A Distinct Gene Module for Dysfunction Uncoupled from Activation in Tumor-Infiltrating T Cells.

Reversing the dysfunctional T cell state that arises in cancer and chronic viral infections is the focus of therapeutic interventions; however, current therapies are effective in only some patients and some tumor types. To gain a deeper molecular understanding of the dysfunctional T cell state, we analyzed population and single-cell RNA profiles of CD8(+) tumor-infiltrating lymphocytes (TILs) and used genetic perturbations to identify a distinct gene module for T cell dysfunction that can be uncoupled from T cell activation. This distinct dysfunction module is downstream of intracellular metallothioneins that regulate zinc metabolism and can be identified at single-cell resolution. We further identify Gata-3, a zinc-finger transcription factor in the dysfunctional module, as a regulator of dysfunction, and we use CRISPR-Cas9 genome editing to show that it drives a dysfunctional phenotype in CD8(+) TILs. Our results open novel avenues for targeting dysfunctional T cell states while leaving activation programs intact.

Induction and transcriptional regulation of the co-inhibitory gene module in T cells.

The expression of co-inhibitory receptors, such as CTLA-4 and PD-1, on effector T cells is a key mechanism for ensuring immune homeostasis. Dysregulated expression of co-inhibitory receptors on CD4+ T cells promotes autoimmunity, whereas sustained overexpression on CD8+ T cells promotes T cell dysfunction or exhaustion, leading to impaired ability to clear chronic viral infections and diseases such as cancer1,2. Here, using RNA and protein expression profiling at single-cell resolution in mouse cells, we identify a module of co-inhibitory receptors that includes not only several known co-inhibitory receptors (PD-1, TIM-3, LAG-3 and TIGIT) but also many new surface receptors. We functionally validated two new co-inhibitory receptors, activated protein C receptor (PROCR) and podoplanin (PDPN). The module of co-inhibitory receptors is co-expressed in both CD4+ and CD8+ T cells and is part of a larger co-inhibitory gene program that is shared by non-responsive T cells in several physiological contexts and is driven by the immunoregulatory cytokine IL-27. Computational analysis identified the transcription factors PRDM1 and c-MAF as cooperative regulators of the co-inhibitory module, and this was validated experimentally. This molecular circuit underlies the co-expression of co-inhibitory receptors in T cells and identifies regulators of T cell function with the potential to control autoimmunity and tumour immunity.

The role of preoperative computerized tomography (CT) scan of the pelvis and groin in the management of clinically early staged vulva squamous cell carcinoma.

INTRODUCTION: Vulvar squamous cell carcinoma (VSCC) commonly metastasises through groin lymphatics. However, the use of pre-operative imaging in detecting inguinal nodal metastasis before staging surgery or to triage patients for sentinel node biopsy remains unclear. Here, we investigated if pre-operative CT scan, the imaging choice in our cancer centre, influences the overall course of VSCC management in those patients without clinical evidence of groin lymphadenopathy. METHOD: The study comprised of a prospective cohort of 225 patients with VSCC who underwent staging surgery within a regional tertiary gynaecological cancer centre. Comprehensive information of the cohort's demography, clinicopathological variables and outcome data were collected and analysed. Findings of pre-operative imaging were compared with histological findings of inguinal lymph nodes following groin lymphadenectomy. Statistical analyses were performed using SPSS V24. RESULTS: Pre-operative CT scan was performed on 116 (56.6%) patients. The sensitivity and specificity of cross-sectional imaging in detecting groin lymphatic metastasis were 59.1% and 77.8%, respectively; while the positive (PPV) and negative predictive value (NPV) were 61.9% and 75.7%, respectively. In patients who had sentinel inguinal nodes biopsy, the sensitivity, specificity, PPV and NPV of CT scan in detecting inguinal node metastasis were 30.0%, 85.7%, 33.3% and 83.7%, respectively. There was no difference in disease-free and overall survival in those who received pre-operative imaging when compared to those who did not. CONCLUSION: Pre-operative CT scan may be omitted in early stage VSCC prior to surgical staging as it does not affect overall management and surgical outcomes.

Functional Anti-TIGIT Antibodies Regulate Development of Autoimmunity and Antitumor Immunity.

