Search

Endogenous antitumor effector T-cell responses and immune-suppressive regulatory T cells (Treg) critically influence the prognosis of patients with cancer, yet many of the mechanisms of how this occurs remain unresolved. On the basis of an analysis of the function, antigen specificity, and distribution of tumor antigen–reactive T cells and Tregs in patients with breast cancer and transgenic mouse tumor models, we showed that tumor-specific Tregs were selectively activated in the bone marrow (BM) and egressed into the peripheral blood. The BM was constantly depleted of tumor-specific Tregs and was instead a site of increased induction and activity of tumor-reactive effector/memory T cells. Treg egress from the BM was associated with activation-induced expression of peripheral homing receptors such as CCR2. Because breast cancer tissues express the CCR2 ligand CCL2, the activation and egress of tumor antigen–specific Tregs in the BM resulted in the accumulation of Tregs in breast tumor tissue. Such immune compartmentalization and redistribution of T-cell subpopulations between the BM and peripheral tissues were achieved by vaccination with adenoviral vector–encoded TRP-2 tumor antigen in a RET transgenic mouse model of spontaneous malignant melanoma. Thus, the BM simultaneously represented a source of tumor-infiltrating Tregs and a site for the induction of endogenous tumor-specific effector T-cell responses, suggesting that both antitumor immunity and local immune suppression are orchestrated in the BM.

Supplemental Figures S1 to S12

Purpose: In glioma—in contrast to various other cancers—the impact of T-lymphocytes on clinical outcome is not clear. We investigated the clinical relevance and regulation of T-cell infiltration in glioma.Experimental Design: T-cell subpopulations from entire sections of 93 WHO°II–IV gliomas were computationally identified using markers CD3, CD8, and Foxp3; survival analysis was then done on primary glioblastomas (pGBM). Endothelial cells expressing cellular adhesion molecules (CAM) were similarly computationally quantified from the same glioma tissues. Influence of prominent cytokines (as measured by ELISA from 53 WHO°II–IV glioma lysates) on CAM-expression in GBM-isolated endothelial cells was determined using flow cytometry. The functional relevance of the cytokine-mediated CAM regulation was tested in a transmigration assay using GBM-derived endothelial cells and autologous T-cells.Results: Infiltration of all T-cell subsets increased in high-grade tumors. Most strikingly, within pGBM, elevated numbers of intratumoral effector T cells (Teff, cytotoxic and helper) significantly correlated with a better survival; regulatory T cells were infrequently present and not associated with GBM patient outcome. Interestingly, increased infiltration of Teff cells was related to the expression of ICAM-1 on the vessel surface. Transmigration of autologous T cells in vitro was markedly reduced in the presence of CAM-blocking antibodies. We found that TGF-β molecules impeded transmigration and downregulated CAM-expression on GBM-isolated endothelial cells; blocking TGF-β receptor signaling increased transmigration.Conclusions: This study provides comprehensive and novel insights into occurrence and regulation of T-cell infiltration in glioma. Specifically, targeting TGF-β1 and TGF-β2 might improve intratumoral T-cell infiltration and thus enhance effectiveness of immunotherapeutic approaches. Clin Cancer Res; 17(13); 4296–308. ©2011 AACR.

Supplementary Figures S1-S1-; Supplementary Tables S1-S2.

Supplementary Figure 3 from Intratumoral Cytokines and Tumor Cell Biology Determine Spontaneous Breast Cancer–Specific Immune Responses and Their Correlation to Prognosis

Spontaneous immune responses in cancer patients have been described. Yet their clinical relevance and the conditions for their generation remain unclear. We characterized conditions that determine immune responses in primary breast cancer patients. We used tetramer analysis, ex vivo IFN-γ ELISPOT, cytotoxicity assays, and ELISA in 207 untreated patients and 12 Her-2/neu–specific CD8 T-cell lines to evaluate tumor-specific T cells (TC) in the bone marrow or MUC1-specific antibodies in the blood. Multiplex analysis was performed to quantify 27 intratumoral cytokines, chemokines, and growth factors. Results were compared with multiple pathologic and clinical parameters of the patients and tumors. Forty percent of the patients showed tumor-specific TC responses. These correlated with tumors of high differentiation, estrogen receptor expression, and low proliferative activity, and with a reduced cancer mortality risk. High tumor cell differentiation correlated with increased intratumoral, but not plasma, concentrations of IFN-α and reduced transforming growth factor (TGF)β1. In an in vitro priming experiment these two cytokines increased or inhibited, respectively, the capacity of dendritic cells to induce tumor-reactive TC. Tumor-specific B-cell responses, mainly of IgM isotype, were detectable in 50% of the patients and correlated with advanced tumor stage, increased TGFβ1, reduced IFN-α, and absence of TC responses. We show here that different types of immune responses are linked to distinct cytokine microenvironments and correlate with prognosis-relevant differences in tumor pathobiology. These findings shed light on the relation between immune response and cancer prognosis. [Cancer Res 2009;69(21):8420–8]

Supplementary Figure 4 from Intratumoral Cytokines and Tumor Cell Biology Determine Spontaneous Breast Cancer–Specific Immune Responses and Their Correlation to Prognosis

Supplementary Figure 2 from Intratumoral Cytokines and Tumor Cell Biology Determine Spontaneous Breast Cancer–Specific Immune Responses and Their Correlation to Prognosis

