Isotype profiles of anti-influenza antibodies in mice bearing the xid defect.

The humoral response to influenza A/PR8 virus was examined in the CBA/N and C3J.xid strains of mice, both of which bear an X-linked genetic defect (xid), and in strains lacking this defect. Hemagglutination-inhibiting antibody titers and measurement of virus-specific antibodies by solid-phase radioimmunoassay indicated that the xid defect does not impair the production of an adequate anti-influenza antibody response. However, investigation of the isotypes of PR8 virus-specific antibodies disclosed a relative decrease in the levels of IgG3 and IgG1 in the xid-bearing strains. This was observed after both intraperitoneal immunization and aerosol infection. The isotype differences were not reflected in the susceptibility of these strains to influenza virus infection.     

Regulation of T15 idiotype dominance. I. Mice expressing the xid immune defect provide normal help to T15+ B cell precursors

The immune response to phosphocholine (PC) in many strains of mice is dominated by the T15 idiotype family of anti-PC antibodies. By introducing the CBA/N X-linked immune defect (xid gene) into these mice, one profoundly alters their ability to make a T15-predominant, IgM anti-PC response. This loss of T15 dominance in mice expressing the xid gene is not due to the presence of suppressor T cells or the lack of T15 idiotype-specific helper cells in these mice. Thus, one can reconstitute a T15 idiotype-dominant response in immune defective mice with B cells from normal mice, and in adoptive transfer assays the primed T helper cells from immune-defective mice provide qualitatively the same help to normal B cells as the T helper cells from normal mice. T15 idiotype dominance appears to be controlled by the expression and activation of Lyb-5+ PC-specific B cells. Thus, the majority of T15+ B cell precursors are restricted to this B cell subset, whereas the Lyb-5- B cell subset contains predominantly T15-, anti-PC B cell precursors, which produce mainly IgG antibodies after activation by PC-containing antigens.     

Cloning of B cells from autoimmune MRL-lpr/lpr and MRL.xid mice

The relationship between colony formation (cloning) of B cells and their activation in murine autoimmunity was investigated in MRL-lpr/lpr and MRL.xid mice. Cells from MRL-lpr/lpr mice showed similar requirements for in vitro growth as normal CBA/J and BALB/c cells, with maximal colony formation in the presence of the supporting factors lipopolysaccharide and sheep red blood cells. The frequency of colony-forming cells from MRL-lpr/lpr spleens or hapten-specific B-cell preparations was slightly greater than the two normal control strains, with this difference significant only for a comparison of BALB/c and MRL-lpr/lpr spleens. In contrast, MRL-lpr/lpr mice bearing the xid gene for B-cell immunodeficiency (MRL.xid) had markedly reduced B-cell colony formation. These mice nevertheless expressed anti-DNA antibodies, although at levels reduced from that of MRL-lpr/lpr controls. These results indicate that enhanced in vitro colony formation need not accompany B-cell hyperactivity in murine autoimmune disease and that autoantibody production can occur in mice with impairment in this growth property.     

Studies of congenic MRL-Ipr/Ipr.xid mice

Highly inbred MRL-Ipr/Ipr.xid congenic mice were bred and compared with their + littermates. The xid-bearing congenics developed lymphadenopathy consisting of dull Ly-1+ T cells and impairment of cellular proliferation and IL 2 production in response to the T cell mitogen Con A. Thus, the lpr gene was fully expressed. The xid gene, however, was also expressed as indicated by the failure to respond to immunization with TNP-Ficoll and flow cytometric analysis of splenic B cells. The xid gene was associated with a marked reduction in IgM anti-ssDNA and anti-nDNA of both classes, and serum Ig-bound gp 70. Kidney disease was markedly retarded as was death from the autoimmune process. These studies suggest that the T cell lymphoproliferation and dysfunction characteristic of MRL-Ipr/Ipr mice is not sufficient to induce accelerated autoimmunity; xid is able to markedly slow the process. The xid gene interferes with the development of a B cell subset necessary for maximum autoantibody production, anti-gp 70 production, and the resultant immune complex renal and cardiac disease. The present finding of protection against accelerated autoimmunity in MRL-Ipr/Ipr mice by xid, coupled with previous demonstrations of protection against autoimmunity in other autoimmune mouse strains, suggests that a common approach to the therapy of systemic lupus may be possible.     

