bluuebunny/crossref_metadata_2025 huggingface dataset:
High-dimensional profiling of tumor-infiltrating CD4+T cells in Human cancer: Are they all tumor-specific?
Authors: Li Shamin , Simoni Yannick , Zhuang Summer , Heit Antje , Tan Iain , Tan WS Daniel , Newell Evan
Published: January 2023

Tumor-specific T cells act as major actors in underpinning effective immune checkpoint-blockade therapies in cancer, but most of the studies focus on CD8+ tumor-infiltrating lymphocytes (TILs). Although roles of CD4+ T cells and especially Tregs in cancer have been established, the basis for their phenotypic heterogeneity in human tumor infiltrates is not well determined. Here we profile ex-vivounexpanded CD4+ TILs in human lung and colorectal cancer using single-cell and mass-cytometry analysis. Surprisingly, up to 80% of CD4+ TILs are composed of non-Treg cells (FoxP3−). Non-Treg CD4+ TILs are highly heterogeneous and consist of different populations of effector (CD45RO+), cytotoxic (Granzyme B+), senescent (CD57+) and exhausted cells at different levels (PD-1+/++, CTLA-4+/−, TIGIT+, CD39+/−). Recently, we showed that the surface marker CD39 can accurately discriminate between bystander (CD39−) and tumor-specific (CD39+) CD8+ TILs. Across all tumors tested, more than 90% of Tregs cells expressed CD39. However, frequencies of CD39+ among non-Treg CD4+ TILs varied dramatically from 0% to 90% and strongly correlated (r2=0.8) with the frequency of CD39+CD8+ TILs in our cohort of patients. These observations suggest the presence of bystander CD4+ TILs and a high degree of coordination between the tumor-specific CD4 and CD8 T cell responses. Using in-vitroassay, we aim to confirm the presence of these bystander CD4+ TILs specific for cancer-unrelated antigens (HCMV, EBV, Influenza). Overall, our findings could highlight that not all CD4+ TILs are tumor-specific and could suggest measuring CD39 expression as a straightforward way to quantify or isolate tumor-specific T cells, thus opening new diagnostic and therapeutic avenues.
In silico assessment of somatic mutation generated T cell epitopes in various subsets of Acute Myeloid Leukemia
Authors: Loo Christopher P
Published: January 2023

The mechanisms of targeted-immunotherapy are still at the beginning stages for blood tumors. In the current era of big data and genomic sequencing, evolving bioinformatic tools are need for the validation and prediction of current and future immunotherapies. In particular, many neoepitope prediction programs do not consider germline context or the potential for co-occurrence of two or more somatic variants on the same mRNA transcript in the context of DNA-seq of complementary tumor and normal patient samples. Without consideration of these phenomena, existing approaches are likely to produce both false positive and false negative results, resulting in an inaccurate and incomplete picture of the cancer neoepitope landscape. In this study, we investigate the neoepitope landscape in largest-to-data dataset on acute myeloid leukemia (AML) from the Beat AML program (lead by OHSU), a cohort of 562 patients. Using a novel computational pipeline Neoepiscope (derived by Reid Thompson, MD PhD, OHSU) to chiefly address this issue for single nucleotide variants (SNVs) and insertions/deletions (indels), and herein illustrate how germline and somatic variant phasing affects neoepitope prediction. We hypothesize that in silico neoepitope prediction will accurately predict viable novel peptides that can be used for target immunotherapy in AML.
Multiplex Immunofluorescence Image Cytometry Combined with Spatially-Resolved UMAP Defines Novel Immune Prognostic Biomarkers in Metastatic Melanoma
Authors: Giraldo-Castillo Nicolas A , Berry Sneha , Stein Julie , Green Benjamin , Nguyen Peter , Soni Abha , Succaria Farah , Ates Deniz , Ogurtsova Aleks , Xu Haiying , Danilova Ludmila , Baras Alex , Taube Janis
Published: January 2023

Multiplex IF (mIF) provides a detailed characterization of spatial relationships and complex cell phenotypes in the tumor microenvironment. However, the data-analysis and visualization is complex and time-consuming. Here, we developed a platform to analyze mIF data through flow cytometry workflows (image cytometry), while maintaining spatial information, and applied it to tissue microarrays of metastatic melanoma specimens (n=93; 6-plex mIF panel: PD-1, PD-L1, CD163, CD8, FoxP3, Sox10/S100). Then, we used a UMAP-based approach driven by cell-to-cell distances (rather than fluorescence intensity) to display and analyze geographic organization and cell interactions. Our pipeline provided equivalent results to the digital pathology gold standard with faster run times (5-fold reduction) and higher reproducibility. We identified key prognostic immune variables, including CD8PD1Low and CD8PD1Neg cells which associated with longer overall survival (OS, both p<0.01), and CD163PDL1Neg cells which associated with shorter OS (p=0.001). The spatial UMAPs showed that PD-L1 and PD-1 intensities were spatially encoded, and their expression on distinct cell subsets was organized in geographic clusters. Specifically, PD-L1Hi cells co-located to areas of CD8 cells, and PD-1Hi cells were observed near dense collections of tumor cells. Spatial UMAP subtraction analysis (survivors vs. non-survivors at 5 years) identified geographic and co-expression signatures associated with improved prognosis, i.e. CD8-driven PD-L1 expression and lacking CD163PDL1Neg macrophages. These data demonstrate the use of image cytometry and spatial UMAPs for improved visualization and interpretation of single-cell, spatially-resolved mIF data.
Myeloid Response Score (MRS): A Simple Signature That Reveals the Myeloid Contexture and Predicts the Prognosis of Hepatocellular Carcinoma
Authors: Wu Chong , Lin Jie , Weng Yulan , Zeng Dan-Ni , Zheng Limin
Published: January 2023

