While viral pathogens are often subdivided into neurotropic and non-neurotropic categories, systemic inflammation caused by non-neurotropic viruses still possesses the ability to alter the central nervous system (CNS). Studies of CNS disease induced by viral infection, whether neurotropic or not, are presented with a unique set of challenges. First, because brain biopsies are rarely necessary to diagnose viral-associated neurological disorders, antemortem tissue samples are not readily available for study and human pathological studies must rely on end-stage, postmortem evaluations. Second, in vitro models fail to fully capture the nuances of an intact immune system, necessitating the use of animal models to fully characterize pathogenesis and identify potential therapeutic approaches. Non-human primates (NHP) represent a particularly attractive animal model in that they overcome many of the limits posed by more distant species and most closely mirror human disease pathogenesis and susceptibility. Here, we review NHP infection models of viruses known to infect and/or replicate within cells of the CNS, including West Nile virus, the equine encephalitis viruses, Zika virus, and herpesviruses, as well as those known to alter the immune status of the brain in the absence of significant CNS penetrance, including human immunodeficiency virus (HIV) in the current era of combination antiretroviral therapy (cART) and the coronavirus of severe acute respiratory syndrome (SARS)-CoV−2. This review focuses on viruses with an established role in causing CNS disease, including encephalitis, meningitis, and myelitis and NHP models of viral infection that are directly translatable to the human condition through relevant routes of infection, comparable disease pathogenesis, and responses to therapeutic intervention.

Hyperglycemia, and exacerbation of pre-existing deficits in glucose metabolism, are manifestations of the post-acute sequelae of SARS-CoV-2. Our understanding of metabolic decline after acute COVID-19 remains unclear due to the lack of animal models. Here, we report a non-human primate model of metabolic post-acute sequelae of SARS-CoV-2 using SARS-CoV-2 infected African green monkeys. Using this model, we identify a dysregulated blood chemokine signature during acute COVID-19 that correlates with elevated and persistent hyperglycemia four months post-infection. Hyperglycemia also correlates with liver glycogen levels, but there is no evidence of substantial long-term SARS-CoV-2 replication in the liver and pancreas. Finally, we report a favorable glycemic effect of the SARS-CoV-2 mRNA vaccine, administered on day 4 post-infection. Together, these data suggest that the African green monkey model exhibits important similarities to humans and can be utilized to assess therapeutic candidates to combat COVID-related metabolic defects.

Objective SLE is an autoimmune disease characterised by persistent inflammation and autoantibody production. Genetic predisposition and environmental factors such as a high-fat diet (HFD) may contribute to lupus development. However, the immune cell profile and gender difference in response to HFD in lupus have not been reported. Here we investigated the impact of HFD on lupus pathogenesis and autoimmunity using lupus-prone mice.Methods Thirty male and 30 female MRL/lymphoproliferation (lpr) mice were fed with regular diet (RD) or HFD. Body weights were recorded weekly. SLE progression was monitored by skin lesion, urine protein, titres of antidouble-strand DNA (dsDNA) and ANA. At week 14, kidney and skin tissue sections were stained with H&E and periodic acid–Schiff to detect histological kidney index and skin score. Splenocytes were identified by immunofluorescence staining and flow cytometry.Results HFD significantly increased body weight and lipid levels compared with RD (p<0.01). Skin lesions were observed in 55.6% of the HFD group compared with 11.1% of the RD group, with greater histopathological skin scores in the female HFD group (p<0.01). Although both male and female mice had higher serum IgG in the HFD group than in the RD group, only the male HFD group showed an increased trend in anti-dsDNA Ab and ANA titres. Kidney pathological changes in the HFD group were more severe in male mice than in female mice (p<0.05), detected by proteinuria, kidney index and glomerular cell proliferation. Significant increases of germinal centre B cells and T follicular helper cells were observed in the spleens of HFD mice (p<0.05).Conclusion HFD induced an accelerated and exacerbated lupus development and autoimmunity in MRL/lpr mice. Our results parallel many known clinical lupus phenotypes and sexual dimorphism in which male patients are likelier to have a severe disease (nephritis) than female lupus patients who may have a broader range of lupus symptoms.

