DUSP4-mediated accelerated T-cell senescence in idiopathic CD4 lymphopenia.

Idiopathic CD4 lymphopenia (ICL) is a rare heterogeneous immunological syndrome of unclear etiology. ICL predisposes patients to severe opportunistic infections and frequently leads to poor vaccination effectiveness. Chronic immune activation, expansion of memory T cells, and impaired T-cell receptor (TCR) signaling have been reported in ICL, but the mechanistic and causative links remain unclear. We show that late-differentiated T cells in 20 patients with ICL displayed defective TCR responses and aging markers similar to those found in T cells from elderly subjects. Intrinsic T-cell defects were caused by increased expression of dual-specific phosphatase 4 (DUSP4). Normalization of DUSP4 expression using a specific siRNA improved CD4(+) T-cell activity in ICL, as this restored TCR-induced extracellular signal-regulated kinase activation and increased the expression of the costimulatory molecules CD27 and CD40L. Conversely, repeated TCR stimulation led to defective signaling and DUSP4 overexpression in control CD4(+) T cells. This was associated with gradual acquisition of a memory phenotype and was curtailed by DUSP4 silencing. These findings identify a premature T-cell senescence in ICL that might be caused by chronic T-cell activation and a consequential DUSP4-dependent dampening of TCR signaling.

Interleukin-24 inhibits the plasma cell differentiation program in human germinal center B cells.

Complex molecular mechanisms control B-cell fate to become a memory or a plasma cell. Interleukin-24 (IL-24) is a class II family cytokine of poorly understood immune function that regulates the cell cycle. We previously observed that IL-24 is strongly expressed in leukemic memory-type B cells. Here we show that IL-24 is also expressed in human follicular B cells; it is more abundant in CD27(+) memory B cells and CD5-expressing B cells, whereas it is low to undetectable in centroblasts and plasma cells. Addition of IL-24 to B cells, cultured in conditions shown to promote plasma cell differentiation, strongly inhibited plasma cell generation and immunoglobulin G (IgG) production. By contrast, IL-24 siRNA increased terminal differentiation of B cells into plasma cells. IL-24 is optimally induced by BCR triggering and CD40 engagement; IL-24 increased CD40-induced B-cell proliferation and modulated the transcription of key factors involved in plasma cell differentiation. It also inhibited activation-induced tyrosine phosphorylation of signal transducer and activator of transcription-3 (STAT-3), and inhibited the transcription of IL-10. Taken together, our results indicate that IL-24 is a novel cytokine involved in T-dependent antigen (Ag)-driven B-cell differentiation and suggest its physiologic role in favoring germinal center B-cell maturation in memory B cells at the expense of plasma cells.

Long term culture of mesenchymal stem cells in hypoxia promotes a genetic program maintaining their undifferentiated and multipotent status.

BACKGROUND: In the bone marrow, hematopietic and mesenchymal stem cells form a unique niche in which the oxygen tension is low. Hypoxia may have a role in maintaining stem cell fate, self renewal and multipotency. However, whereas most studies addressed the effect of transient in vitro exposure of MSC to hypoxia, permanent culture under hypoxia should reflect the better physiological conditions. RESULTS: Morphologic studies, differentiation and transcriptional profiling experiments were performed on MSC cultured in normoxia (21% O2) versus hypoxia (5% O2) for up to passage 2. Cells at passage 0 and at passage 2 were compared, and those at passage 0 in hypoxia generated fewer and smaller colonies than in normoxia. In parallel, MSC displayed (>4 fold) inhibition of genes involved in DNA metabolism, cell cycle progression and chromosome cohesion whereas transcripts involved in adhesion and metabolism (CD93, ESAM, VWF, PLVAP, ANGPT2, LEP, TCF1) were stimulated. Compared to normoxic cells, hypoxic cells were morphologically undifferentiated and contained less mitochondrias. After this lag phase, cells at passage 2 in hypoxia outgrew the cells cultured in normoxia and displayed an enhanced expression of genes (4-60 fold) involved in extracellular matrix assembly (SMOC2), neural and muscle development (NOG, GPR56, SNTG2, LAMA) and epithelial development (DMKN). This group described herein for the first time was assigned by the Gene Ontology program to "plasticity". CONCLUSION: The duration of hypoxemia is a critical parameter in the differentiation capacity of MSC. Even in growth promoting conditions, hypoxia enhanced a genetic program that maintained the cells undifferentiated and multipotent. This condition may better reflect the in vivo gene signature of MSC, with potential implications in regenerative medicine.

