Tumor-Induced Disruption of Proximal TCR-Mediated Signal Transduction in Tumor-Infiltrating CD8+ Lymphocytes Inactivates Antitumor Effector Phase Edwin J. Vazquez-Cintron,* Ngozi R. Monu,*,† and Alan B. Frey* The presence in cancer tissue of Ag-specific, activated tu- increase the frequency of antitumor T cells [in some cases mor infiltrating CD8+ T cells proves that tumors express resulting in a reduced rate of tumor growth (9)], either en- Ags capable of eliciting immune response. Therefore, in dogenous priming of antitumor immune response is insuf- general, tumor escape from immune-mediated clearance ficient to engender successful tumor elimination in patients is not attributable to immunological ignorance. How- receiving no therapy, or the effector phase is suppressed, or ever, tumor-infiltrating lymphocytes are defective in ef- both. Analysis of APCs in murine tumors has shown that fector phase function, demonstrating tumor-induced dendritic cells (DCs) are frequently defective in some aspect immune suppression that likely underlies tumor escape. of priming: Ag capture, cytokine expression, costimulatory Since exocytosis of lytic granules is dependent upon function, or migration to proximal lymph node (10). This TCR-mediated signal transduction, it is a reasonable results in diminished initiation of adaptive response to tumor contention that tumors may induce defective signal Ags. In some cases, tumor DCs have been shown to be not transduction in tumor infiltrating T cells. In this review, only defective at priming but also tolerogenic (11, 12). Why we consider the biochemical basis for antitumor T cell tumor DCs do not function effectively as occurs in response dysfunction, focusing on the role of inhibitory signaling to pathogens in which infection is resolved [e.g., Listeria receptors in restricting TCR-mediated signaling in tumor- monocytogenes (13)] is unclear but may be related to the ki- infiltrating lymphocytes. The Journal of Immunology, netics of tumor growth (i.e., the dose of Ag available for 2010, 185: 7133–7140. priming, continual low amounts, as well as the lack of robust danger signals) (14). Similar observations have been made for mmune response to cancer is apparent; equally apparent DCs isolated from cancer patients (15, 16). An additional is that tumors grow, implying escape from antitumor im- consideration is that, because many tumor Ags are closely I munity (1) or defective antitumor immune responses (2). related to self, cognate TCRs expressed in antitumor T cells Multiple candidate mechanisms to account for failure of anti- that survive thymic selection are likely of low affinity and tumor immunity have been described that involve a variety of likely have enhanced activation requirements. cell types, factors, and mechanistic considerations (3). In mu- Furthermore, two immunosuppressive cell types, regulatory rine models wherein tumor-bearing mice can be immunized T cells (Tregs) and myeloid-derived suppressor cells (MDSCs), with a variety of Ags (4), and patients in whom tumor-reactive have been shown to accumulate in tumors, both of which are Abs and T cells are commonly found (5), cancer does not cause thought to restrict the priming (17) and effector (18, 19) defective systemic immune responses. Thus, tumor itself, or the phases of adaptive immune response. Depletion or inactiva- host response, causes Ag-specific immune tolerance, almost cer- tion of Tregs (20) or MDSCs (21) enhances experimental im- tainly in the priming, and unequivocally in the effector phase of munotherapy in preclinical models, although data from clin- adaptive immunity, primarily in antitumor T cells resident in ical trials are less robust. The basis by which either Tregs or tumor tissue (6–8). MDSCs inhibit priming is not definitively known, but these cells can produce a variety of molecules that are known to Priming of antitumor immune response is ineffectual to eliminate inhibit both DCs and T cell function including: TGFb-1, tumors IL-10, reactive oxygen and nitrogen species, and enzymes that Detectable priming of antitumor T cells occurs during tumor are thought to either deplete the microenvironment of certain growth but, because vaccination of patients can dramatically amino acids [arginine (22), tryptophan (23), and/or cysteine