Infection with the etiological agent of COVID-19, SARS-CoV-2, appears capable of impacting cognition in some patients with post-acute sequelae of SARS-CoV-2 (PASC). To evaluate neuropathophysiological consequences of SARS-CoV-2 infection, we examine transcriptional and cellular signatures in the Brodmann area 9 (BA9) of the frontal cortex and the hippocampal formation (HF) in SARS-CoV-2, Alzheimer's disease (AD), and SARS-CoV-2-infected AD individuals compared to age- and gender-matched neurological cases. Here, we show similar alterations of neuroinflammation and blood-brain barrier integrity in SARS-CoV-2, AD, and SARS-CoV-2-infected AD individuals. Distribution of microglial changes reflected by the increase in Iba-1 reveals nodular morphological alterations in SARS-CoV-2-infected AD individuals. Similarly, HIF-1α is significantly upregulated in the context of SARS-CoV-2 infection in the same brain regions regardless of AD status. The finding may help in informing decision-making regarding therapeutic treatments in patients with neuro-PASC, especially those at increased risk of developing AD.

Neurologic manifestations are among the most frequently reported complications of COVID-19. However, given the paucity of tissue samples and the highly infectious nature of the etiologic agent of COVID-19, we have limited information to understand the neuropathogenesis of COVID-19. Therefore, to better understand the impact of COVID-19 on the brain, we used mass-spectrometry-based proteomics with a data-independent acquisition mode to investigate cerebrospinal fluid (CSF) proteins collected from two different nonhuman primates, Rhesus Macaque and African Green Monkeys, for the neurologic effects of the infection. These monkeys exhibited minimal to mild pulmonary pathology but moderate to severe central nervous system (CNS) pathology. Our results indicated that CSF proteome changes after infection resolution corresponded with bronchial virus abundance during early infection and revealed substantial differences between the infected nonhuman primates and their age-matched uninfected controls, suggesting these differences could reflect altered secretion of CNS factors in response to SARS-CoV-2-induced neuropathology. We also observed the infected animals exhibited highly scattered data distributions compared to their corresponding controls indicating the heterogeneity of the CSF proteome change and the host response to the viral infection. Dysregulated CSF proteins were preferentially enriched in functional pathways associated with progressive neurodegenerative disorders, hemostasis, and innate immune responses that could influence neuroinflammatory responses following COVID-19. Mapping these dysregulated proteins to the Human Brain Protein Atlas found that they tended to be enriched in brain regions that exhibit more frequent injury following COVID-19. It, therefore, appears reasonable to speculate that such CSF protein changes could serve as signatures for neurologic injury, identify important regulatory pathways in this process, and potentially reveal therapeutic targets to prevent or attenuate the development of neurologic injuries following COVID-19.

Neuropathological complications are frequently observed in SARS‐CoV‐2 infection and brain autopsies from human subjects who died from COVID‐19 have revealed significant pathology, including wide‐spread neuroinflammation, hypoxic‐ischemic injury, and microhemorrhages. To begin to understand the neuropathogenesis of SARS‐CoV‐2 infection, we investigated brain from infected non‐human primates (NHP)s for pathological changes consistent with that seen among humans. Eight aged NHPs were inoculated with the 2019‐nCoV/USA‐WA1/2020 strain of SARS‐CoV‐2 via a multi‐route mucosal or aerosol challenge. Hematoxylin and eosin (H&E) and immunohistochemistry (IHC) staining was done on seven brain regions to elucidate general pathology, microhemorrhages, platelet derived thrombi, neuronal apoptosis, microglia and astrocyte morphology, hypoxia, and virus present. Similar to humans, pathology was variable but included wide‐spread neuroinflammation, nodular lesions, neuronal degeneration, and microhemorrhages. Neuronal degeneration was most often seen in the cerebellum and brainstem of infected animals. Neuronal death was confirmed through FluorJade C and cleaved (active) caspase 3 IHC, which showed foci of positivity, particularly among Purkinje cells of the cerebellum. Importantly, this was seen among infected animals that did not develop severe respiratory disease. Hypoxia inducible factor‐1α (HIF‐1α) was observed at a higher intensity around the vasculature within deep brain regions of the infected animals. Microhemorrhages were prevalent among all animals but were less frequently associated with platelet derived thrombi in the infected animals, as compared to mock‐infected controls. Sparse virus was detected in brain endothelial cells but did not associate with the severity of CNS injury. Increased HIF‐1α suggests that brain hypoxia may promote neuronal degeneration within infected brain. Wide‐spread neuroinflammation may also contribute to neuronal injury/death and neurological manifestations seen in the context of infection.

