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The expression and function of ICAM-1 are critical components in the initiation and elicitation of many T cell-mediated responses. Whether ICAM-1 expression is required on the T cells or on the APC during T cell priming remains unclear. To address this issue in alloantigen-specific T cell activation, the priming and function of T cells in response to heart allografts from MHC-mismatched wild-type vs ICAM-1−/− donors were tested. Wild-type C57BL/6 (H-2b) heart allografts were rejected by A/J (H-2a) recipients on days 7–9, whereas B6.ICAM-1−/− allografts survived until days 18–23 post-transplant. On day 7 post-transplant, infiltrating macrophages and CD4+ and CD8+ T cells in the ICAM-1−/− allografts were 20–30% those observed in the wild-type allografts. ELISPOT analyses indicated that the number of alloantigen-specific T cells producing IFN-γ from recipients of ICAM-1-deficient grafts was 60% lower than that from recipients of wild-type allografts. On day 16 post-transplant, these numbers did not markedly increase in ICAM-1-deficient allograft recipients. Consistent with the reduced priming of alloreactive T cells, isolated dendritic cells from ICAM-1−/− mice stimulated allogeneic T cell proliferation poorly compared with wild-type dendritic cells. When A/J mice were primed with wild-type dendritic cells and then received wild-type or ICAM-1-deficient heart allografts 3 days later, the primed recipients rejected the wild-type and ICAM-1−/− allografts on days 5–6 post-transplant. These results indicate that optimal priming of alloreactive T cells requires allograft expression of ICAM-1, but, once primed, recipient T cell infiltration into the allograft is independent of graft ICAM-1 expression.

e14543 Background: Poor infiltration of activated lymphocytes into solid tumors can be a fundamental factor limiting their efficacy. The purpose of this study was to determine whether a cycled tumor-penetrating peptide, iRGD, with a well-defined role in delivering drugs into extravascular tumor tissues, could facilitate lymphocytes infiltration into tumors in both the iRGD combination regimen and conjugated pattern. Methods: We used polyethylene glycol-conjugated phospholipid (PEG-lipid) derivatives, a time-efficient and versatile platform, to immobilize iRGD on T cell membrane, and examined the ability of iRGD to improve lymphocytes infiltration into tumors in multicellular spheroids and two xenograft mouse models. Furthermore, the synergy effect of iRGD modification and PD-1 gene knock out in adoptive T cell transfer immunotherapy was evaluated in a xenograft model of EBV associated gastric cancer. Results: In this study, we first showed that systematic injection of iRGD could double the number of CD3+ T cells in 4T1 tumor in BALB/C mouse and the synthetic iRGD-PEG-lipid, which could spontaneously transfer from solution to cell surfaces, had no effect to cell vitality, proliferation, phenotype and function. In adoptive transfer studies, the infiltration of T cells could be promoted by the co-administration with iRGD while T cells modified with iRGD could spread more extensively throughout both the multicellular spheroids and tumor mass. Furthermore, We demonstrated that iRGD modified T cells had superior antitumor efficiency to iRGD combination regimen due to increased T cell infiltration, and exhibited robust synergy effect with PD-1 gene knock out. Conclusions: Our study offers a simple method to target immune cells into tumors and may have the potential to enhance the clinical efficacy of adoptive T cell immunotherapy.

// Shenying Jin 1, 2 , Bo Xu 1 , Lixia Yu 1 , Yao Fu 3 , Hongyan Wu 3 , Xiangshan Fan 3 , Jia Wei 1 and Baorui Liu 1 1 The Comprehensive Cancer Centre of Drum Tower Hospital, Medical School of Nanjing University & Clinical Cancer Institute of Nanjing University, Nanjing 210008, China 2 The Comprehensive Cancer Center of Drum Tower Hospital, Clinical College of Nanjing Medical University, Nanjing 210008, China 3 Department of Pathology, Nanjing Drum Tower Hospital, The Affiliated Hospital of Nanjing University Medical School, Nanjing 210008, China Correspondence to: Baorui Liu, email: baoruiliu@nju.edu.cn Jia Wei, email: weijia01627@hotmail.com Keywords: programmed cell death protein 1, programmed cell death ligand 1, CD3+ T cell, microsatellite instability, gastric signet-ring cell carcinoma Received: January 09, 2017 Accepted: February 24, 2017 Published: March 21, 2017 ABSTRACT Recent data supports a potentially significant role for immune checkpoint inhibitors in the treatment of gastric cancer. However, there are few data on the clinical implications of immunotherapy markers in gastric signet-ring cell carcinoma (SRCC). We evaluated the expression of programmed cell death protein-1 (PD-1), programmed cell death ligand 1(PD-L1), infiltration by CD3+ T cell, microsatellite instability (MSI), and Epstein-Barr Virus (EBV), and the relationship of each factor to survival in 89 advanced SRCC patients. All patients received 5-FU-based first-line chemotherapy. PD-L1 and PD-1 were expressed in 40.4% and 18.0% of the patients, respectively. There was a significant correlation between PD-L1 and PD-1 expression (r=0.363, p

