Small bowel enteropathy: role of intraepithelial lymphocytes and of cytokines (IL-12, IFN-gamma, TNF) in the induction of epithelial cell death and renewal.

The small bowel mucosa contains within its villus epithelium a large number of intraepithelial lymphocytes (IEL) which upon activation are cytotoxic and release large quantities of IFN-gamma and TNF; these activities are increased by in vitro exposure to IL-12. Mice injected with IL-12 develop severe damage of the villus epithelial cells, in form of apoptosis, necrosis and a third distinct form of cell death, characterized ultrastructurally by progressive cell shrinkage. These lesions are accompanied by a compensatory acceleration of the epithelial renewal, a hallmark of epithelial injury. Use of a variety of mutant mice showed that these lesions require the presence of IEL (all populations being involved, thymus-dependent as well as natural killer-T cell IEL) and the release of IFN-gamma. The critical role of IFN-gamma may result in part from its capacity to induce on epithelial cells the expression of target molecules involved in the different cytotoxic pathways used by IEL. However, injection of IFN-gamma into mutant mice lacking IEL showed that IFN-gamma can directly induce villus epithelial damage as well. On the other hand, injection of TNF induces fulminant apoptosis of villus epithelial cells, starting at the top of the villi; however TNF is not required for IL-12-induced enteropathy, which is unmodified in mutant mice lacking TNF. We propose that, when activated by their cognate ligands and/or IL-12 produced by cells in the lamina propria, IEL eliminate infected and senescent epithelial cells through a combination of cytotoxicity and of IFN-gamma and TNF release. This insures the rapid epithelial renewal of the villi, which in turn helps maintain the functional integrity of the barrier.

The common cytokine receptor gamma chain controls survival of gamma/delta T cells.

We have investigated the role of common gamma chain (gamma c)-signaling pathways for the development of T cell receptor for antigen (TCR)-gamma/delta T cells. TCR-gamma/delta-bearing cells were absent from the adult thymus, spleen, and skin of gamma c-deficient (gamma c-) mice, whereas small numbers of thymocytes expressing low levels of TCR-gamma/delta were detected during fetal life. Recent reports have suggested that signaling via interleukin (IL)-7 plays a major role in facilitating TCR-gamma/delta development through induction of V-J (variable-joining) rearrangements at the TCR-gamma locus. In contrast, we detected clearly TCR-gamma rearrangements in fetal thymi from gamma c- mice (which fail to signal in response to IL-7) and reduced TCR-gamma rearrangements in adult gamma c thymi. No gross defects in TCR-delta or TCR-beta rearrangements were observed in gamma c- mice of any age. Introduction of productively rearranged TCR V gamma 1 or TCR V gamma 1/V delta 6 transgenes onto mice bearing the gamma c mutation did not restore TCR-gamma/delta development to normal levels suggesting that gamma c-dependent pathways provide additional signals to developing gamma/delta T cells other than for the recombination process. Bcl-2 levels in transgenic thymocytes from gamma c- mice were dramatically reduced compared to gamma c+ transgenic littermates. We favor the concept that gamma c-dependent receptors are required for the maintenance of TCR-gamma/delta cells and contribute to the completion of TCR-gamma rearrangements primarily by promoting survival of cells committed to the TCR-gamma/delta lineage.

Cytokines: shared receptors, distinct functions.

That the signal transduction pathways used by the cytokines IL-2 and IL-15 are identical would suggest that these cytokines have redundant roles in lymphoid development; instead, IL-2 is the guardian of thymus-derived T-cell homeostasis, while interleukin-15 promotes extrathymic development of T and NK cells.

Mouse macrophage development in the absence of the common gamma chain: defining receptor complexes responsible for IL-4 and IL-13 signaling.

The common gamma chain (gamma c) forms a critical component of the receptors for interleukins (IL)-2, IL-4, IL-7, IL-9, and IL-15. We analyzed gamma c-deficient mice to define a role for gamma c signaling in the development and function of the macrophage lineage. No major differences in absolute cell numbers, cell surface phenotype, or in vitro function of gamma c- compared to gamma c+ macrophages were observed. We therefore conclude that signaling through the gamma c chain is not essential for the differentiation of mouse macrophages. Although B and T cells require gamma c for IL-4 responses, IL-4 up-regulated major histocompatibility class II molecules and inhibited nitric oxide production from gamma c- macrophages following stimulation with lipopolysaccharide and interferon-gamma. gamma c- macrophages could also respond to IL-13, consistent with the model of a type II IL-4 receptor alpha/IL-13R which can function in the absence of gamma c. Both IL-4 and IL-13 responses could be completely inhibited with the mouse IL-4 antagonist OY, suggesting that all of the observed IL-13 responses pass through the type II receptor, making it the primary signaling receptor complex for IL-13 in mouse macrophages.

