The rational development of CD5-targeting biepitopic CARs with fully human heavy-chain-only antigen recognition domains.

T cell malignancies are a group of hematologic cancers with high recurrence and mortality rates. CD5 is highly expressed in ∼85% of T cell malignancies, although normal expression of CD5 is restricted to thymocytes, T cells, and B1 cells. However, CD5 expression on chimeric antigen receptor (CAR)-T cells leads to CAR-T cell fratricide. Once this limitation is overcome, CD5-targeting CAR-T therapy could be an attractive strategy to treat T cell malignancies. Here, we report the selection of novel CD5-targeting fully human heavy-chain variable (FHVH) domains for the development of a biepitopic CAR, termed FHVH3/VH1, containing FHVH1 and FHVH3, which were validated to bind different epitopes of the CD5 antigen. To prevent fratricide in CD5 CAR-T cells, we optimized the manufacturing procedures of a CRISPR-Cas9-based CD5 knockout (CD5KO) and lentiviral transduction of anti-CD5 CAR. In vitro and in vivo functional comparisons demonstrated that biepitopic CD5KO FHVH3/VH1 CAR-T cells exhibited enhanced and longer lasting efficacy; produced moderate levels of cytokine secretion; showed similar specificity profiles as either FHVH1, FHVH3, or the clinically tested H65; and is therefore suitable for further development.

Trans-signaling of interleukin-6 (IL-6) is mediated by the soluble IL-6 receptor, but not by soluble CD5.

IL-6 exerts its pleiotropic activities on its target cells via the IL-6 alpha-receptor (IL-6R), which is expressed on a limited number of cell types. IL-6 can further signal via soluble forms of its receptor (sIL-6R), a process that has been termed trans-signaling. Recently, CD5 was described as an alternative alpha-receptor for IL-6 on B cells leading to the phosphorylation of the transcription factor STAT3 via the signal-transducing ß-receptor gp130 in a Jak2-dependent manner. In this study, we sought to investigate whether IL-6 was also able to signal via soluble CD5 (sCD5) analogous to IL-6 trans-signaling. We show that IL-6 indeed binds to sCD5, but that this does not lead to the activation of signal transduction or cell proliferation. Furthermore, sCD5 did also not interfere with IL-6 classic signaling, suggesting that the affinity between the two proteins was too weak to provoke a biological effect. Thus, trans-signaling of IL-6 can only occur via sIL-6R, but not sCD5.

Functional requirement of tyrosine residue 429 within CD5 cytoplasmic domain for regulation of T cell activation and survival.

CD5 has been mainly described as a negative regulator of TCR and BCR signaling and recent evidence has shown an important role for this receptor in delivering pro-survival signals. However, the molecular mechanisms underlying these processes remain unresolved. TCR crosslinking leads to phosphorylation of three tyrosine residues within the cytoplasmic tail of CD5 (Y429, Y441 and Y463) leading to the recruitment of signaling molecules like PI3K, c-Cbl and RasGAP; nevertheless, the role of these residues in T cell survival has not yet been assessed. In this study, we show that alanine-scanning mutagenesis of such tyrosine residues, either singly or in combination, leads to an increased thymocyte cell death with or without α-CD3 stimulation. Remarkably, the T-cell death observed with each individual tyrosine mutant was Caspase 3-independent. Furthermore, Y429 mutation resulted in a hyper-phosphorylation of ERK suggesting that this tyrosine residue regulates cell survival through down modulation of TCR signaling. Mutation of Y441 or Y463 did not induce hyper-responsiveness to TCR activation, indicating that they promoted T-cell survival by a TCR signal-independent pathway. Our results show that three tyrosine-based domains within CD5 cytoplasmic tail promote T-cell survival through non-overlapping mechanisms. This study also reveals that Y429 domain of CD5, previously described as a "pseudo ITAM", is functionally an ITIM domain in T cells.

CD5 as a Target for Immune-Based Therapies.

CD5 was one of the first surface receptors described for mouse and human T lymphocytes. Since then, it has been found to be highly expressed by regulatory T cells and a subpopulation of regulatory B cells, to be physically associated with the T- and B-cell antigen receptors, to negatively modulate TCR- and BCR-mediated signals, and to bind certain pathogen-associated molecular patterns. These findings position CD5 as an attractive target for developing immunotherapies aimed at either boosting or dampening ongoing immune responses. Here the available data on the function of CD5 and its involvement in the regulation of immune responses in health and disease are reviewed, as well as the evidence for and future challenges in developing therapeutic strategies aimed at targeting CD5 for autoimmune diseases, cancer, and infections.

The carboxy-terminal region of CD5 is required for c-CBL mediated TCR signaling downmodulation in thymocytes.