Polyclonal activation of xid B cells by auto-Ia-reactive T cell clones

The mechanism of T cell-dependent activation of xid B cells into Ig-producing cells was studied by employing H-2-restricted, antigen-specific T cell clones. Helper factors (B cell stimulatory factors, BSF) released from KLH-specific T cell lines could induce polyclonal Ig production in B cells from (CBA/N X BALB/c)F1 (NBF1) female mice but not from CBA/N or NBF1 male mice. Direct addition of helper T cell lines induced Ig production in xid B cells from CBA/N or NBF1 male mice. A T cell clone, MK6, which was derived from NBF1 male mice and specific against Iad determinant, could activate NBF1 male but not CBA/N B cells. Another clone, CK4, derived from CBA/N mice and having specificity against KLH plus I-Ak determinant could activate both CBA/N and NBF1 male B cells into IgM- and IgG-producing cells in the absence of KLH, and monoclonal anti-I-Ak antibody specifically blocked such activation. These results suggest that xid B cells are able to be activated by the signal provided by the recognition of Ia molecules on B cells by auto-Ia-reactive T cells. Xid B cells from CBA/N mice that had been co-cultured with a T cell line specific against I-Ak determinant for 24 hr became reactive to BSF and capable of differentiating into Ig-producing cells in the presence of BSF. The results showed that even xid B cells could be responsive to BSF if they were in a certain activation stage.     

B lymphocyte production in the bone marrow of mice with X-linked immunodeficiency (xid)

CBA/N mice carry an X-linked recessive immunodeficiency (xid) gene manifested by the absence of a B lymphocyte subpopulation, but the manner in which the xid gene exerts its effect on B lymphocyte development is unknown. The production of B lymphocytes in the bone marrow of CBA/N mice has now been compared with that of normal CBA/J mice by using two in vivo assays: immunofluorescence stathmokinetic studies measured pre-B cell proliferation, whereas radioautographic [3H]thymidine labeling was used to evaluate small lymphocyte turnover. Although the total cellularity of CBA/N mouse bone marrow was greater than normal, the absolute number of marrow small lymphocytes, pre-B cells, and B lymphocytes were all similar to those in CBA/J controls. Furthermore, in the bone marrow of CBA/N mice, the proliferation rate of pre-B cells, calculated from their rate of entry into mitosis, and the turnover rate of small lymphocytes, derived from their rate of [3H]thymidine labeling, were not significantly different from those seen in nondefective mice. The present findings that pre-B cell proliferation and small lymphocyte production proceed at similar rates in the bone marrow of xid and normal mice suggest that the xid gene does not act at the level of primary B cell genesis in the bone marrow. The findings are in accord with the view that the xid gene produces a maturation block or a functional abnormality among B lymphocytes in the peripheral lymphoid tissues rather than the deletion of a sublineage of B lymphocytes in the bone marrow.     

Clonal analysis of the primary and secondary B cell responses of neonatal, adult, and xid mice to (T,G)-A--L

The splenic focus assay was used to clone B cells from neonatal, adult and xid mice in order to examine their primary and secondary responses to (T,G)-A--L. Adult precursor cell frequencies to (T,G)-A--L were achieved late in neonatal ontogeny. Primary xid B cells responded to DNP-HY but not to (T,G)-A--L in the splenic focus assay. The frequency of secondary B cells from (T,G)-A--L-primed xid mice was less than or equal to 10% that of secondary B cells from wild-type (non-xid or X/Xxid heterozygous) mice. Although xid B cells were poorly responsive to (T,G)-A--L in the splenic focus assay, (T,G)-A--L-primed xid mice could provide help as recipients for stimulation of wild-type primary and secondary B cells. It seems likely that the B2 subset contributes most of the splenic focus response to (T,G)-A--L. The fine specificities of antibodies produced by neonatal, xid, and adult (wild-type) B cell clones were analyzed using analogues of (T,G)-A--L. A specificity shift was observed between the adult primary and secondary antibody responses to (T,G)-A--L. Less than 10% of adult primary clones produced antibodies cross-reactive on (Phe,G)-A--L (recognizing A--L determinants or Phe,Glu determinants), whereas more than 70% of primary clones produced Tyr,Glu side-chain specific antibodies cross-reactive on GT. The percentage of clones producing GT-binding antibodies diminished in the secondary response, while the percentage of clones producing antibodies cross-reacting on (Phe,G)-A--L increased. Neonatal clones also produced mostly GT-binding antibodies but gave a higher percentage of (Phe,G)-A--L-cross-reacting antibodies than adult primary clones. The specificities of secondary antibodies produced by xid and wild-type B cell clones were dissimilar. First, xid secondary clones were "primary-like" in that no anti-A--L antibodies were detected. Second, clones whose antibodies bound side-chain determinants but not GT were produced in higher frequency by xid than by wild-type secondary B cells. The differential responsiveness of B cell subsets to antigen and regulatory signals may influence memory B cell generation and the specificity of antibodies produced in the primary vs secondary response.     