IL-24 induces apoptosis of chronic lymphocytic leukemia B cells engaged into the cell cycle through dephosphorylation of STAT3 and stabilization of p53 expression.

Chronic lymphocytic leukemia (CLL) is characterized by the accumulation of long-lived monoclonal B cells mostly arrested at the G(0)/G(1) phase of the cell cycle. CLL cells strongly express intracellular melanoma differentiation-associated gene-7 (MDA7)/IL-24. However, adenovirus-delivered MDA7 was reported to be cytotoxic in several tumor cell lines. We report herein that rIL-24 alone had no effect; however, sequential incubation with rIL-2 and rIL-24 reduced thymidine incorporation by 50% and induced apoptosis of CLL cells in S and G(2)/M phases of the cell cycle, but not of normal adult blood or tonsil B cells. IL-24 stimulated STAT3 phosphorylation in IL-24R1-transfected cells but not in normal or CLL B cells. In contrast, IL-24 reversed the IL-2-induced phosphorylation of STAT3 in CLL, and this effect was neutralized by anti-IL-24 Ab. Phospho- (P)STAT3 inhibition induced by IL-24 was reversed by pervanadate, an inhibitor of tyrosine phosphatases. The addition of rIL-24 to IL-2-activated CLL B cells resulted in increases of transcription, protein synthesis. and phosphorylation of p53. The biological effects of IL-24 were reversed by the p53 inhibitor pifithrin-alpha and partly by the caspase inhibitor zvad. Troglitazone (a protein tyrosine phosphatase, PTP1B activator) phosphatase inhibited PSTAT3 and augmented p53 expression. PSTAT3 is a transcriptional repressor of p53, and therefore IL-24 induction of p53 secondary to PSTAT3 dephosphorylation may be sensed as a stress signal and promote apoptosis in cycling cells. This model explains why IL-24 can protect some resting/differentiated cells and be deleterious to proliferating cells.

Lymphoid differentiation of hematopoietic stem cells requires efficient Cxcr4 desensitization.

The CXCL12/CXCR4 signaling exerts a dominant role in promoting hematopoietic stem and progenitor cell (HSPC) retention and quiescence in bone marrow. Gain-of-function CXCR4 mutations that affect homologous desensitization of the receptor have been reported in the WHIM Syndrome (WS), a rare immunodeficiency characterized by lymphopenia. The mechanisms underpinning this remain obscure. Using a mouse model with a naturally occurring WS-linked gain-of-function Cxcr4 mutation, we explored the possibility that the lymphopenia in WS arises from defects at the HSPC level. We reported that Cxcr4 desensitization is required for quiescence/cycling balance of murine short-term hematopoietic stem cells and their differentiation into multipotent and downstream lymphoid-biased progenitors. Alteration in Cxcr4 desensitization resulted in decrease of circulating HSPCs in five patients with WS. This was also evidenced in WS mice and mirrored by accumulation of HSPCs in the spleen, where we observed enhanced extramedullary hematopoiesis. Therefore, efficient Cxcr4 desensitization is critical for lymphoid differentiation of HSPCs, and its impairment is a key mechanism underpinning the lymphopenia observed in mice and likely in WS patients.

Type-I interferons and systemic lupus erythematosus.