Myeloid cells, including monocytes/macrophages (Mo/Mϕ), neutrophils, and dendritic cells, can mediate divergent immune responses according to different pathological conditions. In cancer, myeloid cells play important roles in disease progression and therapy, suggesting that appraising the overall and exact intratumoral myeloid response may provide valuable information that may help to predict cancer prognosis and tailor more precise therapies. In this study, we screened biomarkers that cover major myeloid subtypes in human tumors, then constructed and validated a simple prognostic signature, called Myeloid Response Score (MRS), based on a dataset from a multi-center cohort of 1,177 HCC patients. Using multiplex immunofluorescence staining, we observed a shift of predominant myeloid population from CD169+, CD169−CD11b−CD14+, and CD169−CD11b−CD163+ Mo/Mϕs in MRSlow HCC to neutrophils and CD11b+CD169− Mo/Mϕs in MRShigh tumor. Transcriptomic and flow cytometric analyses showed that MRShigh HCC exhibited patterns of CD8+ T cell exhaustion including impaired cytotoxicity and increased PD-1 expression, suggesting highly suppressive immune microenvironment. The “climb trajectory” of MRSs between HCC tumors were depicted using the TCGA dataset, and was linked to CD8+ T cell dysfunction and poorer survival of patients. Moreover, MRS-based nomograms were highly discriminative, accurate, and useful in predicting HCC prognosis and we provide additional evidence that MRS may associate with the efficacies of post-recurrence sorafenib and transcatheter arterial chemoembolization (TACE) treatments. Together, we constructed and validated a new myeloid signature with valuable immunological and clinical potential.
Exploring the main mechanisms of anti-tumor and immunotherapy-related adverse events for safer and effective anti-CTLA-4 immunotherapy
Authors: Du Xuexiang , Liu Mingyue , Zhang Yan , Tang Fei , Zheng Pan , Liu Yang
Published: January 2023

Targeting CTLA-4 has shown remarkable long-term benefit and thus remains a valuable tool for cancer immunotherapy if the immunotherapy-related adverse effects (irAEs) can be brought under control. Here, we systematically studied the immunotherapeutic effect of anti-human CTLA-4 mAbs and suggest that Fc/Fc receptor mediated intra-tumor Treg depletion rather than the widely held mechanism of checkpoint blockade contribute to the cancer immunotherapeutic effect, which raised the reappraisal of CTLA-4 checkpoint blockade in cancer immunotherapy. Meanwhile, an animal model, which recapitulates clinical irAE and cancer immunotherapeutic effect (CITE), would be valuable for developing safer CTLA-4-targeting reagents. We established such an irAEs model and found that the Ipilimumab (or TremeIgG1) induced severe irAEs especially when combined with an anti-PD-1 antibody; whereas HL12/HL32 induced comparable CITE with very mild irAE under the same conditions. Furthermore we tried to uncouple the undesired irAEs from the beneficial CITE by exploring the mechanism of irAEs of anti-CTLA-4 antibodies in our irAEs model, and found that while irAE-prone Ipilimumab and TremeIgG1 rapidly direct cell surface CTLA-4 for lysosomal degradation, the non-irAE-prone antibodies we generated, HL12 or HL32, dissociate from CTLA-4 after endocytosis and allow CTLA-4 recycling to cell surface, increasing the pH sensitivity of TremeIgG1 by introducing designed tyrosine-to-histidine mutations prevents antibody-triggered lysosomal CTLA-4 downregulation and dramatically attenuates irAE. Our data establish a new paradigm for cancer research that allows for abrogating irAE while increasing CITE of anti-CTLA-4 antibodies.
A tumor-infiltrating IL23R-expressing CD4+ Treg population represents a safe and potent immunotherapeutic candidate in mice
Authors: Wight Andrew E , Sido Jessica Margaret , Degryse Sandrine , Oseghali Oba , Ao Lin , Nakagawa Hidatoshi , Kim Hye-Jung , Cantor Harvey
Published: January 2023

Interleukin 23 receptor (IL23R) is a signaling protein normally implicated in gut T cell development, but which has also been correlated with poor cancer prognoses. Previous studies have found that interfering with this pathway results in improved cancer outcomes in mice. These studies found that blocking IL23R was more effective than neutralizing IL23, suggesting that an IL23R-expressing cell was responsible for the tumor-protective effects observed in targeting IL23R. Here, we show that Treg-specific targeting of IL23R provides potent tumor immunity that explains previous successes with anti-IL23R therapy in cancer. We show that there is indeed a tumor-infiltrating Treg subset, marked by IL23R expression, that is enriched for markers of potent suppressive activity. Moreover, we find that targeting their IL23R expression is correlated with Treg conversion and increased IFNγ expression on tumor-infiltrating CD8 T cells. Preliminary results suggest that this Treg conversion is associated with enhanced IL12 sensitivity as a result of targeting the IL23R chain. In keeping with the tumor-specific expression pattern of IL23R, we did not find these changes in systemic T cells in the tumor-bearing mice. Ultimately, these Treg-specific IL23R-deleted mice do not display overt autoimmune disease at even advanced ages, suggesting this approach could represent a safe avenue of cancer immunotherapeutic research.
Characterization of novel CD25 antibodies that deplete human regulatory T cells in humanized mouse model
Authors: Buszko Maja A , Ward Kirstin , Christie Darah , Dziegelewski Michael , Blair David , Shevach Ethan
Published: January 2023

