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Supplementary Figure S1. B cells in human PDAC. Supplementary Figure S2. B cells and FcRgamma-positive cells regulate murine PDAC. Supplementary Figure S3. Leukocytes and BTK in human and murine PDAC. Supplementary Figure S4. Macrophage PI3Kgamma activates BTK to promote PDAC progression. Supplementary Figure S5. BTK and PI3Kgamma-inhibition decrease PDAC growth. Supplementary Figure S6. BTK inhibition improves gemcitabine response in late-stage PDAC.

Supplementary Table 1: Primers used for genotyping and qRT-PCR. Supplementary Figure 1: Establishment of murine models of targeted H-RasV12G to basal and suprabasal layers of the epidermis. Supplementary Figure 2: Leukocyte infiltration into K14Ras and InvRas tumors correlates with tumor number and basal cell proliferation. Supplementary Figure 3: Cytokine expression and MDSC suppression assays display distinct immunosuppressive phenotypes in K14Ras mice. Supplementary Figure 4: CD8 or CD8 + B Cell transfers do not restore Rag1+/+ tumor counts or B cell phenotypes. Supplementary Figure 5: Adoptive co-transfer of CD4 T Cells and B cells selectively restores Rag1+/+ cytokine expression. Supplementary Figure 6: Dendritic Cell activity in hyperplastic skin or SDLNs does not significantly contribute to the overall Ras-induced immune response. Supplementary Figure 7: Adoptively transferred B cells migrate directly to the tumor microenvironment following Ras activation.

Heterogeneity in tumor immune responses is a poorly understood yet critical parameter for successful immunotherapy. In two doxycycline-inducible models where oncogenic H-RasG12V is targeted either to the epidermal basal/stem cell layer with a Keratin14-rtTA transgene (K14Ras), or committed progenitor/suprabasal cells with an Involucrin-tTA transgene (InvRas), we observed strikingly distinct tumor immune responses. On threshold doxycycline levels yielding similar Ras expression, tumor latency, and numbers, tumors from K14Ras mice had an immunosuppressed microenvironment, whereas InvRas tumors had a proinflammatory microenvironment. On a Rag1−/− background, InvRas mice developed fewer and smaller tumors that regressed over time, whereas K14Ras mice developed more tumors with shorter latency than Rag1+/+ controls. Adoptive transfer and depletion studies revealed that B-cell and CD4 T-cell cooperation was critical for tumor yield, lymphocyte polarization, and tumor immune phenotype in Rag1+/+ mice of both models. Coculture of tumor-conditioned B cells with CD4 T cells implicated direct contact for Th1 and regulatory T cell (Treg) polarization, and CD40-CD40L for Th1, Th2, and Treg generation, a response not observed from splenic B cells. Anti-CD40L caused regression of InvRas tumors but enhanced growth in K14Ras, whereas a CD40 agonist mAb had opposite effects in each tumor model. These data show that position of tumor-initiating cells within a stratified squamous epithelial tissue provokes distinct B- and CD4 T-cell interactions, which establish unique tumor microenvironments that regulate tumor development and response to immunotherapy. Cancer Immunol Res; 5(3); 198–210. ©2017 AACR.

TGF-β is an immunosuppressive cytokine that is upregulated in colorectal cancer. TGF-β blockade improved response to chemoradiotherapy in preclinical models of colorectal adenocarcinoma. We aimed to test the hypothesis that adding the TGF-β type I receptor kinase inhibitor galunisertib to neoadjuvant chemoradiotherapy would improve pathological complete response rates in patients with locally advanced rectal cancer.This was an investigator-initiated, single-arm, phase 2 study done in two medical centres in Portland (OR, USA). Eligible patients had previously untreated, locally advanced, rectal adenocarcinoma, stage IIA-IIIC or IV as per the American Joint Committee on Cancer; Eastern Cooperative Oncology Group status 0-2; and were aged 18 years or older. Participants completed two 14-day courses of oral galunisertib 150 mg twice daily, before and during fluorouracil-based chemoradiotherapy (intravenous fluorouracil 225 mg/mBetween Oct 19, 2016, and Aug 31, 2020, 38 participants were enrolled. 25 (71%) of the 35 patients who completed chemoradiotherapy proceeded to total mesorectal excision surgery, five (20%) of whom had pathological complete responses. Ten (29%) patients had non-operative management, three (30%) of whom ultimately chose to have total mesorectal excision. Two (67%) of those three patients had pathological complete responses. Of the remaining seven patients in the non-operative management group, five (71%) had clinical complete responses at 1 year after their last modified FOLFOX6 infusion. In total, 12 (32% [one-sided 95% CI ≥19%]) of 38 patients had a complete response. Common grade 3 adverse events during treatment included diarrhoea in six (16%) of 38 patients, and haematological toxicity in seven (18%) patients. Two (5%) patients had grade 4 adverse events, one related to chemoradiotherapy-induced diarrhoea and dehydration, and the other an intraoperative ischaemic event. No treatment-related deaths occurred.The addition of galunisertib to neoadjuvant chemoradiotherapy in patients with locally advanced rectal cancer improved the complete response rate to 32%, was well tolerated, and warrants further assessment in randomised trials.Eli Lilly via ExIST program, The Providence Foundation.