Infection with the etiological agent of COVID-19, SARS-CoV-2, appears capable of impacting cognition, which some patients with Post-acute Sequelae of SARS-CoV-2 (PASC). To evaluate neuro-pathophysiological consequences of SARS-CoV-2 infection, we examine transcriptional and cellular signatures in the Broadman area 9 (BA9) of the frontal cortex and the hippocampal formation (HF) in SARS-CoV-2, Alzheimer’s disease (AD) and SARS-CoV-2 infected AD individuals, compared to age- and gender-matched neurological cases. Here we show similar alterations of neuroinflammation and blood-brain barrier integrity in SARS-CoV-2, AD, and SARS-CoV-2 infected AD individuals. Distribution of microglial changes reflected by the increase of Iba-1 reveal nodular morphological alterations in SARS-CoV-2 infected AD individuals. Similarly, HIF-1α is significantly upregulated in the context of SARS-CoV-2 infection in the same brain regions regardless of AD status. The finding may help to inform decision-making regarding therapeutic treatments in patients with neuro-PASC, especially those at increased risk of developing AD. Teaser SARS-CoV-2 and Alzheimer’s disease share similar neuroinflammatory processes, which may help explain neuro-PASC.

Abstract Background/Purpose Systemic Lupus Erythematosus (SLE) is an autoimmune disease characterized by persistent inflammation, autoantibodies production, and organ damage. Genetic predisposition and environmental factors such as fat diet/obesity contribute to lupus pathogenesis. Here, we investigated the role of immune cells, especially T follicular helper (Tfh) cells and T regulatory (Treg) cells, in bridging obesity and SLE manifestations using lupus prone mice. Methods Fifty MRL/lpr mice were fed and grouped in a regular diet (RD) or high fat diet (HFD). Body weights and skin lesions were recorded weekly. Urine protein, serum IgG, anti-dsDNA antibody (Ab), and anti-nuclear Ab were detected. At week 14, kidney and skin biopsy were collected for H&E and PAS staining for histopathological lesions and quantified as kidney index and skin score. Immune cells in spleen were examined by flow cytometry and confirmed by slides staining. Results HFD induced a significant increase in body weight than RD (p<0.01). SLE features, such as skin lesions, splenomegaly, proteinuria, higher kidney index, increase of anti-dsDNA Ab and IgG titer were observed in HFD mice. There were significant increase of germinal center B cells and plasma cells in the spleen of HFD mice. The percentage of Tfh cells and the ratio of Tfh/Treg were significantly increased in HFD group (p<0.05). Conclusion HFD induced an exacerbated lupus development with dysregulated Tfh/Treg cells associated with increased of anti-dsDNA Ab in MRL/lpr mice, suggesting the central role of Tfh/Treg cells in linking HFD to autoimmunity in SLE. Intervention of healthy diet or restoring balance of Tfh/Treg cells may improve lupus symptoms and outcomes in genetically predisposed individual. Supported by None

