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This study sought to understand how the programmed death ligand 1 (PD-L1) inhibitor durvalumab and the immunomodulatory agent pomalidomide regulate immune cell activation and function in patients with relapsed/refractory (RR) multiple myeloma (MM). Immunologic changes in peripheral blood and bone marrow of patients treated with durvalumab as monotherapy or in combination with pomalidomide with/without dexamethasone were characterized by assessing subsets of immune cells and gene signatures to understand the immunomodulatory effect of the treatment. Soluble PD-L1 levels were elevated at screening in patients with RRMM but did not correlate with response to durvalumab combination therapy. Immune cell subsets were increased in peripheral blood during treatment with durvalumab and pomalidomide, and combination therapy induced significant gene expression changes in the MM tumor microenvironment versus durvalumab alone. Estimation of cell populations based on RNA sequencing data revealed increased monocytes, neutrophils, and natural killer cells with the combination therapy, but not with durvalumab alone. Additionally, multiplex immunofluorescence of bone marrow demonstrated that immune populations were different in responders versus nonresponders to durvalumab plus pomalidomide with dexamethasone therapy. Overall, durvalumab effectively blocked soluble PD-L1; however, durvalumab monotherapy was not associated with immunologic changes, which were observed with combination therapy.

Background The tumor microenvironment (TME) is increasingly appreciated as an important determinant of cancer outcome, including in multiple myeloma (MM). However, most myeloma microenvironment studies have been based on bone marrow (BM) aspirates, which often do not fully reflect the cellular content of BM tissue itself. To address this limitation in myeloma research, we systematically characterized the whole bone marrow (WBM) microenvironment during premalignant, baseline, on treatment, and post-treatment phases. Methods and findings Between 2004 and 2019, 998 BM samples were taken from 436 patients with newly diagnosed MM (NDMM) at the University of Arkansas for Medical Sciences in Little Rock, Arkansas, United States of America. These patients were 61% male and 39% female, 89% White, 8% Black, and 3% other/refused, with a mean age of 58 years. Using WBM and matched cluster of differentiation (CD)138-selected tumor gene expression to control for tumor burden, we identified a subgroup of patients with an adverse TME associated with 17 fewer months of progression-free survival (PFS) (95% confidence interval [CI] 5–29, 49–69 versus 70–82 months, χ2 p = 0.001) and 15 fewer months of overall survival (OS; 95% CI –1 to 31, 92–120 versus 113–129 months, χ2 p = 0.036). Using immunohistochemistry-validated computational tools that identify distinct cell types from bulk gene expression, we showed that the adverse outcome was correlated with elevated CD8+ T cell and reduced granulocytic cell proportions. This microenvironment develops during the progression of premalignant to malignant disease and becomes less prevalent after therapy, in which it is associated with improved outcomes. In patients with quantified International Staging System (ISS) stage and 70-gene Prognostic Risk Score (GEP-70) scores, taking the microenvironment into consideration would have identified an additional 40 out of 290 patients (14%, premutation p = 0.001) with significantly worse outcomes (PFS, 95% CI 6–36, 49–73 versus 74–90 months) who were not identified by existing clinical (ISS stage III) and tumor (GEP-70) criteria as high risk. The main limitations of this study are that it relies on computationally identified cell types and that patients were treated with thalidomide rather than current therapies. Conclusions In this study, we observe that granulocyte signatures in the MM TME contribute to a more accurate prognosis. This implies that future researchers and clinicians treating patients should quantify TME components, in particular monocytes and granulocytes, which are often ignored in microenvironment studies., Author summary Why was this study done? The cells around a tumor, also known as the tumor microenvironment (TME), can help a tumor grow by suppressing the immune system or fight a tumor by mounting an immune response. Most studies of multiple myeloma (MM) have focused on the tumor itself, rather than the bone marrow (BM) TME in which the tumor is growing. We hypothesized that the MM TME held clues that could help us better treat patients. What did the researchers do and find? We used a gene-expression–based computational technique to determine which cell types were present in patient BM. Patients with BM lacking a family of innate immune cells called granulocytes presented with worse outcomes compared to other patients. As MM progresses from a predisease to a cancerous state, the percentage of granulocytes decreases; the patients with the fewest granulocytes had more serious diseases. What do these findings mean? If granulocytes help myeloma patients respond to therapy, then addressing the decline in granulocytes may improve MM treatment. Patients with MM and few granulocytes in their BM should be watched for worse outcomes.