Regulatory T cells (Tregs) are key players in the suppression of immune responses and in the maintenance of immune homeostasis. Due to their central role in pathogenesis of multiple conditions, Tregs are an attractive target for immunomodulatory drug development. Tregs exhibit an activated phenotype and share the expression of many activation antigens with activated conventional T cells (Tconv). Our goal in this project was to generate and characterize monoclonal antibodies (mAbs) that would specifically react with human Tregs. mAbs were raised in mice by immunization with purified, in vitro expanded human Tregs. In contrast to widely available anti-CD25 mAbs that equally bound both activated Tregs and activated Tconv, we identified several mAbs that bound CD25 on expanded Tregs, but failed to bind to CD25 on expanded Tconv. To determine whether a similar selective binding could be demonstrated in vivo, we used the xeno-GVHD model in which NOD-SCID-γc−/− (NSG) mice are engrafted with human peripheral blood mononuclear cells (PBMCs). Several of the mAbs bound to CD25 expressed on Tregs, but not to CD25 expressed on activated Tconv. To test the effect of in vivo administration of the Treg specific mAbs, one of the clones, 2B010, was selected and injected intravenously (i.v) 2 weeks after PBMCs engraftment. Injection of 2B010 resulted in markedly reduced Tregs frequencies and numbers, much less depletion of CD25+ activated Tconv, and no reduction of CD25+CD8+ T cells. Further studies will address the effect of these mAbs at reversing the Treg-mediated protection of xeno-GVHD and enhancing tumor immunity in reconstituted NSG recipients bearing transplanted tumors.
Early Suppression of Immune Regulatory Mechanisms Reduces Tumor Progression in a Murine Model of Urothelial Cancer
Authors: Eaton Valerie L , Fantini Damiano , Yu Yanni , Chiang Ming-Yi , Meeks Joshua , Podojil Joseph R , Miller Stephen D
Published: January 2023

Urothelial cancer (UC) is the fifth most common cancer in the US. While cancer cells may be recognized initially by the immune system, this response is reduced as the tumor escapes immune surveillance over time. An understanding of the immune responses early in cancer progression could identify new therapies that can suppress tumor progression. To that end, we used a murine model of bladder cancer induced by the carcinogen BBN, which replicates the clinical presentation of UC in humans. RNAseq analysis of bladder tumors from mice exposed to BBN revealed an increase in immune regulatory signatures as early as 2 – 4 weeks post-initiation of exposure. These findings were confirmed by FACS analysis of spleen and bladders of mice at 0.5, 1, 2, 3 and 5 months post-initiation of exposure to BBN. The data show a bi-phasic pattern in the immune response, where there is an early increase in the number of Treg and Teff (CD4+ and CD8+) cells within the bladder at 0.5 – 1 months post-initiation of BBN exposure. The Treg and CD8+ T cells populations appear to decrease at 2 months and then increase at 3 and 5 months (corresponding to development of detectible tumor development). To determine if early suppression of Tregs affects tumor development, we depleted Tregs in wildtype C57BL/6 mice with anti-CD25 or diphtheria toxin treatment of Foxp3-DTR mice during the first month of BBN exposure. The data show that the absence of functional Tregs during the first month results in sustained Teff activity at 4 months. The increased Teff cells were found to induce a decrease in overall bladder weight, tumor development, and consequently contained fewer infiltrating immune cells. These findings suggest that early inhibition of Tregs allows for long-lived killing of tumor cells.
The human antibody repertoire genetically encodes for both productive and non-productive targeting solutions against the same broadly neutralizing antibody epitope on influenza virus
Authors: Sangesland Maya , Ghebremichael Musie , Ronsard Larance , Lingwood Daniel
Published: January 2023

A major obstacle to universal influenza vaccine development centers on the ability to elicit high titer responses against functionally conserved but immunologically subdominant features of this virus. Using a transgenic mouse vaccine model wherein antibodies develop with human CDRH3 diversity but are constrained to user defined antibody V genes, we have recently demonstrated that the human VH gene IGHV1-69 encodes for germline B cell receptors (BCRs) that naturally engage a conserved broadly neutralizing epitope on the hemagglutinin (HA) stalk of Group 1 influenza A viruses (IAV). Importantly, this VH endowment enables elicitation of an immunodominant bnAb response against IAV following vaccination with an engineered HA vaccine nanoparticle. However, IGHV1-69 is polymorphic with ~15% of the global population homozygous for alleles that bear a single nucleotide polymorphism (SNP) in CDRH2 (F54L) which confers germline binding to the bnAb target. To test the consequences of this, we applied our transgenic vaccine model to compare the capacity of IGHV1-69 vs IGHV1-69 SNP to elicit bnAb responses following immunization with our HA nanoparticle. We found that while both VH forms endow germline BCR targeting solutions to the same IAV bnAb site, only the WT form is productive following immunization. Germline competition within heterozygous animals (F54/L54) suggests that the capacity for pathway expansion arises via differences in the germline-endowed affinity for the same bnAb target. While globally, most humans utilize at least one copy of IGHV1-69 and would be predicted to support a bnAb response to our vaccine antigen, certain ethnicities show heightened usage of IGHV1-69 SNP and may therefore be limited by non-productive responses.
Fusion protein (M2e-HA2-NP) adjuvanted with γ-PGA/Alum induce the cross-reactivity against heterologous influenza A viruses
Authors: Kwak Chaewon , Nguyen Quyen Thi , Poo Haryoung
Published: January 2023

The epidemic and pandemic influenza viruses result in substantial morbidity and mortality in humans. Vaccination is the most effective way to prevent influenza virus infection. In this study, we cloned the conserved regions of influenza A virus, extracellular domain of matrix protein 2 (M2e), hemagglutinin 2 (HA2) and nucleoprotein (NP). Then, we generated fusion (M2e-HA2-NP) protein as a universal vaccine antigen candidate. γ-PGA/Alum (PA) was used as an adjuvant to enhance the efficacy of fusion protein. In mice challenged with influenza viruses, fusion protein (15 μg) provided 100% protection against A/Puerto Rico/8/1934 (H1N1; PR8) or A/California/04/09 (pH1N1; CA04) and 80% protection against H3N2 (a reassortant virus carrying HA and NA genes of A/Hong Kong/1/68). In dose-sparing effect experiments, low dose (1 or 5 μg) of fusion protein with PA provided the same protection comparable to 15 μg of fusion protein against H1N1, pH1N1 and H3N2. Humoral and cellular immunities against heterologous influenza viruses were significantly enhanced in mice vaccinated with fusion protein mixed with PA compared with alum-adjuvanted fusion protein. Taken together, the fusion protein adjuvanted with PA may be a good vaccine candidate showing heterosubtypic cross-reactivity.
Development of inactivated subunit influenza vaccine endowing with IgA induction and protection against heterologous strain
Authors: Bang Yoo-Jin , Kim Yun-Hee , Lee Yu-Sun , Kim Jae-Yong , Park Hyo-Jung , Ko Hae-Li , Park Sang-In , Hwang Kyung-Ah , Kim Hun , Nam Jae-Hwan
Published: January 2023