3617 Background: Transforming growth factor beta (TGFβ) is an immunosuppressive cytokine upregulated in colorectal cancer. Preclinical data demonstrated improved response to chemoradiation with TGFβ blockade in colorectal adenocarcinoma. Here we report the results of our single arm Phase II study combining the TGFβ type I receptor kinase inhibitor, galunisertib, with neoadjuvant chemoradiation in patients with locally advanced rectal adenocarcinoma. Methods: Eligible patients had T3+ or N+ rectal adenocarcinoma planned for surgical resection. Enrolled patients completed a 14-day course of galunisertib, followed by chemoradiation with continuous infusion 5-fluorouracil or capecitabine with radiation to 50.4-54Gy in 28-30 fractions. On day 30, patients underwent another 14-day course of galunisertib concurrent with ongoing chemoradiation. Five to nine weeks after completing neoadjuvant therapy, patients underwent response assessment. Those with complete response by physical exam, proctoscopy, and MRI, could opt for non-operative management and proceed to mFOLFOX6. Those with less than a complete response underwent surgical resection. The primary endpoint was complete response rate (CR), which was a composite of pathologic complete responses in those patients who proceeded to surgery, and clinical complete responses maintained at 1 year after completion of therapy for those who chose non-operative management. Using a Simon Two-Stage approach with 90% power and α = 0.05, to detect a 20% improvement in complete response rate compared to historical control (15% vs 35%); we reject the null hypothesis if ≥10/38 patients have a CR. 38 patients were enrolled with 35 patients evaluable. Results: Median age was 51y, 68% of patients were male, 87% of patients were Stage III, 97% of patients were node-positive, and 97% were proficient in mismatch repair. 95% of patients completed study therapy. Toxicity attributed to galunisertib was Grade 1-2. 28 patients went to surgery, with a mean neoadjuvant rectal (NAR) score (NAR = 5pN-3(cT-pT)+12]2/9.61) of 11.29, and pCR in 7 patients. 7 patients underwent non-operative management, with 5 achieving cCR at 1 year. Therefore, complete responses were observed in 12 patients. Peripheral immune monitoring revealed percent change in CD3+CD4-CXCR3+ T cells and activated CD8EM T cells correlated with response to therapy. 2y PFS and OS were 81.5% and 97%. Conclusions: The addition of TGFβ inhibition to neoadjuvant chemoradiation in locally advanced rectal cancer markedly improved response to therapy, was well tolerated, and warrants further investigation. Clinical trial information: NCT02688712.

Pancreatic ductal adenocarcinoma (PDAC) has traditionally been thought of as an immunologically quiescent tumor type presumably because of a relatively low tumor mutational burden (TMB) and poor responses to checkpoint blockade therapy. However, many PDAC tumors exhibit T cell inflamed phenotypes. The presence of tertiary lymphoid structures (TLS) has recently been shown to be predictive of checkpoint blockade response in melanomas and sarcomas, and are prognostic for survival in PDAC. In order to more comprehensively understand tumor immunity in PDAC patients with TLS, we performed RNA-seq, single and multiplex IHC, flow cytometry and predictive genomic analysis on treatment naïve, PDAC surgical specimens. Forty-six percent of tumors contained distinct T and B cell aggregates reflective of “early-stage TLS” (ES-TLS), which correlated with longer overall and progression-free survival. These tumors had greater CD8+ T cell infiltration but were not defined by previously published TLS gene-expression signatures. ES-TLS+ tumors were enriched for IgG1 class-switched memory B cells and memory CD4+ T cells, suggesting durable immunological memory persisted in these patients. We also observed the presence of active germinal centers (mature-TLS) in 31% of tumors with lymphocyte clusters, whose patients had long-term survival (median 56 months). M-TLS-positive tumors had equivalent overall T cell infiltration to ES-TLS, but were enriched for activated CD4+ memory cells, naive B cells and NK cells. Finally, using a TCGA-PDAC dataset, ES-TLS+ tumors harbored a decreased TMB, but M-TLS with germinal centers expressed significantly more MHCI-restricted neoantigens as determined by an in silico neoantigen prediction method. Interestingly, M-TLS+ tumors also had evidence of increased rates of B cell somatic hypermutation, suggesting that germinal centers form in the presence of high-quality tumor neoantigens leading to increased humoral immunity that confers improved survival for PDAC patients. AbbreviationsTLS: tertiary lymphoid structures; GC: germinal center(s); PDAC: pancreatic ductal adenocarcinoma; RNA-seq: RNA sequencing; BCRseq: B cell receptor sequencing; HEV: high endothelial venule; PNAd: peripheral node addressin; TMB: tumor mutational burden; TCGA: the cancer genome atlas; PAAD: pancreatic adenocarcinoma; FFPE: formalin fixed paraffin embedded; TIME: tumor immune microenvironment.