e14608 Background: Recent research suggests an important role for Wnt/β-catenin signaling in mediating cancer immune evasion and resistance to immune checkpoint therapy. The mechnism is believed to involve blocking of specific cytokines which trigger immune cell recruitment to the tumor, resulting in the phenomenon of T-cell exclusion and rendering the tumor to a non-inflamed state. Inhibition of β-catenin may be an effective strategy for increasing the low response rate to these effective medicines in numerous cancer populations. DCR-BCAT is an advanced preclinical development candidate that has a potent and specific chemically-optimized RNA interference (RNAi) trigger targeting CTNNB1, the gene that encodes β-catenin, formulated in a tumor-selective lipid nanoparticle. Methods: Syngeneic murine models and transgenic MMTV-Wnt1 mouse models were used in this study. In both cases, a sequential dose regimen was employed where, in each dosing cycle, animals received DCR-BCAT, followed by a combination of anti-PD-1 and anti-CTLA-4 on subsequent days. Pharmacodynamic endpoints included CTNNB1 (β-catenin), CCL4, PD-1, PD-L1 mRNA measurement by quantitative PCR, as well as β-catenin, perforin, and granzyme B immunohistochemistry. Results: In syngeneic models, β-catenin inhibition with DCR-BCAT significantly improved the T-cell infiltration. The combination of DCR-BCAT and immune checkpoint blockade yielded significant tumor growth inhibition compared to monotherapy in B16F10, 4T1, Neuro2A and Renca tumors. The combination therapy was associated with high levels of granzyme B and perforin, strongly suggesting that the mechanism of sensitization to checkpoint therapy was a sharp increase in T-cell mediated cytotoxicity. Finally, when DCR-BCAT was combined with anti-PD-1/CTLA-4 antibodies in mice which develop spontaneous Wnt-driven mammary tumors, checkpoint therapy potentiation yielded complete tumor regressions. Conclusions: These data offer proof-of-concept for conversion of non-inflamed tumors to inflamed tumors by β-catenin inhibition, and support clinical evaluation of this combination approach using a first-in-class RNAi-based agent.

3599 Background: Pre-operative CRT followed by total mesorectal excision (TME) is nowadays the standard of care for patient with LARC (cT3-T4N0 or cTxN+). Currently, pathologic complete response occurs in +/- 15% after CRT. Colorectal cancer T-cell infiltration is a strong prognostic factor for survival after primary tumor resection. Our aim was to determine whether T-cell infiltration in PTB could be predictive of tumor response and relapse after CRT + TME. Methods: Between 1999 and 2012, patients with LARC who underwent CRT + TME and with available clinical follow-up and PTB (with sufficient tumor cells density) were identified at the Cliniques universitaires St-Luc. The density of CD3 (T cells) and CD8 (cytotoxic) was quantified on immunostained PTB slides and analyzed with a dedicated image analysis software on whole-slide imaging. Comparisons were made using the Wilcoxon-Mann-Whitney test. Cumulative disease-free survival (DFS) was performed using the Kaplan-Meier estimator and compared by log-rank tests. Cox regression we used for uni- and multi-variate analysis. P value of less than 0.05 was considered statistically significant. Results: 154 patients (sex ratio M/F 1.8; mean age 65 years-old; upper (20%), mid (29%) and low rectum (51%), synchronous metastases (11%)) were analyzed. High CD3 and CD8 PTB densities were significantly associated with a higher pathological response (Dworak 3-4) and lower ypTNM stage after CRT +TME (p < 0,05). Higher CD3 and CD8 PTB densities were associated with higher patient DFS (CD3: HR = 2,30 (CI95%:1,15-4,59) p = 0,02; CD8: HR = 1,95 (CI95%: 1,01-3,75) p = 0,04). These results were confirmed in uni and multivariate analysis. CD3 and CD8 PTB densities added to pathological response (ypTNM/Dworak) but also clinical response (ycTNM) after CRT + TME increases significantly the accuracy prediction of tumor relapse. Conclusions: Pretherapeutic T-cell infiltration of LARC is predictive of tumor response and relapse after CRT +TME. This biomarker could be helpful for patient treatment decision. It must be validated in larger patient cohorts.

3053 Background: Activation of the Wnt-β-catenin signaling pathway is associated with poor T cell infiltration of tumors. We have previously demonstrated that paracrine Wnt5a-β-catenin signaling is a critical trigger of dendritic cell (DC) tolerization and regulatory T cell (Treg) differentiation in the melanoma microenvironment. In a transgenic BRAFV600EPTEN-/- model, the genetic silencing of melanoma-derived Wnt5a potently enhances infiltrating CD8+T cell effector function and promotes responses to anti-PD-1 Ab therapy. Ipafricept (IPA) is a recombinant Wnt decoy receptor and Vantictumab (VAN) is a Fzd receptor monoclonal Ab. Both molecules inhibit Wnt-β-catenin signaling and have been well-tolerated in ongoing phase I/Ib clinical trials. We explored the ability of IPA/VAN to reverse tumor-mediated immune tolerance and enhance the efficacy of anti-PD-1 Ab immunotherapy in a pre-clinical model that closely recapitulates human melanoma. Methods: Both IPA and VAN were utilized to investigate Wnt-β-catenin inhibition as a strategy for suppressing melanoma-induced DC indoleamine 2,3-dioxygenase (IDO) expression and Treg differentiation in vitro. These agents were further tested for their ability to enhance anti-tumor T cell responses and to augment the efficacy of anti-PD-1 Ab therapy in syngeneic and autochthonous models of BRAFV600EPTEN-/- melanoma. Results: IPA and VAN effectively inhibit Wnt5a and melanoma-induced DC IDO expression and Treg differentiation in vitro. Further studies demonstrate that IPA and VAN significantly augment anti-PD-1 Ab-mediated suppression of primary and metastatic tumor progression in both syngeneic and autochthonous BRAFV600EPTEN-/- melanoma models. These anti-tumor effects correlated with suppressed IDO enzymatic activity, enhanced tumor-infiltrating CD8+T cell/Treg ratios, and increased activation of TRP2 antigen-specific effector T cells. Conclusions: The pharmacological inhibition of paracrine Wnt-β-catenin signaling with IPA and VAN augment the anti-tumor efficacy of anti-PD-1 Ab therapy and represent a promising strategy for further phase I testing in melanoma and other solid tumors.

