Immunological monitoring of the tumor immunoenvironment for clinical trials.

Monitoring of immunotherapeutic clinical trials has undergone a considerable change in the last decade resulting in a general agreement that immune monitoring should guide the development of cancer vaccines. The emphasis on immune cell functions and quantitation of antigen-specific T cells have been playing a major role in the attempts to establish meaningful correlations between therapy-induced alterations in immune responses and clinical endpoints. However, one significant unresolved issue in modern immunotherapy is that when a tumor-specific cellular immune response is observed following the course of immunotherapy, it does not always lead to clinically proven cancer regression. This disappointing lack of a correlation between the tumor-specific cytotoxic immune responses and the clinical efficacy of immunotherapy may be explained, among other reasons, by the notion that the analysis of any single immunological parameter is not sufficient to provide clinically feasible information about the complex interactions between different cell subsets in the peripheral blood and immune, tumor, and stromal cells in the tumor milieu. By contrast, a systemic approach is required for improving the quality of a serial monitoring to ensure that it adequately and reliably measures potential changes induced in patients by administered vaccines or immunomodulators. Comprehensive evaluation of the balance between the immunostimulatory and immunosuppressive compartments of the immune system could be critical for a better understanding of how a given immunotherapy works or does not work in a particular clinical trial. New approaches to characterize tumor-infiltrating leukocytes, their phenotypic, biochemical, and genetic characteristics within the tumor microenvironment need to be developed and validated and should complement current monitoring techniques. These immune-monitoring assays for the local tumor immunoenvironment should be developed, validated, and standardized for reliability and consistency in order to establish the overall performance standards.

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.

Emerging concepts in biomarker discovery; the US-Japan Workshop on Immunological Molecular Markers in Oncology.

Supported by the Office of International Affairs, National Cancer Institute (NCI), the "US-Japan Workshop on Immunological Biomarkers in Oncology" was held in March 2009. The workshop was related to a task force launched by the International Society for the Biological Therapy of Cancer (iSBTc) and the United States Food and Drug Administration (FDA) to identify strategies for biomarker discovery and validation in the field of biotherapy. The effort will culminate on October 28th 2009 in the "iSBTc-FDA-NCI Workshop on Prognostic and Predictive Immunologic Biomarkers in Cancer", which will be held in Washington DC in association with the Annual Meeting. The purposes of the US-Japan workshop were a) to discuss novel approaches to enhance the discovery of predictive and/or prognostic markers in cancer immunotherapy; b) to define the state of the science in biomarker discovery and validation. The participation of Japanese and US scientists provided the opportunity to identify shared or discordant themes across the distinct immune genetic background and the diverse prevalence of disease between the two Nations. Converging concepts were identified: enhanced knowledge of interferon-related pathways was found to be central to the understanding of immune-mediated tissue-specific destruction (TSD) of which tumor rejection is a representative facet. Although the expression of interferon-stimulated genes (ISGs) likely mediates the inflammatory process leading to tumor rejection, it is insufficient by itself and the associated mechanisms need to be identified. It is likely that adaptive immune responses play a broader role in tumor rejection than those strictly related to their antigen-specificity; likely, their primary role is to trigger an acute and tissue-specific inflammatory response at the tumor site that leads to rejection upon recruitment of additional innate and adaptive immune mechanisms. Other candidate systemic and/or tissue-specific biomarkers were recognized that might be added to the list of known entities applicable in immunotherapy trials. The need for a systematic approach to biomarker discovery that takes advantage of powerful high-throughput technologies was recognized; it was clear from the current state of the science that immunotherapy is still in a discovery phase and only a few of the current biomarkers warrant extensive validation. It was, finally, clear that, while current technologies have almost limitless potential, inadequate study design, limited standardization and cross-validation among laboratories and suboptimal comparability of data remain major road blocks. The institution of an interactive consortium for high throughput molecular monitoring of clinical trials with voluntary participation might provide cost-effective solutions.