CD5 functions as a negative regulator of TCR signaling during thymocyte development, however, the molecular mechanisms involved in this process remain elusive. A key molecule involved in the down modulation of TCR signaling is c-Cbl, an ubiquitin ligase that physically associates with CD5. Crosslinking of TCR in thymocytes leads to ubiquitylation and lysosomal/proteasomal degradation of TCR downstream signaling effectors and CD5 itself. The present report shows that co-engagement of CD3 with CD5 enhanced c-Cbl phosphorylation, which was not affected by the deletion of the pseudo-ITAM domain of CD5, the putative binding site for c-Cbl. However, amino acids present in the carboxy-terminal region of CD5, were necessary for this effect, indicating that ITAM-independent sites were involved in the interaction of c-Cbl with CD5. The carboxy-terminal region of CD5 was also required for Vav degradation, a well-known target for c-Cbl-dependent ubiquitylation. These results support the notion that the distal cytoplasmic domain of CD5, including Y463, plays a relevant role in the downmodulation of TCR signals in thymocytes via c-Cbl.

A new pathway of CD5 glycoprotein-mediated T cell inhibition dependent on inhibitory phosphorylation of Fyn kinase.

Triggering of the T cell receptor initiates a signaling cascade resulting in the activation of the T cell. These signals are integrated alongside those resulting from the triggering of other receptors whose function is to modulate the overall response. CD5 is an immunotyrosine-based inhibition motif-bearing receptor that antagonizes the overt T cell receptor activation response by recruiting inhibitory intracellular mediators such as SHP-1, RasGAP, or Cbl. We now propose that the inhibitory effects of CD5 are also mediated by a parallel pathway that functions at the level of inhibition of Fyn, a kinase generally associated with T cell receptor-mediated activation. After CD5 ligation, phosphorylation of the negative regulatory tyrosine (Tyr(531)) of Fyn increases, and this correlates with a substantial reduction in the kinase activity of Fyn and a profound inhibition of ZAP-70 activation. The effect requires the last 23 amino acids of the cytoplasmic domain of the receptor, strongly implying the involvement of a new CD5-interacting signaling or adaptor protein. Furthermore, we show that upon CD5 ligation there is a profound shift in its distribution from the bulk fluid phase to the lipid raft environment, where it associates with Fyn, Lck, and PAG. We suggest that the relocation of CD5, which we also show is capable of forming homodimers, to the proximity of raft-resident molecules enables CD5 to inhibit membrane proximal signaling by controlling the phosphorylation and activity of Fyn, possibly by interfering with the disassembly of C-terminal Src kinase (Csk)-PAG-Fyn complexes during T cell activation.

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.

Characterisation of the CD5 cDNA sequence from the tammar wallaby (Macropus eugenii).

CD5 has previously been identified in marsupial tissues using anti-human CD5. However, despite the known cross-reactivity of the antibody in marsupial tissues, the cDNA sequence has not previously been characterised in any marsupial. This study has identified the CD5 gene in the opossum genome database and has characterised the CD5 cDNA sequence from the tammar wallaby. Both marsupial CD5 sequences have a high level of sequence identity to known eutherian CD5 sequences, are cysteine-rich and have identical structural motifs to their eutherian homologs. CD5 transcripts were strongly expressed in adult tammar wallaby spleen, mammary node and blood, and expressed at a lower level in liver, kidney and heart tissues. Characterisation of CD5 in marsupials allowed a comparison to the epitope sequence of anti-human CD5 and showed a high level of sequence identity.

The CD5 ectodomain interacts with conserved fungal cell wall components and protects from zymosan-induced septic shock-like syndrome.

The CD5 lymphocyte surface receptor is a group B member of the ancient and highly conserved scavenger receptor cysteine-rich superfamily. CD5 is expressed on mature T and B1a cells, where it is known to modulate lymphocyte activation and/or differentiation processes. Recently, the interaction of a few group B SRCR members (CD6, Spalpha, and DMBT1) with conserved microbial structures has been reported. Protein binding assays presented herein indicate that the CD5 ectodomain binds to and aggregates fungal cells (Schizosaccharomyces pombe, Candida albicans, and Cryptococcus neoformans) but not to Gram-negative (Escherichia coli) or Gram-positive (Staphylococcus aureus) bacteria. Accordingly, the CD5 ectodomain binds to zymosan but not to purified bacterial cell wall constituents (LPS, lipotheicoic acid, or peptidoglycan), and such binding is specifically competed by beta-glucan but not by mannan. The K(d) of the rshCD5/(1-->3)-beta-d-glucan phosphate interaction is 3.7 +/- 0.2 nM as calculated from tryptophan fluorescence data analysis of free and bound rshCD5. Moreover, zymosan binds to membrane-bound CD5, and this induces both MAPK activation and cytokine release. In vivo validation of the fungal binding properties of the CD5 ectodomain is deduced from its protective effect in a mouse model of zymosan-induced septic shock-like syndrome. In conclusion, the present results indicate that the CD5 lymphocyte receptor may sense the presence of conserved fungal components [namely, (1-->3)-beta-d-glucans] and support the therapeutic potential of soluble CD5 forms in fungal sepsis.