Supplementary Figure 5 from Intratumoral Cytokines and Tumor Cell Biology Determine Spontaneous Breast Cancer–Specific Immune Responses and Their Correlation to Prognosis

Supplementary Table 2 from Intratumoral Cytokines and Tumor Cell Biology Determine Spontaneous Breast Cancer–Specific Immune Responses and Their Correlation to Prognosis

Supplementary Table 1 from Intratumoral Cytokines and Tumor Cell Biology Determine Spontaneous Breast Cancer–Specific Immune Responses and Their Correlation to Prognosis

Supplementary Figure 1 from Intratumoral Cytokines and Tumor Cell Biology Determine Spontaneous Breast Cancer–Specific Immune Responses and Their Correlation to Prognosis

Carcinoembryonic antigen-related cell adhesion molecule 6 (CEACAM6), a cell surface receptor, is expressed on normal epithelial tissue and highly expressed in cancers of high unmet medical need, such as non-small cell lung, pancreatic, and colorectal cancer. CEACAM receptors undergo homo- and heterophilic interactions thereby regulating normal tissue homeostasis and angiogenesis, and in cancer, tumor invasion and metastasis. CEACAM6 expression on malignant plasma cells inhibits antitumor activity of T cells, and we hypothesize a similar function on epithelial cancer cells. The interactions between CEACAM6 and its suggested partner CEACAM1 on T cells were studied. A humanized CEACAM6-blocking antibody, BAY 1834942, was developed and characterized for its immunomodulating effects in co-culture experiments with T cells and solid cancer cells and in comparison to antibodies targeting the immune checkpoints programmed cell death protein 1 (PD-1), programmed death-ligand 1 (PD-L1), and T cell immunoglobulin mucin-3 (TIM-3). The immunosuppressive activity of CEACAM6 was mediated by binding to CEACAM1 expressed by activated tumor-specific T cells. BAY 1834942 increased cytokine secretion by T cells and T cell-mediated killing of cancer cells. The in vitro efficacy of BAY 1834942 correlated with the degree of CEACAM6 expression on cancer cells, suggesting potential in guiding patient selection. BAY 1834942 was equally or more efficacious compared to blockade of PD-L1, and at least an additive efficacy was observed in combination with anti-PD-1 or anti-TIM-3 antibodies, suggesting an efficacy independent of the PD-1/PD-L1 axis. In summary, CEACAM6 blockade by BAY 1834942 reactivates the antitumor response of T cells. This warrants clinical evaluation.

Nano/mesoporous carbon was prepared from pine wood sawdust via the pretreatment of acid and hydrothermal process, followed by potassium hydroxide (KOH) activation. This study proposed the enhancement of activated carbon (AC) adsorption capacity by utilizing the vacant sites and phenomena of opposite charge attraction via multilayer adsorption of Cr(VI) ions and dyes with positive and negative charges. On the first layer, the maximum adsorption capacities for Cr(VI) ions, methylene blue (MB) molecules, and acid red 18 (AR18) molecules onto AC were found to be 7.91 mg/g, 476.19 mg/g, and 434.78 mg/g, respectively. For multiple adsorption, after Cr(VI) ions uptake saturation, the sequential adsorption of MB and AR18 on the second layer, the maximum adsorption capacity, reached 322.58 mg/g and 333.33 mg/g. After MB and AR18 uptake saturation, the maximum Cr(VI) adsorption capacity reached 2.92 mg/g and 4.39 mg/g.

Endogenous antitumor effector T-cell responses and immune-suppressive regulatory T cells (Treg) critically influence the prognosis of patients with cancer, yet many of the mechanisms of how this occurs remain unresolved. On the basis of an analysis of the function, antigen specificity, and distribution of tumor antigen–reactive T cells and Tregs in patients with breast cancer and transgenic mouse tumor models, we showed that tumor-specific Tregs were selectively activated in the bone marrow (BM) and egressed into the peripheral blood. The BM was constantly depleted of tumor-specific Tregs and was instead a site of increased induction and activity of tumor-reactive effector/memory T cells. Treg egress from the BM was associated with activation-induced expression of peripheral homing receptors such as CCR2. Because breast cancer tissues express the CCR2 ligand CCL2, the activation and egress of tumor antigen–specific Tregs in the BM resulted in the accumulation of Tregs in breast tumor tissue. Such immune compartmentalization and redistribution of T-cell subpopulations between the BM and peripheral tissues were achieved by vaccination with adenoviral vector–encoded TRP-2 tumor antigen in a RET transgenic mouse model of spontaneous malignant melanoma. Thus, the BM simultaneously represented a source of tumor-infiltrating Tregs and a site for the induction of endogenous tumor-specific effector T-cell responses, suggesting that both antitumor immunity and local immune suppression are orchestrated in the BM.

Chemical investigation of a Chinese indigenous Aglaia species, A. abbreviata, yielded a new 3,4-secodammarane triterpenoid (1), the known triterpenoids 2–9, the known flavaglines 10 and 11, and the known amide derivative 12. Structures were assigned to these compounds on the basis of detailed spectroscopic analyses.

