Thomas R. Nirschl

Stereotactic Radiation Therapy Augments Antigen-Specific PD-1–Mediated Antitumor Immune Responses via Cross-Presentation of Tumor Antigen

Sharabi and colleagues show in two mouse tumor models that radiotherapy combined with PD-1 blockade or Treg depletion improves local tumor control by increasing antigen-experienced and effector-memory T cells, antigen-MHC complexes, and T-cell infiltration into tumors via antigen cross-presentation in the tumor-draining lymph node. The immune-modulating effects of radiotherapy (XRT) have gained considerable interest recently, and there have been multiple reports of synergy between XRT and immunotherapy. However, additional preclinical studies are needed to demonstrate the antigen-specific nature of radiation-induced immune responses and elucidate potential mechanisms of synergy with immunotherapy. Here, we demonstrate the ability of stereotactic XRT to induce endogenous antigen-specific immune responses when it is combined with anti–PD-1 checkpoint blockade immunotherapy. Using the small animal radiation research platform (SARRP), image-guided stereotactic XRT delivered to B16-OVA melanoma or 4T1-HA breast carcinoma tumors resulted in the development of antigen-specific T cell– and B cell–mediated immune responses. These immune-stimulating effects of XRT were significantly increased when XRT was combined with either anti–PD-1 therapy or regulatory T cell (Treg) depletion, resulting in improved local tumor control. Phenotypic analyses of antigen-specific CD8 T cells revealed that XRT increased the percentage of antigen-experienced T cells and effector memory T cells. Mechanistically, we found that XRT upregulates tumor-associated antigen–MHC complexes, enhances antigen cross-presentation in the draining lymph node, and increases T-cell infiltration into tumors. These findings demonstrate the ability of XRT to prime an endogenous antigen-specific immune response and provide an additional mechanistic rationale for combining radiation with PD-1 blockade in the clinic. Cancer Immunol Res; 3(4); 345–55. ©2014 AACR.

Sustained CD4+ T cell-driven lymphopenia without a compensatory IL-7/IL-15 response among high-grade glioma patients treated with radiation and temozolomide

Prolonged lymphopenia correlating with decreased survival commonly occurs among glioma patients undergoing radiation therapy (RT) and temozolomide (TMZ) treatment. To better understand the pathophysiology of this phenomenon, we prospectively monitored serum cytokine levels and lymphocyte subsets in 15 high-grade glioma patients undergoing combined radiation and TMZ (referred to as RT/TMZ) treatment. Sufficient data for analysis were acquired from 11 of the patients initially enrolled. Lymphocyte phenotyping data were obtained using cytofluorometric analysis and serum cytokine levels were measured using the a multiplex bead-based assays. Total lymphocyte counts (TLCs) were > 1000 cells per μL peripheral blood in 10/11 patients at baseline, but dropped significantly after treatment. Specifically, after RT/TMZ therapy, the TLCs were found to be < 500 cells/μL in 2/11 patients, 500–1000 cells/μL in 7/11 patients, and > 1000 cells/μL in the remaining 2 patients. Among residual mononuclear blood cells, we observed a proportional drop in B and CD4+ T cells but not in CD8+ T lymphocytes. Natural killer cells remained to near-to-baseline levels and there was a transient and slight (insignificant) increase in regulatory T cells (Tregs). The circulating levels of IL-7 and IL-15 remained low despite marked drops in both the total and CD4+ T lymphocyte counts. Thus, patients with malignant glioma undergoing RT/TMZ treatment exhibit a marked decline in TLCs, affecting both CD4+ T cells and B lymphocytes, in the absence of a compensatory increase in interleukin-7 levels. The failure to mount an appropriate homeostatic cytokine response may be responsible for the prolonged lymphopenia frequently observed in these patients.

Ipilimumab plus nivolumab and DNA-repair defects in AR-V7-expressing metastatic prostate cancer

