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Gene-mediated cytotoxic immunotherapy (GMCI) is an immuno-oncology approach involving local delivery of a replication-deficient adenovirus expressing herpes simplex thymidine kinase (AdV-tk) followed by anti-herpetic prodrug activation that promotes immunogenic tumor cell death, antigen-presenting cell activation, and T cell stimulation. This phase I dose-escalation pilot trial assessed bronchoscopic delivery of AdV-tk in patients with suspected lung cancer who were candidates for surgery. A single intra-tumoral AdV-tk injection in three dose cohorts (maximum 1012 viral particles) was performed during diagnostic staging, followed by a 14-day course of the prodrug valacyclovir, and subsequent surgery 1 week later. Twelve patients participated after appropriate informed consent. Vector-related adverse events were minimal. Immune biomarkers were evaluated in tumor and blood before and after GMCI. Significantly increased infiltration of CD8+ T cells was found in resected tumors. Expression of activation, inhibitory, and proliferation markers, such as human leukocyte antigen (HLA)-DR, CD38, Ki67, PD-1, CD39, and CTLA-4, were significantly increased in both the tumor and peripheral CD8+ T cells. Thus, intratumoral AdV-tk injection into non-small-cell lung cancer (NSCLC) proved safe and feasible, and it effectively induced CD8+ T cell activation. These data provide a foundation for additional clinical trials of GMCI for lung cancer patients with potential benefit if combined with other immune therapies., Graphical Abstract, Predina and colleagues describe a phase I dose-escalation trial using bronchoscopic delivery of a replication-deficient adenovirus expressing herpes simplex thymidine kinase (AdV-tk) in lung cancer patients who underwent surgery. AdV-tk injection proved safe, feasible, and effectively induced CD8+ T cell activation in blood and in resected tumor tissues.

Hardware breakage during orthopaedic surgery especially closed intramedullary nailing is a nightmare for orthopaedic surgeons. During hip fracture surgery a mechanical failure of the guidewire or the reamer poses an additional risk of intrapelvic migration and neurovascular or visceral injury which can lead to devastating complications and litigation. We report a case of removal of the broken guidewire using a cannulated reamer & discectomy forceps and recommend some suggestions for prevention of this catastrophe.

Immune checkpoint inhibitors demonstrate clinical activity in many tumor types, however, only a fraction of patients benefit. Combining CD137 agonists with these inhibitors increases anti-tumor activity preclinically, but attempts to translate these observations to the clinic have been hampered by systemic toxicity. Here we describe a human CD137xPD-L1 bispecific antibody, MCLA-145, identified through functional screening of agonist- and immune checkpoint inhibitor arm combinations. MCLA-145 potently activates T cells at sub-nanomolar concentrations, even under suppressive conditions, and enhances T cell priming, differentiation and memory recall responses. In vivo, MCLA-145 anti-tumor activity is superior to immune checkpoint inhibitor comparators and linked to recruitment and intra-tumor expansion of CD8 + T cells. No graft-versus-host-disease is observed in contrast to other antibodies inhibiting the PD-1 and PD-L1 pathway. Non-human primates treated with 100 mg/kg/week of MCLA-145 show no adverse effects. The conditional activation of CD137 signaling by MCLA-145, triggered by neighboring cells expressing >5000 copies of PD-L1, may provide both safety and potency advantages., The anti-tumour effect of immune checkpoint inhibitors is potentiated by CD137 agonists in preclinical models, but translation of these results to the clinical practice is hampered by toxicity. Authors describe here a human CD137xPD-L1 bispecific antibody with improved anti-cancer activity whilst maintaining low toxicity in non-human primates.

Cancer progression is marked by dysfunctional tumor-infiltrating lymphocytes (TIL) with high inhibitory receptor (IR) expression. Because IR blockade has led to clinical responses in some patients with non–small cell lung cancer (NSCLC), we investigated how IRs influenced CD8+ TIL function from freshly digested early-stage NSCLC tissues using a killing assay and intracellular cytokine staining after in vitro T-cell restimulation. Early-stage lung cancer TIL function was heterogeneous with only about one third of patients showing decrements in cytokine production and lytic function. TIL hypofunction did not correlate with clinical factors, coexisting immune cells (macrophages, neutrophils, or CD4+ T regulatory cells), nor with PD-1, TIGIT, TIM-3, CD39, or CTLA-4 expression. Instead, we found that the presence of the integrin αeβ7 (CD103), characteristic of tissue-resident memory cells (TRM), was positively associated with cytokine production, whereas expression of the transcription factor Eomesodermin (Eomes) was negatively associated with TIL function. These data suggest that the functionality of CD8+ TILs from early-stage NSCLCs may be influenced by competition between an antitumor CD103+ TRM program and an exhaustion program marked by Eomes expression. Understanding the mechanisms of T-cell function in the progression of lung cancer may have clinical implications for immunotherapy.

