Professional development session for early career scientists at SITC 2012.

The Society for Immunotherapy of Cancer (SITC) 2012 Professional Development Session was held as part of the SITC 27th Annual Meeting, Washington, DC, on October 24, 2012. The session was designed as a new opportunity for early career investigators to learn about relevant career development topics in a didactic setting.

Antibodies against insulin measured by electrochemiluminescence predicts insulitis severity and disease onset in non-obese diabetic mice and can distinguish human type 1 diabetes status.

BACKGROUND: The detection of insulin autoantibodies (IAA) aids in the prediction of autoimmune diabetes development. However, the long-standing, gold standard 125I-insulin radiobinding assay (RBA) has low reproducibility between laboratories, long sample processing times and requires the use of newly synthesized radiolabeled insulin for each set of assays. Therefore, a rapid, non-radioactive, and reproducible assay is highly desirable. METHODS: We have developed electrochemiluminescence (ECL)-based assays that fulfill these criteria in the measurement of IAA and anti-insulin antibodies (IA) in non-obese diabetic (NOD) mice and in type 1 diabetic individuals, respectively. Using the murine IAA ECL assay, we examined the correlation between IAA, histopathological insulitis, and blood glucose in a cohort of female NOD mice from 4 up to 36 weeks of age. We developed a human IA ECL assay that we compared to conventional RBA and validated using samples from 34 diabetic and 59 non-diabetic individuals in three independent laboratories. RESULTS: Our ECL assays were rapid and sensitive with a broad dynamic range and low background. In the NOD mouse model, IAA levels measured by ECL were positively correlated with insulitis severity, and the values measured at 8-10 weeks of age were predictive of diabetes onset. Using human serum and plasma samples, our IA ECL assay yielded reproducible and accurate results with an average sensitivity of 84% at 95% specificity with no statistically significant difference between laboratories. CONCLUSIONS: These novel, non-radioactive ECL-based assays should facilitate reliable and fast detection of antibodies to insulin and its precursors sera and plasma in a standardized manner between laboratories in both research and clinical settings. Our next step is to evaluate the human IA assay in the detection of IAA in prediabetic subjects or those at risk of type 1 diabetes and to develop similar assays for other autoantibodies that together are predictive for the diagnosis of this common disorder, in order to improve prediction and facilitate future therapeutic trials.

HER2-affitoxin: a potent therapeutic agent for the treatment of HER2-overexpressing tumors.

PURPOSE: Cancers overexpressing the HER2/neu gene are usually more aggressive and are associated with poor prognosis. Although trastuzumab has significantly improved the outcome, many tumors do not respond or acquire resistance to current therapies. To provide an alternative HER2-targeted therapy, we have developed and characterized a novel recombinant protein combining an HER2-specific Affibody and modified Pseudomonas aeruginosa exotoxin A (PE 38), which, after binding to HER2, is internalized and delivered to the cytosol of the tumor cell, where it blocks protein synthesis by ADP ribosylation of eEF-2. EXPERIMENTAL DESIGN: The effect of the Affitoxin on cell viability was assessed using CellTiter-Glo (Promega). To assess HER2-specific efficacy, athymic nude mice bearing BT-474 breast cancer, SK-OV-3 ovarian cancer, and NCI-N87 gastric carcinoma xenografts were treated with the Affitoxin (HER2- or Tag-specific), which was injected every third day. Affitoxin immunogenicity in female BALB/c mice was investigated using standard antibody production and splenocyte proliferation assays. RESULTS: In vitro experiments proved that HER2-Affitoxin is a potent agent that eliminates HER2-overexpressing cells at low picomolar concentrations. Therapeutic efficacy studies showed complete eradication of relatively large BT-474 tumors and significant effects on SK-OV-3 and NCI-N87 tumors. HER2-Affitoxin cleared quickly from circulation (T(1/2) < 10 minutes) and was well tolerated by mice at doses of 0.5 mg/kg and below. Immunogenicity studies indicated that HER2-Affitoxin induced antibody development after the third injected dose. CONCLUSIONS: Our findings showed that HER2-Affitoxin is an effective anticancer agent and a potential candidate for clinical studies.

Cutting Edge: Tumor-specific CD8+ T cells infiltrating prostatic tumors are induced to become suppressor cells.

