cd5 as a target for immune-based therapies

85

Critical Reviews™ in Immunology, 35(2): 85-115 (2015)

1040-8401/15/$35.00 © 2015 by Begell House, Inc.

CD5 as a Target for Immune-Based TherapiesMarta Consuegra-Fernández,a Fernando Aranda,a Inês Simões,a Marc Orta,a Adelaida Sarukhan,b & Francisco Lozanoa,c,d,*a Group of Immune Receptors of the Innate and Adaptive System, Institut d’Investigacions Biomèdiques August Pi i Sunyer (IDIBAPS), Barcelona, Spain; bInstitut National de la Santé et de la Recherche Médicale (INSERM), Paris, France; cDepartament de Biologia Cellular, Immunologia i Neurociències, Facultat de Medicina, Universitat de Barcelona, Barcelona, Spain; dServei d’Immunologia, Hospital Clínic de Barcelona, Barcelona, Spain

*Address correspondence to: Francisco Lozano, MD, PhD, Centre Esther Koplowitz, Roselló 149-153, 08036 Barcelona, Spain; Tel.: +34 932 275 400 ext. 4217; [email protected]

ABSTRACT: 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.

KEY WORDS: scavenger receptor, CD5, autoimmunity, cancer, infection, immunotherapy

ABBREVIATIONS: AICD: activation-induced cell death; BCR: B-cell receptor; Breg: regulatory/supressor B cell; B-1a: CD5+ B-cell subset; DC: dendritic cell; EAE: experimental autoimmune encephalomyelitis; EAN: experimental autoimmune/allergic neuritis; GvHD: graft-versus-host disease; HCV: hepatitis C virus; Ig: immunoglobulin; IgAN: human IgA nephropathy; IL: in-terleukin; mAb: monoclonal antibody; MS: multiple sclerosis; RA: rheumatoid arthritis; RTA: ricin toxin A chain; sCD5: soluble CD5; SLE: systemic lupus erythematosus; SRCR: scavenger receptor cysteine-rich; TCR: T-cell receptor; TLR: Toll-like recep-tor; Treg: regulatory T cell; UC: ulcerative colitis

I. INTRODUCTION

The mouse and human CD5 glycoprotein was one of the first lymphocyte surface receptors reported, thanks to the technology of monoclonal antibodies (mAbs),1–3 and its official name was assigned during the first International Workshop and Conference on Human Leukocyte Differentiation Antigens (HLDA) held in Paris in 1982. However, CD5 was discovered much earlier (late 1960s) with the descrip-tion of the so-called mouse cell membrane allogenic determinants (CMADs) (Thy-1, Lyt-1, Lyt-2, Lyt-3, Lyt-4, Lyt-5, Lyt-6, etc.), which were used to distin-guish different functional subpopulations of T and B lymphocytes.4 In that context, the mouse Lyt-1 alloantigen (also known as Ly-A or Ly-1 and later known as CD5) emerged as one of the first clearly defined lymphocyte-specific antigenic systems.5 This

was achieved because of the generation of mouse alloantisera against irradiated murine leukemic T cells.6 The characterization of these alloantisera allowed the initial identification of CD5 expression in T cells from thymus, spleen, and lymph nodes. Later, immunization of mice with lymphocytes from different congenic strains led to the identification of diverse strain-specific polymorphisms of the Lyt-1 molecule (Lyt-1.1 and Lyt-1.2 allotypes).4

The advent of hybridoma technology in the late seventies and early eighties soon rendered the generation of the first rat (e.g., 53-7.3) and mouse (e.g., OKT1) mAbs against mouse and human CD5 molecules.3,7 These reagents led to the discovery that CD5 was not only a pan-T-cell marker but also a molecular signature for a small but functionally relevant subset of B cells (B-1a or CD5+ B cells) involved in the production of natural polyreactive

Critical Reviews™ in Immunology

Consuegra-Fernández et al.86

antibodies (e.g., isoagglutinins). Importantly, recent work demonstrated that this B-cell subset includes regulatory/suppressor B cells (Bregs), which are also known as B10 cells because they are a main source of interleukin (IL)-10. A myriad of early stud-ies with anti-CD5 mAbs came to the misleading conclusion that CD5 was a costimulatory molecule for T-cell activation and proliferation in mouse and human.8–10 This notion was later contradicted by data from CD5-deficient mice,11,12 and today there is broad consensus that CD5 is indeed a negative modulator of the activation signals delivered by the antigen-specific receptor expressed by T and B-1a cells. All of this, together with its physical association with such a relevant receptor, positions CD5 as an attractive target for developing novel immunotherapies aimed at potentiating or inhibit-ing ongoing immune responses. Aside from its role in lymphocyte activation, recent evidence indicates that CD5 may also work as a receptor for microbial surface structures present at least in fungi and viruses, which adds a further level of complexity to CD5’s function. The present report reviews basic informa-tion on CD5 physiology relevant to past, current, and future therapeutic interventions aimed at the immunomodulation of, among others, infectious, autoimmune, and neoplastic disorders.

II. CD5 STRUCTURE, EXPRESSION, AND FUNCTION

A. Structure

The CD5 receptor is a type-I membrane glycoprotein of 67 kDa structurally belonging to the ancient and highly conserved scavenger receptor cysteine-rich (SRCR) superfamily.13,14 This family is composed of more than 30 different membrane-bound and/or soluble receptors having in common the presence of at least one SRCR extracellular domain—a protein module of 90–110 amino acids (aas) homologous to the cysteine-rich C-terminal domain first reported for the macrophage type-I class-A scavenger receptor (SR-AI).15 Although there is no unifying biological function for the members of the SRCR superfam-

ily, some of them have been reported to play a role in homo- or heterotypical protein interactions and pathogen-associated molecular pattern (PAMP) rec-ognition16–22 as well as in the development and modu-lation of innate and adaptive immune responses.23

The CD5 gene maps to human chromosome 11 (chromosome 9 in mice) and is telomeric to another highly related SRCR superfamily member, CD6.24,25 CD5 codes for a surface receptor with an extracellular region composed exclusively of three SRCR domains in tandem (D1, D2, and D3), a transmembrane region, and an intracytoplasmatic region adapted to intracel-lular signaling transduction (Fig. 1).13 The N-terminal domains D1 and D2 are heavily N-glycosilated and spaced by a Pro-Ser-Thr–rich polypeptide with O-linked glycosylations (D3 is not glycosylated at all). Although not all of the extracellular region has been crystallized, three-dimensional structural information is already available on the D1 and D3 domains,26,27 showing no large discrepancies with other SRCR domains.28 The 94-aa-long intracytoplasmic region lacks intrinsic catalytic activity, but includes several 11 Ser, 4 Thr, and 4 Tyr residues suitable for phos-phorylation by protein Ser/Thr kinases [protein kinase C, Ca2+/calmodulin-dependent kinase II, and casein kinase 2 (CK2)]29–31 and protein Tyr kinases (Lck, Fyn).29,32 These residues are also suitable for further generation of downstream signaling events, including activation of phosphatidylcholine-specific phospholipase C,33 acidic sphingomyelinase, protein kinase C-ζ, mitogen-activated protein kinase kinase, and c-Jun NH2-terminal kinase.34