101 Background: Cancers of the head and neck (HNC) represent some of the most debilitating and aggressive tumors. Radiotherapy (RT) is the primary treatment modality for locally advanced HNC, but less than 50% of patients treated with RT survive to 5 years. Clinical trials with the immune checkpoint inhibitor PD-L1 have shown considerable promise, but on its own has only yielded a 20% response rate. RT has the potential to transform the tumor microenvironment promoting infiltration of T-cells in poorly immunogenic tumors and activating T-cells in tumors with pre-existing T-cell populations. We hypothesized that the combination of PD-L1 inhibition with radiation can enhance therapeutic efficacy through re-invigoration of exhausted T-cells. Methods: Tumor cells were injected into the right buccal mucosa. Treatment was initiated when established tumors were observed. Forty mice per tumor model were randomized to IgG2b control, anti PD-L1, RT alone or RT+anti PD-L1. Anti PD-L1 was started 3 days before RT (single dose of 10Gy) and maintained for 3 weeks. For mechanistic studies, mice received the same treatment but were euthanized 72 hours after RT. Flow cytometric analysis of T-cell expression of PD-1, CTLA-4, FOXP3, CD44 and IFNG was performed on freshly harvested tumors and regional lymph nodes. Results: Tumor-bearing mice formed aggressive tumors with regional lymph node metastasis. Mice treated with RT or anti PD-L1 had slightly improved survival whereas mice treated with RT+anti-PD-L1 had significantly improved survival (p = 0.0007). Tumor control was significantly improved in the RT+anti-PD-L1 group compared to other groups. Anti PD-L1 alone did not improve tumor control. Average tumor volume on day 23 was 175±60.1 mm3 in the RT+anti-PD-L1 group compared to 623±91.0 mm3 in the PD-L1 group. Flow cytometric analysis revealed that this is mediated by enhancement in T-cell infiltration. Analysis of T-cell activation showed increased expression of CD44 and IFNG. Conclusions: Our results show a critical role for RT in mediating sensitivity to PD-L1 inhibition in HNC. In combination with anti PD-L1, RT significantly increased survival and tumor control compared to either modality alone.

23 Background: The success of immunotherapy has raised new issues regarding the selection of patients, design of combination strategies, and sequencing of various regimens. Sarcomas have poor outcomes in the metastatic setting but may be amenable to immune therapies. However, we currently have limited knowledge of the immunologic profiles of different soft tissue sarcoma (STS) subtypes. Methods: We identified patients with the relatively common STS subtypes: leiomyosarcoma (LMS), undifferentiated pleomorphic sarcoma (UPS), synovial sarcoma (SS) and liposarcoma. Formalin fixed paraffin embedded (FFPE) tumor samples from 81 patients underwent gene expression analysis, immunohistochemistry for PD-1 and PD-L1, and sequencing of the T cell receptor Vβ region. Differences in liposarcoma subsets were also evaluated. Results: UPS and LMS had high expression levels of genes related to antigen presentation and T cell infiltration. UPS had higher levels of PD-L1 (p ≤ 0.001) and PD-1 (p ≤ 0.05) on IHC. UPS also had the highest T cell infiltration based on TCR sequencing, significantly more than SS, which had the lowest (p ≤ 0.05). UPS and LMS both had higher clonality compared with SS and liposarcoma (p ≤ 0.05). A model adjusted for STS histologic subtype found that for all sarcoma T cell infiltration and clonality were highly correlated with PD-1 and PD-L1 staining levels (p ≤ 0.01). Conclusions: In a model adjusted for sarcoma histologic subtypes, T cell infiltration and clonality were highly correlated with PD-1 and PD-L1 expression, consistent with the emerging view of tumor immunity that highly inflamed tumors acquire inhibitory ligands to evade tumor-specific T cells. UPS, which is a more highly mutated STS subtype, provokes a strong immune response evidenced by multiple inflammatory features suggesting that it may be well-suited to checkpoint inhibitor based approaches. SS and liposarcoma subsets are less highly mutated but do express immunogenic self-antigens therefore strategies to improve antigen presentation and T cell infiltration may be valuable for allowing immunotherapeutic success in these tumor types.

