Increased T cell autoreactivity in the absence of CD40-CD40 ligand interactions: a role of CD40 in regulatory T cell development

Mutations in the CD40 ligand (CD40L) gene lead to X-linked immunodeficiency with hyper-IgM, which is often associated with autoimmune diseases. To determine the contribution of defective CD40-CD40L interactions to T cell autoreactivity, we reconstituted CD40-CD40L interactions by transferring T cells from CD40-deficient mice to syngenic athymic nude mice and assessed autoimmunity. T cells from CD40-deficient mice triggered autoimmune diseases accompanied with elevations of various autoantibodies, while those from wild-type mice did not. In CD40-deficient mice, the CD25(+) CD45RB(low) CD4(+) subpopulation which regulates T cell autoreactivity was markedly reduced. CD40-deficient APCs failed to induce T regulatory cells 1 producing high levels of an inhibitory cytokine, IL-10 in vitro. Furthermore, autoimmune development was inhibited when T cells from CD40-deficient mice were cotransferred with CD45RB(low) CD4(+) T cells from wild-type mice or with T regulatory cells 1 induced on CD40-expressing APCs. Collectively, our results indicate that CD40-CD40L interactions contribute to negative regulation of T cell autoreactivity and that defective interactions can lead to autoimmunity.     

Role of CD28/CD80-86 and CD40/CD154 costimulatory interactions in host defense to primary herpes simplex virus infection

Dependence of the primary antiviral immune response on costimulatory interactions between CD28/CD80-86 and between CD40/CD154 (CD40 ligand) has been correlated with the extent of viral replication in two models of systemic infection, lymphocytic choriomeningitis virus and vesicular stomatitis virus. To determine the role of these costimulatory interactions in the context of an acute cytolytic, but locally replicating viral infection, herpes simplex virus (HSV) infection was assessed in mice that had the CD28/CD80-86 or CD40/CD154 interactions disrupted either genetically or with blocking reagents (CTLA4Ig and MR1, respectively). CTLA4Ig treatment greatly reduced paralysis-free survival during primary acute HSV infection. This reflected an almost total ablation of the anti-HSV CD4(+) and CD8(+) T-cell responses due to anergy and reduced cell numbers, respectively. Disruption of CD40/CD154 interactions impaired survival, but the effect was less severe than that observed in CTLA4Ig-treated mice, with reductions observed in the CD4(+) T-cell but not CD8(+) T-cell responses. These two costimulatory pathways functioned in part independently, since disruption of both further impaired survival. The dependence on these costimulatory interactions for the control of primary HSV infection may represent a more widespread paradigm for nonsystemic viruses, which have restricted sites of replication and which employ immunoevasive measures.     

Differential requirement for the CD40-CD154 costimulatory pathway during Th cell priming by CD8 alpha+ and CD8 alpha- murine dendritic cell subsets

Dendritic cells (DCs) regulate the development of distinct Th populations and thereby provoke appropriate immune responses to various kinds of Ags. In the present work, we investigated the role CD40-CD154 interactions play during the process of Th cell priming by CD8 alpha(+) and CD8 alpha(-) murine DC subsets, which have been reported to differently regulate the Th response. Adoptive transfer of Ag-pulsed CD8 alpha(+) DCs induced a Th1 response and the production of IgG2a Abs, whereas transfer of CD8 alpha(-) DCs induced Th2 cells and IgE Abs in vivo. Induction of distinct Th populations by each DC subset was also confirmed in vitro. Although interruption of CD80/CD86-CD28 interactions inhibited Th cell priming by both DC subsets, disruption of CD40-CD154 interactions only inhibited the induction of the Th1 response by CD8 alpha(+) DCs in vivo. CD40-CD154 interactions were not required for the proliferation of Ag-specific naive Th cells stimulated by either DC subset, but were indispensable in the production of IL-12 from CD8 alpha(+) DCs and their induction of Th1 cells in vitro. Taken together, in our immunization model of Ag-pulsed DC transfer, CD40-CD154 interactions play an important role in the development of CD8 alpha(+) DC-driven Th1 responses but not CD8 alpha(-) DC-driven Th2 responses to protein Ags.     

