HLA-DRB1 Allelic Combinations Differentially Shape Dendritic Cell Antigen Presentation Enhanced by Tumour Cell Line Lysate-Pulsing.

Tumor Protection Following Vaccination With Low Doses of Lentivirally Transduced DCs Expressing the Self-antigen erbB2

The relationship between tumor-infiltrating lymphocytes (TILs) and regression in melanoma is unknown. This report describes a large multicenter study assessing the association between TILs and regression. The Sentinel Lymph Node Working Group database was queried from 1993 to 2018 for cases with TILs and regression data. Clinicopathologic factors were correlated with regression and TIL status, sentinel lymph node (SLN) status, and overall survival (OS). The study enrolled 2450 patients. In 1811 cases, TILs (73.9%) were present, with regression present in 328 of these 1811 (18.1%) cases and in 49 (7.7%) of 639 cases without TILs. The presence of TILs was significantly associated with regression (p < 0.0001) as well as a negative SLN (p < 0.05). However, when TILs were stratified by regression status, only absence or presence of both TILs and regression were significantly associated with SLN metastases (p = 0.038). Although the presence of TILs was associated with OS (p < 0.05), regression status by itself was not (p = 0.2058 and 0.252, respectively). Furthermore, when TILs were stratified by regression status, only the presence of TILs with or without regression was significantly associated with improved OS (p = 0.0081 and 0.0137, respectively) versus the absence of both TILs and regression, with regression status not significantly affecting OS for patients with or without TILs (p = 0.2314 and 0.65, respectively). Regression is highly correlated with TILs, but only TILs are significantly associated with SLN metastasis and OS in melanoma patients, whereas regression is not. The impact of regression on outcomes ultimately appears dependent upon the absence or presence of TILs.

Background: Triple negative breast cancer (TNBC) is a highly heterogeneous and aggressive subtype of breast cancer that accounts for approximately 15% of all breast cancer diagnoses and is associated with a poor prognosis. Based on the KEYNOTE-522 (K522) trial, the FDA approved the use of neoadjuvant immunotherapy, Pembrolizumab along with chemotherapy for high-risk early stage TNBC in July 2021. Specifically, the K522 study demonstrated an increased pathological complete response (pCR) rate of 64.8% vs 51.2% and 36-month event-free survival (EFS) of 84.5% vs 76.8% for Pembrolizumab with chemotherapy vs. chemotherapy alone, respectively. The presence of tumor infiltrating lymphocytes (TILs) is a predictive biomarker of pCR and improved survival in patients with TNBC. In this retrospective cohort study, we examine a clinically diverse patient population who received the K522 regimen to determine whether TILs confer a predictive value in response to neoadjuvant pembrolizumab treatment compared to other tumor and clinical factors. Methods: We reviewed electronic medical records of 271 patients with early TNBC who received neoadjuvant chemo immunotherapy with the K522 regimen at a tertiary care university hospital, UT Southwestern (UTSW), and affiliated safety-net hospital, Parkland Memorial (PM) between August 2021 and May 2024. 184 patients completed the neoadjuvant portion of treatment followed by surgery and fit the criteria for our analysis (UTSW: 120, PM: 64). Baseline patient and tumor characteristics were described. Two proportion Z-tests and Chi-squared tests for independence were used to compare pCR rate between patient ethnicities and univariate logistic regression was used to evaluate the association between binary presence of TILs, grade, and residual cancer burden. Results: In total, 57% achieved a pCR; TILs were reported in 52.8% of pathology reports. The presence of TILs was associated with a significantly improved pCR rate of 70% compared to 48% in individuals without TILs (p=0.0027). The median age of our patient population was 51 years. Of which, 34.8% self-identified as Caucasian, 31.0% Hispanic, 25.5% Black, and 8.7% other. There was no statistically significant difference in pCR rate or presence of reported TILs across different ethnicities. Of note, Hispanic patients with TILs had an increased rate of pCR (80.0%) than Hispanic patients without TILs (54.3%) (p=0.0323). No other ethnic group with TILs showed any statistical association with pCR when compared to their non-TIL presenting counterparts. Controlling for tumor grade, patients with TILs were 2.442 times more likely to have a pCR (CI: 1.310—4.553; p=0.0050). Patients with grade 3 TNBC were 2.89 times more likely to have a pCR (CI: 1.275—11.822; p=0.0170). Node positivity in conjunction with TILs also showed statistically significant pCR rates versus all other subgroups (p=0.0051). Conclusions: The K522 trial did not collect racial data as a baseline demographic characteristic, while our study had a significant percentage of Hispanic and Black populations treated with this regimen. This may account for our pCR rate of 57%, which is lower than what was reported on the K522 trial as studies have shown a lower pCR rate in Black population compared to other ethnicities. Our data suggests that TILs can be considered a predictive marker of pCR rate and treatment response in patients with early-stage TNBC. This is particularly relevant in Hispanic patients with TILs achieving a higher rate of pCR than the rest of our population. This may indicate that TILs have potential to guide treatment decisions for certain subgroups. Current College of American Pathologists guidelines do not mandate standardized or uniform reporting of TILs in pathology reports. While TILs could be considered a potential predictive marker in early stage TNBC, more emphasis needs to be placed in standardized reporting of TILs and larger studies are needed for further validation. Citation Format: Riya Albert, Joshua Thomas, Navid Sadeghi, Sangeetha Reddy, Glenda Delgado, Heather McArthur, Samira Syed, Deborah Farr, Nisha Unni. Tumor Infiltrating Lymphocytes (TILs) as a Predictive Marker of Pathological Complete Response (pCR) in a Diverse Patient Population with Early Triple Negative Breast Cancer (TNBC) Treated with the Neoadjuvant KEYNOTE-522 Regimen [abstract]. In: Proceedings of the San Antonio Breast Cancer Symposium 2024; 2024 Dec 10-13; San Antonio, TX. Philadelphia (PA): AACR; Clin Cancer Res 2025;31(12 Suppl):Abstract nr PS1-01.