We previously reported that naive, tumor-specific CD8(+) (TcR-I) T cells transferred into prostate tumor-bearing mice traffic to the prostate where they become tolerized. We now report that TcR-I cells suppress the proliferation of naive T cells. This suppression is mediated at least in part by secreted factors, and the suppressive activity can be blocked by Abs directed against TGF-beta. We further report that TcR-I cells must infiltrate the prostate to acquire suppressive activity. Delivery of tumor-specific CD4(+) T cells prevents the conversion of TcR-I cells into suppressor cells. Taken together, our findings may have critical implications for sustaining T cell responsiveness during immunotherapy, as the development of suppressor cells in the tumor microenvironment may eliminate the potency of T cells primed in the periphery or delivered during adoptive immunotherapy.

Immunity to murine prostatic tumors: continuous provision of T-cell help prevents CD8 T-cell tolerance and activates tumor-infiltrating dendritic cells.

We reported previously that tumor-specific CD8(+) T cells (TcR-I) become tolerant in the transgenic adenocarcinoma of the mouse prostate (TRAMP) model. In this study, we show that CD4(+) TcR transgenic (TcR-II) T cells transferred into TRAMP mice became activated in lymph nodes, trafficked to the prostate, and initially functioned as T(H)1 cells. Although a single cotransfer of TcR-II cells delayed TcR-I cell tolerization, repeated transfer of TcR-II cells was required to prevent TcR-I cell tolerization and significantly slowed progression of TRAMP prostate tumors. After transfer of TcR-II cells, dendritic cells within the tumor expressed higher levels of costimulatory molecules and displayed an enhanced ability to stimulate proliferation of naive T cells. Blockade of CD40-CD40L interactions during TcR-II transfer resulted in a profound reduction in dendritic cell stimulatory capacity and a partial loss of TcR-I effector functions and tumor immunity. These data show that sustained provision of activated tumor-specific CD4(+) T cells alters the immunosuppressive tumor microenvironment, ultimately leading to the control of tumor growth. These findings will assist in the design of more effective immunotherapeutic approaches for cancer.

Application of a flow cytometric cytotoxicity assay for monitoring cancer vaccine trials.

In this study, we evaluated the applicability of a flow cytometry-based cytotoxicity (FC) assay previously developed by our laboratory, for monitoring cancer vaccine trials. The assay simultaneously measures effector cell degranulation and target cell death. Clinically relevant samples consisted of frozen peripheral blood mononuclear cells (PBMC) from vaccinated melanoma patients with known response to the melanoma peptide g209. Both PBMC and 7 day in vitro-stimulated lymphocyte from patient samples were used as effector cells in the FC assay. Activity against the relevant g209 and the control g280 peptide measured in the FC assay was directly compared with results obtained from the Granzyme B enzyme-linked immunosorbent spot assay and the standard 51Cr-release assay run in tandem. The FC assay detected low or no activity when PBMC were used as effector cells. Using cytotoxic T lymphocytes as effector cells, little or no effector cell degranulation or cytotoxicity was measured in the FC assay in prevaccination samples. After vaccination, an increase in both degranulation and target cell death could be determined when target cells were pulsed with g209. No or low reactivity was found against g280 at any time point. Our findings exhibited excellent correlation between CD107a expression and GrB secretion and also Annexin V binding to target cells and specific lysis measured in the 51Cr-release assay. Results obtained from the FC assay were highly reproducible. Therefore, the FC assay may be applied to vaccine trial monitoring and allows the measurement of effector cell degranulation and target cell death simultaneously in a single sample.

New approaches for monitoring CTL activity in clinical trials.

We have developed a modification of the ELISPOT assay that measures Granzyme B (GrB) release from cytotoxic T lymphocytes (CTLs). The GrB ELISPOT assay is a superior alternative to the 51Cr-release assay since it is significantly more sensitive and provides an estimation of cytotoxic effector cell frequency. Additionally, unlike the IFN-gamma ELISPOT assay, the GrB ELISPOT directly measures the release of a cytolytic protein. We report that the GrB ELISPOT can be utilized to measure ex vivo antigen-specific cytotoxicity of peripheral blood mononuclear cells (PBMCs) from cancer patients vaccinated with a peptide-based cancer vaccine. We compare the reactivity of patients' PBMCs in the GrB ELISPOT, with reactivity in the tetramer, IFN-gamma ELISPOT and chromium (51Cr)-release assays. Differences in immune response over all assays tested were found between patients, and four response patterns were observed. Reactivity in the GrB ELISPOT was more closely associated with cytotoxicity in the 51Cr-release assay than the tetramer or IFN-gamma ELISPOT assays. We also optimized the GrB ELISPOT assay to directly measure immune responses against autologous primary tumor cells in vaccinated cancer patients. A perforin ELISPOT assay was also adapted to evaluate peptide-stimulated reactivity of PMBCs from vaccinated melanoma patients. Modifications of the ELISPOT assay described in this chapter allow a more comprehensive evaluation of low-frequency tumor-specific CTLs and their specific effector functions and can provide a valuable insight into immune responses in cancer vaccine trials.