Carcinoembryonic antigens (CD66) on epithelial cells and neutrophils are receptors for Opa proteins of pathogenic neisseriae

Opa protein-expressing pathogenic neisseriae interact with CD66a-transfected COS (African green monkey kidney) and CHO (Chinese hamster ovary) cells. CD66a (BGP) is a member of carcinoembryonic antigen (CEA, CD66) family. The interactions occur at the N-terminal domain of CD66a, a region that is highly conserved between members of the CEA subgroup of the CD66 family. In this study, we have investigated the roles of CD66 expressed on human epithelial cells and polymorphonuclear phagocytes (PMNs) in adhesion mediated via Opa proteins. Using human colonic (HT29) and lung (A549) epithelial cell lines known to express CD66 molecules, we show that these receptors are used by meningococci. A monoclonal antibody, YTH71.3, against the N-terminal domain of CD66, but not 3B10 directed against domains, A1/B1, inhibited meningococcal adhesion to host cells. When acapsulate bacteria expressing Opa proteins were used, large numbers of bacteria adhered to HT29 and A549 cells. In addition, both CD66a-transfected CHO cells and human epithelial cells were invaded by Opa-expressing meningococci, suggesting that epithelial cell invasion may occur via Opa–CD66 interactions. In previous studies we have shown that serogroup A strain C751 expresses three Opa proteins, all of which mediate non-opsonic interactions with neutrophils. We have examined the mechanisms of these interactions using antibodies and soluble chimeric receptors. The results indicate that the nature of their interactions with purified CD66a molecules and with CD66 on neutrophils is alike and that these interactions occur at the N-terminal domain of CD66. Thus, the Opa family of neisserial ligands may interact with several members of the CD66 family via their largely conserved N-terminal domains.     

Deleterious effect of HIV-1 plasma viremia on B cell costimulatory function

HIV infection leads to numerous immunologic defects, including impaired B cell function. An effective humoral response requires bidirectional interactions between B cells and CD4(+) T cells, critical of which are interactions between CD80/CD86 expressed on activated B cells and CD28 expressed on responder CD4(+) T cells. In the present study, we examined the effect of active HIV replication on B cell costimulatory function. Induction of CD80/CD86 on B cells following B cell receptor and CD40 triggering and responsiveness of CD4(+) T cells to activated B cells were investigated in a system where B cells of HIV-infected patients were compared concurrently to B cells of HIV-negative donors. In contrast to HIV-aviremic patients, B cells of HIV-viremic patients were ineffective at stimulating CD4(+) T cells, as measured by the induction of activation markers and proliferation. The importance of interactions of CD80/CD86 and CD28 in activating CD4(+) T cells was clear; the ablation of a normal response following the addition of neutralizing anti-CD86/CD80 Abs mirrored the response of CD4(+) T cells to B cells of HIV-viremic patients, while the addition of exogenous CD28 ligands partially restored the poor CD4(+) T cell response to the B cells of HIV-viremic patients. Ineffective B cell costimulatory function in HIV-viremic patients was associated with low induction of CD80/CD86 expression on B cells. Our findings further delineate the scope of defects associated with cognate B cell-CD4(+) T cell interactions in HIV infection and suggest that therapeutic interventions designed to enhance CD28-dependent costimulatory pathways may help restore immune functions.     

Direct and indirect interactions of the cytoplasmic region of CD244 (2B4) in mice and humans with FYN kinase

Engagement of the receptor CD244 (2B4) by its ligand CD48 has inhibitory and activating potential, and this differs depending on experimental systems in mouse and human. We show that, in both mouse and human upon engagement of its ligand CD48, CD244 can give a negative signal to natural killer cells, implying conservation of function between the two species. The signaling mechanisms used by CD244 in both human and mouse are conserved as shown by quantitative analyses of the direct molecular interactions of the SH2 domains of the adaptors SLAM-associated protein (SAP) and EAT-2 and of FYN kinase with CD244 together with the indirect interactions of the FYN SH2 domain with EAT-2. Functional experiments support the biochemical hierarchy of interactions and show that EAT-2 is not inhibitory per se. The data are consistent with a model in which the mechanism of signal transduction by CD244 is to regulate FYN kinase recruitment and/or activity and the outcome of CD48/CD244 interactions is determined by which other receptors are engaged.     

