New Approaches for Monitoring CTL Activity in Clinical Trials

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Anatoli Malyguine, Susan Strobl, Liubov Zaritskaya, Michael Baseler, and Kimberly Shafer-Weaver

Applied and Developmental Research Support Program, SAIC-Frederick, Inc., NCI-Frederick, Frederick, MD, USA, [email protected]

Abstract. We have developed a modification of the ELISPOT assay that measures Gran-zyme 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-y 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 cytotoxic-ity 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-y 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-Y 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.

1. Immunological Assays for Monitoring Cancer Vaccine Trials

Active specific immunotherapy is a promising but investigational modality in the management of cancer patients. Currently, several cancer vaccine formulations such as peptides, proteins, antigen-pulsed dendritic cells, and whole tumor cells, in combination with various adjuvants and carriers are being evaluated in clinical trials (Wang and Rosenberg 1999; Rosenberg 2001; Kwak 2003). Monitoring T cell responses in the course of clinical trials is widely used to assess the efficacy of cancer immunotherapy. Selection of an ex vivo monitoring method that provides the best measure of immune reactivity is important in determining correlations between clinical and immunological responses to specific immunotherapy. Standard immunological assays, such as cytokine induction and cell proliferation, can detect immune responses in vaccinated patients but are not suitable for evaluation of individual effector cell reactivity. The chromium-release assay (51Cr-release) has long been the standard assay to measure natural killer (NK) and cytotoxic T lymphocyte (CTL) cytotoxicity; however, the traditional 51Cr-release assay provides only semiquantitative data unless limiting dilution assays are performed and has a relatively low level of sensitivity. The tetramer assay identifies the number of epitope-specific CTLs (Altman et al. 1996), but to quan-titate functional cells, it should be combined with intracellular cytokine staining. Assays that can monitor both CTL frequency and function, such as the IFN-y ELISPOT assay, have gained increasing popularity for monitoring clinical trials (Scheibenbogen et al. 1997 a, b; Keilholz et al. 2002). The ELISPOT assay, a modification of the ELISA, utilizes antibody-coated membranes to detect locally secreted cytokines or other immune proteins by individual cells. The release of immune proteins from activated cells results in spot formation. At appropriate cellular concentrations, each spot formed represents a single reactive cell. Thus, the ELISPOT assay provides both qualitative (type of immune protein) and quantitative (number of responding cells) information. The assay is both reliable and highly sensitive as its detection limit is 1/105 peripheral blood mononuclear cells (PBMCs). However, the IFN-y ELISPOT assay is not an exclusive measure of CTL activity as non-cytotoxic cells can also secrete IFN-y (Bachmann et al. 1999). Additionally, CTLs with lytic activity do not always secrete IFN-y. A more relevant approach to assess functional activity of CTLs would be to measure the secretion of molecules associated with lytic activity.

2. Granzyme B ELISPOT Assay as an Alternative to 51Cr Release

One of the major mechanisms of cell-mediated cytotoxicity involves exocytosis of preformed granules from the effector toward the target cell. These granules contain a number of cytotoxic proteins, including the pore-forming protein perforin and a family of serine proteases called granzymes, including Granzyme B (GrB). Gr B and perforin are present mainly in the granules of CD8+ CTL and NK cells and the release of these factors in response to the appropriate target may be used as a surrogate to evaluate cell-mediated cytotoxicity.

Unlike the IFN-y ELISPOT, which is widely utilized, the application of the GrB ELISPOT for monitoring clinical trials has been limited. The GrB ELISPOT assay was previously shown to measure GrB release by GrB-transfected CHO cells, T cell lines, and PBMCs from patients with AIDS (Rininsland et al. 2000; Kleen et al. 2004). Our laboratory assessed whether the GrB ELISPOT assay was a viable alternative to the Cr-release assay for measuring antigen-specific CTLs (Shafer-Weaver et al. 2003). We generated aFMP-CTL as a clinically relevant model system to assess whether the GrB ELISPOT assay can reliably detect effector cell responses to specific peptides. We found excellent correlation between GrB release in the ELISPOT assay and cytotoxicity in the 51Cr-release assay Figure 1.

