A dendritic cell (DC)-based vaccine, Sipuleucel-T, remains the sole FDA-approved cancer vaccine. Despite their established safety and efficacy against cancers and infections in numerous trials, long-term clinical benefits have been modest. Most trials have employed DCs derived from blood monocytes, but emerging evidence underscores the unique role of conventional type 1 DCs (cDC1) in triggering potent antitumor immune responses and their intratumoral infiltration with favorable prognoses in many cancers. However, the scarcity of cDC1s in peripheral blood and the challenges in generating them in vitro have hindered a deeper understanding of their biology and their widespread application as cellular vaccines. In this study, we present a serum-free culture system capable of generating billions of human cDC1s from CD34+ progenitors derived from cord or peripheral blood. The system leverages the requirement of Notch signaling for cDC1 differentiation and generates DCs that closely resemble in vivo cDC1s, exhibiting functions including cellular antigen cross-presentation. This robust protocol enables the scalable production of cDC1s for both fundamental biological research and therapeutic applications.

Understanding the breadth and functional profile of T cell responses is crucial for assessing their role in immune surveillance of emerging SARS-CoV-2 variants. Sampling healthy individuals, we profiled the kinetics and polyfunctionality of T cell immunity elicited by mRNA vaccination. Modeling of anti-spike T cell responses against ancestral and variant strains suggested epitope immunodominance and cross-reactivity as major predictive determinants of T cell immunity. To identify immunodominant epitopes, we comprehensively mapped CD4+ and CD8+ T cell epitopes within non-spike proteins using samples from convalescent patients. We found that immunodominant epitopes mainly resided within regions that were minimally disrupted by emerging mutations. Conservation analysis across human coronaviruses and in silico alanine scanning highlighted the functional importance of mutationally constrained immunodominant regions. Collectively, these findings identify immunodominant T cell epitopes across the SARS-CoV-2 proteome that may enhance immune surveillance against emerging variants and inform next-generation vaccine designs providing broader and more durable protection.

ANOVA with multiple comparisons and Tukey's post-hoc test

Overlapping peptides covering the entire NY-ESO-1 protein

Immunophenotyping of the SDR rat. Analysis of SDR whole blood demonstrates greatly diminished T and B cells compared to the wild-type rat. A, The wild-type rat has a significant population of circulating CD4+, CD8+, and CD4/CD8 double positive T cell populations compared to the SDR rat. B, The wild-type rat has over 20% B220/IgM double positive mature B cells, compared to 3.5% in the SDR rat. C, The SDR rat has slightly less circulating NK cells, unlike what is observed in the spleen. Panels are representative of 3 wild-type and 3 SDR rats. Left panels: wild-type rat. Right panels: SDR rat.

Western Blot analysis for RAG2 protein expression in SDR rats. Rat thymus and spleen lysates were loaded onto a 7.5% SDS-PAGE gel and probed with a RAG2 polyclonal antibody at a dilution of 1:1000.

Descriptions of Supplementary Figures 1, 2, and 3, and supplementary table 1

Karyotype analysis for Spermatogonia at passage 16, before transfection with XTNs demonstrating a normal male 40, XY karyotype.

Pattern recognition receptors (PRRs) protect against microbial invasion by de-tecting specific molecular patterns found in pathogens and initiating an immune response. Although microbial-derived PRR ligands have been extensively charac-terized, the contribution and relevance of endogenous ligands to PRR activation remains overlooked. Here, we characterize the landscape of endogenous ligands that engage RIG-I-like receptors (RLRs) upon infection by different RNA viruses. In each infection, several RNAs transcribed by RNA polymerase III (Pol3) specif-ically engaged RLRs, particularly the family of Y RNAs. Sensing of Y RNAs was dependent on their mimicking of viral secondary structure and their 5'-triphos-phate extremity. Further, we found that HIV-1 triggered a VPR-dependent down -regulation of RNA triphosphatase DUSP11 in vitro and in vivo, inducing a tran-scriptome-wide change of cellular RNA 5'-triphosphorylation that licenses Y RNA immunogenicity. Overall, our work uncovers the contribution of endoge-nous RNAs to antiviral immunity and demonstrates the importance of this pathway in HIV-1 infection.