Systemic lupus erythematosus (SLE) is a prototypical autoimmune disease characterized by the production of autoantibodies directed against nuclear antigens and chronic inflammation affecting multiple tissues. Complex genetic disorders are at the origin of the disease in humans and in SLE-prone mice, leading to the escape of auto-reactive B-lymphocytes from central and peripheral control checkpoints that operate normally in healthy organisms. Although necessary, autoimmune B-cells are not sufficient and additional mechanisms such as T-cell help are clearly needed for the disease to occur. The role of type-I interferons (type-I IFNs), and in particular IFNalpha, as prominent cofactors for SLE was suggested years ago. Leading observations in patients and recent data in SLE-prone mice have now established IFNalpha as a major actor in SLE. Several systemic clinical symptoms and laboratory findings can indeed be interpreted as downstream effects of a high IFNalpha production, and point to this cytokine as a link between the expansion of autoimmune B-cells and the stimulation of other components of the immune system. Consequently, a vicious circle is established with overt immune-cell activation and inflammatory infiltrates culminating in the selective destruction of tissue targets, notably the kidney. These notions can now be transplanted to the clinic and designate IFNalpha as a new promising therapeutic target.

Plasmacytoid dendritic cell reconstitution following bone marrow transplantation: subnormal recovery and functional deficit of IFN-alpha/beta production in response to herpes simplex virus.

Infections with herpesviruses were frequent after bone marrow transplantation (BMT) before the preventive use of antiviral drugs, suggesting a deficit of innate immunity. A retrospective phenotypical and functional study was carried out on 25 patients 1-36 months after allogeneic BMT. Leukocyte counts followed a normal reconstitution, including natural killer (NK) cells and monocytes. Plasmacytoid dendritic cell (PDC) counts increased steadily, although they remained below normal values after 2 years. Most patients produced less interferon- alpha/beta (IFN-alphabeta) in vitro than healthy controls after infection with herpes simplex virus type 1 (HSV-1), whereas they responded normally to Sendai virus (SV). In addition, 6 patients had biologic signs of infection with herpesviruses, confirming a specific immunologic deficit against these viruses. IFN production was not correlated to PDC counts or to the occurrence of graft-versus-host disease (GVHD). Because all patients were under immunosuppressive treatment, we investigated the effect of drugs on IFN production by mononuclear cells. Glucocorticoids and cyclosporine A inhibited IFN production by infected leukocytes, with a predominant action on HSV-1-infected PDC. The inability of transplanted patients to mount an efficient immune response to herpesviruses may be partly related to drug toxicity toward cells of the innate immune system.

Analysis of transcription factors in thymic and CD34+ progenitor-derived plasmacytoid and myeloid dendritic cells: evidence for distinct expression profiles.

OBJECTIVE: The expression of mRNA for pre-Talpha is specific for human plasmacytoid dendritic cells (PDC), a population ontogenically close to T cells. The latter need Gata-3 transcription factor to develop. PU1 and RelB are two transcription factors involved in the development of murine myeloid DC (MDC). To determine the lineage origin of human thymic DC, the expression of these genes was investigated. MATERIALS AND METHODS: Fresh thymic DC, CD34(+)CD1a(-) progenitors, and progenitor-derived DC populations were sorted, analyzed, and compared to blood DC. RESULTS: Three DC populations were found in the thymus. 1) CD123(-/lo)HLA-DR(hi) DC expressing PU1 and RelB; 2) CD123(hi)HLA-DR(+) DC expressing only pre-Talpha, the expression of which was similar to that of MDC and PDC from peripheral blood; and 3) a new mature CD123(hi)HLA-DR(hi) PDC population with pre-Talpha, PU1 and RelB mRNAs. In culture, most CD34(+)CD1a(-) progenitors remained CD1a(-)CD123(-); had a T and natural killer cell differentiation potential; and expressed Gata-3 mRNA contrary to DC precursors. A few cells (10%) became CD1a(+)CD123(+) expressing pre-Talpha, PU1, and RelB mRNAs and were able to differentiate into typical Langerhans cells with transforming growth factor-beta. Coculture of thymic progenitors on a murine cell line generated CD123(hi)CD1a(-) cells with typical PDC morphology, expressing pre-Talpha but not PU1 and RelB transcripts. Activated PDC acquired myeloid antigens, and up-regulated PU1 and RelB mRNAs while down-regulating pre-Talpha mRNA expression. CONCLUSION: Both DC maturation pathways may arise from distinct precursors but are interconnected. DC differentiation seems to occur from Gata-3(-) precursors upstream of T and natural killer precursors.