Introduction: Breast cancer corresponds to 25% of new cancer cases each year. In Brazil, the rate is 29.5% and 59,700 new cases were estimated in 2019. Tumor infiltrating lymphocytes (TIL) are observed in some solid tumors, including breast cancer, the amount of TIL is an indirect marker of the pre-existing antitumor activated T cell response, reflecting the host’s antitumor immunity. Studies show that its presence is an independent predictor of complete pathological response (PRC) to neoadjuvant chemotherapy (QTneo) and its presence provides a favorable prognosis in breast cancer. Objectives: To evaluate the relationship between the presence and value of TIL with the pathological response after QTneo in patients with breast cancer. Materials and methods: An analytical, descriptive, retrospective, case-control study was carried out with 40 patients diagnosed with breast cancer who underwent QTneo between 01/01/2016 to 01/01/2019 at the ISPON complex in the city of Ponta Grossa, Paraná. Data collection was carried out in electronic medical records. The association between variables was tested with the χ2 test and the intensity of the association with the Odds Ratio estimate with a 95% confidence interval. TIL were considered absent when 0, low when 1% to 10%, moderate when 11% to 59%, and high if 60% or more; this division was based on other articles that quantify TIL. Results: The mean age of the patients in the study was 50 years old, 13 of them (32.5%) had PRC, 15 (37.5%) had a partial response, and 13 (30%) had no response to QTneo. Of the 13 that obtained PRC, 7 (53.8%) were triple negative (TN), 5 (38.4%) HER 2, and 1 (7.6%) Luminal. PRC was more frequent in triple negative invasive ductal carcinoma (53.8%), the frequency of PRC in the luminal subtype was significantly lower than that of TN (p=0.03), with the worst response to QTneo. The mean age of patients who had PRC was 48 years. TIL were present in 27 (67.5%) patients, whose value was low in 57.5%, moderate in 3 patients, and high in one case. The presence of TIL, regardless of their value, increased the probability of PRC by 7.7 times when compared to their absence (OR 7.7 CI 1.15‒51.17 p=0.03). When considering its value, it was found that higher values conferred a significant association with PRC (p=0.01). There was a statistically significant association between the clinical stage (CS) and the presence of response to QTneo, patients with locally advanced neoplasia were 6.44 times more likely to respond than patients in an early stage (p=0.015). Conclusion: TIL can be used as a predictor of response to QTneo, being an aid in choosing this treatment and an indicator of better prognosis. The evaluation of TIL should be performed routinely in the preparation of anatomopathological reports, as it is an indication of the investigation of PD-L1 and its blockade, if present.