*Department of Cell Biology and †New York University Cancer Institute, New York mediator; IR, inhibitory signaling receptor; ITSM, immunoreceptor tyrosine-based University Langone Medical Center, New York, NY 10016 switch motif; KIR, killer Ig-related (or Ig-like) receptor; LAIR-1, leukocyte-associated Ig-like receptor-1; LAT, linker for activation of T cells; LILR, leukocyte Ig-like receptor; Received for publication June 30, 2010. Accepted for publication October 6, 2010. LIME, LCK-interacting molecule; LLT1, lectin-like transcript-1; MAFA-1, mast cell This work was supported by National Institutes of Health Grants F31CA136164 (to function-associated Ag-1; MDSC, myeloid-derived suppressor cell; MGL, macrophage E.J.V.-C.) and R01CA108573 (to A.B.F.). galactose-type lectin; PAG, associated with glycosphingolipid-enriched micro- domains; PTP, protein tyrosine phosphatase; SHP, Src homology 2 domain-containing Address correspondence and reprint requests to Dr. Alan B. Frey, Department of Cell tyrosine phosphatase; TIL, tumor-infiltrating lymphocyte; TIM-3, T cell Ig and mucin Biology, Room MSB 623, New York University Langone Medical Center, 550 First domain-3; Treg, regulatory T cell. Avenue, New York, NY 10016. E-mail address: [email protected] Abbreviations used in this paper: Cbp, C-terminal Src kinase binding protein; Csk, Copyright Ó 2010 by The American Association of Immunologists, Inc. 0022-1767/10/$16.00 C-terminal Src kinase; DC, dendritic cell; FasL, ; HVEM, herpesvirus entry www.jimmunol.org/cgi/doi/10.4049/jimmunol.1001157 7134 BRIEF REVIEWS: INHIBITION OF TUMOR-INFILTRATING T CELL FUNCTION

(24)] or produce toxic metabolites (25), therein leading either in tumor tissue (40, 41)], TILs appear to be deficient in to a state of metabolic quiescence or induction of apoptosis Ag-dependent TCR-mediated signal transduction. A detailed in tumor-infiltrating lymphocytes (TILs) [although the notion biochemical analysis of TCR signaling was investigated in of immune modulation by tryptophan metabolites has been a murine model (42) wherein nonlytic TILs were shown to questioned (26)]. Alternatively, or in addition, altered nitro- be triggered in that p56lck is activated upon recognition of gen metabolism in the context of enhanced production of cognate tumor cells (becoming phosphorylated on Y394) but reactive oxygen species is thought to produce highly reactive the activation signal does not penetrate deeper into the sig- oxygen and nitrogen species that are capable of modifying naling cascade because ZAP70 is not activated (37), calcium both the cell surface (27) and enzyme activity within antitu- flux is extinguished (37) and activation of LFA-1–mediated mor T cells (28). Postcoculture with T cells in vitro, MDSC- TIL adhesion is deficient (43) [shown to require p56lck ac- mediated production of reactive nitrogen has been shown to tivity (44)]. After initial p56lck activation, Src homology re- modify TCRs, resulting in diminished recognition by tetra- gion 2 domain-containing phosphatase (SHP)-1 was rapidly mer and reduced Ag-dependent lysis and cytokine release, a activated and localized to the immunological synapse coinci- phenotype that may reflect inhibitory activity of those cells dent with dephosphorylation of p56lck Y394. Reversal of in tumor-draining lymph nodes (27, 29). Soluble bioactive both defective proximal signaling and effector function is molecules produced by Tregs and MDSCs that inhibit adap- rapidly achieved by purification of TILs and brief culture in tive immunity, presumably by direct action on APCs in the vitro in the absence of tumor. In a similar manner, reintro- priming phase, are also capable of potentially impacting other duction of the signaling block is rapidly achieved by coculture antitumor immune cell types in proximity, including NK cells of TILs with tumor [and not with MDSCs or syngeneic but and CD4+ and CD8+ T cells in the effector phase. In a mech- antigenically distinct tumor cells (42)], observations that are anistic variation, purified MDSCs obtained from tumor (30) consistent with a mechanism involving a fast-acting biochem- or peripheral lymphoid tissue (31) have been shown to cause ical inhibitory switch, one that requires contact with cognate apoptosis in activated T cells in vitro, implying a different role tumor for activation (42). for MDSCs