To evaluate specificity of the assay, we tested CTL reactivity against FMP-pulsed C1R.A2 (specific targets) as well as nonpulsed and MART-1-pulsed C1R.A2 cells (nonspecific targets) in the GrB ELISPOT assay. K562 were utilized as a control for NK activity (data not shown). GrB secretion was antigen specific as only wells with CTLs and FMP-pulsed CIR.A2 contained a substantial number of spots Figure 2. To further confirm that we were measuring CTL activity, we removed CD8+ cells from the cultures using anti-CD8 mAbs and magnetic beads. After depletion, the percentage of CD8+ cells in the cultures was decreased from 24.8 ± 2.9% to 2.7 ± 0.4%. GrB secretion was assessed for both total and CD8+-depleted CTL cultures. With CD8+ cell depletion from the CTL cultures, there was almost complete abrogation of GrB measured in the ELISPOT. This demonstrates that CTLs are the main producers of GrB and confirm the specificity of the GrB ELISPOT assay.

Ctl Elispot

Figure 1. Granzyme B release in the ELISPOT assay correlates with cytotoxicity in the 51Cr-release assay. Human anti-FMP-CTLs (7-day culture) were used as effector cells. Target cells were C1R.A2 pulsed with FMP. Closed squares, GrB ELISPOT; open squares, 51Cr-release assay. Representative experiment is shown.

Figure 1. Granzyme B release in the ELISPOT assay correlates with cytotoxicity in the 51Cr-release assay. Human anti-FMP-CTLs (7-day culture) were used as effector cells. Target cells were C1R.A2 pulsed with FMP. Closed squares, GrB ELISPOT; open squares, 51Cr-release assay. Representative experiment is shown.

Release Assay

Figure 2. Specificity of Granzyme B (GrB) secretion by aFMP-CTL in the ELISPOT assay. Human aFMP-CTL (7-day culture, 5 x 103 cells/well) were run alone or against various target cells (5 x 104 cells/well): C1R.A2, C1R.A2 pulsed with 5 ^g/ml FMP or C1R.A2 pulsed with 3 |M MART-1. Effector and target cells were incubated for 4 h at 37°C. Image from the plate scan generated by the CTL Analyzer is shown.

Figure 2. Specificity of Granzyme B (GrB) secretion by aFMP-CTL in the ELISPOT assay. Human aFMP-CTL (7-day culture, 5 x 103 cells/well) were run alone or against various target cells (5 x 104 cells/well): C1R.A2, C1R.A2 pulsed with 5 ^g/ml FMP or C1R.A2 pulsed with 3 |M MART-1. Effector and target cells were incubated for 4 h at 37°C. Image from the plate scan generated by the CTL Analyzer is shown.

3. Advantages of the ELISPOT Method

Although the assays correlated, there are numerous advantages in utilizing the ELISPOT assays over the standard 51Cr-release assay. The ELISPOT assays use a lower number of effector cells to accurately assess activity. The high sensitivity and specificity of the ELISPOT assays are beneficial for monitoring clinical trials where frequently there are limited numbers of patients' cells available. The ELISPOT assays also enumerate antigen - specific lymphocyte frequency by measuring secretion of a specific immune protein. Additionally, problems associated with the labeling efficiency of targets are not a concern with the ELISPOT assays. Therefore, the GrB assay is a superior alternative to the 51Cr-release assay to assess a CTL response. Moreover, unlike the IFN-y ELISPOT assay, the GrB ELISPOT directly measures the release of a cytolytic protein. Detection of low-frequency tumor-specific CTLs and their specific effector functions can provide a valuable insight in to immunologi-cal responses.

4. Application of the Gr B ELISPOT Assay for Monitoring Clinical Samples

4.1. CTL Reactivity to Cancer Vaccine Components

We further investigated whether the GrB ELISPOT assay can be applied to monitor the frequency and activity of CTL in PBMCs from patients with cancer (Shafer-Weaver et al. 2006). The clinical efficacy of cancer vaccines is likely to depend on several factors including the specificity, functional quality, and the magnitude of the induced antitumor T cell response. Therefore, we compared peptide-stimulated reactivity of PMBCs from vaccinated cancer patients in the GrB and IFN-y ELISPOT assays as well as in the tetramer and 51Cr-release assays. PBMCs from melanoma patients vaccinated with an HLA-A2*0201 binding peptide from the gp100 protein (gp100:209-2M) were utilized.

