Expression of immunoreceptor tyrosine-based activation motif (ITAM)-containing signaling proteins is normally restricted to hematopoietic tissues. The basal activity of ITAM-containing proteins is mediated through negative regulation by coreceptors restricted to hematopoietic tissues. We have identified an ITAM signaling domain encoded within the env gene of murine mammary tumor virus (MMTV). Three-dimensional structures derived in vitro from murine cells stably transfected with MMTV env display a depolarized morphology in comparison with control mammary epithelial cells. This effect is abolished by Y>F substitution within the Env ITAM, as well as inhibitors of Syk and Src protein tyrosine kinases. Env-expressing cells bear hallmarks of cell transformation such as sensitivity to apoptosis induced by tumor necrosis factor (TNF)–related apoptosis-inducing ligand (TRAIL) or TNFα, as well as down-regulation of E-cadherin and Keratin-18. Human normal mammary epithelial cells expressing MMTV Env also develop transformed phenotype, as typified by growth in soft agar and Matrigel invasion. These disruptions are abrogated by Y>F substitutions. We conclude that ITAM-dependent signals are generated through MMTV Env and trigger early hallmarks of transformation of mouse and human mammary epithelial cells. Therefore, these data suggest a heretofore unappreciated potential mechanism for the initiation of breast cancer and identify MMTV Env and ITAM-containing proteins in human breast tumors as probable oncoproteins.

Highlights d Early loss of PD-1 leads to overactivation of CD8 + T cells during acute infection d Mice constitutively lacking PD-1 or PD-L develop impaired CD8 + T cell memory d Cell-intrinsic PD-1 signals suppress effector cell expansion and promote memory d Timing of PD-1 blockade determines impact on memory generation

IntroductionBoth normal hematopoiesis and lymphomagenesis are driven, to a large extent, by expression of lineage-specific transcription factors. Many such factors are overtly oncogenic. For example, the hallmark of Burkitt lymphoma and some diffuse large B cell lymphomas (DLBCLs) is the t(8;14) translocation that places the MYC protooncogene under control of the Ig heavy chain gene enhancer (1, 2); a similar translocation has been identified in murine plasmacytomas (3). The causative role of Myc in B-lymphomagenesis has been validated through the generation of various transgenic (4-7) and nontransgenic (8) murine models of non-Hodgkin lymphoma.Other transcription factors are not known to initiate B-lymphomagenesis, but are consistently mutated in full-fledged neoplasms. One such intriguing protein is paired box gene 5 (Pax5), a B cell activator protein (9) with a characteristic paired box DNA-binding motif (10). Inactivation of Pax5 via homologous recombination precludes normal B cell development (11,12), and several lines of evidence implicate Pax5 as an important factor in B-lymphomagenesis as well.

Immunoreceptor tyrosine-based activation motifs (ITAMs) are involved in the transduction of signals necessary for activation, differentiation, and survival in hematopoietic cells. Several viruses have been shown to encode ITAM-containing transmembrane proteins. Although expression of these viral proteins has in some cases been shown to transform nonhematopoietic cells, a causal role for a functional ITAM in this process has not been elucidated. To examine the potential transforming properties of ITAM-containing proteins, a recombinant protein consisting of ITAM-containing cytoplasmic regions of the B-cell antigen receptor was expressed in immortalized murine mammary epithelial and fibroblast cells. Mammary epithelial cells expressing this construct exhibited depolarized morphology in three-dimensional cultures. This transformed phenotype was characterized by a loss of anchorage dependence and hallmarks of epithelial to mesenchymal transition. Fibroblasts expressing this ITAM construct also lost contact inhibition and anchorage dependence. The transformed phenotype seen in both cell types was abrogated upon tyrosine to phenylalanine substitutions of the ITAMs. Inhibition of Syk tyrosine kinase, which associates with the ITAM, also prevented cell transformation. Our results indicate that expression of a nonviral ITAM-containing protein is sufficient for cell transformation. Despite lacking intrinsic enzymatic activity, ITAM-containing proteins can function as potent oncoproteins by scaffolding downstream mediators.

