Impaired light chain allelic exclusion and lack of positive selection in immature B cells expressing incompetent receptor deficient of CD19

Positive signaling is now thought to be important for B cell maturation, although the nature of such signals has not yet been defined. We are studying the regulatory role of B cell Ag receptor (BCR) signaling in mediating positive selection of immature B cells. To do so, we use Ig transgenic mice (3-83Tg) that are deficient in CD19, thus generating a monoclonal immature B cell population expressing signaling-incompetent BCR. Immature 3-83Tg CD19(-/-) B cells undergo developmental arrest in the bone marrow, allowing maturation only to cells that effectively compensate for the compromised receptor by elevated levels of BCR. We find that developmentally arrested 3-83Tg CD19(-/-) B cells fail to impose L chain allelic exclusion and undergo intensive V(D)J recombination to edit their BCR. Furthermore, immature 3-83Tg CD19(-/-) B cells, which were grown in vitro, failed to undergo positive selection and to survive when adoptively transferred into normal recipients. However, elevation of BCR expression levels, obtained by transgene homozygosity, effectively compensated for the compromised BCR and completely restored BCR-mediated Ca(2+) influx, allelic exclusion, and positive selection. Our results suggest that the BCR signaling threshold mediates positive selection of developing B cells, and that a receptor-editing mechanism has an important role in rescuing cells that fail positive selection because of incompetent receptors.     

A CD19-dependent signaling pathway regulates autoimmunity in Lyn-deficient mice

CD19 and the Src family protein tyrosine kinases (PTKs) are important regulators of intrinsic signaling thresholds in B cells. Regulation is achieved by cross-talk between Src family PTKs and CD19; Lyn is essential for CD19 phosphorylation, while CD19 establishes an Src family PTK activation loop that amplifies kinase activity. However, CD19-deficient (CD19(-/-)) B cells are hyporesponsive to transmembrane signals, while Lyn-deficient (Lyn(-/-)) B cells exhibit a hyper-responsive phenotype resulting in autoimmunity. To identify the outcome of interactions between CD19 and Src family PTKs in vivo, B cell function was examined in mice deficient for CD19 and Lyn (CD19/Lyn(-/-)). Remarkably, CD19 deficiency suppressed the hyper-responsive phenotype of Lyn(-/-) B cells and autoimmunity characterized by serum autoantibodies and immune complex-mediated glomerulonephritis in Lyn(-/-) mice. Consistent with Lyn and CD19 each regulating conventional B cell development, B1 cell development was markedly reduced by Lyn deficiency, with further reductions in the absence of CD19 expression. Tyrosine phosphorylation of Fyn and other cellular proteins induced following B cell Ag receptor ligation was dramatically reduced in CD19/Lyn(-/-) B cells relative to Lyn(-/-) B cells, while Syk phosphorylation was normal. In addition, the enhanced intracellular Ca(2+) responses following B cell Ag receptor ligation that typify Lyn deficiency were delayed by the loss of CD19 expression. BCR-induced proliferation and humoral immune responses were also markedly inhibited by CD19/Lyn deficiency. These findings demonstrate that while the CD19/Lyn amplification loop is a major regulator of signal transduction thresholds in B lymphocytes, CD19 regulation of other Src family PTKs also influences B cell function and the development of autoimmunity.     

Identification and characterization of a cell cycle and apoptosis regulatory protein-1 as a novel mediator of apoptosis signaling by retinoid CD437