e14035 Background: The immune response to melanoma has been shown to be locally affected by presence of tumor-infiltrating lymphocytes (TILs), generally divided into brisk (infiltrating the entire base of the invasive tumor), non-brisk (infiltrating only focally) and absent. Several studies showed that greater presence of TILs, especially brisk, in primary melanoma is associated with a better prognosis and a higher survival rate. Since recent studies revealed an association between PD-1/PD-L1 expression levels and tumor response, the aim of our study was to investigate the correlation between plasma PD-1 and presence/absence/class of TILs in metastatic melanoma patients. Methods: The plasma PD-1 levels were analyzed in 28 patients with metastatic melanoma using a specific ELISA assay. The characterization of TILs in tumor tissue was performed by immunohistochemistry. Statistical analysis was assessed using t-Student and ANOVA tests. Survival curves were estimated by using the Kaplan-Meier method and log-rank test to evaluate significant differences among them. Results: 16 out of 28 patients showed the presence of TILs in primary tumor, 10 of which brisk and 6 nonbrisk. The plasma PD-1 levels were analyzed in relation to the presence/absence of TILs (p = 0.022), brisk TILs versus nonbrisk/absent TILs (p = 0.014), and brisk vs nonbrisk vs absent TILs (p = 0.032). In particular, low plasma PD-1 levels have been shown to be associated with brisk TILs in primary melanoma, intermediate values with nonbrisk TILs, and high expression with absent TILs. Although the low number of samples did not allow us to obtain a statistically significant association between the plasma PD-1 expression levels and overall survival (OS) depending on the absence or presence of TILs (brisk/nonbrisk), however the median survival of patients having brisk TILs was five months higher than other 2 groups of patients with absent and nonbrisk TILs, respectively. Conclusions: This work highlights, for the first time, the potential ability of using the plasma PD-1 levels to predict prognosis also in patients with metastatic melanoma at diagnosis for which it is not possible to identify the primary tumor.

OR8 Quantitative assessment of HLA class I presentation in ICT-107, a dendritic cell-based immunotherapy

Background: Tumor infiltrating lymphocytes (TILs) are associated with an improved outcome in triple negative and HER2 breast cancers. Conversely, a recent pioneer study showed that TILs in infiltrating lobular carcinoma (ILC) were associated with a worse prognosis. We aimed at assessing the prognostic impact of TILs in a large cohort of primary surgically treated ILC with a long follow-up in a single institution. Methods: We retrospectively reviewed 459 ILC treated at the Curie Institute between 2005 and 2008. Clinico-pathological patients' characteristics were collected and all archived tumor slides were centrally reviewed for TILs quantification. We analyzed stromal TILs scored as a continuous variable. Cox analyses were performed for relapse free survival (RFS) and for overall survival (OS), and chi-square and Fisher test were performed to evaluate the correlation between TILs and other clinico-pathological variables. For statistical analyses, presence or absence of TILs was considered. Results: Patients had a mean age of 60.3 years and a median follow-up of 8.8 years, during which 74 local and/or distant relapse events occurred, among which 37 deaths due to the disease. The presence of TILs were significantly associated with greater tumor size (pT), positive nodal status (pN), molecular class (HER2 amplified), and specific morphological nuclear features such as high nuclear grade, multinucleation, macronucleoli and high Nottingham Prognostic Index class. Presence of intra-tumoral lymphocytes is significantly associated with a worse RFS (HR: 2.07 p=0.003) and OS (HR: 4.58 p&amp;lt;0.001) in a multivariate analysis, independently of chemotherapy. Relapse Free Survival TILs% (IC 95%)% (IC 95%)No95.3 (92.5-98.2)89.0 (84.3-94.0)Yes89.3 (85.1-93.6)74.0 (67.5-81-1) Overall Survival OS at 5 yearsOS at 10 yearsTILs% (IC 95%)% (IC 95%)No99.5 (97.8-100)95.8 (92.3-99.4)Yes95.3 (92.3-98.1)83.5 (77.8-89.7) Conclusion: TILs are an independent prognostic factor associated with a poor outcome in ILC that are easy to assess on diagnostic biopsies. In order to elucidate the role of TILs in ILC, we will further characterize the immune infiltrate and integrate these results with DNA, RNA and protein analyses of the same carcinomas, in order to gain insight into their molecular features and to facilitate the identification of new therapeutic strategies for these ILC with TILs. Citation Format: Tille J-C, Saint Martin C, Fuhrmann L, De Koning L, Reyal F, Piccart M, Kirova Y, Cottu P, Carton M, Vincent Salomon A. Tumor-infiltrating lymphocytes in invasive lobular breast cancer identify a poor prognostic sub-group [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr P4-02-04.