Interestingly, the cytoplasmic tail of CD5 pres-ents a pseudo-ITAM (immunoreceptor tyrosine–based activation motif ) that serves as a docking site for signaling mediators such as Lck, Fyn, Ras GTPase-activating protein (Ras-GAP), and c-Cbl29; it also presents a membrane-proximal pseudo-ITIM (immunoreceptor tyrosine–based inhibitory motif ) that interacts with SHP-1 (Src homology region 2 domain-containing phosphatase-1).35 Both of these motifs have been linked to the activating or inhibi-tory signaling properties attributed to CD5.36 Also, Y429 from the ITAM-like motif has been reported to be critical for binding the m2 subunit of the AP2 adaptor complex to CD5 and for the internaliza-

Volume 35, Number 2, 2015

CD5-Based Immunotherapies 87

tion of the latter via clathrin-coated pits following monovalent or bivalent ligation.37

In addition to membrane-bound CD5, a circulat-ing soluble form (sCD5) is detected at a picomolar range in sera from healthy individuals as the result of proteolytic cleavage following lymphocyte activation.38 Furthermore, patients with lymphocyte hyperactivation

diseases, such as Sjögren’s syndrome, have increased levels of sCD5 in serum.39 The function of this soluble form is still unknown, but one hypothesis is that it may compete with membrane-bound CD5 for interaction with its ligand(s) and thus interfere with homeostatic lymphocyte responses. Recent data from transgenic mice expressing human sCD5 bear this out.40

FIG. 1: Schematic of the extracellular and intracellular interactions mediated by CD5. AP2: adaptor protein com-plex 2; BCR: B cell receptor; CaMK-IV: calcium/calmodulin-dependent protein kinase IV; c-Cbl: casitas b-lineage lymphoma–related protein; CK2: casein kinase 2; Fyn: proto-oncogene tyrosine-protein kinase Fyn; VHFR: framework region of immunoglobulin heavy chain variable domain; gp40-80: mouse glycoprotein 40-80 kDa; gp150: human glycoprotein 150 kDa; gp200: bovine glycoprotein 200 kDa; HCV: hepatitis C virus; IgVH: immunoglobulin vari-able heavy region; ITAM-like: immune receptor tyrosine-based activation-like motif; ITIM-like: immune receptor tyrosine-based inhibitory-like motif; Lck: lymphocyte specific tyrosine-protein kinase; PKC/G/A: protein kinase C, G, or A; Ras-GAP: GTP-binding proteins of the Ras superfamily; SHP-1: Src homology region 2 domain–containing phosphatase-1; Syk: spleen tyrosine kinase; TCR: T-cell receptor; ZAP70: zeta-chain-associated protein kinase 70.



B. Expression

CD5 is a highly specific lymphoid marker expressed by all mature T cells (both αβ and γδ) but only a small subset of mature B cells (B-1a cells). In thymocytes, it is expressed from the early stages (double negative, or DN) of thymocyte development in a T-cell recep-tor (TCR-) –regulated manner, and consequently its expression increases during the transition from DN to double positive (DP) and later from DP to single positive (SP).41 Moreover, CD5 expression is directly proportional to the avidity of the TCR–ligand interaction.41,42 In general, the expression levels found in peripheral T cells are higher than in B cells43 and are the highest in CD4+TCRαβ cells, followed by CD8+TCRαβ cells and then CD4–CD8–TCRγδ cells.44 Regarding the last, although most spleen γδT cells are CD5+, gut intraepithelial γδT cells are mostly CD5–.45 Interestingly, CD5 expression is found increased in T- and B-lymphocyte subsets with regulatory functions (both Tregs and Bregs); thus,46,47 CD5 overexpression (CD5hi) can be used as a marker of these cell populations. Similarly, anergic T and B cells resulting from chronic stimulation by endogenous or exogenous antigens are characterized by CD5 overexpression.48,49

B-1a cells (CD5+CD23–IgMhiIgDlow) are related to the production of natural polyreactive autoanti-bodies50 and are expanded in several autoimmune disorders such as rheumatoid arthritis,51 systemic lupus erythematosus,52 insulin-dependent diabetes mellitus),53 Graves’ thyroiditis,54 or Sjögren’s syn-drome,55 and chronic hepatitis C virus infection, as well as in some B-cell lymphoproliferative disorders (B-cell chronic lymphocytic leukemia, Mantle zone lymphoma, and hairy cell leukemia).56,57 CD5 is considered an activation marker for conventional mouse B2 cells (CD5–CD23+IgMlowIgDhi) because its expression can be induced following IgM cross-linking.58 This is not the case for human B cells.59 B-cell receptor (BCR-) –induced up-regulation of CD5 was shown to be dependent on nuclear factor of activated T cell (NFAT) binding to an enhancer located 2 kb upstream of the mouse cd5 gene.60 The transcriptional control of CD5 gene expression is, however, only partially known. It mainly involves

regulatory elements mapping to the 5′-flanking region of exon 1 and binding to, among others, members of the Ets family of transcription factors, which are also relevant for other lymphoid-specific genes (e.g., CD4, TCRα, TCRβ).61

C. Ligands

The ultimate nature of the endogenous CD5 ligand(s) is still a controversial matter. Several reported CD5 ligands have been questioned because of the inability of other groups to validate them. They include coun-ter-receptors expressed by B cells such as CD72,62 the framework region of IgVH,63 and gp20064; by B and T cells such as gp40-8065,66 and CD5 itself66; and by lymphoid, myelomonocytic, and epithelial cells such as gp150.68 Only some of these interac-tions have been mapped, and they involve the most amino-terminal domains—that is, D1 for CD5, D2 for the framework region of IgVH, and D1-D2 for gp150 (Fig. 1).

Recent evidence shows that exogenous ligands can also interact with the extracellular region of CD5. This is the case for β-D-glucans, a conserved constitutive component of fungal cell walls.21 The affinity of this interaction has been calculated (KD, 3.7 ± 0.2 nM) and is in the same range as that reported for dectin-1, which is the main known receptor of β-D-glucans in mammalian myeloid cells.69 All three CD5 individual extracellular domains can recognize β-D-glucans, and the interaction seems to be specific given that little or no binding to other conserved fungal (mannan) and bacterial (lipopolysaccharide, peptidoglycan) cell wall constituents is observed. Furthermore, exposure of CD5 transfectants to zymosan (a β-D-glucan–rich fungal particle) was shown to induce mitogen-activated protein kinase (MAPK) pathway activation and IL-8 secretion, which is dependent on the integrity of the CD5 cytoplasmic region. Interestingly, CD5-deficient mice are hypersensitive to zymosan-induced septic shock–like syndrome, as deduced from higher clini-cal scores and serum cytokine levels (Fenutria et.al., unpublished observations).