The bone marrow (BM) of breast cancer patients harbors tumor-reactive memory T cells (TCs) with therapeutic potential. We recently described the immunologic effects of adoptive transfer of ex vivo restimulated tumor-reactive memory TCs from the BM of 12 metastasized breast cancer patients in a clinical phase-I study. In this trial, adoptive T cell transfer resulted in the occurrence of circulating tumor antigen-reactive type-1 TCs. We here describe the long-term clinical outcome and its correlation with tumor-specific cellular immune response in 16 metastasized breast cancer patients, including 12 included in the original study. Sixteen metastatic breast cancer patients with preexisting tumor-reactive BM memory TCs were included into the study. The study protocol involved one transfusion of TCs which were reactivated in vitro with autologous dendritic cells pulsed with lysates of MCF-7 breast cancer cells as source of tumor antigens. The presence of tumor-reactive memory TCs was analyzed by IFN-γ ELISpot assays. Tumor-reactive memory TCs in the peripheral blood were induced de novo in 7/16 patients (44 %) after adoptive TC transfer. These patients were considered immunologic responders to the therapy. Positive adoptive immunotherapy (ADI) response was observed significantly more often in patients without bone metastases (p = 0.0051), in patients with high levels of tumor-reactive BM TCs prior to therapy (p = 0.036) and correlated significantly with the estimated numbers of transferred tumor-reactive TCs (p = 0.0021). After the treatment, we observed an overall median survival of 33.8 months in the total cohort with three patients alive at last follow-up and more than 7 years after ADI. Numbers of transferred tumor-reactive TCs correlated significantly with the overall survival of patients (p = 0.017). Patients with an immunologic response to ADI in the peripheral blood had a significantly longer median survival than nonresponders (median survival 58.6 vs. 13.6 months; p = 0.009). In metastasized breast cancer patients, adoptive transfer of BM TCs can induce the presence of tumor antigen-reactive type-1 TCs in the peripheral blood. Patients with immunologic response after ADI show a significantly longer overall survival. Patients with bone metastases significantly less frequently respond to the treatment and, therefore, might not be optimal candidates for ADI. Although the present study does not yet prove the therapeutic effect of ADI, these findings shed light on the relation between immune response and cancer prognosis and suggest that transfer of reactivated BM TCs might bear therapeutic potential.

Regulatory T cells (Treg) hamper anti-tumor T-cell responses resulting in reduced survival and failure of cancer immunotherapy. Among lymphoid organs, the bone marrow (BM) is a major site of Treg residence and recirculation. However, the process governing the emigration of Treg from BM into the circulation remains elusive. We here show that breast cancer patients harbour reduced Treg frequencies in the BM as compared to healthy individuals or the blood. This was particularly the case for tumor antigen-specific Treg which were quantified by MHCII tumor peptide loaded tetramers. We further demonstrate that decreased Treg distribution in the BM correlated with increased Treg redistribution to tumor tissue, suggesting that TCR triggering induces a translocation of Treg from the BM into tumor tissue. Sphingosine-1-phosphate receptor 1 (S1P1)—which is known to mediate exit of immune cells from lymphoid organs was selectively expressed by tumor antigen-specific BM Treg. S1P1 expression could be induced in Treg by BM-resident antigen-presenting cells (BMAPCs) in conjunction with TCR stimulation, but not by TCR stimulation or BMAPCs alone and triggered the migration of Treg but not conventional T cells (Tcon) to its ligand Sphingosine-1-phosphate (S1P). Interestingly, we detected marked S1P gradients between PB and BM in breast cancer patients but not in healthy individuals. Taken together, our data suggest a role for S1P1 in mediating the selective mobilization of tumor specific Treg from the BM of breast cancer patients and their translocation into tumor tissue. Electronic supplementary material The online version of this article (doi:10.1007/s00262-017-1964-4) contains supplementary material, which is available to authorized users.

Immunotherapy is a promising approach in an effort to control castration resistant prostate cancer. We characterized tumor antigen reactive T cells in patients with prostate cancer and analyzed the suppression of antitumor responses by regulatory T cells.Peripheral blood samples were collected from 57 patients with histologically confirmed prostate cancer, 8 patients with benign prostatic hyperplasia and 16 healthy donors. Peripheral blood mononuclear cells were isolated and antigen specific interferon-γ secretion of isolated T cells was analyzed by enzyme-linked immunospot assay. T cells were functionally characterized and T-cell responses before and after regulatory T-cell depletion were compared. As test tumor antigens, a panel of 11 long synthetic peptides derived from a total of 8 tumor antigens was used, including prostate specific antigen and prostatic acid phosphatase.In patients with prostate cancer we noted a 74.5% effector T-cell response rate compared with only 25% in patients with benign prostatic hyperplasia and 31% in healthy donors. In most patients 2 or 3 tumor antigens were recognized. Comparing various disease stages there was a clear increase in the immune response against prostate specific antigens from intermediate to high risk tumors and castration resistant disease. Regulatory T-cell depletion led to a significant boost in effector T-cell responses against prostate specific antigen and prostatic acid phosphatase.Tumor specific effector T cells were detected in most patients with prostate cancer, especially those with castration resistant prostate cancer. Since effector T-cell responses against prostate specific antigens strongly increased after regulatory T-cell depletion, our results indicate that immunotherapy efficacy could be enhanced by decreasing regulatory T cells.