CD437, a novel retinoid, causes cell cycle arrest and apoptosis in a number of cancer cells including human breast carcinoma (HBC) by utilizing an undefined retinoic acid receptor/retinoid X receptor-independent mechanism. To delineate mediators of CD437 signaling, we utilized a random antisense-dependent functional knockout genetic approach. We identified a cDNA that encodes approximately 130-kDa HBC cell perinuclear protein (termed CARP-1). Treatments with CD437 or chemotherapeutic agent adriamycin, as well as serum deprivation of HBC cells, stimulate CARP-1 expression. Reduced levels of CARP-1 result in inhibition of apoptosis by CD437 or adriamycin, whereas increased expression of CARP-1 causes elevated levels of cyclin-dependent kinase inhibitor p21WAF1/CIP1 and apoptosis. CARP-1 interacts with 14-3-3 protein as well as causes reduced expression of cell cycle regulatory genes including c-Myc and cyclin B1. Loss of c-Myc sensitizes cells to apoptosis by CARP-1, whereas expression of c-Myc or 14-3-3 inhibits CARP-1-dependent apoptosis. Thus, apoptosis induction by CARP-1 involves sequestration of 14-3-3 and CARP-1-mediated altered expression of multiple cell cycle regulatory genes. Identification of CARP-1 as a key mediator of signaling by CD437 or adriamycin allows for delineation of pathways that, in turn, may prove beneficial for design and targeting of novel antitumor agents.     

Th3 cells in peripheral tolerance. II. TGF-beta-transgenic Th3 cells rescue IL-2-deficient mice from autoimmunity

We developed a transgenic (Tg) mouse that expresses TGF-beta under control of the IL-2 promoter to investigate Th3 cell differentiation both in vitro and in vivo. We previously found that repetitive in vitro Ag stimulation results in constant expression of Foxp3 in TGF-beta-Tg Th3 cells that acquire regulatory function independent of surface expression of CD25. To examine the differentiation and function of Th3 cells in vivo and to compare them with thymic-derived CD4(+)CD25(+) regulatory T cells (Treg), we introduced the TGF-beta transgene into T cells of IL-2-deficient (IL-2(-/-)) mice. We found that the induction, differentiation, and function of TGF-beta-derived Foxp3(+) Th3 cells were independent of IL-2, which differs from thymic Tregs. In an environment that lacks functional CD25(+) thymic-derived Tregs, expression of the TGF-beta transgene in IL-2(-/-) mice led to the induction of distinct CD25(-) regulatory cells in the periphery. These cells expressed Foxp3 and efficiently controlled hyperproliferation of T cells and rescued the IL-2(-/-) mouse from lethal autoimmunity. Unlike IL-2(-/-) animals, TGF-beta/IL-2(-/-) mice had normal numbers of T cells, B cells, macrophages, and dendritic cells and did not have splenomegaly, lymphadenopathy, or inflammation in multiple organs. Accumulation of Foxp3(+) cells over time, however, was dependent on IL-2. Our results suggest that TGF-beta-derived Foxp3(+)CD25(+/-) Th3 regulatory cells represent a different cell lineage from thymic-derived CD25(+) Tregs in the periphery but may play an important role in maintaining thymic Tregs in the peripheral immune compartment by secretion of TGF-beta.     

Cyclin D3 compensates for loss of cyclin D2 in mouse B-lymphocytes activated via the antigen receptor and CD40

Cyclin D2 is the only D-type cyclin expressed in mature mouse B-lymphocytes, and its expression is associated with retinoblastoma protein (pRB) and pRB-related protein phosphorylation and induction of E2F activity, as B-cells enter the cell cycle following stimulation via surface IgM and/or CD40. Cyclin D-dependent kinase activity is required for cell proliferation, yet cyclin D2(-/-) mice have normal levels of mature B-lymphocytes. Here we show that B-lymphocytes from cyclin D2(-/-) mice can proliferate in response to anti-IgM and anti-CD40, but the time taken to enter S-phase is longer than for the corresponding cyclin D2(+/+) cells. This is due to the compensatory induction of cyclin D3, but not cyclin D1, which causes pRb phosphorylation on CDK4-specific sites. This is the first demonstration that loss of a D-type cyclin causes specific expression and functional compensation by another member of the family in vivo and provides a rationale for the presence of mature B-lymphocytes in cyclin D2(-/-) mice.     

CD19 can regulate B lymphocyte signal transduction independent of complement activation.