Introduction: Dendritic cells (DCs) that are known as professional antigen-presenting cells have been found to pre-locate in non-inflammatory arterial wall and increasingly accumulate during atherosclerosis progression. Previous findings suggested that residential DCs in the intima are responsible for capturing modified lipids and forming foam cells during the initiation of atherosclerosis. Hypothesis: DC accumulation and enhanced DC-T cell interaction play a critical role in the initiation of atherosclerosis. Methods: We measured plaque formation, vascular DC accumulation and antigen-specific T cell proliferation mediated by isolated aortic cells in ApoE-/- mice, as well as DTR-CD11c/ApoE-/- or DTR-CD11b/ApoE-/- mice for conditional depletion of DCs or macrophages, respectively. A brief high-fat diet for 10 days was used as a model of initial atherosclerosis. Results: In addition to increased intimal DC accumulation and plaque formation in aortic roots, 10 days of HFD induced T cell infiltration in ApoE-/- mice, compared to those without HFD as the control. Isolated aortic cells from mice with 10-day HFD showed stronger capability in inducing antigen-specific T cell proliferation, compare to the control (HFD: 3.14±0.71%; no HFD: 1.56±0.36%; p=0.022). Single diphtheria toxin (DT) injection at day 1 yielded approximately 50% decrease in intimal DC accumulation, as well as 60% attenuation in plaque formation in DTR-CD11c/ApoE-/- mice after 10-day HFD. Capability of stimulating antigen-specific T cell proliferation was also impaired in aortic cells from DC-depleted mice (DT-treated: 1.62±0.30%; PBS-treated: 3.04±0.59%; p= 0.004), along with reduction in indirect conduction of T cell activation. In contrast, no significant changes were found in plaque formation and DC accumulation in DT-injected DTR-CD11b/ApoE-/- mice after 10 days of HFD, compared to control group. Furthermore, depletion of CD11b+ macrophages in either aortas or spleens didn’t alter capability of inducing antigen-specific T cell proliferation in DT-injected mice. Conclusions: These results suggested that vascular DCs rather than macrophages play a more important role in T cell activation and initiation of atherosclerosis.

It is known that astrocytes are susceptible to thiamine deficiency (TD) and involved in TD-induced CNS damage/neuroinflammation. However, TD-induced neuroinflammation not only involves astrocytes, but also neurons, microglia, and endothelia cells. As previously demonstrated, TD produces a mild

Nitric oxide is a critical regulator of blood pressure (BP) and inflammation, and female spontaneously hypertensive rats (SHR) have higher renal nitric oxide bioavailability than males. We hypothesize that female SHR will have a greater rise in BP and renal T cell infiltration in response to nitric oxide synthase (NOS) inhibition than males. Both male and female SHR displayed a dose-dependent increase in BP to the nonspecific NOS inhibitor NG-nitro-l-arginine methyl ester (l-NAME: 2, 5, and 7 mg·kg−1·day−1 for 4 days each); however, females exhibited a greater increase in BP than males. Treatment of male and female SHR with 7 mg·kg−1·day−1 l-NAME for 2 wk significantly increased BP in both sexes; however, prior exposure to l-NAME only increased BP sensitivity to chronic NOS inhibition in females. l-NAME-induced hypertension increased renal T cell infiltration and indices of renal injury in both sexes, yet female SHR exhibited greater increases in Th17 cells and greater decreases in regulatory T cells than males. Chronic l-NAME was also associated with larger increases in renal cortical adhesion molecule expression in female SHR. The use of triple therapy to block l-NAME-mediated increases in BP attenuated l-NAME-induced increases in renal T cell counts and normalized adhesion molecule expression in SHR, suggesting that l-NAME-induced increases in renal T cells were dependent on both increases in BP and NOS inhibition. Our data suggest that NOS is critical in the ability of SHR, females in particular, to maintain BP and limit a pro-inflammatory renal T cell profile.

Colorectal cancer has an extremely poor prognosis due to its high rate of recurrence and metastasis. The present study aimed to investigate the correlations between the B7-H1 and B7-H4 expressions as well as the clinicopathological characteristics and the prognosis of patients with colorectal cancer. We further inferred from these findings whether T lymphocyte co-inhibitory molecules (B7-H1 and B7-H4) led to a poor prognosis in Heilongjiang patients with colorectal cancer. Survival analysis revealed that the poor prognosis of these patients was unrelated to patient age, tumor size or histological grade, or lymph node metastasis, but was associated with TNM stage, high B7-H1 and B7-H4 expression levels. High B7-H1 and B7-H4 expressions were closely correlated with poor prognosis in patients with colorectal cancer. We speculate that the joint detection of these molecules may clinically apply for diagnosing and predicting poor prognosis of patients with colorectal cancer in northeast China’s Heilongjiang province. In addition, intervention of B7-H1 and B7-H4 may be beneficial for enhancement of immunity in these patients.