in dysregulation of the effector phase. However, TILs in situ are not appreciably apoptotic (32), thus arguing TILs express inhibitory receptors that mediate negative signaling and that the proapoptotic activity of either MDSCs or tumor cells effector phase dysfunction is a function of in vitro analysis. Collectively considered, the Considered collectively, the requirement for tumor cell contact extent to which MDSCs inhibit T cell signaling and function to induce the signaling defect and the rapid kinetics of in- may reflect the tissue site of interaction with T cells (lym- duction of the TIL signaling block (42) implies that tumor- phoid or tumor) and as such may contribute to antitumor induced inhibition of TIL signaling involves a ligand–recep- T cell dysfunction in addition to other candidate mechanisms, tor interaction, one similar to that characteristic of inhibitory as described below. receptors expressed on NK cells (45). Cell surface inhibitory signaling receptors (IRs) that contain cytoplasmic ITIMs are The suppressed phenotype of TILs reflects the mechanism of the expressed in a wide variety of immune cells and function in acquired functional defect and involves interference with the homeostatic regulation of immune responses wherein they TCR-mediated signaling pathway negatively regulate signaling mediated by Ag (activating) re- TILs have long been recognized as being deficient in cytokine ceptors. Most IRs function in concert with triggering of the release, proliferation, and lytic function (6). One potential Ag receptor in a manner analogous to that of costimulatory mechanism for blockade of effector phase involves galectins, receptors (e.g., ), with the distinction that the activating a family of carbohydrate binding made in a variety signal is dampened or abrogated, and function in the homeo- of cells that have multiple functions but are thought to restrict stasis of normal responses important during cell differentia- the mobility of cell-surface T cell proteins important in Ag tion and activation (46). recognition and signaling (33). Such a mechanism has re- As a consequence of ligand binding, typically IRs are acti- ceived support from several lines of investigation but is as vated by tyrosine phosphorylation on a consensus structure yet incompletely understood and undoubtedly complex be- ([I/V/L/S]-X-Y-X-X-[L/V]) by a kinase that is associated with cause exposure of immune cells to some galectins induce tol- the Ag receptor. Upon ITIM phosphorylation, phosphatase(s) erance or death (34); in contrast, other galectins change the are recruited and likely activated by the kinase associated with phenotype of T cells (35), whereas yet others enhance DCs the Ag receptor. The activated protein tyrosine phosphatase and CD8+ T cell numbers (36). (PTP) rapidly dephosphorylates proximal substrates, typically Our laboratory has pursued a murine model of TIL dys- the Src family kinase associated with the Ag receptor (e.g., function wherein the defective phenotype was transient, being p56lck, but also additional proximal kinases [ZAP70] or adap- regained upon purification and culture in vitro (37). CD8+ tor proteins [TCRz, Vav-1]). Thus, coordinately with Ag-de- TILs have hallmarks of proximal TCR-mediated signaling pendent activation of p56lck, IRs are tyrosine phosphorylated, blockade, interpreted to be the basis of defective lytic function leading to recruitment of PTP and inactivation of the activa- [in freshly isolated TILs assayed in vitro (38), as judged in- tion signal. In the course of induction of an activating immune ferentially because tumor cells proximal to TILs in situ are not response wherein a sufficiently strong positive signal is pro- TUNEL+ (32) or evaluated by confocal microscopy wherein vided to the T cell such that a sustained triggering event occurs lytic granules and the microtubule organizing center fail to (i.e., a high concentration of cognate Ag presented by an ap- polarize to the immune synapse (38, 39)]. Considered with propriately activated APC), PTP-mediated inactivation of other TIL phenotypes [cell cycle arrest, lack of Ag-dependent proximal signaling occurs following cell activation, reflecting cytokine release in vitro, and the inability to arrest migration the downregulation of T cell activation during differentiation The