T cell regulation of polyclonal B cell responsiveness. II. Evidence for a deficit in T cell function in mice with an X-linked B lymphocyte defect.

In addition to the x-linked B cell maturation deficit previously reported in CBA/N mice, a functional T cell defect has now been observed. T lymphocyte regulation of the polyclonal PFC response was studied within the context of this x-linked immunodeficiency model. The ability of 1) B cells from (CBA X CBA/CaJ)F1, male mice to respond to nonspecific T cell helper signals and 2) T cells from NCF1 male mice to provide such signals was investigated under in vitro conditions by using bacterial lipopolysaccharide (LPS) as the polyclonal activator. B lymphocytes from both male and female NCF1 mice were receptive to T cell help rendered by NCF1 female T cells. Male T cells. however, were unable to augment polyclonal B cell responses of either NCF1 male or female B cells to LPS. Treatment with ATS + C reduced the polyclonal response of female but not male spleen cells to LPS. This deficit could not be overcome by the use of greater numbers of NCF1 male T cells. The observation that this deficiency in T cell regulation is not due to active suppression suggests that the results may be attributable to an intrinsic T cell defect.     

An analysis of the natural killer cell defect in beige mice

Although C57BL/6 bg^J/bg^J mice exhibit very low or undetectable levels of endogenous natural killer cell activity, such activity can be induced by the administration of BCG or tilorone hydrochloride to these animals. This cytotoxic activity has been shown to be due to NK cells by the criteria of nylon-wool nonadherence and of effector cell sensitivity to treatment with either anti-asialo GM₁ serum or high concentrations of anti-Thy 1.2 serum, in the presence of complement. Even after the administration of inducing agents, however, beige mice continue to display significantly less NK activity than do their heterozygous littermates. In an attempt to ascertain what cell might be defective in responding to induction, we utilized an in vitro system in which the induction of NK activity by poly I:C in a nylon-wool nonadherent population is dependent upon plastic-adherent cells. We found that adherent cells from either beige or heterozygous mice were indistinguishable in their ability to restore the NK response of nylon-wool-nonadherent spleen cells stimulated with poly I:C. This was true when either beige or heterozygous mice were used as the source of responder cells. Thus, it appears that the defect in responsiveness to inducing agents may reside in the beige NK cell itself.     

The ontogeny of thymic independent antibody responses in vitro in normal mice and mice with an X-linked B cell defect.

The primary in vitro antibody response of neonatal spleen cells to three thymic independent antigens has been examined. The time of onset of responsiveness to TNP-Brucella abortus and TNP-lipopolysaccharide was significantly earlier than the onset of responsiveness to TNP-Ficoll. This ontologic sequence was not affected by T cell depletion or antigen presentation on adult macrophages. In neonatal mice bearing the X-linked CBA/N defect, the response to TNP-Brucella abortus and TNP-lipopolysaccharide was much delayed and no response to TNP-Ficoll developed. We conclude that different thymic independent antigens address different subpopulations of B cells, one of which appears earlier in ontogeny than the other.     

A new molecular genetic diagnosis of the Btk(xid) mice.

A new method of polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) analysis was developed for genetic typing of a point mutation of the Bruton's tyrosine kinase (Btk) gene in CBA/N mice bearing an X-linked recessive immunodeficiency (xid). Since restriction site useful for RFLP analysis does not exist in the spontaneous mutant Btk(xid) locus, an artificial restriction site was introduced by PCR amplification with a modified primer. The five genotypes of the Btk locus (Btk(xid)/ Btk(xid), Btk(xid) /Btk+ and Btk+/Btk+ females and Btk(xid)/Btk(null) and Btk+/Btk(null) males) could be distinguished by three patterns clearly and easily. This PCR-RFLP analytic method will be useful as a tool in the production of congenic mice and mice with multiple immunodeficient genes.     

Differential endotoxin sensitivity of lymphocytes and macrophages from mice with an X-linked defect in B cell maturation.

The in vitro sensitivity of B lymphocytes and macrophages derived from (CBA/N X DBA/2N) F1 male mice, which carry an X-linked recessive gene that produces defective B cell maturation, was compared to phenotypically normal F1 female mice. B lymphocytes of F1 males exhibit an abnormal mitogenic response to LPS in serum-free culture conditions, which is partially reversed in the presence of serum. In contrast, both resident and thioglycollate-induced peritoneal macrophages of F1 male mice respond normally to LPS. In response to LPS in vitro, F1 male macrophages produce the monokine, lymphocyte-activating factor (LAF) and release prostaglandins. Furthermore, F1 male macrophages are sensitive to the lethal effects of LPS. Therefore, the defective CBA/N gene appears to be expressed only in B lymphocytes and not in macrophages. Since F1 male mice are normally sensitive to the lethal and adjuvant effects of LPS in vivo, these findings suggest that a mature B lymphocyte population is not required for these effects and support the role of the macrophage in the mediation of LPS-induced lethality and adjuvanticity.     