Transforming growth factor beta (TGFβ) is a multipotent immunosuppressive cytokine. TGFβ excludes immune cells from tumors, and TGFβ inhibition improves the efficacy of cytotoxic and immune therapies. Using preclinical colorectal cancer models in cell type-conditional TGFβ receptor I (ALK5) knockout mice, we interrogate this mechanism. Tumor growth delay and radiation response are unchanged in animals with Treg or macrophage-specific ALK5 deletion. However, CD8αCre-ALK5flox/flox (ALK5ΔCD8) mice reject tumors in high proportions, dependent on CD8+ T cells. ALK5ΔCD8 mice have more tumor-infiltrating effector CD8+ T cells, with more cytotoxic capacity. ALK5-deficient CD8+ T cells exhibit increased CXCR3 expression and enhanced migration towards CXCL10. TGFβ reduces CXCR3 expression, and increases binding of Smad2 to the CXCR3 promoter. In vivo CXCR3 blockade partially abrogates the survival advantage of an ALK5ΔCD8 host. These data demonstrate a mechanism of TGFβ immunosuppression through inhibition of CXCR3 in CD8+ T cells, thereby limiting their trafficking into tumors., TGFβ has a role in cancer immunosuppression but the exact mechanisms haven’t been fully elucidated. Here, using mouse models deficient in TGFβ-signaling, the authors show that loss of ALK5 in CD8 + T cells enhances their tumour trafficking and cytotoxicity suggesting that ALK5 inhibitors may have clinical utility.

Pancreatic adenocarcinoma is characterized by a complex tumor environment with a wide diversity of infiltrating stromal and immune cell types that impact the tumor response to conventional treatments. However, even in this poorly responsive tumor the extent of T cell infiltration as determined by quantitative immunohistology is a candidate prognostic factor for patient outcome. As such, even more comprehensive immunophenotyping of the tumor environment, such as immune cell type deconvolution via inference models based on gene expression profiling, holds significant promise. We hypothesized that RNA-Seq can provide a comprehensive alternative to quantitative immunohistology for immunophenotyping pancreatic cancer. We performed RNA-Seq on a prospective cohort of pancreatic tumor specimens and compared multiple approaches for gene expression-based immunophenotyping analysis compared to quantitative immunohistology. Our analyses demonstrated that while gene expression analyses provide additional information on the complexity of the tumor immune environment, they are limited in sensitivity by the low overall immune infiltrate in pancreatic cancer. As an alternative approach, we identified a set of genes that were enriched in highly T cell infiltrated pancreatic tumors, and demonstrate that these can identify patients with improved outcome in a reference population. These data demonstrate that the poor immune infiltrate in pancreatic cancer can present problems for analyses that use gene expression-based tools; however, there remains enormous potential in using these approaches to understand the relationships between diverse patterns of infiltrating cells and their impact on patient treatment outcomes.

Heterogeneity in tumor immune responses is a poorly understood yet critical parameter for successful immunotherapy. In two doxycycline-inducible models where oncogenic H-RasG12V is targeted either to the epidermal basal/stem cell layer with a Keratin14-rtTA transgene (K14Ras), or committed progenitor/suprabasal cells with an Involucrin-tTA transgene (InvRas), we observed strikingly distinct tumor immune responses. On threshold doxycycline levels yielding similar Ras expression, tumor latency, and numbers, tumors from K14Ras mice had an immunosuppressed microenvironment, whereas InvRas tumors had a proinflammatory microenvironment. On a Rag1−/− background, InvRas mice developed fewer and smaller tumors that regressed over time, whereas K14Ras mice developed more tumors with shorter latency than Rag1+/+ controls. Adoptive transfer and depletion studies revealed that B-cell and CD4 T-cell cooperation was critical for tumor yield, lymphocyte polarization, and tumor immune phenotype in Rag1+/+ mice of both models. Coculture of tumor-conditioned B cells with CD4 T cells implicated direct contact for Th1 and regulatory T cell (Treg) polarization, and CD40-CD40L for Th1, Th2, and Treg generation, a response not observed from splenic B cells. Anti-CD40L caused regression of InvRas tumors but enhanced growth in K14Ras, whereas a CD40 agonist mAb had opposite effects in each tumor model. These data show that position of tumor-initiating cells within a stratified squamous epithelial tissue provokes distinct B- and CD4 T-cell interactions, which establish unique tumor microenvironments that regulate tumor development and response to immunotherapy. Cancer Immunol Res; 5(3); 198–210. ©2017 AACR.

Pancreatic ductal adenocarcinoma (PDAC) is characterized by a fibrotic stroma with a poor lymphocyte infiltrate, in part driven by cancer-associated fibroblasts (CAFs). CAFs, which express fibroblast activation protein (FAP), contribute to immune escape via exclusion of anti-tumor CD8(+) T cells from cancer cells, upregulation of immune checkpoint ligand expression, immunosuppressive cytokine production, and polarization of tumor infiltrating inflammatory cells. FAP is a post-proline peptidase selectively expressed during tissue remodeling and repair, such as with wound healing, and in the tumor microenvironment by cancer-associated fibroblasts. We targeted FAP function using a novel small molecule inhibitor, UAMC-1110, and mice with germline knockout of FAP and concomitant knock-in of E. coli beta-galactosidase. We depleted CAFs by adoptive transfer of anti-beta gal T cells into the FAP knockout animals. Established syngeneic pancreatic tumors in immune competent mice were targeted with these 3 strategies, followed by focal radiotherapy to the tumor. FAP loss was associated with improved antigen-specific tumor T cell infiltrate and enhanced collagen deposition. However, FAP targeting alone or with tumor-directed radiation did not improve survival even when combined with anti-PD1 therapy. Targeting of CAFs alone or in combination with radiation did not improve survival. We conclude that targeting FAP and CAFs in combination with radiation is capable of enhancing anti-tumor T cell infiltrate and function, but does not result in sufficient tumor clearance to extend survival.