CD160 promotes NK cell cytotoxicity and IFN-g production, but the function of CD160 on CD8 + T cells remains unclear with some studies supporting a coinhibitory role and others a costimulatory role. In this study, we demonstrate that CD160 has a costimulatory role in promoting CD8 + T cell effector functions needed for optimal clearance of oral Listeria monocytogenes infection. CD160 2/2 mice did not clear oral L. monocytogenes as efficiently as wild type (WT) littermates. WT RAG 2/2 and CD160 2/2 RAG 2/2 mice similarly cleared L. monocytogenes, indicating that CD160 on NK cells does not contribute to impaired L. monocytogenes clearance. Defective L. monocytogenes clearance is due to compromised intraepithelial lymphocytes and CD8 + T cell functions. There was a reduction in the frequencies of granzyme B-expressing intraepithelial lymphocytes in L. monocytogenes-infected CD160 2/2 mice as compared with WT littermate controls. Similarly, the frequencies of granzyme B-expressing splenic CD8 + T cells and IFN-g and TNF-a double-producer CD8 + T cells were significantly reduced in L. monocytogenes-infected CD160 2/2 mice compared with WT littermates. Adoptive transfer studies showed that RAG 2/2 recipients receiving CD160 2/2 CD8 + T cells had a higher mortality, exhibited more weight loss, and had a higher bacterial burden compared with RAG 2/2 recipients receiving WT CD8 + T cells. These findings demonstrate that CD160 provides costimulatory signals to CD8 + T cells needed for optimal CD8 + T cell responses and protective immunity during an acute mucosal bacterial infection. ImmunoHorizons, 2018, 2: 238-250.

Patients treated with chimeric antigen receptor (CAR) T cells targeting CD19 for B cell malignancies have experienced rapid and durable tumor regressions. Manufacture of CAR T cells is challenged by the necessity to produce a unique drug product for each patient. Each treatment requires ex vivo culture of patient T cells to facilitate CAR gene transfer and to achieve therapeutic amounts of T cells. Paradoxically, ex vivo culture with IL-2 also decreases CAR T cell activity. Some investigators have proposed isolating central memory T cells (thought to be enriched for therapeutic T cells), yet isolation techniques are cumbersome and costly to scale commercially. Culture of T cells in IL-7 and IL-15 has also been shown by several investigators to improve therapeutic activity. Here we explored the potential for culture modifications to improve the therapeutic potential of CAR T cells without adding complexity to manufacturing. We tested this hypothesis using CAR T cells specific to B cell maturation antigen (BCMA) manufactured using standard IL-2 culture with an inhibitor of PI3K added to the media, or with IL-7 and IL-15 in place of IL-2. The in vivo activity was studied in NSG mouse models of human Burkitt's lymphoma (Daudi), and multiple myeloma (RPMI-8226), both of which express BCMA. In the lymphoma model, NSG mice were injected intravenously (IV) with 2 x 106 Daudi cells and allowed to accumulate a large tumor burden before being treated with 4 x 106 CAR+ T cells on day 18 post-tumor injection. At this late time point post implantation, mice had highly disseminated Daudi tumor (our goal was to model late stage disease observed in relapsed and refractory lymphoma). In this model of advanced disease, IL-2 cultured anti-BCMA CAR T cells had no effect on tumor growth (p = 0.22) and all mice succumbed to the tumors within two weeks after treatment. Anti-BCMA CAR T cells grown in IL-7 and IL-15 also failed to control tumor growth (p = 0.23). In sharp contrast, all animals treated with anti-BCMA CAR T cells cultured with the PI3K inhibitor survived and experienced complete long-term tumor regression (p=0.003). The same anti-BCMA CAR T cells were used in a model of multiple myeloma. NSG mice were injected subcutaneously (SC) with 107 RPMI-8226 MM cells, and at 22 days post-implantation mice received a single IV administration of anti-BCMA CAR T cells (4 x 105 CAR+ T cells/mouse) cultured under various conditions. In this model, all treatment groups demonstrated tumor regression, regardless of the in vitro culture conditions. To evaluate CAR T cell durability, two weeks after initial tumor clearance, surviving animals were then re-challenged with RPMI-8226 cells on the opposite flank to model tumor relapse. We found that only animals that had been treated with anti-BCMA CAR T cells cultured with PI3K inhibition were immune to subsequent tumor challenge (p=0.005). Given the superior in vivo efficacy of anti-BCMA CAR T cells cultured with PI3K inhibition, we sought to identify phenotypic characteristics associated with the improved therapeutic activity. Anti-BCMA CAR T cells cultured with PI3K inhibition contained an increased frequency of CD62L+ CD8 T cells in the final product (p < 0.001) suggesting improved expansion of a distinct CD8 T cell subset. These data suggest that inhibition of PI3K during ex vivo expansion with IL-2 may generate a superior anti-BCMA CAR T cell product for clinical use. Furthermore, this approach could potentially be used in the manufacture of other T cell therapies. Disclosures Perkins: bluebird bio: Employment, Equity Ownership. Grande:bluebird bio: Employment, Equity Ownership. Hamel:bluebird bio: Employment, Equity Ownership. Horton:bluebird bio: Employment, Equity Ownership. Garrett:bluebird bio: Employment, Equity Ownership. Miller:bluebird bio: Employment, Equity Ownership. Latimer:bluebird bio: Employment, Equity Ownership. Horvath:bluebird bio: Employment, Equity Ownership. Kuczewski:bluebird bio: Employment, Equity Ownership. Friedman:bluebird bio: Employment, Equity Ownership. Morgan:bluebird bio: Employment, Equity Ownership.