The presence of an immunosuppressive tumor microenvironment is a major obstacle in the success of cancer immunotherapies. Because extracellular matrix components can shape the microenvironment, we investigated the role of matrix metalloproteinase 2 (MMP2) in melanoma tumorigenesis. We found that MMP2 signals proinflammatory pathways on antigen presenting cells, and this requires both TLR2 and TLR4. B16 melanoma cells that express MMP2 at baseline have slower kinetics in Tlr2(-/-) Tlr4(-/-) mice, implicating MMP2 in promoting tumor growth. Indeed, Mmp2 overexpression in B16 cells potentiated rapid tumor growth, which was accompanied by reduced intratumoral cytolytic cells and increased M2 macrophages. In contrast, knockdown of Mmp2 slowed tumor growth and enhanced T cell proliferation and NK cell recruitment. Finally, we found that these effects of MMP2 are mediated through dysfunctional DC-T cell cross-talk as they are lost in Batf3(-/-) and Rag2(-/-) mice. These findings provide insights into the detrimental role of endogenous alarmins like MMP2 in modulating immune responses in the tumor microenvironment.

Abstract Given its ability to induce both humoral and cellular immune responses, NY-ESO-1 has been considered a suitable antigen for a cancer vaccine. Despite promising results from early-phase clinical studies in patients with melanoma, NY-ESO-1 vaccine immunotherapy has not been widely investigated in larger trials; consequently, many questions remain as to the optimal vaccine formulation, predictive biomarkers, and sequencing and timing of vaccines in melanoma treatment. We conducted an adjuvant phase I/II clinical trial in high-risk resected melanoma to optimize the delivery of poly-ICLC, a TLR-3/MDA-5 agonist, as a component of vaccine formulation. A phase I dose-escalation part was undertaken to identify the MTD of poly-ICLC administered in combination with NY-ESO-1 and montanide. This was followed by a randomized phase II part investigating the MTD of poly-ICLC with NY-ESO-1 with or without montanide. The vaccine regimens were generally well tolerated, with no treatment-related grade 3/4 adverse events. Both regimens induced integrated NY-ESO-1–specific CD4+ T-cell and humoral responses. CD8+ T-cell responses were mainly detected in patients receiving montanide. T-cell avidity toward NY-ESO-1 peptides was higher in patients vaccinated with montanide. In conclusion, NY-ESO-1 protein in combination with poly-ICLC is safe, well tolerated, and capable of inducing integrated antibody and CD4+ T-cell responses in most patients. Combination with montanide enhances antigen-specific T-cell avidity and CD8+ T-cell cross-priming in a fraction of patients, indicating that montanide contributes to the induction of specific CD8+ T-cell responses to NY-ESO-1.

Microsatellite instability-high (MSI-H) tumors are an important model system for evaluating neoantigen-based immunotherapies given their high tumor mutation burden and response to checkpoint blockade. We identified tumor-specific, frameshift peptides, encoding multiple epitopes that originated from indel mutations shared among patients with MSI-H endometrial, colorectal and stomach cancers. Epitopes derived from these shared frameshifts have high population occurrence rates, wide presence in many tumor subclones and are predicted to bind to the most frequent HLA alleles in the TCGA MSI-H patient cohorts. Neoantigens arising from these mutations are more dissimilar to both self and viral antigens, indicating the creation of peptides, that, when translated, can present truly novel antigens to the immune system. Finally, we validated the immunogenicity of common frameshift peptides from MSI-H endometrial patients in an array of T cell stimulation experiments, using peripheral blood mononuclear cells isolated from healthy donors. Our study describes for the first time the widespread occurrence and strong immunogenicity of tumor-specific antigens, derived from shared frameshift mutations in MSI-H cancer and Lynch syndrome patients, suitable for the design of common preventive “off-the-shelf” cancer vaccines.

