Class II transactivator (CIITA) is known as a coactivator for MHC class II gene expression in antigen-presenting cells. Surprisingly, when CIITA-/- CD4 T cells were stimulated in the presence of IL-12, they produced not only IFNgamma but also high levels of IL-4. The IL-4 production is due to the accumulation of IL-4 gene transcripts in Th1 cells. This transcriptional control is observed in T cells differentiating to the Th1 but not Th2 lineage, consistent with induction of expression of the CIITA gene in T cells by IFNgamma. Thus, in addition to its role in transactivation of genes involved in antigen presentation, CIITA plays a critical role during the T cell differentiation by negatively regulating the IL-4 gene transcription.
The MHC class II transactivator (CIITA) activates the expression of multiple genes involved in Ag presentation, but inhibits Th2-type cytokine production, including IL-4, during Th1 cell differentiation. Th1 cells derived from CIITA-deficient mice produce both Th1- and Th2-type cytokines, and the introduction of CIITA to Th2 cells down-regulates Th2-type cytokine gene transcription. Here we show that the IL-4 promoter is regulated by multiple protein-protein interactions among CIITA, NF-AT, and coactivator CBP/p300. The introduction of CBP/p300 and NF-AT enhances the IL-4 promoter activity, and this activation was repressed by CIITA. Furthermore, our data show that CIITA competes with NF-AT to bind CBP/p300 and that this competition dramatically influences transcriptional activation of the IL-4 promoter. We identified two domains of CIITA that interact with two distinct domains of CBP/p300 that are also recognized by NF-AT. CIITA mutants that retain the ability to interact with CBP/p300 are sufficient to inhibit NF-AT-mediated IL-4 gene expression.
The major histocompatibility complex (MHC) class II transactivator (CIITA) regulates the expression of genes involved in the immune response, including MHC class II genes and the interleukin-4 gene. Interactions between CIITA and sequence-specific, DNA-binding proteins are required for CIITA to function as an activator of MHC class II genes. CIITA also interacts with the coactivators CBP (also called p300), and this interaction leads to synergistic activation of MHC class II promoters. Here, we report that CIITA forms complexes with itself and that a central region, including the GTP-binding domain is sufficient for self-association. Additionally, this central region interacts with the C-terminal leucine-rich repeat as well as the N-terminal acidic domain. LXXLL motifs residing in the GTP-binding domain are essential for self-association. Finally, distinct differences exist among various CIITA mutant proteins with regard to activation function, subcellular localization, and association with wild-type protein and dominant-negative potential.Major histocompatibility complex (MHC) class II molecules present exogenously derived antigenic peptides to CD4 ϩ T cells. The recognition of alien peptide by these T cells allows a host to immunologically respond to foreign pathogens. MHC class II molecules are constitutively expressed on B cells and dendritic cells and inducible upon other cells, such as macrophages, all of which are capable of the uptake and processing of foreign invaders. In the absence of MHC class II molecules, individuals are unable to mount a T-cell-mediated immune response and overwhelming infection ensues. A group of immunodeficient patients which lack MHC class II molecules have been identified, and this disease has been coined bare lymphocyte syndrome (BLS) (8,15). One class of these BLS patients (group A) lack MHC class II molecules on their cellular surfaces due to a defect in the MHC class II transactivator, CIITA (38).The regulation of MHC class II gene expression is primarily at the transcriptional level. The promoters of MHC class II genes contain at least four conserved sequences: the S, X, X2, and Y boxes (reviewed in reference 28). These cis-acting elements are occupied by sequence-specific transcription factors; the heterotrimeric NF-Y complex binds to the Y box (25), the multimeric RFX proteins bind to the S and X boxes (10, 21), and the cyclic AMP response element binding protein (CREB) binds to the X2 box (31). Protein-protein interactions stabilize the binding of these proteins to MHC class II promoter DNA as illustrated by interactions between the RFX complex and 40,41). and the enhancement of RFX complex binding facilitated by CREB (27, 41). However, the binding of all of these transcription factors to their respective cis-acting elements is insufficient to lead to MHC class II promoter activity. CIITA is required for both the constitutive and the gamma interferon-inducible expression of MHC class II genes (4, 38, 39). CIITA does not bind directly to DNA. The exact mechanism of CIIT...