Type I interferon production by plasmacytoid dendritic cells and monocytes is triggered by viruses, but the level of production is controlled by distinct cytokines.

The principal interferon-alpha/beta (IFN-I)-producing cells are plasmacytoid dendritic cell (PDC) precursors belonging to the lymphoid lineage. Monocytes that can differentiate into dendritic cells (DC) also produce IFN-I, although much less than PDC, after interaction with infectious agents. We show that whereas viruses trigger these cells to produce IFN-I, the amount of IFN is tightly controlled by cytokines. Monocytes produced IFN-I in response to Sendai virus (SV) infection, and PDC responded to both SV and herpes simplex virus (HSV). All cytokines tested failed to induce production of IFN-I in the absence of infection. However, among 18 relevant cytokines, incubation of PDC with interleukin-4 (IL-4), IL-15, and IL-7 alone or in combination with IL-3 before infection, enhanced IFN-I secretion. At variance, IL-12 alone or in synergy with granulocyte-macrophage colony-stimulating factor (GM-CSF) was active on SV-infected but not on HSV-infected monocytes. Tumor necrosis factor-alpha (TNF-alpha) and IL-4 inhibited IFN-I production by PDC and monocytes, respectively, and IL-10 strongly inhibited IFN-I production in both cell lineages. The response of PDC to IL-7 and IL-15, which also activate natural killer (NK) cell maturation, further emphasizes the cooperation between these two cell subsets in the control of innate immunity.

Severe herpes virus (HSV-2) infection in two patients with myelodysplasia and undetectable NK cells and plasmacytoid dendritic cells in the blood.

BACKGROUND: How the immune system contains herpes simplex virus (HSV) infections is partly understood. T cells from infected persons proliferate in response to HSV antigens in vitro and may control local relapse rather than primary infection. NK cells have been involved in the control of experimental infections. A potentially important, as yet unexplored, population of interest might be the plasmacytoid dendritic cells (PDC), which contrary to monocytes, produce very high amounts of the major antiviral molecules, type-I interferon (IFN) following interaction with HSV. OBJECTIVES: Measure type-I IFN production, PDC, and NK cells in patients with unusually severe HSV infections. STUDY DESIGN: Two female patients of 33- and 50-year-old, respectively were referred because of severe disseminated HSV2 infection and myelodysplastic marrow. One patient had leukaemia and a primary HSV2 infection whereas the other had systemic lupus erythematosus (SLE) and a chronic HSV2 infection. The following studies were performed at various time points over 18 months: analysis of the lymphocytes and PDC subsets phenotype, lymphocyte proliferation assays to recall antigens; generation of NK cells in cultures, and production of type-I IFN in serum and by HSV-infected and by sendai virus (SV)-infected blood cells. RESULTS AND CONCLUSIONS: PDC and NK cells were undetectable in the blood of both patients and NK cells could not be generated in culture at the time of ongoing infection. PBMC failed to produce IFN after infection with HSV contrasting with a normal T cell proliferation to HSV antigens in patient 1. Our observation suggests that innate immunity, through NK cells and PDC may control HSV infections, and together with IFN-producing capacity, should be investigated in patients with unusually severe HSV infections.

Hypoxia and visualization of the stem cell niche.

It is widely accepted that mammalian stem cells reside in a specialized cellular and a cellular microenvironment called the niche. The niche contrary to other tissues is characterized by a low partial Oxygen pressure (ppO2). This microenvironment protects stem cells from deleterious effects of O2 on proteins and DNA, through the production of reactive oxygen species (ROS). In addition there is now solid evidence that this physiological hypoxia helps stem cells maintaining their major characteristics: multipotency and ability to differentiate and migrate from the niche to specialized tissues in order to fulfill the needs of the organism. Immuno Histological techniques can stain stem cells in situ by specific Abs (such as against CD34 and CD45 for Hematopoietic Stem Cells HSC). However, a universal marker of hypoxia is Hypoxia-Inducible Factor-1, HIF-1, which is stabilized by low ppO2 and acts as a transcription factor to regulate a vast array of genes downstream. HIF-1, together with pimonidazole, a chemical compound interacting with proteins that are reduced in a hypoxic environment, are bona fide markers of the stem cell niche.