We have created the immunodeficient SRG rat, a Sprague-Dawley Rag2/Il2rg double knockout that lacks mature B cells, T cells, and circulating NK cells. This model has been tested and validated for use in oncology (SRG OncoRat®). The SRG rat demonstrates efficient tumor take rates and growth kinetics with different human cancer cell lines and PDXs. Although multiple immunodeficient rodent strains are available, some important human cancer cell lines exhibit poor tumor growth and high variability in those models. The VCaP prostate cancer model is one such cell line that engrafts unreliably and grows irregularly in existing models but displays over 90% engraftment rate in the SRG rat with uniform growth kinetics. Since rats can support much larger tumors than mice, the SRG rat is an attractive host for PDX establishment. Surgically resected NSCLC tissue from nine patients were implanted in SRG rats, seven of which engrafted and grew for an overall success rate of 78%. These developed into a large tumor volume, over 20,000 mm3 in the first passage, which would provide an ample source of tissue for characterization and/or subsequent passage into NSG mice for drug efficacy studies. Molecular characterization and histological analyses were performed for three PDX lines and showed high concordance between passages 1, 2 and 3 (P1, P2, P3), and the original patient sample. Our data suggest the SRG OncoRat is a valuable tool for establishing PDX banks and thus serves as an alternative to current PDX mouse models hindered by low engraftment rates, slow tumor growth kinetics, and multiple passages to develop adequate tissue banks.

Pattern recognition receptors (PRRs) protect against host invasion by detecting specific molecular patterns found in pathogens and initiating an immune response. While microbial-derived PRR ligands have been extensively characterized, the contribution and relevance of endogenous ligands to PRR activation during viral infection remain overlooked. In this work, we characterize the landscape of endogenous ligands that engage RIG-I-like receptors (RLRs) upon infection by a positive-sense RNA virus, a negative-sense RNA virus or a retrovirus. We found that several endogenous RNAs transcribed by RNA polymerase 3 (Pol3) specifically engage RLRs, and in particular the family of small non-coding repeats Y-RNAs, which presents the highest affinity as RIG-I ligands. We show that this recognition is dependent on Y-RNA mimicking viral secondary structure and its 5’-triphosphate extremity. Further, we found that HIV-1 infection triggers a VPR-dependent downregulation of RNA triphosphatase DUSP11 in vitro and in vivo , leading to an increase of Y-RNA 5’-triphosphorylation that enables their immunogenicity. Importantly, we show that altering DUSP11 expression is sufficient to induce a type-I interferon and T cell activation transcriptional program associated with HIV-1 infection. Overall, our work uncovers the critical contribution of endogenous repeat RNAs ligands to antiviral immunity and demonstrates the role of this pathway in HIV-1 infection.

Abstract The rat is the preferred model for toxicology studies, and it offers distinctive advantages over the mouse as a preclinical research model including larger sample size collection, lower rates of drug clearance, and relative ease of surgical manipulation. An immunodeficient rat would allow for larger tumor size development, prolonged dosing and drug efficacy studies, and preliminary toxicologic testing and pharmacokinetic/pharmacodynamic studies in the same model animal. Here, we created an immunodeficient rat with a functional deletion of the Recombination Activating Gene 2 (Rag2) gene, using genetically modified spermatogonial stem cells (SSC). We targeted the Rag2 gene in rat SSCs with TALENs and transplanted these Rag2-deficient SSCs into sterile recipients. Offspring were genotyped, and a founder with a 27 bp deletion mutation was identified and bred to homozygosity to produce the Sprague-Dawley Rag2 - Rag2tm1Hera (SDR) knockout rat. We demonstrated that SDR rat lacks mature B and T cells. Furthermore, the SDR rat model was permissive to growth of human glioblastoma cell line subcutaneously resulting in successful growth of tumors. In addition, a human KRAS-mutant non–small cell lung cancer cell line (H358), a patient-derived high-grade serous ovarian cancer cell line (OV81), and a patient-derived recurrent endometrial cancer cell line (OV185) were transplanted subcutaneously to test the ability of the SDR rat to accommodate human xenografts from multiple tissue types. All human cancer cell lines showed efficient tumor uptake and growth kinetics indicating that the SDR rat is a viable host for a range of xenograft studies. Mol Cancer Ther; 17(11); 2481–9. ©2018 AACR.