CD80 and CD86 Differentially Regulate Mechanical Interactions of T-Cells with Antigen-Presenting Dendritic Cells and B-Cells

Functional T-cell responses are initiated by physical interactions between T-cells and antigen-presenting cells (APCs), including dendritic cells (DCs) and B-cells. T-cells are activated more effectively by DCs than by B-cells, but little is known about the key molecular mechanisms that underpin the particular potency of DC in triggering T-cell responses. To better understand the influence of physical intercellular interactions on APC efficacy in activating T-cells, we used single cell force spectroscopy to characterize and compare the mechanical forces of interactions between DC:T-cells and B:T-cells. Following antigen stimulation, intercellular interactions of DC:T-cell conjugates were stronger than B:T-cell interactions. DCs induced higher levels of T-cell calcium mobilization and production of IL-2 and IFNγ than were elicited by B-cells, thus suggesting that tight intercellular contacts are important in providing mechanically stable environment to initiate T-cell activation. Blocking antibodies targeting surface co-stimulatory molecules CD80 or CD86 weakened intercellular interactions and dampen T-cell activation, highlighting the amplificatory roles of CD80/86 in regulating APC:T-cell interactions and T-cell functional activation. The variable strength of mechanical forces between DC:T-cells and B:T-cell interactions were not solely dependent on differential APC expression of CD80/86, since DCs were superior to B-cells in promoting strong interactions with T-cells even when CD80 and CD86 were inhibited. These data provide mechanical insights into the effects of co-stimulatory molecules in regulating APC:T-cell interactions.     

CD40-CD40 ligand interactions in oxidative stress, inflammation and vascular disease

CD40 ligand (CD40L) and its receptor CD40 participate in numerous inflammatory pathways that contribute to multiple pathophysiological processes. A role for CD40-CD40L interactions has been identified in atherosclerosis, and such interactions are known to destabilize atherosclerotic plaques by inducing the expression of cytokines, chemokines, growth factors, matrix metalloproteinases and pro-coagulant factors. The CD40-CD40L interaction has also been implicated in immune system disorders. Recent studies have suggested that CD40-CD40L interactions regulate oxidative stress and affect various signaling pathways in both the immunological and cardiovascular systems. Here, we discuss the emerging role of CD40-CD40L-mediated processes in oxidative stress, inflammatory pathways and vascular diseases. Understanding the roles and regulation of CD40-CD40L-mediated oxidative signaling in immune and non-immune cells could facilitate the development of therapeutics targeting diverse inflammatory diseases.     

Ecto-Phosphorylation of CD98 Regulates Cell-Cell Interactions

Ecto-phosphorylation plays an important role in many cellular functions. The transmembrane glycoprotein CD98 contains potential phosphorylation sites in its extracellular C-terminal tail. We hypothesized that extracellular signaling through ecto-protein kinases (ePKs) might lead to ecto-phosphorylation of CD98 and influence its multiple functions, including its role in cell-cell interactions. Our results show that recombinant CD98 was phosphorylated in vitro by ePKs from Jurkat cells and by the commercial casein kinase 2 (CK2). Alanine substitutions at serines-305/307/309 or serines-426/430 attenuated CK2-mediated CD98 phosphorylation, suggesting that these residues are the dominant phosphorylation sites for CK2. Furthermore, CD98 expressed in the basolateral membranes of intestinal epithelial Caco2-BBE cells was ecto-phosphorylated by Jurkat cell-derived ePKs and ecto-CK2 was involved in this process. Importantly, cell attachment studies showed that the ecto-phosphorylation of CD98 enhanced heterotypic cell-cell interactions and that the extracellular domain of CD98, which possesses the serine phosphorylation sites, was crucial for this effect. In addition, phosphorylation of recombinant CD98 increased its interactions with Jurkat and Caco2-BBE cells, and promoted cell attachment and spreading. In conclusion, here we demonstrated the ecto-phosphorylation of CD98 by ePKs and its functional importance in cell-cell interactions. Our findings reveal a novel mechanism involved in regulating the multiple functions of CD98 and raise CD98 as a promising target for therapeutic modulations of cell-cell interactions.     