Immune checkpoint blockade therapies fail to induce responses in the majority of cancer patients, so how to increase the objective response rate becomes an urgent challenge. Here, we demonstrate that sufficient T cell infiltration in tumor tissues is a prerequisite for response to PD-L1 blockade. Targeting tumors with tumor necrosis factor superfamily member LIGHT activates lymphotoxin β-receptor signaling, leading to the production of chemokines that recruit massive numbers of T cells. Furthermore, targeting non-T cell-inflamed tumor tissues by antibody-guided LIGHT creates a T cell-inflamed microenvironment and overcomes tumor resistance to checkpoint blockade. Our data indicate that targeting LIGHT might be a potent strategy to increase the responses to checkpoint blockades and other immunotherapies in non-T cell-inflamed tumors.

Tumors are heterogeneous, containing tumor-infiltrating lymphocytes (TILs) which presence has been associated with favorable prognosis in multiple tumor types. Importantly, most of the T-cell specificity for tumor neo-antigen lies in a unique sequence of DNA (CDR3) resulting from the recombination of the V, D, and J segments of the T-cell receptor beta (TCRB clonotype). The Cancer Genome Atlas (TCGA) is the most comprehensive molecular dataset of tumors but does not include detailed information about TILs or tumor immunity in general. In order to characterize the T-cell infiltration in public cancer genomic datasets, we hypothesize that such a unique CDR3 DNA fragment can be recovered from bulk tumor genome-wide DNA or RNA sequencing and that the information can be used for clinical association studies. We first establish the feasibility of the approach by comparing the deep CDR3 sequencing (ImmunoSeq) and CDR3 reads recovered in tumor exome data. The clonotypes were detected using three CDR3 read detection tools and results were compared to the ImmunoSeq results. Each tool identifies between 10 and 38 reads. As expected, most of the CDR3 reads are misaligned to the naïve reference genome (60% clipped). Across all three tools, we identify 26 unique clonotypes, 15 of which are detected in the ImmunoSeq experiment. IMSEQ shows the strongest overlap with the other tools (13/14 clonotypes) and with ImmunoSeq (9/14 clonotypes) showing that our strategy identifies bona fide CDR3 reads from exome datasets. We applied the approach to a cohort of 1,078 TCGA samples. We identified CDR3 reads in 473 samples which are more likely to have a higher fraction of TILs as detected by histological review (p = 1.14×10-8). Using the RNA-seq datasets, 906 samples had CDR3 reads and also associated with higher TIL content (p = 4.41×10-6). Tumors with CDR3 reads in both DNA and RNA datasets tend have a higher TIL fraction. A set of 66 tumors shares a unique TCRâ clonotype with however no association with tumor subtypes or HLA haplotype. When restricted to Her2+ tumors, high CDR3 read counts, but not high fraction of TILs, was associated with better overall survival (p = 0.016), suggesting that CDR3 reads are predictive of anti-Her2 response. In contrast, the presence of CDR3 reads in Her2- tumors is not prognostic. A detailed analysis of 22 immune meta-gene expression signatures in these samples complements CDR3 reads information and suggests mechanisms of immune-tolerance. Our results demonstrate the feasibility of identifying genetic evidence of T-cells in bulk tumor samples using deep sequencing, providing information about the T-cell abundance and clonal identity. This novel type of tumor immune biomarker enriches the comprehensive dataset collected through TCGA, and reveals correlations with other molecular features and clinical outcomes. Citation Format: Olivier Harismendy, Eric D. Levy, Valentine Garate-Calderon, Brian Woo, Ricardo Armisen. Molecular characterization of breast tumor T-cell infiltration in exome datasets. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 847.

3042Background: Mutations in DNA repair genes are common in cancer, and may induce a state of hypermutability. Importantly, mutational burden is linked to enhanced response to immunotherapies, as p...

The aim of this study was to relate the density of tumor infiltrating T cells to cancer-specific survival in colorectal cancer, taking into consideration the CpG island methylator phenotype (CIMP) and microsatellite instability (MSI) screening status. The T-cell marker CD3 was stained by immunohistochemistry in 484 archival tumor tissue samples. T-cell density was semiquantitatively estimated and scored 1-4 in the tumor front and center (T cells in stroma), and intraepithelially (T cells infiltrating tumor cell nests). Total CD3 score was calculated as the sum of the three CD3 scores (range 3-12). MSI screening status was assessed by immunohistochemistry. CIMP status was determined by quantitative real-time PCR (MethyLight) using an eight-gene panel. We found that patients whose tumors were highly infiltrated by T cells (total CD3 score ≥7) had longer survival compared with patients with poorly infiltrated tumors (total CD3 score ≤4). This finding was statistically significant in multivariate analyses (multivariate hazard ratio, 0.57; 95% confidence interval, 0.31-1.00). Importantly, the finding was consistent in rectal cancer patients treated with preoperative radiotherapy. Although microsatellite unstable tumor patients are generally considered to have better prognosis, we found no difference in survival between microsatellite unstable and microsatellite stable (MSS) colorectal cancer patients with similar total CD3 scores. Patients with MSS tumors highly infiltrated by T cells had better prognosis compared with intermediately or poorly infiltrated microsatellite unstable tumors (log rank P=0.013). Regarding CIMP status, CIMP-low was associated with particularly poor prognosis in patients with poorly infiltrated tumors (multivariate hazard ratio for CIMP-low versus CIMP-negative, 3.07; 95% confidence interval, 1.53-6.15). However, some subset analyses suffered from low power and are in need of confirmation by independent studies. In conclusion, patients whose tumors are highly infiltrated by T cells have a beneficial prognosis, regardless of MSI, whereas the role of CIMP status in this context is less clear.

