Insulin-like growth factor-1 controls type 2 T cell-independent B cell response

The IGF-1 receptor (IGF-1R) is expressed on T and B lymphocytes, and the expression of the insulin- and IGF-1-signaling machinery undergoes defined changes throughout lineage differentiation, offering a putative role for IGF-1 in the regulation of immune responses. To study the role of the IGF-1R in lymphocyte differentiation and function in vivo, we have reconstituted immunodeficient RAG2-deficient mice with IGF-1R(-/-) fetal liver cells. Despite the absence of IGF-1Rs, the development and ex vivo activation of B and T lymphocytes were unaltered in these chimeric mice. By contrast, the humoral immune response to the T cell-independent type 2 Ag 4-hydroxy-3-nitrophenyl acetyl-Ficoll was significantly reduced in mice reconstituted with IGF-1R-deficient fetal liver cells, whereas responses to the T cell-dependent Ag 4-hydroxy-3-nitrophenyl acetyl-chicken globulin were normal. Moreover, in an in vitro model of T cell-independent type 2 responses, IGF-1 promoted Ig production potently upon polyvalent membrane-IgD cross-linking. These data indicate that functional IGF-1R signaling is required for T cell-independent B cell responses in vivo, defining a novel regulatory mechanism for the immune response against bacterial polysaccharides.     

Negative regulation of lymphocyte activation by the adaptor protein LAX

The membrane-associated adaptor protein LAX is a linker for activation of T cells (LAT)-like molecule that is expressed in lymphoid tissues. Upon stimulation of T or B cells, it is phosphorylated and interacts with Grb2 and the p85 subunit of PI3K. LAX, however, is not capable of replacing LAT in the TCR signaling pathway. In this study we report that upon T or B cell activation, the LAX protein was up-regulated dramatically. Although disruption of the LAX gene by homologous recombination had no major impact on lymphocyte development, it caused a significant reduction in CD23 expression on mature B cells. Interestingly, naive LAX(-/-) mice had spontaneous germinal center formation. Compared with normal T and B cells, LAX(-/-) T and B cells were hyperresponsive and had enhanced calcium flux, protein tyrosine phosphorylation, MAPK and Akt activation, and cell survival upon engagement of the T or B AgRs. Our data demonstrate that LAX functions as a negative regulator in lymphocyte signaling.     

The Rac2 guanosine triphosphatase regulates B lymphocyte antigen receptor responses and chemotaxis and is required for establishment of B-1a and marginal zone B lymphocytes

We have defined roles for the hemopoietic-specific Rho guanosine triphosphatase, Rac2, in B lymphocyte development and function through examination of rac2(-/-) mice. Rac2-deficient mice displayed peripheral blood B lymphocytosis and marked reductions in peritoneal cavity B-1a lymphocytes, marginal zone B lymphocytes, and IgM-secreting plasma cells as well as reduced concentrations of serum IgM and IgA. The rac2(-/-) B lymphocytes exhibited reduced calcium flux following coligation of B cell AgR and CD19 and reduced chemotaxis in chemokine gradients. T cell-independent responses to DNP-dextran were of reduced magnitude, but normal kinetics, in rac2(-/-) mice, while T-dependent responses to nitrophenyl-keyhole limpet hemocyanin were subtly abnormal. Rac2 is therefore an essential element in regulating B lymphocyte functions and maintaining B lymphocyte populations in vivo.     