Four distinct response patterns were observed among the sixteen patients tested Figure 3. Five of the 16 patients were unresponsive in all four assays. Eleven responsive patients could be further categorized. Four were positive in all four assays, three were positive in the tetramer, IFN-y, and GrB ELISPOT assays, and four were positive in only the tetramer and the IFN-y assays. These data patterns demonstrate that vaccination can elicit differences in immune responses among patients. Correlations between the tetramer, IFN-y GrB ELISPOT, and the 51Cr-release assays differed. The IFN-y and tetramer assays perfectly correlated (Phi Coefficient = 1.00, p < 0.0001), whereas the GrB ELISPOT assay was significantly associated with all three of the other assays (p values of 0.015, 0.015, and 0.0059 with tetramer, IFN-y ELISPOT, and 51Cr-release assays, respectively). Moreover, the 51Cr-release assay significantly correlated only with the GrB ELISPOT. Although a limited number of patients were analyzed and direct comparisons were not made to clinical outcomes, the particular vaccination schema did not seem to correlate to discrete immune responses. Differences in the resulting vaccination-induced immune responses most likely are due to patient variation. Regardless, it may be speculated that vaccination is inducing the generation of different populations of effector cells or that the same effector cells are undergoing differentiation over time due to vaccination. Preliminary findings utilizing in vitro stimulated CTLs in a simultaneous GrB and IFN-y dual-color ELISPOT approach show distinct populations of IFN-y-secreting, GrB-secreting, or dual-secreting (both IFN-y and GrB) populations in response to relevant peptide stimulation (unpublished data).

Previous research has demonstrated shifts in T cell phenotype from naïve to an activated/effector phenotype after continued vaccination. Tetramer-positive CD8+ T cells from melanoma patients vaccinated with g209 peptide maintain an effector memory phenotype after 1 year postvaccination (Powell and Rosenberg 2004). However, only CCR7CD45RACD45RO+ tumor antigen-specific T cells produced IFN-y and lysed tumor cells (Dunbar et al. 2000; Valmori et al. 2002). Although pheno-typic analysis was not performed in our study, the functional data suggest that vaccination induced the generation of effector memory cells in some but not all patients. Significantly more antigen-specific T cells produced IFN-y than GrB,

Blood Cell Phenotype

Figure 3. Granzyme B and IFN-y release in the ELISPOT assays by peripheral blood mononuclear cells (PBMCs) from gp100:209-2M-vaccinated melanoma patients, cytotoxicity in the 51Cr-release assay and tetramer data (Collaboration with Dr. Rosenberg). Patient PBMCs obtained before and 3 weeks after each vaccination (Post 1-4). Tetramer data were obtained by Surgery Branch (NCI) and shown as the number of tetramer-positive cells per 104 CD8bright T cell. IFN-y and GrB ELISPOT values are average number of IFN-y - or GrB-secreting cells per 105 effector cells. The 51Cr-release assay data are presented as percent specific lysis at E:T ratio of 50:1. The data presented are a representative patient for each pattern of response category. Closed squares, tetramer assay; open squares, IFN-y ELISPOT assay; closed triangles, GrB ELISPOT assay; open triangles, 51Cr-release assay.

Figure 3. Granzyme B and IFN-y release in the ELISPOT assays by peripheral blood mononuclear cells (PBMCs) from gp100:209-2M-vaccinated melanoma patients, cytotoxicity in the 51Cr-release assay and tetramer data (Collaboration with Dr. Rosenberg). Patient PBMCs obtained before and 3 weeks after each vaccination (Post 1-4). Tetramer data were obtained by Surgery Branch (NCI) and shown as the number of tetramer-positive cells per 104 CD8bright T cell. IFN-y and GrB ELISPOT values are average number of IFN-y - or GrB-secreting cells per 105 effector cells. The 51Cr-release assay data are presented as percent specific lysis at E:T ratio of 50:1. The data presented are a representative patient for each pattern of response category. Closed squares, tetramer assay; open squares, IFN-y ELISPOT assay; closed triangles, GrB ELISPOT assay; open triangles, 51Cr-release assay.

suggesting that in addition to the magnitude of the immune response, the type of of immune response generated to peptide vaccination may also be important.

