The Prolyl Isomerase Pin1 Modulates Development of CD8+ cDC in Mice

We have identified a novel role for Pin1 as a modulator of CD8+ cDC development. Consistent with reduced numbers of CD8+ cDC in Pin1-null mice, we find that the absence of Pin1 impairs CD8+ T cell proliferation in response to infection with Listeria monocytogenes. These data suggest that, via regulation of CD8+ cDC production, Pin1 may serve as an important modulator of adaptive immunity

31st Annual Meeting and Associated Programs of the Society for Immunotherapy of Cancer (SITC 2016) : part two

Background The immunological escape of tumors represents one of the main ob- stacles to the treatment of malignancies. The blockade of PD-1 or CTLA-4 receptors represented a milestone in the history of immunotherapy. However, immune checkpoint inhibitors seem to be effective in specific cohorts of patients. It has been proposed that their efficacy relies on the presence of an immunological response. Thus, we hypothesized that disruption of the PD-L1/PD-1 axis would synergize with our oncolytic vaccine platform PeptiCRAd. Methods We used murine B16OVA in vivo tumor models and flow cytometry analysis to investigate the immunological background. Results First, we found that high-burden B16OVA tumors were refractory to combination immunotherapy. However, with a more aggressive schedule, tumors with a lower burden were more susceptible to the combination of PeptiCRAd and PD-L1 blockade. The therapy signifi- cantly increased the median survival of mice (Fig. 7). Interestingly, the reduced growth of contralaterally injected B16F10 cells sug- gested the presence of a long lasting immunological memory also against non-targeted antigens. Concerning the functional state of tumor infiltrating lymphocytes (TILs), we found that all the immune therapies would enhance the percentage of activated (PD-1pos TIM- 3neg) T lymphocytes and reduce the amount of exhausted (PD-1pos TIM-3pos) cells compared to placebo. As expected, we found that PeptiCRAd monotherapy could increase the number of antigen spe- cific CD8+ T cells compared to other treatments. However, only the combination with PD-L1 blockade could significantly increase the ra- tio between activated and exhausted pentamer positive cells (p= 0.0058), suggesting that by disrupting the PD-1/PD-L1 axis we could decrease the amount of dysfunctional antigen specific T cells. We ob- served that the anatomical location deeply influenced the state of CD4+ and CD8+ T lymphocytes. In fact, TIM-3 expression was in- creased by 2 fold on TILs compared to splenic and lymphoid T cells. In the CD8+ compartment, the expression of PD-1 on the surface seemed to be restricted to the tumor micro-environment, while CD4 + T cells had a high expression of PD-1 also in lymphoid organs. Interestingly, we found that the levels of PD-1 were significantly higher on CD8+ T cells than on CD4+ T cells into the tumor micro- environment (p < 0.0001). Conclusions In conclusion, we demonstrated that the efficacy of immune check- point inhibitors might be strongly enhanced by their combination with cancer vaccines. PeptiCRAd was able to increase the number of antigen-specific T cells and PD-L1 blockade prevented their exhaus- tion, resulting in long-lasting immunological memory and increased median survival

NF-κB p50-deficient immature myeloid cell (p50-IMC) adoptive transfer slows the growth of murine prostate and pancreatic ductal carcinoma

BackgroundMacrophages and dendritic cells lacking the transcription factor nuclear factor kappa B p50 are skewed toward a proinflammatory phenotype, with increased cytokine expression and enhanced T cell activation; additionally, murine melanoma, fibrosarcoma, colon carcinoma, and glioblastoma grow slower in p50−/− mice. We therefore evaluated the efficacy of p50-negative immature myeloid cells (p50-IMCs) adoptively transferred into tumor-bearing hosts. Immature cells were used to maximize tumor localization, and pretreatment with 5-fluorouracil (5FU) was examined due to its potential to impair marrow production of myeloid cells, to target tumor myeloid cells and to release tumor neoantigens.MethodsWild-type (WT)-IMC or p50-IMC were generated by culturing lineage-negative marrow cells from WT or p50−/− mice in media containing thrombopoietin, stem cell factor and Flt3 ligand for 6 days followed by monocyte colony-stimulating factor for 1 day on ultralow attachment plates. Mice inoculated with Hi-Myc prostate cancer (PCa) cells or K-RasG12D pancreatic ductal carcinoma (PDC)-luciferase cells received 5FU followed 5 days later by three doses of 107 immature myeloid cells (IMC) every 3–4 days.ResultsPCa cells grew slower in p50−/− mice, and absence of host p50 prolonged the survival of mice inoculated orthotopically with PDC cells. IMC from Cytomegalovirus (CMV)-luciferase mice localized to tumor, nodes, spleen, marrow, and lung. 5FU followed by p50-IMC slowed PCa and PDC tumor growth, ~3-fold on average, in contrast to 5FU followed by WT-IMC, 5FU alone or p50-IMC alone. Slowed tumor growth was evident for 93% of PCa but only 53% of PDC tumors; we therefore focused on PCa for additional IMC analyses. In PCa, p50-IMC matured into F4/80+ macrophages, as well as CD11b+F4/80−CD11c+ conventional dendritic cells (cDCs). In both tumor and draining lymph nodes, p50-IMC generated more macrophages and cDCs than WT-IMC. Activated tumor CD8+ T cells were increased fivefold by p50-IMC compared with WT-IMC, and antibody-mediated CD8+ T cell depletion obviated slower tumor growth induced by 5FU followed by p50-IMC.Conclusions5FU followed by p50-IMC slows the growth of murine prostate and pancreatic carcinoma and depends on CD8+ T cell activation. Deletion of p50 in patient-derived marrow CD34+ cells and subsequent production of IMC for adoptive transfer may contribute to the therapy of these and additional cancers

DC development in bone marrow cultures.