SARS-CoV-2 infection impacts multiple organ systems, including the central nervous system (CNS). Multiple reports have described a variety of neurological manifestations associated with infection that may contribute to worsening COVID-19. The neuropathology of SARS-CoV-2 is not well understood, necessitating the development of relevant animal models for investigation. Here, we report marked neuropathology but with limited virus in the CNS of two non-human primate models (NHPs) of SARS-CoV-2 infection. Adult male and female purpose-bred Rhesus macaques (RMs; n = 4) and wild-caught African green monkeys (AGMs; n = 4) were inoculated with the 2019-nCoV/USA-WA1/2020 strain of SARS-CoV-2 via multi-route mucosal or aerosol challenge. SARS-CoV-2 nucleocapsid (SARS-N) mRNA was detected in nasal swabs within the first week of inoculation, demonstrating infection of all study animals. All animals were euthanized at the study endpoint of 4 weeks post-inoculation, with the exception of two AGMs that reached humane endpoints at 8- and 22-days post-challenge. Seven regions of the CNS were investigated for pathology and virus infection. Archival brain tissues from two non-infected adult female RMs were used as aged-matched controls. Mild, but chronic, hypoxemia with impaired gas exchange were suggested by SpO2 values that stayed at or below 95% and elevated blood CO2 in the majority of the study animals. Neuroinflammation was seen throughout the brain and brainstem but with limited virus detection by immunohistochemistry and RNAscope of fixed tissues and viral RNA detection using a highly sensitive CRISPR-fluorescent detection system on RNA extracted from sectioned brain lysates. In addition, neuronal injury and death were suggested by pyknotic and karyolytic nuclei and cellular blebbing. Limited myelin vacuolation was revealed in two infected animals through Luxol Fast Blue staining. Neuronal cleaved caspase 3 positivity was seen at a greater frequency in infected animals compared to controls, suggesting increased apoptosis in infection. Microhemorrhages were larger and more frequent among infected NHPs, as compared to controls. Neuroinflammation, neuronal injury and death, and microhemorrhages were seen in animals that did not develop severe respiratory disease and may suggest neuropathology contributes to on-going symptoms of convalesced patients. Our findings in NHPs are in agreement with human autopsy and neuroimaging studies and demonstrate this is a relevant animal model for investigating neuropathological changes associated with COVID-19. Our results also suggest that hypoxic-ischemic events leading to energy failure and neuronal injury, contribute to the neuropathological consequences of COVID-19. Further studies are warranted to elucidate the mechanisms of SARS-CoV-2 neuropathogenesis.