AR-V7-expressing metastatic prostate cancer is an aggressive phenotype with poor progression-free survival (PFS) and overall survival (OS). Preliminary evidence suggests that AR-V7-positive tumors may be enriched for DNA-repair defects, perhaps rendering them more sensitive to immune-checkpoint blockade. We enrolled 15 metastatic prostate cancer patients with AR-V7-expressing circulating tumor cells into a prospective phase-2 trial. Patients received nivolumab 3 mg/kg plus ipilimumab 1 mg/kg every 3 weeks for four doses, then maintenance nivolumab 3 mg/kg every 2 weeks. Targeted next-generation sequencing was performed to determine DNA-repair deficiency (DRD) status. Outcomes included PSA response rates, objective response rates (ORR), PSA progression-free survival (PSA-PFS), clinical/radiographic PFS and OS. Median age of participants was 65, median PSA was 115 ng/mL, 67% had visceral metastases, and 60% had ≥4 prior systemic therapies. Six of 15 men (40%) had DRD mutations (three in BRCA2, two in ATM, one in ERCC4; none had microsatellite instability). Overall, the PSA response rate was 2/15 (13%), ORR was 2/8 (25%) in those with measurable disease, median PSA-PFS was 3.0 (95%CI 2.1–NR) months, PFS was 3.7 (95%CI 2.8–7.5) months, and OS was 8.2 (95%CI 5.5–10.4) months. Outcomes appeared generally better in DRD+ vs. DRD– tumors with respect to PSA responses (33% vs. 0%; P=0.14, nonsignificant), ORR (40% vs. 0%; P=0.46, nonsignificant), PSA-PFS (HR 0.19; P<0.01, significant), PFS (HR 0.31; P=0.01, significant), and OS (HR 0.41; P=0.11, nonsignificant). There were no new safety concerns. Ipilimumab plus nivolumab demonstrated encouraging efficacy in AR-V7-positive prostate cancers with DRD mutations, but not in the overall study population.

Stereotactic radiotherapy increases functionally suppressive regulatory T cells in the tumor microenvironment

Stereotactic irradiation of implanted murine tumors increased proliferation of regulatory T cells (Tregs) within tumors. These cells were functionally more suppressive than Tregs derived from unirradiated tumors, highlighting a potential counterbalance to the immunogenic effects of radiotherapy. Radiotherapy (RT) enhances innate and adaptive antitumor immunity; however, the effects of radiation on suppressive immune cells, such as regulatory T cells (Treg), in the tumor microenvironment (TME) are not fully elucidated. Although previous reports suggest an increased Treg infiltration after radiation, whether these Tregs are functionally suppressive remains undetermined. To test the hypothesis that RT enhances the suppressive function of Treg in the TME, we selectively irradiated implanted tumors using the small animal radiation research platform (SARRP), which models stereotactic radiotherapy in human patients. We then analyzed tumor-infiltrating lymphocytes (TIL) with flow-cytometry and functional assays. Our data showed that RT significantly increased tumor-infiltrating Tregs (TIL-Treg), which had higher expression of CTLA-4, 4-1BB, and Helios compared with Tregs in nonirradiated tumors. This observation held true across several tumor models (B16/F10, RENCA, and MC38). We found that TIL-Tregs from irradiated tumors had equal or improved suppressive capacity compared with nonirradiated tumors. Our data also indicated that after RT, Tregs proliferated more robustly than other T-cell subsets in the TME. In addition, after RT, expansion of Tregs occurred when T-cell migration was inhibited using Fingolimod, suggesting that the increased Treg frequency was likely due to preferential proliferation of intratumoral Treg after radiation. Our data also suggested that Treg expansion after irradiation was independent of TGFβ and IL33 signaling. These data demonstrate that RT increased phenotypically and functionally suppressive Tregs in the TME. Our results suggest that RT might be combined effectively with Treg-targeting agents to maximize antitumor efficacy. Cancer Immunol Res; 5(11); 992–1004. ©2017 AACR.

Systemic depletion of lymphocytes following focal radiation to the brain in a murine model

ABSTRACT Severe radiation-related lymphopenia is common and associated with decreased survival in patients with several solid tumors. As the mechanisms underlying systemic lymphopenia are poorly understood, we developed an animal model to study the effects of brain radiation on lymphocytes and cytokines. C57 BL/6 and BALB/c mice received focal brain irradiation (4 Gy x 10 fractions or 2 Gy x 30 fractions). Weekly total lymphocyte counts (TLC), lymphocyte subsets and cytokines in blood and lymph nodes were measured. Non-irradiated lymph nodes were collected and examined before, during, and after radiation. We found that systemic TLC decreased rapidly irrespective of mouse strain or radiation schedule. 4 Gy x 10 resulted in a 42% and 75% & 70% and 49% TLC reduction in C57 BL/6 and BALB/c mice respectively. 2 Gy x 30 caused a 70% / 49% decrease in TLC in C57 BL/6 and BALB/c. Similar trends were seen for total T cells, CD4+, regulatory T and CD8+ cells. Changes in lymph node architecture and cellular composition correlated with the development of systemic lymphopenia. Three weeks after radiation, TLC returned to 60–80% of baseline, preceded by increased IL-7 levels in the lymph nodes. Focal brain radiation in mice results in significant systemic lymphodepletion.

Elective Nodal Irradiation Attenuates the Combinatorial Efficacy of Stereotactic Radiation Therapy and Immunotherapy.

