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The mechanisms responsible for common variable immunodeficiency syndrome (CVID) are as yet unknown. In the present study, we show that the B-cell dysfunction in a subset of CVID patients is caused by defective protein tyrosine phosphorylation (PTP). We demonstrated that the PTP level and immunoglobulin (Ig) secretion malfunctions can be successfully repaired when normal plasma membrane components are implanted into these patients' B cells. Stimulation of CVID patients' peripheral blood mononucleated cells with anti-Ig antibody revealed that 7 of 11 patients had lower PTP levels than those found in the normal donor cells. Plasma membrane implantation to the cells of these patients resulted in elevated PTP levels which reached normal levels upon stimulation with anti-human Ig antibody. The results revealed two distinct groups of CVID patients. The first group included patients whose B cells expressed low PTP levels after Ig stimulation. In these patients the plasma membrane implantation restored the normal PTP level as well as the ability to secrete IgM and/or IgG after B-cell stimulation. In the second group, patients whose B cells expressed a normal PTP level after Ig stimulation, with no restoration of their ability to secrete Ig upon plasma membrane implantation and lipopolysaccharide stimulation. We conclude that the first group has an early signal transduction defect located in the B-cell plasma membrane, while in the second group the defect is located elsewhere.

We challenge the theory that the CD40-CD40 ligand is the only explanation for X-linked immunodeficiency in patients with hyper-immunoglobulin M (IgM) syndrome (HIGM1), and we demonstrate an intrinsic defect in the patients' B cells. Patients with HIGM1 have a defective CD40 ligand on their activated T-helper cells; therefore, they cannot receive signals for isotype switching when the cells are activated by T cell-dependent antigens. We activated mononuclear cells from three patients with HIGM1 and from three healthy blood donors with T cell-independent mitogens and studied their proliferative responses and Ig secretion. Normal murine plasma membrane fragments were implanted into peripheral blood mononuclear cells, and the cells were activated with Staphylococcus aureus Cowan I, pokeweed mitogen, and lipopolysaccharide. This implantation significantly augmented the proliferative responses to the mitogens in two patients. However, it augmented IGM secretion in response to B-cell mitogens in only one patient. No IgG or IgA response could be detected in the implanted mononuclear cells that originated from patients with HIGM1, unlike implanted mononuclear cells from healthy donors, which responded by IgM, IgG, and IgA antibody secretion following their stimulation with B-cell mitogens. The data suggest that the B cells of patients with HIGM1 possess an additional defect which prevents Ig isotype switching in response to T cell-independent mitogens. This defect is not located in the membrane receptors or within the membrane enzymes.

Human-human B cell hybridoma JK32.1, constructed from B lymphocytes of a common variable immunodeficient patient and nonsecreting cell line, retains the defects of B cell immunodeficiency. Efforts to clarify whether the defect is located within the plasma membranes of this cell line were carried out by implanting them with plasma membrane fraction derived from normal functional cells via intact non-infectious Sendai virus. The implanted cells were activated with various mitogens and their Ig responses and isotype switching were examined. Restoration of IgM secretion was achieved in the implanted JK32.1 cells following stimulation with SAC, PWM, or retinoic acid. Augmented IgM response was also obtained in the implanted cells treated with retinoic acid and lipopolysaccharide (LPS) despite their unresponsiveness to LPS alone. No IgG or IgA response could be detected in the implanted JK32.1 cells. These data suggest that this immunodeficient cell line possesses at least two different malfunctions, one located within the plasma membrane moiety of the cells and the other located within the cytoplasmic and/or nucleic components. The plasma membrane moiety defect can be repaired temporarily by delivering proper signals via the implanted plasma membranes. However, this manipulation of the cells could not overcome the intrinsic defect of the cells which blocks isotype switching and secretion of IgG, IgE, and IgA antibodies.