B lymphocytes are critically regulated by signals transduced through the CD19-CD21 cell surface receptor complex, where complement C3d binding to CD21 supplies an already characterized ligand. To determine the extent that CD19 function is controlled by complement activation, CD19-deficient mice (that are hyporesponsive to transmembrane signals) and mice overexpressing CD19 (that are hyperresponsive) were crossed with CD21- and C3-deficient mice. Cell surface CD19 and CD21 expression were significantly affected by the loss of CD21 and C3 expression, respectively. Mature B cells from CD21-deficient littermates had approximately 36% higher cell surface CD19 expression, whereas CD21/35 expression was increased by approximately 45% on B cells from C3-deficient mice. Negative regulation of CD19 and CD21 expression by CD21 and C3, respectively, may be functionally significant because small increases in cell surface CD19 overexpression can predispose to autoimmunity. Otherwise, B cell development and function in CD19-deficient and -overexpressing mice were not significantly affected by a simultaneous loss of CD21 expression. Although CD21-deficient mice were found to express a hypomorphic cell surface CD21 protein at low levels that associated with mouse CD19, C3 deficiency did not significantly affect B cell development and function in CD19-deficient or -overexpressing mice. These results, and the severe phenotype exhibited by CD19-deficient mice compared with CD21- or C3-deficient mice, collectively demonstrate that CD19 can regulate B cell signaling thresholds independent of CD21 engagement and complement activation.     

The Nef-mediated AIDS-like disease of CD4C/human immunodeficiency virus transgenic mice is associated with increased Fas/FasL expression on T cells and T-cell death but is not prevented in Fas-, FasL-, tumor necrosis factor receptor 1-, or interleukin-1beta-converting enzyme-deficient or Bcl2-expressing transgenic mice

CD4(+)- and CD8(+)-T-cell death is a frequent immunological dysfunction associated with the development of human AIDS. We studied a murine model of AIDS, the CD4C/HIV transgenic (Tg) mouse model, to assess the importance of the apoptotic pathway in human immunodeficiency virus type 1 (HIV-1) pathogenesis. In these Tg mice, Nef is the major determinant of the disease and is expressed in immature and mature CD4(+) T cells and in cells of the macrophage/myeloid lineage. We report here a novel AIDS-like phenotype: enhanced death, most likely by apoptosis (as assessed by 7-aminoactinomycin D and annexin V/propidium iodide staining), of Tg thymic and peripheral CD4(+) and CD8(+) T cells. The Tg CD4(+) and CD8(+) T cells were also more susceptible to cell death after activation in vitro in mixed lymph node (LN) cultures. However, activation-induced cell death was not higher in Tg than in non-Tg-purified CD4(+) T cells. In addition, expression of Fas and FasL, assessed by flow cytometry, was increased in CD4(+) and CD8(+) T cells from Tg mice compared to that of non-Tg littermates. Despite the enhanced expression of Fas and FasL on Tg CD4(+) and CD8(+) T cells, Fas (lpr/lpr) and FasL (gld/gld) mutant CD4C/HIV Tg mice developed an AIDS-like disease indistinguishable from lpr/+ and gld/+ CD4C/HIV Tg mice, including loss of CD4(+) T cells. Similarly, CD4C/HIV Tg mice homozygous for mutations of two other genes implicated in cell death (interleukin-1beta-converting enzyme [ICE], tumor necrosis factor receptor 1 [TNFR-1]) developed similar AIDS-like disease as their respective heterozygous controls. Moreover, the double-Tg mice from a cross between the Bcl2/Wehi25 and CD4C/HIV Tg mice showed no major protection against disease. These results represent genetic evidence for the dispensable role of Fas, FasL, ICE, and TNFR-1 on the development of both T-cell loss and organ disease of these Tg mice. They also provide compelling evidence on the lack of protection by Bcl2 against Tg CD4(+)-T-cell death. In view of the high resemblance between numerous phenotypes observed in the CD4C/HIV Tg mice and in human AIDS, our findings are likely to be relevant for the human disease.     

