The Lymphocytic Scavenger Receptor CD5 Shows Therapeutic Potential in Mouse Models of Fungal Infection.

Invasive fungal diseases represent an unmet clinical need that could benefit from novel immunotherapeutic approaches. Host pattern recognition receptors (e.g., Toll-like receptors, C-type lectins, or scavenger receptors) that sense conserved fungal cell wall constituents may provide suitable immunotherapeutic antifungal agents. Thus, we explored the therapeutic potential of the lymphocyte class I scavenger receptor CD5, a nonredundant component of the antifungal host immune response that binds to fungal ß-glucans. Antifungal properties of the soluble ectodomain of human CD5 (shCD5) were assessed in vivo in experimental models of systemic fungal infection induced by pathogenic species (Candida albicans and Cryptococcus neoformans). In vitro mechanistic studies were performed by means of fungus-spleen cell cocultures. shCD5-induced survival of lethally infected mice was dose and time dependent and concomitant with reduced fungal load and increased leukocyte infiltration in the primary target organ. Additive effects were observed in vivo after shCD5 was combined with suboptimal doses of fluconazole. Ex vivo addition of shCD5 to fungus-spleen cell cocultures increased the release of proinflammatory cytokines involved in antifungal defense (tumor necrosis factor alpha and gamma interferon) and reduced the number of viable C. albicans organisms. The results prompt further exploration of the adjunctive therapeutic potential of shCD5 in severe invasive fungal diseases.

Protective Effects of Human and Mouse Soluble Scavenger-Like CD6 Lymphocyte Receptor in a Lethal Model of Polymicrobial Sepsis.

Sepsis still constitutes an unmet clinical need, which could benefit from novel adjunctive strategies to conventional antibiotic therapy. The soluble form of the scavenger-like human CD6 lymphocyte receptor (shCD6) binds to key pathogenic components from Gram-positive and -negative bacteria and shows time- and dose-dependent efficacy in mouse models of monobacterial sepsis. The objective of the present work was to demonstrate the effectiveness of infusing mouse and human sCD6 by different systemic routes, either alone or as adjunctive therapy to gold standard antibiotics, in a lethal model of polymicrobial sepsis. To this end, C57BL/6 mice undergoing high-grade septic shock induced by cecal ligation and puncture (CLP; ≥90% lethality) were infused via the intraperitoneal (i.p.) or intravenous (i.v.) route with shCD6 at different doses and time points, either alone or in combination with imipenem/cilastatin (I/C) at a dose of 33 mg/kg of body weight every 8 h. Significantly reduced mortality and proinflammatory cytokine levels were observed by i.p. infusion of a single shCD6 dose (1.25 mg/kg) 1 h pre- or post-CLP. When using the i.v. route, mice survival was significantly extended by starting shCD6 infusion at later time points post-CLP (up to 6 h after CLP). Significant adjunctive effects on mouse survival were observed by i.p. or i.v. infusion of shCD6 in combination with i.p. I/C post-CLP. Similar results were obtained in mice expressing high sustained levels (5 to 10 µg/ml) of mouse sCD6 in serum by means of transduction with hepatotropic adeno-associated virus (AAV). Taken together, the data support the conserved antibacterial effects of human and mouse sCD6 and their use as adjunctive therapy in experimental models of complex and severe polymicrobial sepsis.

Inherited functional variants of the lymphocyte receptor CD5 influence melanoma survival.

Despite the recent progress in treatment options, malignant melanoma remains a deadly disease. Besides therapy, inherited factors might modulate clinical outcome, explaining in part widely varying survival rates. T-cell effector function regulators on antitumor immune responses could also influence survival. CD5, a T-cell receptor inhibitory molecule, contributes to the modulation of antimelanoma immune responses as deduced from genetically modified mouse models. The CD5 SNPs rs2241002 (NM_014207.3:c.671C > T, p.Pro224Leu) and rs2229177 (NM_014207.3:c.1412C > T, p.Ala471Val) constitute an ancestral haplotype (Pro224-Ala471) that confers T-cell hyper-responsiveness and worsens clinical autoimmune outcome. The assessment of these SNPs on survival impact from two melanoma patient cohorts (Barcelona, N = 493 and Essen, N = 215) reveals that p.Ala471 correlates with a better outcome (OR= 0.57, 95% CI = 0.33-0.99, Adj. p = 0.043, in Barcelona OR = 0.63, 95% CI = 0.40-1.01, Adj. p = 0.051, in Essen). While, p.Leu224 was associated with increased melanoma-associated mortality in both cohorts (OR = 1.87, 95% CI = 1.07-3.24, Adj. p = 0.030 in Barcelona and OR = 1.84, 95% CI = 1.04-3.26, Adj. p = 0.037, in Essen). Furthermore survival analyses showed that the Pro224-Ala471 haplotype in homozygosis improved melanoma survival in the entire set of patients (HR = 0.27, 95% CI 0.11-0.67, Adj. p = 0.005). These findings highlight the relevance of genetic variability in immune-related genes for clinical outcome in melanoma.