T cell tolerance to tumors and cancer immunotherapy.

It is widely recognized that the immune system plays a role in cancer progression and that some tumors are inherently immunogenic. The identification of tumor-associated antigens (TAAs) has stimulated research focused on immunotherapies to mediate the regression of established tumors. Cancer-specific immunity has traditionally been aimed at activating CD8+ cytotoxic T lymphocytes (CTLs) directed against major histocompatibility complex (MHC) class I-binding peptide epitopes. Other approaches utilize T cell adoptive therapy where autologous, tumor-specific T cells propagated in vitro are transferred back into recipients. However, these strategies have met with limited success in part due to the regulatory mechanisms of T cell tolerance, which poses a considerable challenge to cancer immunotherapy. Our laboratory utilizes the TRansgenic Adenocarcinoma of the Mouse Prostate (TRAMP) model, a murine model of prostate cancer, to study mechanisms of T cell tolerization to tumor antigens. We previously demonstrated that upon encounter with their cognate antigen in the tumor microenvironment, naive T cell become tolerized. Our ongoing studies are testing whether provision of CD4+ T cells can enhance tumor immunity by preventing CD8+ T cell tolerance. A greater understanding of the interaction between various tumor-specific T cell subsets will facilitate the design of novel approaches to stimulate a more potent antitumor immune response.

Tolerization of tumor-specific T cells despite efficient initial priming in a primary murine model of prostate cancer.

In this report, we studied T cell responses to a prostate cancer Ag by adoptively transferring tumor Ag-specific T cells into prostate tumor-bearing mice. Our findings demonstrate that CD8(+) T cells initially encountered tumor Ag in the lymph node and underwent an abortive proliferative response. Upon isolation from the tumor, the residual tumor-specific T cells were functionally tolerant of tumor Ag as measured by their inability to degranulate and secrete IFN-gamma and granzyme B. We next sought to determine whether providing an ex vivo-matured, peptide-pulsed dendritic cell (DC) vaccine could overcome the tolerizing mechanisms of tumor-bearing transgenic adenocarcinoma of the mouse prostate model mice. We demonstrate that tumor Ag-specific T cells were protected from tolerance following provision of the DC vaccine. Concurrently, there was a reduction in prostate tumor size. However, even when activated DCs initially present tumor Ag, T cells persisting within the tolerogenic tumor environment gradually lost Ag reactivity. These results suggest that even though a productive antitumor response can be initiated by a DC vaccine, the tolerizing environment created by the tumor still exerts suppressive effects on the T cells. Furthermore, our results demonstrate that when trying to elicit an effective antitumor immune response, two obstacles must be considered: to maintain tumor Ag responsiveness, T cells must be efficiently primed to overcome tumor Ag presented in a tolerizing manner and protected from the suppressive mechanisms of the tumor microenvironment.

Application of the granzyme B ELISPOT assay for monitoring cancer vaccine trials.

Granzyme B (GrB) is present in the granules of cytolytic lymphocytes and is a key mediator of cell-mediated target cell death via the granule-mediated pathway. The release of GrB can be used as an indicator of a cytotoxic T lymphocyte response. Herein, we report that the GrB enzyme-linked immunospot assay (ELISPOT) can be used to measure ex vivo antigen-specific cytotoxicity of peripheral blood mononuclear cells from cancer patients vaccinated with a peptide-based cancer vaccine. We compare the reactivity of patients' peripheral blood mononuclear cells in the GrB ELISPOT with reactivity in the tetramer, interferon (IFN)-gamma ELISPOT, and Cr-release assays. Differences in immune response over all assays tested were found between patients and 4 response patterns were observed. Reactivity in the GrB ELISPOT was more closely associated with cytotoxicity in the Cr-release assay than the tetramer or IFN-gamma ELISPOT assays. Moreover, the higher affinity g209-2M peptide (used for vaccination) elicited greater GrB secretion than the native g209 peptide, although this difference was not observed with IFN-gamma secretion. Taken together with the fact that GrB is a specific mediator released by cytotoxic T lymphocytes, these results show that simultaneous use of the GrB ELISPOT assay with other immunologic assays may provide important additional immunologic insight into patient responses to cancer vaccines.