B-cell-mediated strategies to fight chronic allograft rejection.

Solid organs have been transplanted for decades. Since the improvement in graft selection and in medical and surgical procedures, the likelihood of graft function after 1 year is now close to 90%. Nonetheless even well-matched recipients continue to need medications for the rest of their lives hence adverse side effects and enhanced morbidity. Understanding Immune rejection mechanisms, is of increasing importance since the greater use of living-unrelated donors and genetically unmatched individuals. Chronic rejection is devoted to T-cells, however the role of B-cells in rejection has been appreciated recently by the observation that B-cell depletion improve graft survival. By contrast however, B-cells can be beneficial to the grafted tissue. This protective effect is secondary to either the secretion of protective antibodies or the induction of B-cells that restrain excessive inflammatory responses, chiefly by local provision of IL-10, or inhibit effector T-cells by direct cellular interactions. As a proof of concept B-cell-mediated infectious transplantation tolerance could be achieved in animal models, and evidence emerged that the presence of such B-cells in transplanted patients correlate with a favorable outcome. Among these populations, regulatory B-cells constitute a recently described population. These cells may develop as a feedback mechanism to prevent uncontrolled reactivity to antigens and inflammatory stimuli. The difficult task for the clinician, is to quantify the respective ratios and functions of "tolerant" vs. effector B-cells within a transplanted organ, at a given time point in order to modulate B-cell-directed therapy. Several receptors at the B-cell membrane as well as signaling molecules, can now be targeted for this purpose. Understanding the temporal expansion of regulatory B-cells in grafted patients and the stimuli that activate them will help in the future to implement specific strategies aimed at fighting chronic allograft rejection.

Interleukin-24 mediates apoptosis in human B-cells through early activation of cell cycle arrest followed by late induction of the mitochondrial apoptosis pathway.

Interleukin (IL)-24 has death-promoting effects on various proliferating cells including B-cells from chronic lymphocytic leukemia (CLL) and germinal center B-cells, but its molecular mechanisms are poorly understood. Using a B-cell differentiation model and mRNA profiling, we found that recombinant (r)IL-24 stimulated genes of the mitochondrial apoptotic pathway (Bax, Bid, Casp8, COX6C, COX7B) after 36 h, whereas the transcription of genes involved in DNA replication and metabolism was inhibited within 6 h. Unexpectedly, insulin-like growth factor 1 (IGF1), a hormone known to promote cell growth, was stimulated by IL-24. Activated B-cells express receptor for IGF1, to which they become sensitized and undergo apoptosis, a mechanism similar in this respect to IL-24-induced cell death. Furthermore, inhibition of the IGF1 pathway reversed the effects of IL-24. IL-24-mediated apoptosis was also antagonized by pifithrin-alpha, an inhibitor of p53 transactivation. Altogether, these results disclose sequential molecular signals generated by IL-24 in activated B-cells.

CD5: a safeguard against autoimmunity and a shield for cancer cells.

CD5 is a pan T cell marker also expressed at various developmental and activation stages on human B cells. CD5 is a well established negative regulator of TCR and BCR signalling. In the last years, great advances have been made in understanding the molecular mechanisms behind this regulatory function. Using animal models, it was demonstrated that increased expression of CD5 on either T cells or B cells protects against autoimmunity; as a consequence of an increase of the threshold needed for TCR- or a BCR-mediated activation following antigen recognition. CD5-positive cells may also prevent the emergence of autoimmunity by provision of cytokines such as IL-10. Although the physiological role of CD5 might be to control the generation of aberrant immune responses, the expansion of CD5+ lymphocytes may be deleterious. The other side of the coin is that in cancers, CD5 expression plays a role in the fate of tumour-specific T cells, rendering lymphocytes tolerant and unable to recognize and eliminate malignant cells. IL-10-producing B-cells may also inhibit anti-tumoural immune responses. An important issue is therefore to better understand the molecules produced by tolerogenic or immunogenic microenvironments that respectively turn on or downregulate CD5 expression, with potential therapeutic implications.

