IL-2 receptor γ-chain molecule is critical for intestinal T-cell reconstitution in humanized mice.

Intestinal immune cells are important in host defense, yet the determinants for human lymphoid homeostasis in the intestines are poorly understood. In contrast, lymphoid homeostasis has been studied extensively in mice, where the requirement for a functional common γ-chain molecule has been established. We hypothesized that humanized mice could offer insights into human intestinal lymphoid homeostasis if generated in a strain with an intact mouse common γ-chain molecule. To address this hypothesis, we used three mouse strains (non-obese diabetic (NOD)/severe-combined immunodeficient (SCID) (N/S); NOD/SCID γ-chain(-/-) (NSG); and Rag2(-/-) γ-chain(-/-) (DKO)) and two humanization techniques (bone marrow liver thymus (BLT) and human CD34(+) cell bone marrow transplant of newborn mice (hu)) to generate four common types of humanized mice: N/S-BLT, NSG-BLT, NSG-hu, and DKO-hu mice. The highest levels of intestinal human T cells throughout the small and large intestines were observed in N/S-BLT mice, which have an intact common γ-chain molecule. Furthermore, the small intestine lamina propria T-cell populations of N/S-BLT mice exhibit a human intestine-specific surface phenotype. Thus, the extensive intestinal immune reconstitution of N/S-BLT mice was both quantitatively and qualitatively better when compared with the other models tested such that N/S-BLT mice are well suited for the analysis of human intestinal lymphocyte trafficking and human-specific diseases affecting the intestines.

Humanized immune system (HIS) mice as a tool to study human NK cell development.

The study of human hematopoiesis is conditioned by access to nondiseased human tissue samples that harbor the cellular substrates for this developmental process. Technical and ethical concerns limit the availability to tissues derived from the fetal and newborn periods, while adult samples are generally restricted to peripheral blood. Access to a small animal model that faithfully recapitulates the process of human hematopoiesis would provide an important tool. Natural killer (NK) cells comprise between 10% and 15% of human peripheral blood lymphocytes and appear conserved in several species. NK cells are implicated in the recognition of pathogen-infected cells and in the clearance of certain tumor cells. In this chapter, we discuss NK cell developmental pathways and the use of humanized murine models for the study of human hematopoiesis and, in particular, human NK cell development.

TCRA gene rearrangement in immature thymocytes in absence of CD3, pre-TCR, and TCR signaling.

During thymocyte differentiation, TCRA genes are massively rearranged only after productively rearranged TCRB genes are expressed in association with pTalpha and CD3 complex molecules within a pre-TCR. Signaling from the pre-TCR via the CD3 complex is thought to be required to promote TCRA gene accessibility and recombination. However, alphabeta(+) thymocytes do develop in pTalpha-deficient mice, showing that TCRalpha-chain genes are rearranged, either in CD4(-)CD8(-) or CD4(+)CD8(+) thymocytes, in the absence of pre-TCR expression. In this study, we analyzed the TCRA gene recombination status of early immature thymocytes in mutant mice with arrested thymocyte development, deficient for either CD3 or pTalpha and gammac expression. ADV genes belonging to different families were found rearranged to multiple AJ segments in both cases. Thus, TCRA gene rearrangement is independent of CD3 and gammac signaling. However, CD3 expression was found to play a role in transcription of rearranged TCRalpha-chain genes in CD4(-)CD8(-) thymocytes. Taken together, these results provide new insights into the molecular control of early T cell differentiation.

IL-12-independent IFN-gamma production by T cells in experimental Chagas' disease is mediated by IL-18.