Differential role for cyclic AMP response element binding protein-1 in multiple stages of B cell development, differentiation, and survival

CREB-1 is expressed in the bone marrow and in developing B cells. To determine the role of CREB-1 in developing B cells in the bone marrow, several lines of transgenic (Tg) mice overexpressing a dominant-negative Ser(119-ala) phosphomutant CREB-1 in the bone marrow were generated. Analysis of RNA and protein revealed expression of the transgene in the bone marrow. Flow cytometric analysis of bone marrow cells from Tg mice revealed approximately 70% increase in pre-B1 (CD43(+)B220(+)CD24(+(int))) and approximately 60% decreased pre-BII (CD43(+)B220(+)CD24(++(high))) cells, indicating a developmental block in pre-BI to pre-BII transition. Consistent with this, the Tg mice showed approximately 4-fold decrease in immature and mature B cells in the bone marrow. RT-PCR analysis of RNA from Tg mice revealed increased JunB and c-Jun in pre-BII cells associated with decreased S-phase entry. Adoptive transfer of bone marrow cells into RAG-2(-/-) mice resulted in reconstitution of non-Tg but not Tg bone marrow-derived CD43(+)B220(+)CD24(high) population that is normally absent in RAG-2(-/-) mice. In the periphery, the Tg mice exhibited decreased CD21(dim)CD23(high)IgM(+) follicular B cells in the spleen and increased B1a and B1b B cells in the peritoneum. While exhibiting normal Ab responses to T-independent Ags and primary response to the T-dependent Ag DNP-keyhole limpet hemocyanin, the Tg mice exhibited severely impaired secondary Ab responses. These studies provide the first evidence for a differential role for CRE-binding proteins in multiple stages of B cell development, functional maturation, and B1 and B2 B cells.     

Severely impaired B lymphocyte proliferation, survival, and induction of the c-Myc:Cullin 1 ubiquitin ligase pathway resulting from CD22 deficiency on the C57BL/6 genetic background

Understanding the molecular mechanisms through which CD22 regulates B lymphocyte homeostasis, signal transduction, and tolerance is critical to defining normal B cell function and understanding the role of CD22 in autoimmunity. Therefore, CD22 function was examined in vivo and in vitro using B cells from CD22-deficient (CD22(-/-)) mice. Backcrossing of founder CD22(-/-) mice onto the C57BL/6 (B6) genetic background from a B6/129 mixed background resulted in a dramatically reduced B cell proliferative response following IgM ligation, characterized by a paucity of lymphoblasts and augmented apoptosis. Also, the phenotype of splenic B6 CD22(-/-) B cells was uniquely HSA(high) and IgD(low)/CD21(low) with intermediate levels of CD5 expression, although the percentages of mature and transitional B cells were normal. That B6 CD22(-/-) B cells predominantly underwent apoptosis following IgM ligation correlated with this unique tolerant phenotype, as well as defective induction of the c-Myc:Cullin 1 (CUL1) ubiquitin ligase pathway that is necessary for progression to the S phase of cell cycle. CD40 ligation compensated for CD22 deficiency by restoring lymphoblast development, proliferation, c-Myc and CUL1 expression, and protein ubiquitination/degradation in IgM-stimulated B6 CD22(-/-) B cell cultures. Thereby, this study expands our current understanding of the complex role of CD22 during B cell homeostasis and Ag responsiveness, and reveals that the impact of CD22 deficiency is dictated by the genetic background on which it is rendered. Moreover, this study defines CD22 and CD40 as the first examples of lymphocyte coreceptors that influence induction of the c-Myc:CUL1 ubiquitin ligase pathway.     