<p>(A) Representative FACS plots of WT and Pin1-null bone marrow-derived Mac1- cDC generated by culturing with FL. Cells were previously gated as B220-Mac1− (“Mac1−”) and cDC are identified as MHCII^hiCD11c^hi cells. The frequency of Mac1− cDC corresponding to the populations gated in the FACS plots on the left are graphed on the right as percentage of total cells (n = 16). (B) Representative FACS plots of WT and Pin1-null bone marrow-derived Mac1+ cDC generated by culturing with either FL (n = 16) or GM-CSF (n = 11). FL-generated cells were previously gated as B220-Mac1+, and GM-CSF-generated cells were previously gated as B220-Mac1+GR1-. In the plots shown, cDC are identified as MHCII^hiCD11c^hi cells. The frequencies of Mac1+ cDC gated on the left are depicted in the graph on the right as the percentage of total cells. (C) Representative overlaid histograms comparing CD11c expression in WT and Pin1-null B220-Mac1+ cells generated from FL and GM-CSF bone marrow cultures. (D) Representative FACS plots of WT and Pin1-null bone marrow-derived pDC and macrophage (mΦ). Cells were not previously gated. pDC are identified as PDCA1+CD11c^int cells and macrophages are identified as F4/80+Mac1+ cells. The frequencies of WT and Pin1-null bone marrow-derived pDC (n = 16) and macrophages (n = 6) are shown in the graph on the right as percentage of total cells.</p

DC bone marrow progenitors are unaltered in absence of Pin1.

<p>(A) Normalization of total bone marrow cells from WT and Pin1-null mice by weight (grams). WT and Pin1-null mice were weighed upon euthanization. The normalized graph was generated by dividing the total number of bone marrow cells by the body weight (grams) of each mouse (n = 6). (B) Quantitation of normalized numbers of bone marrow progenitors in both WT and Pin1-null mice. KLS, c-kit+Lin-sca1+ stem cells; CLP, common lymphoid progenitor; CMP, common myeloid progenitor; GMP, granulocyte-macrophage progenitor; MDP, macrophage-dendritic cell progenitor; CDP, common dendritic cell progenitor. Total cell numbers were determined by multiplying the frequency of each population by the total number of bone marrow cells. Total population numbers were then normalized to body weight by dividing the number of cells in each population by the body weight (grams) of each mouse (n = 6). (C) Quantitation of normalized numbers of pre-cDC and pDC in the bone marrow of WT and Pin1-null mice. Normalized cell numbers were calculated as described in (B) (n = 6).</p

Pin1-null mice have fewer steady-state spleen cDC and fewer FL-expanded CD8+ cDC <i>in vivo</i>.

<p>(A) Quantitation of the numbers of steady-state spleen DC subsets from healthy WT and Pin1-null mice. Cell population numbers were determined by multiplying the frequency of the cell population by the total number of splenocytes obtained from each mouse (n = 6). Definitions of each cell population can be found in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0029808#s4" target="_blank">Materials and Methods</a>. (B) Quantitation of the numbers of spleen DC subsets from WT and Pin1-null mice that were administered 1 µg Flt3 Ligand (FL) for 9 consecutive days. Cell population numbers were determined by multiplying the frequency of the cell population by the total number of splenocytes obtained from each mouse (n = 5).</p

Cell survival and pre-cDC generation in bone marrow cultures.

<p>(A) The graph on the left shows the quantitation of the total number of WT and Pin1-null bone marrow-derived dendritic cells recovered from bone marrow cultures after 9 days incubation with FL. The graph on the right is a quantitation of the frequency of live WT and Pin1-null bone marrow-derived DC that did not stain with propidium iodide (PI−) (n = 3). (B) The top panel depicts part of the gating strategy used to identify pre-cDC. Previously gated PDCA1-CD11c+ cells were further gated, as shown, to identify PDCA1-CD11c+MHCII-B220+ Sirpα-CD24+ pre-cDC in FL cultured bone marrow cells. The frequencies of pre-cDC from WT and Pin1-null cultures are quantified in the graph below as percentage of total cells (n = 3).</p

Pin1 modulates PU.1 protein stability.

<p>(A) Immunoblot analysis of PU.1 protein expression in WT and Pin1-null FL-cultured bone marrow-derived DC (FLDC) and primary MEF. For FLDC, the immunoblot shown is representative of cells derived from 3 different mice. For MEF, the immunoblot shown is representative of 3 different experiments. (B) Quantitation of PU.1 mRNA expression from WT and Pin1-null FLDC (n = 5) and MEF (n = 3). (C) GST-Pin1 pull down in WT and Pin1-null MEF lysates. 1 mg of total lysate was incubated with GST alone, WT GST-Pin1, or WW GST-Pin1 for 2 hours. After binding, beads were washed, resuspended in SDS-Page sample buffer, boiled, and then analyzed by immunoblot. Membranes were probed for expression of PU.1. (D) PU.1 protein expression in WT and Pin1-null MEFs after being treated with 150 µg/ml cycloheximide (CHX) to inhibit protein synthesis for 2, 4, 6, 8, or 10 hours. The immunoblot shown is representative of two independent experiments. PU.1 protein expression is plotted in the bottom graph as a percentage of total PU.1 protein at time zero, and reflects the values obtained from immunoblots shown directly above.</p