NKG2D-bispecific enhances NK and CD8+ T cell antitumor immunity.

BACKGROUND: Cancer immunotherapy approaches that elicit immune cell responses, including T and NK cells, have revolutionized the field of oncology. However, immunosuppressive mechanisms restrain immune cell activation within solid tumors so additional strategies to augment activity are required. METHODS: We identified the co-stimulatory receptor NKG2D as a target based on its expression on a large proportion of CD8+ tumor infiltrating lymphocytes (TILs) from breast cancer patient samples. Human and murine surrogate NKG2D co-stimulatory receptor-bispecifics (CRB) that bind NKG2D on NK and CD8+ T cells as well as HER2 on breast cancer cells (HER2-CRB) were developed as a proof of concept for targeting this signaling axis in vitro and in vivo. RESULTS: HER2-CRB enhanced NK cell activation and cytokine production when co-cultured with HER2 expressing breast cancer cell lines. HER2-CRB when combined with a T cell-dependent-bispecific (TDB) antibody that synthetically activates T cells by crosslinking CD3 to HER2 (HER2-TDB), enhanced T cell cytotoxicity, cytokine production and in vivo antitumor activity. A mouse surrogate HER2-CRB (mHER2-CRB) improved in vivo efficacy of HER2-TDB and augmented NK as well as T cell activation, cytokine production and effector CD8+ T cell differentiation. CONCLUSION: We demonstrate that targeting NKG2D with bispecific antibodies (BsAbs) is an effective approach to augment NK and CD8+ T cell antitumor immune responses. Given the large number of ongoing clinical trials leveraging NK and T cells for cancer immunotherapy, NKG2D-bispecifics have broad combinatorial potential.

Translational PK/PD and the first-in-human dose selection of a PD1/IL15: an engineered recombinant targeted cytokine for cancer immunotherapy.

Introduction: Interleukin 15 (IL-15) is a potential anticancer agent and numerous engineered IL-15 agonists are currently under clinical investigation. Selective targeting of IL-15 to specific lymphocytes may enhance therapeutic effects while helping to minimize toxicities. Methods: We designed and built a heterodimeric targeted cytokine (TaCk) that consists of an anti-programmed cell death 1 receptor antibody (anti-PD-1) and an engineered IL-15. This "PD1/IL15" selectively delivers IL-15 signaling to lymphocytes expressing PD-1. We then investigated the pharmacokinetic (PK) and pharmacodynamic (PD) effects of PD1/IL15 TaCk on immune cell subsets in cynomolgus monkeys after single and repeat intravenous dose administrations. We used these results to determine the first-in-human (FIH) dose and dosing frequency for early clinical trials. Results: The PD1/IL15 TaCk exhibited a nonlinear multiphasic PK profile, while the untargeted isotype control TaCk, containing an anti-respiratory syncytial virus antibody (RSV/IL15), showed linear and dose proportional PK. The PD1/IL15 TaCk also displayed a considerably prolonged PK (half-life range ∼1.0-4.1 days) compared to wild-type IL-15 (half-life ∼1.1 h), which led to an enhanced cell expansion PD response. The PD was dose-dependent, durable, and selective for PD-1+ lymphocytes. Notably, the dose- and time-dependent PK was attributed to dynamic TMDD resulting from test article-induced lymphocyte expansion upon repeat administration. The recommended first-in-human (FIH) dose of PD1/IL15 TaCk is 0.003 mg/kg, determined based on a minimum anticipated biological effect level (MABEL) approach utilizing a combination of in vitro and preclinical in vivo data. Conclusion: This work provides insight into the complex PK/PD relationship of PD1/IL15 TaCk in monkeys and informs the recommended starting dose and dosing frequency selection to support clinical evaluation of this novel targeted cytokine.

Anti-TIGIT antibody improves PD-L1 blockade through myeloid and Treg cells.

Tiragolumab, an anti-TIGIT antibody with an active IgG1κ Fc, demonstrated improved outcomes in the phase 2 CITYSCAPE trial (ClinicalTrials.gov: NCT03563716 ) when combined with atezolizumab (anti-PD-L1) versus atezolizumab alone1. However, there remains little consensus on the mechanism(s) of response with this combination2. Here we find that a high baseline of intratumoural macrophages and regulatory T cells is associated with better outcomes in patients treated with atezolizumab plus tiragolumab but not with atezolizumab alone. Serum sample analysis revealed that macrophage activation is associated with a clinical benefit in patients who received the combination treatment. In mouse tumour models, tiragolumab surrogate antibodies inflamed tumour-associated macrophages, monocytes and dendritic cells through Fcγ receptors (FcγR), in turn driving anti-tumour CD8+ T cells from an exhausted effector-like state to a more memory-like state. These results reveal a mechanism of action through which TIGIT checkpoint inhibitors can remodel immunosuppressive tumour microenvironments, and suggest that FcγR engagement is an important consideration in anti-TIGIT antibody development.