Cell surface sialic acids do not affect primary CD22 interactions with CD45 and surface IgM nor the rate of constitutive CD22 endocytosis

CD22/Siglec-2 is a B cell-specific molecule modulating surface IgM (sIgM) signaling via cytosolic tyrosine-based motifs. CD22 recognizes alpha2-6-linked sialic acids (Sias) via an amino-terminal Ig-like domain. This Sia-binding site is typically masked by unknown sialylated ligands on the same cell surface, an interaction required for optimal signaling function. We studied the effect of cell surface Sias on specific interactions of CD22 with other molecules and on its turnover via endocytosis. A novel approach for simultaneous biotinylation and cross-linking showed that CD22 associates with CD45 and sIgM at much higher levels than reported in prior studies, possibly involving cell surface multimers of CD22. Sia removal or mutation of a CD22 arginine residue required for Sia recognition did not affect these associations even in human:mouse heterologous systems, indicating that they are primarily determined by evolutionarily conserved protein-protein interactions. Thus masking of the Sia-binding site of CD22 involves many cell surface sialoglycoproteins, without requiring specific ligand(s) and/or is mediated by secondary interactions with Sias on CD45 and sIgM. Abrogating Sia interactions also does not affect constitutive CD22 endocytosis. Sia removal does enhance the much faster rate of anti-CD22 antibody-triggered endocytosis, as well as killing by an anti-CD22 immunotoxin. In contrast to the unstimulated state, sIgM cross-linking inhibits both antibody-induced endocytosis and immunotoxin killing. Thus the signal- modulating activity of CD22 Sia recognition cannot be explained by mediation of primary interactions with specific molecules, nor by effects on constitutive endocytosis. The effects on antibody-mediated endocytosis could be of relevance to immunotoxin treatment of lymphomas.     

NK cells stimulate proliferation of T and NK cells through 2B4/CD48 interactions

Few studies have addressed the consequences of physical interactions between NK and T cells, as well as physical interactions among NK cells themselves. We show in this study that NK cells can enhance T cell activation and proliferation in response to CD3 cross-linking and specific Ag through interactions between 2B4 (CD244) on NK cells and CD48 on T cells. Furthermore, 2B4/CD48 interactions between NK cells also enhanced proliferation of NK cells in response to IL-2. Overall, these results suggest that NK cells augment the proliferation of neighboring T and NK cells through direct cell-cell contact. These results provide new insights into NK cell-mediated control of innate and adaptive immunity and demonstrate that receptor/ligand-specific cross talk between lymphocytes may occur in settings other than T-B cell or T-T cell interactions.     

CD6 recognizes the neural adhesion molecule BEN.

CD6 and its ligand activated leukocyte cell adhesion molecule (ALCAM, CD166) have been detected on various immune cells and in the brain. CD6-ligand interactions have been implicated in the regulation of T cell function. ALCAM shares the same extracellular domain organization and significant sequence homology with the chicken neural adhesion molecule BEN. Although ALCAM's CD6 binding site is only partially conserved in BEN, CD6 specifically binds BEN, albeit with approximately 10-fold lower avidity than ALCAM. Differences in binding avidity are not detected when ALCAM and BEN fusion proteins containing the full-length extracellular regions are tested. Homotypic interactions between full-length forms are likely to account for these observations. The identified cross-species interaction between CD6 and BEN suggests that CD6-ligand interactions are highly conserved.     

Murine B1 B cells require IL-5 for optimal T cell-dependent activation

T helper cell-driven activation of murine B cells has been shown to depend upon CD40-CD40 ligand (CD40L) interactions and a defined set of cytokines. These observations are primarily based on the use of conventional B cells obtained from the spleen. Therefore, it is presently unclear whether all mature B cell subsets found in the mouse have an equal dependence upon CD40-CD40L interactions and use the same T cell-derived cytokines. The present study tested the response of splenic follicular and marginal zone as well as peritoneal B2 and B1 B cells to Th cell stimulation. Splenic and peritoneal B cell subsets were sort purified based on CD23 expression, and cultured with rCD40L and cytokines or Th2 cells. The results demonstrate that follicular, marginal zone, and peritoneal B2 B cells require CD40-CD40L interactions and preferentially use IL-4 for optimal proliferation, differentiation, and isotype switching. In contrast, peritoneal B1 B cells use IL-5 in conjunction with CD40-CD40L interactions for maximal Th cell-dependent responses. Furthermore, B1 B cells are capable of proliferating, differentiating, and isotype switching in the absence of CD40-CD40L interactions. B1 B cells are able to respond to Th2 clones in the presence of anti-CD40L mAb as well as to Th2 clones derived from CD40L(-/-) mice. The CD40-CD40L-independent response of B1 B cells is attributable to the presence of both IL-4 and IL-5, and may explain the residual Ab response to T cell-dependent Ags in CD40L- or CD40-deficient mice, and in X-linked hyper-IgM (X-HIM) patients.     