Three-dimensional solution structure and conformational plasticity of the N-terminal scavenger receptor cysteine-rich domain of human CD5.

The lymphocyte receptor CD5 influences cell activation by modifying the strength of the intracellular response initiated by antigen engagement. Regulation through CD5 involves the interaction of one or more of its three scavenger receptor cysteine-rich domains present in the extracellular region. Here, we present the 3D solution structure of a non-glycosylated double mutant of the N-terminal domain of human CD5 expressed in Escherichia coli (eCD5d1m), which has enhanced solubility compared to the non-glycosylated wild-type (eCD5d1). In common with a glycosylated form expressed in Pichia pastoris, the [(15)N,(1)H]-correlation spectra of both eCD5d1 and eCD5d1m exhibit non-uniform temperature-dependent signal intensities, indicating extensive conformational fluctuations on the micro-millisecond timescale. Although approximately one half of the signals expected for the domain are absent at 298 K, essentially complete resonance assignments and a solution structure could be obtained at 318 K. Because of the sparse nature of the experimental restraint data and the potentially important contribution of conformational exchange to the nuclear Overhauser effect peak intensity, we applied inferential structure determination to calculate the eCD5d1m structure. The inferential structure determination ensemble has similar features to that obtained by traditional simulated annealing methods, but displays superior definition and structural quality. The eCD5d1m structure is similar to other members of the scavenger receptor cysteine-rich superfamily, but the position of the lone alpha helix differs due to interactions with the unique N-terminal region of the domain. The availability of an experimentally tractable form of CD5d1, together with its 3D structure, provides new tools for further investigation of its function within intact CD5.

Antibody-mediated cell labeling of peripheral T cells with micron-sized iron oxide particles (MPIOs) allows single cell detection by MRI.

Labeling cells with iron oxide is a useful tool for MRI based cellular imaging. Here it is demonstrated that peripheral rat T cells can be labeled in whole blood, in vitro, with streptavidin-coated micron-sized iron oxide particles (MPIOs), achieving iron concentrations as high as 60 pg iron per cell. This is 30 times the amount of labeling reported with ultrasmall particles of iron oxide (USPIOs). Labeling was mediated by use of a biotinylated anti-CD5 antibody, which is specific for peripheral T cells. Such labeling allowed the in vitro detection of single lymphocytes by MRI, using conditions well suited for in vivo animal work. Electron microscopic analysis demonstrated that MPIOs remained largely extracellular after labeling, with some evidence of intracellular uptake. Cell viability and early and late cytokine release studies showed no significant differences between labeled and unlabeled cells. Therefore, the use of MPIOs for achieving high iron concentrations for cellular MRI is potentially an effective new modality for non-invasive imaging of lymphocytes.

Expression, purification and crystallization of human CD5 domain III, a nano-scale crystallization example.

The human lymphocyte receptor CD5, a key regulator of immune responses, is involved in the modulation of antigen specific receptor-mediated T cell activation and differentiation signals. CD5 is a membrane glycoprotein which belongs to the group B scavenger receptor cysteine-rich (SRCR) superfamily for which no structural information is available. The most conserved membrane-proximal SRCR domain of CD5 (domain III) has been expressed in HEK-EBNA-293 cells. Although the yield of the purified protein was at the level of micrograms, well diffracting crystals have been obtained. The crystals belong to a tetragonal space group P4(1)22 or P4(3)22. They contain two molecules per asymmetric unit and diffracted to 2.5A resolution using synchrotron radiation. The strategy shown here to produce, isolate and crystallize CD5 domain III can be used for other mammalian proteins difficult to produce for structural or other biophysical studies.

Crystal structure of the third extracellular domain of CD5 reveals the fold of a group B scavenger cysteine-rich receptor domain.

Scavenger receptor cysteine-rich (SRCR) domains are ancient protein modules widely found among cell surface and secreted proteins of the innate and adaptive immune system, where they mediate ligand binding. We have solved the crystal structure at 2.2 A of resolution of the SRCR CD5 domain III, a human lymphocyte receptor involved in the modulation of antigen specific receptor-mediated T cell activation and differentiation signals. The first structure of a member of a group B SRCR domain reveals the fold of this ancient protein module into a central core formed by two antiparallel beta-sheets and one alpha-helix, illustrating the conserved core at the protein level of genes coding for group A and B members of the SRCR superfamily. The novel SRCR group B structure permits the interpretation of site-directed mutagenesis data on the binding of activated leukocyte cell adhesion molecule (ALCAM/CD166) binding to CD6, a closely related lymphocyte receptor homologue to CD5.