Simple Summary The CD38-targeting antibody daratumumab has marked activity in multiple myeloma through direct anti-tumor effects and immunomodulatory activity. However, eventually most patients will develop daratumumab-refractory disease. We hypothesized that daratumumab-resistance could be reversed by the addition of an inhibitor of the PD-1/PD-L1 signaling pathway, resulting in improved T- and NK-cell mediated anti-tumor immune responses. We therefore performed a phase 2 study to investigate the efficacy and safety of adding the PD-L1 checkpoint inhibitor durvalumab to daratumumab at the time of daratumumab failure. The toxicity profile of the daratumumab/durvalumab combination was acceptable, but none of the 18 enrolled patients achieved a clinical response. Immunomonitoring of bone marrow samples at baseline and during treatment showed a reduction of regulatory T-cell numbers and a decrease in the proportion of T-cells expressing LAG3 and CD8+ T-cells expressing TIM-3, whereas tumor cell characteristics were not affected. These results indicate that co-targeting PD-L1 at the time of daratumumab failure is insufficient to reverse daratumumab-resistance. Abstract Daratumumab is active both as a single agent and in combination with other agents in multiple myeloma (MM) patients. However, the majority of patients will develop daratumumab-refractory disease, which carries a poor prognosis. Since daratumumab also has immunomodulatory effects, addition of the PD-L1 blocking antibody durvalumab at the time of progression may reverse daratumumab-resistance. The efficacy and safety of daratumumab and durvalumab in daratumumab-refractory relapsed/refractory MM patients was evaluated in this prospective, single-arm phase 2 study (NCT03000452). None of the 18 enrolled patients achieved PR or better. The frequency of serious adverse events was 38.9%, with one patient experiencing an immune related adverse event (grade 2 hyperthyroidism). No infusion-related reactions were observed. Analysis of tumor- and immune cell characteristics was performed on bone marrow samples obtained at baseline and during treatment. Daratumumab combined with durvalumab reduced the frequency of regulatory T-cells and decreased the proportion of T-cells expressing LAG3 and CD8+ T-cells expressing TIM-3, without altering T- and NK-cell frequencies. Durvalumab did not affect tumor cell characteristics associated with daratumumab resistance. In conclusion, the addition of durvalumab to daratumumab following development of daratumumab-resistance was associated with an acceptable toxicity profile, but was not effective. This indicates that inhibition of the PD-1/PD-L1 signaling pathway at the time of daratumumab-resistance is insufficient to reverse daratumumab-resistance.