Pancreas ductal adenocarcinoma (PDAC) has one of the worst 5-year survival rates of all solid tumors, and thus new treatment strategies are urgently needed. Here, we report that targeting Bruton tyrosine kinase (BTK), a key B-cell and macrophage kinase, restores T cell–dependent antitumor immune responses, thereby inhibiting PDAC growth and improving responsiveness to standard-of-care chemotherapy. We report that PDAC tumor growth depends on cross-talk between B cells and FcRγ+ tumor–associated macrophages, resulting in TH2-type macrophage programming via BTK activation in a PI3Kγ-dependent manner. Treatment of PDAC-bearing mice with the BTK inhibitor PCI32765 (ibrutinib) or by PI3Kγ inhibition reprogrammed macrophages toward a TH1 phenotype that fostered CD8+ T-cell cytotoxicity, and suppressed PDAC growth, indicating that BTK signaling mediates PDAC immunosuppression. These data indicate that pharmacologic inhibition of BTK in PDAC can reactivate adaptive immune responses, presenting a new therapeutic modality for this devastating tumor type. Significance: We report that BTK regulates B-cell and macrophage-mediated T-cell suppression in pancreas adenocarcinomas. Inhibition of BTK with the FDA-approved inhibitor ibrutinib restores T cell–dependent antitumor immune responses to inhibit PDAC growth and improves responsiveness to chemotherapy, presenting a new therapeutic modality for pancreas cancer. Cancer Discov; 6(3); 270–85. ©2015 AACR. See related commentary by Roghanian et al., p. 230. See related article by Pylayeva-Gupta et al., p. 247. See related article by Lee et al., p. 256. This article is highlighted in the In This Issue feature, p. 217

SummaryB cells foster squamous cell carcinoma (SCC) development through deposition of immunoglobulin-containing immune complexes in premalignant tissue and Fcγ receptor-dependent activation of myeloid cells. Because human SCCs of the vulva and head and neck exhibited hallmarks of B cell infiltration, we examined B cell-deficient mice and found reduced support for SCC growth. Although ineffective as a single agent, treatment of mice bearing preexisting SCCs with B cell-depleting αCD20 monoclonal antibodies improved response to platinum- and Taxol-based chemotherapy. Improved chemoresponsiveness was dependent on altered chemokine expression by macrophages that promoted tumor infiltration of activated CD8+ lymphocytes via CCR5-dependent mechanisms. These data reveal that B cells, and the downstream myeloid-based pathways they regulate, represent tractable targets for anticancer therapy in select tumors.

Transforming growth factor beta 1 (TGFβ1) is a pleiotropic cytokine in the skin that can function both as a tumor promoter and suppressor in chemically induced skin carcinogenesis, but the function in ultraviolet B (UVB) carcinogenesis is not well understood. Treatment of SKH1 hairless mice with the activin-like kinase 5 (ALK5) inhibitor SB431542 to block UVB-induced activation of cutaneous TGFβ1 signaling suppressed skin tumor formation but did not alter tumor size or tumor cell proliferation. Tumors that arose in SB-treated mice after 30 weeks had significantly reduced percentage of IFNγ(+) tumor-infiltrating lymphocytes compared with control mice. SB431542 blocked acute and chronic UVB-induced skin inflammation and T-cell activation in the skin-draining lymph node (SDLN) and skin but did not alter UVB-induced epidermal proliferation. We tested the effect of SB431542 on migration of skin dendritic cell (DC) populations because DCs are critical mediators of T-cell activation and cutaneous inflammation. SB431542 blocked (i) UVB-induced Smad2 phosphorylation in dermal DC (dDC) and (ii) SDLN and ear explant migration of CD103(+) CD207(+) and CD207(-) skin DC subsets but did not affect basal or UV-induced migration of Langerhans cells. Mice expressing a dominant-negative TGFβ type II receptor in CD11c(+) cells had reduced basal and UVB-induced SDLN migration of CD103(+) CD207(+) and CD207(-) DC subsets and a reduced percentage of CD86(high) dDC following UVB irradiation. Together, these suggest that TGFβ1 signaling has a tumor-promoting role in UVB-induced skin carcinogenesis and this is mediated in part through its role in UVB-induced migration of dDC and cutaneous inflammation.