Effector memory T cells (T(EM)) have an important role in immunity against infection. However, little is known about the factors regulating T(EM) maintenance and proliferation. In this study, we investigated the role of direct interactions between CD4(+) and CD8(+) T cells (TC) for human T(EM) expansion. Proliferation of separated or mixed CD4(+) and CD8(+)T(EM) populations was analyzed after polyclonal stimulation in vitro. Compared to each isolated subset mixed T(EM) populations showed increased proliferation and expansion of both CD4(+) and CD8(+)T(EM) subpopulations. Combined activation of CD4(+) and CD8(+) memory T cells (Tmem) induced an increased expression of CD40L and CD40 on both populations. Subsequently, CD40/CD40L caused a bi-directional stimulation of CD40(+)CD4(+)T(EM) by CD40L(+)CD8(+)T(EM) and of CD40(+)CD8(+)T(EM) by CD40L(+)CD4(+)T(EM). Blocking of CD40L on activated CD8(+)T(EM) selectively inhibited proliferation of CD4(+)T(EM), while blocking of CD40L on CD4(+)T(EM) abrogated proliferation of CD8(+)T(EM). Taken together, we demonstrate for the first time that the expression of CD40L is exploited on the one hand by CD8(+)T(EM) to increase the proliferation of activated CD4(+)T(EM) and on the other hand by CD4(+)T(EM) to support the expansion of activated CD8(+)T(EM). Thus, efficient T(EM) expansion requires bi-directional interactions between CD4(+) and CD8(+)T(EM) cells.

CEACAM6 (CD66c) was previously shown to act as a novel immune checkpoint regulator suppressing the activity of effector T cells against tumors (Witzens-Harig et al., Blood 2013). CEACAM6 is a GPI-linked protein that is strongly expressed at the tumor cell surface in multiple cancer indications such as non-small cell lung adenocarcinoma (NSCLC), colorectal carcinoma (CRC), gastric adenocarcinoma and pancreatic cancer. In general, elevated CEACAM6 expression is associated with advanced tumor stages and poor prognosis. In vitro experiments showed that engagement of T-cells with CEACAM6, either expressed on tumor cells or presented on beads, resulted in suppression of TCR-mediated T-cell activation and ZAP70 phosphorylation. Based on these findings, we hypothesized that antibodies targeting CEACAM6 may be employed to enhance T-cell responses against CEACAM6-expressing cancers. Here we report the generation and characterization of BAY 1834942, a humanized monoclonal antibody selectively blocking the inhibitory impact of CEACAM6 on human T cells. There is no rodent ortholog of CEACAM6 precluding in vivo efficacy studies. In tumor cell / T cell co-culture systems, BAY 1834942 increased secretion of T-cell cytokines and effector molecules (e.g. IFNγ, TNFα, IL-2, granzyme B) and resulted in improved tumor cell killing. The effects of BAY 1834942 were dose-dependent, only observed in the context of CEACAM6-expressing tumor cells and could be reproduced in experiments using tumor cell lines and T-cell preparations from different sources, including T cells derived from tumor infiltrating lymphocytes from pancreatic cancer. BAY 1834942 is cross-reactive with the cynomolgus CEACAM6 ortholog and was well-tolerated in monkey toxicology studies. In summary, BAY 1834942 is a novel checkpoint inhibitor with potential for the treatment of patients with CEACAM6 expressing cancers, both as single agent and in combination with other checkpoint inhibitors. First-in-man trials are expected to commence in 2018. Citation Format: Joerg Willuda, Mark Trautwein, Jessica Pinkert, Wolf-Dietrich Doecke, Hans-Henning Boehm, Florian Wessel, Yingzi Ge, Eva Maria Gutierrez, Joerg Weiske, Christoph Freiberg, Uwe Gritzan, Julian Glueck, Dieter Zopf, Sven Golfier, Oliver von Ahsen, Ruprecht Zierz, Sabine Wittemer-Rump, Heiner Apeler, Ziegelbauer Karl, Rienk Offringa, Bertolt Kreft, Beckhove Philipp. BAY 1834942 is an immunotherapeutic antibody blocking the novel immune checkpoint regulator CEACAM6 (CD66c) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1771.

Background & Aims Chronic pancreatitis is characterized by alternating phases of acute inflammation and quiescent disease. Involvement of T-cell responses has been suggested, but pancreatitis-specific T cells have not been described. Methods We characterized T-cell responses against pancreatitis, pancreatic carcinoma-associated antigens, and tetanus toxoid in the bone marrow, blood, and/or pancreatitis lesions of patients with pancreatitis, pancreatic cancer, and healthy individuals. T cells were functionally characterized by antigen-dependent secretion of interferon (IFN)-γ, interleukin (Il)-4, and IL-10, which indicate type 1, type 2, or regulatory T-cell responses, respectively. Regulatory T cells were characterized by multicolor flow cytometry. Isolated regulatory T cells were tested for their capacity to recognize pancreatitis-associated antigens and to suppress conventional T cells in an antigen-dependent manner. T cell-derived cytokines in tissue lesions were quantified by enzyme-linked immunosorbent assay. Results Chronic pancreatitis patients showed similar to pancreatic cancer patients and healthy individuals type 1 T-cell responses against tetanus toxoid; however, they exhibited strong IL-10-based T-cell responses against pancreatitis-associated but not pancreatic carcinoma-associated antigens. T cells from pancreatic cancer patients responded to pancreatic cancer-associated but not pancreatitis-associated antigens with IFN-γ secretion. Pancreatitis-specific IL-10 responses were mediated by IL-10+IFN-γ−FoxP3+ regulatory T cells, which were expanded in the blood, bone marrow, and pancreatitis lesions and possessed the potential to suppress the proliferation of autologous conventional T cells in an antigen-specific manner. Pancreatitis lesions, in comparison with pancreatic carcinomas, contained increased concentrations of IL-10 and reduced levels of IFN-γ, suggesting pancreatitis-specific activity of regulatory T cells in situ. Conclusions Chronic pancreatitis is associated with disease-specific regulatory T-cell responses.