Several studies have shown that vaccine therapy using dendritic cells (DCs) pulsed with specific tumor antigen peptides can effectively induce antitumor immunity. Peptide-pulsed DC therapy is reported to be effective against melanoma, while it is still not sufficient to show the antitumor therapeutic effect against epithelial solid tumors such as gastrointestinal malignancies. Recently, it has been reported that vaccine therapy using DCs transduced with a surrogate tumor antigen gene can elicit a potent therapeutic antitumor immunity. In this study, we investigated the efficacy of vaccine therapy using DCs transduced with the natural tumor antigen in comparison with peptide-pulsed DCs. DCs derived from murine bone marrow were adenovirally transduced with murine endogenous tumor antigen gp70 gene, which is expressed in CT26 cells, or DCs were pulsed with the immunodominant peptide AH-1 derived from gp70. We compared these two cancer vaccines in terms of induction of antigen-specific cytotoxic T lymphocyte (CTL) responses, CD4+ T cell response against tumor cells, migratory capacity of DCs and therapeutic immunity in vivo. The cytotoxic activity of splenocytes against CT26 and Meth-A pulsed with AH-1 in mice immunized with gp70 gene-transduced DCs was higher than that with AH-1-pulsed DCs. CD4+ T cells induced from mice immunized with gp70 gene-transduced DCs produced higher levels of IFN-γ by stimulation with CT26 than those from mice immunized with AH-1-pulsed DCs (p < 0.0001), and it was suggested that DCs transduced with tumor-associated antigen (TAA) gene induced tumor-specific CD4+ T cells, and those CD4+ T cells played a critical role in the priming phase of the CD8+ T cell response for the induction of CD8+ CTL. Furthermore, DCs adenovirally transduced with TAA gene showed an enhancement of expression of CC chemokine receptor 7 and improved the migratory capacity to draining lymph nodes. In subcutaneous models, the vaccination using gp70 gene-transduced DCs provided a remarkably higher therapeutic efficacy than that using AH-1-pulsed DCs. These results suggested that vaccine therapy using DCs adenovirally transduced with TAA gene can elicit potent antitumor immunity, and may be useful for clinical application.

Enhanced Induction of HIV-specific Cytotoxic T Lymphocytes by Dendritic Cell-targeted Delivery of SOCS-1 siRNA

Dendritic cells are unique in their ability to stimulate naive T cells. These investigators have developed a prostate cancer vaccine using autologous dendritic cells as a vehicle to present prostate antigens to T cells in vivo.