The LEOPARD trial evaluated lenalidomide and alternate day prednisolone (RAP) as post ASCT maintenance in newly diagnosed transplant eligible MM patients (TE NDMM). 60 patients were recruited. Estimated median potential follow-up was 44 months (IQR 26m - 52m). Median PFS from time of commencing RAP was 38.3m (95% CI, 25.8 to 54.8); median OS was not reached (71.4% of patients were alive at 36 months). Here we present the findings from correlative immune studies of this trial. Aims: To undertake mass cytometry (CyTOF) based immune profiling in patients with TE NDMM treated with RAP maintenance post ASCT. Methods: The LEOPARD trial was a phase II, multi centre, open label, single arm study of RAP maintenance after a single melphalan conditioned (200mg/m2) ASCT as part of up-front therapy. Patients were restaged at D+42 ASCT, and if eligible, were commenced on RAP maintenance (LEN 10mg daily increasing to 15mg daily after 8 weeks and alternate day prednisolone 50mg) within 8 weeks of D+0 of the ASCT. Therapy continued until toxicity/progression. CyTOF was performed in sequential samples in two selected groups of patients: long runners (LR, n=7), defined as those with PFS > 36 months (median) and early relapsers (ER, n=8), defined as those who progressed/died before reaching the lower quartile of PFS. [All patients had peripheral blood collected at baseline (pre-ASCT), 6w post-ASCT and weeks 4, 8, 12, 20, 28 and 40 of RAP]. Cells were barcoded using the Cell-ID 20-Plex Pd barcoding kit (Fluidigm) followed by staining with sub-set/function defining antibodies (targeting myeloid, B, T and NK cells: CD45, CD3, CD19, CD5, CD1c, CD226. CD8, CD11c, CD16, CD127, CD138, CD123, NKG2A, TIGIT, TIM3, CD45RA. CD274, CD27, CD197, CD28, Ki67, CD66b, CD183, KLRG1, CD43, NKG2D, CD38, CD278/ICOS, CD25, HLA-DR, CD4, CD57, GramB, PD-1, CD14, CD56, CD11b, Tbet, CD33). Samples were acquired on the Helios instrument. Supervised analysis was performed to determine differences in canonical immune cell populations. Unsupervised analysis was then performed: data were clustered in the VORTEX package. Significant differences in cluster frequency were assessed by Mann-Whitney test for statistical significance. Cluster phenotypes were determined and validated via multiple visualisation approaches. Results: Median age was 56yrs for LR versus 63yrs for ER. Median PFS for LR was 46.3m (38.4 - 51.5m) versus 10.2 m (2.1 - 21.3m) for ER. Supervised analysis was performed on all samples, dichotomized into baseline and last time point sampled for each patient. At baseline, Ki67+CD8+ T cells, ICOS+CD8+ T cells, HLA-DR+CD4+ T cells and CD11c+ myeloid cells were enriched in LR compared to ER. At the last timepoint sampled, Ki67+CD8+ T cells and ICOS+CD8+ T cells were again enriched in LR compared to ER. Conversely, B-reg-like cells (CD19+CD5+CD43-) were enriched in ER compared to LR at the last timepoint sampled. Unsupervised analysis was performed on all samples (all timepoints were pooled). Five clusters were significantly enriched in LR compared to ER. Four of these clusters represented activated/cytotoxic NK cells: CD56 dim, CD16-, NKG2A(CD159a)+, NKG2D(CD314)+, Granzyme B+ and CD38+, and additional expression of CD57 on one cluster; one cluster represented a mature myeloid population, with high expression of HLA-DR, CD11b and CD11c and low expression of CD33. One cluster was significantly enriched in ER compared to LR, representing activated neutrophils, with high expression of CD66b, CD11b and CD16. The clusters that were enriched were then assessed longitudinally over all time points. There was no difference in the kinetics of these populations between groups. Conclusions Significant differences in both T-cell and NK cell populations were demonstrable at baseline in LR versus ER patients. Subsequently, durable responses to post-ASCT lenalidomide maintenance were associated with a cytotoxic, controlled immune response whereas early relapse was characterised by a more uncontrolled inflammatory response and the emergence of B-reg-like cells prior to relapse. We conclude that immune profiling at baseline and after initiation of therapy may help to predict a more sustained response to lenalidomide maintenance enabling pre-emptive tailored treatment decisions. Disclosures Kalff: Roche: Honoraria; Janssen: Honoraria; Amgen: Honoraria; CSL: Honoraria; Celgene: Honoraria. Young:Bristol Meyers Squibb: Current Employment, Current equity holder in publicly-traded company. Pierceall:Bristol Myers Squibb: Current Employment, Current equity holder in publicly-traded company. Thakurta:Bristol Myers Squibb: Current Employment, Current equity holder in publicly-traded company; Oxford University: Other: visiting professor. Oppermann:Bristol Meyers Squibb: Research Funding. Guo:Bristol Meyers Squibb: Research Funding. Reynolds:Novartis AG: Current equity holder in publicly-traded company. Spencer:AbbVie, Celgene, Haemalogix, Janssen, Sanofi, SecuraBio, Specialised Therapeutics Australia, Servier and Takeda: Consultancy; AbbVie, Amgen, Celgene, Haemalogix, Janssen, Sanofi, SecuraBio, Specialised Therapeutics Australia, Servier and Takeda: Honoraria; Amgen, Celgene, Haemalogix, Janssen, Servier and Takeda: Research Funding; Celgene, Janssen and Takeda: Speakers Bureau.