T-cell development in the absence of the pre-T-cell receptor.

The development of pre-T-cells with productive T-cell receptor beta (TCR beta) rearrangements can be furthered by each of the pre-T-cell receptors (pre-TCR), the alpha beta TCR as well as the gamma delta TCR, albeit by distinct mechanisms. While the gamma delta TCR affects CD4-8- precursor cells irrespective of their TCR beta rearrangement status both the pre-TCR and the alpha beta TCR select only cells with productive TCR beta genes for expansion and maturation. The alpha beta TCR is much less effective than the pre-TCR because of the paucity of TCR alpha proteins in TCR beta positive precursors.

Role of different T cell receptors in the development of pre-T cells.

The development of pre-T cells with productive TCR-beta rearrangements can be mediated by each the pre-T cell receptor (pre-TCR), the TCR-alphabeta as well as the TCR-gammadelta, albeit by distinct mechanisms. Although the TCR-gammadelta affects CD4-8- precursor cells irrespective of their rearrangement status by TCR-beta mechanisms not involving TCR-beta selection, both the pre-TCR and the TCR-alphabeta select only cells with productive TCR-beta genes for expansion and maturation. The TCR-alphabeta appears to be much less effective than the pre-TCR because of the paucity of TCR-alpha proteins in TCR-beta-positive precursors since an early expressed transgenic TCR-alphabeta can largely substitute for the pre-TCR. Thus, the TCR-alphabeta can assume a role not only in the rescue from programmed cell death of CD4+8+ but also of CD4-8- thymocytes. In evolution this double function of the TCR-alphabeta may have been responsible for the maturation of alphabeta T cells before the advent of the pre-TCR-alpha chain.

Deregulated TCR alpha beta T cell population provokes extramedullary hematopoiesis in mice deficient in the common gamma chain.

Deficiency of the cytokine receptor common gamma chain (gamma c) results in abnormal lymphoid development and a severe immunodeficiency disease due to the combined loss of the receptors for interleukins (IL)-2, -4, -7, -9, and -15. We have observed the development of secondary hematopoiesis with circulating hematopoietic progenitor cells in adult mice harboring a null mutation in gamma c. These extramedullary changes were not secondary to bone marrow failure or to an inability to maintain circulating blood counts. These results suggested that gamma c-dependent cytokine signaling pathways modulate hematopoietic development. An intrinsic defect in gamma c- hematopoietic stem cell commitment appeared unlikely, as fetal liver hematopoiesis was unaltered in gamma c- embryos. Furthermore, the absence of natural killer cells in gamma c- mice was not responsible for the observed hematopoietic changes. Peripheral TCR alpha beta T cells from gamma c- mice were characterized by an activated phenotype (CD62Llo, CD44hi, CD69hi) and showed increased levels of transcripts for hematopoietic stimulating cytokines, including IL-3 and granulocyte/macrophage-colony-stimulating factor. A predominance of these cells was detected in the bone marrow, suggesting a role for residual T cells in the enhanced hematopoiesis. Strikingly, the elimination of residual T cells from gamma c- mice reduced splenic and circulating hematopoietic precursor frequencies to normal levels. These results clearly implicate a deregulated TCR alpha beta T cell population in the observed hematopoietic changes in gamma c- mice, and emphasize the importance of gamma c-dependent cytokine interactions in modulating mature T cell responses.

Lineage relationships and differentiation of natural killer (NK) T cells: intrathymic selection and interleukin (IL)-4 production in the absence of NKR-P1 and Ly49 molecules.