Abstract Background: One of the recently understood mechanisms associated with the progression of cancer is the immune checkpoint pathway. Many tumors can stimulate the expression of immune checkpoint molecules, resulting in a phenotype of exhausted T cells that cannot restrain tumor progression. One such inhibitory ligand and receptor pair in solid tumors are the programmed death-ligand 1 (PD-1) and programmed death receptor-1 (PD-L1). They prevent the killing of cancer cells by cytotoxic T-lymphocytes. PD-1 receptor is expressed by activated T cells among other cells, while PD-L1 is overexpressed on many tumor types including colorectal carcinoma (CRC). We have developed a humanized patient-derived orthotopic xenograft (hPDOX) model for CRC. Here, we investigate the potential efficacy of combination of immune checkpoint inhibitors (ICIs) and fluorouracil (5FU) in this CRC hPDOX model. Methods: Humanized mice were established by IP injection of donor peripheral blood mononuclear cells (PBMCs) into recombinase activating gene 2 (Rag2) and common cytokine receptor gamma chain gene (IL2Rγ) double knockout (Rag2-/-/IL2Rγ-/-) Rag2 mice. Luciferase-tagged patient tumor CoCaPt302 cells were injected intrarectally into Rag2 mice. Groups of mice (n=7-10) received ICIs, anti-PD-1 and anti-PD-L1 antibodies (nivolumab, 200 μg/mouse and atezolizumab, 200 μg/mouse), and 5FU (200 mg/kg) once a week for 4 weeks, IV injection alone or in combination. Tumor growth was measured weekly by bioluminescent imaging (BLI). At necropsy, tumor weights were measured. Human HLA-ABC+CD45+ hematopoietic cells and CD4+ or CD8+ T cells were detected in peritoneal lavage, blood, and spleen by flow cytometry. The presence of human immune cells (humanization) and tumor-infiltrating lymphocytes was confirmed by immunohistochemistry staining. Results: Humanization was evidenced by mouse blood containing more than 45% HLA-ABC+CD45+ human cells when tested by flow cytometry. Tumor weight showed significant reduction in group treated with combination of ICIs and 5FU compared with untreated controls (p=.0038). Tumors were also significantly smaller than ICIs (p=.0123) or 5FU (p=.0427) monotherapy groups. In addition, the lung and liver metastasis detected by ex vivo BLI were reduced from 50% of mice in control group to 33% and 11%, respectively, and exhibited better antitumor response as well as less development of metastases. CD3+ T cells, especially CD4+, CD8+ T cells in blood and spleen, were reduced in the ICIs only and combination treatment groups, but not in 5FU group compared to the controls. Conclusion: Our study provides preclinical evidence of establishment of humanized Rag2 mice to be used as a hPDOX model for CRC; and treatment efficacy including inhibiting tumor growth and distant organ metastasis was significantly enhanced through ICI and 5FU combination therapy. The effect of the combination therapy using CRC patient-derived specimens and PBMCs from MHC matched donors or autologous PBMCs will be further investigated. Citation Format: Xin Zhang, Grace Maresh, Linh Hellmers, Henry Yip, Lara McKean Baste, Heather Green, David Margolin, Li Li. Combination therapy using the programmed death receptor-1 and the programmed death-ligand 1 inhibitors and Fluorouracil in human colorectal carcinoma in a humanized patient-derived orthotopic mouse model [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 B85.

To evaluate therapeutic efficacy of immune checkpoint blockade (ICB) and conventional chemotherapy in pre-clinical models, we developed humanized patient-derived orthotopic xenograft (PDOX) models for colorectal cancer (CRC) and renal cell carcinoma (RCC). Rag2 mice were humanized by donor peripheral blood mononuclear cells (PBMC) transfer. Luciferase-tagged CRC and RCC tumor cells were injected intra-rectally and intra renal subcapsular, respectively. Groups of mice (n=5–10) received ICB, anti-PD-1 and anti-PD-L1 antibodies (Nivolumab and Atezolizumab, 200 μg each/mouse) for 4 weeks, and conventional therapy of 5FU (200 mg/kg, for CRC) alone or in combination. Tumor growth was measured by weekly bioluminescent image (BLI) and tumor weight at necropsy. The presence of human immune cells and tumor infiltrating lymphocytes were confirmed by flow cytometry and immunohistochemistry staining. Humanization was evidenced by >45% HLA-ABC+CD45+ circulating human cells in both CRC and RCC models. CRC tumor weight showed significant reduction in group treated with combination of ICB and 5FU compared with untreated controls or 5FU monotherapy group (p<0.05) using patient tumor cell CoCa302. The lung/liver metastasis detected by ex vivo BLI were reduced from 50% of mice in control group to 33% and 11%, respectively. For RCC, tumor size was reduced by >30% from controls in ICB treated group using patient tumor cell KiCa118 and cell line SN12K1. The later had 20% reduction of liver metastasis. The combination of ICB and conventional chemotherapies for CRC and RCC can be tested in our humanized PDOX mouse models. The effect of the combination therapy using MHC matched donors or patient autologous PBMC will be further investigated.