IL-12p35-deficient (IL-12p35(-/-)) mice were highly susceptible to Trypanosoma cruzi infection and succumbed during acute infection, demonstrating the crucial importance of endogenous IL-12 in resistance to experimental Chagas' disease. Delayed immune responses were observed in mutant mice, although comparable IFN-gamma and TNF-alpha blood levels as in wild-type mice were detected 2 wk postinfection. In vivo and in vitro analysis demonstrated that T cells, but not NK cells, were recruited to infected organs. Analysis of mice double deficient in the recombinase-activating gene 2 (RAG2) and IL-12p35, as well as studies involving T cell depletion, identified CD4(+) T cells as the cellular source for IL-12-independent IFN-gamma production. IL-18 was induced in IL-12p35(-/-) mice and was responsible for IFN-gamma production, as demonstrated by in vivo IL-18 neutralization studies. In conclusion, evidence is presented for an IL-12-independent IFN-gamma production in experimental Chagas' disease that is T cell and IL-18 dependent.

Intraembryonic, but not yolk sac hematopoietic precursors, isolated before circulation, provide long-term multilineage reconstitution.

The relative contribution of yolk sac and intraembryonic precursors to hematopoiesis has been a matter of long-standing controversy. As reconstitution activity has so far only been found in embryonic tissues after the onset of circulation, the origin of reconstituting cells could not be formally established. Here, we separated yolk sac and intraembryonic splanchnopleura prior to circulation and maintained the explants in organ culture before transfer. Precursors derived from the intraembryonic site generated multilineage hematopoietic progeny in adult mice for more than 6 months. Yolk sac cells only provided myeloid short-term reconstitution. The results reveal a differential hematopoietic capacity of precirculation embryonic tissues in vivo, and indicate that the only cells capable of adult long-term hematopoiesis are of intraembryonic origin.

Stress-induced ClpP serine protease of Listeria monocytogenes is essential for induction of listeriolysin O-dependent protective immunity.

The stress-induced protease ClpP is required for virulence of the facultative intracellular pathogen Listeria monocytogenes. We previously found that in the absence of ClpP, the virulence of this pathogen was strongly reduced, mainly due to the decreased production of functional listeriolysin O (LLO), a major immunodominant virulence factor promoting intracellular growth. In this work, a clpP deletion mutant of L. monocytogenes was used to study the generation of anti-Listeria protective immunity. We found that ClpP is required for the intracellular growth of L. monocytogenes in resident macrophages in vivo. Mice infected with doses as high as 10(6) clpP mutant bacteria were not protected against a lethal challenge of wild-type bacteria and did not develop any detectable LLO-specific cytolytic T cells or antibodies, suggesting that the amount of LLO produced in infected mice under these conditions was too low to induce a specific immune response. However, in contrast to the results obtained with a mutant with a disrupted hly gene, this lack of protection was overcome by inoculation of very high infecting doses of clpP mutant bacteria (5 x 10(8)), thus producing sufficient amounts of LLO to stimulate anti-Listeria immunity. The role of ClpP was confirmed by showing that anti-Listeria immunity was restored in mice infected with a clpP-complemented mutant. These results indicate that the stress-induced serine protease ClpP is a potential target for modulating the presentation of protective antigens such as LLO and thereby the immune response against L. monocytogenes.

Identification of committed NK cell progenitors in adult murine bone marrow.

Natural killer (NK) cells play important roles in innate immunity by lysing tumor and virally infected cells and by producing cytokines including interferon-gamma. While NK cell progenitors have been described in the fetal thymus, NK cell generation from hematopoietic stem cells (HSC) in the bone marrow (BM) occurs throughout life, and in athymic mice and humans. Interleukin (IL)-15 promotes NK development in vitro and is essential for the generation of normal numbers of NK cells in vivo. By characterizing BM cells expressing IL-15 receptor components, we found marked heterogeneity within the IL-2 receptor beta chain(+) (CD122(+)) subset, which included cells uniquely committed to the NK lineage. These CD122(+) NK cell precursors (NKP) are negative for markers used to identify mature NK cells, including NK1.1, DX5 and members of Ly-49 family, and fail to demonstrate natural cytotoxicity against susceptible target cells. In vitro culture of NKP generates mature lytic NK1.1(+) cells at high frequencies, while they do not give rise to T, B, myeloid or erythroid cells under appropriate conditions. NKP lack transcripts associated with early B and T cell differentiation (pTalpha, lambda5 and CD3epsilon), but express a group of genes (IL-15Ralpha, Id2, GATA-3 and Ets-1) and the 2B4 marker, which may define NK cell commitment. We propose that NKP represent the earliest adult BM precursor uniquely restricted to the NK cell lineage.