In this report, we have assessed the lineage relationships and cytokine dependency of natural killer (NK) T cells compared with mainstream TCR-alphabeta T cells and NK cells. For this purpose, we studied common gamma chain (gamma c)-deficient mice, which demonstrate a selective defect in CD3- NK cell development relative to conventional TCR-alphabeta T cells. NK thymocytes differentiate in gamma c- mice as shown by the normal percentage of TCR Vbeta8+ CD4-CD8- cells and the normal quantity of thymic Va14-Jalpha281 mRNA that characterize the NK T repertoire. However, gamma c-deficient NK thymocytes fail to coexpress the NK-associated markers NKR-P1 or Ly49, yet retain characteristic expression of the cytokine receptors interleukin (IL)-7R alpha and IL-2Rbeta. Despite these phenotypic abnormalities, gamma c- NK thymocytes could produce normal amounts of IL-4. These results define a maturational progression of NK thymocyte differentiation where intrathymic selection and IL-4-producing capacity can be clearly dissociated from the acquisition of the NK phenotype. Moreover, these data suggest a closer ontogenic relationship of NK T cells to TCR-alphabeta T cells than to NK cells with respect to cytokine dependency. We also failed to detect peripheral NK T cells in these mice, demonstrating that gamma c-dependent interactions are required for export and/or survival of NK T cells from the thymus. These results suggest a stepwise pattern of differentiation for thymically derived NK T cells: primary selection via their invariant TCR to confer the IL-4-producing phenotype, followed by acquisition of NK-associated markers and maturation/export to the periphery.

Abnormal CD40-mediated activation pathway in B lymphocytes from patients with hyper-IgM syndrome and normal CD40 ligand expression.

The CD40-mediated activation pathway of B cells from 10 patients with hyper-IgM syndrome and normal expression of CD40 ligand was studied. In all 10 cases, B cells were found to be defective for IgG, IgA, and IgE production after stimulation by anti-CD40 mAbs and cytokines. In the patients tested, neither B cell proliferation (n = 6) nor CD23 molecule expression (n = 5) were observed in cultures stimulated with anti-CD40 mAb. These results point to an intrinsic B cell deficiency and a defect in the CD40-triggered B cell activation pathway; this conclusion was supported by a lack of detectable germinal centers in the spleen of two patients. CD40-triggered activation events, i.e., phosphatidylinositol 3 (PI3) kinase activation and induction of transcription factors NF-kappaB and AP-1, were next analyzed in B cell lines derived from five patients. Three distinct patterns were observed: an absence of detectable abnormalities (n = 1), defective PI3 kinase activation with normal induction of NF-kappaB and AP-1 (n = 3), and defects in both PI3 kinase activation and induction of NF-kappaB and AP-1 (n = 1). In three B cell lines, each exhibiting one of the CD40-mediated activation patterns, sequences of CD40 and CD40 binding protein coding regions were normal. The coding region of TNF receptor-associated factor 2 (TRAF2), which is known to interact with CD40 for NF-kappaB induction, was also found to be normal in B cell lines deficient in NF-kappaB induction. Altogether, these results suggest that CD40 ligand-positive hyper-IgM syndrome could be genetically heterogeneous, although phenotypic variability is not excluded, and that an early defect in the CD40-triggered activation cascade can account for defective Ig class switching in some patients with CD40 ligand-positive hyper-IgM syndrome.

Pro-thymocyte expansion by c-kit and the common cytokine receptor gamma chain is essential for repertoire formation.

Growth factors have been implicated in thymocyte development, but mutants lacking cytokines, or their receptors, have failed to reveal essential roles for growth/differentiation factors in the thymus. Mutations in the receptor tyrosine kinase c-kit and the common cytokine receptor gamma chain (gamma c) reduce cellularity, but are permissive for thymocyte development. We now report that thymocyte development is completely abrogated in mice lacking both c-kit and gamma c (c-kit-gamma c-). Thymic hypocellularity is so severe that the T cell receptor repertoire fails to form except for monoclonal or oligoclonal beta chain DJ rearrangements. B lymphopoiesis is only mildly reduced in c-kit-gamma c- as compared with c-kit+gamma c- mice, and hematological values are identical comparing c-kit-deficient and c-kit-gamma c- mice. These experiments reveal essential, overlapping, and synergistic functions for two distinct signaling pathways, one utilizing c-kit and the other cytokine receptor gamma c complexes coupling to Janus kinases and signal transducers and activators of transcription.

Histological studies of gene-ablated mice support important functional roles for natural killer cells in the uterus during pregnancy.

Maternal lymphocytes having a large and granulated morphology accumulate at healthy implantation sites in normal mice. Insight into the functions of these cells has come from a previous study of two independent lines of mice deficient in natural killer (NK) cells. In pregnant Tg epsilon 26 mice, vascular pathology was found that led to the major complications of either fetal death or intrauterine growth retardation. In pregnant p56lck null x IL-2R beta null mice, extensive distension of the decidua was observed that separated the placenta from the myometrium and appeared to be interstitial edema. To strengthen assignment of uterine large granulated lymphocytes to the NK cell lineage and to understand which aspects of NK cell biology may be important for a uterine-based, pregnancy-associated subset, mid-gestation implantation sites from a new series of mice having gene deletions which alter NK cells (IL-2R gamma null, Stat4 null, IL-12 p40 null, beta 7 integrin null and Muc-1 null) have been examined histologically. The findings support the assignment of pregnancy-associated large granulated cells of mice to the NK cell lineage and suggest that the primary functions of these tissue-based NK cells are to support normal development of the decidua and/or its vasculature using pathways that involve IL-12 mediated signal transduction.