Das Mammakarzinom wird als systemische Erkrankung mit primar lokoregionarer Komponente verstanden. Ziel der Therapie ist neben der operativen Resektion sowie der Strahlentherapie eine Elimination von disseminierten, mikrometastatischen Tumorzellen durch zytostatische bzw. hormonelle Therapie. Trotzdem erleidet ein Grosteil der Patientinnen systemische Rezidive in Form von Fernmetastasen. Ein Grund hierfur kann der Verlust des immunologischen Gleichgewichts („tumor dormancy“) im Rahmen der intensiven Auseinandersetzung von T-Zellen und Tumorzellen im Knochenmark sein. Daher rucken unter Berucksichtigung der immunologischen Besonderheiten des Mammakarzinoms weitere supportive Therapien immer starker in den klinischen Fokus. Tumorspezifische Gedachtnis-T-Zellen (TMC) sind bei einem Grosteil der Mammakarzinompatientinnen im Knochenmark nachweisbar. Eine vergleichbare spezifische T-Zell-Reaktivitat im peripheren Blut kann nicht belegt werden. TMC des Knochenmarks lassen sich in vitro zu Interferon-γ-produzierenden, zytotoxischen Effektorzellen reaktivieren. In NOD/SCID-Mausen konnen tumorreaktive TMC des Knochenmarks nach spezifischer Restimulation mittels autologer dendritischer Zellen xenotransplantierte, autologe Tumoren abstosen. Die Tumorregression beruht vor allem auf einer selektiven Infiltration von CD45RO+ TMC und CD11+ dendritischen Zellen. Die adoptive Immuntherapie (ADI) bei metastasierten Mammakarzinompatientinnen mit aktivierten, autologen TMC des Knochenmarks erwies sich im Rahmen einer Phase-I-Studie als nebenwirkungsarme Therapieform ohne relevante Toxizitat. Posttherapeutisch konnten bei der Halfte der Patientinnen tumorantigenreaktive T-Zellen im peripheren Blut nachgewiesen werden (ADI-Response). Bei einem Teil der Patientinnen wurde ein objektivierbarer Abfall der Tumormarker beobachtet. Trotzdem zeigten alle Patientinnen in der klinischen Verlaufskontrolle eine Progredienz der Erkrankung. Hier gilt es, die tatsachliche Effektivitat der ADI in einer langfristigen Phase-II-Studie zu untersuchen. Moglicherweise ergibt sich ein vorteilhafteres Verhaltnis von Tumorzellen zu transfundierbaren, zytotoxischen Effektor-T-Zellen zum Zeitpunkt der geringsten Tumorlast direkt post operationem. Perspektivisch bleibt abzuwarten, ob die ADI – beispielsweise in der adjuvanten Situa - tion – neben bereits etablierten Therapieformen der betroffenen Patientin nachweisbar klinischen Nutzen bringen kann.

Breast cancer patients frequently harbour tumour-reactive memory T cells in their bone marrow (BM) but not in the blood. After reactivation ex-vivo these cells rejected autologous breast tumours in xenotransplanted mice demonstrating therapeutic potential upon reactivation and mobilization into the blood. We conducted a clinical pilot study on metastasized breast cancer patients to investigate if ex-vivo reactivation of tumour-reactive BM memory T cells and their adoptive transfer is feasible and increases the frequencies of tumour-reactive T cells in the blood.The study protocol involved one transfusion of T cells which were reactivated in vitro with autologous dendritic cells pulsed with lysate of MCF7 breast cancer cells as source of tumour antigens. Immunomonitoring included characterization of T cell activation in vitro and of tumour-specific T cells in the blood by interferon (IFN)-gamma ELISPOT assay, HLA-tetramers and antigen-induced interleukin (IL)-4 secretion.Twelve patients with pre-existing tumour-reactive BM memory T cells were included into the study. In all cases, the treatment was feasible and well tolerated. Six patients (responders) showed by ELISPOT assay de-novo tumour antigen-specific, IFN-gamma-secreting T cells in the blood after 7 days. In contrast, non responders showed in the blood tumour antigen-induced IL-4 responses. All responders received more than 6.5 x 10(3) tumour-reactive T cells. In contrast, all non responders received lower numbers of tumour antigen-reactive T cells. This was associated with reduced activation of memory T cells in activation cultures, increased amounts of CD4(+) CD25(high) regulatory T cells in the BM and increased tumour antigen-dependent IL-10 secretion. The latter was prevented by preceding depletion of regulatory T cells suggesting that regulatory T cells in the BM can inhibit reactivation of tumour-specific T cells.Taken together, adoptive transfer of ex-vivo re-stimulated tumour-reactive memory T cells from BM of metastasized breast cancer patients can induce the presence of tumour antigen-reactive type-1 T cells in the peripheral blood.

