Munc13-4 deficiency with CD5 downregulation on activated CD8+ T cells.

Familial hemophagocytic lymphohistiocytosis (FHL) is characterized by uncontrolled activation of T cells and macrophages and hypercytokinemia. We have recently described a significant increase in a subpopulation of CD8(+) T cells with downregulation of CD5 during the acute phase of FHL type2 (FHL2; perforin deficiency), which declines after successful treatment, with a concomitant reduction in serum cytokine level. This unusual subset of CD8(+) T cells, however, has not been characterized in patients with other subtypes of FHL. Herein, we describe a patient with FHL3 (Munc13-4 deficiency) carrying compound heterozygous mutations in the UNC13D gene. He had high serum levels of pro-inflammatory cytokines and significantly increased activated CD8(+) T cells with downregulation of CD5 during the acute phase, similar to that found in FHL2. This immunophenotypic feature may serve as a useful marker of immune dysregulation in FHL3 in addition to FHL2.

Role of scavenger receptors in the pathophysiology of chronic liver diseases.

Scavenger receptors comprise a large family of structurally diverse proteins that are involved in many homeostatic functions. They recognize a wide range of ligands, from pathogen-associated molecular patterns (PAMPs) to endogenous, as well as modified host-derived molecules (DAMPs). The liver deals with blood micro-organisms and DAMPs released from injured organs, thus performing vital metabolic and clearance functions that require the uptake of nutrients and toxins. Many liver cell types, including hepatocytes and Kupffer cells, express scavenger receptors that play key roles in hepatitis C virus entry, lipid uptake, and macrophage activation, among others. Chronic liver disease causes high morbidity and mortality worldwide. Hepatitis virus infection, alcohol abuse, and non-alcoholic fatty liver are the main etiologies associated with this disease. In this context, continuous inflammation as a result of liver damage leads to hepatic fibrosis, which frequently brings about cirrhosis and ultimately hepatocellular carcinoma. In this review, we will summarize the role of scavenger receptors in the pathophysiology of chronic liver diseases. We will also emphasize their potential as biomarkers of advanced liver disease, including cirrhosis and cancer.

CD5-dependent CK2 activation pathway regulates threshold for T cell anergy.

CD5 activates casein kinase 2 (CK2), a serine/threonine kinase that constitutively associates with the CK2-binding domain at the end of its cytoplasmic tail. To determine the physiological significance of CD5-dependent CK2 activation in T cells, we generated a knock-in mouse that expresses a CD5 protein containing a microdeletion with selective inability to interact with CK2 (CD5ΔCK2BD). The levels of CD5 on developing and mature T cell populations from CD5ΔCK2BD mice and CD5 wild-type (WT) mice were similar. The thymus of CD5ΔCK2BD mice contained fewer double-positive thymocytes than did that of both CD5WT and CD5 knockout (KO) mice, although the numbers of all other immature and mature T cell populations were unaltered. CD5ΔCK2BD T cells hypoproliferated and exhibited enhanced activation-induced cell death when stimulated with anti-CD3 or cognate peptide in comparison with CD5WT T cells. We also found that functional CD5-dependent CK2 signaling was necessary for efficient differentiation of naive CD4+ T cells into Th2 and Th17 cells, but not Th1 cells. We previously showed that experimental autoimmune encephalomyelitis (EAE) in CD5KO mice was less severe and delayed in onset than in CD5WT mice. Remarkably, CD5ΔCK2BD mice recapitulated both EAE severity and disease onset of CD5KO mice. Increasing the immunization dose of myelin oligodendrocyte glycoprotein 35-55 peptide, a model that mimics high-dose tolerance, led to decreased severity of EAE in CD5WT mice but not in CD5KO or CD5ΔCK2BD mice. This property was recapitulated in in vitro restimulation assays. These results demonstrate that CD5-CK2 signaling sets the threshold for T cell responsiveness and is necessary for efficient generation of Th2 and Th17 cells.

DNA methylation and B-cell autoreactivity.