B cells have recently been appreciated as paracrine mediators of solid tumor development. Their ability to influence various hallmarks of cancer development, aside from antigen presentation, can be attributed to the diversity of soluble mediators they express, including cytokines and immunoglobulins, that can act directly and indirectly on the diversity of leukocyte subsets that infiltrate developing tumors, evolving neoplastic cells, as well as select T cell populations in secondary lymphoid organs and within tumor stroma. Herein, we review the literature supporting these interactions and discuss novel approaches to ameliorate protumoral B cell effects for anti-cancer therapy.

Overexpression of transforming growth factor-beta1 (TGFbeta1) in the normal epidermis can provoke an inflammatory response, but whether this occurs within a developing tumor is not clear. To test this, we used an inducible transgenic mouse to overexpress TGFbeta1 in premalignant squamous lesions. Within 48 hours of TGFbeta1 induction, there was an increase in IL-17 production by both CD4(+) and gammadelta(+) T cells, together with increased expression of T-helper-17 (Th17)-polarizing cytokines. Induction of TGFbeta1 in premalignant primary keratinocytes elevated the expression of proinflammatory and Th17-polarizing cytokines, and the keratinocyte-conditioned media caused IL-17 production by naive T cells that was dependent on T-cell TGFbeta1 signaling. Microarray analysis showed significant upregulation of proinflammatory genes 2 days after TGFbeta1 induction, and this was followed by increased MPO(+), F4/80(+), and CD8(+) cells in tumors, increased CD8(+) effectors and IFNgamma(+) cells in skin-draining LNs, and tumor regression. In parallel, the percentage of tumor CD11b(+)Ly6G(+) neutrophils was reduced. Neutralization of IL-17 blocked TGFbeta1-induced CD11b(+) Ly6G(-) tumor infiltration but did not alter the reduction of neutrophils or tumor regression. Thus, TGFbeta1 overexpression causes IL-17-dependent and IL-17-independent changes in the premalignant tumor inflammatory microenvironment.

Heterogeneity in tumor immune responses is a poorly understood yet critical parameter for successful immunotherapy. We used doxycycline-inducible epidermal squamous cancer models driven by oncogenic Ras to test whether the origin of the tumor-initiating cell influenced the tumor immune response. Threshold doxycycline induced expression of H-RasG12V in either the basal/stem cell layer with a Keratin14-rtTA transgene (K14Ras), or committed progenitor/suprabasal cells with an Involucrin-tTA transgene (InvRas), caused distinct immune responses despite similar levels of Ras, tumor latency, and tumor numbers. End stage K14Ras tumors had an immunosuppressed microenvironment with abundant Tregs and Bregs which evolved from an initially Th1 dominant environment in the earliest detectable lesions. In contrast InvRas tumors had a Th2 pro-inflammatory microenvironment, with significantly fewer Tregs or Bregs at any time point. This difference in immune microenvironment between the tumor models was also found in hyperplastic skin after 5 days of maximal Ras expression, indicating that it was not simply a property of the end stage tumor. Surprisingly, adaptive immunity had opposite roles in tumor development. InvRasRag1-/- mice developed fewer and smaller tumors that regressed while K14RasRag1-/- mice developed more tumors with shorter latency than Rag1+/+ controls. In both models adoptive transfer and depletion studies showed that cooperation between B and CD4 T cells drove the opposing tumor responses, lymphocyte polarization, and tumor immune phenotype. Co-culture of tumor-conditioned but not splenic B cells from each model with naive CD4 T cells showed that direct contact and CD40-CD40L ligation was required for opposite polarization towards either a Th2 or Treg phenotype. Importantly, we found that anti-CD40L mAb caused regression of preexisting InvRas tumors but enhanced growth of K14Ras tumors. In contrast, CD40 agonist mAb enhanced growth of preexisting InvRas tumors, and suppressed growth of K14Ras tumors. Thus in an in vivo setting the type of tumor immune microenvironment and opposing role of CD40-CD40L in tumor development generates distinct responses to therapeutic antibodies. Together these data show that the position of a tumor initiating cell within the stem cell hierarchy in a stratified squamous epithelia has important consequences for the type of tumor immune microenvironment and response to checkpoint therapy. Citation Format: Adam B. Glick, Jacob T. Bailey, Andrew Gunderson, Kyle Breech, Michael Podolsky. Divergent therapeutic responses to CD40L blockade or CD40 activation in Ras-driven skin cancers determined by origin of tumor initiating cell [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 2673. doi:10.1158/1538-7445.AM2017-2673

