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

The recently published article by Bessa Pereira et al. reports that the human SSc5D receptor physically interacts with some bacterial species (1), thus basically confirming previous available information on its mouse homolog (S5D-SRCRB) (2). The interspecies conservation of such a basic innate immune function (bacterial binding) has been noticed for other members of the scavenger receptor cysteine-rich superfamily (SRCR-SF) (e.g., human Spα and its mouse homolog AIM/Api6/CD5L) (3, 4). This advocates for its functional physiological relevance in innate defense of body surfaces as it has been proposed for the urogenital tract (5). A substantive part of the work by Bessa Pereira et al. is also devoted to explore putative qualitative and/or quantitative differences on the bacterial-binding properties of SSc5D with other human SRCR-SF proteins, namely, CD5, Spα, and CD6 by using conventional protein-bacteria binding assays and surface plasmon resonance-based assays. They were chosen based on previously reported information showing that Spα (4) and CD6 (6-8) but not CD5 (9) exhibit broad bacterial-binding properties. While the authors confirmed the work on Spα and CD5, they were unable to replicate that on CD6. Exclusively based on a single experimental evidence, the authors cast doubt on the well-documented bacterial-binding properties of CD6 (6-8). These properties were unveiled by using a recombinant soluble form of human CD6 (rshCD6) encompassing from D25 to M400 and, indistinctly, produced in different mammalian cell expression systems (NSO, HEK293-EBNA, and CHO cells). Further confirmation was obtained by demonstrating similar properties displayed by a natural soluble CD6 form isolated from human serum, as well as by Jurkat cell transfectants expressing a membrane-bound full-length form of CD6 (6). Accordingly, it was later reported that rshCD6 infusion significantly reduces mouse mortality following septic shock induced by intraperitoneal monobacterial infection of Gram-positive (S. aureus) or Gram-negative (A. baumannii) origin (7). More recently, new evidence shows that not only rshCD6 but also adenovirally expressed mouse sCD6 have protective survival effects on polymicrobial septic shock induced by cecal ligation and puncture (8), the gold standard model for experimental sepsis

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.Ministerio de Economía y Competitividad (España) SAF2013-46151-R PCIN-2015-070Instituto de Salud Carlos III RD12/0015/0018European Development Regional Fund RD12/0015/0018Fundació La Marató TV3 201319-30-3

Conserved bacterial-binding peptides of the scavenger-like 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

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

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

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

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

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 (T-reg, 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(+)CD23(hi)) and T-reg cells. Interestingly, functional analysis of in vivo-generated CD6(-/-)T(reg) 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

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