Coinhibitory receptors, such as CTLA-4 and PD-1, play a critical role in maintaining immune homeostasis by dampening T cell responses. Recently, they have gained attention as therapeutic targets in chronic disease settings where their dysregulated expression contributes to suppressed immune responses. The novel coinhibitory receptor TIGIT (T cell Ig and ITIM domain) has been shown to play an important role in modulating immune responses in the context of autoimmunity and cancer. However, the molecular mechanisms by which TIGIT modulates immune responses are still insufficiently understood. We have generated a panel of monoclonal anti-mouse TIGIT Abs that show functional properties in mice in vivo and can serve as important tools to study the underlying mechanisms of TIGIT function. We have identified agonistic as well as blocking anti-TIGIT Ab clones that are capable of modulating T cell responses in vivo. Administration of either agonist or blocking anti-TIGIT Abs modulated autoimmune disease severity whereas administration of blocking anti-TIGIT Abs synergized with anti-PD-1 Abs to affect partial or even complete tumor regression. The Abs presented in this study can thus serve as important tools for detailed analysis of TIGIT function in different disease settings and the knowledge gained will provide valuable insight for the development of novel therapeutic approaches targeting TIGIT.

Does sites of recurrence impact survival in secondary cytoreduction surgery for recurrent epithelial ovarian cancer?

Outcomes of secondary cytoreduction surgery (SCS) were evaluated for morbidity, progression free survival (PFS) and overall survival (OS) and factors influencing results were explored. Retrospective analysis of all cases of SCS for epithelial ovarian cancer (EOC) was performed from October 2010 to December 2017. 62 patients were prospectively identified as candidates for SCS and 57 underwent SCS. 20(35%) patients required bowel resection/s, 24(42%) had nodal resections and 11(19%) had extensive upper abdominal surgery. 51(89%) achieved complete cytoreduction. After a median follow-up of 30 months (range 9-95 months), median PFS was 32 months (CI 17-76 months) and median OS has not reached. Seventeen patients have died and 32 have progressed. Three patients had Clavien-Dindo grade-3 and two had grade-4 morbidity. Patients who had multi-site recurrence had shorter median PFS (p = 0.04) and patients who required bowel resections had lower median OS (p = 0.009) compared to rest of the cohort.IMPACT STATEMENTWhat is already known on this subject? Retrospective studies have confirmed survival advantage for recurrence in epithelial ovarian cancer and recommend SCS for carefully selected patients. This finding is being evaluated in randomised control trials currently.What do the results of this study add? This study presents excellent results for survival outcomes after SCS and highlights importance of careful selection of patients with a goal to achieve complete cytoreduction. In addition, for the first time in literature, this study also explores various factors that may influence results and finds that there are no differences in survival outcomes whether these patients had early stage or advanced stage disease earlier. Patients who have multisite recurrence tend to have shorter PFS but no difference were noted for overall survival. Patients who have recurrence in bowels necessitating resection/s have a shorter median OS compared to rest of cohorts, however, still achieving a good survival time.What are the implications of these findings for clinical practice and/or further research? These findings will raise awareness for the clinicians and patients while discussing surgical outcomes and would set an achievable standard to improve cancer services. The pattern of recurrence and associated outcomes also point towards difference in biological nature of recurrent disease and could provide an opportunity for scientists to study the biological makeup of these recurrent tumours.

Socioeconomic differences impact overall survival in advanced ovarian cancer (AOC) prior to achievement of standard therapy.

PURPOSE: Survival difference between socioeconomic groups with ovarian cancer has persisted in the United Kingdom despite efforts to reduce disparities in care. Our aim was to delineate critical episodes in the patient journey, where deprivation has most impact on survival. METHODS: A retrospective review of 834 patients with advanced ovarian cancer (AOC) between 16/8/07-16/2/17 at a large cancer centre serving one of the most deprived areas of the UK. Using the Index of Multiple Deprivation (IMD), patients were categorised into five groups. RESULTS: Surgery was more common in less deprived patients (p < 0.00001). Across IMD groups, there were no differences in complete (R0) cytoreduction rate (r = 0.18, p > 0.05), age, or comorbidity. The R0/total cohort rate increased with increasing IMD group (p < 0.0001). Patients refusing any intervention belonged exclusively to the three most deprived groups; 5/7 patients who refused surgery belonged to the most deprived IMD group. Overall survival in the total patient group was less in IMD group 1-2 compared to 9-10 (p = 0.002). On multivariate analysis, IMD group was not an independent predictor of survival (p > 0.05). CONCLUSIONS: Socioeconomic differences in survival manifest in patients not receiving surgical treatment for AOC and are not purely explained by comorbidity, age, stage, or histological factors.