CD6 regulates T-cell responses through activation-dependent recruitment of the positive regulator SLP-76.

Deciphering the role of lymphocyte membrane proteins depends on dissecting the role of a protein in the steady state and on engagement with its ligand. We show that expression of CD6 in T cells limits their responsiveness but that engagement by the physiological ligand CD166 gives costimulation. This costimulatory effect of CD6 is mediated through phosphorylation-dependent binding of a specific tyrosine residue, 662Y, in its cytoplasmic region to the adaptor SLP-76. A direct interaction between SLP-76 and CD6 was shown by binding both to a phosphorylated peptide (equilibrium dissociation constant [K(D)] = 0.5 muM at 37 degrees C) and, using a novel approach, to native phosphorylated CD6. Evidence that CD6 and SLP-76 interact in cells was obtained in coprecipitation experiments with normal human T cells. Analysis of human CD6 mutants in a murine T-cell hybridoma model showed that both costimulation by CD6 and the interaction between CD6 and SLP-76 were dependent on 662Y. The results have implications for regulation by CD6 and the related T-cell surface protein, CD5.

A new epitope on swine CD5 molecule detected by monoclonal antibody 5F12/9.

This paper describes the production and characterization of a monoclonal antibody (MAb), 5F12/9, that recognizes a new epitope on porcine CD5. Conformation of its CD5 specificity was obtained by means of sequential immunoprecipitation and Western blot experiments in combination with anti-CD5 MAb 1H6/8, whereas cross-blocking experiments with both MAbs showed that they reacted with different epitopes.

Isolation of Ly-1+/CD5+ B cells by cell sorting.

Ly-1/CD5 is a 68-kDa glycoprotein that was originally thought to mark the helper subset of T cells. Later it was shown to be present on all T cells and, more recently, on a subset of B cells. Although its function remains the subject of speculation, CD5 expression serves as a useful marker for a functionally distinct population of B cells that has attracted a considerable amount of interest from investigators of both murine and human immune systems. Of critical importance in much of this work is the isolation of pure populations of CD5+ B cells along with appropriate control populations. A flow cytometry technique is presented in this unit, which results in the selection and isolation of two populations of cells from a complex mixture based on physical properties (e.g., size and internal granularity) and correlated expression of several surface molecules.

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.

CD2 physically associates with CD5 in rat T lymphocytes with the involvement of both extracellular and intracellular domains.

T lymphocytes can be activated and induced to proliferate through stimulation of the CD2 glycoprotein with functional combinations of CD2 antibodies. However, this mechanism of signal transduction via CD2 is still not fully understood. We have investigated which molecules on the T cell surface preferentially associate in Cis with CD2 and may regulate its signaling properties. Though a quantification method we found that CD5 represents the antigen capable of co-precipitating a larger proportion of CD2. Using co-capping assays and immunoprecipitations from cell lysates, we show that an association between CD2 and CD5 can be found in rat thymocytes, T lymphocytes and in a thymoma cell line. Possibly, this interaction is a direct one, since CD2 and CD5 transiently expressed in Cos7 cells co-precipitate each other. Furthermore, using CD2 chimeric proteins containing different domains of CD2, expressed in Cos7 cells as well as in stably transfected Jurkat cells, we show that the interaction between CD2 and CD5 is held at both the intra- and extracellular levels, but does not involve the transmembrane domain. The fact that both the extracellular and the cytoplasmic domains of CD2 interact with CD5 suggests a specific and tight association between the two molecules, possibly relevant for the fine-tuning of signal transduction in T lymphocytes.

Origins and functions of B-1 cells with notes on the role of CD5.

Whether B-1a (CD5+) cells are a distinct lineage derived from committed fetal/neonatal precursors or arise from follicular B-2 cells in response to BCR ligation and other, unknown signals remains controversial. Recent evidence indicates that B-1a cells can derive from adult precursors expressing an appropriate specificity when the (self-) antigen is present. Antibody specificity determines whether a B cell expressing immunoglobulin transgenes has a B-2, B-1a or marginal zone (MZ) phenotype. MZ cells share many phenotypic characteristics of B-1 cells and, like them, appear to develop in response to T independent type 2 antigens. Because fetal-derived B cell progenitors fail to express terminal deoxynucleotidyl transferase (TdT) and for other reasons, they are likely to express a repertoire that allows selection into the B-1a population. As it is selected by self-antigen, the B-1 repertoire tends to be autoreactive. This potentially dangerous repertoire is also useful, as B-1 cells are essential for resistance to several pathogens and they play an important role in mucosal immunity. The CD5 molecule can function as a negative regulator of BCR signaling that may help prevent inappropriate activation of autoreactive B-1a cells.

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.