In this issue of Blood, Fhu et al report that Reed-Sternberg cell-derived lymphotoxin-{alpha} activates endothelial cells to enhance T-cell recruitment in classical Hodgkin lymphoma (cHL), a process that is regulated by cyclooxygenase/nuclear factor-{kappa}B/activator protein 1 signaling pathways.

Cancer cells produce galectin-1 as a tumor promoting protein. Thiodigalactoside (TDG) as a non-metabolised small drug, is shown to suppress tumor growth by inhibiting multiple cancer enhancing activities of galectin-1, including immune cell dysregulation, angiogenesis and protection against oxidative stress. Thus, using B16F10 melanoma and 4T1 orthotopic breast cancer models, intratumoral injection of TDG significantly raised the levels of tumor-infiltrating CD8+ lymphocytes and reduced CD31+ endothelial cell content, reducing tumor growth. TDG treatment of tumors in Balb/c nude mice (defective in T cell immunity) reduced angiogenesis and slowed tumor growth by a third less than in immunocompetent mice. Knocking down galectin-1 expression (G1KD) in both cancer cell types significantly impeded tumor growth and the sensitivity of the G1KD tumors to TDG was severely reduced, highlighting a specific role for galectin-1. Endothelial cells were protected by galectin-1 from oxidative stress-induced apoptosis induced by H2O2, but TDG inhibited this antioxidant protective effect of galectin-1 and reduced tube forming activity in angiogenic assays. We show for the first time that the single agent, TDG, concurrently prevents many tumor promoting effects of galectin-1 on angiogenesis, immune dysregulation and protection against oxidative stress, providing a potent and novel small molecule as an anti-cancer drug. Electronic supplementary material The online version of this article (doi:10.1007/s10456-011-9213-5) contains supplementary material, which is available to authorized users.

The relation between monoclonal antibodies and polyneuropathy is best supported for polyneuropathy associated with IgM monoclonal anti-myelin associated glycoprotein (anti-MAG) antibodies. These anti-MAG antibodies are reactive against peripheral nerve autoantigen, thereby causing an autoimmune mediated polyneuropathy. To further discriminate clinical entities, we analyzed the presence of T cells in sural nerve biopsies in primary demyelinating and primary axonal polyneuropathies associated with monoclonal gammopathy. Increased T cell densities were found in: 1) patients with progressive demyelinating polyneuropathy associated with IgG monoclonal gammopathy, with T cell densities (T cells/mm2) comparable to densities in patients with chronic inflammatory demyelinating polyneuropathy (CIDP); 2) patients with progressive axonal polyneuropathy associated with monoclonal gammopathy. The pathologic features in the biopsies of these patients resembled those found in vasculitic neuropathy. In polyneuropathy associated with IgM monoclonal gammopathy, antibodies to myelin-associated glycoprotein (MAG), sulfoglucuronyl paragloboside (SGPG) and sulfatide have been associated with specific clinical and electrophysiologic features. However, it is not known whether the results of antibody tests provide additional information for the individual patient (and the neurologist) on future neurologic deficit or outcome. Therefore, we studied the potential prognostic value of clinical, electrophysiologic, pathologic and antibody determination (Chapter 3). In multivariate analysis initial sensory symptoms of the feet is associated with a slowly progressive disease course and less disability at four years, demyelination on electrophysiologic examination is prognostic for development of weakness and symptoms of upper extremities at four years. Half of the patients with polyneuropathy associated with IgM monoclonal gammopathy have anti-MAG antibodies. Since patients with IgM monoclonal gammopathy without anti-MAG antibodies have similar symptoms and comparable response to treatment, it is most likely that these patients have antibody reactivity against other components of the nerve. We found monoclonal anti-ganglioside antibodies in half of the patients with polyneuropathy associated with IgM monoclonal gammopathy without anti-MAG antibodies (Chapter 3). Monoclonal IgM anti-GQ1b antibodies were associated with ataxia. Monoclonal gammopathy is of undetermined significance (MGUS) as long as no hematologic malignancy, like multiple myeloma or lymphoma is found. In patients with polyneuropathy associated with monoclonal gammopathy the frequency of hematologic malignancy is high (22%). A follow-up study of patients with polyneuropathy associated with MGUS showed that the incidence of malignant transformation is higher (2.7/100 patient years, 95% CI 1.52-4.22) than in patient with MGUS without polyneuropathy (1/100 patient years, 95% CI 0.85-1.24). Progression of the polyneuropathy, weight loss, unexplained fever or night sweats and M-protein level were independent predictors (Chapter 4). To find an explanation for the increased frequency of hematologic malignancies in polyneuropathy associated with monoclonal gammopathy we studied the occurrence of genetic aberrations (Chapter 5) by using interphase fluorescence in situ hybridisation (FISH), multiplex ligation-dependent probe amplification (MLPA) assay and genome-wide array-based comparative genomic hybridisation (CGH). We identified different genetic aberrations in patients with polyneuropathy associated with IgM monoclonal gammopathy. Half of the patients had chromosome 14 translocations, which are also frequently identified in multiple myeloma and B cell malignancies. The variable part of the immunoglobulin is responsible for the antigen binding and consists of V, D, J gene segments. We determined the immunoglobulin gene usage in patients with polyneuropathy associated with IgM monoclonal gammopathy (Chapter 6). We found a preferential use of immunoglobulin genes associated with immune responses to encapsulated bacteria (preferentially VH3-23). In patients with polyneuropathy and monoclonal gammopathy, after excluding other causes, the disease course leads the diagnostic process and the additional investigations (Chapter 7, flow chart). We advise a yearly follow-up of all patients with polyneuropathy associated with monoclonal gammopathy, including M-protein level determination. A hematologic screening, including bone marrow examination should be performed if the patient has clinical risk factors of malignant transformation (weight loss, night sweats, bone pain, anemia, rise of M-protein level) or a progressive polyneuropathy.