Vaccination is known to be the best way to prevent and control seasonal influenza infections. Among the various available influenza vaccines, an inactivated vaccine shows improved protective effects associated with greater safety. However, since the influenza virus is continuously evolving, its response to inactivated vaccines becomes increasingly difficult to predict, leading to complete or partial loss of protection against the virus. In addition, immunogenicity is lower than other types and Th2-biased immune responses have been reported. In this study, we have investigated the role of the single-stranded RNA (ssRNA) adjuvant derived from the intergenic region of internal ribosome entry site of Cricket Paralysis virus in seasonal inactivated subunit influenza vaccine (ISIV). We found that the ssRNA adjuvant stimulated balanced cellular (indicated by IgG2a, IFN-γ, IL-2, and TNF-α) and humoral (indicated by IgG1 and HI) responses, along with mucosal (indicated by IgA) immune response. Moreover, the ssRNA adjuvant formulated ISIV enhanced viral clearance and improved lung pathology after homologous and even heterologous influenza virus infections. The proportion of memory CD4+ and CD8+ T cells, important for long term immunity, was also observed to increase. Therefore, the ssRNA adjuvant formulated ISIV is effective in inducing humoral and cellular immune responses, cross protection, and long-term immunity.
Immunology of vaccine adjuvants enhancing respiratory viral vaccine efficacy
Authors: Kang Sang-Moo , Ko Eun-Ju , Lee Youri , Kim Ki-Hye
Published: January 2023

We investigated the adjuvant effects of MPL (TLR4 agonist) and CpG (TLR9 agonist) on promoting protection after vaccination of C57BL/6 and BALB/c mice with inactivated influenza split virus or human respiratory syncytial virus (RSV) vaccines. Adjuvant effects of combination MPL and CpG (MPL+CpG) on improving protective vaccine efficacy were evident by higher levels of protective humoral and cellular immune responses to vaccination, more effective control of lung viral replication, prevention of weight loss, and inflammatory cytokines and cellular infiltrates in vaccinated mice after virus challenge. Vaccine-enhanced respiratory disease, a well-known phenomenon due to atypical immune priming by RSV vaccination after challenge, could be prevented by MPL + CpG adjuvants in RSV vaccination, shaping toward Th1 and IgG2a isotype immune responses. In contrast, aluminum hydroxide and squalene oil-in-water adjuvants (AddaVax) were not effective in conferring vaccine-induced protection against influenza and RSV in BALB/c mice after vaccination and challenge. Combination of MPL and CpG was found to exhibit distinct effects on modulating inflammatory micro-environment with cytokines, chemokines, and innate infiltrates in vivo within a day as well as stimulating dendritic cells in vitro to secrete IL-12p70 and TNF-α cytokines. In summary, the findings suggest that a combination adjuvant of different TLR agonists exhibits a unique pattern of innate and adaptive immune responses, contributing to protection by inactivated influenza split virus and subunit RSV vaccination.
Safety, immunogenicity, and protective efficacy of a recombinant vaccine against RSV: Evidence from murine and bovine models
Authors: Diaz Fabian E , Guerra-Maupome Mariana , Soto Jorge A , Gálvez Nicolás M S , Ríos Mariana , McGill Jodi L , Kalergis Alexis M
Published: January 2023

The human Respiratory Syncytial virus (RSV) is a leading cause of lower respiratory tract infection in infants worldwide. Despite its health burden, there are no licensed vaccines to prevent human RSV-caused disease. In mice, immunization with a single subcutaneous dose of 3 × 105 CFUs of a recombinant BCG expressing hRSV N protein (rBCG-N-hRSV) is safe, and induces robust, long-lived humoral and cellular Th1/Th17 immunity, which prevents lung damage after an intranasal hRSV challenge. Vaccinated mice display reduced weight loss, less neutrophil airway infiltration, and reduced lung viral loads, when compared to unimmunized controls. To further explore the safety, immunogenicity and efficacy of this formulation, we carried out a vaccination and challenge study on a bovine RSV - neonatal calf infection model. Five day old calves received a subcutaneous dose of the rBCG-N-hRSV vaccine and a booster vaccination after 14 days. On day 28, calves were inoculated via aerosol challenge with ~104 TCID50/mL of BRSV strain 375. Clinical data was registered daily until euthanasia on day 7 post infection. Virus-specific IgA and neutralization activity increased in vaccinated calves at 7dpi, and the animals mounted a robust virus-specific IFN-γ/IL-17 response observed after PBMC antigen recall assays. Following bRSV challenge, we observed reduced clinical disease compared to unvaccinated control calves, without signs of vaccine-enhanced disease. Reduction trends in viral shedding and lung viral load were observed in vaccinated animals, as well as a significantly reduced neutrophil infiltration in BAL. These results suggest that the rBCG-N-hRSV vaccine would be safe for neonatal use and support further investigation in clinical studies.
Chitosan adjuvantation improves protection elicited by single, low dose influenza A virus recombinant protein vaccination
Authors: Lampe Anna T , Farris Eric J , Ballweg Matthew D , Pannier Angela K , Brown Deborah M
Published: January 2023