Purpose: In the proper context, radiotherapy can promote antitumor immunity. It is unknown if elective nodal irradiation (ENI), a strategy that irradiates tumor-associated draining lymph nodes (DLN), affects adaptive immune responses and combinatorial efficacy of radiotherapy with immune checkpoint blockade (ICB). Experimental Design: We developed a preclinical model to compare stereotactic radiotherapy (Tumor RT) with or without ENI to examine immunologic differences between radiotherapy techniques that spare or irradiate the DLN. Results: Tumor RT was associated with upregulation of an intratumoral T-cell chemoattractant chemokine signature (CXCR3, CCR5-related) that resulted in robust infiltration of antigen-specific CD8+ effector T cells as well as FoxP3+ regulatory T cells (Tregs). The addition of ENI attenuated chemokine expression, restrained immune infiltration, and adversely affected survival when combined with ICB, especially with anti-CLTA4 therapy. The combination of stereotactic radiotherapy and ICB led to long-term survival in a subset of mice and was associated with favorable CD8 effector-to-Treg ratios and increased intratumoral density of antigen-specific CD8+ T cells. Although radiotherapy technique (Tumor RT vs. ENI) affected initial tumor control and survival, the ability to reject tumor upon rechallenge was partially dependent upon the mechanism of action of ICB; as radiotherapy/anti-CTLA4 was superior to radiotherapy/anti-PD-1. Conclusions: Our results highlight that irradiation of the DLN restrains adaptive immune responses through altered chemokine expression and CD8+ T-cell trafficking. These data have implications for combining radiotherapy and ICB, long-term survival, and induction of immunologic memory. Clinically, the immunomodulatory effect of the radiotherapy strategy should be considered when combining stereotactic radiotherapy with immunotherapy. Clin Cancer Res; 24(20); 5058–71. ©2018 AACR.

MP65-06 INTRAVESICAL BCG INDUCES CD4+ T CELL EXPANSION BUT NOT ACTIVATION IN A CLINICALLY RELEVANT IMMUNE COMPETENT MODEL OF BLADDER CANCER

respect to the GSTT2B genotypes. The impact of BCG instillation (numbers) on recurrence was analyzed in a subset of patients for whom complete 10y follow-up data was available. Analysis was performed using SPSS 23.0 and p<0.05 was taken to be significant. RESULTS: GSTT2 was silenced in MGH cells (GSTT2B homozygous full length (GSTT2B)) and overexpressed in UMUC3 and U937 cells (GSTT2B homozygous deleted (GSTT2B)). A 2h exposure to BCG resulted in decreased ROS in GSTT2 silenced cells (p<0.05) and increased ROS in GSTT2 overexpressing cells (p<0.05). There was no difference in cellular cytotoxicity to BCG with respect to GSTT2 expression. However, intracellular BCG survival increased at 2 hours when GSTT2 was silenced (p<0.05) and decreased when GSTT2 was overexpressed (p<0.05). There was no significant difference between these groups at 24h. The majority of patients with complete 10y follow-up data, completed a 6+3 BCG schedule (n1⁄463) and n1⁄422 had less than 8 instillations. Patients with GSTT2B genotype (n1⁄46) who received 8 or less BCG instillations, were recurrence free (Likelihood ratio 1⁄4 0.040, p1⁄40.054). In the group that received at least 9 instillations of BCG, the GSTT2B was associated with earlier recurrence. CONCLUSIONS: GSTT2 expression decreases cellular ROS and BCG survival. GSTT2B was associated with lower likelihood of recurrence for patients who received 8 or less BCG instillations. In contrast patients with GSTT2B who received 9 or more instillations of BCG had earlier recurrences. Hence GSTT2B could be used as a marker for patients who will do well with less BCG therapy.

Combining intratumoral Treg depletion with androgen deprivation therapy (ADT): preclinical activity in the Myc-CaP model

BackgroundImmune checkpoint blockade has shown promising antitumor activity against a variety of tumor types. However, responses in castration-resistant prostate cancer remain relatively rare—potentially due to low baseline levels of infiltration. Using an immunocompetent cMyc-driven model (Myc-CaP), we sought to understand the immune infiltrate induced by androgen deprivation therapy (ADT) and to leverage that infiltration toward therapeutic benefit.MethodsUsing flow cytometry, qPCR and IHC, we quantified ADT-induced immune infiltration in terms of cell type and function. Preclinical treatment studies tested the combinatorial effects of ADT and immune checkpoint blockade using tumor outgrowth and overall survival as end points.ResultsADT induces a complex pro-inflammatory infiltrate. This pro-inflammatory infiltrate was apparent in the early postcastration period but diminished as castration resistance emerged. Combining ADT with tumor-infiltrating regulatory T cell (Treg) depletion using a depleting anti-CTLA-4 antibody significantly delayed the development of castration resistance and prolonged survival of a fraction of tumor-bearing mice. Immunotherapy as a monotherapy failed to provide a survival benefit and was ineffective if not administered in the peri-castration period.ConclusionsThe immune infiltrate induced by ADT is diverse and varies over time. Therapeutic strategies focusing on depleting Tregs in the peri-castration period are of particular interest.