A systematic approach to biomarker discovery; preamble to "the iSBTc-FDA taskforce on immunotherapy biomarkers".

The International Society for the Biological Therapy of Cancer (iSBTc) has initiated in collaboration with the United States Food and Drug Administration (FDA) a programmatic look at innovative avenues for the identification of relevant parameters to assist clinical and basic scientists who study the natural course of host/tumor interactions or their response to immune manipulation. The task force has two primary goals: 1) identify best practices of standardized and validated immune monitoring procedures and assays to promote inter-trial comparisons and 2) develop strategies for the identification of novel biomarkers that may enhance our understating of principles governing human cancer immune biology and, consequently, implement their clinical application. Two working groups were created that will report the developed best practices at an NCI/FDA/iSBTc sponsored workshop tied to the annual meeting of the iSBTc to be held in Washington DC in the Fall of 2009. This foreword provides an overview of the task force and invites feedback from readers that might be incorporated in the discussions and in the final document.

The role of ascorbate in the modulation of HIF-1alpha protein and HIF-dependent transcription by chromium(VI) and nickel(II).

Molecular oxygen is involved in hydroxylation and subsequent degradation of HIF-1alpha, a subunit of HIF-1 transcription factor; therefore oxygen shortage (hypoxia) stabilizes this protein. However, HIF-1alpha can also be stabilized by transition metal ions in the presence of oxygen, suggesting that a different mechanism is involved in metal-induced hypoxic stress. Recently, we showed that the depletion of intracellular ascorbate by metals may lead to the inhibition of hydroxylases. Because nickel(II) has similarity to iron(II), an alternative hypothesis suggests that iron substitution for nickel in the enzyme inhibits hydroxylase activity. Here we investigated the induction of HIF-1 by another metal, chromium, which cannot replace iron in the enzyme. We show that chromium(VI), but not chromium(III), can oxidize ascorbate both in cells and in a cell-free system. In agreement with these data chromium(VI) stabilizes HIF-1alpha protein in cells only until it is reduced to chromium(III). In contrast, nickel(II) was found to be a catalyst, which facilitated continuous oxidation of ascorbate by ambient oxygen. These data correlate with extended stabilization of HIF-1alpha after acute exposure to nickel(II). The HIF-1-dependent reporter assays revealed that 20-24 h was required to fully develop the HIF-1 transcriptional response, and the acute exposure to nickel(II), but not chromium(VI), meets this requirement. However, repeated (chronic) exposure to chromium(VI) can also lead to extended stabilization of HIF-1alpha. Thus, the obtained data emphasize the important role of ascorbate in regulation of HIF-1 transcriptional activity in metal-exposed human lung cells.

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.

The proinflammatory cytokine interleukin-18 alters multiple signaling pathways to inhibit natural killer cell death.

The proinflammatory cytokine, interleukin-18 (IL-18), is a natural killer (NK) cell activator that induces NK cell cytotoxicity and interferon-gamma (IFN-gamma) expression. In this report, we define a novel role for IL-18 as an NK cell protective agent. Specifically, IL-18 prevents NK cell death initiated by different and distinct stress mechanisms. IL-18 reduces NK cell self-destruction during NK-targeted cell killing, and in the presence of staurosporin, a potent apoptotic inducer, IL-18 reduces caspase-3 activity. The critical regulatory step in this process is downstream of the mitochondrion and involves reduced cleavage and activation of caspase-9 and caspase-3. The ability of IL-18 to regulate cell survival is not limited to a caspase death pathway in that IL-18 augments tumor necrosis factor (TNF) signaling, resulting in increased and prolonged mRNA expression of c-apoptosis inhibitor 2 (cIAP2), a prosurvival factor and caspase-3 inhibitor, and TNF receptor-associated factor 1 (TRAF1), a prosurvival protein. The cumulative effects of IL-18 define a novel role for this cytokine as a molecular survival switch that functions to both decrease cell death through inhibition of the mitochondrial apoptotic pathway and enhance TNF induction of prosurvival factors.