The presence of an immunoreceptor tyrosine-based activation motif (ITAM) makes immunoreceptors different from other signaling receptors, like integrins, G-coupled protein receptors, chemokine receptors, and growth factor receptors. This unique motif has the canonical sequence D/Ex(0-2)YxxL/Ix(6-8)YxxL/I, where x represents any amino acid and is present at least once in all immunoreceptor complexes. Immunoreceptors can promote survival, activation, and differentiation by transducing signals through these highly conserved motifs. Traditionally, ITAM signaling is thought to occur in response to ligand-induced aggregation, although evidence indicates that ligand-independent tonic signaling also provides functionally relevant signals. The majority of proteins containing ITAMs are transmembrane proteins that exist as part of immunoreceptor complexes. However, oncogenic viruses also have ITAM-containing proteins. In this review, we discuss what is known about tonic signaling by both cellular and viral ITAM-containing proteins and speculate what we might learn from each context.

Introduction: B-cell maturation antigen (BCMA) is primarily expressed by malignant and normal plasma cells, making it an attractive target for the treatment of multiple myeloma (MM). bb21217 is a BCMA-directed chimeric antigen receptor (CAR) T cell therapy that uses the same CAR molecule as idecabtagene vicleucel (ide-cel, bb2121), but adds the PI3K inhibitor bb007 during manufacturing to enrich the drug product (DP) for memory-like T cells, thereby reducing the proportion of highly differentiated or senescent T cells. We conducted correlative analyses to investigate the mechanistic hypothesis that CAR+ T cells with memory like phenotypes may persist and function longer, which may be one determinant of duration of response (DOR). Methods: An ongoing phase I clinical study (CRB-402; NCT03274219) is assessing safety and efficacy of bb21217 in relapsed/refractory MM patients. A total of 44 patients had PBMCs, collected from apheresis, and DP characterized by RNA sequencing (RNAseq) and mass Cytometry (CyTOF). The correlation of T cell phenotype with peak expansion, response and DOR per IMWG Uniform Response Criteria was explored. P-values were determined by Wilcoxon test, Spearman correlation, or Cox PH regression on DOR with categorical marker values (high/low). Results: In this patient population, substantial cross patient heterogeneity in T cell phenotypes was observed both in PBMCs and DP. Late differentiation/senescent markers in PBMCs were negatively correlated with clinical response. In particular, patients whose DP had higher expression of CD57 had lower peak expansion (p&lt;0.0001), experienced more early relapse by month 6 (M6) (p&lt;0.05) and had lower DOR (p&lt;0.05) compared to those with lower CD57 expression. Paired analysis of PBMCs and DP demonstrated bb21217 DP is enriched for memory-like T cells (LEF-1+ (median increase 310%, p&lt;0.0001), CD27+ (median increase 84.7%, p&lt;0.0001), CCR7+ (median increase 188.3%, p&lt;0.0001) and depleted of highly differentiated or senescent CD57+ T cells (median decrease 87.3%, p&lt;0.0001), relative to PBMCs. As CAR+ T cell peak expansion is associated with initial clinical response, we investigated the relationship between peak expansion and DP phenotype. Expression of early memory T cell markers (eg, LEF1, CD27, CCR7) in DP were positively correlated with peak expansion, while markers of terminally differentiated effector cell markers in DP (eg, CD57, GZMA, GZMB) were negatively correlated with peak expansion. RNAseq also showed a significant enrichment in naïve/early memory gene signatures and a decrease in late differentiation gene signatures in DP from patients with high peak expansion (&gt;2x105copies/ug), consistent with the CYTOF findings. Early activation markers (CD38 p&lt;0.001, IRF4 p&lt;0.01) were expressed in the DP from patients with high peak expansion, suggesting that an early activation phenotype may contribute to robust expansion. We assessed the relationship between T cell memory markers in DP and sustained response by comparing DP from patients with or without progressive disease (PD) by M6. RNAseq analysis showed a significant enrichment for early memory (CCR7, p&lt;0.05, SELL, p&lt;0.05) and early activation (CD38 p&lt;0.05, IRF4 p&lt;0.05) T cell phenotypes and a significant reduction in late differentiation/senescence (CD57 p&lt;0.05, GZMB p&lt;0.01. KLRG1 p&lt;0.01) T cell phenotypes in DP from patients without PD at M6. Gene set enrichment analysis