Interleukin-24: a molecule with potential anti-cancer activity and a cytokine in search of a function.

The gene encoding Melanoma-differentiation antigen-7 (MDA-7) was discovered more than 10 years ago. Its potential anti-cancer activity was surmised because its expression is inversely correlated with the cell proliferation status. Indeed adenoviral delivery of this gene proved to be efficient in killing several cancer cell lines and great strides have been made concerning its molecular ways of action. Later it was shown that mda7 encoded a secreted cytokine which belongs to the IL-10, class-II family of cytokines. We recently found that this molecule exerted apoptotic activity on stimulating but not on resting lymphocytes from a B cell leukaemia. This activity is distinct from that of intracellular MDA-7, and may pave the way for using the cytokine in cancers provided that they express the IL-24 Receptors; in this respect, melanomas are insensitive to the recombinant cytokine due to the lack of IL-24 receptors at their surface. In contrast to its anti-cancer activity, the immunological role of IL-24 is still unclear, with differences between mice and human. If however it is demonstrated that IL-24 can inhibit the function of STAT3 in normal lymphocytes as it is the case in leukemic cells, and given that STAT3 is needed for the differentiation of several lymphocyte subsets, this will give us hints as to the potential role of this cytokine in the immune system.

Natural phosphorylation of CD5 in chronic lymphocytic leukemia B cells and analysis of CD5-regulated genes in a B cell line suggest a role for CD5 in malignant phenotype.

Chronic lymphocytic leukemia (CLL) results in the accumulation of B cells, presumably reflecting the selection of malignant cell precursors with Ag combined with complex alterations in protein activity. Repeated BCR stimulation of normal B cells leads to anergy and CD5 expression, both of which are features of CLL. Because CD5 is phosphorylated on tyrosine following BCR engagement and negatively regulates BCR signaling in normal B cells, we investigated its phosphorylation status and found it to be naturally phosphorylated on tyrosine but not on serine residues in CLL samples. To analyze the role of CD5, we established a B cell line in which CD5 is phosphorylated. Gene profiling of vector vs CD5-transfected B cells pointed out gene groups whose expression was enhanced: Apoptosis inhibitors (BCL2), NF-kappaB (RELB, BCL3), Wnt, TGFbeta, VEGF, MAPKs, Stats, cytokines, chemokines (IL-10, IL-10R, IL-2R, CCL-3, CCL-4, and CCR7), TLR-9, and the surface Ags CD52, CD54, CD70, and CD72. Most of these gene groups are strongly expressed in CLL B cells as compared with normal B cells. Unexpectedly, metabolic pathways, namely cholesterol synthesis and adipogenesis, are also enhanced by CD5. Conversely, CD5 inhibited genes involved in RNA splicing and processing, ribosome biogenesis, proteasome, and CD80 and CD86 Ags, whose expression is low in CLL. Comparison of CD5- vs tailless CD5-transfected cells further demonstrated the role of CD5 phosphorylation in the regulation of selected genes. These results support a model where CLL cells are chronically stimulated, leading to CD5 activation and cell survival. In addition to CD5 itself, we point to several CD5-induced genes as potential therapeutic targets.

Cyclosporin-A inhibits ERK phosphorylation in B cells by modulating the binding of Raf protein to Bcl2.

Extracellular signal-related kinase (ERK) signaling is regulated by sequential phosphorylation of upstream kinases including Raf. We report herein that ERK phosphorylation is inhibited by a short incubation with Cyclosporin-A (CsA) in anti-IgM activated Daudi B cells. As Bcl2, through its BH4 domain, was previously shown to bind both Calcineurin (Can) and Raf proteins, we hypothesized that CsA inhibited Can binding to Bcl2 allowing the latter to bind more Raf at the mitochondria thereby diverting it from activating the ERK cascade. In support of this less Bcl2 coprecipitated with Can heterodimer in total lysates of cells treated with CsA as compared to controls. In parallel, Raf1 was augmented in both the mitochondrial fractions of cells treated with CsA and in Bcl2 immunoprecipitates under CsA. Finally, introduction of a Bcl2 BH4 domain into Daudi cells augmented ERK phosphorylation in unstimulated cells and this augmentation was unsensitive to CsA. We therefore suggest that CsA indirectly inhibited ERK activation through sequestration of Raf1, at the mitochondria.