Abstract Background Rheumatoidarthritis (RA) is a autoimmune disease characterized by the accumulation of inflammatory cells in the joints, leading to hyperproliferation of synovial cells and tissue destruction. Our previous data showed autoreactive B cells could be induced by T follicular helper (Tfh) cells to undergo clonal expansion with in germinal center (GC), and ultimately differentiate into autoantibody producing plasma cells in RA. Here, we further investigated the role of Tfh cells in RA pathogenesis and the therapeutic effect of a small molecule inhibitor targeting Tfh cells (SMI-Tfh) in collagen-induced arthritis (CIA)mouse model. Methods CIA model was induced by immunization with chicken type II collagen in DBA/1 mice. Disease progression was monitored daily by the paw swollenness. Following the onset of clinical arthritis, mice were treated with SMI-Tfh. At the end of the study, blood, spleen, and affected paws were collected. Pathological changes were examined by H&E staining of tissue sections. Immunofluorescent histochemistry (IHC) staining and flow cytometry analysis were used to identify Tfh cells (CD4+CXCR5+ICOS+) in spleen and blood. Results Arthritis onset was developed at day 21 and with peak on day 42 after initial immunization. Destruction of articular cartilage, increased inflammatory cells and Tfh infiltration were observed in the synovial tissue. Mice treated with SMI-Tfh had significantly reduced Tfh cells in GC of the spleen (p<0.01) and less arthritis feature (p<0.05) in CIA mouse. Conclusion SMI-Tfh selectively inhibits GC-Tfh cells in the spleen and abrogates the severity of inflammatory arthritis. It may serve as a novel therapy by interfering with autoreactive GC-Tfh cells development in RA.

Elevated CUB-domain containing protein 1 (CDCP1) is predictive of colorectal cancer (CRC) recurrence and poor patient survival. While CDCP1 expression identifies stem cell populations that mediate lung metastasis, mechanisms underlying the role of this cell surface receptor in CRC have not been defined. We sought to identify CDCP1 regulated processes in CRC using stem cell populations, enriched from primary cells and cell lines, in extensive in vitro and in vivo assays. These experiments, demonstrating that CDCP1 is functionally important in CRC tumor initiation, growth and metastasis, identified CDCP1 as a positive regulator of Wnt signaling. Detailed cell fractionation, immunoprecipitation, microscopy, and immunohistochemical analyses demonstrated that CDCP1 promotes translocation of the key regulators of Wnt signaling, β-catenin, and E-cadherin, to the nucleus. Of functional importance, disruption of CDCP1 reduces nuclear localized, chromatin-associated β-catenin and nuclear localized E-cadherin, increases sequestration of these proteins in cell membranes, disrupts regulation of CRC promoting genes, and reduces CRC tumor burden. Thus, disruption of CDCP1 perturbs pro-cancerous Wnt signaling including nuclear localization of β-catenin and E-cadherin.

Cancer patients have poor prognoses when lymph node (LN) involvement is present in both high-grade urothelial cell carcinoma (HG-UCC) of the bladder and colorectal cancer (CRC). More than 50% of patients with muscle-invasive UCC, despite curative therapy for clinically-localized disease, will develop metastases and die within 5 years, and metastatic CRC is a leading cause of cancer-related deaths in the US. Xenograft models that consistently mimic UCC and CRC metastasis seen in patients are needed. This study aims to generate patient-derived orthotopic xenograft (PDOX) models of UCC and CRC for primary tumor growth and spontaneous metastases under the influence of LN stromal cells mimicking the progression of human metastatic diseases for drug screening. Fresh UCC and CRC tumors were obtained from consented patients undergoing resection for HG-UCC and colorectal adenocarcinoma, respectively. Co-inoculated with LN stromal cell (LNSC) analog HK cells, luciferase-tagged UCC cells were intra-vesically (IB) instilled into female non-obese diabetic/severe combined immunodeficiency (NOD/SCID) mice, and CRC cells were intra-rectally (IR) injected into male NOD/SCID mice. Tumor growth and metastasis were monitored weekly using bioluminescence imaging (BLI). Upon sacrifice, primary tumors and mouse organs were harvested, weighed, and formalin-fixed for Hematoxylin and Eosin and immunohistochemistry staining. In our unique PDOX models, xenograft tumors resemble patient pre-implantation tumors. In the presence of HK cells, both models have high tumor implantation rates measured by BLI and tumor weights, 83.3% for UCC and 96.9% for CRC, and high distant organ metastasis rates (33.3% detected liver or lung metastasis for UCC and 53.1% for CRC). In addition, both models have zero mortality from the procedure. We have established unique, reproducible PDOX models for human HG-UCC and CRC, which allow for tumor formation, growth, and metastasis studies. With these models, testing of novel therapeutic drugs can be performed efficiently and in a clinically-mimetic manner.