A novel flow cytometric assay for evaluating cell-mediated cytotoxicity.

Comprehensive evaluation of cell-mediated cytotoxicity is very important, especially in the clinical setting, when a surrogate immunologic endpoint that correlates with a clinical outcome needs to be defined. With the objective of simultaneously evaluating target cell death and effector cell frequency, the authors combined the measuring of the expression of the degranulation marker CD107a by effector cells with the apoptosis marker annexin V binding to target cells. Using human cytotoxic T lymphocytes, the authors found a significant incubation time-dependent increase of surface CD107a expression on effector cells due to degranulation. A parallel increase of annexin V binding to target cells due to target cell apoptosis was also found. These two parameters have shown excellent correlation in all effector/target cell systems studied. To find possible connections between effector cell degranulation (CD107a expression), granzyme B secretion, and target cell death (annexin V binding), the GrB ELISPOT assay and flow cytometric assay were performed and excellent cross-correlation was found between all three parameters. The specificity of the assay was also shown. These data show that this novel assay allows measurement of cytolytic cell activation and frequency as well as target cell death in the same sample.

IL-27 mediates complete regression of orthotopic primary and metastatic murine neuroblastoma tumors: role for CD8+ T cells.

We have shown previously that IFN-gamma-inducing cytokines such as IL-12 can mediate potent antitumor effects against murine solid tumors. IL-27 is a newly described IL-12-related cytokine that potentiates various aspects of T and/or NK cell function. We hypothesized that IL-27 might also mediate potent antitumor activity in vivo. TBJ neuroblastoma cells engineered to overexpress IL-27 demonstrated markedly delayed growth compared with control mice, and complete durable tumor regression was observed in >90% of mice bearing either s.c. or orthotopic intra-adrenal tumors, and 40% of mice bearing induced metastatic disease. The majority of mice cured of their original TBJ-IL-27 tumors were resistant to tumor rechallenge. Furthermore, TBJ-IL-27 tumors were heavily infiltrated by CD8(+) T cells, and draining lymph node-derived lymphocytes from mice bearing s.c. TBJ-IL-27 tumors are primed to proliferate more readily when cultured ex vivo with anti-CD3/anti-CD28 compared with lymphocytes from mice bearing control tumors, and to secrete higher levels of IFN-gamma. In addition, marked enhancement of local IFN-gamma gene expression and potent up-regulation of cell surface MHC class I expression are noted within TBJ-IL-27 tumors compared with control tumors. Functionally, these alterations occur in conjunction with the generation of tumor-specific CTL reactivity in mice bearing TBJ-IL-27 tumors, and the induction of tumor regression via mechanisms that are critically dependent on CD8(+), but not CD4(+) T cells or NK cells. Collectively, these studies suggest that IL-27 could be used therapeutically to potentiate the host antitumor immune response in patients with malignancy.

Evaluating the cytotoxicity of innate immune effector cells using the GrB ELISPOT assay.

BACKGROUND: This study assessed the Granzyme B (GrB) ELISPOT as a viable alternative to the 51Cr-release assay for measuring cytotoxic activity of innate immune effector cells. We strategically selected the GrB ELISPOT assay because GrB is a hallmark effector molecule of cell-mediated destruction of target cells. METHODS: We optimized the GrB ELISPOT assay using the human-derived TALL-104 cytotoxic cell line as effectors against K562 target cells. Titration studies were performed to assess whether the ELISPOT assay could accurately enumerate the number of GrB-secreting effector cells. TALL-104 were treated with various secretion inhibitors and utilized in the GrB ELISPOT to determine if GrB measured in the ELISPOT was due to degranulation of effector cells. Additionally, CD107a expression on effector cells after effector-target interaction was utilized to further confirm the mechanism of GrB release by TALL-104 and lymphokine-activated killer (LAK) cells. Direct comparisons between the GrB ELISPOT, the IFN-gamma ELISPOT and the standard 51Cr-release assays were made using human LAK cells. RESULTS: Titration studies demonstrated a strong correlation between the number of TALL-104 and LAK effector cells and the number of GrB spots per well. GrB secretion was detectable within 10 min of effector-target contact with optimal secretion observed at 3-4 h; in contrast, optimal IFN-gamma secretion was not observed until 24 h. The protein secretion inhibitor, brefeldin A, did not inhibit the release of GrB but did abrogate IFN-gamma production by TALL-104 cells. GrB secretion was abrogated by BAPTA-AM (1,2-bis-(2-aminophenoxy)ethane-N,N,N', N'-tetraacetic acid tetra(acetoxymethyl) ester), which sequesters intracellular Ca2+, thereby preventing degranulation. The number of effector cells expressing the degranulation associated glycoprotein CD107a increased after interaction with target cells and correlated with the stimulated release of GrB measured in the ELISPOT assay. CONCLUSIONS: Because of its high sensitivity and ability to estimate cytotoxic effector cell frequency, the GrB ELISPOT assay is a viable alternative to the 51Cr-release assay to measure MHC non-restricted cytotoxic activity of innate immune cells. Compared to the IFN-gamma ELISPOT assay, the GrB ELISPOT may be a more direct measure of cytotoxic cell activity. Because GrB is one of the primary effector molecules in natural killer (NK) cell-mediated killing, detection and enumeration of GrB secreting effector cells can provide valuable insight with regards to innate immunological responses.