Background: The availability of chimeric antigen receptor (CAR)-modified T cells (CAR T) has profoundly increased therapeutic options for patients (pts) with B cell malignancies, including DLBCL. Liso-cel is an investigational, anti-CD19, defined composition, 4-1BB, CAR T cell product administered at a target dose of CD4+ and CD8+ CAR T cells. To understand tumor microenvironmental (TME) factors affecting short-term and durable responses in pts with R/R DLBCL who received liso-cel in the TRANSCEND NHL 001 study, we conducted multiplexed IF analyses of 111 DLBCL biopsies for 83 pts obtained at baseline (n=58) and approximately 11 days (D11) (n=53; 28 paired) after liso-cel infusion (NCT02631044). Methods: We employed three 5-plex IF panels, consisting of antibodies detecting (1) B cell (CD19, CD20) and T cell lineage (CD4, CD8, EGFR) markers, (2) immunosuppressive markers (CD163, FoxP3, CD73, IDO1, PD-L1), and (3) functional markers (CD3, Ki67, GZMB, PD-1, EGFR). Liso-cel expresses a truncated EGFR (EGFRt), and fluorescent anti-EGFR was used to identify CAR T cells within the tumor biopsies. We also performed bulk tumor RNA profiling for an overlapping subset of 50 baseline biopsies and 37 D11 biopsies (11 paired). We investigated the association of differences in marker densities for pts with best overall response (BOR) of complete response (CR), and progressive disease (PD). Baseline and D11 biopsy findings were correlated with early responses at ~1 month (M1) posttreatment (PD n=16; CR n=42) and durable responses at ~9 months (M9) posttreatment (PD n=76; CR n=32; 55 pts evaluated at both M1 and M9). We investigated how baseline and D11 densities, with spatial distinction between tumoral and peritumoral regions, correlated with early and durable responses. All comparisons describe differences in median densities, and have statistical significance reported with uncorrected P values assessed via the (unpaired) Wilcoxon-Mann-Whitney nonparametric test. Results: Signals in baseline biopsies that correlated with early (M1) response differed from those that correlated with durable (M9) CR. A 21% higher baseline presence of PD-1+ T cells was associated with pts who achieved early CR at M1 vs pts who had PD at M1 (P=0.007). Pts with durable CR at M9 had 39% lower baseline levels of CD163+ macrophages (P=0.019) and 270% higher levels of CD73+ cells (P=0.028) than those with PD at M9. On-treatment (D11) tumors of pts with both early and durable CR had 28% higher levels of EGFRt+ (CAR T) CD8+ T cells (P=0.022), and 810% higher EGFRt- (non-CAR T) CD4+ (but notably, not CD8+; P=0.28) T cells (P=0.009). We also investigated changes in marker densities between baseline and on-treatment (D11) biopsies, and found that pts with durable CR at M9 had decreased on-treatment B cell densities (P=0.029), and increased densities of CD8+ GZMB+, Ki67+, and/or PD-1+ CAR (P=0.001) as well as non-CAR T (P=0.017) cells. Pts with durable CR also had a 29% increase in tumor-associated CD163+ macrophages at D11 relative to baseline (P=0.033). While the accessibility of spatial arrangements and multilabeled cells from IF enables a more nuanced picture of the TME, many of the general trends described above are concordant with those observed in bulk tumor RNA sequencing. Lower baseline expression of CD163 (P=0.021) and higher expression of CD73 (P=0.054) were seen in pts with durable CR. Additionally, elevated on-treatment (D11) expression of CD3E, CD4, and liso-cel (P Conclusions: Overall, these data suggest that increased infiltration of tumor-specific CAR T cells upon initial treatment with liso-cel helped establish an active immune response, and that recruitment of additional functional endogenous (particularly CD4+) T cells correlated with durable response. Higher numbers of activated/functional T cells and lower numbers of macrophages prior to treatment also correlated with durable response to liso-cel. Thus, tumors in responders may already have had a baseline TME in which T cells could infiltrate and respond to antigen. This may have promoted the success of CAR T cell entry into tumors and the subsequent recruitment and activation of endogenous lymphocytes that support their function. Disclosures Reiss: Celgene Corporation: Employment, Equity Ownership. Do:Juno Therapeutics, a Celgene Company: Employment, Equity Ownership. Kuo:Juno Therapeutics, a Celgene Company: Employment, Equity Ownership. Gray:Celgene Corporation: Employment, Equity Ownership. Olson:Celgene Corporation: Employment, Equity Ownership. Lee:Celgene Corporation: Employment, Equity Ownership. Young:Celgene Corporation: Employment, Equity Ownership. Srinivasan:Juno Therapeutics, a Celgene Company: Employment, Equity Ownership. Gray:Celgene: Employment, Equity Ownership. Fox:Celgene Corporation: Employment, Equity Ownership. Couto:Celgene Corporation: Employment, Equity Ownership. Dubovsky:Celgene: Employment. Schmitz:Celgene Corporation: Employment, Equity Ownership. Newhall:Celgene Corporation: Employment, Equity Ownership.

Introduction Hindlimb unloading-induced muscle atrophy is often assessed after a homeostatic state is established, thus overlooking the early adaptations that are critical to developing this pattern of atrophy. Methods Muscle function and physiology were characterized at 0, 1, 3, 7, and 14 days of hindlimb suspension (HS). Results Reductions in muscle mass were maximal by Day 14 of HS. Functional strength and isolated muscle strength were reduced. MyHC-I and -IIa expressing fibers were reduced in size by Day 7 in the soleus and by Day 14 in the gastrocnemius (MyHC-I fibers only). Atrogin-1 and MuRF1 expression was increased by Day 1 in both the calf and tibialis anterior while IGF-1 expression was significantly reduced on Day 3. Phosphorylation of Akt was reduced on Day 14. Conclusions Insight into these early changes in response to HS improves understanding of the molecular and functional changes that lead to muscle atrophy. Muscle Nerve 48: 393–402, 2013

Introduction:In vitro or in situ methods to assess neuromuscular performance in rodents are invasive and inadequate to fully assess large hindlimb muscles. Methods: An in vivo hindlimb exertion force test (HEFT) was developed to quantify muscle function peak force (PF), peak rate of force development (PRFD), and short- and long-latency reaction times (SLRT and LLRT, respectively) in C57BL/6J mice. Results: PF did not change with one- and three-times-per-week repeated HEFT trials, demonstrating assessment reproducibility. However, PRFD decreased with trial, indicating that mice modified response behavior while achieving the same PF. Separately, mice were subjected to 14 days of hindlimb suspension (HS) to induce muscle atrophy. Concomitant with decreased lean carcass and individual muscle masses, HS mice showed reduced PF and LLRT. Conclusions: The results demonstrate that HEFT is an effective tool for evaluating in vivo hindlimb neuromuscular performance due to disuse muscle atrophy and potentially for other disease and injury models. Muscle Nerve, 2012