4.2. CTL Reactivity to Primary Tumors

The ELISPOT assay has been primarily used for the detection of T cell responses against vaccine components by using peptide- or protein-pulsed antigen-presenting cells as surrogate T cell targets. However, demonstration of reactivity to vaccine components does not necessarily equate to recognition and elimination of tumor cells. 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). We tested antigen-specific CTL responses against autologous primary tumor cells in vaccinated cancer patients. We utilized PBMCs directly isolated from follicular lymphoma patients vaccinated with tumor-derived idiotype (Id) protein as a model system because Id vaccination has been

Pre Post

Figure 4. Granzyme B (GrB) secretion in ELISPOT assay by patients' peripheral blood mononuclear cells (PBMCs) vaccinated with Id and stimulated in vitro with CD40L-activated autologous follicular lymphoma cells. Pre- and postvaccine PBMC samples (1 x 105 cells/well) were cocultured with either autologous sCD40Lt-activated follicular lymphoma tumor cells or activated normal B cells (2 x 105 cells/well) for 24 h (GrB) or 48 h (IFN-y) ELISPOT assays. Black bars, response to normal autologous B cells; gray bars, response to tumor cells. Data are presented as spots per 1 x 105 PBMCs and is representative of three separate experiments.

shown to induce tumor-specific T cell responses, including IFN-y secretion in the ELISPOT assay (Malyguine et al. 2004) in these patients.

In this study, PBMCs isolated prior to vaccination and at subsequent time points throughout the vaccination schema were stimulated with patient tumor cells (relevant) or normal B cells (irrelevant) in the GrB assay. Our findings demonstrated that the GrB ELISPOT assay can be successfully applied to enumerate CTLs that can recognize and mount an immune response to tumor cells. PBMCs from several cancer patients vaccinated with Id secreted significant amounts of GrB when stimulated with autologous tumor cells but not normal B cells (Figure 4). Direct cyotoxicity was not assessed for these samples because primary tumor cells cannot be labeled efficiently with 51Cr. Therefore, the GrB ELISPOT is a viable alternative to measure CTL cytotoxic ability against primary tumors.

4.3. Additional Applications of GrB ELISPOT

The role of GrB in immune surveillance and rejection of tumors is still controversial. Rejection of spontaneous or experimental tumors by CTLs in mice was recently shown to be independent of GrB secretion (Smyth et al. 2000, 2003). These findings are in contrast to numerous studies in which GrB contributes to controlling tumors in vivo (Sayers et al. 1992; Motyka et al. 2000; Davis et al. 2001; Medema et al. 2001; Smyth et al. 2001). Regardless of the role of GrB in cell-mediated killing, GrB expression is mainly restricted to cytolytic cells, and therefore, the release of GrB is a more specific measure of CTL than IFN-y.

Currently, limited studies have utilized GrB ELISPOT assay to measure immune responses in cancer patients. In one study, CD4+ T lymphocytes from one melanoma patient vaccinated with MHC II-restricted MART-1 peptide secreted GrB in the ELISPOT assay after ex vivo stimulation (Wong et al. 2004). In another study, CD8+ T lymphocytes from two of five nonvaccinated hepatocellular carcinoma patients produced GrB against the NY-ESO-1b peptide (Shang, et al. 2004). Additionally, immunization of patients with acute myeloid leukemia led to increased GrB secretion in the ELISPOT assay but only after patients' PBMCs were prestimulated for 8 days with specific peptide or Id (Hasenkamp et al. 2006).

Our study utilized the GrB ELISPOT assay to evaluate the response of PBMCs (no preactivation) from cancer patients vaccinated with an MHC I-restricted peptide. We demonstrated that antigen-specific CTLs can be enumerated directly from peripheral blood samples without further in vitro manipulation including cell enrichment, expansion, or prolonged stimulation. This is an important finding because in vitro stimulation can hinder the accuracy of monitoring the frequency, phenotype and functions of T cells elicited to vaccination in vivo (Faure et al. 1998; thor Straten et al. 2000). Therefore, the GrB ELISPOT can directly assess vaccine-specific T cell frequency in vivo. Additionally, the 51Cr-release assay significantly correlated only with the GrB ELISPOT, demonstrating that the GrB ELISPOT is a viable alternative for clinical monitoring of T cell cytolytic ability.