Human CD5 promotes B-cell survival through stimulation of autocrine IL-10 production.

CD5 is a negative regulator of B-cell receptor (BCR) signaling that is up-regulated after BCR stimulation and likely contributes to B-cell tolerance in vivo. However, CD5 is constitutively expressed on the B-1 subset of B cells. Contrary to CD5(-) B-2 B cells, B-1 B cells are long-lived because of autocrine interleukin-10 (IL-10) production through unknown mechanisms. We demonstrate herein a direct relationship between CD5 expression and IL-10 production. Human peripheral blood CD5(+) B cells produce more IL-10 than CD5(-) B cells after BCR activation. Introducing CD5 into CD5(-) B cells induces the production of IL-10 by activating its promoter and the synthesis of its mRNA. The cytoplasmic domain of CD5 is sufficient for this process. CD5 also protects normal human B cells from apoptosis after BCR stimulation while reducing the BCR-induced Ca(2+) response. We conclude that CD5 supports the survival of B cells by stimulating IL-10 production and by concurrently exerting negative feedback on BCR-induced signaling events that can promote cell death.

CD5-negative regulation of B cell receptor signaling pathways originates from tyrosine residue Y429 outside an immunoreceptor tyrosine-based inhibitory motif.

CD5 is a cell surface receptor that negatively regulates B cell function, but whose relationship to the immunoreceptor tyrosine-based inhibitory motif (ITIM) family of B cell inhibitory receptors is unclear. Using Fcgamma type IIB receptor-CD5 chimeras encompassing the cytoplasmic domain of CD5, we previously showed that a particular region of the molecule containing two tyrosine residues, Y429 and Y441, in an amino acid stretch similar to the Src autophosphorylation motif and a putative ITIM, respectively, antagonized early signaling events triggered through the B cell receptor (BCR). In this study, we provide evidences that only Y429 is mandatory for the inhibition by CD5 of the calcium response activated via the BCR. This residue also efficiently controls inhibition of the Ras/extracellular signal-related kinase-2 pathway. Analyzing the membrane translocation of the AKT protooncogene using its 3'-phosphoinositide-specific pleckstrin homology domain fused to the green fluorescent protein as a probe, we also show that CD5 strongly impairs its cellular redistribution and demonstrate the role played by Y429 in this process. We finally report that Y429 controls almost exclusively CD5 phosphorylation as well as inhibition of BCR-triggered IL-2 production upon coaggregation of the two receptors. Thus, CD5 uses an ITIM-independent strategy, centered on Y429, the major tyrosine-phosphorylated residue in its cytoplasmic domain, to inhibit BCR activation.

HIV infection rapidly induces and maintains a substantial suppression of thymocyte proliferation.

The supply of naive T cells by the thymus normally requires precursor T cell proliferation within the thymus and would be particularly important in the setting of HIV infection when both naive and memory T cells are progressively depleted. As a robust, quantitative index of intrathymic proliferation, the ratio of different T cell receptor excision circles (TRECs), molecular markers of distinct T cell receptor rearrangements occurring at different stages of thymocyte development, was measured in peripheral blood-mononuclear cells (PBMCs). This ratio has the virtue that it is a "signature" of thymic emigrants throughout their entire life and, thus, can be measured in peripheral cell populations that are easy to obtain. Using the new assay, we evaluated the effect of HIV infection on intrathymic precursor T cell proliferation by longitudinal analysis of PBMCs from recently infected individuals. Our findings reveal a substantial reduction in intrathymic proliferation. The analysis also indicates the existence of a compensatory mechanism acting to sustain the numbers of recent thymic emigrants (RTEs) in the periphery.