Determination of lymphoid cell fate is dependent on the expression status of the IL-7 receptor

Signaling through the IL-7 receptor (IL-7R) is necessary for the development of the earliest B- and T-lineage cells. IL-7R is first expressed on common lymphoid progenitor cells and is not detected on primitive common myeloid progenitors. In this study, we show that enforced expression of IL-7R on multipotential stem cells does not influence lymphoid versus myeloid cell fate. T cell development was compatible with sustained IL-7R expression; however, we observed a near complete block in B cell development at the onset of B-lineage commitment. Unlike pre-proB cells from control animals, developmentally-arrested IL-7R(+)B220(+)CD19(-)NK1.1(-)Ly-6C(-) cells failed to express EBF and Pax5. These results suggest that transient downregulation of IL-7R signaling is a necessary event for induction of EBF and Pax5 expression and B-lymphocyte commitment.     

Experimental murine schistosomiasis in the absence of B7 costimulatory molecules: reversal of elicited T cell cytokine profile and partial inhibition of egg granuloma formation

The granulomatous inflammation in infection with the helminth Schistosoma mansoni represents a cellular hypersensitivity reaction mediated by, and dependent upon, MHC class II-restricted CD4+ Th cells sensitized to parasite egg Ags. The current work examines the role and significance of the B7:CD28/CTLA-4 pathway in providing the costimulation necessary for the activation of these pathogenic T cells. In vitro T cell responses in B7-1-/- mice, 7-8 wk postinfection, were no different from wild-type controls, but the absence of B7-2 molecules resulted in a decrease in egg Ag-induced proliferation with increased IFN-gamma production. Both B7-1-/- and B7-2-/- mice exhibited intact granuloma formation. In contrast, CD4+ Th cells from B7-1/2 double-deficient mice displayed a dramatic loss of proliferative capacity upon stimulation with egg Ag. Most strikingly, these T cells secreted only IFN-gamma, but not IL-4 and IL-10, a pattern entirely opposite to that displayed by wild-type controls. Despite these major differences in T cell reactivity, B7-1/2-/- mice had only a limited reduction of granuloma size and fibrosis, without appreciable difference in cellular composition. These results show that substantial granuloma formation can occur under conditions of limited T cell expansion and restricted Th1-type cytokine production. They also support the notion that the combined effect of B7 signaling is not as critical for Th1 cell activation as it is for the development of the Th2 dominant environment characteristic of the evolving schistosome infection in H-2b mice.     

The role of IL-5 for mature B-1 cells in homeostatic proliferation, cell survival, and Ig production

B-1 cells, distinguishable from conventional B-2 cells by their cell surface marker, anatomical location, and self-replenishing activity, play an important role in innate immune responses. B-1 cells constitutively express the IL-5R alpha-chain (IL-5Ralpha) and give rise to Ab-producing cells in response to various stimuli, including IL-5 and LPS. Here we report that the IL-5/IL-5R system plays an important role in maintaining the number and the cell size as well as the functions of mature B-1 cells. The administration of anti-IL-5 mAb into wild-type mice, T cell-depleted mice, or mast cell-depleted mice resulted in reduction in the total number and cell size of B-1 cells to an extent similar to that of IL-5Ralpha-deficient (IL-5Ralpha(-/-)) mice. Cell transfer experiments have demonstrated that B-1 cell survival in wild-type mice and homeostatic proliferation in recombination-activating gene 2-deficient mice are impaired in the absence of IL-5Ralpha. IL-5 stimulation of wild-type B-1 cells, but not IL-5Ralpha(-/-) B-1 cells, enhances CD40 expression and augments IgM and IgG production after stimulation with anti-CD40 mAb. Enhanced IgA production in feces induced by the oral administration of LPS was not observed in IL-5Ralpha(-/-) mice. Our results illuminate the role of IL-5 in the homeostatic proliferation and survival of mature B-1 cells and in IgA production in the mucosal tissues.     