Histone/protein deacetylases (HDACs) are frequently upregulated in human malignancies and have therefore become therapeutic targets in cancer therapy. However, inhibiting certain HDAC isoforms can have protolerogenic effects on the immune system, which could make it easier for tumor cells to evade the host immune system. Therefore, a better understanding of how each HDAC isoform affects immune biology is needed to develop targeted cancer therapy. Here, we studied the immune phenotype of HDAC5(-/-) mice on a C57BL/6 background. While HDAC5(-/-) mice replicate at expected Mendelian ratios and do not develop overt autoimmune disease, their T-regulatory (Treg) cells show reduced suppressive function in vitro and in vivo. Likewise, CD4(+) T-cells lacking HDAC5 convert poorly to Tregs under appropriately polarizing conditions. To test if this attenuated Treg formation and suppressive function translated into improved anticancer immunity, we inoculated HDAC5(-/-) mice and littermate controls with a lung adenocarcinoma cell line. Cumulatively, lack of HDAC5 did not lead to better anticancer immunity. We found that CD8(+) T cells missing HDAC5 had a reduced ability to produce the cytokine, IFN-γ, in vitro and in vivo, which may offset the benefit of weakened Treg function and formation. Taken together, targeting HDAC5 weakens suppressive function and de-novo induction of Tregs, but also reduces the ability of CD8(+) T cells to produce IFN-γ.

Purpose: Tumor-infiltrating lymphocytes (TILs) become hypofunctional, although the mechanisms are not clear. Our goal was to generate a model of human tumor-induced TIL hypofunction to study mechanisms and to test anti-human therapeutics. Experimental Design: We transduced human T cells with a published, optimized T-cell receptor (TCR) that is directed to a peptide within the cancer testis antigen, NY-ESO-1. After demonstrating antigen-specific in vitro activity, these cells were used to target a human lung cancer line that expressed NY-ESO-1 in the appropriate HLA context growing in immunodeficient mice. The ability of anti-PD1 antibody to augment efficacy was tested. Results: Injection of transgenic T cells had some antitumor activity, but did not eliminate the tumors. The injected T cells became profoundly hypofunctional accompanied by upregulation of PD1, Tim3, and Lag3 with coexpression of multiple inhibitory receptors in a high percentage of cells. This model allowed us to test reagents targeted specifically to human T cells. We found that injections of an anti-PD1 antibody in combination with T cells led to decreased TIL hypofunction and augmented the efficacy of the adoptively transferred T cells. Conclusions: This model offers a platform for preclinical testing of adjuvant immunotherapeutics targeted to human T cells prior to transition to the bedside. Because the model employs engineering of human T cells with a TCR clone instead of a CAR, it allows for study of the biology of tumor-reactive TILs that signal through an endogenous TCR. The lessons learned from TCR-engineered TILs can thus be applied to tumor-reactive TILs. Clin Cancer Res; 22(2); 436–47. ©2015 AACR. See related commentary by Yang, p. 275