Studies on immunity in hybridoma-bearing mice. B. Immunity against the hybridoma. II. The phenotype and specificity of the immune cell

When appropriate numbers of anti-dinitrophenyl (DNP) immunoglobulin (Ig) E-secreting hybridoma (B 53) cells were injected s.c. into normal BALB/c mice, some of the recipients rejected the tumors. These mice were shown to be immune to B 53 as they withstood, without any ill effect, the i.p. injection of lethal doses of B 53 cells. In previous studies, it was shown that the spleen cells of these mice protected against the growth of B 53 cells. In this study, the characteristics and specificity of the immune spleen cells were examined. The cells responsible for this immunity were shown to be T cells that express Ly-2 on their surface. These cells were shown in in vivo and in vitro assays to limit the growth of the immunizing hybridoma, as well as some but not all BALB/c plasmacytomas and hybridomas.     

A heritable defect in IL-12 signaling in B10.Q/J mice. I. In vitro analysis

B10.Q mice are normally susceptible to the induction of collagen-induced arthritis. We noted that one subline of B10.Q mice, B10.Q/J, was completely resistant to disease induction when immunized with collagen in CFA. B10.Q/J mice have a global defect in the generation of Th1 responses, and Ag-specific T cells derived from this strain failed to produce IFN-gamma. Because T cells from these mice could produce normal amounts of IFN-gamma when activated by IL-12/IL-18-independent stimuli, the defect appeared to be a failure to respond to IL-12. This defect extended to NK cells, which also failed to produce IFN-gamma when stimulated by IL-12. The capacity of NK cells, but not activated T cells, to produce IFN-gamma in response to IL-12 could be partially restored by IL-18. The expression of the IL-12R beta1- and beta2-chains on T cells and NK cells from B10.Q/J mice was normal. However, activated T cells from B10.Q/J mice did not signal normally through the IL-12R and manifested a defect in their capacity to phosphorylate Stat4. This defect was partial in that it could be overcome by increasing both the concentration of IL-12 and the incubation times in the Stat4 phosphorylation assays. Because Stat4 function is apparently intact in B10.Q/J mice, the defect in IL-12 signaling can be localized between the IL-12R complex and Stat4. This subtle abnormality in IL-12 responsiveness results in a profound defect in the generation of Th1 cells and the development of autoimmune disease.     

Genetic control of lymphocyte suppression. I. Lack of suppression in aged NZB mice is due to a B cell defect.

Con A-activated cells from old NZB mice were found capable of inhibiting the polyclonal response of cells from young NZB and BALB/c animals. Furthermore, Con A-preactivated spleen cells from young NZB and BALB/c mice did not significantly affect the response of spleen cells from old NZB mice. These results suggest that the defective suppressive activity in old NZB mice may be traced to a defect at the B cell level.     

An analysis of in vitro B cell immune responsiveness in human lymphatic filariasis

The immunoregulatory mechanisms involved in B cell function in patients with varying clinical manifestations of bancroftian filariasis were examined by studying the ability of peripheral blood mononuclear cells (PBMC) or PBMC subpopulations from patients with elephantiasis, asymptomatic microfilaremia (MF), and acute tropical pulmonary eosinophilia (TPE) to produce polyclonal and parasite-specific antibody in vitro, both spontaneously and in response to a mitogen (PWM) and to parasite antigen. When the spontaneous or mitogen-driven polyclonal responses were examined, all groups produced significant amounts of IgM and IgG; those with TPE produced extremely high levels. However, when in vitro parasite antigen-specific responses were examined, those with MF were unable to produce filaria-specific antibody either spontaneously or in response to PWM or parasite antigen; in contrast, patients with chronic lymphatic obstruction or TPE produced large quantities. Removal of neither adherent cells nor T8+ T cells affected the parasite-specific B cell anergy seen in those with MF. This absent or severely diminished capacity to produce antibody on parasite antigenic stimulation in patients with MF is likely responsible for the low levels of parasite-specific antibody seen in this most common clinical manifestation of bancroftian filariasis. Its inability to be reversed by the removal of "suppressor elements" suggests a state of B cell unresponsiveness to the parasite.