Background: Several studies show that the presence of TILs has prognostic significance in TNBC, with greater lymphocytic infiltration associated with clinical outcomes. In the phase 2 KEYNOTE-086 study, TIL levels were a surrogate marker of preexisting antitumor immunity and were independent predictors of response to pembrolizumab (pembro) monotherapy. Here, we analyzed the relationship between the presence of TILs and outcomes in the phase 3 KEYNOTE-119 (NCT02555657) study of pembro monotherapy vs single-agent chemotherapy (chemo) in patients (pts) with previously treated mTNBC. Methods: From October 2015 to April 2017, 622 pts from 31 countries were enrolled. Key eligibility criteria included centrally confirmed TNBC, 1 or 2 prior systemic treatments for metastatic disease, documented progression on most recent therapy, prior treatment with an anthracycline and/or a taxane, and provision of a tumor sample for central determination of triple-negative status and PD-L1 expression. Pts with active brain metastases were excluded. Pts were stratified by PD-L1 status (positive vs negative; PD-L1 positivity was defined as a CPS ≥1) and history of prior neoadjuvant/adjuvant treatment vs de novo metastatic disease at initial diagnosis. Pts were randomly assigned 1:1 to pembro 200 mg Q3W or single-agent chemo per investigator’s choice of capecitabine, eribulin, gemcitabine, or vinorelbine, with a maximum enrollment cap of 60% for each, administered per local product label, until progression, intolerable toxicity, or investigator/patient decision. Response was assessed every 9 wk for 1 y and every 12 wk thereafter. Primary end points were OS in the PD-L1 CPS ≥10, CPS ≥1, and total populations. Secondary end points were PFS, ORR, duration of response, and DCR in the PD-L1 CPS ≥10, CPS ≥1, and total populations, and safety. The presence of TILs in archival tumor samples was assessed by light microscopy of hematoxylin and eosin-stained sections using a predefined method. The relationship between TILs and OS and PFS was analyzed using Cox regression; the relationship between TILs and ORR was analyzed using logistic regression. The interaction between treatment and TILs was assessed. Results: The study was negative for the primary end point. TILs were evaluable in 536/622 (86.2%) treated pts (273 [pembro]; 263 [chemo]). The median TILs distribution was 5% (IQR, 14%). TIL levels were significantly higher in responders vs nonresponders in the pembro arm, but not the chemo arm. TIL levels as a continuous variable were significantly (P&amp;lt; 0.05) associated with all clinical outcomes tested in the pembro but not the chemo arm (Table). For pts with TILs &amp;lt;5%, assessment of OS yielded a HR of 1.50 (95% CI, 1.14-1.97); for pts with TILs ≥5%, the HR was 0.75 (95% CI, 0.59-0.96). Median OS and 18-month OS rate (pembro vs chemo) for pts with TILs &amp;lt;5% was 5.9 vs 8.8 mo and 15% vs 27%, respectively; for pts with TILs ≥5%, this was 12.5 vs 11.3 mo and 35% vs 27%. The correlation between TILs vs CPS was moderate at 0.45; multivariate modeling of TILs and CPS showed independent predictive value. Conclusions: High TILs were significantly associated with better clinical outcomes with pembro but not chemo. Efficacy estimates at the prespecified median TIL cut point (≥5%) suggest that a subset of later-line advanced pts with TNBC can derive prolonged survival benefit from pembro over chemo. Association of TILs With Clinical OutcomesTreatment ArmContinuousBORDCRPFSOSVariableORRP*DCRP*Event RateP*Event RateP*Pembro (n=273),TILs25 (9.2)0.000732 (11.7)0.0058241 (88.3)0.0002239 (87.5)0.0001n (%)Chemo (n=263),TILs29 (11.0)0.180950 (19.0)0.3026191 (72.6)0.234236 (89.7)0.3321n (%)*P values are 1-sided for pembro and 2-sided for chemo.The stratification variable, prior (neo)adjuvant therapy vs de novo metastatic disease at initial diagnosis, was used as a covariate in the model.TILs was square root transformed.BOR, best overall response; chemo, chemotherapy; DCR, disease control rate; OS, overall survival; pembro, pembrolizumab; PFS, progression-free survival; TILs, tumor-infiltrating lymphocytes. Citation Format: Sherene Loi, Eric Winer, Oleg Lipatov, Seock-Ah Im, Anthony Goncalves, Javier Cortes, Keun S Lee, Peter Schmid, Laura Testa, Isabell Witzel, Shoichiro Ohtani, Nicholas Turner, Stefania Zambelli, Nadia Harbeck, Fabrice Andre, Rebecca Dent, LingKang Huang, Jaime Mejia, Vassiliki Karantza, Roberto Salgado. Relationship between tumor-infiltrating lymphocytes (TILs) and outcomes in the KEYNOTE-119 study of pembrolizumab vs chemotherapy for previously treated metastatic triple-negative breast cancer (mTNBC) [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr PD5-03.