Multi-color panel design can be difficult due to the many factors which impact signal detection including the fluorescence detection capability of the cytometer, target molecule density and the spectral characteristics of available fluorophores (fluors). We demonstrate that a flow cytometer with a fixed and standardized fluorescence measurement system simplifies design of multi-color flow experiments, using a common conundrum researchers face as illustration: several different fluor conjugates detectable by the cytometer are available for a given clone. Which is best to use? First we show that by locking down optical alignment and optimizing fluorescence detection at the time of manufacture the BD Accuri C6 gives reproducible, predictable fluorescence measurements both for individual instruments over time and between instruments. We also show that this design leads to predictable fluorescence spillover for stable fluors. Since fluorescence spillover due to bright fluors and/or highly expressed epitopes can mask dimmer signals in neighboring channels, successfully predicting spillover greatly simplifies multi-color panel design. We illustrate this by comparing the spillover of four fluors optimally detected in FL3 of the BD Accuri™ C6 (PerCP, PerCP-Cy™5.5, PE-Cy7, BD Horizon™ PE-CF594), when conjugated to antibody clones of a high (CD45) and low (CD19) epitope-number antigen. We also quantitate the impact of the spillover on detection of weak signals in neighboring channels.
The development of immuno-oncology agents against human targets is actively pursued in the preclinical space using immune deficient mice with reconstituted human immune systems. We utilized a human peripheral blood mononuclear cell (hPBMC) based humanized mouse model to investigate treatment response in human pancreatic and non-small cell lung cancers. Three human PBMC donors were identified that exhibited consistent T cell engraftment, allowing for a four-week treatment window, and robust growth of two human xenograft models in NSG mice, MiaPaCa-2 (pancreatic) and A549 (non-small cell lung). Anti-tumor efficacy of pembrolizumab (Keytruda®) targeting human PD-1 was also assessed in these humanized models. All animal work was performed in an AAALAC accredited facility, in alignment with applicable animal welfare regulations and with predetermined humane euthanasia criteria on all studies. Control growth of MiaPaCa-2 and A549 tumors were similar with and without hPBMC engraftment. Both tumor models progressed uniformly to a mean tumor volume of 1000 mm3 without apparent hPBMC impact. Engraftment was assessed at 28 and 38 days (MiaPaCa-2) or 42 days (A549) after hPBMC administration by immunophenotypic analysis using flow cytometry in peripheral blood. At all timepoints, hCD45+ cells were detected. While intragroup variability occurred, engraftment remained constant or increased over time. hPBMC engraftment was reproducibly higher for animals harboring A549 compared to MiaPaCa-2 tumors, with hCD45+ cells in blood ranging from 31-52% and 19-33% of total live cells, respectively. It is unknown if this was a result of normal variability or whether this finding is model dependent. Pembrolizumab treatment in the hPBMC-engrafted mice did not inhibit A549 or MiaPaCa-2 tumor growth under the conditions tested (median ΔT/ΔC of 76-118% (Day 41) and 60-106% (Day 22), respectively). However, in vitro, CD8+ T cells from all donors responded to CD3/CD28 stimulation, demonstrating at least 36-fold upregulation of the Ki67 proliferation marker and at least 9-fold upregulation of interferon gamma release, indicative of cytotoxic activity. This result indicated that the hPBMCs were healthy and responsive, but the extent of activation varied by donor. Investigation of human T cell infiltration into human tumors following treatment with pembrolizumab are currently being performed. Despite the lack of response of pembrolizumab against A549 or MiaPaCa-2, these models are promising translational tools for assessing efficacy of novel therapeutics using rational combination strategies with pembrolizumab. This humanized mouse model represents a powerful preclinical platform to examine the effects of novel agents that harness human T cells to direct anti-tumor activity both as monotherapy and in combination with FDA approved or investigational agents. Citation Format: Sheri Barnes, David Draper, Stacey Roys, Philip Lapinski, Maryland Rosenfeld Franklin. Modeling pancreatic and non-small cell lung cancer using a humanized mouse platform [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2711.