Background: The immune system plays an important role in tumor immune surveillance and progression. Immune checkpoint blockade is a new approach for cancer immunotherapy. The programmed death receptor-1 (PD-1) and the programmed death-ligand 1 (PD-L1) are immune checkpoint molecules and their expression results in negative regulation of T-cells primarily within the tumor microenvironment by preventing the killing of cancer cells by cytotoxic T-lymphocytes. Antibody blocking of the PD-1/PD-L1 signal exhibits promising therapeutic effects in non-small cell lung cancer and melanoma in patients. Due to its early success, more trials have been conducted to evaluate their efficacy for different tumors. We have developed a patient-derived orthotopic xenograft mouse model for colorectal carcinoma (CRC). Here, we investigate the potential efficacy of PD-1/PD-L1 blockade in our humanized orthotopic mouse models for human CRC. Methods: All studies were conducted under approved guidelines of the Institutional Animal Care and Use Committee and the Investigative Review Board of Ochsner Clinic Foundation. Humanized mice were established by intraperitoneal injection of donor human peripheral blood mononuclear cells (PBMCs) into recombinase activating gene 2 (Rag2) and common cytokine receptor gamma chain gene (IL2Rγ) double knockout (Rag2-/-/IL2Rγ-/-) Rag2 mice. Luciferase-tagged CRC cells were injected intra-rectally into Rag2 mice. One group of mice received a combination of anti-PD-1 and anti-PD-L1 antibodies (nivolumab, 200 µg/mouse and atezolizumab, 200 µg/mouse, intravenous injection) once a week for 3 weeks. Tumor growth was measured weekly by bioluminescent imaging (BLI). At necropsy, the CRC tumor weights were measured. Human CD45+ hematopoietic cells, CD4+ and CD8+ T-cells, and CD20+ B cells were detected in peritoneal lavage, blood, and tumor by flow cytometry. The presence of human immune cells (humanization) and tumor-infiltrating human lymphocytes was further confirmed by immunohistochemistry staining on paraffin-embedded tissue slides of mouse spleen and tumor, respectively. Results: Blood from mice receiving human PBMCs contained on average 31.4% CD45+ human cells (n=9) when tested by FACS analysis. Smaller tumor growth was observed in mice given PBMCs. This may be due to alloreactivity based on the recognition of MHC alloantigens in the transplanted tumor cells. However, mice further treated with the combination therapy of anti-PD-1 and anti-PD-L1 antibodies exhibited better antitumor response as well as less development of lung metastases compared to untreated controls. On average, tumor weight was reduced by 36% and lung metastases measured by ex-vivo BLI was reduced by 82% (n=5). In addition, circulating CD326+ tumor cells were reduced by 21% and CD3, CD4, CD8 and CD20 positive human lymphocytes were also reduced in the combination treatment group. Conclusion: Our study provides preclinical evidence of establishment of humanized Rag2 mice to be used as an orthotopic xenograft model for CRC; and treatment benefit for CRC through targeting immune checkpoint molecules PD-1 and PD-L1. The effect of immune checkpoint blockade in combination with conventional chemotherapy using CRC patient-derived specimens and PBMCs from MHC matched donors or autologous PBMCs will be further investigated. Citation Format: Li Li, Xin Zhang, Grace Maresh, Linh Hellmers, Avi Patel, Ravan Moret, Sarah Cohen, David Margolin. Antitumor effects of the programmed death receptor-1 and the programmed death-ligand 1 blockade in human colorectal carcinoma in a humanized orthotopic mouse model [abstract]. In: Proceedings of the Fourth CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; Sept 30-Oct 3, 2018; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2019;7(2 Suppl):Abstract nr B027.