Targeting of key pathogenic factors from gram-positive bacteria by the soluble ectodomain of the scavenger-like lymphocyte receptor CD6.

Gram-positive bacteria cause a broad spectrum of infection-related diseases in both immunocompetent and immunocompromised hosts, ranging from localized infections to severe systemic conditions such as septic and toxic shock syndromes. This situation has been aggravated by the recent emergence of multidrug-resistant strains, thus stressing the need for alternative therapeutic approaches. One such possibility would be modulating the host's immune response. Herein, the potential use of a soluble form of the scavenger-like human lymphocyte receptor CD6 (shCD6) belonging to an ancient family of innate immune receptors has been evaluated. shCD6 can bind to a broad spectrum of gram-positive bacteria thanks to the recognition of highly conserved cell wall components (lipoteichoic acid [LTA] and peptidoglycan [PGN]), which are essential for their viability and pathogenicity and are not amenable to antibiotic resistance. shCD6 has in vitro inhibitory effects on both bacterial growth and Toll-like receptor-mediated inflammatory response induced by LTA plus PGN. In vivo infusion of shCD6 improves survival on mouse models of septic shock by Staphylococcus aureus (either multidrug-resistant or -sensitive) or their endotoxins (LTA + PGN) or exotoxins (TSST-1). These results support the use of shCD6 and/or other scavenger-like immune receptors in the treatment of severe gram-positive-induced infectious conditions.

The membrane adaptor LAT is proteolytically cleaved following Fas engagement in a tyrosine phosphorylation-dependent fashion.

Engagement of the TCR (T-cell receptor) induces tyrosine phosphorylation of the LAT (linker for the activation of T-cells) adaptor, and thereby it recruits several cytosolic mediators for downstream signalling pathways. The Fas protein is essential for T-lymphocyte apoptosis, and following Fas engagement, many proteins are proteolytically cleaved, including several molecules that are important for the transduction of TCR intracellular signals. In the present study, we demonstrate that the adaptor LAT is also subject to a proteolytic cleavage in mature T-lymphocytes and thymocytes in response to Fas engagement, and also on TCR stimulation, and we identify three aspartic acid residues at which LAT is cleaved. Interestingly, these aspartic acid residues are located in proximity to several functionally important tyrosine residues of LAT, raising the possibility that their phosphorylation could modulate LAT cleavage. Consistent with that hypothesis, we show that induction of phosphorylation by pervanadate or H2O2 in Jurkat cells and thymocytes inhibits Fas-mediated cleavage of LAT. Moreover, we show that LAT proteolysis is also enhanced during anergy induction of primary human T-cells, suggesting that LAT cleavage may act as a regulator of TCR-mediated activation of T-cells and not only as a transducer of cell death promoting stimuli.

Evolutionary and functional evidence for positive selection at the human CD5 immune receptor gene.