Abstract PR03: Prophylactic nodal irradiation abrogates the synergy of tumor radiotherapy and immune checkpoint blockade

Background: Prophylactic nodal irradiation (PNI) is a strategy used to treat early stage cancers with the potential to enhance local control and prevent metastatic spread. It remains unclear whether irradiation of tumor-associated draining lymph nodes (LN) influences anti-tumor T cell responses. Our aim was to investigate the in vivo effects of nodal irradiation on the phenotype and function of tumor infiltrating lymphocytes (TIL) and the impact of PNI on the anti-tumor effects of immunotherapy. Methods: The Small Animal Radiation Research Platform (SARRP) delivered imaged-guided stereotactic radiation (RT) to tumor (T-only) or tumor and LN (T+LN). Syngeneic tumors (MC38 colon, B16 melanoma) were implanted in C57BL/6 mice and irradiated (12Gy x1) and/or treated with immune checkpoint blockade with αCTLA-4 IgG2a (depleting) or αPD-1 IgG1. The composition of the tumor microenvironment (TME) was assayed by flow cytometry. To query tumor-antigen specific T cell responses, adoptive transfer experiments were performed using OVA-specific CD8+ T-cells from Rag-/- OT-1 mice and OVA-MHC class I tetramer (SIINFEKL). Tumor lysate was also collected for chemokine analysis, and tumor outgrowth was quantified over time. Results: T-only RT resulted in a significant increase (P Conclusions: We have successfully developed a SARRP-based early stage cancer model with the ability to target or spare the tumor-associated LN. Results to date demonstrate significant immunological differences that are contingent upon inclusion/exclusion of the LN. Long-term survival experiments suggest response and survival advantages with T-only RT, particularly in combination with αCTLA-4 blockade. Taken together, these data suggest that PNI may dampen anti-tumor immune responses and that Tumor-only RT might be a better strategy in combination immunotherapy regimens, although these results should be explored in carefully designed clinical trials. This abstract is also being presented as Poster B41. Citation Format: Ariel E. Marciscano, Ali Ghasemzadeh, Thomas R. Nirschl, Brian J. Francica, Debebe Theodros, Esteban Velarde, J Wong, Daniel LJ Thorek, Theodore L. DeWeese, Charles G. Drake. Prophylactic nodal irradiation abrogates the synergy of tumor radiotherapy and immune checkpoint blockade. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2016 Oct 20-23; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2017;5(3 Suppl):Abstract nr PR03.

Abstract 1611: Intravesical BCG induces CD4 T Cell expansion in an immune competent model of bladder cancer

Intravesical BCG Immunotherapy is the standard of care in treating non-muscle invasive bladder cancer (NMIBC), yet its mechanism of action remains elusive. Both innate and adaptive immune responses have been implicated in BCG activity. While prior research has indirectly demonstrated the importance of T cells and shown a rise in CD4+ T cells in bladder tissue after BCG, T cell subpopulations have not been fully characterized. We investigated the relationship between effector and regulatory T cells in an immune competent, clinically relevant rodent model of bladder cancer. Fischer 344 rats aged 7 weeks received 1.5mg/kg N-Nitroso-N-methylurea (MNU) every other week for 6 weeks (4 doses). Bladder dysplasia began by week 8 and by week 16 the majority of rats had a NMIBC phenotype. Beginning week 8 following the first MNU dose, rats were intravesically administered 0.3ml of BCG (Tice), cisplatin (1mg/ml), Mitomycin C (2mg/ml), MMC+ BCG, or saline (n=10 for all groups) weekly for 6 total doses. Animals were sacrificed at week 16, and bladders were processed for histopathology and digested into single cell suspensions for flow cytometry. Whole transcriptome expression profiling was then performed on sorted CD4 and CD8 cells of post-BCG tumors vs untreated tumors to assess T cell differentiation after BCG. Our data demonstrate that cancer progression in the MNU rat model of bladder cancer is characterized by a decline in the CD8/FoxP3 ratio, consistent with decreased adaptive immunity. By contrast, treatment with intravesical BCG leads to a large, transient rise in the CD4+ T cell population in the urothelium, and is both more effective and immunogenic compared to intravesical chemotherapy. Interestingly, whole transcriptome expression profiling of post-treatment intravesical CD4+ and CD8+ T cells revealed minimal differences in gene expression after BCG treatment. Together, our results suggest that while BCG induces T cell recruitment to the bladder, the T cell phenotype does not markedly change, implying that combining T cell activating agents with BCG might improve clinical activity. Citation Format: Max Kates, Thomas Nirschl, Nikolai Sopko, Noah Hahn, David McConkey, Alex Baras, Charles Drake, Trintiy Bivalacqua. Intravesical BCG induces CD4 T Cell expansion in an immune competent model of bladder cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1611. doi:10.1158/1538-7445.AM2017-1611