It was recently reported that CD5 is a relevant receptor for hepatitis C virus (HCV) entry into both



primary and leukemic human T cells.70 This explains the lymphoid reservoir reported for HCV and opens the possibility that CD5 may act as a receptor for other related and unrelated viruses. This is not surpris-ing because viral recognition is a property reported for other related members of the SRCR superfamily such as salivary agglutinin/DMBT1/gp340 [gp120 HIV-1 (human immunodeficiency virus type 1), influenza A virus)] and CD163 [porcine reproductive and respiratory syndrome virus (PRRSV), simian hemorrhagic fever virus (SHFV)].71–74

D. Polymorphism

The existence of a biallelic polymorphism for the extracellular region of CD5 has been reported in some mammalian species,4,75 with most of the nucleotide changes mapping to D1 in mouse.76 A number of nonsynonymous variants can be found for human CD5, with only two of them present at relatively high frequencies in Caucasians: rs2241002, involving a Pro to Leu substitution at position 224 in the extracellular D2 domain; and rs2229177, involving Ala to Val substitution at position 471 just C-terminal to the cytoplasmic ITAM-like motif (Fig. 1).77 Interestingly, the CD5 SNP rs2229177 was identified among the gene signatures for recent positive selection in human populations, particularly East Asians and Native Americans.78 Although the mechanism by which such a substitution would have been favored by selection remains unclear, evidence for its functional relevance comes from observed differences regarding CD5’s signaling capabilities and lymphocyte proliferative responses following zymosan exposure or CD3 cross-linking among Ala- and Val-expressing individuals.77,79

E. Function

1. Regulation of T- and B-Cell Activation

CD5 is physically associated with the antigen-specific receptor of T and B cells (TCR and BCR) and becomes part of the macromolecular complex in the central area of the mature immune synapse of T cells.80–82 This feature positions it as a potential

modulator of lymphocyte activation or cell death signals following antigen-specific binding.

CD5 has long been considered an accessory molecule involved in either positive or negative regulation of lymphocyte activation depending on cell type and maturation stage.36 An early myriad of in vitro studies using anti-CD5 mAbs, either alone or in combination with anti-CD3 or anti-CD28 mAbs, led to the notion that CD5 is a costimula-tory molecule for lymphocyte activation.8–10,83,84 This notion was later challenged by in vivo experiments with CD5-deficient mice that showed that CD5 acts as a negative modulator of activation and differentia-tion signals mediated by antigen-specific receptors in both thymocytes and B-1a cells.11,12 Accordingly, thymocytes from CD5-deficient mice are hyper-responsive to TCR/CD3 cross-linking, showing enhanced proliferative responses, intracellular Ca2+ mobilization, and tyrosine phosphorylation of PLC-γ1, TCRζ, LAT, and Vav.11 In turn, membrane IgM cross-linking of B-1a cells from the same mice also induces increased Ca2+ mobilization, resistance to apoptosis, and enhanced cell proliferation.12

The studies cited suggested that CD5 very likely influences the selection and maturation process of T and B-1a cells. Indeed, studies with TCR-transgenic mice showed that CD5 negatively modulates the development of CD4+ T cells.85 Further work also showed that developmental expression of CD5 paral-lels TCR signal intensity and avidity and that it plays a key role in the fine-tuning of TCR signaling.41,86 Thus, CD5-mediated signal inhibition during thy-mocyte selection increases when the avidity of the positively selecting TCR–ligand interaction (and TCR signal strength) is relatively high. Conversely, the requirement for CD5-mediated signal inhibition decreases when the avidity of the positively selecting TCR–ligand interaction (and TCR signal strength) is relatively low.86

Although CD5 expression correlates with self-reactivity and is highly expressed by regulatory and anergic T cells, two recent studies showed that CD5hi T cells are better positioned to respond to foreign antigens because of their enhanced antipathogen reactivity.87,88 A similar situation applies to B cells, in which membrane CD5 negatively regulates Ig



receptor signaling and inhibits autoimmune B-cell responses.49 Interestingly, a new role for IL-10–pro-ducing CD5+ B cells was recently proposed in which this cell subset inhibits IgE-mediated mast cell activa-tion and anaphylaxis in mice in an IL-10–dependent and cell-to-cell contact manner.89

2. Regulation of Survival

CD5 has been shown to function as a regulator of T- and B-1a-cell survival.90 In T cells, the mecha-nism by which CD5 mediates survival is not fully understood. Nevertheless, the PI3K/Akt and CK2 signaling pathways seem to be involved in CD5-mediated lymphocyte survival by inducing expres-sion of prosurvival molecules (Bcl-2 and Bcl-xl) or by inhibiting proteins associated with apoptotic pathways (caspases and Bid).91–93 In B cells, CD5 protects against apoptosis by stimulating IL-10 production upon BCR stimulation while reduc-ing the BCR-induced Ca2+ mobilization response that would eventually send death signals.94 Thus, membrane-bound CD5 might support survival and eventually maintain homeostasis of B-1a cells that are susceptible to activation-induced cell death (AICD).90

3. Tolerance Induction

CD5 plays a role in the establishment of immuno-logical tolerance.90 First, the CK2-binding region in the intracellular tail of CD5 was shown to be neces-sary for inducing antigen-specific unresponsiveness in a myelin oligodendrocyte glycoprotein (MOG-) –induced experimental allergic encephalomyelitis (EAE) model.95 The researchers proposed that CD5 contributes to tolerance by setting the threshold of T-cell responsiveness. In addition, the absence of CD5 results in increased Treg numbers and sup-pressive activity by mechanisms that remain to be defined.46,96

A recently published report on the role of CD5 in peripheral T-cell homeostasis proposed that CD5 promotes the development of extrathymic Treg cells by blocking mTOR-dependent signals induced by effector-differentiating cytokines to inhibit Treg

induction.97 Accordingly, CD5 would be determinant in T-cell fate by facilitating Treg cell conversion from T cells not only when recognizing high-affinity self-pMHC in the thymus but also when tolerizing antigens presented by DCs in the periphery.97

4. Pathogen Recognition

The fact that CD5 can recognize pathogen-associated structures present in fungi and viruses opens the question of its putative physiological relevance. The expression of pattern recognition receptors by cells outside the innate immune system, such as T and B lymphocytes, is well documented. This is the case with CD6, a closely related member of the SRCR superfamily that recognizes bacterial pathogen–asso-ciated molecular patterns (PAMPs) (LPS, LTA, and PGN).19,22 It is also the case with different Toll-like receptors (TLRs).98,99 Increasing evidence demon-strates that the binding of microbial components to Pattern Recognition Receptors (PRRs) expressed by lymphocytes modulates the latter’s functions.100–104 Although the ultimate consequences of such binding remain unknown, PAMP binding to surface CD5 (or CD6) engages intracellular signaling cascades.21,77 Based on these results, together with the role of CD5 in negative regulation of lymphocyte antigen receptor signaling, it has been hypothesized that sensing of the external milieu for microbial products by scavenger-like lymphocyte receptors (namely CD5) is beneficial because (1) it prevents autoimmune responses triggered by infectious agents (microbial binding to CD5 would provide inhibitory signals to low-affinity T/B-cell clones that could otherwise become autoreactive during infection) and (2) it optimizes antimicrobial immune responses (CD5 inhibitory signals would increase the threshold for activation of microbe-specific lympho-cytes, allowing expansion of high- but not low-affinity T/B clones).105 Another, nonexcluding, hypothesis is that CD5 may be expressed by nonlymphoid cell types and thus exert its pathogen-related effects through activation of innate immune responses. In line with this is the observation of CD5 expression on certain endothelial cells,106 macrophages, and peripheral blood and vaginal dendritic cell (DC) subsets.107–110



III. INVOLVEMENT OF CD5 IN CLINICAL AND EXPERIMENTAL DISORDERS AND ITS POTENTIAL IMMUNOTHERAPEUTIC USE

In view of the key role played by CD5 in modulat-ing T- and B1-a-cell responses, this report reviews the available information on the involvement of CD5hi cells in different immune disorders as well as the different therapeutic approaches based on CD5 targeting, all of which are summarized in Table 1.