The role of B cells in the development of CD4 effector T cells during a polarized Th2 immune response

Previous studies have suggested that B cells promote Th2 cell development by inhibiting Th1 cell differentiation. To examine whether B cells are directly required for the development of IL-4-producing T cells in the lymph node during a highly polarized Th2 response, B cell-deficient and wild-type mice were inoculated with the nematode parasite, Nippostrongylus brasiliensis. On day 7, in the absence of increased IFN-gamma, IL-4 protein and gene expression from CD4 T cells in the draining lymph nodes were markedly reduced in B cell-deficient mice and could not be restored by multiple immunizations. Using a DO11.10 T cell adoptive transfer system, OVA-specific T cell IL-4 production and cell cycle progression, but not cell surface expression of early activation markers, were impaired in B cell-deficient recipient mice following immunization with N. brasiliensis plus OVA. Laser capture microdissection and immunofluorescent staining showed that pronounced IL-4 mRNA and protein secretion by donor DO11.10 T cells first occurred in the T cell:B cell zone of the lymph node shortly after inoculation of IL-4-/- recipients, suggesting that this microenvironment is critical for initial Th2 cell development. Reconstitution of B cell-deficient mice with wild-type naive B cells, or IL-4-/- B cells, substantially restored Ag-specific T cell IL-4 production. However, reconstitution with B7-1/B7-2-deficient B cells failed to rescue the IL-4-producing DO11.10 T cells. These results suggest that B cells, expressing B7 costimulatory molecules, are required in the absence of an underlying IFN-gamma-mediated response for the development of a polarized primary Ag-specific Th2 response in vivo.     

TIRC7 deficiency causes in vitro and in vivo augmentation of T and B cell activation and cytokine response

The membrane protein T cell immune response cDNA 7 (TIRC7) was recently identified and was shown to play an important role in T cell activation. To characterize the function of TIRC7 in more detail, we generated TIRC7-deficient mice by gene targeting. We observed disturbed T and B cell function both in vitro and in vivo in TIRC7(-/-) mice. Histologically, primary and secondary lymphoid organs showed a mixture of hypo-, hyper-, and dysplastic changes of multiple lymphohemopoietic compartments. T cells from TIRC7(-/-) mice exhibited significantly increased proliferation and expression of IL-2, IFN-gamma, and IL-4 in response to different stimuli. Resting T cells from TIRC7(-/-) mice exhibited decreased CD62L, but increased CD11a and CD44 expression, suggesting an in vivo expansion of memory/effector T cells. Remarkably, activated T cells from TIRC7(-/-) mice expressed lower levels of CTLA-4 in comparison with wild-type cells. B cells from TIRC7-deficient mice exhibited significantly higher in vitro proliferation following stimulation with anti-CD40 Ab or LPS plus IL-4. B cell hyperreactivity was reflected in vivo by elevated serum levels of various Ig classes and higher CD86 expression on B cells. Furthermore, TIRC7 deficiency resulted in an augmented delayed-type hypersensitivity response that was also reflected in increased mononuclear infiltration in the skin obtained from TIRC7-deficient mice food pads. In summary, the data strongly support an important role for TIRC7 in regulating both T and B cell responses.     

Stat5 is essential for early B cell development but not for B cell maturation and function

The two closely related Stat5 (Stat5A and Stat5B) proteins are activated by a broad spectrum of cytokines. However, with the complication of the involvement of Stat5A/5B in stem cell function, the role of Stat5A/5B in the development and function of lymphocytes, especially B cells, is not fully understood. In this study, we demonstrated that Stat5A/5B(-/-) fetal liver cells had severe diminution of B cell progenitors but clearly had myeloid progenitors. Consistently, the mutant fetal liver cells could give rise to hemopoietic progenitors and myeloid cells but not B cells beyond pro-B cell progenitors in lethally irradiated wild-type or Jak3(-/-) mice. Deletion of Stat5A/5B in vitro directly impaired IL-7-mediated B cell expansion. Of note, reintroduction of Stat5A back into Stat5A/5B(-/-) fetal liver cells restored their abilities to develop B cells. Importantly, CD19-Cre-mediated deletion of Stat5A/5B in the B cell compartment specifically impaired early B cell development but not late B cell maturation. Moreover, the B cell-specific deletion of Stat5A/5B did not impair splenic B cell survival, proliferation, and Ig production. Taken together, these data demonstrate that Stat5A/5B directly control IL-7-mediated early B cell development but are not required for B cell maturation and Ig production.     