VEGFR-2 is expressed on tumor vasculature and a target for anti-angiogenic intervention. VXM01 is a first in kind orally applied tumor vaccine based on live, attenuated Salmonella bacteria carrying an expression plasmid, encoding VEGFR-2. We here studied the safety, tolerability, T effector (Teff), T regulatory (Treg) and humoral responses to VEGFR2 and anti-angiogenic effects in advanced pancreatic cancer patients in a randomized, dose escalation phase I clinical trial. Results of the first 3 mo observation period are reported. Locally advanced or metastatic, pancreatic cancer patients were enrolled. In five escalating dose groups, 30 patients received VXM01 and 15 placebo on days 1, 3, 5, and 7. Treatment was well tolerated at all dose levels. No dose-limiting toxicities were observed. Salmonella excretion and salmonella-specific humoral immune responses occurred in the two highest dose groups. VEGFR2 specific Teff, but not Treg responses were overall increased in vaccinated patients. We furthermore observed a significant reduction of tumor perfusion after 38 d in vaccinated patients together with increased levels of serum biomarkers indicative of anti-angiogenic activity, VEGF-A, and collagen IV. Vaccine specific Teff responses significantly correlated with reductions of tumor perfusion and high levels of preexisting VEGFR2-specific Teff while those showing no antiangiogenic activity had low levels of preexisting VEGFR2 specific Teff, showed a transient early increase of VEGFR2-specific Treg and reduced levels of VEGFR2-specific Teff at later time points - pointing to the possibility that early anti-angiogenic activity might be based at least in part on specific reactivation of preexisting memory T cells.

Regulatory T cells (Tregs) play an important role in controlling antitumor T-cell responses and hence represent a considerable obstacle for cancer immunotherapy. The abundance of specific Treg populations in cancer patients has been poorly analyzed so far. Here, we demonstrate that in breast cancer patients, Tregs often control spontaneous effector memory T-cell responses against mammaglobin, a common breast tissue-associated antigen that is overexpressed by breast carcinoma. Using functional assays, we identified a HLA-DRB1*04:01- and HLA-DRB1*07:01-restricted epitope of mammaglobin (mam34–48) that was frequently recognized by Tregs isolated from breast cancer patients. Using mam34–48-labeled HLA Class II tetramers, we quantified mammaglobin-specific Tregs and CD4+ conventional T (Tcon) cells in breast carcinoma patients as well as in healthy individuals. Both mammaglobin-specific Tregs and Tcon cells were expanded in breast cancer patients, each constituting approximately 0.2% of their respective cell subpopulations. Conversely, mammaglobin-specific Tregs and CD4+ Tcon cells were rare in healthy individuals (0.07%). Thus, we provide here for the first time evidence supporting the expansion of breast tissue-specific Tregs and CD4+ Tcon cells in breast cancer patients. In addition, we substantiate the potential implications of breast tissue-specific Tregs in the suppression of antitumor immune responses in breast cancer patients. The HLA Class II tetramers used in this study may constitute a valuable tool to elucidate the role of antigen-specific Tregs in breast cancer immunity and to monitor breast cancer-specific CD4+ T cells.

High regulatory T cell (Treg) infiltration in breast tumors is associated with reduced survival. However, the source of tumor infiltrating Treg and signals underlying their migration from lymphoid organs to the tumor tissue remain elusive. We here demonstrate that pronounced Treg infiltration in human breast tumors correlates with a selective reduction of tumor antigen specifc Treg from the bone marrow. Furthermore using MHC-II tumor peptide tetramers we show that tumor antigen specific bone marrow Treg selectively express the egress receptor Sphingosine-1-phosphate receptor 1 (S1P1) and CCR2. S1P1 and CCR2 were upregulated in Treg upon TCR stimulation and triggered selective Treg but not Tcon cell migration in response to S1P and CCL2 gradients between bone marrow and blood which we found to be significantly increased in breast cancer patients. Taken together, our data suggest that tumor antigen specific Treg population in the bone marrow are mobilized into the blood after TCR stimulation through elevated S1P and CCL2 gradients in breast cancer patients.

Background The investigational oral DNA vaccine VXM01 targets the vascular endothelial growth factor receptor 2 (VEGFR-2) and uses Salmonella typhi Ty21a as a vector. The immune reaction elicited by VXM01 is expected to disrupt the tumor neovasculature and, consequently, inhibit tumor growth. VXM01 potentially combines the advantages of anti-angiogenic therapy and active immunotherapy. Methods/Design This phase I trial examines the safety, tolerability, and immunological and clinical responses to VXM01. The randomized, placebo-controlled, double blind dose-escalation study includes up to 45 patients with locally advanced and stage IV pancreatic cancer. The patients will receive four doses of VXM01 or placebo in addition to gemcitabine as standard of care. Doses from 106 cfu up to 1010 cfu of VXM01 will be evaluated in the study. An independent data safety monitoring board (DSMB) will be involved in the dose-escalation decisions. In addition to safety as primary endpoint, the VXM01-specific immune reaction, as well as clinical response parameters will be evaluated. Discussion The results of this study shall provide the first data regarding the safety and immunogenicity of the oral anti-VEGFR-2 vaccine VXM01 in cancer patients. They will also define the recommended dose for phase II and provide the basis for further clinical evaluation, which may also include additional cancer indications. Trial registration EudraCT No.: 2011-000222-29, NCT01486329, ISRCTN68809279