To examine how baseline tumor immunophenotyes can influence therapeutic outcome, we used the CT26 colorectal and 4T1-Luc (4T1) breast cancer models, which are immunologically warm and cold tumors, respectively. We treated tumor-bearing mice with control or anti-mCTLA-4 antibodies (anti-CTLA-4), or left them untreated, and excised tumors to measure immune cell infiltrates, TIL cytokine/granzyme B production, and activation marker expression. Baseline analysis in untreated mice revealed that CD8+ T-cell infiltrates were similar between models (5-6% of total CD45+ cells). NKT cells were reduced in CT26 (0.5% vs. 1.2%). The myeloid compartment in CT26 was mostly M2 macrophages (43% of CD45+ cells), whereas G-MDSCs dominated in 4T1 (40% of CD45+ cells). CD69 and PD-1 activation markers were expressed on >90% of CD8+ T cells from CT26 tumors compared to 4T1, which were <40% positive for both markers. Following ex vivo stimulation, the IFNγ response in CD8+ T cells was higher in CT26-derived cells. Treatment with anti-CTLA4 triggered distinct responses in the two models. Delayed tumor growth was observed in CT26 (74% TGI), but no effect in 4T1 was shown. In CT26 tumors, CD8+ T-cell and NKT cell infiltration increased, with a corresponding decrease in M2 macrophages. In 4T1 tumors, only increased NKT cell infiltration occurred. Several pharmacodynamic changes were observed in immune cell activation in CT26 but absent in 4T1. IFNγ production increased in CT26-derived CD4+ and CD8+ T cells, as well as NKT cell subsets (70%, 25%, and 17%, respectively compared to control). This included an increase in polyfunctional T cells that produced both IFNγ and TNFα. Moreover, we found that tumors with the highest frequency of IFNγ/TNFα double positive T cells had slower growth, suggesting that polyfunctional T-cell differentiation correlates with tumor growth delay. Granzyme B was 31% reduced in NKT cells in CT26. Together with increased CD107a expression on these cells, the observed granzyme B reduction was likely due to treatment-induced degranulation. Notably, a correlation between granzyme B expression and delayed tumor growth was also observed, albeit indirect. In summary, untreated CT26 tumors contained TILs that expressed high levels of CD69/PD-1 and moderately expressed IFNγ, and subsequently responded to anti-CTLA-4 treatment with increased polyfunctional T-cell recruitment, NKT cell activity, and reduced M2 macrophage infiltration. In contrast, 4T1 tumors contained low levels of both CD69/PD-1 expression and TIL cytokine expression, which is characteristic of an immunosuppressive tumor microenvironment, and did not display any remarkable response to therapy. Collectively, these results suggest baseline tumor profiles can provide insight into responsiveness to therapy and assist in model selection for preclinical research. Future studies to more broadly profile baseline TIL functional states across different syngeneic models should prove valuable during study design for targeted I/O therapy development. Citation Format: David Draper, Philip Lapinski, Stacey Roys, Scott Wise, Maryland Rosenfeld Franklin. Comparative immunophenotypic analysis of immunogenically warm and cold syngeneic tumor models at baseline and after anti-mCTLA-4 treatment [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2019 Nov 17-20; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2020;8(3 Suppl):Abstract nr A86.
Ovarian cancer (OC) is a gynecological malignancy with a high mortality rate of over 14,000 deaths annually in the US. Although ~ 80% of OC patients initially go into remission after 1st line treatment (surgery and chemotherapy), more than 60% relapse within 16-18 months. Low neoantigen burden and the immunologically “cold” nature of OC makes it a challenging malignancy. Therefore, novel methods such as immunotherapy and/or combined therapies are essential to improve clinical outcome of patients. This work evaluated the ID8-Luc murine ovarian carcinoma model to determine sensitivity to immunomodulatory agents. C57BL/6 albino mice implanted intraperitoneally with ID8-Luc cells were evaluated for growth and sensitivity to checkpoint inhibitors anti-PD-1, anti-PD-L1, anti-CTLA-4 and the chemotherapy agent paclitaxel. Disease progression was monitored weekly by in vivo bioluminescence imaging (BLI). Baseline immune profile of the model was determined by collecting ascites from untreated mice and analyzed with panels for myeloid and lymphoid cell populations by flow cytometry. ID8-Luc cells successfully established tumors with 7-8 days doubling time, as determined by BLI, and a median overall survival time of ~ 40-50 days. Formation of ascites resulted in weight gain and extended abdomen at advanced disease state. Necropsy showed solid tumor nodules within the peritoneal cavity including involvement of pancreas, liver, spleen and abdominal wall. Baseline immune profiling of ascites indicated presence of B cells (23%), granulocytic myeloid derived suppressor cells (G-MDSCs; 15%), TAMs (25%) and a low percentage of T cells (3-4%). This was suggestive of a possibly “cold” tumor model. Paclitaxel is a front-line chemotherapy option in OC. We find that the ID8-Luc model has 60% tumor free survivors (TFS) following treatment. To determine how this model responds to immune checkpoint inhibition we tested response to anti-PD-1, anti-PD-L1 and anti-CTLA-4 antibodies. Treatment was initiated at either 7 (early) or 14 (late) days post tumor cell implant and we found that the overall response varied depending on the timing of treatment initiation. Early treatment with either anti-PD-1 or anti-PD-L1 resulted in complete regression of tumors (100% TFS) while the model was refractory to anti-CTLA-4 treatment. Late treatment initiation of anti-PD-1 or anti-PD-L1 resulted in an “all or nothing” response (40% complete response and 60% non-response). In addition, reducing the antibody dose from 10mg/kg to 5mg/kg did not make a material impact on the anti-tumor activity of these agents in the late treatment regimen. New models to further the preclinical testing of agents for OC is vital. This work characterizes the baseline immune profile of the murine ID8-Luc model along with response to checkpoint inhibitors to enable the rational design of combination strategies in the preclinical setting. Citation Format: Sumithra Urs, Sheri Barnes, Stacey Roys, Maryland R. Franklin. ID8-Luc syngeneic ovarian cancer model for preclinical evaluation of immunomodulatory molecules [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3718.
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