Journal of Immunology 7135 of naive cells into effector cells. It makes sense conceptually Functionally defective T cells in viral infection or cancer that IRs on effector T cells restrict inadvertent expression of have been shown to express IRs (e.g., PD-1, LAG-3, or CTLA- effector phase functions until the T cell recognizes a target cell 4), and experimental therapeutic intervention based upon expressing cognate Ag (as discussed below). Thus, IRs function blocking IRs is extant. Tumor cells often express ligands (coun- as part of a system that integrates positive (activating) and terreceptors) for IRs and, as such, when the tumor cell is rec- negative (inhibitory) signals, therein both maintaining tonic ognized by Ag-specific CTL, deliver a negative signal that balance and influencing cell activation thresholds. We hypoth- blocks (or partially blocks) the TIL activation signal, thus esize that the activity of IRs can be considered as cell- or organ- restricting effector phase functions, to the detriment of the specific rheostats that control the magnitude or extent of T cell host. The phenotype of suppression of T cell activation by activation, and, because there are .100 human contain- IRs was shown in 1997 in a mouse model wherein cytokine ing ITIM sequences, it is likely that IRs play an important role release and cytotoxicity was blocked upon engagement of in regulation of immune responses. A compilation of IRs that NKG2A/CD94 by tumor MHC class I (47). Biochemical can be expressed in T cells is shown in Table I. analysis of signaling showed that TCRz was phosphorylated, The majority of IRs are transmembrane plasma membrane demonstrating the cells were triggered, but ZAP70 was not proteins for which the extracellular portion contains recognition activated, thus explaining how the functional defect was in- elements that govern ligand interaction. The variety of ligand– duced in that delivery of the activation signal deeper into the receptor interaction is considerable in terms of both the num- TCR signaling pathway was abrogated. [The detailed bio- ber of IRs and the number and type of ligands, leading to the chemical phenotype and functional defect was demonstrated consideration that T cell activation is under the constant influ- more recently in a study of TILs (42)]. ence, if not control, by this regulatory system. Because some IR The factors that influence the basis of IR-mediated con- ligands are widespread (e.g., MHC class I, sialic acid, collagen, trol of T cell activation are incompletely known but likely in- and certain integrins), the notion of constant involvement of volve the following considerations. First, expression of IR IR in the control of T cell activation seems plausible. Some IRs ligands on the various cells that T cells contact—DCs during mediate homophilic interaction (e.g., CD31), therein restrict- priming, endothelia during transit to the tissues, and ulti- ing activation to a limited number of cell types; others in- mately target cells—is undoubtedly important in that receptor teract with MHC Ag-presenting molecules both class I ligation is required for function and likely influenced by the (CD85, CD158, Ly49) or class II (CD223), implying en- differentiation and activation status of the cell. We hypothe- hanced function during interaction with APCs. size that interaction of IR with cognate ligands (on DCs, Many IRs belong to families that share certain structural endothelia, or tumor cells) functions to recruit/stabilize IR motifs, such as the Ig superfamily (e.g., CD31, CD66a, into proximity with the Ag receptor/associated p56lck so that, CD152) or the Siglec family (e.g., CD22, CD33, CD170). in turn, the recruited PTP will be in proximity to the target Because many IRs share related ligands (e.g., sialic acid), it is kinase (Fig. 1). During activation of a productive immune reasonable to consider that there exists overlap or redundancy response, DCs receive appropriate danger signals leading to in the types of target cells that can inhibit a given T cell. Almost their state of full competency, and we hypothesize that this all IRs contain at least one ITIM motif in their cytoplasmic includes modest (or repressed) expression of IR ligands: either domain, which, when phosphorylated, recruits SHP-1 (e.g., levels of a given ligand, or