The p56lck SH2 domain mediates recruitment of CD8/p56lck to the activated T cell receptor/CD3/zeta complex.

The CD4 or CD8 co-receptors and the T cell receptor (TCR) are though to interact with the same antigen-presenting major histocompatibility complex molecule in a stable ternary complex. Therefore, the TCR and its co-receptor need to come into close proximity on the surface of the T cell. We have previously shown that the interaction of the p56lck SH2 domain with zeta-associated, tyrosine phosphorylated ZAP-70 and Syk kinases leads to an enhanced association of CD4 with TCR/CD3/zeta complex after CD3 stimulation of Jurkat cells. In this report, we analyzed whether a similar mechanism can mediate recruitment of the CD8 alpha alpha and CD8 alpha beta isoforms to the TCR. We demonstrate in vivo in association of CD8 alpha alpha/p56lck with the tyrosine kinase ZAP-70 after CD3 stimulation of Jurkat cells. A phosphopeptide competing in vitro for the binding of tyrosine phosphorylated proteins to the SH2 domain of p56lck specifically impedes the association of ZAP-70 with CD8 alpha alpha/p56lck without affecting the zeta/ZAP-70 interaction. The same peptide is able to compete for the activation-dependent association of the CD8 alpha alpha or CD8 alpha beta isoform with the TCR/CD3/zeta complex. Moreover, co-precipitation of the TCR with both CD8 isoforms was observed after CD3 stimulation. These findings strongly suggest that the p56lck SH2 domain mediates recruitment of CD8/p56lck to the activated TCR/CD3/zeta complex.

Critical role for the common cytokine receptor gamma chain in intrathymic and peripheral T cell selection.

The common cytokine receptor gamma chain (gammac), which is a functional subunit of the receptors for interleukins (IL)-2, -4, -7, -9, and -15, plays an important role in lymphoid development. Inactivation of this molecule in mice leads to abnormal T cell lymphopoiesis characterized by thymic hypoplasia and reduced numbers of peripheral T cells. To determine whether T cell development in the absence of gammac is associated with alterations of intrathymic and peripheral T cell selection, we have analyzed gammac-deficient mice made transgenic for the male-specific T cell receptor (TCR) HY (HY/gammac- mice). In HY/gammac- male mice, negative selection of autoreactive thymocytes was not diminished; however, peripheral T cells expressing transgenic TCR-alpha and -beta chains (TCR-alphaT/betaT) were absent, and extrathymic T cell development was completely abrogated. In HY/gammac- female mice, the expression of the transgenic TCR partially reversed the profound thymic hypoplasia observed in nontransgenic gammac- mice, generating increased numbers of thymocytes in all subsets, particularly the TCR-alphaT/betaT CD8+ single-positive thymocytes. Despite efficient positive selection, however, naive CD8+ TCR-alphaT/betaT T cells were severely reduced in the peripheral lymphoid organs of HY/gammac- female mice. These results not only underscore the indispensible role of gammac in thymocyte development, but also demonstrate the critical role of gammac in the maintenance and/or expansion of peripheral T cell pools.

[Gene therapy for hereditary immunodeficiencies]. / Thérapie génique des déficits immunitaires héréditaires.

There are numerous inherited immunodeficiencies characterized by either defects in T, B lymphocytes, phagocytic cells or the complement system. About 20 genes involved in inherited immunodeficiencies have now been identified. This opens theorically the possibility to consider gene therapy for the most severe of the diseases. A logical approach consists in attempting gene transfer into hematopoietic stem cells in order to achieve a definitive cure. However, the presently available vectors, i.e. retroviruses induce only stable gene integration and possibly expression into cycling cells while most stem cells are in G0/G1. This precludes at this time efficient gene therapy for many inherited immunodeficiencies. Nevertheless in instances, where there is an early block in cell differentiation like in adenosine desaminase deficiency (ADA) or X-L severe combined immunodeficiency (IL2 R gamma deficiencies), a selective advantage could be provided to the few transduced stem cells enabling progressive lymphocyte differentiation. This hypothesis sets the basis for the ongoing clinical studies in patients with ADA deficiency and will be assessed in available animal model of XL SCID.