Reporting 'Denominator' data is essential for benchmarking and quality standards in ovarian cancer.

OBJECTIVE: Combined surgery and platinum-based chemotherapy is the internationally agreed standard therapy for advanced ovarian cancer (AOC). However international cancer registry datasets demonstrate a significant proportion of patients do not receive both or either therapies. Our objective was to evaluate the effect of total patient cohort data ('Denominator') on median overall survival (OS) and determine how frequently this was reported in literature. METHODS: We retrospectively reviewed OS outcomes for 593 patients diagnosed with AOC for 77 months at a regional cancer centre. Patients were stratified into five progressively overlapping categories based on treatment received - Primary debulking surgery (PDS), PDS or Interval debulking (IDS), all surgery and those considered for IDS, patients receiving any treatment and total patient cohort. A systematic search of literature was performed. RESULTS: Median OS progressively decreased from 54.5 months in patients receiving PDS, 38.7 months in the PDS+IDS group, 35.4 months in the PDS/IDS+patients considered for IDS, 33.3 months in patients receiving any treatment and 30.2 months in the total patient cohort. OS in the surgically treated group was statistically significantly different from the OS in the total patient cohort (Denominator)(p=0.000353). Denominator descriptors were identified in 11% of studies. CONCLUSIONS: Denominator data is critical to understanding selection and OS in AOC. Published outcomes of selected cohorts should routinely incorporate outcomes for all women managed within the reporting Centre. This is essential for benchmarking and quality assurance in gynaecological cancer and should be an integral part of any publication on outcomes from AOC.

Is routine adnexal scanning for postmenopausal bleeding of value? Observational study of 2101 women.

Our objective is to assess the merits of adnexal scanning during the investigation of women with postmenopausal bleeding (PMB) in terms of adnexal cancer diagnosis. This observational study was designed utilising an institutional PMB database in a teaching hospital, analysing a sample of 2101 consecutive women with PMB seen between 16th February 2012 and 12th August 2014 looking at the prevalence of cancer in adnexal masses identified on Trans-vaginal ultrasound scanning (TVS) in these PMB women. This study suggests that routine adnexal scanning in women with PMB may provide no benefit. It could be exposing women to unnecessary surgery or surveillance with the associated risks and cost implications. Most of the women who underwent surgery presented with palpable masses. Those with negative clinical examination had either benign masses which may have remained inconsequential or non-suspicious scan findings. A well-designed randomised controlled trial is needed to confirm the findings. Impact statement Trans-vaginal ultrasound scanning (TVS) is the standard first line investigation for women presenting with postmenopausal bleeding (PMB) primarily to assess the endometrial thickness. This has led to a widespread practice of opportunistic adnexal scanning, which generated a debate amongst gynaecologists about the value of such practice. This observational study, assessing the merits of routine adnexal scanning in these women in terms of adnexal cancer diagnosis, suggests that this practice may provide no benefit to women with isolated self-limiting PMB and unremarkable bimanual examination. It could be exposing women to unnecessary surgery or surveillance with the associated risks and cost implications when insignificant adnexal masses are identified on the scan. A well-designed randomised controlled trial is needed to elucidate if clinical examination in combination with endometrial scanning only is more effective and cost-effective than clinical examination followed by systematic pelvic scanning to detect cases of ovarian cancer in women with PMB.

Predictive value of the age-adjusted Charlston co-morbidity index on peri-operative complications, adjuvant chemotherapy usage and survival in patients undergoing debulking surgery after neo-adjuvant chemotherapy for advanced epithelial ovarian cancer.