An analysis of cell lines representing different stages of the B-cell differentiation pathway indicated that about 50% of the cell lines examined expressed exclusively wild type p53 protein. These lines therefore offer a convenient system to study the involvement of p53 in cell differentiation. When 70Z/3, a pre-B cell line which expresses wild type p53, was treated with the differentiation inducer lipopolysaccharide (LPS), it was seen that increased levels of p53 mRNA preceded specific changes in kappa (kappa) immunoglobulin expression. This increased expression of kappa specific mRNA, which was evaluated by specific PCR analysis, was blocked following transfection with mutant p53 coding plasmids. Treatment of 13A60, another cell line which endogenously expresses wild type p53, with LPS caused a secretion of IgA antibodies, also accompanied by increased p53 mRNA expression. The conclusion was that induction of B-cell differentiation involves the transcription of the p53 gene. This was further substantiated by experiments showing that differentiation of stable clones derived from the 70Z/3 cell line, harboring a p53-promoter-CAT plasmid, induced increased CAT activity. Furthermore, wild type p53 transactivated the promoter control sequences of the kappa light chain gene. Taken together, these results suggest that p53 is involved in B-cell differentiation, a pathway which involves DNA rearrangements that may be accompanied by generation of faulty DNA. The fact that wild type p53 was shown to function as a transcriptional factor, coupled with the notion that it is associated with DNA repair systems, may designate p53 as a control protein in the B-cell differentiation pathway.

An osteogenic member, MBA-15 cell line, from marrow stroma compartment, was examined for its ability to regulate mature lymphoma B cell line functions. The effect of MBA-15 cell secreting factor/s on cell proliferation, immunoglobulin secretion and isotype switch of 29M4.1 IgM+ mature B cell line was examined. The factor causes temporal arrest in cell proliferation, augments immunoglobulin secretion and has no effect on isotype switching. The results revealed a direct effect of the MBA-15 factors on the B cell functions. Factors secreted by other osteoblastic cells (ROS 17/2.8 and MC-3T3-E1) possess the same biological activities. In contrast, factors secreted by an endothelial adipocyte stromal cell line 14F1.1 lack these properties.

The direct linkage of the B cell maturation process and infiltration of the thymus with mature B cells was studied in SJL mice. Phenotypically and functionally, B cells in the thymus of old SJL mice are mature B cells; IgM+, IgD+, Ly-1-, and evince a high proliferative response to lipopolysaccharide and a low one to dextran sulphate. Memory B cells can be found in the thymus of mice immunized with T-dependent or T-independent antigens. Chronic depletion and B cell maturation arrest induced by fractionated total lymphoid irradiation or by neonatal splenectomy eliminate B cells from the thymus and block their migration from the periphery to the thymus. When examined in adoptive transfer experiments, thymus B cells were found to possess a normal migration pattern and homing receptors; their migration pattern did not differ from that of lymph node or splenic B cells. It is evident, therefore, that the large number of normal functioning B cells in the thymus of SJL mice reflects a massive infiltration of the thymus by mature B cells from the periphery due to thymus dysfunction rather than to an abnormal in situ differentiation of intrathymic B cell precursors.

Previously reported studies revealed that extensive immunization with the Methanol Extraction Residue (MER) of BCG tubercle bacillus in Incomplete Freund's Adjuvant (IFA) induced marked suppression of T and B cell functions in vivo and in vitro. The purpose of the present work was to determine whether immunosuppression induced by hyperimmunization with MER is correlated with changes in morphological characteristics and in phenotype pattern of spleen and peritoneal lymphoid cells. Hyperimmunization with MER resulted in a marked increase in the number of spleen cells and in enlargement and granulation of spleen and peritoneal cells. Similar changes in size and granulation were also observed in isolated fractions of splenic T and B cells. Extensive immunization with MER also induced marked decrease in the total number of T cells (Thy 1,2 positive). The decrease in T cells was observed in all three T-cell subsets investigated: Lyt-1, Lyt-2 and L3T4 positive. Although the number of splenic B cells was decreased in samples (10,000 cells), taken from MER hyperimmunized mice, this decrease was compensated by overall increase in the number of spleen cells. The marked decrease in the percentage of splenic T cells was counterbalanced by marked increase in the splenic macrophage population: increase in MAC-1, MAC-2 and MAC-3 positive cells. It is concluded that extensive immunization with MER induces morphological changes in spleen and peritoneal cells, marked decrease in the number of splenic T cells and marked increase of the splenic macrophagic population. It is postulated that these changes are correlated with induction of immunosuppression by a similar procedure of extensive immunization with the agent.