As T-cell receptor-major histocompatibility complex (MHC) class I/self peptide interaction regulates T-cell development in the thymus, we reasoned that presentation of peptides by self dendritic cells (DC) to developing T cells in the thymus might induce acquired thymic tolerance. This hypothesis is based on the finding that intrathymic injection of allopeptides in the adult animal induces acquired tolerance. To examine this hypothesis, we studied the effects of intrathymic (IT) injection of a single immunodominant Wistar-Furth (WF) MHC class I (RT1.Au) peptide-pulsed host DC on islet allograft survival in the WF-to-ACI rat combination. Bone marrow-derived ACI DC expressing MHC class I and II, OX62, and ED2 present allopeptides to naive and specifically peptide-primed syngeneic T cells in mixed lymphocyte reaction. Host DC pulsed with RT1.Au peptide 5 (residues 93-109) were injected into the thymus of streptozotocin-induced diabetic ACI that were transplanted 7 days later with donor-type (WF) or third-party (Brown Norway [BN]) islets. Whereas IT injection of 300 microg of peptide 5 alone led to normoglycemia and permanent islet survival in three of six diabetic ACI recipients, similar treatment combined with simultaneous intraperitoneal injection of 0.5 ml of anti-lymphocyte serum (ALS) on day -7 led to 100% permanent islet allograft survival (>200 days) compared to a mean survival time of 15.0+/-2.3 days in controls treated with ALS alone. In contrast, similarly prepared animals rejected the third-party (BN) islets in an acute fashion. To address the question of indirect allorecognition in acquired thymic tolerance, we examined the effect of peptide-pulsed host DC on graft survival. Whereas IT injection of peptide-pulsed host DC alone resulted in permanent islet survival in two of five animals, IT injection of peptide-pulsed host DC combined with 0.5 ml of ALS induced 100% donor-specific permanent islet allograft survival in the WF-to-ACI rat combination. These results suggest that thymic DC take up, process, and present the administered peptide to the developing T cells by the indirect allorecognition pathway in the induction of acquired thymic tolerance. We have demonstrated a novel approach to inducing transplant tolerance to islet allografts with IT injection of allopeptide-pulsed host DC. This finding suggests that immunization strategies using DC expressing MHC allopeptides or peptide analogue might be potentially useful in the treatment of autoimmune diabetes mellitus.

BackgroundCervical cancer is the second most common gynecological cancer amongst women world-wide. Despite optimized protocols, standard treatments still face several disadvantages. Therefore, research aims at the development of immune-based strategies using tumor antigen-loaded dendritic cells for the induction of cellular anti-tumor immunity.ResultsIn this study, we used dendritic cells loaded with the HLA-A2-restricted HPV type 16 E711–20 peptide in order to induce an in vitro CD8+ T cell response. For this purpose, peptide-pulsed dendritic cells were co-cultured with autologous CD8+ T cells. After 5 weekly stimulations with peptide-pulsed mature dendritic cells, cultured T cells were analyzed for antigen specificity by an IFN-γ ELISPOT assay. Using this ELISPOT assay, we were able to detect E7-specific IFN-γ-secreting CD8+ T cells in 5/5 healthy donors.ConclusionWe show that peptide-pulsed mature dendritic cells are able to stimulate a HPV type 16 E7 peptide-specific immune response in vitro. These experiments describe an efficient culture protocol for antigen-specific T cells for use in pre-clinical vaccination research and confirm the need for sensitive T cell assays for detection of tumor-specific immune responses in vitro.