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.

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.

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.

Study of diverse mechanisms of cell-mediated cytotoxicity in gene-targeted mice using flow cytometric cytotoxicity assay.

Cytotoxic lymphocytes kill tumor or virus-infected target cells utilizing two mechanisms-(1) release of lytic granules (containing perforin and granzymes), and (2) Fas ligand (FasL)/Fas or TNF-initiated apoptosis. We have examined mechanisms of target cell lysis by effector T cells from gene-targeted and mutant mice using a new Flow Cytometric Cytotoxicity Assay (FC Assay). Target cells were labeled with PKH67 dye. Cell death was estimated by 7-AAD inclusion and annexin V-PE binding. A direct correlation has been found between the percentage of dead target cells in FC Assay and the results of 111In release cytotoxicity assay when effector T cells from either Pfp -/- (perforin knockout) or gld (non-functional Fas Ligand) mice were used. As shown by the 4 h FC assay, the granule-mediated mechanism was utilized by T cells from gld mice. In contrast, T cells from Pfp -/- mice used death receptor-mediated lysis. Therefore, cytotoxic cells from gene-targeted and mutant mice can serve as valuable tools for studying different mechanisms of cell-mediated cytotoxicity, and the FC assay could be applied irrespective of which cytotoxic effector pathway is involved.

ELISPOT Assay for Monitoring Cytotoxic T Lymphocytes (CTL) Activity in Cancer Vaccine Clinical Trials.

The profiling and monitoring of immune responses are key elements in the evaluation of the efficacy and development of new biotherapies, and a number of assays have been introduced for analyzing various immune parameters before, during, and after immunotherapy. The choice of immune assays for a given clinical trial depends on the known or suggested immunomodulating mechanisms associated with the tested therapeutic modality. Cell-mediated cytotoxicity represents a key mechanism in the immune response to various pathogens and tumors. Therefore, the selection of monitoring methods for the appropriate assessment of cell-mediated cytotoxicity is thought to be crucial. Assays that can detect both cytotoxic T lymphocytes (CTL) frequency and function, such as the IFN-γ enzyme-linked immunospot assay (ELISPOT) have gained increasing popularity for monitoring clinical trials and in basic research. Results from various clinical trials, including peptide and whole tumor cell vaccination and cytokine treatment, have shown the suitability of the IFN-γ ELISPOT assay for monitoring T cell responses. However, the Granzyme B ELISPOT assay and Perforin ELISPOT assay may represent a more direct analysis of cell-mediated cytotoxicity as compared to the IFN-γ ELISPOT, since Granzyme B and perforin are the key mediators of target cell death via the granule-mediated pathway. In this review we analyze our own data and the data reported by others with regard to the application of various modifications of ELISPOT assays for monitoring CTL activity in clinical vaccine trials.

Conference overview: Cancer Immunotherapy and Immunomonitoring (CITIM): moving forward.

The immune system is a critical element involved in the control of tumor development and progression. While professionals have learned how to manipulate the immune system to generate tumor-specific immune responses, cancer immunotherapy has not yet delivered substantial clinical benefits. It has become increasingly clear that tumor-induced abnormalities in the immune system not only hamper tumor immunosurveillance, but also limit the efficacy of cancer immunotherapy. Meanwhile, the results of recent studies allow the belief that one is on the edge of a real breakthrough in this promising direction in cancer therapy. The 2(nd) International Conference 'Cancer Immunotherapy and Immunomonitoring (CITIM)' was the second meeting in Eastern Europe to specifically focus on the issue of immune regulation in the tumor environment, cancer immunotherapy, and immunomonitoring of immunotherapeutic clinical trials. This CITIM Conference held in Budapest, Hungary, was comprised from 12 plenary sessions, Best Abstract Award session, Poster session, and four Keynote lectures. Outstanding presentations and numerous productive discussions summarized the current place of the field and opened new directions for improving monitoring and therapy for patients with cancer.

Conference Scene: Immunotherapy reaches new milestones in cancer eradication.