Although not exclusive, mounting evidence supports the fact that DNA methylation at CpG dinucleotides controls B-cell development and the progressive eliminati or inactivation of autoreactive B cell. Indeed, the expression of different B ce specific factors, including Pax5, rearrangement of the B-cell receptor (BCR) and cytokine production are tightly controlled by DNA methylation. Among normal B cells, the autoreactive CD5+ B cell sub-population presents a reduced capacity to methylate its DNA that leads to the expression of normally repressed genes, such as the human endogenous retrovirus (HERV). In systemic lupus erythematosus (SLE) patients, the archetype ofautoimmune disease, autoreactive B cells are characterized by their inability to induce DNA methylation that prolongs their survival. Finally, treating B cells with demethylating drugs increased their autoreactivity. Altogether this suggests that a deeper comprehension ofDNA methylation in B cells may offer opportunities to develop new therapeutics to control autoreactive B cells.

B-cell: a logical target for treatment of rheumatoid arthritis.

The interest for B-cells in rheumatoid arthritis (RA) is currently being revived. They are involved in the development and activation of lymphoid architecture by regulating dentritic cell and T-cell function through cytokine production. Receptor editing an revising are also essential in B-cells and aid in preventing autoimmunity. Abnormalities in the subset distribution and a default in any task assigned to the B-cells may favor autoimmunity. Beneficied responses to B-cell depletion in RA by anti-CD20 monoclonal antibody rituximab illustrate the importance of B-lymphocytes in the pathogenesis of this disease. A new avenue has thus been opened, whereby B-lymphocytes return as a significant contributor to autoimmune disorders.

Human CD5 protects circulating tumor antigen-specific CTL from tumor-mediated activation-induced cell death.

We previously characterized several tumor-specific T cell clones from PBL and tumor-infiltrating lymphocytes of a lung cancer patient with identical TCR rearrangements and similar lytic potential, but with different antitumor response. A role of the TCR inhibitory molecule CD5 to impair reactivity of peripheral T cells against the tumor was found to be involved in this process. In this report, we demonstrate that CD5 also controls the susceptibility of specific T cells to activation-induced cell death (AICD) triggered by the tumor. Using a panel of tumor-infiltrating lymphocytes and PBL-derived clones expressing different levels of CD5, our results indicate that T lymphocyte AICD in response to the cognate tumor is inversely proportional to the surface expression level of CD5. They also suggest a direct involvement of CD5 in this process, as revealed by an increase in tumor-mediated T lymphocyte AICD following neutralization of the molecule with specific mAb. Mechanistically, our data indicate that down-regulation of FasL expression and subsequent inhibition of caspase-8 activation are involved in CD5-induced T cell survival. These results provide evidence for a role of CD5 in the fate of peripheral tumor-specific T cells and further suggest its contribution to regulate the extension of CTL response against tumor.

CD5 does not regulate the signaling triggered through BCR in B cells from a subset of B-CLL patients.

CD5 is a transmembrane protein expressed on all T lineage cells and a subset of B cells. It is known that CD5 is physically associated with the T-cell receptor and B-cell receptor (BCR), inhibiting the signaling triggered by both of them. CD5 is also characteristic of B-chronic lymphocytic leukemia (B-CLL) B cells, although its implication in the development of this lymphoproliferative disorder has not been studied. In the present study, we examined the effect of CD5 in apoptosis, cell viability and global protein tyrosine phosphorylation mediated by BCR in B cells from B-CLL patients. As opposed to tonsil B cells, we did not observe an increase in the apoptotic or viability signals induced by anti-immunoglobulin M or SAC/interleukin-2 when CD5 was dissociated from BCR in leukemic cells of the majority of patients. We also observed that CD5 did not regulate the BCR-induced phosphotyrosine pattern in B-CLL B cells. These findings suggest that CD5 does not inhibit properly the BCR-mediated signaling in leukemic cells. This defect in inhibiting the BCR might contribute to the enhanced survival of B-CLL B cells.

CD5+ B cells are preferentially expanded in rabbit appendix: the role of CD5 in B cell development and selection.