We investigated the ability of IgM-, IgD-, and IgG-bearing cells from the spleens of (BALB/c x C57BL/Ka)F1 mice primed to dinitrophenyl-bovine serum albumin (DNP-BSA) to restore the adoptive secondary anti-BSA and anti-DNP antibody responses. A rabbit anti-mouse IgD antiserum was prepared and the specificity documented by radioimmunoprecipitation, and cell surface staining. Purified populations of IgM-, IgD-, and IgG-bearing cells were prepared by immunofluorescent staining with isotype-specific reagents, and sorting on the fluorescence activated cell sorter. Bright or dull cells were transferred to irradiated syngeneic recipients which were challenged with DNP-BSA in saline. Unfractionated spleen cells restored an adoptive secondary serum antibody response which was all IgG (2-mercaptoethanol resistant). Purified IgM- or IgD-bearing cells restored both the secondary IgM and IgG antibody response. IgG-bearing cells restored only the IgG response. In addition, the IgG-bearing cells appear to suppress the adoptive secondary IgM response, since depletion of IgG-bearing cells from transferred spleen cells results in a marked increase in the adoptive IgM response.

We investigated the ability of IgM-, IgD-, and IgG-bearing cells from the spleens of unprimed (BALB/c x C57BL/Ka)F1 mice to restore the adoptive primary anti-BSA and anti-DNP antibody responses. Purified populations of isotype-specific cells were prepared by immunofluorescent staining and sorting on the fluorescence activated cell sorter. Bright or dull cells were transferred to irradiated syngeneic recipients which were challenged with DNP-BSA in complete Freund's adjuvant. Unfractionated spleen cells as well as IgM- and IgD-bearing cells restored the adoptive primary IgM and IgG antibody response. IgG-bearing cells restored a vigorous adoptive response which was all IgG (2-mercaptoethanol resistant). Depletion of IgG-bearing cells markedly increased the adoptive IgM response, and depletion of IgM-bearing cells markedly increased the IgG response. However, depletion of IgD-bearing cells resulted in a considerable reduction in the IgG response. The latter finding indicates that there is a subpopulation of IgD-bearing cells which express little or no surface IgM and which make a considerable contribution to the adoptive primary IgG response.

Total lymphoid irradiation (TLI) results in long-lasting changes in the characteristics of both T and B cells suggestive of arrested maturation. A characteristic feature of immature B cells is their high susceptibility to tolerance induction. This study examines the susceptibility to tolerance to bovine serum albumin (BSA) of TLI-treated mice. Two experimental protocols were designed. In the first, tolerance to BSA was induced in TLI-treated adult (BALB/c X C57BL/6)F1 mice, and the ability of B cells of those mice to respond to BSA was assessed in an adoptive transfer system. In the second experimental protocol, tolerance was induced in adoptive hosts reconstituted with purified B cells originating from TLI-treated mice and with splenic T cells of normal, untreated mice. Results obtained in these two systems clearly demonstrated that splenic B cells of TLI-treated mice are highly susceptible to tolerance induction. This high susceptibility of B cells is linked neither with the elevation of immature T cells nor with induced T suppressor cells which arise due to the long-term malfunction of the thymus. Tolerance could be induced in TLI cells even 4 months after termination of the treatment. Thus, maturation processes of B cells in TLI-treated mice are arrested for long periods of time.

The effect of fractionated total lymphoid irradiation (TLI) on the induction of cells blocking in vivo B cell maturation was examined. (BALB/c x C57BL/6)F1 mice received 200 rds lymphoid irradiation daily for 8 days. One month after termination of the irradiation splenic T cells of TLI-treated mice were transferred, together with normal bone marrow cells, to lethally irradiated hosts. In addition, splenic cells or splenic B cells of TLI mice were transferred to irradiated hosts. Two months later, we checked the reconstituted mice for their ability to produce in vivo anti-trinitrophenyl (TNP)-Ficoll antibodies and their capacity to respond in vitro to dextran sulfate and lipopolysaccharide. In parallel, stained spleen cells were analyzed on the fluorescence-activated cells sorter using monoclonal anti-mu, anti-delta and anti-B220 surface marker antibodies. The results indicate that splenic T cells originating from TLI-treated mice cause maturation arrest of normal and TLI-treated B cells, since normal bone marrow cells injected together with those cells have immature phenotypic and functional features. In addition, neither splenic cells nor splenic B cells of TLI-treated mice can mature upon transfer to normal irradiated hosts. The B cells are IgM+, IgD-; they respond with a high proliferation rate to dextran sulfate and low proliferation rate to lipopolysaccharide, and poorly to TNP-Ficoll challenge.