Continued development of effective vaccines requires investigation of new adjuvants that are able to direct and enhance immune responses. Adjuvants have potential to allow for fine-tuning of immune responses against pathogens for which vaccines are suboptimal, or non-existent. Despite this capability, few adjuvants are currently used in human vaccines. Biomaterials provide a pool of potential adjuvants that have been vetted for biocompatibility and adverse effects. One such material is chitosan (CS), a linear, positively charged, partially deacylated derivative of chitin. Varying molecular weights of CS were examined for their ability to improve influenza A virus vaccination. It was observed that mice receiving CS during single, low dose protein vaccination experienced significantly less weight loss after homologous challenge than mice receiving protein alone. CS elicited enhanced protection from weight loss was accompanied by elevated IgG and a trend of increased IgG1. When T cell activation was examined two days after challenge, CS adjuvantation resulted in no significant changes in IFN-γ or IL-4 production in the DLN, however CS induced IL-4 production by CD4 T cells in the lung at day five. This effect appeared to be dependent on the molecular weight of the CS. Additionally, CS adjuvantation resulted in elevated CD69+/CD103+ CD4 T cells in the lung two days after homologous challenge, potentially mediating improved protection against infection. Further, we observed enhanced in vitro cytokine production and IRF pathway activation in antigen presenting cells treated with CS. Taken together, these results suggest that CS is able to act as a vaccine adjuvant and may be used to instruct immune responses and improve vaccine efficacy.
PD1 blockade augments adoptive T cell therapy via endogenous T cells rather than direct enhancement of transferred T cells
Authors: Davies John S , Karimipour Farrah , Zhang Ling , Serna Carylinda , Hinrichs Christian
Published: January 2023

Adoptive T cell therapy (ACT) has yielded promising clinical results but improvements are needed. Cell therapy efficacy may be improved by blockade of the inhibitory receptor PD1. Independent T cell models demonstrated cooperation between ACT and α-PD1. The cooperative effect of α-PD1 was abolished in three separate hosts devoid of polyclonal T cells (Rag1-KO, Trac-KO and irrelevant TCRtg hosts). Moreover, endogenous CD4 and CD8 T cells in the tumor increased and transferred T cells decreased in number following α-PD1 treatment. Efficacy of ACT was not improved when transferred cells were engineered with PD1-targeting miRNA or over-expression of PD1 without the cytoplasmic signaling domain. However, ACT with PD1-KO T cells was improved with the combination of α-PD1. Additionally, transferred T cells did not respond to α-PD1 when they were repeatedly stimulated until high co-expression of PD1+LAG3+TIM3+ (85% vs 34%) prior to transfer, when they were allowed prolonged in vivo tumor engagement prior to α-PD1 administration, or when they were targeting tumors with enforced expression of PDL1. Lastly, α-PD1 therapy showed no efficacy during the treatment of less immunogenic tumors, engineered with ubiquitin-tagged antigen constructs rather than RFP-antigen constructs. This finding established that PD1-sensitive endogenous T cells targeted immunogenic antigens within RFP. The role of endogenous T cells was further corroborated by TCR clonotype skewing when α-PD1 was administered. These data demonstrated that blocking the PD1 axis did not directly benefit adoptively transferred T cells. Rather, the benefit of α-PD1 was dependent on the presence of endogenous T cells specific for immunogenic tumor antigens.
Modulation of thymic architecture and T cell function by Mesenchymal stromal cell extracellular vesicles in a model of neonatal hyperoxia
Authors: Reis Monica , Willis Gareth R. , Fernandez-Gonzalez Angeles , Yeung Vincent , Taglauer Elizabeth , Liu Xianlan , Mitsialis S. Alex , Kourembanas Stella
Published: January 2023

Therapies based on mesenchymal stem/stromal cell extracellular vesicles (MEx) have shown promise in experimental models of bronchopulmonary dysplasia (BPD), a disease of disrupted lung development that can result from neonatal exposure to hyperoxia. The effects of oxygen exposure on other developing organs in the neonatal period remain poorly understood and whether MEx treatment may modify these responses is not known. Our research aimed at understanding the effects of neonatal exposure to hyperoxia and the immunomodulatory properties of MEx on thymic architecture and T cell development. Using a mouse model of neonatal hyperoxia, we showed that, in addition to lung injury resulting in BPD, there was involution of the thymic medulla with altered T cell function. Administration of a single dose of MEx resulted in preservation of thymic architecture, and restoration of normal thymic function through modulation of the thymic medullary antigen presentation network and T cell phenotypes. Consequently, MEx reinstated the mechanisms of thymic-driven central tolerance that are perturbed upon hyperoxia exposure, as demonstrated by increased FoxP3+ regulatory T cell generation and decreased T cell autoreactivity. Overall, our study demonstrates that MEx represent a promising cell-free therapeutic option to restore thymic architecture and ensure the development of central tolerance by preventing thymic injury associated with neonatal hyperoxia exposure.
TLR enhanced GVAX elicits tumor-specific tissue resident memory T cells independent of T cell priming
Authors: Korrer Michael J , Taylor David R , Kim Young Jun
Published: January 2023

GVAX, a genetically modified whole cell vaccine, is thought to work by recruiting and activating APCs which then traffic to the draining lymph node and elicit tumor-specific T cells. GVAX has little therapeutic benefit as a monotherapy, but when combined with a Toll-like receptor (TLR) 4 adjuvant, TLR Enhanced GVAX (TEGVAX) significantly reduces tumor burden in mouse models. Paradoxically, the increased therapeutic benefit of TEGVAX corresponds with a decrease in delivery of tumor antigen to the dLN suggesting a different mechanism of tumor suppression than GVAX.