Although only a small proportion of mouse and human B cells are CD5(+), most adult rabbit B cells express CD5. However, CD5 was not detectable on the majority of B cells in neonatal appendix 1 and 3days after birth. Cell trafficking studies demonstrated that CD5(+) and CD5(-) CD62L(+) B cells from bone marrow migrated into appendix. There, CD5(+) B cells were preferentially expanded and predominated by approximately 2weeks of age. In mutant ali/ali rabbits, VHa2(+) B cells develop through gene conversion-like alteration of rearranged VH genes upstream of deleted VH1a2. Correlated appearance of individual CD5(+) germinal centers and VHa2(+) B-cells in mutant appendix suggests that CD5 binding positively selects cells with a2(+) framework regions that bind CD5. Following negative and positive selection, cells with diversified rearranged heavy- and light-chain sequences exit appendix, migrate to peripheral tissues and constitute the preimmune repertoire of CD5(+) B cells that encounter foreign antigens.

Expression of RAGs in peripheral B cells outside germinal centers is associated with the expression of CD5.

Previous studies have indicated that mature B cells reactivate secondary V(D)J recombination inside and outside the germinal center (GC) of peripheral lymphoid organs. The nature of the B cells undergoing Ig rearrangement before they enter GC is unknown. In this study, we present evidence that activated mature CD5-positive human tonsil B cells coexpress both RAG1 and RAG2 mRNA and protein, and display DNA cleavage resulting from their recombinase activity. Furthermore, in vitro activation of CD5-negative naive mature B cells by IgR and CD40 cross-linking induces expression of CD5 on a subset of cells, and leads to the up-regulation of RAG1 and RAG2 only in cells turned positive for CD5. Thus, RAG gene expression is closely related to CD5 expression outside GCs. These data suggest that CD5 is associated with receptor revision in activated mature B cells and likely to promote expression of suitable IgR capable of initiating the GC reaction.

Mechanisms of peritoneal B-1a cells accumulation induced by murine lupus susceptibility locus Sle2.

The abundance of B-1a cells found in the peritoneal cavity of mice is under genetic control. The lupus-prone mouse New Zealand Black and New Zealand White (NZB x NZW)F(1) and its derivative NZM2410 are among the strains with the highest numbers of peritoneal B1-a cells. We have previously identified an NZM2410 genetic locus, Sle2, which is associated with the production of large numbers of B-1a cells. In this paper, we examined the mechanisms responsible for this phenotype by comparing congenic C57BL/6 mice with or without Sle2. Fetal livers generated more B-1a cells in B6.Sle2 mice, providing them with a greater starting number of B-1a cells early in life. Sle2-expressing B1-a cells proliferated significantly more in vivo than their B6 counterparts, and reciprocal adoptive transfers showed that this phenotype is intrinsic to Sle2 peritoneal B cells. The rate of apoptosis detected was significantly lower in B6.Sle2 peritoneal cavity B-1a cells than in B6, with or without exogenous B cell receptor cross-linking. Increased proliferation and decreased apoptosis did not affect Sle2 peritoneal B-2 cells. In addition, a significant number of peritoneal cavity B-1a cells were recovered in lethally irradiated B6.Sle2 mice reconstituted with B6.Igh(a) bone marrow, showing radiation resistance in Sle2 B-1a cells or its precursors. Finally, B6.Sle2 adult bone marrow and spleen were a significant source of peritoneal B-1a cells when transferred into B6.Rag2(-/-) mice. This suggests that peritoneal B-1a cells are replenished throughout the animal life span in B6.Sle2 mice. These results show that Sle2 regulates the size of the B-1a cell compartment at multiple developmental checkpoints.

The Scavenger Receptor Cysteine-Rich (SRCR) domain: an ancient and highly conserved protein module of the innate immune system.