Determination of carbohydrate structures N-linked to soluble CD154 and characterization of the interactions of CD40 with CD154 expressed in Pichia pastoris and Chinese hamster ovary cells.

CD40-CD154 (CD40 ligand) interactions are essential for the development of protective immunity. Previous studies have described the CD40 binding site as a shallow groove formed between two monomers of CD154. However, these studies have not examined the structure or biological function of the carbohydrate on CD154. Human CD154 contains a single N-linked glycosylation site at asparagine 240. We have characterized the interactions between CD40 and soluble (s) CD154 in which sCD154 contains different types of carbohydrates. Detailed carbohydrate analysis revealed high-mannose structures on sCD154 purified from Pichia pastoris, whereas CD154 purified from Chinese hamster ovary E1A contained heterogeneous populations of complex carbohydrates. sCD154 purified from either system was trimeric, it bound to CD40 with similar affinities of 10-30 nM, and it functionally induced CD69 and CD95 expression on primary B cells. Together, these results indicate that the presence of varied types of N-linked glycans on asparagine 240 of CD154 does not play a significant role in the CD40-CD154 interactions.     

Discovery of selective small-molecule CD80 inhibitors

Protein-protein interactions are widely found in biological systems controlling diverse cellular events. Because these interactions are implicated in many diseases such as autoimmunity and cancer, regulation of protein-protein interactions provides ideal targets for drug intervention. The CD80-CD28 costimulatory pathway plays a critical role in regulation of the immune response and thus constitutes an attractive target for therapeutic manipulation of autoimmune diseases. The objective of this study is to identify small compounds disrupting these pivotal protein-protein interactions. Compounds that specifically blocked binding of CD80 to CD28 were identified using a strategy involving a cell-based scintillation proximity assay as the initial step. Secondary screening (e.g., by analyzing the direct binding of these compounds to the target immobilized on a biosensor surface) revealed that these compounds are highly selective CD80 binders. Screening of structurally related derivatives led to the identification of the chemical features required for inhibition of the CD80-CD28 interaction. In addition, the optimization process led to a 10-fold increase in binding affinity of the CD80 inhibitors. Using this approach, the authors identify low-molecular-weight compounds that specifically and with high potency inhibit the interaction between CD80 and CD28. These compounds serve as promising starting points for further development of CD80 inhibitors as potential immunomodulatory drugs.     

Solution Characterization of the Extracellular Region of CD147 and Its Interaction with Its Enzyme Ligand Cyclophilin A

The CD147 receptor plays an integral role in numerous diseases by stimulating the expression of several protein families and serving as the receptor for extracellular cyclophilins; however, neither CD147 nor its interactions with its cyclophilin ligands have been well characterized in solution. CD147 is a unique protein in that it can function both at the cell membrane and after being released from cells where it continues to retain activity. Thus, the CD147 receptor functions through at least two mechanisms that include both cyclophilin-independent and cyclophilin-dependent modes of action. In regard to CD147 cyclophilin-independent activity, CD147 homophilic interactions are thought to underlie its activity. In regard to CD147 cyclophilin-dependent activity, cyclophilin/CD147 interactions may represent a novel means of signaling since cyclophilins are also peptidyl-prolyl isomerases. However, direct evidence of catalysis has not been shown within the cyclophilin/CD147 complex. In this report, we have characterized the solution behavior of the two most prevalent CD147 extracellular isoforms through biochemical methods that include gel-filtration and native gel analysis as well as directly through multiple NMR methods. All methods indicate that the extracellular immunoglobulin-like domains are monomeric in solution and, thus, suggest that CD147 homophilic interactions in vivo are mediated through other partners. Additionally, using multiple NMR techniques, we have identified and characterized the cyclophilin target site on CD147 and have shown for the first time that CD147 is also a substrate of its primary cyclophilin enzyme ligand, cyclophilin A.     