Functional dichotomy in natural killer cell signaling: Vav1-dependent and -independent mechanisms.

The product of the protooncogene Vav1 participates in multiple signaling pathways and is a critical regulator of antigen-receptor signaling in B and T lymphocytes, but its role during in vivo natural killer (NK) cell differentiation is not known. Here we have studied NK cell development in Vav1-/- mice and found that, in contrast to T and NK-T cells, the absolute numbers of phenotypically mature NK cells were not reduced. Vav1-/- mice produced normal amounts of interferon (IFN)-gamma in response to Listeria monocytogenes and controlled early infection but showed reduced tumor clearance in vivo. In vitro stimulation of surface receptors in Vav1-/- NK cells resulted in normal IFN-gamma production but reduced tumor cell lysis. Vav1 was found to control activation of extracellular signal-regulated kinases and exocytosis of cytotoxic granules. In contrast, conjugate formation appeared to be only mildly affected, and calcium mobilization was normal in Vav1-/- NK cells. These results highlight fundamental differences between proximal signaling events in T and NK cells and suggest a functional dichotomy for Vav1 in NK cells: a role in cytotoxicity but not for IFN-gamma production.

Natural killer cell-dependent apoptosis of peripheral murine hematopoietic progenitor cells in response to Fas cross-linking: involvement of tumor necrosis factor-alpha.

Recently, a marked extramedullary myelopoiesis in Fas/CD95- or FasL/CD95L-deficient mice has been reported. In the present in vitro study, the mechanisms underlying Fas-induced apoptosis of normal peripheral colony-forming unit-C (CFU-C) progenitors in the spleen were analyzed. Surprisingly, it was found that clonogenic progenitors were protected from gammaIFN plus Fas-induced programmed cell death when Lin(+) cells were removed from cultured splenocytes. The cells that rendered CFU-C sensitive to the activation of the Fas pathway did not belong to the T or the myelocytic-monocytic lineage but comprised a non-B-cell subset expressing the activation marker B220. Among CD19(-) B220(+) splenocytes, nearly half were natural killer (NK) 1.1(+) cells whose in vivo depletion or deficiency in RAG2-gamma(c)(-/-) mice abrogated the effect of Fas cross-linking. NK cells exerted their accessory function, at least in part, through tumor necrosis factor-alpha (TNF-alpha), which they readily produced during pretreatment with the anti-Fas/CD95 monoclonal antibody and IFN-gamma and whose addition could compensate for the loss of sensitivity. In conclusion, this study provides evidence that peripheral clonogenic progenitors are not directly responsive to Fas cross-linking, even in the presence of IFN-gamma, but require NK cells as a source of TNF-alpha to make them susceptible to this death pathway.

A new immunodeficient mouse model for human myoblast transplantation.

Design of efficient transplantation strategies for myoblast-based gene therapies in humans requires animal models in which xenografts are tolerated for long periods of time. In addition, such recipients should be able to withstand pretransplantation manipulations for enhancement of graft growth. Here we report that a newly developed immunodeficient mouse carrying two known mutations (the recombinase activating gene 2, RAG2, and the common cytokine receptor gamma, gammac) is a candidate fulfilling these requirements. Skeletal muscles from RAG2(-/-)/gammac(-/-) double mutant mice recover normally after myotoxin application or cryolesion, procedures commonly used to induce regeneration and improve transplantation efficiency. Well-differentiated donor-derived muscle tissue could be detected up to 9 weeks after transplantation of human myoblasts into RAG2(-/-)/gammac(-/-) muscles. These results suggest that the RAG2(-/-)/gammac(-/-) mouse model will provide new opportunities for human muscle research.

Differential requirement for the transcription factor PU.1 in the generation of natural killer cells versus B and T cells.