Background & Aims Premalignant lesions and early stage tumors contain immunosuppressive microenvironments that create barriers for cancer vaccines. Kras G12D/+ ;Trp53 R172H/+ ;Pdx-1-Cre (KPC) mice, which express an activated form of Kras in pancreatic tissues, develop pancreatic intraepithelial neoplasms (PanIN) that progress to pancreatic ductal adenocarcinoma (PDA). We used these mice to study immune suppression in PDA. Methods We immunized KPC and Kras G12D/+ ; Pdx-1-Cre mice with attenuated intracellular Listeria monocytogenes (which induces CD4 + and CD8 + T-cell immunity) engineered to express Kras G12D (LM-Kras). The vaccine was given alone or in sequence with an anti-CD25 antibody (PC61) and cyclophosphamide to deplete T-regulatory (Treg) cells. Survival times were measured; pancreatic and spleen tissues were collected and analyzed by histologic, flow cytometry, and immunohistochemical analyses. Results Interferon γ–mediated, CD8 + T-cell responses were observed in KPC and Kras G12D/+ ; Pdx-1-Cre mice given LM-Kras, but not in unvaccinated mice. Administration of LM-Kras to KPC mice 4–6 weeks old (with early stage PanINs), depleted of Treg cells, significantly prolonged survival and reduced PanIN progression (median survival, 265 days), compared with unvaccinated mice (median survival, 150 days; P = .002), mice given only LM-Kras (median survival, 150 days; P = .050), and unvaccinated mice depleted of Treg cells (median survival, 170 days; P = .048). In 8- to 12-week-old mice (with late-stage PanINs), LM-Kras, alone or in combination with Treg cell depletion, did not increase survival time or slow PanIN progression. The combination of LM-Kras and Treg cell depletion reduced numbers of Foxp3 + CD4 + T cells in pancreatic lymph nodes, increased numbers of CD4 + T cells that secrete interleukin 17 and interferon γ, and caused CD11b + Gr1 + cells in the pancreas to acquire an immunostimulatory phenotype. Conclusions Immunization of KPC mice with Listeria monocytogenes engineered to express Kras G12D , along with depletion of Treg cells, reduces progression of early stage, but not late-stage, PanINs. This approach increases infiltration of the lesion with inflammatory cells. It might be possible to design immunotherapies against premalignant pancreatic lesions to slow or prevent progression to PDA.

Tumors from stratified epithelium contain proliferating and differentiating compartments, but it is not clear how tumor cells in different layers alternatively engage the immune system. We have established two doxycycline inducible Ras models in which oncogenic RasV12G is targeted to either the epidermal basal/stem cell layer with a Keratin14-rtTA transgene (K14Ras), or suprabasal differentiating cells with an Involucrin-tTA transgene (InvRas). On threshold doxycycline levels yielding similar tumor numbers and Ras expression over 30 days, mice with basal cell targeted Ras developed focal squamous cell carcinoma while suprabasal targeting caused benign squamous papilloma formation. On a Rag1-/- background InvRas mice developed fewer tumors that regressed over time while K14Ras mice developed more tumors with shorter latency than Rag1+/+ controls. Depletion and adoptive transfer studies revealed that naïve B and CD4 T cells together, but not alone, suppressed tumor formation in K14Ras mice but restored tumor numbers in InvRas mice. Tumors developing in K14Ras mice showed a loss of proinflammatory CD4 T and B cells and increased percentages of regulatory cells infiltrating the tumor tissue. In vivo cotransfers show that B and CD4 T cells reciprocally prime each other towards a regulatory phenotype in the K14Ras tumor microenvironment, but in the InvRas tumor microenvironment proinflammatory CD4 T and B cell phenotypes result. Coculture of tumor-conditioned B cells with stimulated naïve CD4 T cells showed an importance of direct contact and CD40/CD40L interactions for the generation of regulatory T cells (Tregs) by B cells in K14Ras mice. In contrast, tumor-conditioned B cells from InvRas mice support generation of proinflammatory CD4 T cells, and antagonize Treg development. This function is restricted to tumor-conditioned B cells, as splenic B cells from tumor-bearing mice had no effect on CD4 T cell phenotype. In vivo blockade of CD40L in K14Ras mice resulted in significantly increased tumor counts as well as reduced B and Treg prevalence. Blockade of CD40L in InvRas mice significantly reduced tumor number, increasing Treg cell count and decreasing neutrophil infiltration into the tumor tissue. Time-course experiments suggest a protective role of Tregs in late stages of K14Ras tumors, and a tumor-promoting role of proinflammatory CD4 T cells in InvRas tumors. Thus, basal/stem cell expression of Ras provokes a regulatory cell inducing microenvironment, suppressing tumor-promoting inflammation in late-stage tumors. Ras expression in differentiating cells activates a tumor promoting proinflammatory phenotype in B and CD4 T cells without provoking immunosurveillance. Taken together, these data show that tumor cell position within a stratified epithelium differentiation hierarchy provokes distinct B and CD4 T cell interactions with opposing effects on tumor development. Citation Format: Michael A. Podolsky, Carrie J. Oakes, Andrew Gunderson, Kyle Breech, Jacob Bailey, Adam B. Glick. Location of oncogene expression within a stratified squamous epithelium drives distinct B and CD4 T-cell crosstalk to dictate the tumor immune response. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 530.