The aim of this study was to determine whether the age-adjusted Charlston co-morbidity index (ACCI) can predict post-operative complications, adjuvant chemotherapy usage and overall survival (OS) in patients with advanced epithelial ovarian cancer (AOC) treated with neoadjuvant chemotherapy (NACT). A review was performed of all cytoreductive surgeries performed between 16/8/07-3/2/14 for AOC at a UK Cancer Centre. All surgeries were stratified by ACCI into three groups: Low (0-1), Intermediate (2-3) and High (≥4). Of the 293 cases the ACCI distribution was: 74 (25.26%) low, 164 (55.97%) intermediate and 55 (18.77%) high. Patients with a high ACCI were less likely to receive adjuvant chemotherapy (p = .023), more likely to receive fewer adjuvant cycles (p = .0057) but no more likely to experience complications. Median OS for patients with a low, intermediate and high ACCI was 44.58 (95%CI 36.98-52.19), 34.65 (95%CI 29.48-39.82) and 33.37 (95%CI 17.47-49.27) months. ACCI was associated with OS (p < .01) confirmed on multivariate analysis (p = .03). The ACCI is, therefore, a marker of survival in these patients and predicts adjuvant chemotherapy usage. Impact statement The Age-Adjusted Charlston Co-morbidity Index has previously been identified as a predictor of survival in both medical and surgical conditions. Recently it has also been validated in patients undergoing primary cytoreductive surgery for advanced ovarian cancer. This study is the first to validate the Age-Adjusted Charlston Co-morbidity Index in patients undergoing cytoreductive surgery following neoadjuvant chemotherapy. Our findings demonstrate that it can be used to not only predict overall survival in women undergoing debulking surgery after neo-adjuvant chemotherapy but also predicts the uptake and commencement of adjuvant chemotherapy. Such findings are important considerations to enable an informed patient choice regarding interval surgery in the more co-morbid patients. More importantly, although the ACCI can be used as a marker of overall survival, even in the most co-morbid of patients there remains a significant survival advantage following surgery to the extent that it should not be contraindicated in this cohort. The ACCI is being increasingly incorporated into various clinical trials as a standard demographic measure and this study validates its inclusion in patients undergoing interval debulking surgery.

Patient-Reported Outcomes After Extensive (Ultraradical) Surgery for Ovarian Cancer: Results From a Prospective Longitudinal Feasibility Study.

BACKGROUND: Extensive (ultraradical) surgery may facilitate complete cytoreduction in ovarian cancer with potential survival benefit but with greater morbidity. Currently, patient-reported outcomes (PROs) from such surgery are unknown. We conducted the Surgery in Ovarian Cancer Quality of life Evaluation Research study (SOCQER 1), a prospective study investigating the feasibility of collection of serial PROs in patients who had extensive surgery and standard surgery for ovarian cancer. METHODS: Ninety-three patients were recruited for 33 months to complete serial PRO assessments using the validated EORTC QLQ-C30 and the ovarian cancer-specific QLQ-OV28 questionnaires preoperatively, at 6 weeks, and at 3, 6, and 9 months postoperatively. Aletti Surgical Complexity Score of 3 or lower was considered standard surgery; a Surgical Complexity Score of 4 or higher was considered extensive surgery. Prospective data collection was obtained from the hospital electronic database, including patient demographics, American Society of Anaesthesiologists grade, preoperative serum CA125 and albumin levels, chemotherapy regimen, and surgical morbidity. RESULTS: Three cohorts of patients--32 benign, 32 undergoing standard surgery, and 24 undergoing extensive surgery--completed the questionnaires. Median questionnaire completion rate in this study was 64%, demonstrating the feasibility of longitudinal quality of life (QoL) assessment after surgery. Patient-reported outcomes revealed a falling trend in QoL in the short-term (6 weeks-3 months) after surgery, which gradually returned to baseline at 6 to 9 months; this trend was more marked after extensive surgery. CONCLUSIONS: This study provides useful insight into the impact of extensive surgery on patients. Further multicenter studies are needed to evaluate the impact of extensive surgery on patient's QoL and survival.

Tissue-specific modifier alleles determine Mertk loss-of-function traits.

Knockout (KO) mouse models play critical roles in elucidating biological processes behind disease-associated or disease-resistant traits. As a presumed consequence of gene KO, mice display certain phenotypes. Based on insight into the molecular role of said gene in a biological process, it is inferred that the particular biological process causally underlies the trait. This approach has been crucial towards understanding the basis of pathological and/or advantageous traits associated with Mertk KO mice. Mertk KO mice suffer from severe, early-onset retinal degeneration. MERTK, expressed in retinal pigment epithelia, is a receptor tyrosine kinase with a critical role in phagocytosis of apoptotic cells or cellular debris. Therefore, early-onset, severe retinal degeneration was described to be a direct consequence of failed MERTK-mediated phagocytosis of photoreceptor outer segments by retinal pigment epithelia. Here, we report that the loss of Mertk alone is not sufficient for retinal degeneration. The widely used Mertk KO mouse carries multiple coincidental changes in its genome that affect the expression of a number of genes, including the Mertk paralog Tyro3. Retinal degeneration manifests only when the function of Tyro3 is concomitantly lost. Furthermore, Mertk KO mice display improved anti-tumor immunity. MERTK is expressed in macrophages. Therefore, enhanced anti-tumor immunity was inferred to result from the failure of macrophages to dispose of cancer cell corpses, resulting in a pro-inflammatory tumor microenvironment. The resistance against two syngeneic mouse tumor models observed in Mertk KO mice is not, however, phenocopied by the loss of Mertk alone. Neither Tyro3 nor macrophage phagocytosis by alternate genetic redundancy accounts for the absence of anti-tumor immunity. Collectively, our results indicate that context-dependent epistasis of independent modifier alleles determines Mertk KO traits.