9050 Background: GMCI is a tumor-specific immuno-oncology approach implemented through local delivery of aglatimagene besadenovec(AdV-tk) followed by anti-herpetic prodrug. This leads to immunogenic tumor cell death, antigen presenting cell activation, and T cell stimulation resulting in CD8+ T cell dependent protection, as demonstrated in preclinical models and clinical trials in other tumor types. This is the first study to assess endobronchial delivery of AdV-tk for NSCLC. Methods: This Phase I dose escalation trial enrolled patients with suspected NSCLC who were candidates for surgery. A single AdV-tk injection was performed by endobronchial ultrasound (n = 11) or mediastinoscopy (n = 1) during the diagnostic staging procedure 3 weeks prior to surgery. Three dose levels were evaluated: 2.5x 1011, 5x1011, and 1x1012 vector particles (vp) in a 3+3 design. Valacyclovir was administered for 14 days, starting the day after AdV-tk injection. To assess the local and systemic effects of GMCI, immune biomarkers were evaluated in blood and tumor samples before and after GMCI. Results: From 2017-2019, 12 patients (9 men, 3 women, median age 65 [range 55-80]) received GMCI followed by surgery. Average tumor size was 5.1 cm (largest diameter) and final pathologic stage was I (n = 4), II (n = 3), and III (n = 5). Treatment-related adverse events were CTC grade 1 fever (n = 1), flu-like symptoms (n = 1) and nausea/vomiting/diarrhea (n = 1). The only > grade 2 lab abnormality was transient grade 3 lymphopenia (n = 2). A measurable reduction in tumor size was observed in one patient. The average amount of tumor necrosis was 29.4%. Significant infiltration of CD8+T cells (5.2-fold compared to baseline, p = 0.001) was found in tumor 19-22 days after AdV-tk injection. Within the CD8+tumor infiltrating lymphocytes, there was increased expression of CD38 (2.5-fold, p = 0.002), Ki67 (4.8-fold, p = 0.02), PD1 (1.9-fold, p = 0.002), CD39 (2.9-fold, p = 0.04) and CTLA-4 (4.8-fold, p < 0.001), without significant detected differences in Tim3 or TIGIT. Simultaneously, peripheral blood CD8+ cells displayed significant increases in CD38 (3.4-fold, p = 0.006), HLA-DR (4.2-fold, p = 0.002), and Ki67 (5.8-fold, p = 0.017). Conclusions: Intratumoral injection of AdV-tk into lung tumors was safe and feasible. Further, AdV-tk effectively induced peripheral blood and intra-tumoral CD8 T cell activation. Consequent upregulation of inhibitory receptors suggests a potential benefit for combination therapies. Clinical trial information: NCT03131037.

Exhausted CD8 T (Tex) cells are immunotherapy targets in chronic infection and cancer, but a comprehensive assessment of Tex cell diversity in human disease is lacking. Here, we developed a transcriptomic- and epigenetic-guided mass cytometry approach to define core exhaustion-specific genes and disease-induced changes in Tex cells in HIV and human cancer. Single-cell proteomic profiling identified 9 distinct Tex cell clusters using phenotypic, functional, transcription factor, and inhibitory receptor co-expression patterns. An exhaustion severity metric was developed and integrated with high-dimensional phenotypes to define Tex cell clusters that were present in healthy subjects, common across chronic infection and cancer or enriched in either disease, linked to disease severity, and changed with HIV therapy. Combinatorial patterns of immunotherapy targets on different Tex cell clusters were also defined. This approach and associated datasets present a resource for investigating human Tex cell biology, with implications for immune monitoring and immunomodulation in chronic infections, autoimmunity, and cancer. Exhausted T (Tex) cells have poor function in chronic infections and cancer but can be therapeutically re-invigorated. Bengsch et al. use genes modified epigenetically during exhaustion and high-dimensional CyTOF profiling to define Tex cell heterogeneity in humans with HIV or lung cancer and link Tex cell features to disease progression and response to immunotherapy.

Given the growing interest and promising preliminary results of immunotherapy in malignant pleural mesothelioma (MPM), it has become important to more fully understand the immune landscape in this tumor. This may be especially relevant in deciding who might benefit most from checkpoint blockade or agonist antibody therapy. Since the phenotype of tumor infiltrating lymphocytes (TILs) in MPM has not been fully described and their function has not been carefully assessed, we collected fresh tumor and blood from 22 patients undergoing surgical resection and analysed single cell suspensions by flow cytometry. The functionality of TILs was assessed by measurement of cytokine expression (IFN-γ) following overnight stimulation ex vivo. Results showed low numbers of CD8+ TILs whose function was either moderately or severely suppressed. The degree of TIL hypofunction did not correlate with the presence of co-existing macrophages or neutrophils, nor with expression of the inhibitory receptors PD-1, CD39 and CTLA-4. Hypofunction was associated with higher numbers of CD4 regulatory T cells (Tregs) and with expression of the inhibitory receptor TIGIT. On the other hand, presence of tissue-resident memory (Trm) cells and expression of TIM-3 on CD8+ cells were positively associated with cytokine production. However, Trm function was partially suppressed when the transcription factor Eomesodermin (Eomes) was co-expressed. Understanding the function of TILs in malignant mesothelioma may have clinical implications for immunotherapy, especially in choosing the best immunotherapy targets. Our data suggests that Treg cell blocking agents or TIGIT inhibitor antibodies might be especially valuable in these patients.