PU.1 is a member of the Ets family of transcription factors required for the development of various lymphoid and myeloid cell lineages, but its role in natural killer (NK) cell development is not known. The study shows that PU.1 is expressed in NK cells and that, on cell transfer into alymphoid Rag2/gammac(-/-) mice, hematopoietic progenitors of PU.1(-/-) fetal liver cells could generate functional NK cells but not B or T cells. Nevertheless, the numbers of bone marrow NK cell precursors and splenic mature NK cells were reduced compared to controls. Moreover, PU.1(-/-) NK cells displayed reduced expression of the receptors for stem cell factor and interleukin (IL)-7, suggesting a nonredundant role for PU.1 in regulating the expression of these cytokine receptor genes during NK cell development. PU.1(-/-) NK cells also showed defective expression of inhibitory and activating members of the Ly49 family and failed to proliferate in response to IL-2 and IL-12. Thus, despite the less stringent requirement for PU.1 in NK cell development compared to B and T cells, PU.1 regulates NK cell differentiation and homeostasis.

Inhibition of fibroblast growth factor/fibroblast growth factor receptor activity in glioma cells impedes tumor growth by both angiogenesis-dependent and -independent mechanisms.

We undertook a series of systematic studies to address the role of fibroblast growth factor/fibroblast growth factor receptor (FGF/FGFR) activity in tumor growth and angiogenesis. We expressed dominant-negative FGFR2 (FGFR2-DN) or FGFR1 (FGFR1-DN) in glioma C6 cells by using constitutive or tetracycline-regulated expression systems. Anchorage-dependent or independent growth was inhibited in FGFR-DN-expressing cells. Tumor development after xenografting FGFR-DN-expressing cells in immunodeficient mice or after transplantation in rat brain was strongly inhibited. Quantification of microvessels demonstrated a significant decrease in vessel density in tumors derived from FGFR-DN-expressing cells. Furthermore, in a rabbit corneal assay, the angiogenic response after implantation of FGFR-DN-expressing cells was decreased. In tumors expressing FGFR-DN, vascular endothelial growth factor expression was strongly inhibited as compared with control tumor. These results indicate that inhibition of FGF activity may constitute a dominant therapeutic strategy in the treatment of FGF-producing cerebral malignancies and may disrupt both angiogenesis-dependent and -independent signals required for glioma growth and invasion.

Cytokines: IL-21 joins the gamma(c)-dependent network?

The discovery of the cytokine IL-21 adds another member to the ever-growing list of small secreted molecules that have potent effects on lymphoid cells. Initial characterization of the IL-21 receptor complex suggests that IL-21 may belong to the cytokine family whose receptors share the common gamma chain, gamma(c).

Differential requirement of the cytoplasmic subregions of gamma c chain in T cell development and function.

The common cytokine receptor gamma chain (gammac), a shared component of the receptors for IL-2, IL-4, IL-7, IL-9, and IL-15, is critical for the development and function of lymphocytes. The cytoplasmic domain of gammac consists of 85 aa, in which the carboxyl-terminal 48 aa are essential for its interaction with and activation of the Janus kinase, Jak3. Evidence has been provided that Jak3-independent signals might be transmitted via the residual membrane-proximal region; however, its role in vivo remains totally unknown. In the present study, we expressed mutant forms of gammac, which lack either most of the cytoplasmic domain or only the membrane-distal Jak3-binding region, on a gammac null background. We demonstrate that, unlike gammac or Jak3 null mice, expression of the latter, but not the former mutant, restores T lymphopoiesis in vivo, accompanied by strong expression of Bcl-2. On the other hand, the in vitro functions of the restored T cells still remained impaired. These results not only reveal the hitherto unknown role of the gammac membrane-proximal region, but also suggest the differential requirement of the cytoplasmic subregions of gammac in T cell development and function.

Interferon gamma contributes to initiation of uterine vascular modification, decidual integrity, and uterine natural killer cell maturation during normal murine pregnancy.