Purpose: In glioma—in contrast to various other cancers—the impact of T-lymphocytes on clinical outcome is not clear. We investigated the clinical relevance and regulation of T-cell infiltration in glioma. Experimental Design: T-cell subpopulations from entire sections of 93 WHO°II–IV gliomas were computationally identified using markers CD3, CD8, and Foxp3; survival analysis was then done on primary glioblastomas (pGBM). Endothelial cells expressing cellular adhesion molecules (CAM) were similarly computationally quantified from the same glioma tissues. Influence of prominent cytokines (as measured by ELISA from 53 WHO°II–IV glioma lysates) on CAM-expression in GBM-isolated endothelial cells was determined using flow cytometry. The functional relevance of the cytokine-mediated CAM regulation was tested in a transmigration assay using GBM-derived endothelial cells and autologous T-cells. Results: Infiltration of all T-cell subsets increased in high-grade tumors. Most strikingly, within pGBM, elevated numbers of intratumoral effector T cells (Teff, cytotoxic and helper) significantly correlated with a better survival; regulatory T cells were infrequently present and not associated with GBM patient outcome. Interestingly, increased infiltration of Teff cells was related to the expression of ICAM-1 on the vessel surface. Transmigration of autologous T cells in vitro was markedly reduced in the presence of CAM-blocking antibodies. We found that TGF-β molecules impeded transmigration and downregulated CAM-expression on GBM-isolated endothelial cells; blocking TGF-β receptor signaling increased transmigration. Conclusions: This study provides comprehensive and novel insights into occurrence and regulation of T-cell infiltration in glioma. Specifically, targeting TGF-β1 and TGF-β2 might improve intratumoral T-cell infiltration and thus enhance effectiveness of immunotherapeutic approaches. Clin Cancer Res; 17(13); 4296–308. ©2011 AACR.

A wide variety of cancer types has been associated with paraneoplastic autoimmune disorders and with the induction of autoimmunity against several autoantigens, among them self-antigens that are also expressed by tumor cells. This raises the question of autoimmune disorders as a result of immune reactions to the tumor. To date, however, requirements for the generation of autoimmune reactions in cancer patients remain largely unclear. In this study, we characterized conditions in altogether 131 patients, which determine autoimmune responses in primary breast cancer patients. We used ex vivo IFN-γ EliSpot assays against autologous tumor or skin lysates to evaluate tumor- and auto-reactive T-cells (TCs) in the bone marrow (BM) as well as ELISA, ECLIA, and turbidimetric immunoassays for the detection of auto-reactive antibodies in the peripheral blood and compared results to intratumoral cytokine concentrations and pathobiological features of the primary tumor tissue. We here demonstrate a significant correlation between anti-tumor BMTC responses and cellular autoimmune reactivity in primary breast cancer patients (P = 0.002). Humoral autoimmune reactions, however, were negatively correlated with anti-tumor TC immunity (P = 0.039). We observed auto-reactive BMTCs especially in patients with well-differentiated, hormone receptor-positive carcinomas (P = 0.009). Furthermore, elevated concentrations of intratumoral IFN-α significantly correlated with the induction of cellular autoimmune reactivity (P = 0.0002), while humoral autoimmune reactions correlated with increased levels of intratumoral IL-12 (P = 0.04). Altogether, these data indicate a significant role of the tumor microenvironment and particularly that of IFN-α and IL-12 in the induction of systemic autoimmune responses and imply that the primary tumor tissue represents an integral site of autoimmune regulation in cancer patients.

Spontaneous immune responses in cancer patients have been described. Yet their clinical relevance and the conditions for their generation remain unclear. We characterized conditions that determine immune responses in primary breast cancer patients. We used tetramer analysis, ex vivo IFN-γ ELISPOT, cytotoxicity assays, and ELISA in 207 untreated patients and 12 Her-2/neu–specific CD8 T-cell lines to evaluate tumor-specific T cells (TC) in the bone marrow or MUC1-specific antibodies in the blood. Multiplex analysis was performed to quantify 27 intratumoral cytokines, chemokines, and growth factors. Results were compared with multiple pathologic and clinical parameters of the patients and tumors. Forty percent of the patients showed tumor-specific TC responses. These correlated with tumors of high differentiation, estrogen receptor expression, and low proliferative activity, and with a reduced cancer mortality risk. High tumor cell differentiation correlated with increased intratumoral, but not plasma, concentrations of IFN-α and reduced transforming growth factor (TGF)β1. In an in vitro priming experiment these two cytokines increased or inhibited, respectively, the capacity of dendritic cells to induce tumor-reactive TC. Tumor-specific B-cell responses, mainly of IgM isotype, were detectable in 50% of the patients and correlated with advanced tumor stage, increased TGFβ1, reduced IFN-α, and absence of TC responses. We show here that different types of immune responses are linked to distinct cytokine microenvironments and correlate with prognosis-relevant differences in tumor pathobiology. These findings shed light on the relation between immune response and cancer prognosis. [Cancer Res 2009;69(21):8420–8]