The field of cancer immunotherapy has been re-energized by the application of chimeric antigen receptor (CAR) T cell therapy in cancers. These CAR T cells are engineered to express synthetic receptors that redirect polyclonal T cells to surface antigens for subsequent tumor elimination. Many CARs are designed with elements that augment T cell persistence and activity. To date, CAR T cells have demonstrated tremendous success in eradicating hematologic malignancies (e.g., CD19 CARs in leukemias). However, this success has yet to be extrapolated to solid tumors, and the reasons for this are being actively investigated. We characterize some of the challenges that CAR T cells have to surmount in the solid tumor microenvironment and new approaches that are being considered to overcome these hurdles.

Introduction Given the growing interest and promising preliminary results of immunotherapy in mesothelioma, it has become important to more fully understand the immune landscape in this tumour. This may be especially important for deciding who might benefit most from checkpoint blockade or agonist antibody therapy. Although the phenotype of tumour infiltrating lymphocytes (TILs) in mesothelioma is being increasingly well described, there is very little functional analysis of mesothelioma TILs. Material and methods Fresh mesothelioma tissue and blood were sampled from patients enrolled in an on-going clinical trial of radical pleurectomy vs. radical pleurectomy and photodynamic therapy (PDT) at our institution. Following processing, single cell suspensions were used for phenotypic using flow cytometry. Functionality of TILs was accessed by measurement of cytokine (IFN-γ production) following overnight anti-CD3 antibody stimulation ex vivo . These data were compared to TILs from early stage lung cancer patients. Results and discussions Flow cytometric analysis of 19 patient samples so far shows that the numbers of CD8-positive TILs are markedly low at around 3%–10% of total live cells. TILs have an effector memory phenotype enriched for CD103 +tissue memory cells, they show high expression of inhibitory receptors PD-1 (25%–80%) and TIGIT (50%–80%), and have an Eomes hi T-Bet low phenotype. Unlike TILs from early stage lung cancers, which are generally able to make cytokines, TILs from advanced stage mesotheliomas show profound hypofunction in their ability to produce IFNγ ( Conclusion Our results show that TILs from mesothelioma tumours are profoundly hypofunctional. Areas of active interest are to determine the mechanisms and best markers of T cell hypofunction. Such information might allow us to understand with TILs might be activated by checkpoint inhibitors. We also plan to correlate T cell function with clinical responses in our trial.

CTL-associated antigen 4 (CTLA4) is a costimulatory molecule expressed on activated T cells that delivers an inhibitory signal to these T cells. CTLA4 blockade with antibody treatment has been shown to augment antitumor immunity in animal models and is being developed as a treatment for cancer patients. As has been seen in preclinical models, combining CTLA4 blockade and granulocyte macrophage colony-stimulating factor (GM-CSF)–based immunotherapies can enhance the antitumor efficacy of this approach. We therefore examined whether CTLA4 blockade could be combined with GM-CSF administration. We treated 24 patients with metastatic, castration-resistant prostate cancer in a phase I trial where sequential cohorts were treated with increasing doses of ipilimumab, a fully human anti-CTLA4 antibody. Study subjects also received s.c. injections of GM-CSF at a fixed dose. Of the six patients treated at the highest dose level, three had confirmed PSA declines of >50%, including one patient that had a partial response in visceral metastases. Expansion of activated, circulating CD25+ CD69+ CD8+ T cells occurred more frequently at higher doses of treatment and was greater in magnitude than was seen in patients who received the same doses of either ipilimumab or GM-CSF alone. By screening sera with protein arrays, we showed that our treatment can induce antibody responses to NY-ESO-1. These results show that this combination immunotherapy can induce the expansion not only of activated effector CD8 T cells in vivo but also of T cells that are specific for known tumor-associated antigens from the endogenous immune repertoire. [Cancer Res 2009;69(2):609–15]

Antitumor treatments based on the infusion of T cells expressing chimeric antigen receptors (CAR T cells) are still relatively ineffective for solid tumors, due to the presence of immunosuppressive mediators [such as prostaglandin E2 (PGE2) and adenosine] and poor T-cell trafficking. PGE2 and adenosine activate protein kinase A (PKA), which then inhibits T-cell receptor (TCR) activation. This inhibition process requires PKA to localize to the immune synapse via binding to the membrane protein ezrin. We generated CAR T cells that expressed a small peptide called the “regulatory subunit I anchoring disruptor” (RIAD) that inhibits the association of PKA with ezrin, thus blunting the negative effects of PKA on TCR activation. After exposure to PGE2 or adenosine in vitro, CAR-RIAD T cells showed increased TCR signaling, released more cytokines, and showed enhanced killing of tumor cells compared with CAR T cells. When injected into tumor-bearing mice, the antitumor efficacy of murine and human CAR-RIAD T cells was enhanced compared with that of CAR T cells, due to resistance to tumor-induced hypofunction and increased T-cell infiltration of established tumors. Subsequent in vitro assays showed that both mouse and human CAR-RIAD cells migrated more efficiently than CAR cells did in response to the chemokine CXCL10 and also had better adhesion to various matrices. Thus, the intracellular addition of the RIAD peptide to adoptively transferred CAR T cells augments their efficacy by increasing their effector function and by improving trafficking into tumor sites. This treatment strategy, therefore, shows potential clinical application for treating solid tumors. Cancer Immunol Res; 4(6); 541–51. ©2016 AACR.