Stimulation of the B cell receptor, CD86 (B7-2), and the beta 2-adrenergic receptor intrinsically modulates the level of IgG1 and IgE produced per B cell

Our findings using B cells from either wild-type, CD86-deficient, or beta 2-adrenergic receptor (beta2AR)-deficient mice suggest three mechanisms by which the level of IgG1 and IgE production can be increased on a per cell basis. Trinitrophenyl-specific B cells enriched from unimmunized mouse spleens were pre-exposed to Ag and/or the beta 2AR ligand terbutaline for 24 h before being activated by either a beta 2AR-negative Th2 cell clone or CD40 ligand/Sf9 cells and IL-4 in the presence or absence of an anti-CD86 Ab. Data suggest that the first mechanism involves a B cell receptor (BCR)-dependent up-regulation of CD86 expression that, when CD86 is stimulated, increases the amount of IgG1 and IgE produced in comparison to unstimulated cells. The second mechanism involves a BCR- and beta 2AR-dependent up-regulation of CD86 to a level higher than that induced by stimulation of either receptor alone that, when CD86 is stimulated, further increases the amount of IgG1 and IgE produced. The third mechanism is BCR-independent and involves a beta 2AR-dependent increase in the ability of a B cell to respond to IL-4. Flow cytometric and limiting dilution analyses suggest that the increase in IgG1 and IgE occurs independently from the isotype switching event. These findings suggest that the BCR, the beta 2AR, and CD86 are involved in regulating IL-4-dependent IgG1 and IgE production.     

A profound deficiency in thymic progenitor cells in mice lacking Jak3

Humans and mice with genetic deficiencies that lead to loss of signaling through common gamma-chain (gammac)-containing cytokine receptors have severe defects in B and T lymphocytes. In humans, these deficiencies lead to a complete absence of T cells, whereas in mice, small thymuses give rise to normal numbers of peripheral T cells. We have examined the first wave of developing T cells in Jak3-/-, IL-7-/-, and IL-7Ralpha-/- fetal mice, and have found a near absence of thymic progenitor cells. This deficiency is highlighted by the complete inability of Jak3-/- progenitor cells to reconstitute T cell development in the presence of competing wild-type cells. These data clearly demonstrate a strong common basis for the T cell deficiencies in mice and humans lacking gammac/Jak3 signaling pathways.     

The absence of IL-6 does not affect Th2 cell development in vivo, but does lead to impaired proliferation, IL-2 receptor expression, and B cell responses

The role of IL-6 in Th2 cell differentiation and response development after the injection of eggs from Schistosoma mansoni was investigated using wild-type (WT) and IL-6-/- mice. IL-6 was induced in the lymph nodes (LN) of WT mice within 24 h of egg injection, and IL-4 production by WT LN cells and CD4 T cells isolated 24 h after egg injection and stimulated in vitro was observed. In the absence of IL-6, this early production of IL-4 by LN cells and purified CD4 T cells was not abolished; although the level of IL-4 produced by IL-6-/- LN cells was similar to WT, IL-4 production by purified IL-6-/- CD4 T cells was reduced compared with WT. Despite the difference in CD4 T cell production of IL-4, the development of egg-specific Th2 cells 7 days after egg injection was not affected by the absence of IL-6. Nevertheless, Ab production was impaired and the in vitro proliferative response of whole LN cell populations, CD4 and CD8 T cells, and B cells from IL-6-/- mice was poor compared with WT. The proliferative defect in the IL-6-/- cells correlated with decreased IL-2R expression, and addition of exogenous IL-6 enhanced IL-2R expression as well as proliferation of LN cells from IL-6-/- mice. These studies demonstrate that Th2 differentiation and response development in vivo is not dependent on IL-6, but that Th-dependent and independent B cell responses are. Our results also emphasize the importance of IL-6 for lymphoproliferation, possibly through induction of IL-2R expression.     

CD19 function in early and late B cell development. II. CD19 facilitates the pro-B/pre-B transition

Proliferative expansion of pro-B cells is an IL-7-dependent process that allows for the rearrangement of H chain genes and the expression of the pre-B cell receptor (pre-BCR). Further B cell differentiation is dependent upon signals elicited through the pre-BCR, which are thought to be responsible for allelic exclusion, induced L chain gene rearrangement, and continued proliferation. CD19 promotes the proliferation and survival of mature B cells, but its role in early B cell development is less well understood. Here we identify and characterize impairments in early B cell development in CD19(-/-) mice. Following sublethal irradiation, we found decreased numbers of autoreconstituted early B cells, which was first evident in the large cycling pre-B cell fraction. Reduced cell progression due to a defect in proliferation was made evident from cell cycle analysis and bromodeoxyuridine labeling of bone marrow cells from CD19(-/-) and wild-type mice. Studies of IL-7-dependent pre-B cell cultures derived from wild-type and CD19(-/-) mouse bone marrow suggested that CD19 has little affect on IL-7 signaling. By contrast, signaling through the pre-BCR was impaired in the absence of CD19, as demonstrated by reduced activation of Bruton's tyrosine kinase and extracellular signal-regulated kinase/mitogen-activated protein kinase. Thus, in addition to promoting mature B cell homeostasis and Ag-induced responses, the early onset of CD19 expression acts to enhance B cell generation.     