Abstract Mouse models of human cancer have paved the way for studying cancer biology and genomics and their effects on cancer growth kinetics, propensity for metastasis, and treatment response. In addition, human cancer xenografts provide the opportunity to study cancer cell interactions with host stroma and tumor morphology. A plethora of genetically immunodeficient mouse models exist with different immune phenotypes, resulting in significant variability in tumor take rates and growth kinetics for a wide range of human cancer cell lines and patient-derived xenografts (PDX). Inconsistent or poor growth in these immunodeficient models have made downstream analysis and drug efficacy testing difficult. As a result, a significant number of mice are needed for drug efficacy screening to achieve a cohort of animals with tumors of similar size with similar tumor growth kinetics for treatment. It is possible that these cell lines might grow more consistently in a different immunodeficient model, such as an immunodeficient rat. Until recently, the only immunodeficient rat that existed was the nude (NIH-Foxn1rnu; RNU) rat. This rat lacks T cells, but maintains a normal repertoire of all other immune cells, including B and NK cells. As such, there are a limited number of human cancer cell lines that can survive in the nude rat. We have created a genetically modified rat with a functional mutation of the Rag2 gene (Sprague Dawley – Rag2 null; SDR), resulting in a loss of mature B and T cells. In addition, we have created a Rag2/Il2rg double knockout rat (Sprague Dawley – Rag2; Il2rg null; SRG) that lacks mature B cells, T cells, and has fewer NK cells than wild-type Sprague Dawley rats. We have shown that the SDR rat is permissive for solid tumor growth of the human acute lymphocytic leukemia REH cell line, human glioblastoma U87MG cell line, human non-small cell lung cancer H358 cell line, and cell lines derived from ovarian and endometrial PDX samples. In some cases, tumor growth kinetics are superior in the SDR rat compared with immunodeficient mouse models. We have demonstrated that the human prostate cancer cell line, VCaP, which has poor engraftment efficiency and growth kinetics in the mouse, grows well in the SRG rat. The SRG rat is currently being validated for growth kinetics of other human cancer cell lines and PDX tissues. In addition, we are developing several disseminated human leukemia models in the SRG rat and creating immune-humanized mice and rats to be used in conjunction with human tumor xenografts for immunotherapy efficacy studies. Citation Format: Fallon K. Noto, Kamesh Ravi, Angela Arey, Christopher McClain, Wei Zhang, Goutham Narla, Jack Crawford, Tseten Yeshi. Novel immunodeficient rat models capable of supporting the growth of human tumor xenografts [abstract]. In: Proceedings of the AACR Special Conference: Advances in Modeling Cancer in Mice: Technology, Biology, and Beyond; 2017 Sep 24-27; Orlando, Florida. Philadelphia (PA): AACR; Cancer Res 2018;78(10 Suppl):Abstract nr B36.