Introduction Hip fractures are becoming an increasing public health issue due to an ageing population (1). Total hip replacements (THR) produce better outcomes in certain patients who were functioning independently before the injury (2). We aimed to assess whether the management of intracapsular hip fracture is carried out in accordance with the National Institute for Health and Care Excellence (NICE) hip fracture guidance (1) and the outcomes with regards to performing THRs on those patients who fulfil the described criteria. Method Data was collected retrospectively from the 1st April 2012 to 31st March 2013 from all fractured neck of femur patients admitted to our hospital. Results Of the 382 patients fit for an operation, 78 (20.4%) met with the NICE hip fracture guidance for a total hip replacement. Of those eligible, 32 (41.0%) did receive a THR and 4 (2.8%) patients of the 142 not eligible for a total hip replacement also received a THR. Discussions Of those eligible for a THR, the patients who underwent that procedure had a significantly lower mortality rate compared to those who underwent a hemiarthroplasty (0% versus 19.6% at 1 year, p = 0.007). However, those who did not meet the NICE criteria but underwent a THR had the worst mortality rate (50% at 30 days and 1 year). The provision rate of THR in displaced intracapsular hip fracture is low at 41.0% for those who met the NICE criteria. The results suggest that the decision process when determining if a patient should undergo THR for a fractured neck of femur is multifactorial.

Inhibitors of VEGF signaling can block angiogenesis and reduce tumor vascularity, but little is known about the reversibility of these changes after treatment ends. In the present study, regrowth of blood vessels in spontaneous RIP-Tag2 tumors and implanted Lewis lung carcinomas in mice was assessed after inhibition of VEGF receptor signaling by AG-013736 or AG-028262 for 7 days. Both agents caused loss of 50%-60% of tumor vasculature. Empty sleeves of basement membrane were left behind. Pericytes also survived but had less alpha-SMA immunoreactivity. One day after drug withdrawal, endothelial sprouts grew into empty sleeves of basement membrane. Vessel patency and connection to the bloodstream followed close behind. By 7 days, tumors were fully revascularized, and the pericyte phenotype returned to baseline. Importantly, the regrown vasculature regressed as much during a second treatment as it did in the first. Inhibition of MMPs or targeting of type IV collagen cryptic sites by antibody HUIV26 did not eliminate the sleeves or slow revascularization. These results suggest that empty sleeves of basement membrane and accompanying pericytes provide a scaffold for rapid revascularization of tumors after removal of anti-VEGF therapy and highlight their importance as potential targets in cancer therapy.