Enforced expression of EBF in hematopoietic stem cells restricts lymphopoiesis to the B cell lineage

Mice deficient in early B cell factor (EBF) are blocked at the progenitor B cell stage prior to immunoglobulin gene rearrangement. The EBF-dependent block in B cell development occurs near the onset of B-lineage commitment, which raises the possibility that EBF may act instructively to specify the B cell fate from uncommitted, multipotential progenitor cells. To test this hypothesis, we transduced enriched hematopoietic progenitor cells with a retroviral vector that coexpressed EBF and the green fluorescent protein (GFP). Mice reconstituted with EBF-expressing cells showed a near complete absence of T lymphocytes. Spleen and peripheral blood samples were >95 and 90% GFP+EBF+ mature B cells, respectively. Both NK and lymphoid-derived dendritic cells were also significantly reduced compared with control-transplanted mice. These data suggest that EBF can restrict lymphopoiesis to the B cell lineage by blocking development of other lymphoid-derived cell pathways.     

The role of CTLA-4 in regulating Th2 differentiation.

To examine the role of CTLA-4 in Th cell differentiation, we used two newly generated CTLA-4-deficient (CTLA-4-/-) mouse strains: DO11. 10 CTLA-4-/- mice carrying a class II restricted transgenic TCR specific for OVA, and mice lacking CTLA-4, B7.1 and B7.2 (CTLA-4-/- B7.1/B7.2-/- ). When purified naive CD4+ DO11.10 T cells from CTLA-4-/- and wild-type mice were primed and restimulated in vitro with peptide Ag, CTLA-4-/- DO11.10 T cells developed into Th2 cells, whereas wild-type DO11.10 T cells developed into Th1 cells. Similarly, when CTLA-4-/- CD4+ T cells from mice lacking CTLA-4, B7. 1, and B7.2 were stimulated in vitro with anti-CD3 Ab and wild-type APC, these CTLA-4-/- CD4+ T cells produced IL-4 even during the primary stimulation, whereas CD4+ cells from B7.1/B7.2-/- mice did not produce IL-4. Upon secondary stimulation, CD4+ T cells from CTLA-4-/- B7.1/B7.2-/- mice secreted high levels of IL-4, whereas CD4+ T cells from B7.1/B7.2-/- mice produced IFN-gamma. In contrast to the effects on CD4+ Th differentiation, the absence of CTLA-4 resulted in only a modest effect on T cell proliferation, and increased proliferation of CTLA-4-/- CD4+ T cells was seen only during secondary stimulation in vitro. Administration of a stimulatory anti-CD28 Ab in vivo induced IL-4 production in CTLA-4-/- B7.1/B7.2-/- but not wild-type mice. These studies demonstrate that CTLA-4 is a critical and potent inhibitor of Th2 differentiation. Thus, the B7-CD28/CTLA-4 pathway plays a critical role in regulating Th2 differentiation in two ways: CD28 promotes Th2 differentiation while CTLA-4 limits Th2 differentiation.     

B7 expression on T cells down-regulates immune responses through CTLA-4 ligation via T-T interactions [corrections

Although B7 on APCs has a well-recognized role in T cell costimulation, little is known about the functional significance of constitutive and activation-induced B7 expression that also occurs on T cells. To analyze the role of B7 on T cells, B7-1/B7-2-deficient mice (B7 double knockout) and mice overexpressing B7-2 exclusively on T cells (B7-2 transgenic) were used as T cell donors for allogeneic transplant recipients, and graft-vs-host disease (GVHD) was assessed. B7 double-knockout T cells resulted in significant GVHD acceleration compared with wild-type T cells. Conversely, B7-2 transgenic donor T cells mediated reduced GVHD mortality compared with wild-type T cells. Data indicated that B7 expression on T cells down-regulated alloresponses through CTLA-4 ligation. This study is the first to provide definitive in vivo data illustrating the importance of T cell-associated B7 as a negative regulator of immune responses in a clinically relevant murine model of GVHD. The up-regulation of B7 on T cells may be an important component of normal immune homeostasis.     