TIGIT-CD226-PVR axis: advancing immune checkpoint blockade for cancer immunotherapy.

Recent advances in understanding the roles of immune checkpoints in allowing tumors to circumvent the immune system have led to successful therapeutic strategies that have fundamentally changed oncology practice. Thus far, immunotherapies against only two checkpoint targets have been approved, CTLA-4 and PD-L1/PD-1. Antibody blockade of these targets enhances the function of antitumor T cells at least in part by relieving inhibition of the T cell costimulatory receptor CD28. These successes have stimulated considerable interest in identifying other pathways that may bte targeted alone or together with existing immunotherapies. One such immune checkpoint axis is comprised of members of the PVR/nectin family that includes the inhibitory receptor T cell immunoreceptor with Ig and immunoreceptor tyrosine-based inhibitory domains (TIGIT). Interestingly, TIGIT acts to regulate the activity of a second costimulatory receptor CD226 that works in parallel to CD28. There are currently over two dozen TIGIT-directed blocking antibodies in various phases of clinical development, testament to the promise of modulating this pathway to enhance antitumor immune responses. In this review, we discuss the role of TIGIT as a checkpoint inhibitor, its interplay with the activating counter-receptor CD226, and its status as the next advance in cancer immunotherapy.

Mechanistic convergence of the TIGIT and PD-1 inhibitory pathways necessitates co-blockade to optimize anti-tumor CD8+ T cell responses.

Dual blockade of the PD-1 and TIGIT coinhibitory receptors on T cells shows promising early results in cancer patients. Here, we studied the mechanisms whereby PD-1 and/or TIGIT blockade modulate anti-tumor CD8+ T cells. Although PD-1 and TIGIT are thought to regulate different costimulatory receptors (CD28 and CD226), effectiveness of PD-1 or TIGIT inhibition in preclinical tumor models was reduced in the absence of CD226. CD226 expression associated with clinical benefit in patients with non-small cell lung carcinoma (NSCLC) treated with anti-PD-L1 antibody atezolizumab. CD226 and CD28 were co-expressed on NSCLC infiltrating CD8+ T cells poised for expansion. Mechanistically, PD-1 inhibited phosphorylation of both CD226 and CD28 via its ITIM-containing intracellular domain (ICD); TIGIT's ICD was dispensable, with TIGIT restricting CD226 co-stimulation by blocking interaction with their common ligand PVR (CD155). Thus, full restoration of CD226 signaling, and optimal anti-tumor CD8+ T cell responses, requires blockade of TIGIT and PD-1, providing a mechanistic rationale for combinatorial targeting in the clinic.

Peripheral T cell expansion predicts tumour infiltration and clinical response.

Despite the resounding clinical success in cancer treatment of antibodies that block the interaction of PD1 with its ligand PDL11, the mechanisms involved remain unknown. A major limitation to understanding the origin and fate of T cells in tumour immunity is the lack of quantitative information on the distribution of individual clonotypes of T cells in patients with cancer. Here, by performing deep single-cell sequencing of RNA and T cell receptors in patients with different types of cancer, we survey the profiles of various populations of T cells and T cell receptors in tumours, normal adjacent tissue, and peripheral blood. We find clear evidence of clonotypic expansion of effector-like T cells not only within the tumour but also in normal adjacent tissue. Patients with gene signatures of such clonotypic expansion respond best to anti-PDL1 therapy. Notably, expanded clonotypes found in the tumour and normal adjacent tissue can also typically be detected in peripheral blood, which suggests a convenient approach to patient identification. Analyses of our data together with several external datasets suggest that intratumoural T cells, especially in responsive patients, are replenished with fresh, non-exhausted replacement cells from sites outside the tumour, suggesting continued activity of the cancer immunity cycle in these patients, the acceleration of which may be associated with clinical response.

CD96 functions as a co-stimulatory receptor to enhance CD8+ T cell activation and effector responses.