A. Autoimmunity

Autoimmunity is defined as a pathological immune-based disorder caused by the peripheral activation of T and B cells against self-tissue components, in the absence or presence of infection, tissue injury, or other discernible origin. Even though failures in the mechanisms maintaining self-tolerance increase the risk of autoimmune diseases, disease progression is regulated by control mechanisms at so-called immunological tolerance checkpoints.111 In this context, both Treg and Breg subpopulations play an important role in the establishment and maintenance of peripheral tolerance. Intriguingly, both regulatory subpopulations express high levels of CD5.

In fact, early reports in mice showed that CD4+CD5hi cells have a suppressive effect on lym-phocyte activation.112 Later on, these cells were pheno-typically better characterized as CD4+CD25hiFoxP3+ and designated as Tregs.113 It was reported that the absence of Tregs increased autoimmunity whereas their adoptive transfer reversed it.114 In humans, Treg defects contribute to autoimmunity because genetic defects in FoxP3, for instance, can lead to autoimmunity (immunodysregulation polyendocrinopathy enteropa-thy X-linked syndrome, or IPEX).115 However, despite numerous studies there is very little evidence linking defects in Treg or Breg numbers to autoimmune disorders, except for systemic lupus erythematosus, in humans.116 It is thus likely that functional changes rather than alterations in Treg and/or Breg numbers are involved in the breakdown of self-tolerance and the progression to autoimmune disease.

1. Systemic Lupus Erythematosus

Systemic lupus erythematosus (SLE) is a chronic autoimmune disorder whose main features are B-cell hyperactivity, spontaneous lymphocyte proliferation, and production of pathogenic antibodies to self-antigens. B-cell abnormalities in SLE also include excess cytokine production, self-antigen presentation to T cells, and modulation of the functions of other immune cells.117 SLE is a very complex disorder that involves genetic and environmental factors, hormones, and immunological mechanisms. The precise origin of the disease is not known, although increased expression of genes regulated by type-I interferon (IFN), termed IFN signature, has been reported in patients with SLE as well as in those with other autoimmune disorders.118

SLE was traditionally treated with immunosup-pressive drugs, such as corticosteroids, mycophenolate, azathioprine, cyclophosphamide, and cyclosporine A, which eventually inhibit T- and B-cell prolifera-tion but cause several adverse side effects.119 More recent strategies focus on selective B-cell depletion, targeting specific B-cell surface receptors and cyto-kines. As an example, rituximab, a cytotoxic anti-CD20 mAb, causes selective short-term depletion of mature B cells and gives encouraging results in murine models, but a review found it to be unsuc-cessful in recent controlled trials.120 Also, the use of ocrelizumab (anti-CD20), epratuzumab (anti-CD22), and certain drugs targeting soluble mediators such as BlyS (B-lymphocyte stimulator) and APRIL (a proliferation-inducing ligand) to inhibit B-cell growth and function has yielded promising results, but needs further long-term clinical studies to discard potential side effects.121–123

Other novel therapies specifically targeting IL-6 and IFN-α are on the horizon, as well as the block-ing of costimulatory interactions between immune cells (CD40–CD40 ligand), interference with cell-signaling pathways, and even the targeting of anti-dsDNA mAbs.124,125

The contribution of CD5+ circulating B cells to autoantibody secretion in SLE has been much discussed and is still an open question, with some studies reporting increased numbers and others



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not.55,126–128 Some researchers propose that increased levels of CD5+ B cells lead to augmented levels of autoantibodies by undergoing uncontrolled Ig VDJ recombination as a result of their expression of recom-bination activation genes (RAG 1 and 2).129,130 Other researchers support the idea that CD5+ cells exhibit an immunosuppressive function involving IL-21–mediated secretion of Granzyme B.131 Renaudineau and colleagues provided evidence for the pivotal role of CD5 in maintaining anergy in autoreactive B cells.132 The level of CD5 on the B-cell surface is determined by the relative level of two alternative CD5 isoforms: CD5-E1A, which is expressed on the membrane, and CD5-E1B, which is retained in the cytoplasm.133 B cells from SLE patients were shown to have decreased DNA methylation, leading to an increased expression of the truncated CD5-E1B form, which in turn results in reduced levels of membrane CD5.132 Because CD5 negatively regulates BCR-mediated signaling, the researchers proposed that lower levels of membrane CD5 can promote the activation and expansion of autoreac-tive B cells in SLE patients. Moreover, the observed DNA hypomethylation was further enhanced by IL-6, suggesting therapeutic benefits of anti-IL-6 treatment.132 Accordingly, the use of tocilizumab, a blocking anti-IL-6 receptor, was shown to improve abnormal B- and T-cell homeostasis and is being used in preliminary clinical trials.134,135

A few pilot studies in human SLE patients have been conducted using an immunoconjugate composed of a murine anti-CD5 mAb bound to ricin toxin A chain (RTA) (zolimomab aritox) to induce depletion of differenT cell subsets expressing CD5.136,137 This immunoconjugate induced modest T-cell depletion, which persisted for months, and a transient decrease in CD5+ B cells but no persistent depletion of total B-cell numbers. Unfortunately, the drug was not successful because of its powerful cytotoxic effect and multiple adverse reactions.

2. Multiple Sclerosis

Demyelinating diseases are characterized by degen-eration and loss of the myelin with a relative pres-ervation of axons.138 Among the many different

etiologies that can cause axonal demyelination, the focus here is on those resulting from inflamma-tory immune-mediated processes: multiple sclerosis (MS), postinfectious encephalomyelitis, and acute hemorrhagic leukoencephalitis. The relationship of CD5+ B-cell subsets and MS has been extensively investigated. Although their role in mediating MS’s inflammatory and degenerative processes is not well understood, it is broadly considered that CD5+ B cells are important in disease pathogenesis.