To investigate the morphological characterization of tumor infiltrating dendritic cells (TIDCs) and tumor infiltrating lymphocytes (TILs) in human rectal cancer. Light and electron microscopy as well as immunohistochemistry were used to observe the distributive and morphological changes of TIDCs and TILs. TIDCs were mainly located in tumor-surrounding tissue. The number of TIDCs in the earlier stage was higher than that in the later stage (P<0.01). TILs were mainly seen in adjacent tissue of cancers and tumor-surrounding tissue. There were more TILs in the earlier stage than that in the later stage (P<0.01). Under electron microscope, TIDCs were irregular in shape and exhibited many dendritic protrusions. It is not obvious that cancer cells perforated the basement membrane and TILs were arranged along the basement membrane in the earlier stage. In the later stage, it is explicit that cancer cells perforated the basement membrane and surrounded by TILs. There were contacts among TIDCs, TILs and tumor cell. One TIDCs contacted one or several TILs which clustered around TIDCs. Glycogen granules were seen between TIDCs and TILs. The number of TIDCs and TILs is related with tumor progression There exist close relationships among TIDCs, TILs and tumor cell.

For successful dendritic cell (DC) therapy of cancer, it is important to choose the optimal method to load DCs with antigens to induce higher anti-tumor immunity, particularly tumor-specific CTL responses. The co-incubation (Co) method is conventionally used to load DCs with tumor antigens in DC therapy. Although DCs could uptake tumor antigens by endocytosis in this method, they are thought to be processed in lysosome and presented mainly on MHC class II, but not efficiently on MHC class I. Electroporation (Ep) is one of the promising antigen-loading method because it is reported that DCs could uptake much larger amount of FITC-dextran by Ep in comparison with Co method. In this study, we performed side-by-side comparisons of these two antigen loading methods using murine bone marrow derived DCs. First, we compared antigen presentation using purified ovalbumin (OVA) and OVA-specific OT-I CD8 and OT-II CD4 T cells. To this end, DCs were loaded with OVA by either Co or Ep, and subsequently cultured with T cells. Two or three days later, T cell expansion and cytokine production were evaluated. As expected, OVA-Co-DCs induced moderate expansion and cytokine production of OT-II T cells, but had little effect on OT-I T cells. In contrast, OVA-Ep-DCs potently stimulated both OT-I and OT-II T cells to expand and produce cytokines, such as IL-2 and IFN-γ. Using monoclonal antibody specific for MHC class I/OVA peptide complex, we confirmed efficient presentation of the OVA peptide on MHC class I by OVA-Ep-DCs. Furthermore, when we used the lysate of EG7 tumor cell line (OVA gene-transfected) as antigen instead of purified OVA, Ep-DCs induced expansion of both OT-I and OT-II T cells efficiently compared with Co-DCs. In the case of Alexa488-OVA, unexpectedly, similar amounts of uptake were seen in Ep-DCs and Co-DCs by FCM analysis. By using confocal microscopy, however, there was a critical difference of intracellular localization of Alexa488-OVA. Although Alexa488-OVA was localized in intracellular vesicles of both DCs, the cytosolic localization was observed only in Ep-DCs, suggesting that antigen loaded by Ep entered cytosol of DCs and would be processed by proteasome and presented on MHC class I efficiently. Finally, to determine the ability of these DCs to induce anti-tumor immunity in vivo, mice were subcutaneously immunized twice at 7 days intervals with 3-5 x 105 cells of either OVA-Ep-DCs or OVA-Co-DCs and then challenged with 3 x 106 of EG7 cells at 7 days after the 2nd immunization. When OVA-Co-DCs were used for immunization, tumor formations were partially prevented. On the contrary, mice immunized with OVA-Ep-DCs were almost completely protected from tumor formations. These results demonstrate that DCs loaded with tumor antigens by Ep have greater antigen presentation capacity, especially through MHC class I pathway, and could induce stronger anti-tumor immunity in vivo than those loaded by Co. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1562. doi:1538-7445.AM2012-1562