Transgenic expression of insulin receptor substrate 2 in murine B cells alters the cell density-dependence of IgE production in vitro and enhances IgE production in vivo

Previous studies have shown that insulin receptor substrate (IRS)1 and IRS2 mediate proliferative and antiapoptotic signaling through the IL-4R in 32D cells; however their role in regulating normal B cell responses is not clear. To investigate the role of IRS2 in normal B cell function, we developed IRS2 transgenic (Tg) mice on the C57BL/6 background. Western blot analysis revealed a 2-fold elevation in IRS2 protein levels in Tg(+) mice compared with littermate controls and a 3-fold increase in basal tyrosine phosphorylated IRS2 in the absence of IL-4 stimulation. IL-4-induced tyrosine phosphorylation of IRS2 was elevated in Tg(+) B cells, whereas IL-4-induced phosphorylation of STAT6 was similar between Tg(+) and Tg(-) B cells. Tg expression of IRS2 had little effect on IL-4-mediated proliferation and no effect on protection from apoptosis. However, production of IgE and IgG1 by Tg(+) B cells using standard in vitro conditions was diminished 50-60%. Because Ig production in vitro is known to be highly cell concentration-dependent, we performed experiments at different cell concentrations. Interestingly, at very low B cell concentrations (1000-5000 B cells/well), IgE and IgG1 production by Tg(+) B cells was greater than that of controls, whereas at higher cell concentrations (10,000-20,000 cells/well) Ig production by Tg(+) B cells was less than controls. Furthermore, in vivo immunization with OVA-alum or goat anti-IgD resulted in elevated serum IgE levels in the Tg(+) mice. These results indicate that overexpression of IRS2 alters the B cell intrinsic density-dependence of IgE and IgG1 production in vitro and enhances IgE responses in vivo.     

Overexpression of the Ctla-4 isoform lacking exons 2 and 3 causes autoimmunity

CTLA-4 is a potent inhibitor of T cell activation, primarily upon binding to its costimulatory ligands (B7.1 and B7.2) expressed on APCs. However, variants of CTLA-4 can also function independently of B7 molecules. 1/4CTLA-4 is a highly conserved isoform encoded by exons 1 and 4 of the Ctla4 gene that lacks the ligand-binding and the transmembrane domains, and as yet, its function is not known. To investigate the function of 1/4CTLA-4, we generated transgenic (Tg) mice overexpressing this variant. Cytokine production by 1/4CTLA-4 Tg T cells was elevated compared with wild type T cells. The frequency of CD44(high) memory T cells in 1/4CTLA-4 Tg mice was increased, and as the mice aged, the frequency further increased. 1/4CTLA-4 Tg mice >1 y old had increased expression of T cell activation markers and developed spontaneous autoimmunity, including elevated production of autoantibodies. In contrast with young 1/4CTLA-4 Tg mice, aged 1/4CTLA-4 Tg mice had elevated frequencies of Foxp3(+) regulatory T cells, but the regulatory T cells from these mice were not able to inhibit colitis development. Collectively, these data suggest that the function of the 1/4CTLA-4 isoform is distinct from that of CTLA-4 in that it enhances T cell activation and promotes autoimmunity rather than inhibiting immune responses.     

The role of the Ets2 transcription factor in the proliferation,maturation, and survival of mouse thymocytes

In this study, we investigated the effects of Ets2 expression on the proliferation, maturation, and survival of thymocytes by establishing transgenic mice that specifically express Ets2 or a dominant negative form of Ets2, Deltaets2, in the thymus. We show that, in young animals, there are fewer T cells in Deltaets2 transgenic thymi and that the maturation of these T cells is affected at the CD4(-)CD8(-) double-negative to CD4(+)CD8(+) double-positive transition compared with wild-type littermate mice. Partial recovery in the number of thymocytes and full T cell maturation are restored with increasing age of Deltaets2 transgenic animals. However, thymocytes from adult Deltaets2 transgenic mice cultured ex vivo are more sensitive to cell death and to glucocorticoid-induced apoptosis than are T cells from control littermate mice. We also show that T cells from adult ets2 transgenic mice proliferate faster than their wild-type littermates. The proliferation and survival of these T cells are clearly affected upon apoptotic signals: glucocorticoid-induced apoptosis induces T cells from ets2 transgenic mice to continue to proliferate in vivo and to survive better ex vivo than T cells from control littermates. It has been shown that c-Myc expression is required for thymic proliferation and improves thymocyte survival of dexamethasone-treated animals. We show that the expression of c-Myc, an Ets2 target, is elevated in T cells freshly isolated from thymi of ets2 transgenic mice pretreated with dexamethasone. Together, these results show that Ets2 plays a role in the proliferation and survival of thymocytes, implicating a Myc-dependent pathway.     