CD5 is a lymphocyte surface coreceptor of still incompletely understood function. Currently available information indicates that CD5 participates not only in cell-to-cell immune interactions through still poorly defined endogenous ligands expressed on hemopoietic and nonhemopoietic cells but also in recognition of exogenous and highly conserved microbial structures such as fungal ß-glucans. Preceding single nucleotide polymorphism (SNP) data analysis provided evidence for a recent selective sweep in East Asia and suggested a nonsynonymous substitution at position 471 (A471V; rs2229177) of the cytoplasmatic region of the CD5 receptor as the most plausible target of selection. The present report further investigates the role of natural selection in the CD5 gene by a resequencing approach in 60 individuals representing populations from 3 different continents (20 Africans, 20 Europeans and 20 East Asians) and by functionally assaying the relevance of the A471V replacement on CD5 signaling. The high differentiation pattern found at the nonsynonymous A471V site together with the low diversity, most of the performed neutrality tests (Tajima's D, Fu and Li's F* and D*, and Fu's Fs) and the predominance of a major haplotype in East Asians strongly argue in favor of positive selection for the A471V site. Importantly, anti-CD5 monoclonal antibody cross-linking unveiled significant differences among A471V variants regarding the mitogen-activated protein kinase (MAPK) cascade activation on COS7 and on human peripheral blood mononuclear cells. Similar differences on MAPK activation and IL-8 cytokine release were also observed upon exposure of HEK293 cell transfectants expressing the A471V variants to Zymosan, a ß-glucan-rich fungal particle. Taken together, the results provide evidence for the hypothesis of an adaptive role of the A471V substitution to environmental challenges, most likely infectious pathogens, in East Asian populations.

CD6 deficiency impairs early immune response to bacterial sepsis.

CD6 is a lymphocyte-specific scavenger receptor expressed on adaptive (T) and innate (B1a, NK) immune cells, which is involved in both fine-tuning of lymphocyte activation/differentiation and recognition of bacterial-associated molecular patterns (i.e., lipopolysaccharide). However, evidence on CD6's role in the physiological response to bacterial infection was missing. Our results show that induction of monobacterial and polymicrobial sepsis in Cd6 -/- mice results in lower survival rates and increased bacterial loads and pro-inflammatory cytokine levels. Steady state analyses of Cd6 -/- mice show decreased levels of natural polyreactive antibodies, concomitant with decreased cell counts of spleen B1a and marginal zone B cells. Adoptive transfer of wild-type B cells and mouse serum, as well as a polyreactive monoclonal antibody improve Cd6 -/- mouse survival rates post-sepsis. These findings support a nonredundant role for CD6 in the early response against bacterial infection, through homeostatic expansion and functionality of innate-related immune cells.

Conserved Bacterial-Binding Peptides of the Scavenger-Like Human Lymphocyte Receptor CD6 Protect From Mouse Experimental Sepsis.

Sepsis is an unmet clinical need constituting one of the most important causes of death worldwide, a fact aggravated by the appearance of multidrug resistant strains due to indiscriminate use of antibiotics. Host innate immune receptors involved in pathogen-associated molecular patterns (PAMPs) recognition represent a source of broad-spectrum therapies alternative or adjunctive to antibiotics. Among the few members of the ancient and highly conserved scavenger receptor cysteine-rich superfamily (SRCR-SF) sharing bacterial-binding properties there is CD6, a lymphocyte-specific surface receptor. Here, we analyze the bacterial-binding properties of three conserved short peptides (11-mer) mapping at extracellular SRCR domains of human CD6 (CD6.PD1, GTVEVRLEASW; CD6.PD2 GRVEMLEHGEW; and CD6.PD3, GQVEVHFRGVW). All peptides show high binding affinity for PAMPs from Gram-negative (lipopolysaccharide; Kd from 3.5 to 3,000 nM) and Gram-positive (lipoteichoic acid; Kd from 36 to 680 nM) bacteria. The CD6.PD3 peptide possesses broad bacterial-agglutination properties and improved survival of mice undergoing polymicrobial sepsis in a dose- and time-dependent manner. Accordingly, CD6.PD3 triggers a decrease in serum levels of both pro-inflammatory cytokines and bacterial load. Interestingly, CD6.PD3 shows additive survival effects on septic mice when combined with Imipenem/Cilastatin. These results illustrate the therapeutic potential of peptides retaining the bacterial-binding properties of native CD6.

Genetic and experimental evidence for the involvement of the CD6 lymphocyte receptor in psoriasis.

