a defect in B-lymphocyte maturation to respond to phosphorycholine on immunogenic carriers.

Mice with the CBA/N defect are unresponsive to the hapten phosphorylcholine (PC) even when presented on a variety of immunogenic carriers. Since these mice have the variable region gene for PC, their inability to respond may reflect deletion or suppression of the line of B lymphocytes which is responsible for the anti-PC response.

Activation of mouse lymphocytes by anti-immunoglobulin. II. A thymus-independent response by a mature subset of B lymphocytes.

Mouse spleen cells can be stimulated to proliferate in vitro by purified anti-mu or anti-gamma,kappa antibodies. These responses can be obtained in cell populations bearing membrane immunoglobulin (Ig), purified by the fluorescence activated cell sorter (FACS), but they are not observed in FACS-purified Ig- cell populations. Furthermore, treatment of spleen cell populations with anti-Thy 1.2 and complement does not impair the response, nor does addition of nylon wool-purified T lymphocytes enhance it. These results indicate that B lymphocytes respond to anti-Ig and that their response does not require T cells. On the other hand, cells from athymic nude (nu/nu) mice respond slightly less well to anti-mu than do cells from heterozygous littermate (nu/+) controls; nu/nu cells are almost unresponsive to anti-gamm,kappa and addition of nylon wool-purified T cells from nu/+ controls does not restore the response. This suggests that T lymphocytes or the thymus may control the appearance of cells responsive to anti-gamma,kappa. Responsiveness of normal mice to anti-mu does not appear until 4 wk of age and does not reach maximum levels until 8 wk of age. Acquisition of full responsiveness to anti-gamma,kappa is even more delayed. This, together with the failure of mice with the CBA/N B-cell defect to respond to anti-Ig, suggests that cells stimulated to proliferate by anti-Ig are a mature subset of B cells. Depletion of adherent cells by Sephadex G-10 treatment or by treatment with carbonyl iron and exposure to a magnetic field does not diminish anti-mu or anti-gamma,kappa responses, suggesting that the responsiveness does not require the presence of macrophages. Thus, activation of B-cell proliferation by anti-Ig appears to be a T-cell independent, macrophage-independent process in which membrane Ig plays a direct role in signal generation.