Lymphoid development in mice with a targeted deletion of the interleukin 2 receptor gamma chain.

The interleukin 2 receptor gamma chain (IL-2R gamma) is a component of the receptors for IL-2, IL-4, IL-7, and IL-15. Mutations in IL-2R gamma in man appear responsible for the X chromosome-linked immunodeficiency SCIDX1, characterized by a defect in T-cell and natural killer (NK)-cell differentiation with the presence of poorly functioning B cells. To explore at which level IL-2R gamma affects lymphoid development in vivo, we have analyzed mice derived from embryonic stem (ES) cells with mutant IL-2R gamma loci generated by Cre/loxP-mediated recombination. In the peripheral blood of chimeric animals, lymphoid cells derived from IL-2R gamma- ES cells were not detected, although control ES cells carrying an IL-2R gamma gene with embedded loxP sites gave rise to T-, B-, and NK-cell lineages. Germline IL-2R gamma-deficient male animals, however, developed some mature splenic B and T cells, although the absolute number of lymphocytes was almost 10-fold reduced. In contrast, there was a complete disappearance of NK cells (over 350-fold reduction). Development of gut-associated intraepithelial lymphocytes was also severely diminished, and Peyer's patches were not detected. In vitro mitogenic responses of thymocytes, IL-4-directed immunoglobulin class switch of splenocytes, and NK activity were defective. Thus, IL-2R gamma facilitates mainstream B- and T-cell generation and function and also appears to be essential for NK-cell development.

The murine interleukin-2 receptor gamma chain gene: organization, chromosomal localization and expression in the adult thymus.

Defects in the interleukin-2 receptor gamma (IL-2R gamma) chain in the man result in an X-linked severe combined immunodeficiency, SCIDX1, characterized by an absence of T-cell differentiation. This phenotype may result from pertubations in IL-2, IL-4-, IL-7- or IL-15-mediated signaling, as the IL-2R gamma chain forms an integral component of these receptor systems. We have isolated and characterized cDNA and genomic clones for the murine IL-2R gamma. The gene (Il2rg) is well conserved between mouse and man with respect to overall structure and size, and contains regions of high conservation in the promoter region as well. Il2rg maps to mouse X chromosome region 40, in a region of synteny with human Xq12-13.1. We have also explored the expression of the IL-2R gamma during thymocyte development. IL-2R gamma transcripts are detected in the earliest thymocyte precursor cells and persist throughout intrathymic development into the mature peripheral compartment. Genomic clones for the murine IL-2R gamma will allow for further studies on the regulation and function of this gene in vivo.

Endocytosis of the beta chain of interleukin-2 receptor requires neither interleukin-2 nor the gamma chain.

Interleukin-2 (IL-2) and IL-2 receptors (IL-2R) critically regulate the magnitude and duration of T cell expansion required in an immune response. Modulation occurs at the level of receptor number and affinity. IL-2R is a multisubunit receptor which contains at least three chains, IL-2R alpha (p55), IL-2R beta (p70) and IL-2R gamma (p64). Some components of high-affinity receptors (alpha beta gamma) are continuously internalized in the absence as well as in the presence of IL-2. From studies on other receptors, it is known that endocytosis of ligand-receptor complexes is due to an intrinsic property of the receptor. However, the specific chains responsible for endocytosis of high-affinity IL-2 receptors have not been fully elucidated. IL-2R gamma has been reported to be necessary for IL-2 internalization, based on the fact that fibroblasts transfected with IL-2R alpha and -beta do not internalize IL-2. However, IL-2 dissociates too rapidly from IL-2R alpha beta receptors to allow for its internalization. From the reported results on IL-2 internalization in transfected fibroblasts, it cannot be concluded as to the respective roles of IL-2R beta and/or IL-2R gamma in endocytosis. As modulation of receptor number is important for biological activity, we have attempted to define the chains responsible for receptor internalization. In this work, we have studied the endocytic properties of IL-2R beta. We demonstrate that IL-2R beta is constitutively endocytosed in a B cell line, derived from a X-linked severe combined immunodeficiency patient, which lacks expression of IL-2R gamma. IL-2R beta was also constitutively internalized in T and natural killer cell lines independently of IL-2R gamma. These results suggest that IL-2R beta is endowed with endocytic capacity and carries internalization signals.