SummaryThe antigen receptor for natural killer T cells (NKT TCR) binds CD1d-restricted microbial and self-lipid antigens, although the molecular basis of self-CD1d recognition is unclear. Here, we have characterized NKT TCR recognition of CD1d molecules loaded with natural self-antigens (Ags) and report the 2.3 Å resolution structure of an autoreactive NKT TCR-phosphatidylinositol-CD1d complex. NKT TCR recognition of self- and foreign antigens was underpinned by a similar mode of germline-encoded recognition of CD1d. However, NKT TCR autoreactivity is mediated by unique sequences within the non-germline-encoded CDR3β loop encoding for a hydrophobic motif that promotes self-association with CD1d. Accordingly, NKT cell autoreactivity may arise from the inherent affinity of the interaction between CD1d and the NKT TCR, resulting in the recognition of a broad range of CD1d-restricted self-antigens. This demonstrates that multiple self-antigens can be recognized in a similar manner by autoreactive NKT TCRs.

SummaryNatural killer T (NKT) cells respond to a variety of CD1d-restricted antigens (Ags), although the basis for Ag discrimination by the NKT cell receptor (TCR) is unclear. Here we have described NKT TCR fine specificity against several closely related Ags, termed altered glycolipid ligands (AGLs), which differentially stimulate NKT cells. The structures of five ternary complexes all revealed similar docking. Acyl chain modifications did not affect the interaction, but reduced NKT cell proliferation, indicating an affect on Ag processing or presentation. Conversely, truncation of the phytosphingosine chain caused an induced fit mode of TCR binding that affected TCR affinity. Modifications in the glycosyl head group had a direct impact on the TCR interaction and associated cellular response, with ligand potency reflecting the t1/2 life of the interaction. Accordingly, we have provided a molecular basis for understanding how modifications in AGLs can result in striking alterations in the cellular response of NKT cells.

Introduction Relapsed and refractory multiple myeloma (RRMM) remains a challenging disease to treat due to its heterogeneity and complexity. There is an urgent need for novel combination strategies, including immunotherapy. The study of the tumour and immune microenvironment before and after treatment with combination therapy is a crucial part of understanding the underpinning of disease response. Methods Longitudinal samples of bone marrow aspirates and whole blood were collected from a phase II clinical trial, MEDI4736-MM-003 (NCT02807454) where daratumumab and durvalumab naïve patients were exposed simultaneously to both these drugs. A combination of mass cytometry (CyTOF), RNAseq and flow cytometry were performed on a subset of samples from these subjects. Specifically, paired bone marrow mononuclear cells (BMMC) samples from nine patients taken at screening and six weeks post-treatment were analysed by mass cytometry (CyTOF) using a 37-marker pan-immune panel that included both lineage and functional intracellular/extracellular markers. In addition, whole blood sample specimens were collected at screening and on treatment (8, 15, 30, and 45 days after treatment) and analysed by flow cytometry. Flow cytometry panels were designed to allow interrogation of the abundance and activation status of immune cell subsets. Finally, RNA from bone marrow aspirates at screening and C2D15 were analysed by RNA sequencing. Expression profiles from the aspirates were used to estimate cell proportions by computational deconvolution. Individual cell types in these microenvironments were estimated using the DCQ algorithm and a gene expression signature matrix based on the published LM22 leukocyte matrix (Newman et al., 2015) augmented with 5 bone marrow- and myeloma-specific cell types. Results In a heavily pre-treated population with RRMM, treatment with durvalumab and daratumumab leads to shifts in a number of key immunological populations when compared to pre-treatment. In the bone marrow, CD8 and CD4 populations rise (by CyTOF and RNAseq), while NK, DC and B cell populations fall (by CyTOF). In the bone marrow within CD8+ T lymphocyte populations, we observed a post-treatment rise in markers of degranulation (granzyme p=0.0195, perforin p=0.0078, Wilcoxon signed-rank test). This is also accompanied by a fall in PD1 expression (p=0.0078) and rise in the co-stimulatory receptor DNAM1 (p=0.0273). These changes are most marked on cells with an effector memory CD45RA+ CD8+ T cell phenotype. In the blood, similar to the bone marrow, CD8+ T cells proliferate over the course of treatment (flow cytometry). A fall in both naïve and active NK cell populations is seen following treatment in bone marrow. NK cells express high levels of CD38 and are therefore depleted by daratumumab. Those NK cells which remain have an active phenotype with increased expression of TNFa (p=0.0039) and IFNg (p=0.0195) following treatment. Across the time points sampled in peripheral blood, NK cells were also decreased and those that remained were proliferating. Dendritic cells with a tolerogenic phenotype can be identified prior to treatment and are seen to fall in abundance following treatment with durvalumab and daratumumab. Conclusions The combination of durvalumab and daratumumab leads to several immune microenvironment changes that biologically portend clinical effect. We see increases in the abundance of cell populations with functional anti-tumour activity, including granzyme B+ CD8 T cells and a reduction in PD1high T cells. Despite the treatment expectedly reducing NK cell numbers, many functionally competent NK cells remain, as evidenced by the presence of anti-tumour cytokines. This combination strategy also reduces immunosuppressive tolerogenic DCs, which suppress CD4 and CD8 T cell activity. Taken together, this suggests that this chemotherapy free, doublet treatment has the potential to up-regulate anti-tumour immunological responses, which may restore immunosurveillance mechanisms critically needed in these highly refractory patients. Disclosures Seymour: Celgene: Research Funding. Young:Celgene Corporation: Employment, Equity Ownership. Tometsko:Celgene Corporation: Employment, Equity Ownership. Cavenagh:Celgene: Honoraria, Research Funding, Speakers Bureau; Janssen: Honoraria, Speakers Bureau; Takeda: Research Funding, Speakers Bureau; Novartis: Honoraria, Speakers Bureau; Amgen: Honoraria, Speakers Bureau. Thompson:Celgene Corporation: Employment, Equity Ownership. Whalen:Celgene Corporation: Employment, Equity Ownership. Danziger:Celgene Corporation: Employment, Equity Ownership. Fitch:Celgene Corporation: Employment, Equity Ownership. Fox:Celgene Corporation: Employment, Equity Ownership. Dervan:Celgene Corporation: Employment, Equity Ownership. Foy:Celgene Corporation: Employment, Equity Ownership. Newhall:Celgene Corporation: Employment, Equity Ownership. Gribben:Acerta Pharma: Honoraria, Research Funding; Cancer Research UK: Research Funding; TG Therapeutics: Honoraria; Roche: Honoraria; NIH: Research Funding; Medical Research Council: Research Funding; Celgene: Consultancy, Honoraria, Research Funding; Abbvie: Honoraria; Kite: Honoraria; Pharmacyclics: Honoraria; Novartis: Honoraria; Janssen: Honoraria, Research Funding; Wellcome Trust: Research Funding; Unum: Equity Ownership.