5. Development of a Perforin ELISPOT Assay

As mentioned earlier, one of the major mechanisms of cell-mediated cytotoxicity involves exocytosis of cytoplasmic granules from the effector toward the target cell. The granules contain, among others, the pore-forming protein perforin that is present mainly in the granules of CD8+ CTL and NK cells (Trinchieri and Perussia 1984; Masopust et al. 2001), and therefore, similar to GrB, the release of this factor in response to the appropriate target may also be used to evaluate cell-mediated cyto-toxicity by specific antitumor CTLs generated by vaccination. Since the release of perforin facilitates GrB entry into the target cell and its subsequent cytoxic effector functions (Froelich et al. 1996; Jans et al. 1996; Shi et al. 1997; Smyth et al. 2001; Trapani and Smyth 2002), perforin may be a better measure of overall cytolytic capability. Currently, the perforin ELISPOT assay has only been used to evaluate anti-viral immunity (Zuber et al. 2005; Burton et al. 2006). We compared peptide-stimulated reactivity of PMBCs from vaccinated cancer patients in the Perforin and GrB ELISPOT assays. PBMCs from melanoma patients vaccinated with an HLA-A2*0201 binding peptide from the gp100 protein (gp100:209-2M) were utilized as effectors. Our preliminary data show that perforin release by PBMCs from vaccinated melanoma patients correlated with GrB release Figure 5. Further comparison of these assays using more clinical samples may help us understand their relative values for immunological monitoring.

Pre Post Post 2

Vaccination course

Figure 5. Secretion of perforin and Granzyme B in the ELISPOT assays by PBMCs from gp100:209-2M vaccinated melanoma patients. Patient PBMC obtained before (Pre) and 3 weeks after each vaccination (Post 1-2). ELISPOT values are average number of perforin- or GrB-secreting cells per 1 x 105 effector cells. Representative experiment shown.

Pre Post Post 2

Vaccination course

Figure 5. Secretion of perforin and Granzyme B in the ELISPOT assays by PBMCs from gp100:209-2M vaccinated melanoma patients. Patient PBMC obtained before (Pre) and 3 weeks after each vaccination (Post 1-2). ELISPOT values are average number of perforin- or GrB-secreting cells per 1 x 105 effector cells. Representative experiment shown.

6. Conclusions

It is important to emphasize that the 51Cr-release assay and the ELISPOT assays measure different aspects of cell-mediated killing—target cell death and effector cell function, respectively. Therefore, these assays do not replace each other but could be used in concert. We believe even though the GrB and perforin ELISPOT assays indirectly measure cytolytic activity, they are still preferential for clinical monitoring because they provide both quantitative and qualitative information, are more sensitive than 51Cr-release assay, utilize lower numbers of cells, and do not require target cell labeling that could be problematic in the clinical setting.

The particular immunological responses following vaccination needed for an effective response against cancer have not been fully elucidated. Detailed knowledge of specific immune responses that correlate with positive clinical outcomes will help identify better strategies to effectively activate the immune system against tumors. Taken together with published data, our results show that the simultaneous use of the ELISPOT assay with other immunological assays may provide additional immunological insight into patient responses to cancer vaccines.

Acknowledgments

The authors would like to thank Dr. Steven Rosenberg (NIH) and Dr. Larry Kwak (NIH) for providing clinical samples. This project has been funded in whole or in part with federal funds from the National Cancer Institute, National Institutes of

Health, under contract N01-CO-12400. The content of this publication does not necessarily reflect the views or policies of the Department of Health and Human Services, nor does it mention of trade names, commercial products, or organizations imply endorsement by the U.S. Government.

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10 Ways To Fight Off Cancer

10 Ways To Fight Off Cancer

Learning About 10 Ways Fight Off Cancer Can Have Amazing Benefits For Your Life The Best Tips On How To Keep This Killer At Bay Discovering that you or a loved one has cancer can be utterly terrifying. All the same, once you comprehend the causes of cancer and learn how to reverse those causes, you or your loved one may have more than a fighting chance of beating out cancer.

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