Effects of T3R alpha 1 and T3R alpha 2 gene deletion on T and B lymphocyte development

Thyroid hormones bind to several nuclear receptors encoded by T3R alpha and T3R beta genes. There is now accumulating evidence that thyroid hormones act on the immune system. Indeed, mice deficient for thyroid hormones show a reduction in lymphocyte production. However, the mechanisms involved and, in particular, the role of the different thyroid hormone receptors in lymphocyte development have not been investigated. To address that question, we have studied lymphocyte development in mice deficient for the T3R alpha 1 and T3R alpha 2 gene products. A strong decrease in spleen cell numbers was found compared with wild-type littermates, B lymphocytes being more severely affected than T lymphocytes. A significant decrease in splenic macrophage and granulocyte numbers was also found. In bone marrow, a reduction in CD45+/IgM- pro/pre-B cell numbers was found in these mice compared with wild-type littermates. This decrease seems to result from a proliferation defect, as CD45+/IgM- cells incorporate less 5-bromo-2'-deoxyuridine in vivo. To define the origin of the bone marrow development defect, chimeric animals between T3R alpha-/- and Rag1-/- mice were generated. Results indicate that for B cells the control of the population size by T3R alpha 1 and T3R alpha 2 is intrinsic. Altogether, these results show that T3R alpha 1 or T3R alpha 2 gene products are implicated in the control of the B cell pool size.     

A lymphotoxin-IFN-beta axis essential for lymphocyte survival revealed during cytomegalovirus infection

The importance of lymphotoxin (LT) betaR (LTbetaR) as a regulator of lymphoid organogenesis is well established, but its role in host defense has yet to be fully defined. In this study, we report that mice deficient in LTbetaR signaling were highly susceptible to infection with murine CMV (MCMV) and early during infection exhibited a catastrophic loss of T and B lymphocytes, although the majority of lymphocytes were themselves not directly infected. Moreover, bone marrow chimeras revealed that lymphocyte survival required LTalpha expression by hemopoietic cells, independent of developmental defects in lymphoid tissue, whereas LTbetaR expression by both stromal and hemopoietic cells was needed to prevent apoptosis. The induction of IFN-beta was also severely impaired in MCMV-infected LTalpha(-/-) mice, but immunotherapy with an agonist LTbetaR Ab restored IFN-beta levels, prevented lymphocyte death, and enhanced the survival of these mice. IFN-alphabetaR(-/-) mice were also found to exhibit profound lymphocyte death during MCMV infection, thus providing a potential mechanistic link between type 1 IFN induction and lymphocyte survival through a LTalphabeta-dependent pathway important for MCMV host defense.     

CD83 expression is a sensitive marker of activation required for B cell and CD4+ T cell longevity in vivo

CD83 is a surface marker that differentiates immature and mature human dendritic cell populations. Thymic epithelial cell expression of CD83 is also necessary for efficient CD4+ T cell development in mice. The altered phenotypes of peripheral B and CD4+ T cells, and the reduction of peripheral CD4+ T cells in CD83-/- mice, suggest additional functions for CD83. To assess this, a panel of mAbs was generated to characterize mouse CD83 expression by peripheral leukocytes. As in humans, activation of conventional and plasmacytoid murine dendritic cell subsets led to rapid up-regulation of CD83 surface expression in mice. In primary and secondary lymphoid compartments, a subset of B cells expressed low-level CD83, while CD83 was not detected on resting T cells. However, CD83 was prominently up-regulated on the majority of spleen B and T cells within hours of activation in vitro. In vivo, a low dose of hen egg lysozyme (1 microg) induced significant CD83 but not CD69 expression by Ag-specific B cells within 4 h of Ag challenge. Although B cell development appeared normal in CD83-/- mice, B and CD4+ T cell expression of CD83 was required for lymphocyte longevity in adoptive transfer experiments. Thus, the restricted expression pattern of CD83, its rapid induction following B cell and T cell activation, and its requirement for B cell and CD4+ T cell longevity demonstrate that CD83 is a functionally significant and sensitive marker of early lymphocyte activation in vivo.     