To determine if TEGVAX alters the priming of CD8 T cells, we performed longitudinal studies of OT-1 CD8 T cell proliferation in vivo. GVAX induced rapid proliferation of OT-1 cells, whereas TEGVAX failed to induce any. We then determined if TEGVAX required myeloid or NK cells to function. TEGVAX reduced B16-mOVA tumor burden in NK depleted, but not myeloid cell depleted mice. To determine if T cell circulation from the dLN to the tumor was necessary, we administered FTY720, which sequesters circulating T cells in lymph nodes, prior to vaccination. TEGVAX significantly reduced B16-mOVA tumor growth even in the presence of FTY720 treatment, suggesting T cell priming was not required. Immune phenotyping of TEGVAX treated B16 tumors and showed the presence of activated tumor infiltrating tissue-resident memory (Trm) T cells.

Our results demonstrate that combining a TLR4 agonist with GVAX completely alters the immune response to GVAX vaccination. TEGVAX primes naïve T cells poorly and does not require T cell circulation to reduce tumor growth. These findings suggest that TEGVAX functions by eliciting a tumor-specific Trm T cell response independent of lymph node priming.
Cytokine driven effector functions of CD8 cells: application to protease-activated cytokine therapy for cancer
Authors: Norville Karli , Skrombolas Denise , Ferry Shannon , Corson Nancy , Frelinger John G.
Published: January 2023

Cytokines are potent immune mediators and play a crucial role in the differentiation and activation of effector CD8 T cells which are critical in anti-tumor responses. Here we tested the ability of IL-2, IL-12, and IL-18 to drive expression of gamma interferon (IFNg) in the absence of intentional antigen stimulation. We show in vitro that spleen cells from both BALB/c and C57BL/6 mice produced large amounts of IFNg and downstream mediators in response to these cytokines individually or synergistically in combination. Moreover, CD8 cells purified from Colon38 tumors can be similarly activated, demonstrating that they are not permanently inactivated or exhausted. These data illustrate innate-like function of CD8 cells, including those in the tumor microenvironment (TME). To address concerns of severe toxicities that can accompany systemic delivery of cytokines, we designed protease-activated cytokine fusion proteins (FPs) which exploit the overexpression of proteases in tumors to release active cytokine in the TME. Using gene transfection techniques, we show that an IL-2FP with a matrix metalloproteinase 2, 9 cleavage sequence can be activated in the TME and reduce tumor growth. In contrast, a FP with a scrambled non-cleavable sequence does not reduce tumor growth, illustrating the specificity of cleavage of the FP in vivo. Additionally, we demonstrate that systemic expression of the IL-2FP elicits little systemic toxicity in striking contrast to the delivery of free IL-2. Collectively, these data support the development of cytokine fusions proteins as a new class of therapeutics to promote anti-tumor effectors with limited detrimental cytokine side effects.
The impact of glycoyslation on filovirus vaccine immunogenicity in mice
Authors: Clarke Elizabeth C , Bradfute Steven
Published: January 2023

Filoviruses are highly virulent pathogens which cause severe hemorrhagic fevers, including Ebola virus disease (EVD). There is currently an ongoing large outbreak in the Democratic Republic of Congo, and while investigational vaccines have been used in trials during the outbreak, there are no licensed vaccines to EVD, or the related Marburg virus disease (MVD). The glycoproteins of filoviruses are the only virally expressed proteins on the virion surface and are required for receptor binding and as such, they are the main candidate vaccine antigen. A major component of the glycoprotein are the N- and O-linked glycans, but these glycans vary in number, distribution and type depending on the cell type producing the glycoprotein. Both mammalian and insect cell lines are commonly used to produce filovirus proteins used in vaccine development, but produce proteins with very different glycosylation. Here, we describe the impact of the filovirus glycoprotein glycans on the immunogenicity of the transmembrane-deleted glycoprotein (GPdTM) vaccine in mice using insect-produced, mammalian-produced, and deglycosylated filovirus glycoproteins. Results suggest differences in antibody titer and T cell epitopes induced between glycoproteins with different glycosylation patterns. Studies are underway to test how these changes affect survival against live virus challenge with mouse-adapted Ebola and Marburg.
Unique regulation of the DNA methylation machinery in natural hosts contributes to CD4 gene repression and SIV disease non-progression
Authors: Mudd Joseph C , Lai Stephen , Shah Sanjana , Rahmberg Andrew R , Flynn Jacob , Perikins Molly R , Ransier Amy , Starke Carly E , Douek Danny C , Hirsch Vanessa M , Cameron Mark J , Brenchley Jason M
Published: January 2023

African green monkeys (AGMs) are natural hosts of Simian immunodeficiency virus (SIV) that post-thymically downregulate CD4 to maintain a large population of CD4−CD8aa+ virus-resistant T cells which retain T-helper functionality. AGMs can become aviremic and apparently cured of SIV by down-regulating CD4 to completion. To understand the mechanisms of this process, purified CD4+ T cells from four AGMs, closely-related Patas monkeys, and rhesus macaques were stimulated with SEB for 5 days and RNAseq was performed on divided cells induced to downregulate CD4 (AGM, Patas) and those that maintain CD4 expression (rhesus). 1,917 DEGs were revealed to be common among divided, CD4-downregulated AGM and Patas T cells, yet unique from divided rhesus CD4+ T cells. Genes well-known to be regulated in natural hosts were selectively present in this dataset, including CD4, CD8A, and CXCR6 (p= 1.27e−27, 2.68e−5, 6.72e−15, respectively). Pathway analysis of DEGs revealed proteins involved in DNA methylation to be enriched in CD4-downregulated AGM and Patas T cells (p=0.013). Inhibition of DNA methyltransferases (DNMT) with 5-aza-2 deoxycitidine inhibited CD4 downregulation in AGM CD4+ T cells induced to divide in vitro (p=0.005), indicating CD4 can be pharmacologically manipulated in natural hosts. Cytosine residues within the AGM CD4 promoter region became methylated during CD4 downregulation in vitro (p= < 0.0001), and were stably inherited in AGM CD4−CD8aa+ T cells sorted directly ex vivo. These results suggest AGMs employ epigenetic mechanisms to durably silence the CD4 gene. Manipulation of these mechanisms could provide avenues for modulating SIV/HIV-1 entry receptor expression in hosts that become progressively HIV-1/SIV-infected.
The novel innate immune-antagonistic effects of the multifunctional ectromelia virus C15 protein
Authors: Peauroi Elise , Forsyth Katherine S , Eisenlohr Laurence C.
Published: January 2023