Psoriasis is a chronic inflammatory skin disease with a strong genetic background and is triggered by environmental factors. Available evidence supports CD6, a lymphocyte surface receptor mostly expressed by T cells, as a putative target in autoimmunity. Accordingly, a humanized anti-CD6 antibody has been assayed for the treatment of certain autoimmune disorders, including psoriasis. Here, we present novel evidence in mice and humans for a direct involvement of CD6 in psoriasis pathophysiology. First, an attenuated form of imiquimod-induced psoriasis-like skin inflammation was demonstrated in CD6-deficient mice, as deduced from lower epidermal thickness and local reduced production of pro-inflammatory cytokines, namely, interleukin-17A. Thus, isolated CD4+CD62L+ T cells from CD6-deficient mice displayed decreased in vitro T-helper type 17 polarization. Second, a statistically significant association between CD6 single-nucleotide polymorphisms (rs17824933, rs11230563 and rs12360861) and more severe forms of psoriasis was demonstrated in a cohort of 304 patients at three public hospitals from the metropolitan area of Barcelona. Taken together, these results provide new supportive evidence of the contribution of the CD6 lymphocyte receptor in psoriasis at both experimental and clinical levels.

Corrigendum: Commentary: The Scavenger Receptor SSc5D Physically Interacts with Bacteria through the SRCR-Containing N-Terminal Domain.

[This corrects the article on p. 366 in vol. 8, PMID: 28396672.].

Oral Administration of Pentoxifylline Reduces Endometriosis-Like Lesions in a Nude Mouse Model.

INTRODUCTION: Recent reports consider endometriosis to be an immunological disorder, thus suggesting potential efficacy of immunomodulators for its treatment. The aim of this study was to assess the effects of oral administration of pentoxifylline on endometriosis-like lesions in a heterologous mice model. STUDY DESIGN: Human endometrial tissue obtained from women (n = 5) undergoing surgery for benign conditions was implanted in nude female mice (n = 30). The animals were distributed into 3 experimental groups receiving: saline 0.1 mL/d (control, group 1); pentoxifylline 100 mg/kg/d (group 2), and pentoxifylline 200 mg/kg/d (group 3). After 28 days, the number of implants and the total volume of surgically extracted tissue were recorded. Immunohistochemical analysis was performed to assess the area of endometriosis and vascularization of endometriosis-like lesions. Cytokine levels in peritoneal fluid samples were measured. RESULTS: Macroscopic quantification showed a trend to dose-dependent reduction in the number of the endometriosis-like lesions after 28 days. The volume was significantly reduced in group 3 versus group 2 and controls (399.10 ± 120.68 mm3 vs 276.75 ± 94.30 mm3 and 145.33 ± 38.20 mm3, respectively; P = .04). Similarly, the mean area of endometriosis was significantly lower in group 3 (0.12 ± 0.08 mm2) versus group 2 (1.35 ± 0.43 mm2) and control (2.84 ± 0.60 mm2; P = .001). Vascularization and cytokine levels were also reduced posttreatment. CONCLUSION: Our results suggest that the oral administration of pentoxifylline may be an alternative to current therapies for endometriosis. Nonetheless, further studies are required.

Relevance of CD6-Mediated Interactions in the Regulation of Peripheral T-Cell Responses and Tolerance.

The CD6 lymphocyte receptor has been involved in the pathophysiology of different autoimmune disorders and is now considered a feasible target for their treatment. In vitro data show the relevance of CD6 in the stabilization of adhesive contacts between T-cell and antigen-presenting cells, and the modulation of T-cell receptor signals. However, the in vivo consequences of such a function are yet undisclosed due to the lack of suitable genetically modified animal models. Here, the in vitro and in vivo challenge of CD6-deficient (CD6-/-) cells with allogeneic cells was used as an approach to explore the role of CD6 in immune responses under relative physiological stimulatory conditions. Mixed lymphocyte reaction (MLR) assays showed lower proliferative responses of splenocytes from CD6-/- mice together with higher induction of regulatory T cells (Treg, CD4+CD25+FoxP3+) with low suppressive activity on T and B-cell proliferation. In line with these results, CD6-/- mice undergoing a lupus-like disorder induced by chronic graft-versus-host disease (cGvHD) showed higher serum titers of anti-double-stranded DNA and nucleosome autoantibodies. This occurred together with reduced splenomegaly, which was associated with lower in vivo bromodesoxyuridine incorporation of spleen cells and with increased percentages of spleen follicular B cells (B2, CD21+CD23hi) and Treg cells. Interestingly, functional analysis of in vivo-generated CD6-/- Treg cells exhibited defective suppressive activity. In conclusion, the data from MLR and cGvHD-induced lupus-like models in CD6-/- mice illustrate the relevance of CD6 in T (and B) cell proliferative responses and, even more importantly, Treg induction and suppressive function in the in vivo maintenance of peripheral tolerance.