Introduction The multistep progression of multiple myeloma from a normal plasma cell to a system with the features of invasive cancer provides a unique opportunity to understand the co-evolution of the malignant clone within its microenvironment. Understanding these changes is becoming increasingly important as we attempt to design early intervention strategies and to precisely leverage emerging immunotherapeutic modalities to prevent and treat disease progression. In this work, we used mass cytometry (CyTOF) to generate a high-resolution map of the BM microenvironment and how it changes during the transition from health through pre-malignancy to disease. This approach allows us to both understand microenvironmental patterns that correlate with rapid disease progression as well as to generate new hypotheses about permissive and protective immune-phenotypes that might reveal novel immunologic drug targets. Methods To understand the immunologic characteristics of monoclonal gammopathy of undetermined significance (MGUS), smoldering multiple myeloma (SMM), newly diagnosed multiple myeloma (NDMM) and relapsed-refractory multiple myeloma (RRMM), we profiled BM aspirates from 79 patients using mass cytometry by time of flight (CyTOF). Furthermore, we compared the BM compartment of pre-malignant, malignant, and relapsed disease states to the BM of healthy donors using a 37-marker pan-immune panel. In this panel, we used antibodies against several immune lineages, tumor antigens, and functional surface markers, including co-stimulatory and co-inhibitory receptors. Cell clusters defined by Citrus analysis of CyTOF data were combined into an evolutionarily optimized decision tree by evtree to identify cluster interactions that strongly partition patient samples. Results During MGUS, when the tumor plasma cells are Conclusions Immune dysregulation is thought to be a major contributor to the progression and outcome of patients with MGUS, SMM, and MM. Using CyTOF, we have begun to benchmark the content of the immune microenvironment across the myeloma continuum. Based on this cross-sectional analysis we hypothesize that it is important to further interrogate whether the losses in the CD4 memory and effector populations we described correlate with outcomes after therapy with either CAR T or T cell engager trials that are currently ongoing, and whether reconstituting these cell types could provide a meaningful treatment strategy. Disclosures Young: Celgene Corporation: Employment, Equity Ownership. Danziger:Celgene Corporation: Employment, Equity Ownership. Fitch:Celgene Corporation: Employment, Equity Ownership. Schmitz:Celgene Corporation: Employment, Equity Ownership. Gockley:Celgene Corporation: Employment. McConnell:Celgene Corporation: Employment. Reiss:Celgene Corporation: Employment, Equity Ownership. Copeland:Celgene Corporation: Employment, Equity Ownership. Newhall:Celgene Corporation: Employment, Equity Ownership. Hershberg:Celgene Corporation: Employment, Equity Ownership, Patents & Royalties. Foy:Celgene Corporation: Employment, Equity Ownership. Ratushny:Celgene Corporation: Employment, Equity Ownership. Dervan:Celgene Corporation: Employment, Equity Ownership. Morgan:Takeda: Consultancy, Honoraria; Janssen: Research Funding; Celgene: Consultancy, Honoraria, Research Funding; Bristol-Myers Squibb: Consultancy, Honoraria.