Background: Immunotherapy using checkpoint blockade has demonstrated impressive, durable responses in select patients with solid malignancies. This is especially true for the blockade of programmed death 1 (PD1). Currently, efforts are focused on understanding non-response. One hypothesis is that multiple inhibitory receptors (IRs) are upregulated on hypofunctional tumor-infiltrating lymphocytes (TILs) and necessitates blocking multiple IRs to increase the response rate. We describe data from a unique xenograft model of human lung cancer where tumor-reactive human T cells become hypofunctional and upregulate multiple IRs including PD1 and TIM3 (T cell immunoglobulin and mucin-domain containing-3). Combining a single IV dose of tumor-reactive T cells with blockade of both PD1 and TIM3 led to greater control of tumor growth than combining it with blockade of either alone. Materials/Methods: The A549 human lung cancer cell line was transduced to stably express the cancer-testis antigen, NYESO1, expressed in the contact of HLA-A2 (A549-A2-ESO). 5x106 A549-A2-ESO cells were injected subcutaneously into the flanks NSG mice. Activated T cells from healthy donors were lentivirally transduced with a TCR targeting NYESO1 (Ly95). After two weeks when tumors were established and measured ~150mm3, the mice were randomly assigned to one of the following treatments: 1) untreated, 2) non-transduced (NTD) T cells plus anti-PD1 and anti-TIM3 Ab, 3) Ly95 T cells, 4) Ly95 T cells + anti-PD1 Ab, 5) Ly95 T cells + anti-TIM3 Ab, 6) Ly95 T cells + anti-PD1 and anti-TIM3 Ab. T cells were injected IV at a single dose of 10x106/mouse. Abs were injected intraperitonealy (IP) at 10mg/kg every 5 days from the time of T cell injection. Tumor volumes were measured serially. At the end, the mice were sacrificed and the tumors were harvested, digested, and processed into single cell suspension. Flow cytometry was performed to look at degree of TIL infiltration and expression of surface markers. Ficoll gradient was used to isolate the T cells to conduct functional analyses. Results/Conclusion: By twenty days post T cell injection, the tumors in the Ly95 T cell group (3) were ~30% smaller than that of the untreated (1) and the NTD T cell + Ab group (2). Anti-PD1 Ab augmented Ly95 T cell control of tumor (4) (further decrease in size by ~30%) (4). Anti-TIM3 Ab had no effect on Ly95 T cell tumor control (5). Anti-PD1 plus anti-TIM3 Abs had the greatest augmentation on Ly95 T cell control of tumor size (6) (greater than 50% reduction in tumor size compared Ly95 T cells alone) The greatest TIL infiltration and ex-vivo TIL function were seen in tumors that received Ly95 T cell + anti-PD1 and anti-TIM3 Abs. Flow cytometry analysis revealed dynamic, adaptive upregulation of one IR when another IR was blocked. This explained why augmentation of Ly95 T cell control of tumor was greatest with combinatorial Ab blockade. Citation Format: Edmund Moon, Soyeon Kim, Shaun O'Brien, Naomi Saint Jean, Raluca Verona, Linda Snyder, Yangbing Zhao, Steven Albelda. Dual antibody blockade of TIM3 and PD1 on NYESO1 redirected human T cells leads to augmented control of lung cancer tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1641. doi:10.1158/1538-7445.AM2017-1641

Background: Immunotherapy using chimeric antigen receptor (CAR) T cells has demonstrated profound, durable success in hematologic malignancies. Solid tumors present hurdles to the successful application of CAR T cells. One is the upregulation of inhibitory receptors (IRs), like PD1 and CTLA4, many of which rely on shared signaling molecules to shut off T cell activation. One such molecule is SHP1 (Src homology region 2 domain-containing phosphatase-1) which dephosphorylates key components of T cell receptor (TCR) signaling. We have engineered a unique dominant-negative SHP1 (dnSHP1) that is able to augment CAR T cell control of PDL1 positive solid tumors. Materials and Methods: The human mesothelioma cell line, EMP, was transduced to express high levels of mesothelin and PDL1 (EMMESO-PDL1). Activated human T cells from healthy donors were lentivirally transduced to express a mesothelin-directed CAR (mesoCAR) with and without a dnSHP1. MesoCAR and mesoCAR/dnSHP1 T cells were cocultured with tumor cells x 18hrs and specific lysis was measured. These T cells were also restimulated with plate-bound anti-CD3 overnight and were subjected to intracellular flow cytometry staining (ICS) of cytokines. NSG mice were injected subcutaneously in the flanks with 5x106 EMMESO-PDL1 tumor cells. After tumors established and grew to ~150mm3, mice were randomly assigned to one of the following treatments: 1) non-transduced (NTD) T cells, 2) mesoCAR T cells, 3) mesoCAR T cells + sodium stibogluconate (SSG; a chemical inhibitor of SHP1), 4) mesoCAR/dnSHP1 T cells. T cells were injected IV once at a dose of 10x106 T cells/mouse. SSG was administered IM at 20mg/kg every 2 days. Tumors were measured serially. At the end, mice were sacrificed, tumors were harvested, digested, processed into single cell suspension, and subjected to flow cytometry analysis. The tumor infiltrating lymphocytes (TILs) were also isolated and tested for function ex-vivo. Results/Conclusion: In vitro, mesoCAR T cells demonstrated suppressed lysis of EMMESO-PDL1 tumor cells compared to EMMESO cells. MesoCAR/dnSHP1 T cells were able to lyse EMMESO-PDL1 and EMMESO tumor cells with similar efficiency. Anti-CD3 restimulation of T cells revealed enhanced secretion of TNF-alpha and IL2 by mesoCAR/dnSHP1 vs. mesoCAR T cells as measured by ICS. In vivo, SSG injections had minimal impact on mesoCAR T cell control of tumors, whereas mesoCAR/dnSHP1 T cells demonstrated significantly enhanced control of EMMESO-PDL1 tumor growth compared to mesoCAR T cells (60% greater decrease in tumor volume compared to mesoCAR T cells). TIL infiltration was 3-fold higher in tumors harvested from mice that received mesoCAR/dnSHP1 T cells compared to other groups. Isolated mesoCAR/dnSHP1 TILs demonstrated the greatest ex-vivo lysis of fresh tumor cells. DnSHP1 engineering is a powerful and novel way of blocking the suppression of CAR T cells by PD1 and other similar IRs. Citation Format: Edmund Moon, Soyeon Kim, Naomi Saint Jean, Shaun O'Brien, Steven Maceyko, Jing Sun, Carl June, Steven Albelda. Genetic blockade of the protein tyrosine phosphatase SHP1 augments CAR T cell activity against PDL1 expressing solid tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3749. doi:10.1158/1538-7445.AM2017-3749