Spontaneous antitumor T cell responses in cancer patients are strongly controlled by Tregs, and increased numbers of tumor-infiltrating Tregs correlate with reduced survival. However, the tumor antigens recognized by Tregs in cancer patients and the impact of these cells on tumor-specific T cell responses have not been systematically characterized. Here we used a broad panel of long synthetic peptides of defined tumor antigens and normal tissue antigens to exploit a newly developed method to identify and compare ex vivo the antigen specificities of Tregs with those of effector/memory T cells in peripheral blood of colorectal cancer patients and healthy subjects. Tregs in tumor patients were highly specific for a distinct set of only a few tumor antigens, suggesting that Tregs exert T cell suppression in an antigen-selective manner. Tumor-specific effector T cells were detectable in the majority of colorectal cancer patients but not in healthy individuals. We detected differences in the repertoires of antigens recognized by Tregs and effector/memory T cells in the majority of colorectal cancer patients. In addition, only effector/memory T cell responses against antigens recognized by Tregs strongly increased after Treg depletion. The selection of antigens according to preexisting T cell responses may improve the efficacy of future immunotherapies for cancer and autoimmune disease.

High regulatory T cell (Treg) infiltration in breast tumors is associated with reduced survival. However, the source of tumor infiltrating Treg and signals underlying their migration from lymphoid organs to the tumor tissue remain elusive. We here demonstrate that pronounced Treg infiltration in human breast tumors correlates with a selective reduction of tumor antigen specifc Treg from the bone marrow. Using MHC-II tumor peptide tetramers we furthermore show that tumor specific bone marrow Treg selectively express Sphingosine-1-phosphate receptor 1 (S1P1), the receptor that mediates cell egress. S1P1 was upregulated in Treg upon TCR stimulation mediated by bone marrow resident antigen presenting cells and triggered selective Treg but not conventional T cell migration in response to S1P gradients between bone marrow and blood which we found to be significantly increased in breast cancer patients. Taken together, our data suggests a crucial role for bone marrow antigen presenting cells in regulating the surface expression of S1P1 on tumor specific Treg in the bone marrow which may represent an important source of tumor infiltrating Treg.

Increased expression and secretion of heparanase (Hpa) by tumor cells promotes tumor invasion through extracellular matrices, tissue destruction, angiogenesis, and metastasis. Here, we show the existence in breast cancer patients of Hpa-specific T lymphocytes by fluorescence-activated cell sorting flow cytometry using Hpa peptide-MHC class I tetramers. We furthermore show memory T-cell responses in a high proportion of breast cancer patients to Hpa-derived HLA-A2-restricted peptides, leading to production of IFN-γ and to generation of antitumor CTLs lysing breast cancer cells. Such CTLs recognized endogenously processed respective Hpa peptides on Hpa-transfected and Hpa-expressing untransfected breast carcinoma cells. According to these results and to the fact that such cells were not found in healthy people, Hpa seems to be an attractive new tumor-associated antigen and its HLA-A2-restricted peptides ought to be good candidates for peptide vaccination to reactivate memory immune responses to invasive and metastatic cancer cells. (Cancer Res 2006; 66(15): 7716-23)

3090 Background: VXM01 is an orally available, bacterially transmitted DNA vaccine targeting VEGFR-2. Pre-clinically, VXM01 showed anti-tumor activity in multiple tumor types. This first-in-human study was designed to evaluate the safety and tolerability of VXM01. Secondary endpoints included VEGFR-2 specific T-cell responses, tumor perfusion changes, and related biomarkers. Methods: A randomized, double-blinded, placebo-controlled, dose-escalation study was conducted in 45 patients with advanced pancreatic cancer. VXM01 or placebo was given on days 1, 3, 5, and 7. Doses were escalated from 10(6) CFU to 10(10) CFU over 5 dose groups, each including 6 VXM01 and 3 placebo patients. VEGFR-2 specific T-cell activity was monitored by ELISpot and T(reg) specificity assays before, during and after the vaccination course. Tumor perfusion was assessed by DCE-MRI on days 0 and 38. Biomarkers included CA19-9, VEGF-A and collagen IV. Results: Patients were enrolled from 12/2011 to 10/2012. Most commonly observed AEs were leukopenia, abdominal pain, and diarrhea, which were all equally distributed between treatment and placebo group. While a mild elevation in average blood pressure was observed in the VXM01 group over the placebo group, the hypertension adverse event rate did not differ between both groups. No DLTs were observed. VEGFR-2 specific effector T-cell response was increased in 57% of evaluable VXM01 treated patients, during and after the vaccination course. In 25% of the VXM01 group, the T-cell response score post-vaccination was higher than maximum placebo levels. In contrast, VEGFR-2 specific T(reg) responses were overall reduced in vaccinated patients. DCE-MRI data indicated a >33% drop in K(trans)/tumor perfusion in 35% of evaluable VXM01 treated patients vs. 10% in the placebo group. Mean changes were -4% (VXM01) and +15% (placebo). Reduced tumor perfusion correlated with VEGFR-2 specific T-cell responses and biomarker responses. Conclusions: VXM01 appeared safe and was well tolerated without DLTs across 5 tested dose levels. The data suggest further that VXM01 induces and enhances a VEGFR-2 specific T-cell response and impacts tumor perfusion. Clinical trial information: ISRCTN68809279.