The Scavenger Receptor Cysteine-Rich (SRCR) domain is an ancient and highly conserved protein module of ~100-110 amino acids, which defines a superfamily (SRCR-SF) of either soluble or membrane-bound receptors expressed by hematopoietic and nonhematopoietic cells, at either embryonic or adult stages. The existence of two types of SRCR domains allows the division of the SRCR-SF into two groups. Members of group A contain SRCR domains with 6 cysteine residues and are encoded by two exons, whereas those of group B usually contain 8 cysteines and are encoded by a single exon. Group A members usually present as multidomain mosaic proteins containing single SRCR domains associated to other functional domains, such as enzymatic (protease) domains or collagenous regions. On the contrary, group B members generally present as proteins exclusively composed of tandem repeats of SRCR domains, with or without the presence of CUB and ZP domains thought to be involved in oligomerization but never associated to protease domains. Representatives of either group are found in different animal species, from low invertebrates (sponges) to high vertebrates (mammals). Although no unifying function has been defined for SRCR-SF members, accumulated data, together with the high degree of structural and phylogenetic conservation of SRCR domains indicates that they might subserve basic homeostatic functions, including innate immune defense.

CD5 inhibits signaling at the immunological synapse without impairing its formation.

Physiologically, Ag detection by T cells occurs at the immunological synapse (IS) formed at the interface with an APC. CD5 is considered as an inhibitory molecule for Ag receptor-mediated signals in T cells. However, the influence of CD5 at the IS on synapse formation and functioning has not yet been reported. We demonstrate here that CD5 is recruited and tightly colocalized with CD3 in different human and murine IS. Following transfection in a CD5-negative T cell line of CD5 fused to the green fluorescent protein, we show that CD5 recruitment includes a fast Ag-independent and a slower Ag-dependent component. In video-imaging recordings of doubly transfected cells, the movements of CD3 and CD5 show similar kinetics, and the amount of CD3 recruited to the synapse is unaffected by CD5 expression. Moreover, APC-T cell adhesion is unchanged in CD5-expressing cells. Despite this, the extent of tyrosine phosphorylation at the synapse and the amplitude of calcium responses induced by Ag recognition are both decreased by CD5. These inhibitions increase with CD5 membrane levels. They also requires the pseudo-immunoreceptor tyrosine-based activation motif expressed in the cytoplasmic domain of the molecule. Thus, CD5 is rapidly recruited at the IS and lowers the T cell response elicited by Ag presentation by targeting downstream signaling events without affecting IS formation.

B-1 cell (CD5+/CD11b+) numbers and nIgM levels are elevated in physically active vs. sedentary rats.

Habitual, moderate exercise is associated with improved health, including reductions in illness. These benefits may stem, in part, from immune function improvements. We have previously reported that daily wheel running increases serum and peritoneal natural IgM (nIgM) in pathogen-free Sprague-Dawely rats. B-1 cells, which primarily reside in the peritoneal cavity, produce nIgM in the absence of antigen stimulation. This study examined whether physical activity would also increase B-1 cell numbers in the peritoneal cavity, mesenteric lymph nodes, and spleen. Male, pathogen-free Fischer 344 rats were sedentary (standard cages) or physically active (running wheel access) for 6-7 wk. Peritoneal cavity, mesenteric lymph nodes, and spleen cells were taken, and the number of CD5+/CD11b+ (B-1) cells were measured by using two-color flow cytometry. The results were that physically active animals had increased numbers of CD5+/CD11b+ cells in the peritoneal cavity. In addition, physically active animals had increased serum and peritoneal nIgM, thus replicating our previous observations. These results indicate that voluntary running selectively increases the B-1 cell population, which is most likely responsible for the elevated serum and peritoneal nIgM in active rats. Because B-1 cells are important in host defense, these changes may contribute to the health benefits of exercise.

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-mediated inhibition of TCR signaling during intrathymic selection and development does not require the CD5 extracellular domain.

CD5 functions as a negative regulator of TCR signaling during intrathymic T cell development, but it is not known if this negative regulatory function requires CD5 engagement of an extracellular ligand. The present study has specifically examined the role of the CD5 extracellular domain in T cell development by introducing into CD5-/- mice a chimeric CD5 molecule in which the extracellular domain of CD5 is replaced with the extracellular domain of human IL-2R p55 (Tac) for which no ligand exists in the mouse. We now report that CD5 mediated down-regulation of TCR signaling during thymocyte development does not require the CD5 extracellular domain and, consequently, does not involve CD5 binding of an extracellular ligand in the thymus.