CD96 is a member of the poliovirus receptor (PVR, CD155)-nectin family that includes T cell Ig and ITIM domain (TIGIT) and CD226. While CD96, TIGIT, and CD226 have important roles in regulating NK cell activity, and TIGIT and CD226 have also been shown to regulate T cell responses, it is unclear whether CD96 has inhibitory or stimulatory function in CD8+ T cells. Here, we demonstrate that CD96 has co-stimulatory function on CD8+ T cells. Crosslinking of CD96 on human or mouse CD8+ T cells induced activation, effector cytokine production, and proliferation. CD96 was found to transduce its activating signal through the MEK-ERK pathway. CD96-mediated signaling led to increased frequencies of NUR77- and T-bet-expressing CD8+ T cells and enhanced cytotoxic effector activity, indicating that CD96 can modulate effector T cell differentiation. Antibody blockade of CD96 or genetic ablation of CD96 expression on CD8+ T cells impaired expression of transcription factors and proinflammatory cytokines associated with CD8+ T cell activation in in vivo models. Taken together, CD96 has a co-stimulatory role in CD8+ T cell activation and effector function.

Regulation of Tumor-Associated Myeloid Cell Activity by CBP/EP300 Bromodomain Modulation of H3K27 Acetylation.

Myeloid-derived suppressor cells (MDSCs) are found in most cancer malignancies and support tumorigenesis by suppressing immunity and promoting tumor growth. Here we identify the bromodomain (BRD) of CBP/EP300 as a critical regulator of H3K27 acetylation (H3K27ac) in MDSCs across promoters and enhancers of pro-tumorigenic target genes. In preclinical tumor models, in vivo administration of a CBP/EP300-BRD inhibitor (CBP/EP300-BRDi) alters intratumoral MDSCs and attenuates established tumor growth in immunocompetent tumor-bearing mice, as well as in MDSC-dependent xenograft models. Inhibition of CBP/EP300-BRD redirects tumor-associated MDSCs from a suppressive to an inflammatory phenotype through downregulation of STAT pathway-related genes and inhibition of Arg1 and iNOS. Similarly, CBP/EP300-BRDi decreases differentiation and suppressive function of human MDSCs in vitro. Our findings uncover a role of CBP/EP300-BRD in intratumoral MDSCs that may be targeted therapeutically to boost anti-tumor immunity.

CD226 regulates natural killer cell antitumor responses via phosphorylation-mediated inactivation of transcription factor FOXO1.

Natural killer (NK) cell recognition of tumor cells is mediated through activating receptors such as CD226, with suppression of effector functions often controlled by negative regulatory transcription factors such as FOXO1. Here we show that CD226 regulation of NK cell cytotoxicity is facilitated through inactivation of FOXO1. Gene-expression analysis of NK cells isolated from syngeneic tumors grown in wild-type or CD226-deficient mice revealed dysregulated expression of FOXO1-regulated genes in the absence of CD226. In vitro cytotoxicity and stimulation assays demonstrated that CD226 is required for optimal killing of tumor target cells, with engagement of its ligand CD155 resulting in phosphorylation of FOXO1. CD226 deficiency or anti-CD226 antibody blockade impaired cytotoxicity with concomitant compromised inactivation of FOXO1. Furthermore, inhibitors of FOXO1 phosphorylation abrogated CD226-mediated signaling and effector responses. These results define a pathway by which CD226 exerts control of NK cell responses against tumors.

TIGIT: A Key Inhibitor of the Cancer Immunity Cycle.

Immunotherapies that harness the activity of the immune system against tumors are proving to be an effective therapeutic approach in multiple malignancies. Indeed, through accumulation of genetic mutations, many tumors express antigens that can potentially elicit specific tumor immunity. However, tumors can also suppress these responses by activating negative regulatory pathways and checkpoints such as PD-1/PD-L1 and CTLA-4. Blocking these checkpoints on T cells has provided dramatic clinical benefit, but only a subset of patients exhibit clear and durable responses, suggesting that other mechanisms must be limiting the immune response. We discuss here the role of TIGIT, an inhibitory receptor expressed by lymphocytes, in limiting antitumor responses and we review its mechanisms of action during the cancer immunity cycle.

Dimerization of LTßR by LTα1ß2 is necessary and sufficient for signal transduction.