The non-linear impact of data handling on network diffusion models.

Many computational models rely on real-world data, and the steps required in moving from data collection, to data preparation, to model calibration, and input are becoming increasingly complex. Errors in data can lead to errors in model output that might invalidate conclusions in extreme cases. While the challenge of errors in data collection have been analyzed in the literature, here we highlight the importance of data handling in the modeling and simulation process, and how particular data handling errors can lead to errors in model output. We develop a framework for assessing the impact of potential data errors for models of spreading processes on networks, a broad class of models that capture many important real-world phenomena (e.g., epidemics, rumor spread, etc.). We focus on the susceptible-infected-removed (SIR) and Threshold models and examine how systematic errors in data handling impact the predicted spread of a virus (or information). Our results demonstrate that data handling errors can have significant impact on model conclusions especially in critical regions of a system.

Sympathetic nervous tone limits the development of myeloid-derived suppressor cells.

Sympathetic nerves that innervate lymphoid organs regulate immune development and function by releasing norepinephrine that is sensed by immune cells via their expression of adrenergic receptors. Here, we demonstrate that ablation of sympathetic nervous system (SNS) signaling suppresses tumor immunity, and we dissect the mechanism of such immune suppression. We report that disruption of the SNS in mice removes a critical α-adrenergic signal required for maturation of myeloid cells in normal and tumor-bearing mice. In tumor-bearing mice, disruption of the α-adrenergic signal leads to the accumulation of immature myeloid-derived suppressor cells (MDSCs) that suppress tumor immunity and promote tumor growth. Furthermore, we show that these SNS-responsive MDSCs drive expansion of regulatory T cells via secretion of the alarmin heterodimer S100A8/A9, thereby compounding their immunosuppressive activity. Our results describe a regulatory framework in which sympathetic tone controls the development of innate and adaptive immune cells and influences their activity in health and disease.

En bloc resection of the external iliac vein along with broad ligament leiomyosarcoma: A case report.

Outcomes following the excision of the external iliac vein during gynaecological oncology surgery are poorly documented. This is because most gynaecological oncologists consider tumours with vascular involvement inoperable. We describe a patient whose right external iliac vein was transected during the removal of a large broad ligament leiomyosarcoma invading the right external iliac vein. The patient's recovery following surgery was uneventful, and she remained disease-free 6 months postoperatively, with minimal morbidity. In describing this case, we hope to educate and inform other gynaecological oncologists facing a similar surgical challenge. We also propose that resection of the external iliac vessels in such cases is safe and feasible, and summarise the anatomical course of venous collaterals, which develop when the external iliac veins are obstructed.

Diaphragm disease in advanced ovarian cancer: Predictability of pre-operative imaging and safety of surgical intervention.