Background Subcutaneous emphysema of a limb after acute injury is classically associated with gas gangrene. Delayed management can result in amputation and death. Typically caused by a clostridial infection, patients are unwell, with rapidly spreading clinical signs, abnormal laboratory results, and cultures positive. There are reports of widespread subcutaneous emphysema of a limb in well-appearing patients, with blood parameters within normal limits; however, the optimum management of this type of case is unclear. Objective Our objectives were to present 4 new cases of acute subcutaneous emphysema in well-appearing patients managed with early surgery, review the literature, and discuss the management decisions in cases of acute subcutaneous emphysema in clinically well patients. Case reports Here we present a case series of 4 patients, all with penetrating injuries to the upper limb resulting in widespread subcutaneous emphysema within 24 h of injury. Mean age was 33 years. All were fit and well, with the exception of one with type 1 diabetes, no cardiorespiratory compromise, and no significant derangement of laboratory investigations. X-ray studies showed widespread gas within the soft tissues. All were treated aggressively with immediate surgical fasciotomy of the upper limb, thorough debridement, and washout as required. Gram stains revealed pus cells (polymorphonuclear leucocytes) in all, but organisms in only one case (Gram-positive cocci and bacilli). Prolonged culture grew organisms in all. All patients had a second washout and closure plus 6 weeks of antibiotics. All survived and had fully functioning limbs. Why should an emergency physician be aware of this? We recommend having a low threshold for rapid referral to an appropriate surgical speciality, allowing prompt and radical surgical management of this type of presentation, even in the presence of a well patient.

A fit and well 36-year-old male presented to his general practitioner with a 10-day history of pain and swelling in his right leg following a football injury. He had sustained blunt trauma to the lateral aspect of his right thigh and described it as a “dead leg”. A clinical diagnosis of deep vein thrombosis (DVT) was made and the patient was advised to attend the DVT outpatient clinic. In line with hospital protocol, he was commenced on low molecular weight heparin (LMWH enoxaparin) as an outpatient pending urgent ultrasound scan. Following his second dose of enoxaparin, he developed worsening pain in his thigh and was admitted for urgent ultrasound scan which showed a large haematoma (15/5 cm) in the thigh (figure 1). A diagnosis of acute compartment syndrome was made and the patient was taken to theatre for an emergency right thigh fasciotomy and decompression of the haematoma.

Our recent adoptive T cell transfer trial for mesothelioma revealed a striking anti-tumor response in a patient which was accompanied by anaphylaxis, suggesting endogenous histamine release. In animal models, histamine has direct and immune-based anti-tumor effects. In fact, histamine dihydrochloride is approved in Europe in combination with interleukin-2 (IL2) for leukemia treatment. We thus explored the anti-tumor role of histamine in mesothelioma. An in vitro MTT assay was used to measure cell proliferation. Daily doses of histamine (1 mg/kg) – alone, or in combination with other immunotherapies – was used to treat four subcutaneous flank tumor models in three different mouse strains. Flow cytometric analyses were subsequently performed on tumors. Histamine had in vitro anti-tumor activity against all five cell lines tested with an IC50 of 1.8 mM. Histamine also significantly reduced tumor volume in all four in vivo models without toxic side effects. Immunodepletion studies revealed that the effect was partially due to T cells. When combined with two immunotherapy approaches (adenovirally-delivered interferon-α or a tumor antigen), anti-tumor efficacy was augmented. Histamine resulted in more interferon-γ and IL2 production by tumor-infiltrating lymphocytes (TIL), downregulation of CD206 expression on tumor-infiltrating macrophages, and reduced numbers of T regulatory cells (Tregs). Histamine has direct anti-tumor effects and immune-stimulatory effects within the tumor microenvironment (as seen with macrophage polarization, increase in cytokine production, and reduction in Tregs). This pre-clinical data suggests that histamine alone, or in combination with immunotherapy, should be explored in the treatment of mesothelioma. Citation Format: Kheng Newick, Shaun O'Brien, Veena Kapoor, Liang-chuan Wang, Edmund Moon, Andrew Haas, Evan Alley, Daniel Sterman, Steven Albelda. Histamine dihydrochloride in the treatment of mesothelioma. [abstract]. In: Proceedings of the AACR Special Conference: Tumor Immunology and Immunotherapy: A New Chapter; December 1-4, 2014; Orlando, FL. Philadelphia (PA): AACR; Cancer Immunol Res 2015;3(10 Suppl):Abstract nr B78.