Homotrimeric TNF superfamily ligands signal by inducing trimers of their cognate receptors. As a biologically active heterotrimer, Lymphotoxin(LT)α1ß2 is unique in the TNF superfamily. How the three unique potential receptor-binding interfaces in LTα1ß2 trigger signaling via LTß Receptor (LTßR) resulting in lymphoid organogenesis and propagation of inflammatory signals is poorly understood. Here we show that LTα1ß2 possesses two binding sites for LTßR with distinct affinities and that dimerization of LTßR by LTα1ß2 is necessary and sufficient for signal transduction. The crystal structure of a complex formed by LTα1ß2, LTßR, and the fab fragment of an antibody that blocks LTßR activation reveals the lower affinity receptor-binding site. Mutations targeting each potential receptor-binding site in an engineered single-chain variant of LTα1ß2 reveal the high-affinity site. NF-κB reporter assays further validate that disruption of receptor interactions at either site is sufficient to prevent signaling via LTßR.

Controlling herpetic stromal keratitis by modulating lymphotoxin-alpha-mediated inflammatory pathways.

Herpes simplex virus 1 infection of the eye can result in stromal keratitis, a chronic immunoinflammatory lesion that is a significant cause of human blindness. A key to controlling the severity of lesions is to identify cellular and molecular events responsible for tissue damage. This report evaluates the role of lymphotoxin-α, a proinflammatory cytokine that could be involved during stromal keratitis. We demonstrate that after infection, both lymphotoxin-α and lymphotoxin-ß transcripts are detectable at high levels 48 h postinfection, suggesting roles for the secreted homotrimer lymphotoxin-α3 and the membrane-bound lymphotoxin-α1ß2 heterotrimer in stromal keratitis. Using a corneal stromal fibroblast cell line, lymphotoxin-α3 and lymphotoxin-α1ß2 were found to have proinflammatory roles by stimulating production of chemokines. Treatment of mice with a depleting anti-lymphotoxin-α mAb during the clinical phase of the disease significantly attenuated stromal keratitis lesions. In treated mice, expression of proinflammatory molecules and chemokines was reduced, as were numbers of cornea-infiltrating proinflammatory cells, particularly Th1 cells. The protective effect of anti-lymphotoxin-α mAb was highly reduced with a mutant version of the mAb that lacks Fc receptor binding activity, indicating that depletion of lymphotoxin-expressing cells was mainly responsible for efficacy, with LT-α3 contributing minimally to inflammation. These data demonstrate that lymphotoxin-expressing cells, such as Th1 cells, mediate stromal keratitis.

In vivo depletion of lymphotoxin-alpha expressing lymphocytes inhibits xenogeneic graft-versus-host-disease.

Graft-versus-host disease (GVHD) is a major barrier to successful allogeneic hematopoietic cell transplantation and is largely mediated by activated donor lymphocytes. Lymphotoxin (LT)-α is expressed by subsets of activated T and B cells, and studies in preclinical models demonstrated that targeted depletion of these cells with a mouse anti-LT-α monoclonal antibody (mAb) was efficacious in inhibiting inflammation and autoimmune disease. Here we demonstrate that LT-α is also upregulated on activated human donor lymphocytes in a xenogeneic model of GVHD and targeted depletion of these donor cells ameliorated GVHD. A depleting humanized anti-LT-α mAb, designated MLTA3698A, was generated that specifically binds to LT-α in both the soluble and membrane-bound forms, and elicits antibody-dependent cellular cytotoxicity (ADCC) activity in vitro. Using a human peripheral blood mononuclear cell transplanted SCID (Hu-SCID) mouse model of GVHD, the anti-human LT-α mAb specifically depleted activated LT-expressing human donor T and B cells, resulting in prolonged survival of the mice. A mutation in the Fc region, rendering the mAb incapable of mediating ADCC, abolished all in vitro and in vivo effects. These data support a role for using a depleting anti-LT-α antibody in treating immune diseases such as GVHD and autoimmune diseases.

Immune complex-mediated cell activation from systemic lupus erythematosus and rheumatoid arthritis patients elaborate different requirements for IRAK1/4 kinase activity across human cell types.

IL-1R-associated kinases (IRAKs) are important mediators of MyD88-dependent signaling by the TLR/IL-1R superfamily and facilitate inflammatory responses. IRAK4 and IRAK1 function as active kinases and as scaffolds for protein-protein interactions. We report that although IRAK1/4 kinase activity is essential for human plasmacytoid dendritic cell (pDC) activation, it is dispensable in B, T, dendritic, and monocytic cells, which is in contrast with an essential active kinase role in comparable mouse cell types. An IRAK1/4 kinase inhibitor abrogated TLR7/9-induced IFN-α responses in both mouse and human pDCs, but other human immune cell populations activated via TLR7/9 or IL-1R were refractory to IRAK4 kinase inhibition. Gene ablation experiments using small interfering RNA demonstrated an essential scaffolding role for IRAK1 and IRAK4 in MyD88-dependent signaling. Finally, we demonstrate that autoimmune patient (systemic lupus erythematosus and rheumatoid arthritis) serum activates both pDC and B cells, but IRAK1/4 kinase inhibition affects only the pDC response, underscoring the differential IRAK1/4 functional requirements in human immune cells. These data reveal important species differences and elaborate cell type requirements for IRAK1/4 kinase activity.