showed enrichment of naïve/memory gene signatures in patients without PD at M6. ScRNAseq analyses from a subset of PBMC/DP as well as immunophenotyping data from clinical samples, including memory phenotypes, and their relationship to clinical outcome, will also be presented. Conclusion: As seen with other CAR T cell studies, the quality of incoming PBMCs, in particular the fraction of T cells with a late differentiation/senescent phenotype, influences initial and sustained clinical response. The analyses reported here support the mechanistic hypothesis of bb21217, suggesting the presence of early memory like T cells in PBMC and/or DP may contribute to high peak expansion and prolonged DOR, while presence of highly differentiated or senescent T cells may negatively impact these measures. Further clinical evaluation of bb21217 and robust correlative analyses will be important to help contextualize the influence of patient and product characteristics on clinical outcomes. Disclosures Finney: bluebird bio: Current Employment, Current equity holder in publicly-traded company. Yeri:bluebird bio: Current Employment, Current equity holder in publicly-traded company. Mao:bluebird bio: Current Employment, Current equity holder in publicly-traded company. Pandya:bluebird bio: Current Employment, Current equity holder in publicly-traded company. Alonzo:bluebird bio: Current Employment, Current equity holder in publicly-traded company. Hopkins:bluebird bio: Current Employment, Current equity holder in publicly-traded company. Hymson:bluebird bio: Current Employment, Current equity holder in publicly-traded company. Hu:bluebird bio: Current Employment, Current equity holder in publicly-traded company. Foos:bluebird bio: Current Employment, Current equity holder in publicly-traded company. Bhadoriya:bluebird bio: Current Employment, Current equity holder in publicly-traded company. Hintzen:bluebird bio: Current Employment, Current equity holder in publicly-traded company. Gioia:bluebird bio: Current Employment, Current equity holder in publicly-traded company. Timm:bluebird bio: Current Employment, Current equity holder in publicly-traded company. Massaro:bluebird bio: Current Employment, Current equity holder in publicly-traded company. Hause:Bristol-Myers Squibb Company: Current Employment, Current equity holder in publicly-traded company. Kaiser:BMS: Current Employment, Current equity holder in publicly-traded company. Martin:BMS: Current Employment, Current equity holder in publicly-traded company. Shah:BMS, Janssen, Bluebird Bio, Sutro Biopharma, Teneobio, Poseida, Nektar: Research Funding; GSK, Amgen, Indapta Therapeutics, Sanofi, BMS, CareDx, Kite, Karyopharm: Consultancy. Raje:Caribou: Membership on an entity's Board of Directors or advisory committees; Astrazeneca: Consultancy; Karyopharm: Consultancy; Janssen: Consultancy; Celgene: Consultancy; BMS: Consultancy; Immuneel: Membership on an entity's Board of Directors or advisory committees; Takeda: Consultancy; Bluebird, Bio: Consultancy, Research Funding; Amgen: Consultancy. Berdeja:Novartis: Research Funding; Lilly: Research Funding; Legend: Consultancy; Takeda: Consultancy, Research Funding; Servier: Consultancy; Teva: Research Funding; Vivolux: Research Funding; Bluebird: Research Funding; Acetylon: Research Funding; Amgen: Consultancy, Research Funding; Abbvie: Research Funding; BMS: Consultancy, Research Funding; Bioclinica: Consultancy; CRISPR Therapeutics: Consultancy, Research Funding; Constellation: Research Funding; Cellularity: Research Funding; Celgene: Consultancy, Research Funding; Glenmark: Research Funding; Genentech, Inc.: Research Funding; EMD Sorono: Research Funding; CURIS: Research Funding; Kite Pharma: Consultancy; Kesios: Research Funding; Karyopharm: Consultancy; Janssen: Consultancy, Research Funding; Prothena: Consultancy; Poseida: Research Funding. Grande:bluebird bio: Current Employment, Current equity holder in publicly-traded company. Bitter:Novartis AG, Predicant Biosciences, Biospect, F Hofmann-La Roche: Ended employment in the past 24 months; bluebird bio: Current Employment, Current equity holder in publicly-traded company; Novartis: Ended employment in the past 24 months, Patents & Royalties. Petrocca:bluebird, bio: Current Employment, Current equity holder in publicly-traded company. Friedman:bluebird bio: Current Employment, Current equity holder in publicly-traded company. Sangurdekar:bluebird bio: Current Employment, Current equity holder in publicly-traded company; Biogen: Ended employment in the past 24 months.