Expression of human complement receptor type 2 (CD21) in mice during early B cell development results in a reduction in mature B cells and hypogammaglobulinemia

Complement receptor (CR) type 2 (CR2/CD21) is normally expressed only during the immature and mature stages of B cell development. In association with CD19, CR2 plays an important role in enhancing mature B cell responses to foreign Ag. We used a murine Vlambda2 promoter/Vlambda2-4 enhancer minigene to develop transgenic mice that initiate expression of human CR2 (hCR2) during the CD43(+)CD25(-) late pro-B cell stage of development. We found peripheral blood B cell numbers reduced by 60% in mice expressing high levels of hCR2 and by 15% in mice with intermediate receptor expression. Splenic B cell populations were altered with an expansion of marginal zone cells, and basal serum IgG levels as well as T-dependent immune responses were also significantly decreased in transgenic mice. Mice expressing the highest levels of hCR2 demonstrated in the bone marrow a slight increase in B220(int)CD43(+)CD25(-) B cells in association with a substantial decrease in immature and mature B cells, indicative of a developmental block in the pro-B cell stage. These data demonstrate that stage-specific expression of CR2 is necessary for normal B cell development, as premature receptor expression substantially alters this process. Alterations in B cell development are most likely due to engagement of pre-B cell receptor-mediated or other regulatory pathways by hCR2 in a CD19- and possibly C3 ligand-dependent manner.     

Complementary roles for CD19 and Bruton's tyrosine kinase in B lymphocyte signal transduction

CD19 and Bruton's tyrosine kinase (Btk) may function along common signaling pathways in regulating intrinsic and B cell Ag receptor (BCR)-induced signals. To identify physical and functional interactions between CD19 and Btk, a CD19-negative variant of the A20 B cell line was isolated, and CD19-deficient (CD19(-/-)) and CD19-overexpressing mice with the X-linked immunodeficient (Xid; Btk) mutation were generated. In A20 cells, Btk physically associated with CD19 following BCR engagement. CD19 and Btk interactions were not required for initial Btk phosphorylation, but CD19 expression maintained Btk in an activated state following BCR engagement. In primary B cells, CD19 signaling also required downstream Btk function since CD19-induced intracellular Ca(2+) ([Ca(2+)](i)) responses were modest in Xid B cells. In addition, CD19 overexpression did not normalize the Xid phenotype and most phenotypic and functional hallmarks of CD19 overexpression were not evident in these mice. However, CD19 and Btk also regulate independent signaling pathways since their combined loss had additive inhibitory effects on BCR-induced [Ca(2+)](i) responses and CD19 deficiency induced a severe immunodeficiency in Xid mice. Thus, CD19 expression amplifies or prolongs Btk-mediated signaling, rather than serving as a required agent for Btk activation. Consistent with this, phosphatidylinositol 3-monophosphate kinase and Akt activation were normal in CD19(-/-) B cells following IgM engagement, although their kinetics of activation was altered. Thus, these biochemical and compound gene dosage studies indicate that Btk activation and [Ca(2+)](i) responses following BCR engagement are regulated through multiple pathways, including a CD19/Src family kinase-dependent pathway that promotes the longevity of Btk signaling.     