Targeted depletion of lymphotoxin-alpha-expressing TH1 and TH17 cells inhibits autoimmune disease.

Uncontrolled T helper type 1 (T(H)1) and T(H)17 cells are associated with autoimmune responses. We identify surface lymphotoxin-alpha (LT-alpha) as common to T(H)0, T(H)1 and T(H)17 cells and employ a unique strategy to target these subsets using a depleting monoclonal antibody (mAb) directed to surface LT-alpha. Depleting LT-alpha-specific mAb inhibited T cell-mediated models of delayed-type hypersensitivity and experimental autoimmune encephalomyelitis. In collagen-induced arthritis (CIA), preventive and therapeutic administration of LT-alpha-specific mAb inhibited disease, and immunoablated T cells expressing interleukin-17 (IL-17), interferon-gamma and tumor necrosis factor-alpha (TNF-alpha), whereas decoy lymphotoxin-beta receptor (LT-betaR) fusion protein had no effect. A mutation in the Fc tail, rendering the antibody incapable of Fcgamma receptor binding and antibody-dependent cellular cytotoxicity activity, abolished all in vivo effects. Efficacy in CIA was preceded by a loss of rheumatoid-associated cytokines IL-6, IL-1beta and TNF-alpha within joints. These data indicate that depleting LT-alpha-expressing lymphocytes with LT-alpha-specific mAb may be beneficial in the treatment of autoimmune disease.

The role of structurally conserved class I MHC in tumor rejection: contribution of the Q8 locus.

The mouse multimember family of Qa-2 oligomorphic class I MHC genes is continuously undergoing duplications and deletions that alter the number of the two "prototype" Qa-2 sequences, Q8 and Q9. The frequent recombination events within the Q region lead to strain-specific modulation of the cumulative Qa-2 expression levels. Q9 protects C57BL/6 hosts from multiple disparate tumors and functions as a major CTL restriction element for shared tumor-associated Ags. We have now analyzed functional and structural properties of Q8, a class I MHC that differs significantly from Q9 in the peptide-binding, CTL-interacting alpha(1) and alpha(2) regions. Unexpectedly, we find that the extracellular domains of Q8 and Q9 act similarly during primary and secondary rejection of tumors, are recognized by cross-reactive antitumor CTL, have overlapping peptide-binding motifs, and are both assembled via the transporter associated with the Ag processing pathway. These findings suggest that shared Ag-presenting functions of the "odd" and "even" Qa-2 loci may contribute to the selective pressures shaping the haplotype-dependent quantitative variation of Qa-2 protein expression.

Protective immunity against disparate tumors is mediated by a nonpolymorphic MHC class I molecule.

Current peptide-based immunotherapies for treatment of model cancers target tumor Ags bound by the classical MHC class I (class Ia) molecules. The extensive polymorphism of class Ia loci greatly limits the effectiveness of these approaches. We demonstrate in this study that the murine nonpolymorphic, nonclassical MHC class I (class Ib) molecule Q9 (Qa-2) promotes potent immune responses against multiple syngeneic tumors. We have previously shown that ectopic expression of Q9 on the surface of class Ia-negative B78H1 melanoma led to efficient CTL-mediated rejection of this tumor. In this study, we report that surface-expressed Q9 on 3LLA9F1 Lewis lung carcinoma and RMA T cell lymphoma also induces potent antitumor CTL responses. Importantly, CTL harvested from animals surviving the initial challenge with Q9-positive 3LLA9F1, RMA, or B78H1 tumors recognized and killed their cognate tumors as well as the other cancer lines. Furthermore, immunization with Q9-expressing 3LLA9F1 or RMA tumor cells established immunological memory that enhanced protection against subsequent challenge with a weakly immunogenic, Q9-bearing melanoma variant. Collectively, the generation of cross-reactive CTL capable of eliminating multiple disparate Q9-expressing tumors suggests that this nonpolymorphic MHC class I molecule serves as a restriction element for a shared tumor Ag(s) common to lung carcinoma, T cell lymphoma, and melanoma.