Given that autoantibodies appear to be involved in a major subgroup of patients with MS,139–141 stud-ies have addressed the contribution of CD5+ B cells with sometimes contradictory results.142 One study, for example, reported an increased percentage of CD5+ B cells in cerebrospinal fluid and associated it with a higher MS risk.143–145 In fact, another study proposed that a high percentage of CD5+ B cells in blood is predictive of earlier conversion to the dis-ease,146 and some studies have found an association between expression levels of CD5 in B cells and the occurrence and duration of relapsing-remitting MS.147–149 However, yet another study found CD5 expression on B cells to be decreased in patients with secondary progressive MS (a stage following relapsing-remitting MS).150 A later study described increased percentages of CD5– B cells and sug-gested that CD5+ B cells are associated with a lower prevalence of antimyelin antibody production.151 Because of such conflicting results, CD5 expression in B cells is considered a double-edged sword that can either worsen the course of MS or result in its lower prevalence. This “janus” facet to CD5 seems to hold true in animal models of MS as well.152–157

The experimental autoimmune/allergic neuritis (EAN) model has been used as an animal approach to inflammatory peripheral demyelinating neuropathy; the experimental autoimmune encephalomyelitis (EAE) model has been used as the corresponding disease model in the central nervous system. Both are considered CD4+ T-cell–mediated models of MS: CD4+ TH17 are the major effector cells, whereas TH2 and Treg cells have a protective role.91,158–160 Both models have been widely studied in rats and mice to determine the involvement of CD5 in MS pathogenesis. For example, a good amount of evidence points toward a protective



role for CD5 or CD5+ B cells in EAE—in one study, targeting myelin oligodendrocyte glycoprotein (MOG) at steady-state DC induced overexpression of CD5 on T cells and proliferative unresponsiveness to antigen rechallenge in vivo and in vitro.152

In a series of studies, adoptively transferred CD5+CD19+ B cells protected against EAE, an effect amplified by the addition of glatiramer acetate (a therapeutic treatment for relapsing MS).153 The cell transfer down-regulated inflammation through the increased production of immunoregulatory cytokines and the down-regulation of the CXCR5 chemo-kine receptor. Along the same lines, oral antibiotic treatment of mice at EAE induction resulted in an increased CD5+ B-cell population with regulatory features (e.g., IL-10 secretion) that conferred pro-tection against the disease. Furthermore, transfer of CD5+ B cells from antibiotic-treated mice conferred protection against EAE.154

Given the results just discussed, the outcome of EAE in CD5-deficient mice came as a surprise. Contrary to what was expected, in one study mice lacking CD5 exhibited significantly delayed EAE onset and decreased severity.155 The researchers pro-posed that this was due to the prosurvival activity of CD5 in T cells via engagement of the serine/kinase CK2 that binds to the receptor. Indeed, transgenic mice lacking the site of interaction between intra-cytoplasmic CD5 and CK2 were also resistant to EAE, which was attributed to increased AICD and decreased populations of cells coexpressing IFN-γ and IL-17.91 The same group later showed that the CK2-CD5 pathway is necessary for efficient differ-entiation of naïve CD4+ T cells to TH2 and TH17 and for induction of T cell anergy.95

Few studies have been conducted on the targeting of CD5 to treat EAE. In one, a mouse anti-CD5 mAb (OX19, IgG1) was used to treat rats showing clinical signs of EAN at different dosing schedules.156 The results indicated that OX19 given at the immu-nization time partly prevented clinical signs of EAN; when given shortly before the expected onset of the disease or during its height, however, OX19 drastically exaggerated disease symptoms. These results illustrate that anti-T/B-lymphocyte antibodies (OX19) might exert opposite effects on autoimmune diseases when

given at different phases of disease development, and they are in line with the results of more recent studies using anti-CD20 mAbs to deplete B cells.156 When given at disease initiation, treatment with anti-CD20 mAbs exacerbated EAE reportedly because of a decrease in CD5+IL-10+ Breg cells. In contrast, B-cell depletion during the disease phase suppressed symptoms. This was also seen in other studies using transgenic mice constitutively expressing a sCD5, which likely acted as a decoy receptor to block the interaction of CD5 and its natural ligand(s). EAE severity was enhanced in these mice, and the same effect was reproduced by repeated administration of recombinant human sCD5 from disease initiation.40

The results just discussed apparently contradict previous reports by Raman’s group showing that the infection of mice with adenovirus expressing CD5-immunoglobulin fusion protein (CD5-Fc) when clinical signs were apparent promoted recovery from EAE.155 It could be that differences in the time at which recombinant human sCD5 (day 0) and CD5-Fc (day 14) were administered were the reason for such contradictory outcomes. If confirmed, this would indicate that EAE may be improved by agonist targeting of CD5 during the disease initiation phases whereas CD5 blockade may be useful during symptomatic and more advanced phases.

3. Rheumatoid Arthritis

Rheumatoid arthritis (RA) is a chronic autoimmune disease characterized by systemic inflammation and synovitis that eventually causes cartilage damage and bone loss as a result of unequal bone formation and resorption.157 Although the pathogenesis of the dis-ease remains unclear, both antibody- and cell-medi-ated immune responses, as well as many cytokines, chemokines, and growth factors, have been reported to be involved in its development. Genetic studies reveal that RA is a complex genetic disease involving the influences of a long list of risk loci shared with other autoimmune disorders.161 Interestingly, CD5 was identified among 14 new susceptibility loci for RA in populations of European ancestry,162 with the nonsynonymous SNP rs229177 (Ala471Val) being a strong candidate for the causal variant.



Although TNF-α, IL-6, and IL-17 have been reported to enhance bone resorption,163 tumor growth factor beta (TGF-b) seems to play a dual role in both bone resorption and formation.164 Treg cells appar-ently inhibit bone resorption (osteoclastogenesis), whereas the role of B cells (including Bregs) is not well understood. The contribution of CD5+ B cells to RA pathogenesis has been investigated for many years. One of the first reports showed increased percentages of CD5+ B cells in peripheral blood from 17 patients with RA compared to normal con-trols.165 Other reports showed increased CD5+ B cell numbers in patients with RA and spondylarthritis and concluded that activated CD5+ B cells could be responsible for autoantibody production.52,166,167 In these studies, higher levels of CD5+ B cells correlated with increased amounts of polyreactive antibodies, increased levels of rheumatoid factor (RF), and the clinical features of a severe disease.168 However, other studies failed to show a correlation between the percentages of CD5+ B cells and the levels of RF or C-reactive protein.169

It has been proposed that the number of CD5+ B lymphocytes reflects individual genetic back-grounds.170–172 Recent studies on Breg populations in RA not only support the idea of a negative cor-relation between Bregs and RA but suggest this cell subset as a potential target in future therapeutic strategies.173–175 Current therapies used in RA, the so-called disease-modifying anti-rheumatic drugs (DMARDs), involve immunosuppressants such as methotrexate, glucocorticoids, and blocking mAbs that target TNF-a, IL-6, or CD19, among many oth-ers, in order to block autoantibody production.176,177

Some RA clinical trials using depleting or block-ing anti-CD5 mAbs have been reported. Clinical studies performed with an anti-CD5 mAb linked to RTA revealed inhibition of IL-2–induced prolifera-tion of synovial-fluid T cells in some patients treated with this immunotoxin.178–182 Despite positive initial results, presumably obtained by the elimination of pathogenic B cells that contribute to inflammation, the use of depleting anti-CD5 mAbs was finally stopped when a wide double-blind placebo-controlled multicenter trial demonstrated no clinical benefits or significant differences between groups.183,184 More

recent studies have shown therapeutic benefits from depleting B cells with an anti-CD20 mAb (rituximab) in RA patients.185–189

Anti-CD5 mAbs have been tested in animal RA models, such as collagen-induced arthritis (CIA), which is the most common experimental mouse model for human RA. One relevant study showed benefits after the administration of a nondepleting anti-CD5 mAb190: a rat IgG2a mAb specific for CD5 (TIB104) was administered in DBA/1 mice that presented clinical arthritis after CIA induction. The results demonstrated a significant decrease in disease severity in 60% of the mice and supported a T-cell–mediated mechanism given that levels of circulating antibodies to native collagen II were unaltered and that the amelioration of disease sever-ity appeared six days after mAb treatment. Overall, this outcome suggests that the blockade of CD5+, rather than the depletion of CD5 populations, is a potential therapeutic strategy in RA that needs more investigation.