Mice lacking the CARD of CARMA1 exhibit defective B lymphocyte development and impaired proliferation of their B and T lymphocytes

CARMA1 (originally called CARD11) is a membrane-associated guanylate kinase family member that is required for T cell receptor (TCR)-induced NF-kappa B activation in T cell leukemia lines. It uses its N-terminal caspase activation and recruitment domain (CARD) to interact with the CARD in the downstream adaptor Bcl-10. We show that primary B and T lymphocytes from knock-in mice expressing only a CARDless form of CARMA1 (Delta CARD) are defective at mitogen-induced NF-kappa B activation and fail to proliferate. CARMA1 mutant mice exhibited normal T but impaired B cell development; CD5(+) peritoneal B cells were absent, and serum immunoglobulin levels were markedly reduced. A lacZ reporter gene knocked into the CARMA1 locus confirmed lymphocyte-specific expression of CARMA1. Thus, CARMA1 has an essential role in mediating B and T lymphocyte proliferation and requires its CARD to engage downstream signaling components.     

Human BLyS facilitates engraftment of human PBL derived B cells in immunodeficient mice

The production of fully immunologically competent humanized mice engrafted with peripheral lymphocyte populations provides a model for in vivo testing of new vaccines, the durability of immunological memory and cancer therapies. This approach is limited, however, by the failure to efficiently engraft human B lymphocytes in immunodeficient mice. We hypothesized that this deficiency was due to the failure of the murine microenvironment to support human B cell survival. We report that while the human B lymphocyte survival factor, B lymphocyte stimulator (BLyS/BAFF) enhances the survival of human B cells ex vivo, murine BLyS has no such protective effect. Although human B cells bound both human and murine BLyS, nuclear accumulation of NF-kappaB p52, an indication of the induction of a protective anti-apoptotic response, following stimulation with human BLyS was more robust than that induced with murine BLyS suggesting a fundamental disparity in BLyS receptor signaling. Efficient engraftment of both human B and T lymphocytes in NOD rag1(-/-) Prf1(-/-) immunodeficient mice treated with recombinant human BLyS is observed after adoptive transfer of human PBL relative to PBS treated controls. Human BLyS treated recipients had on average 40-fold higher levels of serum Ig than controls and mounted a de novo antibody response to the thymus-independent antigens in pneumovax vaccine. The data indicate that production of fully immunologically competent humanized mice from PBL can be markedly facilitated by providing human BLyS.     

Regulation of B cell differentiation and plasma cell generation by IL-21, a novel inducer of Blimp-1 and Bcl-6

IL-21 is a type I cytokine whose receptor is expressed on T, B, and NK cells. Within the B cell lineage, IL-21 regulates IgG1 production and cooperates with IL-4 for the production of multiple Ab classes in vivo. Using IL-21-transgenic mice and hydrodynamics-based gene delivery of IL-21 plasmid DNA into wild-type mice as well as in vitro studies, we demonstrate that although IL-21 induces death of resting B cells, it promotes differentiation of B cells into postswitch and plasma cells. Thus, IL-21 differentially influences B cell fate depending on the signaling context, explaining how IL-21 can be proapoptotic for B cells in vitro yet critical for Ag-specific Ig production in vivo. Moreover, we demonstrate that IL-21 unexpectedly induces expression of both Blimp-1 and Bcl-6, indicating mechanisms as to how IL-21 can serve as a complex regulator of B cell maturation and terminal differentiation. Finally, BXSB-Yaa mice, which develop a systemic lupus erythematosus-like disease, have greatly elevated IL-21, suggesting a role for IL-21 in the development of autoimmune disease.     

Cutting edge: the ontogeny and function of Va14Ja18 natural T lymphocytes require signal processing by protein kinase C theta and NF-kappa B

The rapid and robust immunoregulatory cytokine response of Va14Ja18 natural T (iNKT) cells to glycolipid Ags determines their diverse functions. Unlike conventional T cells, iNKT lymphocyte ontogeny absolutely requires NF-kappa B signaling. However, the precise role of NF-kappa B in iNKT cell function and the identity of upstream signals that activate NF-kappa B in this T cell subset remain unknown. Using mice in which iNKT cell ontogeny has been rescued despite inhibition of NF-kappa B signaling, we demonstrate that iNKT cell function requires NF-kappa B in a lymphocyte-intrinsic manner. Furthermore, the ontogeny of functional iNKT cells requires signaling through protein kinase C theta, which is dispensable for conventional T lymphocyte development. The unique requirement of protein kinase C theta implies that signals emanating from the TCR activate NF-kappa B during iNKT cell development and function. Thus, we conclude that NF-kappa B signaling plays a crucial role at distinct levels of iNKT cell biology.     