The success of poxviruses as pathogens depends upon their extensive antagonism of host immune responses by a large arsenal of immunomodulatory proteins. The C15 protein of ectromelia virus (ECTV, the agent of mousepox) is the largest of the ECTV immunomodulatory proteins and is a member of a well-conserved poxviral family previously studied as inhibitors of T cell activation. We have recently determined that C15 also facilitates early viral spread in vivo, suggesting a second non-adaptive function of C15. Accordingly, we sought to further investigate this new function and identify the cellular target. We first found that C15 maintains its replication-promoting effect in RAG KO but not RAG IL2RG double KO mice, implying the targeting of NK cells. Nevertheless, the impact of C15 is drastically less in RAG KO than WT mice, suggesting that C15 also targets a RAG-dependent immune component. Further studies in various immune deficient strains implicate type I and III interferons, perforin and the NK gene complex in this novel function. The results prompt further in vitro and in vivo studies into the impact of C15 on NK cell and γδ T cell function, as the RAG-independent and -dependent targets respectively, and lead us to propose that, unique among currently identified viral virulence factors, C15 selectively antagonizes both the adaptive and innate immune responses.
HIV-1 Replication is Metabolically Regulated in an Ex Vivo Human Tonsil Histoculture Model of Infection
Authors: Newcombe Kevin Luna , Nixon Douglas F. , Furler Robert Lance
Published: January 2023

Current in vitro HIV-1 infection models use CD4+ T cell lines, primary CD4+ T cell subsets, or PBMC. However, these lack the 3-dimensional structure of lymphoid tissues, where HIV-1 replication primarily occurs. We use an ex vivo tonsil histoculture model of HIV-1 infection to measure the effects of metabolism on HIV-1 replication kinetics. Histocultures were infected with a CCR5-tropic HIV-1 isolate, ADA, in the presence or absence of inhibitors of glycolysis, oxidative phosphorylation, glutaminolysis, or fatty acid oxidation (2-Deoxy-D-glucose (2-DG), Oligomycin, 6-Diazo-5-Oxo-L-Norleucine (DON), and Etomoxir, respectively). HIV-1 replication was measured using p24 ELISA every 3–4 days for a minimum of 3 weeks. Four tonsil sections (~1 mm3 each) were placed on a collagen support in each well of a 6-well plate. 2-DG and Oligomycin significantly decreased HIV-1 production compared to untreated in this system. However, while DON and Etomoxir initially suppressed HIV-1 replication, viral production increased over the course of the experiments. Following T cell activation, increased aerobic glycolysis and oxidative phosphorylation occurs to meet the biomolecule and energy needs of a growing and/or proliferating cell. Inhibiting these pathways in our ex vivo histoculture model significantly reduced HIV-1 production. Next, we will test if these metabolic inhibitors can drive latency in our model and compare our approach to other published cytokine-driven in vitro models.
The replication-competent HIV reservoir is detectable in circulating monocytes and is increased in ART-suppressed chronic HIV-infected individuals with cognitive impairment
Authors: Mitchell Brooks I. , Laws Elizabeth I. , Souza Scott A. , Paul Robert H. , Milne Cris I.P. , Nakamoto Beau K. , Shikuma Cecilia M. , Ndhlovu Lishomwa C.
Published: January 2023

Background

Measuring replication-competent HIV (rcHIV) in monocytes (MO) in the setting of antiretroviral therapy (ART)-suppression, remains a topic of intense interest, particularly due to the continued presence of cognitive (cog.) decline, as well as in our pursuit for an HIV cure.

Methods

PBMCs from ART-suppressed chronic HIV-infected donors, whom had available cog. performance scores, were cell sorted using flow cytometry to obtain ultra pure MO and CD4 T cells confirmed by subsequent flow analyses. Using a novel modified TZM-bl co-culture assay, frequencies of cells producing rcHIV (IUPM) were calculated. Total p24 associated with rcHIV produced by MO or CD4 T cells was quantified using a relative light unit to rcHIV-associated p24 standard curve. The infectious potential (IP) was calculated as a ratio of total rcHIV-associated p24 and IUPM. Non-parametric tests assessed group comparisons.

Results

All HIV-infected donors in cog. impaired (n=7) and cog. normal (n=9) groups were male, on ART > 3 months, and virally-suppressed. IUPMs in MO were detectable in 11 of 16 donors (median IUPM=1.32). IUPMs in CD4 T cells were detectable in 13 of 16 donors (median IUPM=7.22) and were higher as compared to MO (p=0.004). Median IP was slightly higher in MO as compared to CD4 T cells (0.443 vs. 0.104, p=0.271). Cog. impaired donors had higher IUPMs in MO as compared to cog. normal donors (4.43 vs. 0.31, p=0.015). IP between cog. groups were similar for both cellular compartments.