Molecular mechanisms underlying differential contribution of CD28 versus non-CD28 costimulatory molecules to IL-2 promoter activation.

T cell costimulation via CD28 and other (non-CD28) costimulatory molecules induces comparable levels of [(3)H]TdR incorporation, but fundamentally differs in the contribution to IL-2 production. In this study, we investigated the molecular basis underlying the difference between CD28 and non-CD28 costimulation for IL-2 gene expression. Resting T cells from a mutant mouse strain generated by replacing the IL-2 gene with a cDNA encoding green fluorescent protein were stimulated with a low dose of anti-CD3 plus anti-CD28 or anti-non-CD28 (CD5 or CD9) mAbs. CD28 and non-CD28 costimulation capable of inducing potent [(3)H]TdR uptake resulted in high and marginal levels of green fluorescent protein expression, respectively, indicating their differential IL-2 promoter activation. CD28 costimulation exhibited a time-dependent increase in the binding of transcription factors to the NF-AT and NF-kappaB binding sites and the CD28-responsive element of the IL-2 promoter, whereas non-CD28 costimulation did not. Particularly, a striking difference was observed for the binding of NF-kappaB to CD28-responsive element and the NF-kappaB binding site. Decreased NF-kappaB activation in non-CD28 costimulation resulted from the failure to translocate a critical NF-kappaB member, c-Rel, to the nuclear compartment due to the lack of IkappaBbeta inactivation. These observations suggest that unlike CD28 costimulation, non-CD28 costimulation fails to sustain IL-2 promoter activation and that such a failure is ascribed largely to the defect in the activation of c-Rel/NF-kappaB.

CD5-induced apoptosis of B cells in some patients with chronic lymphocytic leukemia.

Although B chronic lymphocytic leukemia (B-CLL) is characterized by prolonged survival of CD5(+) B cells in vivo, these cells apoptose spontaneously in vitro. The effect of CD5 ligation on apoptosis was studied in 27 newly diagnosed patients with B-CLL, in relation to the expression of surface IgM (sIgM), CD79b, CD38, CD72 and CD19. B cells from 15 patients (group I) were resistant to anti-CD5-induced apoptosis, whereas apoptosis above spontaneous levels was seen in the remaining 12 studied (group II). Group II was then subdivided on the basis of differences in the time required to reach maximum apoptosis: whilst B cells from seven patients underwent apoptosis by 18 h, those from the remaining five needed 36 h to apoptose. The expression of sIgM, CD5, CD79b and CD38 was higher in group II than group I, suggesting that signaling for apoptosis might operate via CD79, and that CD38 expression was required. As shown by flow cytometry and confirmed by Western blotting, apoptosis was associated with a decrease in the ratios of Bcl-2/Bax and Bcl(XL)/Bax, due to an increase in the level of Bax, but no change in that of Bcl-2. This heterogeneous apoptotic response to CD5 ligation offers an explanation for the incomplete success of anti-CD5 monoclonal therapy, and might help identify patients who would respond to such treatment.

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.

CD5 (OKT1) augments CD3-mediated intracellular signaling events in human T lymphocytes.

CD5 is expressed on thymocytes, all mature T cells, and a subset of mature B cells, and probably contributes to T-cell-B-cell adhesion. We assessed whether CD5-crosslinking by mAb augments T-cell stimulation. Plate-bound anti-CD5 or anti-CD3 mAb alone had no effect on any of the assessed activation parameters of resting T cells. However, concomitant signaling through both CD5 and CD3 by plate-bound antibodies resulted in marked increases in T-cell surface CD69 expression and T-cell metabolism, as assessed by the T cell's ability to reduce 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxylmethoxyphenyl)-2-(4-sulphophenyl)-2H-tetrazolium (MTS) to formazen. In addition, simultaneous cross-linking of CD5 and CD3 caused a significant (p < 0.001) increase in phosphatidylinositol hydrolysis in resting T cells compared to stimulation with anti-CD3 mAb alone or anti-CD3 mAb plus anti-CD5 isotype control antibody. These results indicate that CD5 augments signaling through CD3 and consequently functions as a costimulatory molecule for resting T cells.