Biotherapy is widely considered as the fourth treatment modality for patients with cancer, and uses the constantly increasing knowledge in molecular biology, cell biology and immunology. Biotherapy uses naturally occurring biological molecules (e.g., cytokines and antibodies) or works by the manipulation of normal biological mechanisms (controlling or inhibiting tumor growth). Important achievements in anticancer drug development are immunotherapeutic strategies recently approved by the US FDA as well as clinical data of the cancer patients treated in clinical trials. There is a need to expand these novel cancer immunotherapeutic modalities for cancer patients all over the world. To meet that goal, it is essential to spread the information, to summarize the new clinical data and to draw the conclusions from the clinical and preclinical investigations. These frontline tasks can be well advanced by organizing international conferences in this domain in less scientifically developed countries, with a significant tumor burden statistics. Therefore, special efforts were done to organize the 2nd International Cancer Immunotherapy and Immunomonitoring Conference (CITIM-2011) in Hungary.

Novel peptides based on HIV-1 gp120 sequence with homology to chemokines inhibit HIV infection in cell culture.

The sequential interaction of the envelope glycoprotein of the human immunodeficiency virus type 1 (HIV-1) with CD4 and certain chemokine coreceptors initiates host cell entry of the virus. The appropriate chemokines have been shown to inhibit viral replication by blocking interaction of the gp120 envelope protein with the coreceptors. We considered the possibility that this interaction involves a motif of the gp120 that may be structurally homologous to the chemokines. In the amino acid sequences of most chemokines there is a Trp residue located at the beginning of the C-terminal α-helix, which is separated by six residues from the fourth Cys residue. The gp120 of all HIV-1 isolates have a similar motif, which includes the C-terminal part of a variable loop 3 (V3) and N-terminal part of a conserved region 3 (C3). Two synthetic peptides, derived from the relevant gp120 sequence inhibited HIV-1 replication in macrophages and T lymphocytes in sequence-dependent manner. The peptides also prevented binding of anti-CXCR4 antibodies to CXCR4, and inhibited the intracellular Ca(2+) influx in response to CXCL12/SDF-1α. Thus these peptides can be used to dissect gp120 interactions with chemokine receptors and could serve as leads for the design of new inhibitors of HIV-1.

Recommendations from the iSBTc-SITC/FDA/NCI Workshop on Immunotherapy Biomarkers.

PURPOSE: To facilitate development of innovative immunotherapy approaches, especially for treatment concepts exploiting the potential benefits of personalized therapy, there is a need to develop and validate tools to identify patients who can benefit from immunotherapy. Despite substantial effort, we do not yet know which parameters of antitumor immunity to measure and which assays are optimal for those measurements. EXPERIMENTAL DESIGN: The iSBTc-SITC (International Society for Biological Therapy of Cancer-Society for Immunotherapy of Cancer), FDA (Food and Drug Administration), and NCI (National Cancer Institute) partnered to address these issues for immunotherapy of cancer. Here, we review the major challenges, give examples of approaches and solutions, and present our recommendations. RESULTS AND CONCLUSIONS: Although specific immune parameters and assays are not yet validated, we recommend following standardized (accurate, precise, and reproducible) protocols and use of functional assays for the primary immunologic readouts of a trial; consideration of central laboratories for immune monitoring of large, multi-institutional trials; and standardized testing of several phenotypic and functional potential potency assays specific to any cellular product. When reporting results, the full QA (quality assessment)/QC (quality control) should be conducted and selected examples of truly representative raw data and assay performance characteristics should be included. Finally, to promote broader analysis of multiple aspects of immunity, and gather data on variability, we recommend that in addition to cells and serum, RNA and DNA samples be banked (under standardized conditions) for later testing. We also recommend that sufficient blood be drawn to allow for planned testing of the primary hypothesis being addressed in the trial, and that additional baseline and posttreatment blood is banked for testing novel hypotheses (or generating new hypotheses) that arise in the field.