Overexpression of transforming growth factor beta-1 (TGFβ1) in mouse epidermis causes cutaneous inflammation and keratinocyte hyperproliferation. Here we examined acute effects of TGFβ1 overproduction by keratinocytes on skin dendritic cells (DCs). TGFβ1 induction for 2 and 4 days increased the numbers and CD86 expression of B220 + plasmacytoid DCs (pDCs) and CD207 + CD103 + , CD207 − CD103 − CD11b + , and CD207 − CD103 − CD11b − dermal DCs (dDCs) in skin-draining lymph nodes (SDLNs). The dermis of TGFβ1-overexpressing mice had significantly more pDCs, CD207 + CD103 + dDCs, and CD207 − CD11b + dDCs in the absence of increased dermal proliferation. Application of dye, tetramethyl rhodamine iso-thiocyanate (TRITC), in dibutylpthalate (DBP) solution after TGFβ1 induction increased the numbers of TRITC + CD207 − dDCs in SDLNs, and augmented TRITC/DBP-induced Langerhans cell (LC) migration 72hours post TRITC treatment. Consistent with this, LC migration was increased in vitro by TGFβ1 overexpression in skin explants and by exogenous TGFβ1 in culture media. Transient TGFβ1 induction during DNFB sensitization increased contact hypersensitivity responses by 1.5-fold. Thus, elevated epidermal TGFβ1 alone is sufficient to alter homeostasis of multiple cutaneous DC subsets, and enhance DC migration and immune responses to contact sensitizers. These results highlight a role for keratinocyte-derived TGFβ1 in DC trafficking and in the initiation of skin inflammation.

Experimental results generated from human pancreatic ductal adenocarcinoma (PDAC) specimens have produced a broad knowledge of pancreatic tumors. However, successful personalized treatment of pancreatic disease will require an exceptionally deep knowledge of the molecular and cellular diversity of tumors, as well as their evolution through the course of disease progression, therapeutics, relapse, and terminal disease burden. To address this need, we have conducted diverse but complementary experimental analyses on adjacent regions of fresh tumor specimens collected from over 70 primary pancreas specimens obtained during Whipple and RAMPS procedures. Coordination between surgical and research teams has ensured that only a minimum time elapses between surgical resection and specimen processing, thus limiting potential degradation by pancreatic enzymes. Researchers with diverse cancer biology expertise obtain adjacent dissections from three regions (i.e., tumor, dysplastic, and normal) within each pancreas specimen. An annotated image of the whole pancreas specimen records the location of each dissected portion relative to one another. Fresh tissues are immediately used to 1) characterize the phenotypic diversity of tumor and non-tumor cells in the microenvironment, 2) isolate and culture cancer stem cells, 3) perform high resolution imaging including 3D-SEM, 4) “bioprint” and culture three-dimensional, multi-cell-type structures, and 5) propagate tumors in mouse avatars. Genomics, epigenomics, and transcriptomics analyses are also performed on laser-captured specimens. In addition to primary PDAC tumors, other fresh pancreatic cancer specimens are being added to the collection including core needle biopsies obtained from treatment-naïve patients, metastases removed during primary tumor resection or recurrence, and tumor specimens from a rapid autopsy program. Importantly, all specimens are fully clinically annotated as patients are followed through the course of their disease. By comprehensively characterizing individual pancreatic tumors from many patients we hope to produce a unique body of information – an “Atlas” of pancreatic disease – that will inform the research community of new molecular and cellular features that contribute to the progression and therapeutic resistance of this devastating disease. Research data and clinical information in the Atlas will be accessible through a user-friendly customizable database. We hope that this resource will support the development of molecularly targeted early detection, therapeutics, and prevention in order to improve patient care. Funding for this initiative comes from philanthropic support to the Brenden-Colson Center for Pancreatic Care at Oregon Health and Science University. Citation Format: Jason M. Link, Brittany Allen-Petersen, Andrew Gunderson, Danielle Jorgens, Craig Dorrell, Jody Hooper, Philip Streeter, Markus Grompe, Lisa Coussens, Joe Gray, Hope Hardaker, Charles D. Lopez, Rosalie C. Sears, Brett C. Sheppard. Developing a molecular and cellular atlas of pancreatic disease. [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer: Innovations in Research and Treatment; May 18-21, 2014; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2015;75(13 Suppl):Abstract nr B118.