A nonclassical MHC class I molecule restricts CTL-mediated rejection of a syngeneic melanoma tumor.

Although CTL and polymorphic, classical MHC class I molecules have well defined roles in the immune response against tumors, little is currently known regarding the participation of nonpolymorphic, nonclassical MHC class I in antitumor immunity. Using an MHC class I-deficient melanoma as a model tumor, we demonstrate that Q9, a murine MHC class Ib molecule from the Qa-2 family, expressed on the surface of tumor cells, protects syngeneic hosts from melanoma outgrowth. Q9-mediated protective immunity is lost or greatly diminished in mice deficient in CTL, including beta(2)-microglobulin knockout (KO), CD8 KO, and SCID mice. In contrast, the Q9 antitumor effects are not detectably suppressed in CD4 KO mice with decreased Th cell activity. Killing by antitumor CTL in vitro is Q9 specific and can be blocked by anti-Q9 and anti-CD8 Abs. The adaptive Q9-restricted CTL response leads to immunological memory, because mice that resist the initial tumor challenge reject subsequent challenges with less immunogenic tumor variants and show expansion of CD8(+) T cell populations with an activated/memory CD44(high) phenotype. Collectively, these studies demonstrate that a MHC class Ib molecule can serve as a restriction element for antitumor CTL and mediate protective immune responses in a syngeneic setting.

Correction of defects responsible for impaired Qa-2 class Ib MHC expression on melanoma cells protects mice from tumor growth.

One of the principal mechanisms of tumor immune evasion is alteration of class I MHC expression. We have identified defects contributing to down-regulation of class I MHC expression in the widely studied murine B16 melanoma and its variants B16F1, B16F10, BL6-2, BL6-8 and B78H1. Transcription of the nonclassical class I MHC genes Q8 and Q9 (Qa-2 Ags) has been switched off in the entire panel of melanoma lines, suggesting that this event occurred early during tumor progression. B78H1, unlike B16F1 and B16F10 sublines, is also selectively devoid of TAP2 and low molecular weight protein 7 as well as classical class I MHC K(b) and D(b) transcripts. Cotransfection of B78H1 with TAP2 and class I H chain genes is sufficient to reconstitute surface expression of exogenously delivered class I MHC without concomitant re-expression of endogenous beta(2)-microglobulin-associated class I. The serological absence of endogenous class Ia and Ib at the surface of TAP2-negative as well as TAP2-transfected B78H1 makes this system a suitable model for studying the properties of isolated class I proteins in tumors. We used this system to demonstrate that B78H1 cells genetically manipulated to re-express Q9 Ag have reduced tumor potential in syngeneic B6 mice compared with TAP2-transfected parental melanoma. Both NK cells and CTLs appear to collaborate in restraining growth of Q9-positive tumors. The results implicate Qa-2 in antitumor responses and illustrate the utility of the B78H1 system for identifying in vivo interactions between class I MHC molecules of interest and immune cells of innate and/or adaptive immunity.

The nonclassical major histocompatibility complex molecule Qa-2 protects tumor cells from NK cell- and lymphokine-activated killer cell-mediated cytolysis.

The cytotoxic activity of NK cells is regulated by class I MHC proteins. Although much has been learned about NK recognition of class I autologous targets, the mechanisms of NK self-tolerance are poorly understood. To examine the role of a nonpolymorphic, ubiquitously expressed class Ib Ag, Q9, we expressed it on class I-deficient and NK-sensitive B78H1 melanoma. Presence of this Qa-2 family member on tumor cells partially protected targets from lysis by bulk lymphokine-activated killer (LAK) cells. H-2K(b)-expressing B78H1 targets also reduced LAK cell activity, while H-2D(b) offered no protection. Importantly, blocking with F(ab')(2) specific for Q9 or removal of this GPI-attached molecule by phospholipase C cleavage restored killing to the level of vector-transfected cells. Experiments with LAK cells derived from H2(b) SCID and B6 mice established that NK1.1(+)TCR(-) NK and NK1.1(+)TCR(+) LAK cells were the prevalent cytolytic populations inhibitable by Q9. Treatment of mice with poly(I:C) also resulted in generation of Q9-regulated splenic cytotoxicity. LAK cells from different mouse strains responded to Q9, suggesting that the protective effect of this molecule is not detectably influenced by Ly49 polymorphisms or the presence/absence of Q9 in NK-harboring hosts. We propose that Q9 expressed on melanoma cells serves as a ligand for yet unidentified NK inhibitory receptor(s) expressed on NK1.1(+) NK/T cells.