4. Insulin-Dependent Diabetes Mellitus

Insulin-dependent diabetes mellitus (IDDM) is an organ-specific autoimmune disease in which T-cell–mediated pancreatic β-cell destruction leads to absolute insulin deficiency.191 Specific autoanti-bodies to insulin (IAAs), glutamic acid decarboxylase (GADA/GAA), and protein tyrosine phosphatase IA2 (IA-2AA) have been related to IDDM.191 Although there is no definitive correlation between expanded CD5+ B lymphocytes and the presence of circulating ICA and IAA autoantibodies, increased proportions of this cell subset were reported in early IDDM phases.53,192

Two studies explored the therapeutic benefits of anti-CD5 mAbs conjugated to RTA in human and mouse autoimmune diabetes.193,194 In the first one, patients with recent-onset IDDM were subjected to anti-T-cell therapy with the immunoconjugate CD5-Plus® (anti-CD5 H65 mAbs bound to RTA). It was reported that the therapy was tolerated and resulted in reversible T-cell depletion together with a dose-dependent preservation of β-cell function.193 In the second study, mice undergoing low-dose streptozocin/



IFN-γ–induced autoimmune diabetes and treated with anti-Ly-1 mAbs linked to RTA were protected against diabetes onset in a dose-dependent manner requiring higher doses and a longer schedule than that required by an anti-CD3 RTA.194 Although the mechanisms underlying anti-CD5-RTA–mediated protection remain poorly defined, both studies show that anti-CD5 immunotoxins may be useful for the in vivo treatment of diabetes and other T-cell–medi-ated autoimmune diseases.

5. Autoimmune Nephropathy

Glomerulonephritis defines a group of kidney autoimmune diseases characterized by glomerular inflammation and leukocyte accumulation. T and B lymphocytes have been reported to play a relevant role in the pathogenesis of proliferative and nonpro-liferative nephritis.195–198 Although T cells are thought to enhance antibody production against glomerular structures and to mediate cellular cytotoxicity through macrophage activation, the precise mechanisms underlying glomerular injury and lymphocyte infiltra-tion are not fully understood. Experimental animal models, such as antiglomerular basement membrane (GBM) glomerulonephritis in rats, have been largely used as an approach to the human diseases. In the rat model, anti-CD5 mAb (OX19) was administered as a pan-T-cell–depleting treatment, in combination with anti-CD4 and anti-CD8 mAbs or even alone, and showed reduced proteinuria and ameliora-tion of glomerular lesions.199–202 Similar beneficial effects were reported for a CD5-Fc chimera in a murine model of antibody-mediated membranous glomerulonephritis.65 Infusion of CD5-Fc was found to abrogate disease development by interfering with the CD5–CD5 ligand interaction.

Regarding the involvement of CD5+ B cells, it has been reported that these cells contribute to the pathology of some human nephropathies such as steroid-dependent nephrotic syndrome, cryo-globulinemic glomerulonephropathy, and human IgA nephropathy (IgAN).203–206 For example, num-bers of CD19+CD5+ cells inversely correlated with response to treatment in IgAN, and CD19+CD5+ cells isolated from untreated patients expressed higher

levels of IgA and IFN-γ and were more resistant to CD95L-induced apoptosis. This suggests that CD5+ B cells play a prominent role in IgAN. Indeed, higher numbers of CD19+CD5+ cells have also been found in patients with IgAN.207

Targeted B-cell–depleting therapies, in particular rituximab, are increasingly being used for the treatment of a number of glomerular diseases.208,209 However, new treatments specifically targeting CD5+CD19+ cells, which should avoid long-term consequences of pan-B-cell–depleting mAbs, remain to be explored.

6. Inflammatory Bowel Disease

Ulcerative colitis (UC) and Crohn’s disease (CD) are inflammatory bowel diseases (IBDs) character-ized by mucosal inflammation due to exacerbated activation of the immune system that affects only the colon (UC) or any region of the intestinal tract (CD). Oral administration of dextran sulfate sodium (DSS) solution has largely been used as the mouse model that best mimics IBD in human UC patients. Although the precise mechanisms of the disease remain unknown, several studies point to the role of Breg and Treg subsets in its modulation.96,210 B cells and IL-10 have been reported to play important inhibitory roles in the development of colitis. For example, a genetic deficiency of IL-10 results in spontaneous UC while its mAbs-induced blockade causes increased severity of IBDs generally.211,212

A recent study observed that, in contrast to RA, an anti-CD20 mAb (rituximab) had deleteri-ous effects in UC by blocking IL-10–producing B cells and stressing the importance of their anti-inflammatory role over their proinflammatory role.213 This observation was experimentally supported by adoptive transfer assays showing that regulatory B10 cells (CD1dhiCD5+IL-10+) from wild-type mice reduced DSS-induced intestinal injury in an IL-10–dependent manner.210 DSS-induced colitis was also shown to be less severe in CD5-deficient mice, which was attributed to increased levels of FoxP3 mRNA in the mouse colon and the enhanced suppressive activity of CD5–/– Tregs.96 Based on these observations, the use of CD5 antagonists in delaying or attenuating human UC deserves further study.



B. Infection

1. Viral Infection

CD5 has often been mentioned in studies aimed at deciphering the mechanisms by which viruses invade immune cells and/or modulate immune responses. Examples are hepatitis C virus (HCV), hepatitis B virus (HBV), HIV-1, Epstein-Barr virus (EBV), and equine infectious anemia (EIA).214–218

HCV infection is a global health problem and one of the main causes of cirrhosis, chronic hepatitis, and liver cancer. HCV infects hepatocytes as well as other cell types, in particular the cells of the immune system such as lymphocytes, monocytes/macrophages, and DCs.219,220 Although the cell invasion mechanism remains incompletely understood, it has been proposed that several surface molecules act as viral receptors mediating virion internalization. In hepatocytes, human scavenger receptor class B type 1 (SR-B1), a lipoprotein receptor responsible for the uptake of cholesteryl esters from high-density lipoproteins, has been reported to mediate cell invasion through its interaction with the HCV E2 glycoprotein.

In addition to SR-B1, LDLr, CD81 tetraspanin, and a few other molecules have been found to be necessary for HCV entry in hepatocytes.221 Different receptors seem to be required for HCV entry into T cells, one of them being CD5.70,214 Only primary T cells and T-cell lines expressing CD5 are suscep-tible to HCV infection, suggesting that CD5 is an important molecule for HCV entry into primary human T lymphocytes.70 The authors70 propose that, while CD81 may contribute to broad recognition of cells by HCV, CD5 facilitates HCV tropism specifi-cally toward lymphocytes. The role of CD5 in HCV entry into B cells has not been established.