The relation between monoclonal antibodies and polyneuropathy is best supported for polyneuropathy associated with IgM monoclonal anti-myelin associated glycoprotein (anti-MAG) antibodies. These anti-MAG antibodies are reactive against peripheral nerve autoantigen, thereby causing an autoimmune mediated polyneuropathy. To further discriminate clinical entities, we analyzed the presence of T cells in sural nerve biopsies in primary demyelinating and primary axonal polyneuropathies associated with monoclonal gammopathy. Increased T cell densities were found in: 1) patients with progressive demyelinating polyneuropathy associated with IgG monoclonal gammopathy, with T cell densities (T cells/mm2) comparable to densities in patients with chronic inflammatory demyelinating polyneuropathy (CIDP); 2) patients with progressive axonal polyneuropathy associated with monoclonal gammopathy. The pathologic features in the biopsies of these patients resembled those found in vasculitic neuropathy. In polyneuropathy associated with IgM monoclonal gammopathy, antibodies to myelin-associated glycoprotein (MAG), sulfoglucuronyl paragloboside (SGPG) and sulfatide have been associated with specific clinical and electrophysiologic features. However, it is not known whether the results of antibody tests provide additional information for the individual patient (and the neurologist) on future neurologic deficit or outcome. Therefore, we studied the potential prognostic value of clinical, electrophysiologic, pathologic and antibody determination (Chapter 3). In multivariate analysis initial sensory symptoms of the feet is associated with a slowly progressive disease course and less disability at four years, demyelination on electrophysiologic examination is prognostic for development of weakness and symptoms of upper extremities at four years. Half of the patients with polyneuropathy associated with IgM monoclonal gammopathy have anti-MAG antibodies. Since patients with IgM monoclonal gammopathy without anti-MAG antibodies have similar symptoms and comparable response to treatment, it is most likely that these patients have antibody reactivity against other components of the nerve. We found monoclonal anti-ganglioside antibodies in half of the patients with polyneuropathy associated with IgM monoclonal gammopathy without anti-MAG antibodies (Chapter 3). Monoclonal IgM anti-GQ1b antibodies were associated with ataxia. Monoclonal gammopathy is of undetermined significance (MGUS) as long as no hematologic malignancy, like multiple myeloma or lymphoma is found. In patients with polyneuropathy associated with monoclonal gammopathy the frequency of hematologic malignancy is high (22%). A follow-up study of patients with polyneuropathy associated with MGUS showed that the incidence of malignant transformation is higher (2.7/100 patient years, 95% CI 1.52-4.22) than in patient with MGUS without polyneuropathy (1/100 patient years, 95% CI 0.85-1.24). Progression of the polyneuropathy, weight loss, unexplained fever or night sweats and M-protein level were independent predictors (Chapter 4). To find an explanation for the increased frequency of hematologic malignancies in polyneuropathy associated with monoclonal gammopathy we studied the occurrence of genetic aberrations (Chapter 5) by using interphase fluorescence in situ hybridisation (FISH), multiplex ligation-dependent probe amplification (MLPA) assay and genome-wide array-based comparative genomic hybridisation (CGH). We identified different genetic aberrations in patients with polyneuropathy associated with IgM monoclonal gammopathy. Half of the patients had chromosome 14 translocations, which are also frequently identified in multiple myeloma and B cell malignancies. The variable part of the immunoglobulin is responsible for the antigen binding and consists of V, D, J gene segments. We determined the immunoglobulin gene usage in patients with polyneuropathy associated with IgM monoclonal gammopathy (Chapter 6). We found a preferential use of immunoglobulin genes associated with immune responses to encapsulated bacteria (preferentially VH3-23). In patients with polyneuropathy and monoclonal gammopathy, after excluding other causes, the disease course leads the diagnostic process and the additional investigations (Chapter 7, flow chart). We advise a yearly follow-up of all patients with polyneuropathy associated with monoclonal gammopathy, including M-protein level determination. A hematologic screening, including bone marrow examination should be performed if the patient has clinical risk factors of malignant transformation (weight loss, night sweats, bone pain, anemia, rise of M-protein level) or a progressive polyneuropathy.