Immunodeficient mouse models seek to recapitulate the clinical features of advanced human cancers. However, drug efficacy testing and downstream analysis such as pharmacokinetic (PK) /pharmacodynamic (PD)-based studies are limited because of variability in tumor growth and kinetics, limited tumor growth potential, requiring the enrollment of larger numbers of mice in each treatment group to achieve required cohort sizes. Considering the distinctive advantages of rats in terms of their larger size, and ease of sampling at different time points for drug efficacy and clinical testing, we have developed unique immunodeficient rat models: the Rag2 knockout SDRTM rat (absence of mature B and T cells) and the Rag2/Il2rg double knockout SRGTM rat (absence of mature B and T cells, and lower NK cells) on a Sprague- Dawley background. Our group has previously demonstrated higher engraftment, better tumor kinetics, and increased tumor volume of xenografts in these rat models compared to available immunodeficient mouse strains for various cancer xenograft models. The purpose of the present study is to demonstrate the feasibility of drug efficacy studies using targeted molecular agents directed against cancer-associated drivers in our newly developed SDR and SRG rat models. We tested the efficacies of a combination treatment of an AKT inhibitor (MK2206) and MEK inhibitor (AZD6244), and a small-molecule activator of PP2A (SMAP) (DT-061) in a SDR rat xenograft model of human KRAS-mutant non-small cell lung cancer. 1 x 106 H358 cells were transplanted subcutaneously on the left hind flank in SDR rats. When tumor size reached 100-150 mm3, the rats were randomized to either control or treatment groups. The treatment groups received either a combination of AZD6244 with MK2206, or DT-061, and the vehicle-only control received n, n-dimethylacetamide + Solutol®/Kolliphor® HS 15, twice daily for 30 days by oral gavage. Both the kinase-inhibitors combination and SMAP treatment inhibited the growth of lung tumor xenograft in the SDR rats as shown by decreased mean tumor volume and fold changes in tumor volume compared to the control group. The combination treatment dose was just as effective at half the dose reported in previous mouse studies, demonstrating that lower dosing is sufficient for efficacy testing in rats. Importantly, no mortality, behavioral abnormalities, or changes in body weight were observed during the study, indicating a favorable tolerability profile in this model. We have also demonstrated that a human prostate cancer cell line VCaP, which has very poor engraftment and growth profiles in existing mouse models, grows well in our SRG rats. The SRG rat is now being used to study the chemotherapeutic efficacy of the benchmark androgen receptor inhibitor enzalutamide, in this VCaP tumor xenograft model. In addition, weekly blood sampling to measure PSA levels in the serum is being performed in the VCaP tumor xenograft efficacy study to demonstrate the flexibility of frequent sampling in the rat model to evaluate correlations between tumor growth and serum PSA levels. Citation Format: Kamesh Ravi, Fallon Noto, Christopher McClain, Angela Arey, Goutham Narla, Jack Crawford, Tseten Yeshi. Novel immunodeficient rat models offer a unique platform for drug efficacy studies in human tumor xenografts [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2017 Oct 26-30; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2018;17(1 Suppl):Abstract nr B170.

Abstract Animal models of human cancer offer the potential to study human tumor growth kinetics, genetic variance among human cancers, and provide in vivo platforms for drug efficacy testing. In particular, immunodeficient mouse models have been invaluable in modeling a wide range of human cancers. However, some cancer lines don’t grow well in the available mouse models or show variability in growth kinetics from mouse to mouse, making drug efficacy studies difficult due to differences in tumor size at the onset of treatment. These challenges are also seen in patient derived xenograft (PDX) models, in addition to long timeframes to obtain sufficient mice with PDX tissue growth for drug efficacy studies. Mice are also limited in tumor growth potential with regard to humane endpoints and small size also limits the volume of blood that can be collected for analysis. An immunodeficient rat model could provide a solution to some of these issues. A rat model would allow for larger tumor size, easier surgical manipulation, and greater volume of tissue and blood sampling for downstream analysis. In addition, large tumors from rats could be serially transplanted into mice for drug efficacy testing and could provide a large number of transplanted mice in a shorter period of time compared with serially transplanting from mouse to mouse. We have created an immunodeficient rat model with a functional deletion of the Rag2 gene. This knockout, created using spermatogonial stem cells, lacks mature B and T cells. To assess the capability of the Rag2 knockout rat to accept human xenografts, we transplanted 2 commercially available human cancer cell lines into our animals. The human REH acute lymphocytic leukemia cell line was transplanted via intravenous injection and the human glioblastoma cell line U87MG was transplanted subcutaneously. Both cell lines survived in the Rag2 knockout rat and resulted in the growth of tumors comprised of human cells. Studies are underway to characterize the Rag2 knockout rat’s ability to grow other human cell lines, including those that do not grow well in mice, and PDX tissues. Citation Format: Fallon K. Noto, Angela Arey, Christopher McClain, Wei Zhang, Tseten Yeshi. A novel immunodeficient rat for modeling human 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 807. doi:10.1158/1538-7445.AM2017-807

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