The role of CD40-CD154 interaction in antiviral T cell-independent IgG responses

Polyomavirus (PyV) infection elicits protective T cell-independent (TI) IgG responses in T cell-deficient mice. The question addressed in this report is whether CD40 signaling plays a role in this TI antiviral IgG response. Because CD40 ligand (CD40L) can be expressed on numerous cell types in addition to activated T cells, it is possible that cells other than T cells provide CD40L to signal through CD40 on B cells and hence positively influence the antiviral TI IgG responses. In this study we show, by blocking CD40-CD40L interactions in vivo with anti-CD40L Ab treatment in TCR betaxdelta-/- mice and by using SCID mice reconstituted with CD40-/- B cells, that the lack of CD40 signaling in B cells results in a 50% decrease in TI IgG secreted in response to PyV. SCID mice reconstituted with CD40L-/- B cells also responded to PyV infection with diminished IgG secretion compared with that of SCID mice reconstituted with wild-type B cells. This finding suggests that B cells may provide the CD40L for CD40 signaling in the absence of T cell help during acute virus infection. Our studies demonstrate that, although about half of the TI IgG responses to PyV are independent of CD40-CD40L interactions, these interactions occur in T cell-deficient mice and enhance antiviral TI Ab responses.     

Local glucocorticoid production in lymphoid organs of mice and birds: Functions in lymphocyte development

Circulating glucocorticoids (GCs) are powerful regulators of immunity. Stress-induced GC secretion by the adrenal glands initially enhances and later suppresses the immune response. GC targets include lymphocytes of the adaptive immune system, which are well known for their sensitivity to GCs. Less appreciated, however, is that GCs are locally produced in lymphoid organs, such as the thymus, where GCs play a critical role in selection of the T cell antigen receptor (TCR) repertoire. Here, we review the roles of systemic and locally-produced GCs in T lymphocyte development, which has been studied primarily in laboratory mice. By antagonizing TCR signaling in developing T cells, thymus-derived GCs promote selection of T cells with stronger TCR signaling. This results in increased T cell-mediated immune responses to a range of antigens. We then compare local and systemic GC patterns in mice to those in several bird species. Taken together, these studies suggest that a combination of adrenal and lymphoid GC production might function to adaptively regulate lymphocyte development and selection, and thus antigen-specific immune reactivity, to optimize survival under different environmental conditions. Future studies should examine how lymphoid GC patterns vary across other vertebrates, how GCs function in B lymphocyte development in the bone marrow, spleen, and the avian bursa of Fabricius, and whether GCs adaptively program immunity in free-living animals.     

The role of tumor-specific Lyt-1+2- T cells in eradicating tumor cells in vivo. I. Lyt-1+2- T cells do not necessarily require recruitment of host's cytotoxic T cell precursors for implementation of in vivo immunity

The present study determines the Ly phenotype of T cells mediating tumor cell rejection in vivo and investigates some of cellular mechanisms involved in the in vivo protective immunity. C3H/HeN mice were immunized to syngeneic X5563 plasmacytoma by intradermal (i.d.) inoculation of viable X5563 tumor cells, followed by the surgical resection of the tumor. Spleen cells from these immune mice were fractionated by treatment with anti-Lyt antibodies plus complement, and each Lyt subpopulation was tested for the reconstituting potential of in vivo protective immunity in syngeneic T cell-depleted mice (B cell mice). When C3H/HeN B cell mice were adoptively transferred with Lyt-1-2+ T cells from the above tumor-immunized mice, these B cell mice exhibited an appreciable cytotoxic T lymphocyte (CTL) response to the X5563 tumor, whereas they failed to resist the i.d. challenge of X5563 tumor cells. In contrast, the adoptive transfer of Lyt-1+2- anti-X5563 immune T cells into B cell mice produced complete protection against the subsequent tumor cell challenge. Although no CTL or antibody response against X5563 tumors was detected in the above tumor-resistant B cell mice, these mice were able to retain Lyt-1+2- T cell-mediated delayed-type hypersensitivity (DTH) responses to the X5563 tumor. These results indicate that Lyt-1+2- T cells depleted of the Lyt-2+ T cell subpopulation containing CTL or CTL precursors are effective in in vivo protective immunity, and that these Lyt-1+2- T cells implement their in vivo anti-tumor activity without inducing CTL or antibody responses. The mechanism(s) by which Lyt-1+2- T cells function in vivo for the implementation of tumor-specific immunity is discussed in the context of DTH responses to the tumor-associated antigens and its related Lyt-1+2- T cell-mediated lymphokine production.     