Single-molecule analysis of CD9 dynamics and partitioning reveals multiple modes of interaction in the tetraspanin web

Tetraspanins regulate cell migration, sperm–egg fusion, and viral infection. Through interactions with one another and other cell surface proteins, tetraspanins form a network of molecular interactions called the tetraspanin web. In this study, we use single-molecule fluorescence microscopy to dissect dynamics and partitioning of the tetraspanin CD9. We show that lateral mobility of CD9 in the plasma membrane is regulated by at least two modes of interaction that each exhibit specific dynamics. The majority of CD9 molecules display Brownian behavior but can be transiently confined to an interaction platform that is in permanent exchange with the rest of the membrane. These platforms, which are enriched in CD9 and its binding partners, are constant in shape and localization. Two CD9 molecules undergoing Brownian trajectories can also codiffuse, revealing extra platform interactions. CD9 mobility and partitioning are both dependent on its palmitoylation and plasma membrane cholesterol. Our data show the high dynamic of interactions in the tetraspanin web and further indicate that the tetraspanin web is distinct from raft microdomains.     

CD4+ T cell responses to CD40-deficient APCs: defects in proliferation and negative selection apply only with B cells as APCs.

During T-APC interactions in vivo, interfering with CD40-CD154 interactions leads to reduced T cell priming, defects in effector function, and, in some cases, T cell tolerance. As shown here, however, presentation of conventional peptide Ags by CD40-deficient spleen APC in vitro leads to normal CD4+ T cell proliferative responses. By contrast, responses to the same peptides presented by purified B cells were markedly reduced in the absence of CD40. Thus, the requirement for CD40-CD154 interactions appears to be strongly influenced by the type of APC involved. Analysis of responses to endogenous superantigens, which are known to be strongly dependent on B cells for presentation, indicated that CD4+ responses to strong Ags are less dependent on CD40 than are responses to weak Ags. Similar findings applied to negative selection in the thymus. Thus, deletion of potentially autoreactive cells depended on CD40 expression when B APC were involved, and this requirement was most pronounced when negative selection was directed to weak Ags.     

CD40/CD40 ligand interactions are critical for elicitation of autoimmune-mediated fibrosis in the lung

Pulmonary interstitial fibrosis (PIF), associated with persistent inflammation and increased collagen deposition in the interstitium, is often considered an autoimmune disease. Hapten immune PIF (HIPIF), a model for PIF, is elicited in the lung by a single intratracheal (i.t.) challenge in mice sensitized with hapten (2,4,6-trinitrobenzene sulfonic acid, TNBS). In this study, we characterized the role of CD40/CD40 ligand (CD40L) interactions in the elicitation of secondary cell-mediated immune responses that lead to development of fibrosis in the lung using an adoptive transfer model of HIPIF. The expression of CD40 was detected on bronchoalveolar lavage (BAL) cells 1-3 days after i.t. challenge with hapten in the HIPIF lung, but not lungs from the control mice. The CD40(bright) BAL cells morphologically resembled infiltrating monocytes. Furthermore, blocking CD40/CD40L interactions with blocking Ab decreased BAL production of Th1-mediators (IL-12 and TNF-alpha). Moreover, either blocking CD40/CD40L interactions with the Ab or using IL-12 knockout recipient mice prevented the increased collagen deposition (accumulation of hydroxyproline) in the lungs during HIPIF induction. We conclude that second signals (CD40/CD40L interactions) are required for elicitation of secondary immune responses that lead to PIF in vivo. The results support the notion that CD40/CD40L interactions are involved in the pathogenesis of an ongoing autoimmune disease.     

A ligand for CD5 is CD5

Recognition by scavenger receptor cysteine-rich domains on membrane proteins regulates innate and adaptive immune responses. Two receptors expressed primarily on T cells, CD5 and CD6, are linked genetically and are structurally similar, both containing three scavenger receptor cysteine-rich domains in their extracellular regions. A specific cell surface interaction for CD5 has been difficult to define at the molecular level because of the susceptibility of CD5 protein to denaturation. By using soluble CD5 purified at neutral pH to preserve biological activity, we show that CD5 mediates species-specific homophilic interactions. CD5 domain 1 only is involved in the interaction. CD5 mAbs that have functional effects in humans, rats, and mice block homophilic binding. Ag-specific responses by mouse T cells in vitro were increased when engagement of human CD5 domain 1 was inhibited by mutation or by IgG or Fab fragment from a CD5 mAb. This showed that homophilic binding results in productive engagement. Enhancement of polyclonal immune responses of rat lymph node cells by a Fab fragment from a CD5 mAb shown to block homophilic interactions provided evidence that the extracellular region of CD5 regulates inhibition in normal cells. These biochemical and in vitro functional assays provide evidence that the extracellular region of CD5 regulates immunity through species-specific homophilic interactions.