the type/number of IR ligands, or CD22), although some IRs contain sequence motif variants both. A testable corollary of this notion is that suboptimal (e.g., immunoreceptor tyrosine-based switch motif, or ITSM), DC activation, such as that leading to differentiation of DCs which are associated with recruitment of a related phosphatase having an inhibitory or tolerogenic phenotype, may lead to SHP-2 (e.g., CD152 and CD279). In addition, some IRs have enhanced expression of inhibitory receptor ligands. Putative several closely related variants (e.g., CD66), some of which are involvement of IR ligands in suppression of DC function may restricted in terms of cell expression or lack a canonical ITIM be in addition to expression of other mediators of inhibition sequence (e.g., CD152 or CD160). Variant IRs lacking ITIM (e.g., IDO) and insufficient levels of costimulatory molecules can be activating rather than inhibitory in function, possibly and activation-associated factors (IL-12). due to contributions to T cell–target adhesion. Secondly, in a similar manner, the differentiation and ac- There are examples of IRs that contain no evident cyto- tivation state of the T cell may control the type and number b plasmic motifs for recruitment of a PTP (e.g., 1 integrins or of IRs that are expressed. For example, perhaps naive T cells CD160, which is also unusual in being GPI anchored that can express a different repertoire of IR than do effector or memory be released in soluble form), but which nonetheless function T cells, reflecting differing activation requirements of T cells to recruit SHP-1. In these cases, perhaps the IR interacts with in different differentiation states. According to this notion, for a cytoplasmic adapter protein that in turn is responsible for example, a memory T cell may express an IR for which the binding and recruiting a PTP into proximity with regulatory cognate ligand is expressed on endothelial cells that the T cell proteins in the signaling cascade wherein cell activation can be must traverse en route to interaction with its cognate Ag- inhibited. In addition to their major role as rheostats of Ag- expressing tissue/target cell. Thus, a T cell may express mul- dependent signaling, there may be other nonsignaling func- tiple IRs, some or any of which may not function until inter- tions of IRs that affect the behavior or activity of immune action with its cognate ligand occurs. Expression and activity cells. For example, several IRs are mediators of adhesion of IRs in this context can be considered to provide another b (e.g., CD22, CD66a, 1 integrins), a function that may pre- level of safety against inadvertent cytokine release or degranu- dominate in situations in which a T cell interacts with cells lation of lytic cells that may occur during extravasation that that express ligands for a given IR but do not express cog- is mediated by activated adhesion molecules that, if they use nate Ag. inside-out signaling, may stimulate inadvertent effector phase 7136 BRIEF REVIEWS: INHIBITION OF TUMOR-INFILTRATING T CELL FUNCTION

Table I. IR expression in T cells

IR Family Ligand Features Reference CD5 Scavenger gp150, CD72 Has a pseudo-ITAM; regulates FasL/caspase 8 activation; 54–58 (cysteine-rich) inhibits Ca flux and ERK activation; no PTP involvement CD22 (Siglec-2) Siglec Sialic acid Multiple ligands, ITIM 59, 60 a b CD31 (PECAM-e) Ig superfamily CD31, CD38, v 3 CD31 is expressed widely, ITIM 61 integrin CD33 (Siglec-3) Siglec Sialic acid ITIM 62 CD38* Cyclic ADP ribose CD31 No ITIM or PTP involvement, enzyme is soluble in serum, 63 hydrolase has both + and 2 function CD66a (CEACAM-1) Ig superfamily Multiple 18 genes in family; is expressed widely; multiple splice variants; 64–67 has both + and 2 function; those with ITIM are inhibitory; function depends on oligomerization CD72 Cysteine-rich CD5, CD100 ITIM 68 CD73* Ecto-59-nucleotidase GPI-linked, adenosine production from tumor 69 CD85 (PIR-A/B LILR MHC class I Some family members lack ITIM 70 in mice) CD94/ NKG2A C-type lectin HLA-E CD94 has two forms: 39 kDa lacks ITIM, 43 kDa 45 (KLRD1) has ITIM (and recruits SHP-1); is expressed on NK cells, memory T cells, and DCs CD152 (CTLA-4) Ig superfamily B7-1/2 Also is expressed on Tregs; no ITIM (has YVKM that 23, 71–74 mediates biding of PI3K, PP2A, and