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.
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).
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.
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.
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.
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Allergic reactions occur when IgE molecules become crosslinked by antigens such as food proteins. Here we create the 'AllerScan' programmable phage display system to characterize the binding ...specificities of anti-allergen IgG and IgE antibodies in serum against thousands of allergenic proteins from hundreds of organisms at peptide resolution. Using AllerScan, we identify robust anti-wheat IgE reactivities in wheat allergic individuals but not in wheat-sensitized individuals. Meanwhile, a key wheat epitope in alpha purothionin elicits dominant IgE responses among allergic patients, and frequent IgG responses among sensitized and non-allergic patients. A double-blind, placebo-controlled trial shows that alpha purothionin reactivity, among others, is strongly modulated by oral immunotherapy in tolerized individuals. AllerScan may thus serve as a high-throughput platform for unbiased analysis of anti-allergen antibody specificities.
BackgroundProstate cancer responds poorly to current immunotherapies. Epigenetic therapies such as BET Bromodomain inhibition can change the transcriptome of tumor cells, possibly making them more ...immunogenic and thus susceptible to immune targeting.MethodsWe characterized the effects of BET bromodomain inhibition using JQ1 on PD-L1 and HLA-ABC expression in two human prostate cell lines, DU145 and PC3. RNA-Seq was performed to assess changes on a genome-wide level. A cytotoxic T cell killing assay was performed in MC38-OVA cells treated with JQ1 to demonstrate increased immunogenicity. In vivo experiments in the Myc-Cap model were conducted to show the effects of JQ1 administration in concert with anti-CTLA-4 checkpoint blockade.ResultsHere, we show that targeting BET bromodomains using the small molecule inhibitor JQ1 decreased PD-L1 expression and mitigated tumor progression in prostate cancer models. Mechanistically, BET bromodomain inhibition increased MHC I expression and increased the immunogenicity of tumor cells. Transcriptional profiling showed that BET bromodomain inhibition regulates distinct networks of antigen processing and immune checkpoint molecules. In murine models, treatment with JQ1 was additive with anti-CTLA-4 immunotherapy, resulting in an increased CD8/Treg ratio.ConclusionsBET Bromodomain inhibition can mediate changes in expression at a genome wide level in prostate cancer cells, resulting in an increased susceptibility to CD8 T cell targeting. These data suggest that combining BET bromodomain inhibition with immune checkpoint blockade may have clinical activity in prostate cancer patients.
Previous studies suggest that androgen deprivation therapy (ADT) promotes antitumor immunity in prostate cancer. Whether a vaccine-based approach can augment this effect remains unknown.
We conducted ...a neoadjuvant, randomized study to quantify the immunologic effects of a GM-CSF-secreting allogeneic cellular vaccine in combination with low-dose cyclophosphamide (Cy/GVAX) followed by degarelix versus degarelix alone in patients with high-risk localized prostate adenocarcinoma who were planned for radical prostatectomy.
Both Cy/GVAX plus degarelix and degarelix alone led to significant increases in intratumoral CD8
T-cell infiltration and PD-L1 expression as compared with a cohort of untreated, matched controls. However, the CD8
T-cell infiltrate was accompanied by a proportional increase in regulatory T cells (Treg), suggesting that adaptive Treg resistance may dampen the immunogenicity of ADT. Although Cy/GVAX followed by degarelix was associated with a modest improvement in time-to-PSA progression and time-to-next treatment, as well as an increase in PD-L1, there was no difference in the CD8
T-cell infiltrate as compared with degarelix alone. Gene expression profiling demonstrated that
, a macrophage marker, was differentially upregulated with Cy/GVAX plus degarelix compared with degarelix alone.
Our results highlight that ADT with or without Cy/GVAX induces a complex immune response within the prostate tumor microenvironment. These data have important implications for combining ADT with immunotherapy. In particular, our finding that ADT increases both CD8
T cells and Tregs supports the development of regimens combining ADT with Treg-depleting agents in the treatment of prostate cancer.
Despite advancements in targeting the immune checkpoints program cell death protein 1 (PD-1), programmed death ligand 1 (PD-L1), and cytotoxic T lymphocyte-associated protein 4 (CTLA-4) for cancer ...immunotherapy, a large number of patients and cancer types remain unresponsive. Current immunotherapies focus on modulating an antitumor immune response by directly or indirectly expanding antitumor CD8 T cells. A complementary strategy might involve inhibition of Tregs that otherwise suppress antitumor immune responses. Here, we sought to identify functional immune molecules preferentially expressed on tumor-infiltrating Tregs. Using genome-wide RNA-Seq analysis of purified Tregs sorted from multiple human cancer types, we identified a conserved Treg immune checkpoint signature. Using immunocompetent murine tumor models, we found that antibody-mediated depletion of 4-1BB-expressing cells (4-1BB is also known as TNFRSF9 or CD137) decreased tumor growth without negatively affecting CD8 T cell function. Furthermore, we found that the immune checkpoint 4-1BB had a high selectivity for human tumor Tregs and was associated with worse survival outcomes in patients with multiple tumor types. Thus, antibody-mediated depletion of 4-1BB-expressing Tregs represents a strategy with potential activity across cancer types.
Novel perioperative strategies are needed to reduce recurrence rates in patients undergoing nephrectomy for high-risk, non-metastatic clear cell renal cell carcinoma (ccRCC). We conducted a ...prospective, phase I trial of neoadjuvant nivolumab prior to nephrectomy in 15 evaluable patients with non-metastatic ccRCC. We leveraged tissue from that cohort to elucidate the effects of PD-1 inhibition on immune cell populations in ccRCC and correlate the evolving immune milieu with anti-PD-1 response. We found that nivolumab durably induces a pro-inflammatory state within the primary tumor, and baseline immune infiltration within the primary tumor correlates with nivolumab responsiveness. Nivolumab increases CTLA-4 expression in the primary tumor, and subsequent nephrectomy increases circulating concentrations of sPD-L1, sPD-L3 (sB7-H3), and s4-1BB. These findings form the basis to consider neoadjuvant immune checkpoint inhibition (ICI) for high-risk ccRCC while the tumor remains in situ and provide the rationale for perioperative strategies of novel ICI combinations.
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.
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This study evaluated the expression of PD-L1 and markers of immune mediated resistance in human medulloblastoma (MB), the most common malignant pediatric brain tumor.
Overall levels of PD-L1 in human ...MB were low; however, some cases demonstrated robust focal expression associated with increased immune infiltrates. The case with highest PD-L1 expression was a sonic hedgehog (SHH) MB. In cell lines, SHH MB, which are low-MYC expressing, demonstrated both constitutive and inducible expression of PD-L1 while those in Group 3/4 that expressed high levels of MYC had only inducible expression.
, IFN-γ robustly stimulated the expression of PD-L1 in all cell lines while radiation induced variable expression. Forced high MYC expression did not significantly alter PD-L1.
Human MB tumor samples were evaluated for expression of PD-L1 and immune cell markers in relation to molecular subgroup assignment. PD-L1 expression was functionally analyzed under conditions of interferon gamma (IFN-γ), radiation, and MYC overexpression.
MB expresses low levels of PD-L1 facilitating immune escape. Importantly, T
1 cytokine stimulation appears to be the most potent inducer of PD-L1 expression
suggesting that an inflamed tumor microenvironment is necessary for PD-1 pathway activation in this tumor.
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