Mouse type I natural killer T cell receptors (iNKT TCRs) use a single V alpha 14-J alpha 18 sequence and V beta s that are almost always V beta 8.2, V beta 7, or V beta 2, although the basis of this differential usage is unclear. We showed that the V beta bias occurred as a consequence of the CDR2 beta loops determining the affinity of the iNKT TCR for CD1d-glycolipids, thus controlling positive selection. Within a conserved iNKT-TCR-CD1d docking framework, these inherent V beta-CD1d affinities are further modulated by the hypervariable CDR3 beta loop, thereby defining a functional interplay between the two iNKT TCR CDR beta loops. These V beta biases revealed a broadly hierarchical response in which V beta 8.2 > V beta 7 > V beta 2 in the recognition of diverse CD1d ligands. This restriction of the iNKT TCR repertoire during thymic selection paradoxically ensures that each peripheral iNKT cell recognizes a similar spectrum of antigens.

The Ikaros family of transcription factors is critical for normal T cell development while limiting malignant transformation. Mature CD8 T cells express multiple Ikaros family members, yet little is known about their function in this context. To test the functions of this gene family, we used retroviral transduction to express a naturally occurring, dominant negative (DN) isoform of Ikaros in activated CD8 T cells. Notably, expression of DN Ikaros profoundly enhanced the competitive advantage of activated CD8 T cells cultured in IL-12, such that by 6 days of culture, DN Ikaros-transduced cells were 100-fold more abundant than control cells. Expression of a DN isoform of Helios, a related Ikaros-family transcription factor, conferred a similar advantage to transduced cells in IL-12. While DN Ikaros-transduced cells had higher expression of the IL-2 receptor alpha chain, DN Ikaros-transduced cells achieved their competitive advantage through an IL-2 independent mechanism. Finally, the competitive advantage of DN Ikaros-transduced cells was manifested in vivo, following adoptive transfer of transduced cells. These data identify the Ikaros family of transcription factors as regulators of cytokine responsiveness in activated CD8 T cells, and suggest a role for this family in influencing effector and memory CD8 T cell differentiation.

Mucosal-associated invariant T cells are a unique population of T cells that express a semi-invariant αβ TCR and are restricted by the MHC class I-related molecule MR1. MAIT cells recognize uncharacterized ligand(s) presented by MR1 through the cognate interaction between their TCR and MR1. To understand how the MAIT TCR recognizes MR1 at the surface of APCs cultured both with and without bacteria, we undertook extensive mutational analysis of both the MAIT TCR and MR1 molecule. We found differential contribution of particular amino acids to the MAIT TCR-MR1 interaction based upon the presence of bacteria, supporting the hypothesis that the structure of the MR1 molecules with the microbial-derived ligand(s) differs from the one with the endogenous ligand(s). Furthermore, we demonstrate that microbial-derived ligand(s) is resistant to proteinase K digestion and does not extract with common lipids, suggesting an unexpected class of antigen(s) might be recognized by this unique lymphocyte population.

Hindlimb unloading-induced muscle atrophy is often assessed after a homeostatic state is established, thus overlooking the early adaptations that are critical to developing this pattern of atrophy.Muscle function and physiology were characterized at 0, 1, 3, 7, and 14 days of hindlimb suspension (HS).Reductions in muscle mass were maximal by Day 14 of HS. Functional strength and isolated muscle strength were reduced. MyHC-I and -IIa expressing fibers were reduced in size by Day 7 in the soleus and by Day 14 in the gastrocnemius (MyHC-I fibers only). Atrogin-1 and MuRF1 expression was increased by Day 1 in both the calf and tibialis anterior while IGF-1 expression was significantly reduced on Day 3. Phosphorylation of Akt was reduced on Day 14.Insight into these early changes in response to HS improves understanding of the molecular and functional changes that lead to muscle atrophy.