SHP-2); CD276-B7-H3 and CD276-B7-H4 are orphan ligands CD155 (TIGIT) Ig superfamily Poliovirus receptor Enhances DC-mediated Treg production; ITIM 75 CD158a KIR MHC class I 14 genes in family; different members have + or 2 function; 76, 77 ITIM CD159a (NKG2A) Ig superfamily Pairs with CD94 Is a multigene family; some lack ITIM 78 CD160 Ig superfamily HVEM GPI-anchored, lacks ITIM; is expressed on CD44+ 79 memory cells CD161 (NKR-P1A) C-type lectin LLT1 Is expressed on NK and CD8+ T cells 80 CD169 (Siglec-1) Siglec Sialic acid Lacks ITIM; expressed on macrophage and activated DCs 81 (may be activating on macrophage but inhibitory on DCs) CD170 (Siglec-5) Siglec Sialic acid CD33 related; ITIM 82 CD172a (SIRP-A) Ig superfamily CD47 Recruits SHP-1/2; may interact with: Csk, SLAP-130, Grb2; 83, 84 is expressed in DCs and mast cells; some forms are activating; ITIM CD223 (LAG-3) Ig superfamily MHC class II Widely expressed on lymphocytes; is related to CD4; effect 85 is reversed by IL-2 CD244 (2B4) CD2 family CD48 and CD244 Is expressed on NK cells and T cells; has ITSM; binding 86, 87 to CD48 is inhibitory; binding to CD244 is activating CD272 (BTLA) Ig superfamily HVEM ITIM 88, 89 CD279 (PD-1) Ig superfamily PDL-1 (CD274 = Recruits SHP-1/2, ITIM, and ITSM 90, 91 B7H1) and PDL-2 (CD273 = B7DC) CD300a (IRp60) Ig superfamily Widely expressed; ITIM 92, 93 CD305 (LAIR-1) Ig superfamily Collagen Recruits SHP-1/2; may interact with Csk; ITIM 94 CD328 (Siglec-7) Siglec Sialic acid Expressed in myeloid, NK cells, subsets of T cells; ITIM 95 a b gp49B-1 Ig superfamily v 3 integrin ITIM 96 KLRG1 (MAFA-1) C-type lectin Cadherin Expressed on T cells and NK cells; ITIM 97–99 Ly49 C-type lectin MHC class I Part of a multigene family; some are activating; ITIM 100 MGL C-type lectin CD45 Expressed on immature DCs 101 Galectin-3 Galectin b-galactoside 15-member family; intracellular but translocates to surface 102 upon activation b 1 integrin Integrin VCAM-1, soluble Recruits SHP-1; when bound to soluble integrin or ligated 103 integrins with monomeric Ab is inhibitory TIM-3 Ig superfamily Galectin-9 Multigene family; some activate (e.g., TIM-1); expressed 104 on various immune cell types T cell Src-binding May sequester p56lck away from functional signaling proteins complexes, therein reducing T cell responsiveness LAT Modulates inhibitory feedback loop via Dok2/SHIP 105, 106 binding; homolog (LAB) induced upon activation PAG/Cbp Ubiquitous; recruits Csk, ERM, RasGAP; also affects Ras 107 LIME Deletion has only a modest effect on peripheral T and 108, 109 B cells SIT Interacts with SHP-2 110 TSAd Rapidly induced in primary T cells by activation 111–113 The common names of receptors are noted in parentheses. CD38 and CD73 (marked with *) are not true IRs per se but are involved in local production of adenosine thought to be immunosuppressive. Although not all IRs contain canonical ITIM elements (e.g., CTLA-4), an ITIM or a functional homolog on the prototypic IR functions to recruit SHP-1 to phosphorylated ITIM and thus into proximity with PTP targets. Cbp, C-terminal Src kinase binding protein; Csk, C-terminal Src kinase; FasL, Fas ligand; HVEM, herpesvirus entry mediator; KIR, killer Ig-related (or Ig-like) receptor; KLRG1, killer cell lectin-like receptor G-1; LAIR-1, leukocyte-associated Ig-like receptor-1; LAT, linker for activation of T cells; LILR, leukocyte Ig-like receptor; LIME, LCK-interacting molecule; LLT1, lectin-like transcript-1; MAFA-1, mast cell function-associated Ag-1; MGL, macrophage galactose-type lectin; PAG, protein associated with glycosphingolipid- enriched microdomains; TIM-3, T cell Ig and mucin domain-3. The Journal of Immunology 7137