Our recent adoptive T cell transfer trial for mesothelioma revealed a striking anti-tumor response in a patient which was accompanied by anaphylaxis, suggesting endogenous histamine release. In animal models, histamine has direct and immune-based anti-tumor effects. In fact, histamine dihydrochloride is approved in Europe in combination with interleukin-2 (IL2) for leukemia treatment. We thus explored the anti-tumor role of histamine in mesothelioma and lung cancer. An in vitro MTT assay was used to measure cell proliferation. Daily doses of histamine (1 mg/kg) - alone, or in combination with other immunotherapies - was used to treat four subcutaneous flank tumor models in three different mouse strains. Flow cytometric analyses were subsequently performed on tumors. Histamine had in vitro anti-tumor activity against all five cell lines tested with an IC50 of 0.1 mg/ml. Histamine also significantly reduced tumor volume in all four in vivo models without toxic side effects. Immunodepletion studies revealed that the effect was partially due to T cells. When combined with two immunotherapy approaches (adenovirally-delivered interferon-α or a tumor antigen), anti-tumor efficacy was augmented. Histamine resulted in more interferon-γ and IL2 production by tumor-infiltrating lymphocytes, downregulation of CD206 expression on tumor-infiltrating macrophages, and reduced numbers of T regulatory cells (Tregs). This pre-clinical data suggests that histamine alone, or in combination with immunotherapy, should be explored in the treatment of mesothelioma, lung cancer, and perhaps other cancers. Citation Format: Kheng Newick, Shaun O'Brien, Veena Kapoor, Liang-chuan Wang, Edmund Moon, Daniel Sterman, Steven Albelda. Histamine dihydrochloride as an immune modulator for solid tumors. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4293. doi:10.1158/1538-7445.AM2015-4293

CTLA-4 is a costimulatory molecule expressed on activated T cells that delivers an inhibitory signal to these T cells. CTLA4 blockade with antibody treatment has been shown to augment T cell responses and anti-tumor immunity in animal models and is being developed as an immunotherapy for cancer patients. We performed a phase I trial in patients with metastatic, hormone refractory prostate cancer (HRPC) where sequential cohorts of 3-6 patients were treated with escalating doses (0.5, 1.5 or 3 mg/kg) of ipilimumab, a fully human anti-CTLA-4 antibody (Medarex/BMS), given IV on day 1 of each 28-day cycle x 4 cycles. Patients also received GM-CSF (sargramostim, Berlex) 250 mg/m2/d SC on days 1-14 of the 28-day cycles. Patients were monitored for toxicity as well as for T cell activation. PSA and radiographic tests were performed at baseline and through therapy to evaluate for clinical response. 24 patients have been treated in the initial phase of this study. Of 6 patients treated on the highest dose level (3.0 mg/kg x 4), 3 (50%) had confirmed PSA declines of >50%, and one of these patients had a partial response in hepatic metastases. Immune-related adverse events associated with ipilimumab treatment were also seen more frequently at higher doses of treatment. Expansion of CD25+CD69+ CD8 T cells was also seem primarily at the highest dose level. The treatment also induced an antibody and CD8 T cell immune response to NY-ESO-1. These results demonstrate that CTLA-4 blockade induces not only the expansion of activated effector CD8 T cells in vivo in cancer patients, but also can induce from the endogenous T cell repertoire of these patients T cells that are specific for tumor-associated antigens. CD8 T cell activation, toxicity, and clinical responses also appear to be dose-dependent. Supported by NIH P50 CA89520.