Levine, David; Hellmers, Linh; Maresh, Grace PhD; Zhang, Xin MD, PhD; Moret, Ravan MS; Green, Heather J. MS; Margolin, David A. MD; Li, Li MD, PhD Author Information

Abstract Background Rheumatoidarthritis (RA) is an inflammatory autoimmune disease characterized by T cell infiltration in the joints and autoantibody production. T follicular helper (Tfh) cells are a unique subset of CD4+ T cells regulating antibody production in B cell follicle. Our previous studies have showed that increased circulating Tfh cells were correlated with anti-CCP antibody titer and disease activity in active RA patients, indicating that Tfh cells may play an important role in RA pathogenesis. Here we investigate the therapeutic potential of a small molecule inhibitor targeting Tfh cells (SMI-Tfh) in mice with collagen-induced arthritis (CIA). Methods CIA was induced in DBA/1 mice by immunization with chicken type II collagen. Following the onset of clinical arthritis, mice were treated with SMI-Tfh (50mg/kg/day) for 10 days. Arthritis progression was monitored daily and recorded by the paw swollenness. Blood, spleen, and affected paws were collected at the end of the study. Tfh cells were examined and defined by CD4+CXCR5+ICOS+via flow cytometry and further confirmed by immunohistochemistry staining in spleen. Results Mice developed arthritis four weeks after immunization with type II collagen. Treatment with SMI-Tfh significantly reduced the disease progression/activity in mice with CIA. SMI-Tfh significantly inhibited the frequency of Tfh cells in spleen (P<0.01), but not the frequency of circulating Tfh cells in CIA mice (P>0.05). Conclusion The small molecule inhibitor SMI-Tfh selectively inhibits Tfh cells and abrogates progression/activity of inflammatory arthritis in CIA mouse model. Treatment with SMI-Tfh in CIA mice provides a potential strategy for joint protection and may be beneficial in RA patients.

High-grade urothelial cell carcinoma of the bladder has a poor prognosis when lymph nodes are involved. Despite curative therapy for clinically-localized disease, over half of the muscle-invasive urothelial cell carcinoma patients will develop metastases and die within 5 years. There are currently no described xenograft models that consistently mimic urothelial cell carcinoma metastasis. To develop a patient-derived orthotopic xenograft model to mimic clinical urothelial cell carcinoma progression to metastatic disease, the urothelial cell carcinoma cell line UM-UC-3 and two urothelial cell carcinoma patient specimens were doubly tagged with Luciferase/RFP and were intra-vesically (IB) instilled into NOD/SCID mice with or without lymph node stromal cells (HK cells). Mice were monitored weekly with bioluminescence imaging to assess tumor growth and metastasis. Primary tumors and organs were harvested for bioluminescence imaging, weight, and formalin-fixed for hematoxylin and eosin and immunohistochemistry staining. In this patient-derived orthotopic xenograft model, xenograft tumors showed better implantation rates than currently reported using other models. Xenograft tumors histologically resembled pre-implanted primary specimens from patients, presenting muscle-invasive growth patterns. In the presence of HK cells, tumor formation, tumor angiogenesis, and distant organ metastasis were significantly enhanced in both UM-UC-3 cells and patient-derived specimens. Thus, we established a unique, reproducible patient-derived orthotopic xenograft model using human high-grade urothelial cell carcinoma cells and lymph node stromal cells. It allows for investigating the mechanism involved in tumor formation and metastasis, and therefore it is useful for future testing the optimal sequence of conventional drugs or the efficacy of novel therapeutic drugs.