FIGURE 1. Negative signaling in T cells mediated by IRs. T cells express a given inhibitory receptor (or multiple IRs, although only PD-1 is shown) for which the ligand can be expressed on DCs, endothelial cells, or tumor cells. When bound by its cognate ligand, IRs are phosphorylated on their ITIM and/ or ITSM (which in turn reveals a PTP binding site, typically SHP-1 but pos- sibly SHP-2) and brought into proximity to a Src kinase and become bound by PTP. PTP becomes activated by tyrosine phosphorylation, likely also by Src, and dephosphorylates proximal substrates, including the activating Src. Di- FIGURE 2. Expression of IRs in nonlytic and lytic MCA38-derived TILs. minished p56lck-mediated activation of downstream signaling raises the acti- Splenocytes or TILs were prepared from control or MCA38 tumor-bearing vation threshold for the T cell. mice as described (42) and analyzed by flow cytometry for expression of the indicated IRs. Isotype-matched nonimmune Ig was used as labeling con- trol, shown in each panel as the black tracing. Staining of nonlytic TILs for activity. In other words, IR activity may be protective against expression of additional IRs was made (anti-CD22, anti-CD31, anti-Ly49 tissue damage by raising the T cell activation threshold, therein [C/I,G2, A, and D], and anti-CD66a) and were consistently negative (dns). restraining effector phase function until an authentic target cell is engaged. Supporting this notion, it was recently reported tified IR controls TIL function. Also of interest is the obser- that multiple IRs are coexpressed on CD8+ T cells in chron- vation that multiple IRs are expressed in normal splenocytes ically infected mice (48). Both of the IRs in that example (Fig. 2), raising the possibility that quiescent naive (or (PD-1 and LAG-3) were functional, as shown by in vivo memory) cells depend on IRs to maintain tonic balance, pos- blockade experiments. sibly permitting a rapid response to a strong activation signal. In a murine tumor model (MCA38), lytic defective CD8+ Several molecules are included in the list of IRs (Table I) antitumor TILs were shown to express several IRs (Fig. 2), even though they are not, strictly speaking, IRs, having very further supporting the notion that tumor-induced blockade of small extracellular domains unlikely to permit interaction with CTL signaling suppresses the effector phase and thus abets an extracellular ligand and no canonical ITIM. However, func- tumor escape. The observation that multiple distinct IRs tionally, these proteins (LAT, PAG, LIME, SIT, and TSAd) are expressed in TILs illustrates the apparent redundancy of may play a similar role as that of ITIM-bearing IRs in that by their potential functional regulation (CD5, CD85, CD94- binding to p56lck its kinase function may be repressed, therein NKG2A, and CD279). Curiously, postrecovery of TIL sig- having the equivalent inhibitory effect on TIL function as does naling and lytic function, the level of those IRs remained recruitment and activation of a PTP. unchanged in purified lytic TILs, arguing against a role for Several unanswered questions arise in consideration of the any of those IRs in mediating TIL signaling defects. A similar role of IRs in tumor escape from antitumor immune response. observation was made in a pathogen model wherein multiple For example, if tumors express ligands for IRs that are capable IRs were expressed in CD8+ T cells, but lytic function was not of mediating blockade of effector functions at the site of the inhibited (49), suggesting that if any of the IRs detected by tumor, why can adoptively transferred T cells eliminate tumor? our flow analysis influence signaling in nonlytic TILs, perhaps Although not definitively known, we hypothesize that priming the context of expression relative to target cell interaction may or culture conditions for T cells in vitro cause downregulation be important in their activity. This may be an important con- of IRs, thus permitting effector phase functions upon adoptive sideration because some IRs are expressed at elevated levels transfer. Another conundrum arises in consideration of why upon T cell activation. It is also possible that the IRs observed transplantable regressor tumors fail to grow. Again, although expressed in TILs do not function in the tumor environment; not known, we hypothesize that either regressor tumors lack perhaps instead their ligands are expressed on endothelial cells expression of IR ligands (and thus do not impede the effector where they can participate in restriction of the TIL lytic phase phase), or priming of antitumor immune response during early- (or on other cells that contact the TIL during its transit into stage growth of this class of tumor does not elicit expression of the tumor). Thus, it is possible that another as yet uniden- IRs. 7138 BRIEF REVIEWS: INHIBITION OF TUMOR-INFILTRATING T CELL FUNCTION

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