Studies in mouse models and human cancers show that B cells can both promote and inhibit cancer development, but the mechanisms generating these alternate outcomes are not well understood. Here we have developed a mouse model of Ras-driven epidermal tumor development in which opposite effects of B cells are manifest, dependent on the differentiation state of cells expressing oncogenic Ras. We used a doxycycline regulatable bitransgenic system to target oncogenic H-RasV12G to either the basal/stem cell (Keratin14-rTAxtetORasV12G, K14Ras) or differentiating (Involucrin-tTA x tetORasV12G, InvRas) compartment of the epidermis. K14Ras mice develop focal squamous cell carcinoma while InvRas mice develop squamous papillomas within 30 days on threshold levels of doxycycline resulting in ∼10 tumors/mouse. On a Rag1-/- background we observed opposite tumor responses, with InvRasRag1-/- mice developing fewer tumors and K14RasRag1-/- mice developing more tumors with shorter latency than Rag1+/+ controls. Depletion of CD4+ T cells caused an overall increase in tumor burden compared to isotype controls in both models, while adoptive transfer of naïve CD4+ T cells (purity ≥ 93%) increased tumor burden in K14RasRag1-/- mice, but suppressed tumors in InvRasRag1-/- mice. Tumors from CD4 transferred K14RasRag1-/- mice had a predominant Th1 infiltrate with few Tregs and increased tumor-infiltrating neutrophils compared to mock transferred controls. Conversely, CD4+ T cells adoptively transferred to InvRasRag1-/- mice displayed a regulatory phenotype with low IFNγ and high Foxp3, which was associated with a decrease in neutrophilic infiltration. In contrast, B cell depletion with anti-CD20 caused a significant suppression of tumor outgrowth in InvRas mice but enhanced tumor burden in K14Ras mice. Furthermore, adoptive transfer of magnetically purified splenic B cells into K14RasRag1-/- mice suppressed tumor formation, and co-transfer of B cells with CD4+ T cells reduced tumor development to a greater extent than B cells alone. Tumor-infiltrating B cells from co-transferred mice had greater percentage of B regulatory cells, and increased costimulatory molecule expression compared to B cells alone, resembling K14RasRag1+/+ controls. B cell co-transfer significantly increased Foxp3+ Treg percentage to levels similar to K14RasRag1+/+ mice as measured by flow cytometry. Taken together these results show that B cells are the critical lymphocyte driving both tumor suppression or promotion in this model, that the differentiation state of the tumor initiating cell determines the B cell response, and that interactions between CD4+ T cells and B cells may be important for the development of a proper anti-tumor immune response. Citation Format: Michael A. Podolsky, Andrew Gunderson, Kyle Breech, Adam B. Glick. Opposing roles of B cells in Ras-driven squamous tumor development dependent on tissue compartment of oncogene expression and tumor phenotype. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4083. doi:10.1158/1538-7445.AM2014-4083

Human pancreatic ductal adenocarcinoma (PDA) remains a fatal disease. Biologically, it is naturally resistant to therapy and characterized by an intense desmoplastic reaction to the tumor. Genetically-engineered mouse models of spontaneously developing PDA tumors mimic the multistep progression from pre-cancer to invasive cancer in humans, and are ideal for studying the tumor microenvironment. We investigated the cellular and molecular signals underlying pancreatic intra-epithelial neoplasm (PanIN) progression, and examined the changes in these signals, PanIN progression, and survival following immunotherapy. The KrasG12D/+; Trp53R172H/+; Pdx-1-Cre mouse model of pancreatic ductal adenocarcinoma combines a Kras dominant active mutation with a p53 loss of function mutation, specifically expressed in the pancreas under the control of the Pdx-1 promoter. Mice begin developing PanINs at several weeks old, progressing to PDA and accompanied by the characteristic desmoplastic reaction seen in human PDA. We evaluated the effects of an attenuated Listeria monocytogenes expressing mutated Kras (LM-Kras) vaccine capable of inducing a Kras-specific T cell responses together with regulatory T cell (Treg)-depleting therapy (Cyclophosphamide and anti-CD25 monoclonal antibody). Combination therapy given to mice with early stage PanINs increases survival and delays PanIN progression, whereas intervention in older mice or with single agent immunotherapy fails to alter the course of tumor progression. Combination treatment administered during early PanINs correlated with enhanced CD8+ T cell infiltration and activation. In addition, combination treatment induced a Th1/Th17 cytokine expression pattern in CD4+ T cells and decreased infiltration by CD4+Foxp3+ Tregs. Local, rather than systemic, changes in T cell activation correlated with delay in PanIN progression. Immunohistochemistry revealed granulocytes, myeloid cells, and macrophages infiltrating untreated PanINs and PDA. Analysis of upregulated genes in laser capture micro-dissected specimens comparing advanced PanIN stages and invasive PDA included those associated with recruitment of MDSCs, induction of a suppressive phenotype in these cells, and mediators secreted by cells with a M2 or N2 phenotype. We are further investigating the effect of combination immunotherapy on the granulocyte and myeloid populations with the hypothesis that these signals will be altered with treatment. Thus, vaccination targeting an early genetic event in PDA can slow progression from early PanINs to invasive cancer when Tregs are also altered. Additional studies are ongoing to determine whether PanIN progression can be delayed when vaccination is administered at a time of advanced PanIN development if additional suppressive populations of immune cells within the tumor microenvironment are also inhibited. This abstract is also presented as Poster A20. Citation Format: Bridget Keenan, Yvonne Saenger, Michel Kafrouni, Ashley Leubner, Peter Lauer, Anirban Maitra, Andrew Gunderson, Lisa Coussens, Todd Armstrong, Elizabeth Jaffee. Prevention of pancreatic intra-epithelial neoplasm progression by a Listeria monocytogenes vaccine targeting mutated Kras, an early genetic event in pancreatic tumor development. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology: Multidisciplinary Science Driving Basic and Clinical Advances; Dec 2-5, 2012; Miami, FL. Philadelphia (PA): AACR; Cancer Res 2013;73(1 Suppl):Abstract nr PR6.

Meeting abstracts Human Papilloma Virus (HPV)-positive head and neck squamous cell carcinoma (HNSCC) is clinically distinct from HPV-negative HNSCC, and as such requires differential therapeutic approaches. Accumulating evidence indicates a significant linkage between the immune response within the