Human CD6 Down-Modulation following T-Cell Activation Compromises Lymphocyte Survival and Proliferative Responses.

Available evidence indicates that the CD6 lymphocyte surface receptor is involved in T-cell developmental and activation processes, by facilitating cell-to-cell adhesive contacts with antigen-presenting cells and likely modulating T-cell receptor (TCR) signaling. Here, we show that in vitro activation of human T cells under different TCR-ligation conditions leads to surface downregulation of CD6 expression. This phenomenon was (i) concomitant to increased levels of soluble CD6 (sCD6) in culture supernatants, (ii) partially reverted by protease inhibitors, (iii) not associated to CD6 mRNA down-regulation, and (iv) reversible by stimulus removal. CD6 down-modulation inversely correlated with the upregulation of CD25 in both FoxP3- (Tact) and FoxP3+ (Treg) T-cell subsets. Furthermore, ex vivo analysis of peripheral CD4+ and CD8+ T cells with activated (CD25+) or effector memory (effector memory T cell, CD45RA-CCR7-) phenotype present lower CD6 levels than their naïve or central memory (central memory T cell, CD45RA-CCR7+) counterparts. CD6lo/- T cells resulting from in vitro T-cell activation show higher apoptosis and lower proliferation levels than CD6hi T cells, supporting the relevance of CD6 in the induction of proper T-cell proliferative responses and resistance to apoptosis. Accordingly, CD6 transfectants also showed higher viability when exposed to TCR-independent apoptosis-inducing conditions in comparison with untransfected cells. Taken together, these results provide insight into the origin of sCD6 and the previously reported circulating CD6-negative T-cell subset in humans, as well as into the functional consequences of CD6 down-modulation on ongoing T-cell responses, which includes sensitization to apoptotic events and attenuation of T-cell proliferative responses.

Activation of T lymphocytes and the role of the adapter LAT.

The adapter molecule LAT (Linker for the Activation of T cells) is a membrane protein that becomes phosphorylated on conserved tyrosine residues upon TCR/CD3 complex engagement in T lymphocytes. Tyrosine phosphorylation of this adapter recruits to the membrane many signaling proteins through the interaction with the phosphotyrosine binding domains of these proteins, allowing the activation of several intracellular signaling pathways. Initial studies performed in T cell lines suggested that the adapter LAT acts primarily as a platform for the distribution of activation signals coming from the TCR/CD3 complex, and the phenotype of LAT deficient mice, in which T cell development is arrested at an early stage, supported this "activatory" function. However, the analysis of several knock-in mice strains in which some tyrosine residues have been mutated, has revealed the development of lymphoproliferative disorders caused by polyclonal T lymphocytes producing high titers of T helper-type 2 (T(H)2) cytokines. Very recently, it has been demonstrated that raft localization of LAT is altered in anergic T lymphocytes. Therefore, LAT show unexpected regulatory functions in T cell development and homeostasis.

Pattern Recognition by CD6: A Scavenger-Like Lymphocyte Receptor.

CD6, one of the first antigens to be identified on T cells, is a membrane glycoprotein that physically associates with the antigen receptor complex. Because of this, its main function seems to involve the modulation of TCR-mediated signaling pathways. However, growing evidence indicates that this ancient and conserved scavenger-like receptor may also play a role as pattern recognition receptor (PRR), similar to other members of the scavenger receptor cysteine rich superfamily (SRCR-SF). Here, we discuss the functional interactions of CD6 with microbe- and damage-associated signals and the potential use of soluble forms of CD6 in the therapeutic treatment of bacterial infections, in particular multi-drug resistant bacterial strains. Importantly, microbe recognition by CD6 may also have functional consequences on T cell activation and differentiation, which remain to be explored.

CD6 modulates thymocyte selection and peripheral T cell homeostasis.