Although there is no reported evidence for direct interaction between CD5 and other known pathogenic viruses, CD5+ T- and B-cell subsets have been proposed to play a role in the progression of some viral infections. On the one hand, patients with HBV were reported to have increased numbers of CD5+ B cells and Treg cells compared to healthy donors.215,222 On the other hand, it was reported that a cytotoxic CD5–CD8+ T cell subpopulation

expanded in patients with HIV-1 and was also found in inflammatory infiltrates of patients undergoing HCV reinfection.216,223 Down-regulation of CD5 on activated CD8+ T cells has been proposed a diagnostic marker of dysregulated T cells in EBV-associated hemophagocytic lymphohistiocytosis as well.217,224,225

The efficacy of specifically depleting CD5+ T lymphocytes in vivo with a murine IgG2a mAb (HB19A) was tested for control of chronic EIA lentiviral infection.218 Horses given this mAb (25–50 mg/day intravenously for 11 days) showed sustained depletion of peripheral blood CD5+ T cells but still maintained a residual (~15%) CD5–CD2+ T-cell population (either CD4+ or CD8+). Following CD5+ T cell depletion, EIA-infected horses did not develop recrudescent viremia or disease, unlike horses receiving corticosteroids.218

2. Fungal Infection

Although most clinical cases of sepsis are caused by gram-positive bacteria followed by endotoxin-pro-ducing gram-negative bacteria, fungi can also cause septic shock. The most recent study on sepsis etiology in the United States noted the dramatic increase in fungal sepsis over the last years,226 with infection by Candida albicans still the most common.227,228 This increase may reflect the efficient bacterial treatments currently in place in contrast to current antifungal treatment regimens that require prolonged admin-istration of medications with significant toxicity. Research on new treatments has focused on the use of vaccines and/or immunotherapy for the active treatment or prevention of specific fungal pathogens.

The pathogen-binding capabilities of the CD5 ectodomain were found to be beneficial for the control of fungal-induced experimental septic shock.21 Thus, a recombinant sCD5 equivalent to the soluble variant present in human serum38,39 was efficient not only in binding but also in aggregating both pathogenic (Can-dida albicans, Cryptococcus neoformans) and saprophytic (Saccharomyces Pombe) fungi through recognition of β-D-glucans, which is a major component of fungal cell walls.21 Pretreatment of mice undergoing zymosan-induced septic shock–like syndrome with a single dose (1.25 mg/Kg, intraperitoneal) of recombinant sCD5



(but not recombinant sCD6 or bovine seroalbumin) induced significant reductions in mortality, toxicity score, serum levels of proinflammatory cytokines, and leukocyte infiltration. Importantly, the beneficial effects were still evident when recombinant sCD5 was administered 1–3 hours post-zymosan challenge. These results open the possibility of developing new antifungal biotherapies based on CD5 alone or in com-bination with other well-studied β-D-glucan receptors such as TLR2. Indeed, TLR2-deficient mice were shown to be more resistant to Candida albicans dis-semination because of an impaired anti-inflammatory response (decreased IL-10 secretion, enhanced IFN-γ secretion),229 while dectin-1–deficient mice became more susceptible to infection because of an impaired proinflammatory response.230 The combination of a TLR2-blocking therapy and recombinant sCD5 is a strategy that should be looked into.

Another issue to look at is the relevance of CD5 polymophisms in individual susceptibility to fungal infections. As demonstrated in a recent report, cells expressing the more recently derived V471 vari-ant showed enhanced IL-8 cytokine secretion and mitogen-activated protein kinase (MAPK) activation after zymosan exposure when compared with the ancestral A471 variant.77

Overall, the data show that the CD5 molecule is involved in the modulation of inappropriate immune responses during fungal infections.

C. Transplantation

1. Hemopoietic Stem Cell Transplantation

Hemopoietic stem cell transplantation is a potentially curative immunotherapy used in several hematologic malignancies. However, graft-versus-host disease (GvHD) continues to limit its success. Even minor donor–host differences in major histocompatibility complex (MHC) and/or non-MHC antigens can lead to T-cell alloreactivity, which is mainly responsible for the pathogenesis of acute or chronic GvHD.231 Donor T-cell alloactivation leads to secretion of Th1 cytokines such as IL-2 and IFN-γ, which eventually activate cytotoxic lymphocytes and effector B cells, and induce macrophage recruitment as well as clonal

expansion. Moreover, common chemotherapy/radio-therapy pretreatment of host tissues can activate host immune populations and increase the release of proinflammatory cytokines (TNF-a and IL-1) that increase the surface expression of MHC antigens. Regarding CD5, expansion of a small population of peripheral CD3+CD5– cytolytic T cells normally present in healthy individuals has been reported after bone marrow transplantation and correlates well with the incidence of GvHD.232,233

Therapeutic strategies used in both acute and chronic GvHD mainly involve immunosuppressive regimes, including cyclosporin A, corticosteroids, and methotrexate. Depleting treatments targeting T-cell or even B-cell receptors are also being consid-ered, as is the blocking of anti-TNF-α and anti-IL-2 receptor mAbs.234 Because most immunosuppressive drugs are reported to cause long-term side effects and are not efficient for chronic GvHD, the main purpose in transplantation now is to induce host-specific tolerance. In this regard, special interest in the specific populations that contribute to the maintenance of long-standing tolerance, such as T and B regulatory subsets and inhibitory cytokines, has emerged.

An increased number of B cells expressing CD5 and CD1d were found in patients who main-tained stable kidney graft function in the absence of immunosuppression as compared to patients with chronic rejection and healthy volunteers.235 Thus, this B-cell population is believed to display an inhibitory profile. In contrast, IL-10, a cytokine secreted by CD19+CD5+ B cells, has been reported to play a dual role: (1) inhibiting both proliferation of antigen-presenting-cell (APC-) –dependent T cells and production of IL-2 T cells236 and (2) accelerat-ing T-cell–mediated GvHD lethality in vivo in a dose-dependent manner.237 Given that both T and B regulatory populations express high levels of CD5 and may be crucial for inducing tolerance after allo-genic transplantation, CD5-based therapies may be a feasible approach to positively immunomodulating these populations and eventually stimulating long-term tolerance. Unfortunately, given that CD5 is a pan-T-cell marker, depleting anti-CD5 mAbs have mostly been used as immunosuppressive drugs.



The first uses of anti-CD5 mAbs in preventing GvHD were in humans 30 years ago. An anti-CD5 mAb (T101) was first used with anti-CD2 (35.1) and anti-CD3 (UCHT-1) mAbs in what was called the TUT cocktail.238 These reagents together inhibited T-cell proliferation in vitro more effectively than did any one of them alone. For this reason, researchers proposed the TUT cocktail as a potential T-cell–depleting therapy. To avoid side effects derived from toxic drugs used in vivo, the cocktail was developed to purge ex vivo GvHD-producing cells from bone marrow before allogeneic marrow engraftment.239–241 However, few beneficial effects were observed in clinical assays.242 Later on, T101 was conjugated to an immunotoxin (T101-IT) and optimized for T-cell depletion from human bone marrow grafts.239,243–245 Clinical studies using T101-IT in ex vivo bone mar-row transplantation in patients with acute leukemia showed not only low effectiveness in preventing GvHD but also increased severity in some cases.246–249

Despite the unsuccessful results reported in the use of ex vivo anti-CD5–depleting therapy, an anti-CD5-RTA conjugate was used in later clinical studies in vivo following bone marrow transplanta-tion. Not surprisingly, patients receiving the drug showed increased risk of rejections, higher morbidity and mortality associated with viral infections, and greater risk of developing post-transplant lympho-proliferative disorder derived from anti-CD5-RTA toxicity.250–255 Toxicities observed in preclinical and clinical trials were also seen in mice and rats receiv-ing anti-CD5-RTA against mouse and rat CD5, respectively, after GvHD induction.256–258

The effect of nondepleting CD5-based inter-ventions in GvHD on the modulation of B and T regulatory populations and host tolerance induction urgently requires investigation.