The dominant lymphocytes in human and murine implantation sites are transient, pregnancy-associated uterine natural killer (uNK) cells. These cells are a major source of interferon (IFN)-gamma. Implantation sites in mice lacking uNK cells (alymphoid recombinase activating gene [RAG]-2(-/)- common cytokine receptor chain gamma [gamma(c)](-/)-) or IFN-gamma signaling (IFN-gamma(-/)- or IFN-gammaRalpha(-/)-) fail to initiate normal pregnancy-induced modification of decidual arteries and display hypocellularity or necrosis of decidua. To investigate the functions of uNK cell-derived IFN-gamma during pregnancy, RAG-2(-/)-gamma(c)(-/)- females were engrafted with bone marrow from IFN-gamma(-/)- mice, IFN-gamma signal-disrupted mice (IFN-gammaRalpha(-/)- or signal transducer and activator of transcription [Stat]-1(-/)-), or from mice able to establish normal uNK cells (severe combined immunodeficient [SCID] or C57BL/6). Mated recipients were analyzed at midgestation. All grafts established uNK cells. Grafts from IFN-gamma(-/)- mice did not reverse host vascular or decidual pathology. Grafts from all other donors promoted modification of decidual arteries and decidual cellularity. Grafts from IFN-gammaRalpha(-/)- or Stat-1(-/)- mice overproduced uNK cells, all of which were immature. Grafts from IFN-gamma(-/)-, SCID, or C57BL/6 mice produced normal, mature uNK cells. Administration of murine recombinant IFN-gamma to pregnant RAG-2(-/)-gamma(c)(-/)- mice initiated decidual vessel modification and promoted decidual cellularity in the absence of uNK cells. These in vivo findings strongly suggest that uNK cell-derived IFN-gamma modifies the expression of genes in the uterine vasculature and stroma, which initiates vessel instability and facilitates pregnancy-induced remodeling of decidual arteries.

A new look at Syk in alpha beta and gamma delta T cell development using chimeric mice with a low competitive hematopoietic environment.

The Syk protein tyrosine kinase (PTK) is essential for B, but not T or NK, cell development, although certain T cell subsets (i.e., gamma delta T cells of intestine and skin) appear to be dependent on Syk. In this report, we have re-evaluated the role of Syk in T cell development in hematopoietic chimeras generated by using Syk-deficient fetal liver hematopoietic stem cells (FL-HSC). We found that Syk-/- FL-HSC were vastly inferior to wild-type FL-HSC in reconstituting T cell development in recombinant-activating gene 2 (RAG2)-deficient mice, identifying an unexpected and nonredundant role for Syk in this process. This novel function of Syk in T cell development was mapped to the CD44-CD25+ stage. According to previous reports, development of intestinal gamma delta T cells was arrested in Syk-/- -->RAG2-/- chimeras. In striking contrast, when hosts were the newly established alymphoid RAG2 x common cytokine receptor gamma-chain (RAG2/gamma c) mice, Syk-/- chimeras developed intestinal gamma delta T cells as well as other T cell subsets (including alpha beta T cells, NK1.1+ alpha beta T cells, and splenic and thymic gamma delta T cells). However, all Syk-deficient T cell subsets were reduced in number, reaching about 25-50% of controls. These results attest to the utility of chimeric mice generated in a low competitive hematopoietic environment to evaluate more accurately the impact of lethal mutations on lymphoid development. Furthermore, they suggest that Syk intervenes in early T cell development independently of ZAP-70, and demonstrate that Syk is not essential for the intestinal gamma delta T cell lineage to develop.

Stable and functional lymphoid reconstitution of common cytokine receptor gamma chain deficient mice by retroviral-mediated gene transfer.