Toll-like receptors (TLRs) are an essential part of the innate immune system. Their function on antigen presenting cells helps drive efficient T cell responses to pathogens. However, T cells also express several TLRs. Additionally, many of the downstream signaling components of TLRs are shared with those of the T cell receptor. Our hypothesis is that these two pathways work in concert to exert unique and potent T cell responses. We have found that several TLRs are able to enhance T cell responses, including TLR3, TLR4, TLR7, and TLR9. We have found that TLR7 signaling, in particular, synergizes with TCR activation and improves T cell effector functions. Priming T cells with synthetic and natural TLR7 ligands before TCR ligation further boosts T cell responses. The combination of TLR7 and TCR stimulation in T cells enhances cytokine production and proliferation in T cells. We identified several overlapping components between TLR7 and TCR activation pathways by gene expression, which may provide combinatorial increases in T cell activation. In addition, we found increases in anti-viral response pathways that are atypical in T cells. This response was unique to the combination of TLR and TCR stimuli. Thus, we wanted to determine if TLR7 signaling in T cells is important during an acute infection. During influenza, T cell-specific loss of TLR7 alters infection dynamics in the T cell population, resulting in significant activation delays and abnormalities. These findings show that TLR7 is important in normal T cell responses to viral infections. Understanding the contribution of TLR signaling to T cell activation will be useful in guiding enhanced vaccine strategies.

In vitro or in situ methods to assess neuromuscular performance in rodents are invasive and inadequate to fully assess large hindlimb muscles.An in vivo hindlimb exertion force test (HEFT) was developed to quantify muscle function peak force (PF), peak rate of force development (PRFD), and short- and long-latency reaction times (SLRT and LLRT, respectively) in C57BL/6J mice.PF did not change with one- and three-times-per-week repeated HEFT trials, demonstrating assessment reproducibility. However, PRFD decreased with trial, indicating that mice modified response behavior while achieving the same PF. Separately, mice were subjected to 14 days of hindlimb suspension (HS) to induce muscle atrophy. Concomitant with decreased lean carcass and individual muscle masses, HS mice showed reduced PF and LLRT.The results demonstrate that HEFT is an effective tool for evaluating in vivo hindlimb neuromuscular performance due to disuse muscle atrophy and potentially for other disease and injury models.

The recognition of lipid antigens by T cells is a complex and fascinating phenomenon. The MHC-like molecules of the CD1 family have evolved to present a wide variety of both self and foreign lipids for recognition by T-cell receptors. While much progress has been made in our understanding of the NKT cells that recognize lipids presented by CD1d molecules, our knowledge of the T-cell populations directed at the related group 1 CD1 molecules, i.e. CD1a, CD1b and CD1c, has lagged behind. In this issue of the European Journal of Immunology, a study identifies a surprisingly large portion of human peripheral blood T cells as being autoreactive to the group 1 CD1 proteins. This work and other recent developments highlight the presence of a substantial number of unconventional T cells as part of our normal T-cell repertoire. This interesting finding is discussed in details in this commentary.

Summaryαβ T cell receptors (TCRs) bind specifically to foreign antigens presented by major histocompatibility complex proteins (MHC) or MHC-like molecules. Accumulating evidence indicates that the germline-encoded TCR segments have features that promote binding to MHC and MHC-like molecules, suggesting coevolution between TCR and MHC molecules. Here, we assess directly the evolutionary conservation of αβ TCR specificity for MHC. Sequence comparisons showed that some Vβs from distantly related jawed vertebrates share amino acids in their complementarity determining region 2 (CDR2). Chimeric TCRs containing amphibian, bony fish, or cartilaginous fish Vβs can recognize antigens presented by mouse MHC class II and CD1d (an MHC-like protein), and this recognition is dependent upon the shared CDR2 amino acids. These results indicate that features of the TCR that control specificity for MHC and MHC-like molecules were selected early in evolution and maintained between species that last shared a common ancestor more than 400 million years ago.

An extensive material characterization facility has been developed to support a modern epitaxial silicon growth laboratory. The growth laboratory provides material for producing impurity band conduction (IBC) IR detector arrays. The laboratory consists of two sections. Material from the research section is used for producing advanced IBC detectors. Material from the manufacturing section is used for producing detector arrays for producibility studies The characterization facility currently includes a computer-controlled variable temperature Hall effect system, a spreading resistance system, and an electrochemical capacitance-voltage dopant profiling device. A low frequency capacitance-voltage system has been assembled from components for specific use with heavily doped silicon exhibiting impurity band conduction. The variable temperature Hall effect measurement allows determination of majority dopant species and concentration, carrier mobility, and total compensation concentration. Spreading resistance yields rapid doping profile measurement of .1 μm to 100 μm thick epitaxial layers. The electrochemical C-V system allows accurate measurement of sharp doping transitions with the capability of 10 A resolution. The low frequency C-V system can perform measurements on test samples or completed devices to determine compensation concentration for material doped so heavily that impurity band conduction interferes with the ability of the Hall measurement to provide this information. Representative data from each system will be shown and discussed. Application of each measurement to implement material quality control as well as material development will be examined.