The CD6 glycoprotein is a lymphocyte surface receptor putatively involved in T cell development and activation. CD6 facilitates adhesion between T cells and antigen-presenting cells through its interaction with CD166/ALCAM (activated leukocyte cell adhesion molecule), and physically associates with the T cell receptor (TCR) at the center of the immunological synapse. However, its precise role during thymocyte development and peripheral T cell immune responses remains to be defined. Here, we analyze the in vivo consequences of CD6 deficiency. CD6(-/-) thymi showed a reduction in both CD4(+) and CD8(+) single-positive subsets, and double-positive thymocytes exhibited increased Ca(2+) mobilization to TCR cross-linking in vitro. Bone marrow chimera experiments revealed a T cell-autonomous selective disadvantage of CD6(-/-) T cells during development. The analysis of TCR-transgenic mice (OT-I and Marilyn) confirmed that abnormal T cell selection events occur in the absence of CD6. CD6(-/-) mice displayed increased frequencies of antigen-experienced peripheral T cells generated under certain levels of TCR signal strength or co-stimulation, such as effector/memory (CD4(+)TEM and CD8(+)TCM) and regulatory (T reg) T cells. The suppressive activity of CD6(-/-) T reg cells was diminished, and CD6(-/-) mice presented an exacerbated autoimmune response to collagen. Collectively, these data indicate that CD6 modulates the threshold for thymocyte selection and the generation and/or function of several peripheral T cell subpopulations, including T reg cells.

Pattern of soluble CD5 and CD6 lymphocyte receptors in critically ill patients with septic syndromes.

PURPOSE: Soluble forms of CD5 and CD6 lymphocyte surface receptors (sCD5 and sCD6) are molecules that seem to prevent experimental sepsis when exogenously administered. The aim of this study was to assess sCD5 and sCD6 levels in patients with septic syndromes. MATERIALS AND METHODS: The study population consisted of 218 patients admitted to the medical intensive care unit (ICU) presenting either septic syndromes or noninfectious systemic inflammatory response syndrome at admission or within the first 48 hours. The sCD5 and sCD6 levels were analyzed by sandwich enzyme-linked immunosorbent assay. RESULTS: Almost 50% of the patients had undetectable levels of sCD5 or sCD6, with no differences in clinical or biological variables with detectable patients. There was a correlation between the delta Sequential Organ Failure Assessment score and both sCD6 and sCD5 levels in all groups. Patients with sCD5 or sCD6 levels greater than 1500 ng/mL presented a higher in-ICU mortality (P < .05). Logistic regression analysis showed that increased sCD6 levels were associated with an increased risk of in-ICU mortality. CONCLUSIONS: Levels of sCD5 and sCD6 in critically ill patients with systemic inflammatory response syndrome present a high variation and an elevated proportion of undetectability. Levels of sCD6 are associated with an increased risk of mortality in these patients.

The macrophage soluble receptor AIM/Api6/CD5L displays a broad pathogen recognition spectrum and is involved in early response to microbial aggression.

Apoptosis inhibitor of macrophages (AIMs), a homologue of human Spα, is a mouse soluble member of the scavenger receptor cysteine-rich superfamily (SRCR-SF). This family integrates a group of proteins expressed by innate and adaptive immune cells for which no unifying function has yet been described. Pleiotropic functions have been ascribed to AIM, from viability support in lymphocytes during thymic selection to lipid metabolism and anti-inflammatory effects in autoimmune pathologies. In the present report, the pathogen binding properties of AIM have been explored. By using a recombinant form of AIM (rAIM) expressed in mammalian cells, it is shown that this protein is able to bind and aggregate Gram-positive and Gram-negative bacteria, as well as pathogenic and saprophytic fungal species. Importantly, endogenous AIM from mouse serum also binds to microorganisms and secretion of AIM was rapidly induced in mouse spleen macrophages following exposure to conserved microbial cell wall components. Cytokine release induced by well-known bacterial and fungal Toll-like receptor (TLR) ligands on mouse splenocytes was also inhibited in the presence of rAIM. Furthermore, mouse models of pathogen-associated molecular patterns (PAMPs)-induced septic shock of bacterial and fungal origin showed that serum AIM levels changed in a time-dependent manner. Altogether, these data suggest that AIM plays a general homeostatic role by supporting innate humoral defense during pathogen aggression.