Mutations in the gene encoding the common cytokine receptor gamma chain (gamma(c)) are responsible for human X-linked severe combined immunodeficiency disease (SCIDX1). We have used a gamma(c)-deficient mouse model to test the feasibility and potential toxicity of gamma(c) gene transfer as a therapy for SCIDX1. A retrovirus harboring the murine gamma(c) chain was introduced into gamma(c)-deficient bone marrow cells, which were then transplanted into alymphoid RAG2/gamma(c) double-deficient recipient mice. Circulating lymphocytes appeared 4 weeks postgraft and achieved steady-state levels by 8 weeks. The mature lymphocytes present in the grafted mice had integrated the gamma(c) transgene, expressed gamma(c) transcripts, and were able to proliferate in response to gamma(c)-dependent cytokines. The gamma(c)-transduced animals demonstrated (1) normal levels of immunoglobulin subclasses, including immunoglobulin G1 (IgG1) and IgG2a (which are severely decreased in gamma(c)(-) mice); (2) the ability to mount an antigen-specific, T-dependent antibody response showing effective in vivo T-B cell cooperation, and (3) the presence of gut-associated cryptopatches and intraepithelial lymphocytes. Importantly, peripheral B and T cells were still present 47 weeks after a primary graft, and animals receiving a secondary graft of gamma(c)-transduced bone marrow cells demonstrated peripheral lymphoid reconstitution. That gamma(c) gene transfer to hematopoietic precursor cells can correct the immune system abnormalities in gamma(c)(-) mice supports the feasibility of in vivo retroviral gene transfer as a treatment for human SCIDX1.

Role of Qa-1(b)-binding receptors in the specificity of developing NK cells.

NK cells acquire the ability to recognize MHC class I molecules during development. Studies with Qa-1(b) tetramers (Qa-1 tetramers) showed that nearly all NK1.1(+) cells from newborn C57BL/6 mice express Qa-1-binding receptors. Cytotoxic activity of these cells is fully inhibited by Qa-1 ligands on target cells. In contrast, neither receptors for H-2K(b) nor H-2D(b) were observed on NK1.1(+) cells from newborn mice. After birth, frequencies of Qa-1 tetramer(+)/ NK1.1(+) cells gradually decrease as the number of Ly49(+) /NK1.1(+) cells increases. Cell transfer studies showed that Qa-1 tetramer(+) cells from newborn mice do not lose expression of Qa-1 receptors, but that they further acquire expression of Ly49 molecules. Acquisition of Qa-1-binding receptors appears largely independent of host MHC class I molecules, as observed in studies using beta2-microglobulin-deficient (beta2m(-/-)) mice as well as K(b)/ D(b-/-) and K(b)/D(b)/beta2m(-/-) mice. The present results suggest that Qa-1-binding receptors play an important role in the specificity of developing NK cells, and suggest that these cells rely mainly on inhibitory receptors specific for non-classical MHC class I molecules to maintain self tolerance during the first weeks of life.

To be or not to be a pro-T?

Recent studies have begun to unravel some of the molecular pathways that appear to control the processes of T cell determination in the earliest thymocyte precursors. In addition, the analyses of mouse mutants with an entirely alymphoid thymus have shed light on the developmental relationship of pro-T cells and thymic dendritic cells, revealing that development of thymocytes and thymic dendritic cells can be dissociated.

The receptor tyrosine kinase c-kit provides a critical signal for survival, expansion, and maturation of mouse natural killer cells.

Fetal liver kinase ligands (flk2L/flt3L) and stem cell factor (SCF) have been shown to promote natural killer (NK) cell differentiation from hematopoietic stem cell (HSC) precursors in vitro. However, the contribution of signaling through the receptors for these growth factors for in vivo NK cell development remains ill-defined. We have analyzed the role of the SCF receptor c-kit in NK cell differentiation by reconstituting NK-deficient mice with fetal liver (FL) HSCs of c-kit(-/-) (W/W) mice. Although c-kit(-/-)NK cells were generated in W/W chimeras, they were reduced in number, contained a lower percentage of CD45R (B220)(+) cells, and were poorly cytolytic. In vitro experiments showed that generation of NK cells from FL precursors was reduced in the absence of c-kit signaling and that SCF promoted the survival of peripheral c-kit(+) NK cells. We conclude that c-kit/SCF interactions in vivo are dispensable for the commitment of HSC to the NK lineage, but they provide essential signals for generating normal numbers of fully mature NK cells.