Zhe Ji et al. ([1][1]) reported that experimental autoimmune encephalomyelitis (induced either by MOG peptide immunization or by MOG-immune T cell transfer) was more severe in mice with thiamine deficiency (TD) than in mice without TD, and concluded that TD influences neuroinflammation by

5543 Background: CD8 positive intra-epithelial tumor infiltrating T cells (TIL) are associated with improved outcomes in ovarian cancer, particularly in HGSC. Radiotherapy (XRT) has immunologic eff...

Metastatic cell heterogeneity presents a significant obstacle to the development of targeted molecular and immunotherapeutics. Profiling of melanocyte differentiation antigens has revealed a nonstochastic, site-specific pattern of expression in metastases that was highest in brain, intermediate in soft tissues/lymph nodes, and lowest in visceral sites. Site-specific antigen heterogeneity, thus, is an important confounding factor to consider when assessing the potential efficacy of antigen-specific therapies.

e22129 Background: Ovarian cancer is diagnosed in more than 190,000 new patients every year and is known to have the highest mortality rate among gynaecologic cancers. The type of immune cells that are present within the tumor microenvironment can play a crucial role in the survival of patients. However, little is known about the dynamics of the tumor-infiltrating immune cells during disease progression. Methods: We studied the immune cells infiltrating the tumor tissue of ovarian cancer patients at different stages of disease. We analysed the patterns of T lymphocytes in fresh tumor tissue as well as blood samples of 44 newly diagnosed ovarian cancer patients by flow cytometry. To evaluate whether regulatory T cells (Tregs) develop in situ or migrate to tumor tissue, we measured a concentration of chemokine CCL22 in tumor cell culture supernatants. We also determined the expression of CCR4 on circulating as well as tumor-infiltrating Tregs by flow cytometry. Results: The early stages of development of ovarian carcinomas were characterized by a strong Th17 immune response, whereas in stage II patients, recruitment of high numbers of Th1 cells was observed. In disseminated tumors (stage III-IV), we detected a dominant population of Helios+ activated regulatory T cells along with high numbers of macrophages and immature myeloid dendritic cells. Tumor-infiltrating Tregs had markedly lower expression of CCR4 than circulating Tregs, and the numbers of tumor-infiltrating Tregs significantly correlated with the levels of CCL22 in ovarian tumor cell culture supernatants, suggesting their recruitment via a CCR4/CCL22 interaction. CCL22 was mainly produced by tumor cells, macrophages and mDCs in the primary ovarian tumors, and its expression markedly increased in response to IFNgamma. Conclusions: Taken together, the specific recruitment of Tregs, probably triggered by inflammatory stimuli, leads to a significant immune suppression in the advanced stages of ovarian cancer.

8501 Background: Despite frequent induction of tumor antigen-specific T cells in melanoma patients following vaccination, tumor regressions remain rare. This observation prompted systematic analysis of the melanoma tumor microenvironment to identify factors that may influence the effector phase of the anti-tumor immune response. Methods: Gene expression profiling using the Affymetrix platform was performed on a series of melanoma metastases, melanoma cell lines, and primary melanocyte cell lines. Confirmatory assays were done by real-time RT-PCR, protein array, immunohistochemistry (IHC), and in vitro chemokine migration assays. Results: Non- supervised hierarchical clustering revealed 3 major subsets of tumors, with the main clustering based on differential expression of T cell-derived transcripts. The presence of CD8+ T cells was confirmed by IHC. Tumors that contained T cells uniquely expressed high levels of multiple chemokines. Protein array confirmed high expression of CCL2, CCL4, and CCL5; real-time RT-PCR additionally confirmed relatively high levels of CXCL9, CXCL10, and CCL3 transcripts. Transwell assays confirmed that each of these 6 chemokines recruited CD8+ effector cells in vitro. Conclusions: We have identified a set of 6 chemokines that likely regulates recruitment of activated T cells into melanoma metastases. Tumors that lack such chemokines might not be capable of supporting the effector phase of the anti-tumor immune response. We suggest that chemokine profiling of tumor sites should be performed in clinical trials of active immunotherapy. No significant financial relationships to disclose.

The mechanisms underlying immune tolerance during pregnancy are poorly understood. We recently proposed a crucial role of T regulatory cells (Treg) in avoiding immunological rejection of the fetus, since we observed diminished number and function of Treg in abortion-prone mice. We further confirmed the protective role of Treg during pregnancy by transferring pregnancy-induced Treg into abortion-prone mice, which prevented rejection. Here, we analyzed the mechanisms involved in Treg-mediated protection. We transferred mice undergoing abortion with Treg from mice having normal pregnancies, including normal pregnant and abortion-prone mice as controls. We evaluated decidual lymphocytes for their cytokine profile. The mRNA levels for several tolerance-associated cytokines were also analyzed. As expected, Treg therapy prevented abortion, while expanding the peripheral and thymic Treg population. Locally, augmented foxp3 levels could be observed. Surprisingly, after therapy the decidual levels of the Th1 cytokines IFN-γ and TNF-α were not diminished. The transfer of Treg dramatically up-regulated the expression of LIF, TGF-β and HO-1 at the fetal-maternal interface. Our data suggest that Treg-treatment can not prevent T cell infiltration or high Th1 levels but is able to create a privileged tolerant microenvironment at the fetal-maternal interface, further shedding light onto the molecular mechanisms involved in pregnancy tolerance.