2. Solid Organ Transplantation

As far as is known, little has been done on the use of CD5-based therapies in solid organ transplanta-tion. Increased survival of renal allografts has been reported in recipients with high titers of pre-existing alloantibodies after preoperational treatment with anti-CD5 immunotoxin (a conjugate of anti-CD5

monoclonal antibody with RTA). In this case, additional pretreatment of renal transplants with anti-CD5-RTA caused a reduction in passenger lymphoid cells, which ultimately improved trans-plantation success.259

D. Cancer

The last decade has seen great progress in under-standing the cellular and molecular interrelationship of immune cells and tumor cells. This improved knowledge has led to the development of a wide range of immunotherapies with greater antitumor efficacy.260 Since Burnet and Thomas first proposed the hypothesis of immunosurveillance in cancer,261 the immune response has been considered to play a relevant role in eradication of tumors. However, tumor cells are usually poorly immunogenic and are able to create suppressive environments262–264 that promote the activation, proliferation, and migration of regulatory cells such as Tregs,265 myeloid-derived suppressor cells (MDSC), tumor-associated macro-phages (TAM), and other subtypes of mature and immature DCs that produce immunosuppressive cytokines (IL-10, IL-4, TGF-β).266 This tumor microenvironment suppresses the immune effector response against tumor cells.

The destruction of tumor cells by the immune system is controlled by receptors that tightly regulate, positively or negatively, T-cell effector functions. Some current immunotherapeutic strategies in cancer are based on blocking the inhibitory receptor for B7, cytotoxic T-lymphocyte antigen 4 (CTLA-4), whose role is to shut off responses against self-antigens.267 Very encouraging results were recently obtained in patients with different types of tumors by blocking another inhibitory receptor, programmed death-1 (PD-1), which prevents T-cell activation.268 Along this line, CD5 also behaves as a negative modulator of activation and differentiation signals mediated by the T-cell receptor, and it has been shown to contribute to the regulation of antitumor immune responses.11,12 An early study in mice reported that a nondepleting mAb against CD5 was efficacious in the treatment of lymphoid (EL-4 leukemia) and nonlymphoid (Lewis lung carcinoma) tumors.269 In



previous work, the researchers had demonstrated that anti-Lyt-1 mAbs augmented the production of IL-2, thus improving T-cell proliferation.270

Much more recently, a careful analysis of human tumor-infiltrating T lymphocytes (TILs) showed that CD5 expression is inversely related to TIL antitumor activity. In this study, certain T-cell clones obtained from human lung carcinoma TILs showed dramatically lower CD5 expression com-pared to peripheral blood lymphocytes from the same patients.271 Increased AICD in lymphocytes with low or undetectable levels of CD5 was also reported,272 suggesting that these lymphocytes are more activated than are their high-expressing coun-terparts and that CD5, much like PD-1 or CTLA-4, may hinder the activation or survival of anti-tumor T cells. This hypothesis was validated by studies in CD5-deficient mice, which showed slower tumor growth in a B16F10 melanoma model.273 The results are in full agreement with observations using trans-genic mice expressing sCD5, which was expected to act as a decoy receptor and to block the binding of cell-associated CD5 to its natural ligand(s).40 Using a nonorthotopic melanoma model (B16), the mice showed slower tumor growth when compared to their nontransgenic littermates. Similar results were achieved by repeated administration of recombinant human sCD5 protein to wild-type mice after tumor injection. The increased antitumor response observed was associated with a basal reduced proportion of Tregs and peritoneal IL-10–producing B cells and an increased percentage of natural killer T cells.40

The targeting of new immunological checkpoints such as CTLA-4 and PD-1/PD1 ligand in cancer has yielded promising results in clinical trials, especially in patients with metastatic melanoma.274 The avail-able data suggest that CD5 represents a new cancer checkpoint molecule, opening up new therapeutic possibilities based on neutralizing anti-CD5 mAbs or sCD5 proteins alone or in combination with other immunomodulatory treatments.

Early phase-I studies have demonstrated rapid but transient responses with the use of an unmodified anti-CD5 mAb (T101) in patients with cutaneous T-cell lymphoma and chronic lymphocytic leukemia with minimal toxicity.275 This has prompted further

phase I studies using radiolabeled (90Y) versions of the same mAb, in which similar partial and transient responses were observed.276

IV. CONCLUSION

Since its discovery more than 50 years ago, much progress has been made toward establishing CD5’s role in the fine-tuning of antigen-mediated responses by T and B cells. For this reason, it is clear that CD5 is an attractive target for developing novel immunotherapies aimed at potentiating or inhibit-ing ongoing immune responses. However, important gaps remain concerning the identification of CD5’s endogenous ligand(s), the role of its soluble form, and the functional consequences of its ligation by pathogen-associated molecular patterns. Further investigation will allow a better interpretation of the sometimes conflicting results concerning the role of CD5+ cells in different autoimmune, infectious, and neoplastic disorders. It will also permit researchers to go beyond initial strategies using pan-T-cell–deplet-ing CD5 antibodies to treat such disorders (e.g., autoimmunity, GvHD) and to design nondepleting reagents that target specific subpopulations to down-modulate self-reactive cells or boost regulatory cell functions. In this regard, attention should also be paid to the disease stage at which those therapies will be introduced to avoid absent or even unwanted effects.

Recent evidence obtained with experimental tumor models suggests that CD5 is a new check-point molecule that can be neutralized to enhance current immunotherapies for cancer. Furthermore, the recently described pathogen (fungal, viral) recognition capacities of CD5 open new venues in the treatment of either acute or chronic infections.

ACKNOWLEDGMENTS

The authors’ work is supported by grants from the Association for International Cancer Research (14-1275) and the Spanish Ministerio de Economía y Competitividad (Plan Nacional de I+D+i, SAF2010-19717 and SAF2013-46151-R), the Instituto de



Salud Carlos III (Spanish Network for Research in Infectious Diseases, REIPI RD06/0008/1013 and RD12/0015/0018), the Fundació La Marató TV3 (201319-30), and the Generalitat de Catalunya (2009SGR1101). IS is the recipient of a fellowship from the Portuguese Fundação para a Ciência e a Tec-nologia (SFRH/BD/75738/2011). MO is the recipi-ent of a FPI fellowship from the Spanish Ministerio de Economía y Competitividad. FL is ad honorem scientific advisor at ImmunNovative Developments, a spin-off company of the University of Barcelona.

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cd5 as a target for immune-based therapies

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