Abnormal Th1 cell differentiation and IFN-gamma production in T lymphocytes from motheaten viable mice mutant for Src homology 2 domain-containing protein tyrosine phosphatase-1

Src homology 2 domain-containing protein tyrosine phosphatase-1 (SHP-1) plays an important role in T and B lymphocyte signaling; however, the function of SHP-1 in Th cell differentiation, in particular, the Th1 response, has not been defined. In this study, we provide evidence that SHP-1 phosphatase negatively regulates Th1 cell development and IFN-gamma production. Compared with the wild-type control, anti-CD3-activated mouse T lymphocytes carrying the motheaten viable mutation in the SHP-1 gene produced a significantly increased amount of IFN-gamma in the presence of IL-12. This increase was also seen at the basal level without IL-12 addition. Similarly, Th1 cell differentiation and proliferation of anti-CD3-activated SHP-1 mutant lymph node cells in the presence or absence of IL-12 were markedly enhanced, indicating a negative role for SHP-1 phosphatase in such lymphocyte activities. Interestingly, IL-12-induced activation of Jak2 and STAT4, critical components for IL-12-mediated cellular responses, was shortened or attenuated in mutant T cells. Together these results suggest that SHP-1 negatively regulates Th1 cell development and functions through a mechanism that is not directly related to IL-12 signaling.     

The related adaptors, adaptor in lymphocytes of unknown function X and Rlk/Itk-binding protein, have nonredundant functions in lymphocytes

Adaptors play a critical role in regulating signaling pathways that control lymphocyte development and activation. Adaptor in lymphocytes of unknown function X (ALX) and Rlk/Itk-binding protein (RIBP) are adaptors related by structure and sequence, coexpressed in T cells. Mice deficient for each adaptor demonstrated that ALX and RIBP, respectively, negatively and positively regulate T cell activation in response to TCR/CD28 stimulation. However, these results did not preclude that they may function redundantly in other cell populations, or in response to other stimuli. Therefore, to understand the relationship between these related adaptors, ALX/RIBP-deficient mice were generated. We demonstrate that although ALX and RIBP are expressed throughout T cell development, T cell development occurs normally in these mice. Using the H-Y TCR transgenic model, positive and negative selection were found to proceed unimpeded in the absence of ALX and RIBP. We demonstrate that RIBP is also expressed in B cells; however, RIBP- and ALX/RIBP-deficient mice had normal B cell development, and responded equivalently to wild type in response to IgM, CD40, B cell-activating factor/B lymphocyte stimulator, CpG, and LPS. Interestingly, T cells deficient in both ALX and RIBP behaved similarly to those deficient in ALX alone during T cell activation in response to TCR/CD28, exhibiting increased IL-2 production, CD25 expression, and proliferation, thus showing that ALX deficiency masked the effect of RIBP deficiency. ALX/RIBP-deficient T cells did not have any alterations in either activation-induced cell death or Th1/2 polarization. Therefore, we did not find any functional redundancy or synergy during lymphocyte development, selection, activation, or survival in ALX/RIBP-deficient mice, demonstrating that these molecules function independently.