Phosphatidylinositol 3-kinase-dependent mitogen-activated protein/extracellular signal-regulated kinase kinase 1/2 and NF-kappa B signaling pathways are required for B cell antigen receptor-mediated cyclin D2 induction in mature B cells

Phosphatidylinositol 3-kinase (PI-3K) has been linked to promitogenic responses in splenic B cells following B cell Ag receptor (BCR) cross-linking; however identification of the signaling intermediates that link PI-3K activity to the cell cycle remains incomplete. We show that cyclin D2 induction is blocked by the PI-3K inhibitors wortmannin and LY294002, which coincides with impaired BCR-mediated mitogen-activated protein/extracellular signal-related kinase kinase (MEK)1/2 and p42/44ERK phosphorylation on activation residues. Cyclin D2 induction is virtually absent in B lymphocytes from mice deficient in the class I(A) PI-3K p85alpha regulatory subunit. In contrast to studies with PI-3K inhibitors, which inhibit all classes of PI-3Ks, the p85alpha regulatory subunit is not required for BCR-induced MEK1/2 and p42/44ERK phosphorylation, suggesting the contribution of another PI-3K family members in MEK1/2 and p42/44ERK activation. However, p85alpha(-/-) splenic B cells are defective in BCR-induced IkappaB kinase beta and IkappaBalpha phosphorylation. We demonstrate that NF-kappaB signaling is required for cyclin D2 induction via the BCR in normal B cells, implicating a possible link with the defective IkappaB kinase beta and IkappaBalpha phosphorylation in p85alpha(-/-) splenic B cells and their ability to induce cyclin D2. These results indicate that MEK1/2-p42/44ERK and NF-kappaB pathways link PI-3K activity to Ag receptor-mediated cyclin D2 induction in splenic B cells.     

Host NKT cells can prevent graft-versus-host disease and permit graft antitumor activity after bone marrow transplantation

Allogeneic bone marrow transplantation is a curative treatment for leukemia and lymphoma, but graft-vs-host disease (GVHD) remains a major complication. Using a GVHD protective nonmyeloablative conditioning regimen of total lymphoid irradiation and antithymocyte serum (TLI/ATS) in mice that has been recently adapted to clinical studies, we show that regulatory host NKT cells prevent the expansion and tissue inflammation induced by donor T cells, but allow retention of the killing activity of donor T cells against the BCL1 B cell lymphoma. Whereas wild-type hosts given transplants from wild-type donors were protected against progressive tumor growth and lethal GVHD, NKT cell-deficient CD1d-/- and Jalpha-18-/- host mice given wild-type transplants cleared the tumor cells but died of GVHD. In contrast, wild-type hosts given transplants from CD8-/- or perforin-/- donors had progressive tumor growth without GVHD. Injection of host-type NKT cells into Jalpha-18-/- host mice conditioned with TLI/ATS markedly reduced the early expansion and colon injury induced by donor T cells. In conclusion, after TLI/ATS host conditioning and allogeneic bone marrow transplantation, host NKT cells can separate the proinflammatory and tumor cytolytic functions of donor T cells.     

Modification of ligand-independent B cell receptor tonic signals activates receptor editing in immature B lymphocytes

Maturation of B lymphocytes strictly depends on the signaling competence of the B cell antigen receptor (BCR). Autoreactive receptors undergo negative selection and can be replaced by receptor editing. In addition, the process of maturation of non-self B cells and migration to the spleen, referred to as positive selection, is limited by the signaling competence of the BCR. Using 3-83Tg mice deficient of CD19 we have shown that signaling incompetence not only blocks positive selection but also activates receptor editing. Here we study the role of ligand-independent BCR tonic tyrosine phosphorylation signals in activation of receptor editing. We find that editing, immature 3-83Tg B cells deficient of CD19 have elevated BCR tonic signals and that lowering these tonic signals effectively suppresses receptor editing. Furthermore, we show that elevation of BCR tonic signals in non-editing, immature 3-83Tg B cells stimulates significant receptor editing. We also show that positive selection and developmental progression from the bone marrow to the spleen are limited to cells capable of establishing appropriate tonic signals, as in contrast to immature cells, splenic 3-83Tg B cells deficient of CD19 have BCR tonic signals similar to those of the control 3-83Tg cells. This developmental progression is accompanied by activation of molecules signaling for growth and survival. Hence, we suggest that ligand-independent BCR tonic signals are required for promoting positive selection and suppressing the receptor-editing mechanism in immature B cells.     