OBJECTIVES: To establish the positive predictive values of pre-operative identification with CT imaging of metastatic diaphragm disease in surgically managed cases of advanced ovarian cancer (AOC). Additionally, we have assessed the post-operative morbidity and survival following diaphragmatic surgical intervention in a large regional cancer centre in the United Kingdom. STUDY DESIGN: A retrospective review of all cases of AOC with metastatic diaphragm disease surgically treated at the Pan-Birmingham Gynaecological Cancer Centre, UK between 1st August 2007 and 29th February 2016. RESULTS: A total of 536 women underwent surgery for primary AOC. Diaphragm disease was evident intra-operatively in 215/536 (40.1%) and 85/536 women (15.9%) underwent a procedure involving their diaphragm. Of these 85 cases, 38 peritoneal strippings (38/85, 44.7%), 31 partial diaphragmatic resections (31/85, 35.6%) and 16 electro-surgical ablations (16/85, 18.9%) were performed. There were no significant differences in post-operative complications between the three different diaphragmatic surgical groups. Of those patients who underwent peritoneal stripping or partial diaphragm resection, 12% were upstaged to stage 4A by virtue of pleural invasion. The positive predictive value for pre-operative radiological identification of diaphragmatic disease was 78.6%. CT imaging failed to detect diaphragmatic involvement despite obvious diaphragm disease during surgery in 29.4% of cases, giving a low negative predictive value of 64.8%. The sensitivity and specificity for CT imaging in detecting diaphragm disease was 44.3% and 93.8%, respectively. CONCLUSIONS: Diaphragmatic disease is often discovered in AOC. However, pre-operative assessment with CT imaging is not reliable in accurately detecting diaphragm involvement. Therefore, all patients with AOC should be regarded as in potential need for diaphragm surgery and their operation undertaken in cancer centres with adequate expertise in upper abdominal surgery. If there is a suspicion of diaphragm muscle invasion during diaphragmatic peritonectomy, the muscle should be partially resected. This will lead to potential upstaging of disease to stage 4A and therefore, to suitability for targeted therapy. In our Centre, the surgical removal of diaphragmatic disease did not significantly increase surgical morbidity.

Complete cytoreduction after five or more cycles of neo-adjuvant chemotherapy confers a survival benefit in advanced ovarian cancer.

OBJECTIVES: To assess the impact of 5 or more cycles of neoadjuvant chemotherapy (NACT) and cytoreductive outcomes on overall survival (OS) in patients undergoing interval debulking surgery (IDS) for advanced ovarian cancer. METHODS: A retrospective review of patients receiving NACT followed by IDS between 2007 and 2017. Patients were analysed according to number of NACT cycles received: group 1 consisted of patients receiving ≤4 cycles and group 2 consisted of those receiving ≥5 cycles. Outcomes were stratified by cytoreductive outcome, surgical complexity, stage and chemotherapy exposure. RESULTS: 231 patients in group 1 and 167 in group 2 were identified. In group 1, the OS for those achieving Complete (R0), Optimal<1 cm (R1) and Suboptimal (R2) was 51.1, 36.1, and 34.3 months respectively. Statistically significant differences in survival were seen in patients achieving R0vR2 (p < 0.019) but not in R0vR1 (p = 0.125) or R1vR2 (p = 0.358). In group 2, the OS for those achieving R0, R1 and R2 was 53.0, 24.7, and 22.1 months respectively. Statistically significant differences were seen between R0vR1 and R0vR2 (p < 0.00001) but not between R1vR2 (p = 0.917). No difference in OS was seen between groups 1 and 2. In patients achieving R1, there was a trend towards decreasing OS with increasing exposure to NACT from 36.1 (95%CI 32.0-40.2)months with 3 cycles to 24.3 (95%CI 14.4-34.2)months with ≥6 cycles. CONCLUSIONS: Surgery with utilisation of cytoreductive procedures to achieve complete clearance should be offered to all patients even after ≥5 cycles if R0 can be achieved. R1 cytoreduction has questionable value in those receiving ≤4 cycles and no value in those receiving ≥5 cycles.

Blockade of Tim-3 binding to phosphatidylserine and CEACAM1 is a shared feature of anti-Tim-3 antibodies that have functional efficacy.

Both in vivo data in preclinical cancer models and in vitro data with T cells from patients with advanced cancer support a role for Tim-3 blockade in promoting effective anti-tumor immunity. Consequently, there is considerable interest in the clinical development of antibody-based therapeutics that target Tim-3 for cancer immunotherapy. A challenge to this clinical development is the fact that several ligands for Tim-3 have been identified: galectin-9, phosphatidylserine, HMGB1, and most recently, CEACAM1. These observations raise the important question of which of these multiple receptor:ligand relationships must be blocked by an anti-Tim-3 antibody in order to achieve therapeutic efficacy. Here, we have examined the properties of anti-murine and anti-human Tim-3 antibodies that have shown functional efficacy and find that all antibodies bind to Tim-3 in a manner that interferes with Tim-3 binding to both phosphatidylserine and CEACAM1. Our data have implications for the understanding of Tim-3 biology and for the screening of anti-Tim-3 antibody candidates that will have functional properties in vivo.