IL-23 is required for long-term control of Mycobacterium tuberculosis and B cell follicle formation in the infected lung

IL-23 is required for the IL-17 response to infection with Mycobacterium tuberculosis, but is not required for the early control of bacterial growth. However, mice deficient for the p19 component of IL-23 (Il23a(-/-)) exhibit increased bacterial growth late in infection that is temporally associated with smaller B cell follicles in the lungs. Cxcl13 is required for B cell follicle formation and immunity during tuberculosis. The absence of IL-23 results in decreased expression of Cxcl13 within M. tuberculosis-induced lymphocyte follicles in the lungs, and this deficiency was associated with increased cuffing of T cells around the vessels in the lungs of these mice. Il23a(-/-) mice also poorly expressed IL-17A and IL-22 mRNA. These cytokines were able to induce Cxcl13 in mouse primary lung fibroblasts, suggesting that these cytokines are likely involved in B cell follicle formation. Indeed, IL-17RA-deficient mice generated smaller B cell follicles early in the response, whereas IL-22-deficient mice had smaller B cell follicles at an intermediate time postinfection; however, only Il23a(-/-) mice had a sustained deficiency in B cell follicle formation and reduced immunity. We propose that in the absence of IL-23, expression of long-term immunity to tuberculosis is compromised due to reduced expression of Cxcl13 in B cell follicles and reduced ability of T cells to migrate from the vessels and into the lesion. Further, although IL-17 and IL-22 can both contribute to Cxcl13 production and B cell follicle formation, it is IL-23 that is critical in this regard.     

B cell-deficient (mu MT) mice have alterations in the cytokine microenvironment of the gut-associated lymphoid tissue (GALT) and a defect in the low dose mechanism of oral tolerance

Peripheral immune tolerance following i.v. administration of Ag has been shown to occur in the absence of B cells. Because different mechanisms have been identified for i.v. vs low dose oral tolerance and B cells are a predominant component of the gut-associated lymphoid tissue (GALT) they may play a role in tolerance induction following oral Ag. To examine the role of B cells in oral tolerance we fed low doses of OVA or myelin oligodendrocyte glycoprotein to B cell-deficient ( microMT) and wild-type C57BL/6 mice. Results showed that the GALT of naive wild-type and microMT mice was characterized by major differences in the cytokine microenvironment. Feeding low doses of 0.5 mg OVA or 250 microg myelin oligodendrocyte glycoprotein resulted in up-regulation of IL-4, IL-10, and TGF-beta in the GALT of wild-type but not microMT mice. Upon stimulation of popliteal node cells, in vitro induction of regulatory cytokines TGF-beta and IL-10 was observed in wild-type but not microMT mice. Greater protection against experimental autoimmune encephalomyelitis was found in wild-type mice. Oral tolerance in microMT and wild-type mice was found to proceed by different mechanisms. Anergy was observed from 0.5 mg to 250 ng in microMT mice but not in wild-type mice. Increased Ag was detected in the lymph of microMT mice. No cytokine-mediated suppression was found following lower doses from 100 ng to 500 pg in either group. These results demonstrate the importance of the B cell for the induction of cytokine-mediated suppression associated with low doses of Ag.     

IL-7 is required for the development of the intrinsic function of marginal zone B cells and the marginal zone microenvironment

The characteristic microarchitecture of the marginal zone (MZ), formed by locally interacting MZ-specific B cells, macrophages, and endothelial cells, is critical for productive marginal zone B cell (MZB cell) Ab responses. Reportedly, IL-7-deficient mice, although severely lymphopenic, retain small numbers of CD21(high)CD23(low) B cells consistent with MZB cell phenotype, suggesting that IL-7 signaling is not exclusively required for MZB cell lymphopoiesis. In this study, we investigated the function of IL-7(-/-) MZB cells and the IL-7(-/-) microenvironment using a model of hamster heart xenograft rejection, which depends exclusively on MZB cell-mediated production of T cell-independent IgM xenoantibodies (IgMXAb). C57BL/6-IL-7(-/-) mice accepted xenografts indefinitely and failed to produce IgMXAb, even after transfer of additional IL-7(-/-) or wild-type C57BL/6 MZB cells. Transfer of wild-type but not IL-7(-/-) B cells enabled SCID mice to produce IgMXAb. When transferred to SCID mice, wild-type but not IL-7(-/-) B cells formed B cell follicles with clearly defined IgM(+), MOMA-1(+), and MAdCAM-1(+) MZ structures. Conversely, adoptively transferred GFP(+) C57BL/6 B cells homed to the MZ area in a SCID but not an IL-7(-/-) environment. Naive IL-7(-/-) mice showed absent or aberrant splenic B cell structures. We provide evidence that IL-7 is critical for the development of the intrinsic function of MZB cells in producing rapidly induced IgM against T cell-independent type II Ags, for their homing potential, and for the development of a functional MZ microanatomy capable of attracting and lodging MZB cells.