Primary human immunodeficiency virus type 1 nef alleles show major differences in pathogenicity in transgenic mice

We previously reported that the human immunodeficiency virus type 1 NL4-3 Nef is necessary and sufficient to induce a severe AIDS-like disease in transgenic (Tg) mice when the protein is expressed under the regulatory sequences of the human CD4 gene. We have now assayed additional Nef alleles (SF2, JR-CSF, YU10x, and NL4-3 [T71R] Nef alleles), including some from long-term nonprogressors (AD-93, 032an, and 039nm alleles) in the same Tg system and compared their pathogenicities. All these Nef alleles downregulated cell surface CD4 in human cells in vitro and also, with the exception of Nef(YU10x), in Tg CD4(+) T cells. Depletion of double-positive and single-positive thymocytes occurred with all alleles but was less pronounced in Nef(YU10x) Tg mice. A loss of peripheral CD4(+) T cells was observed with all alleles but was minimal in Nef(YU10x) Tg mice. In Nef(032an) and Nef(SF2) Tg mice, T-cell loss was severe despite lower levels of Tg expression, suggesting a higher virulence of these alleles. All Nef alleles except the Nef(YU10x) and Nef(NL4-3(T71R)) alleles induced an enhanced activated memory (CD25(+) CD69(+) CD44(high) CD45RB(low) CD62L(low)) and apoptotic phenotype. Also, all could interact with and/or activate PAK2 except the Nef(JR-CSF) allele. Organ (lung and kidney) diseases were present in Nef(NL4-3(T71R)), Nef(032an), Nef(039nm), and Nef(SF2) Tg mice, despite very low levels of Tg expression for the last strain. However, no organ disease or minimal organ disease developed in Nef(YU10x) and Nef(AD-93) Tg mice and Nef(JR-CSF) Tg mice, respectively, despite high levels of Tg expression. Our data show that important differences in the pathogenicities of various Nef alleles can be scored in Tg mice. Interestingly, our results also revealed that some phenotypes can segregate independently, such as CD4(+) T-cell depletion and activation, as well as severe depletion of thymic CD4(+) T cells and peripheral CD4(+) T cells. Therefore, expression of Nef alleles in Tg mice under the CD4C regulatory elements represents a novel assay for measuring their pathogenicity. Because of the very high similarity of this murine AIDS-like disease to human AIDS, this assay may have a predictive value regarding the behavior of Nef in infected humans.     

Lupus-specific antiribonucleoprotein B cell tolerance in nonautoimmune mice is maintained by differentiation to B-1 and governed by B cell receptor signaling thresholds

Systemic lupus erythematosus is an autoimmune disease characterized by the presence of autoantibodies. One of the unique targets of the immune system in systemic lupus erythematosus is Sm, a ribonucleoprotein present in all cells. To understand the regulation of B cells specific to the Sm Ag in normal mice, we have generated an Ig H chain transgenic mouse (2-12H Tg). 2-12H Tg mice produce B cells specific for the Sm that remain tolerant due to ignorance. We demonstrate here that anti-Sm B cells of 2-12H Tg mice can differentiate into Sm-specific peritoneal B-1 cells that remain tolerant. Differentiation to B-1 and tolerance are governed by the strength of B cell receptor signaling, since manipulations of the B cell receptor coreceptors CD19 and CD22 affect anti-Sm B cell differentiation and autoantibody production. These results suggest a differentiation scheme in which peripheral ignorance to Sm is maintained in mice by the differentiation of anti-Sm B cells to B-1 cells that have increased activation thresholds.