We determined whether primed and unprimed B cells in the spleen of (BALB/c x C57BL/Ka)F(1) mice contain subpopulations that express a predominant surface Ig isotype. Spleen cells were stained for surface isotypes and sorted on the fluorescence-activated cell sorter (FACS) in order to obtain B cells bearing predominantly IgM (mup cells), IgD (deltap cells), or IgG (gammap cells). Each population was assayed for its capacity to restore the adoptive primary and secondary anti-bovine serum albumin (BSA) antibody response in irradiated syngeneic recipients. In addition, the adoptive response restored by isotype-predominant cells was compared to that restored by isotype- positive cells (B cells bearing a given surface isotype alone or in combination with others). The experimental results show that mup cells restore the adoptive primary and secondary IgM and IgG responses to BSA, and gammaP cells restore only the primary and secondary IgG response. Deltap Cells restored the adoptive secondary IgG response, but failed to restore the adoptive primary response at the cell doses tested. GammaP Cells but not deltap cells suppressed the IgM response of the mu(+) and delta(+) cells. The contribution of isotype-predominant cells to both the adoptive primary and secondary anti-BSA response was smaller than that of B cells bearing a combination of surface isotypes. Differences in the Ig isotype pattern expressed on the surface of primed and unprimed B cells are discussed.

The pre-thymic cell phenotypes arising in spleen and bone marrow (BM) of fractionated total lymphoid irradiation (TLI)-treated mice are characterized in this paper. Different strains of mice received 200 rad lymphoid irradiation daily for 8 to 10 days. At different time intervals after treatment, BM or spleen cells of treated mice were stained with a battery of antisera directed specifically to TL and Thy-1 antigens. Analysis of the stained cells on a fluorescence-activated cell sorter (FACS) apparatus revealed that 10 to 30% of spleen or BM cells of TLI-treated mice were TL+ cells whereas no TL+ cells could be found in the same organs in the normal untreated mice. The appearance of TL+ cells in the spleen and BM of TLI-treated mice was found to be thymic independent, as thymectomy of mice neither prevented nor altered the kinetics of TL+ cell appearance. The sequence of antigenic marker appearance reveals that TL markers precede Thy-1 markers and that TLaa markers precede TL4 antigen. The relevance of the TL4+ cells to the development of leukemias is discussed.

The effect of fractionated total lymphoid irradiation (TLI) on the maturation of B cells was examined in mice. At various times after irradiation of the mice, their spleen cells were tested for the mitogenic responses to dextran sulfate and lipopolysaccharide. In parallel the cells were stained with fluorescent anti-Thy-1 and various anti-Ig antisera, and were analyzed on a fluorescence-activated cell sorter (FACS). By comparison with normal spleen cells the cells of TLI-treated mice gave a high response to dextran sulfate and a low response to lipopolysaccharide. FACS analysis revealed that TLI-treated spleen contains elevated numbers of B cells bearing high IgM and low IgD on the surface. The B cells of TLI-treated mice retained these immature characteristics even two to four months after the last irradiation. These findings indicate that TLI causes a longlasting blockage in B cell maturation processes.

The role of IgD (delta) in the induction of tolerance in murine B lymphocytes was explored in the in vivo adoptive antibody response to bovine serum albumin (BSA). Delta+ or delta- B cells purified on the fluorescence-activated cell sorter were injected, with or without purified T cells, into lethally irradiated intermediate hosts, which were rendered tolerant by an injection of deaggregated BSA one day after the cell transfer. Spleen cells from the intermediate hosts were treated with anti-Thy-1.2 antiserum and complement mixed with normal T cells and were transferred to final hosts. The ability of the B cells of the final hosts to respond to the tolerogen BSA and to dinitrophenylated human gamma globulin antigen was examined. This approach enabled examination of the function of the B cell subpopulations in an environment free of tolerogen and of induced T suppressor cells. The results revealed that in the presence of T cells the delta+ subpopulation was highly resistant to tolerance induction, whereas the delta- subpopulation was highly susceptible to tolerance induction. However, there was no difference in susceptibility to tolerance induction between the two subpopulations when tolerance was induced in the absence of T cells.