A modified human ELISPOT assay to detect specific responses to primary tumor cell targets.

BACKGROUND: The desired outcome of cancer vaccination is to induce a potent T cell response which can specifically recognize and eliminate autologous tumor cells in vivo. Accordingly, immunological assays that demonstrate recognition of native tumor cells (tumor-specific) may be more clinically relevant than assays that demonstrate recognition of tumor protein or peptide (antigen-specific). METHODS: Towards this goal, we adapted the IFN-gamma ELISPOT assay to measure immune responses against autologous primary tumor cells in vaccinated cancer patients. As a model system to develop the assay, we utilized peripheral blood mononuclear cells (PBMC) directly isolated from follicular lymphoma patients vaccinated with tumor-derived idiotype protein. RESULTS: After optimizing several variables, we demonstrated that the modified IFN-gamma ELISPOT assay could be used to reliably and reproducibly determine the tumor-reactive T cell frequency in the PBMC of these patients. The precursor frequency of tumor-reactive T cells was significantly higher in the postvaccine PBMC, compared with prevaccine samples in all patients tested. Furthermore, the specificity of these T cells was established by the lack of reactivity against autologous normal B cells. CONCLUSIONS: These results demonstrate the feasibility of quantitating tumor-specific T cell responses when autologous, primary tumor cells are available as targets.

The Granzyme B ELISPOT assay: an alternative to the 51Cr-release assay for monitoring cell-mediated cytotoxicity.

BACKGROUND: The interferon-gamma (IFN-gamma) ELISPOT assay is one of the most useful techniques for immunological monitoring of cancer vaccine trials and has gained increased application as a measure of specific T cell activation. However, it does not assess cell-mediated cytotoxicity directly as IFN-gamma secretion is not limited to only cytolytic cells. Granzyme B (GrB) is a key mediator of target cell death via the granule-mediated pathway. Therefore, the release of GrB by cytolytic lymphocytes upon effector-target interaction may be a more specific indicator of CTL and NK cytotoxic ability than IFN-gamma secretion. METHODS: We assessed whether the GrB ELISPOT assay is a viable alternative to the 51Cr-release and IFN-gamma ELISPOT assays for measuring antigen-specific CTL cytotoxicity. Direct comparisons between the three assays were made using human CTL cell lines (alphaEN-EBV and alphaJY) and an in vitro stimulated anti-Flu matrix peptide (FMP)-specific CTL. RESULTS: When the GrB ELISPOT was directly compared to the IFN-gamma ELISPOT and 51Cr-release assays, excellent cross-correlation between all three assays was shown. However, measurable IFN-gamma secretion in the ELISPOT assay was observed only after 1 hour of incubation and cytotoxicity assessed via the 51Cr-release assay after 4 hours, whereas GrB secretion was detectable within 10 min of effector-target contact with significant secretion observed after 1 h. Titration studies demonstrated a strong correlation between the number of effector cells and GrB spots per well. Irrelevant targets or antigens did not induce significant GrB secretion. Additionally, GrB secretion was abrogated when CTL cultures were depleted of CD8+ cells. CONCLUSION: Our findings demonstrate that the GrB ELISPOT assay is a superior alternative to the 51Cr-release assay since it is significantly more sensitive and provides an estimation of cytotoxic effector cell frequency. Additionally, unlike the IFN-gamma ELISPOT assay, the GrB ELISPOT directly measures the release of a cytotolytic protein. Detection of low frequency tumor-specific CTL and their specific effector functions can provide valuable insight with regards to immunological responses.