Conclusions

Despite ART-suppression, circulating MO are capable of producing rcHIV and may contribute to cog. impairment, as well as pose a challenge for HIV remission efforts. Monitoring and targeting HIV persistence in MO during ART-suppression are warranted.
PD-L1 enhances the efficacy of the oncolytic virus VSVΔ51
Authors: Hodgins Jonathan , Park Maria M , Bell John C , Bourgeois-Daigneault Marie-Claude , Ardolino Michele
Published: January 2023

Oncolytic viruses (OVs) are a novel immunotherapy showing great promise in the treatment of cancer. Due to the impaired anti-viral response in cancer cells, OVs such as VSVΔ51 preferentially replicate in cancer cells relative to healthy cells. PD-L1 is a surface protein that binds to the inhibitory checkpoint receptor PD-1 to inhibit anti-cancer immunity in vivo. Previous work has suggested that PD-L1 has the ability to inhibit type I interferon signaling. Given that the type I interferon pathway is responsible for inducing the cellular anti-viral response, we hypothesized that PD-L1 will affect the replication of the OV VSVΔ51 in cancer cells. To this end, a PD-L1 knockout line was generated from the PD-L1-expressing mouse prostate cancer cell line TRAMP-C2 by CRISPR/Cas9 (TRAMP-C2 Cd274−/−). Indeed, WT TRAMP-C2 is more susceptible to VSVΔ51 infection and oncolysis compared to TRAMP-C2 Cd274−/− cells, and TRAMP-C2 Cd274−/− cells exhibit severe defects in viral replication and virion production. Similar results have been observed in a different cell type/line. Mechanistically, TRAMP-C2 Cd274−/− secrete greater amounts of IFN-β compared to WT TRAMP-C2 post-infection (with subsequent enhanced expression of IFN-stimulated anti-viral genes), and have altered signaling in response to IFN-β stimulation and VSVΔ51 infection. Similar results are observed following treatment with the viral mimic, poly(I:C). Importantly, all differences in infection between WT TRAMP-C2 and TRAMP-C2 Cd274−/− are abolished when the activity of IFNAR is blocked. Lastly, preliminary evidence suggests that CD80 surface expression is required for the function of PD-L1. Ultimately, we aim to characterize PD-1-independent functions of PD-L1.
Oncolytic poliovirus immunotherapy for breast cancer
Authors: Frazier Victoria N , Holl Eda , Brown Michael , Boczkowski David , Landa Karenia , Hwang Shelley , Gromeier Matthias , Nair Smita K
Published: January 2023

Oncolytic poliovirus PVSRIPO is a recombinant, non-pathogenic polio:rhinovirus chimera that enters cells via the poliovirus receptor CD155. CD155 expression is virtually universal in solid cancers, including breast cancer. CD155 is also expressed on myeloid cells. PVSRIPO infection of antigen-presenting cells (APC), including dendritic cells (DC) and macrophages, induces type I interferon and APC activation. Intratumor injection of PVSRIPO results in robust T cell infiltration and generates an immune-engaged tumor microenvironment. PVSRIPO is therefore an ideal therapeutic complement to immune checkpoint inhibitors, such as anti-PD1/PDL1.

We tested the hypothesis that blocking PD1/PDL1 in conjunction with PVSRIPO will potentiate durable antitumor immunity in the murine E0771 orthotopic breast cancer immunotherapy model. Intratumor injection of PVSRIPO in 5–7 mm orthotopic tumors induces rapid recruitment of neutrophils followed by infiltration of DC, T cells and B cells. Next, we investigated PVSRIPO with PD1/PDL1 blockade. All treatment groups significantly inhibited tumor growth compared to PBS. There were no significant differences in tumor growth inhibition between PVSRIPO and anti-PD1/PDL1 monotherapies. Combination PVSRIPO+PD1/PDL1 blockade was significantly more effective than the monotherapies at controlling tumor growth.

We also tested neoadjuvant PVSRIPO in E0771-tumor bearing mice. We observed that intratumor PVSRIPO followed by surgery effectively controls tumor growth and prevents tumor regrowth following tumor rechallenge, suggesting induction of a systemic immune response. We are now testing neoadjuvant PVSRIPO with anti-PD1/PDL1 in murine models of cancer.
Human immunity to chikungunya infection
Authors: NAYAK KAUSTUV , Jain Vineet , Kaur Manpreet , Khan Naushad , Rai Ramesh Chandra , Dixit Kritika , Sagar Rohit , Gupta Shipra , Islamuddin Mohammad , Verma Anil , Maheshwari Deepti , Aggarwal Charu , Chawla Yadya , Reddy Elluri Seetharami , Panda Harekrushna , Sharma Pragati , Bhatnagar Priya , Singh Prabhat , Ratageri Vinod H , Chandele Anmol , Ray Pratima , Muralikrishna Kaja
Published: January 2023

Chikungunyna virus is expanding globally and continue to cause major public health threat to Indian populations. Vaccine efforts are underway, and it is hoped that these will eventually progress to human evaluation. However, currently we have little understanding of the phenotypes and functions of the human T cells in chikungunya patients, a knowledge that is essential for improving vaccine design/testing and evaluation efforts. Here, we provide a detailed analysis of the CD8 T cell responses in chikungunya patients from India. We found that CD38+ HLADR+ CD8 T cell subset expanded dramatically in chikungunya febrile patients with frequencies averaging about 20% of the total CD8 T cells, and reaching as high as 50% of the CD8 T cells in some patients. The frequencies of these activated CD8 T cells were substantially low and barely above background levels in afebrile patients reporting to the clinic with persistent arthralgia/arthritis that was lasting for more than 30 days. These massively expanding CD8 T cells observed in the acute febrile patients were highly proliferating (KI67 ), robustly expressing markers indicative strong Th1 differentiation (T-bet), cytotoxic functions (Perforin) and inflammatory/synovial tissue homing characteristics (CX3CR1 and CXCR4). Interestingly, antigen-stimulation mediated IFN-g producing functions of these cells was highly compromized, reminiscent of the “cytokine stunned” phenotype. Taken together, these results suggest that these highly differentiated effector CD8 T cell that were massively expanding during acute chikungunya febrile infection might be involved in protection by homing to infected tissues and eliminating infected targets rather than causing inflammation.