bluuebunny/crossref_metadata_2025 huggingface dataset:
VEN-α, an engineered mammalian milk protein, demonstrates potent proinflammatory cytokine neutralization implicated in Inflammatory Bowel Disease
Authors: Alfano Randall W. , Simon Andrew , Akoyev Vladimir , Fein Seymour
Published: January 2023

Inflammatory Bowel Disease, which is comprised of Crohn’s disease (CD) and ulcerative colitis (UC), are chronic and debilitating diseases. Traditional therapies, which consist of anti-inflammatories and immunomodulators, are palliative at best and do not alter the progressive course of the disease. Advances in the understanding IBD pathology have led to new therapeutic strategies that are centered around targeted biologics to significantly alter disease progression. Although these targeted biologics demonstrate significant improvements in the quality of life for patients, systemic administration can have significant negative side effects such as risk of infection and malignancies.

Ventria Bioscience utilizes a nonanimal-based expression system known as ExpressTec to express at commercially viable levels a recombinant human milk protein that has potent anti-inflammatory activity. This protein, known as VEN-α, exhibits a protective effect when T84 monolayers are exposed to proinflammatory cytokines TNF-alpha and Interferon-gamma in the presence of VEN-α in vitro. Further, when orally delivered once daily to mice subjected to DSS treatment ad libitum, VEN-α improves clinical and histopathological outcomes that is comparable to cyclosporin.

Oral delivery of VEN-α may reduce unwanted effects associated with systemic administration and at the same time concentrate the active biologic at the site of pathology. Future studies planned will focus on further detailing efficacy in other models of IBD and understanding the pharmacokinetic profile of this orally delivered biologic. However, collectively the data to date suggests the orally delivered VEN-α has potential as a novel targeted biologic in the treatment of IBD.
Fumaric acid esters hypermethylate the miR21 locus and prevent the formation of CCR6+ CD4 T cells in multiple sclerosis.
Authors: Ntranos Achilles , Ntranos Vasilis , Bonnefil Valentina , Liu Jia , Zhu Yunjiao , Watson Corey , Katz-Sand Ilana , Casaccia Patrizia
Published: January 2023

Background

Dimethylfumarate (DMF) is known to be converted to monomethylfumarate (MMF) by esterases and then metabolized to fumarate and citric acid via the Krebs cycle. In cancer cells, accumulation of fumarate has been shown to inhibit DNA demethylation and that prompted us to investigate whether the immunomodulatory effects of fumaric acid esters (FAEs) in MS patients could also be related to changes of DNA methylation in CD4 T cells.

Methods

PBMCs were collected for immunophenotyping and CD4 T cell isolation from 54 Naive and 36 DMF treated RRMS patients. In addition, CD4 T cells were also isolated from 7 patients before and after DMF treatment. DNA methylation was analyzed using the Infinium Methylation EPIC BeadChip array. Methylation changes were validated in naïve and memory CD4 T cell cultures in vitro using MassArray EpiTYPER. qPCR was performed to assess the effect of DNA methylation on transcript levels in CD4 T cells.

Results

DMF treatment was associated with a significant reduction of CCR6+ CD4 T cells and a strong DNA hypermethylation signature in CD4 T cells. The highest levels of DNA methylation in DMF treated patients compared to controls were detected in a DNA region that was spanning the promoter of microRNA-21 (miR21) in both the cross-sectional and longitudinal cohorts. This region was also found to be hypermethylated in naïve CD4 T cells after treatment with MMF in vitro. Naïve CD4 T cells that were treated with MMF also exhibited reduced miR21 and CCR6 transcript levels.

Conclusions

Our results suggest that by modulating DNA methylation in CD4 T cells, FAEs reduce the expression of miR21 and prevent the production of CCR6+ CD4 T cells, which in recent studies were identified as potentially encephalitogenic in MS patients.
Host-derived CD27 suppresses graft versus host disease by limiting donor T cell expansion in a caspase dependent mechanism
Authors: ONeill Rachel , Leigh Nicholas , Du Wei , Chen George , McCarthy Philip , Cao Xuefang
Published: January 2023

Along with TCR signaling, costimulatory pathways are involved in T cell activation. CD27 is a TNF receptor family member expressed on T−, B−, NK and hematopoietic progenitor cells and its ligand, CD70, is known to be expressed on activated antigen-presenting cells, T−, B− and NK cells. Interaction of CD27 and CD70 in inflammatory conditions has been shown to cause immune dysregulation and immunopathology. However, the role of this pathway in an allogeneic transplantation setting is previously unknown. Host-derived CD27 inhibits GVHD as CD27−/− hosts show significantly increased GVHD. This is evidenced by reduced survival, more severe weight loss, and increased clinical GVHD score. In addition, CD27−/− hosts have higher levels of proinflammatory cytokines TNF-α, IFN-γ, IL-2, and IL-17. Moreover, accumulation of donor CD4+ and CD8+ effector T cells is increased in CD27−/− versus wild-type hosts. Mechanistic analyses suggest that CD27 expressed by host hematopoietic cells is involved in the control of alloreactive T cell apoptosis and expansion. Overall, our findings demonstrate that host CD27 serves as a unique negative regulator of allogeneic T cell response by contributing to donor T cell apoptosis and inhibiting expansion of donor effector T cells.
Eosinophils alleviate lung allograft rejection through their modulation of CD8+ T Cells
Authors: Onyema Oscar Okwudiri , Guo Yizhan , Wang Qing , Gelman Andrew E. , Kreisel Daniel , Jacobsen Elizabeth A. , Krupnick Alexander Sasha
Published: January 2023

Introduction

Based on the accumulation of eosinophils (eos) in rejected lung grafts, it was widely held that eos perpetuate lung graft rejection. However, we showed recently in a mechanistic study that eos mediate lung graft tolerance. As eos are also present in rejected lungs, we therefore explore the possibility of a different role of eos in lung graft rejection.

Method

Using orthotopic left lung transplantation of MHC-1 mismatched Balb/c (H2Kd) donors to C57BL/6J (H2Kb) recipients, with or without co-stimulatory blockade immunosuppression, we quantified the number of eos in lung grafts at day 7 post-transplant (D7). Th1, Th2 and Th17 cytokine profiles of D4 lung grafts were analyzed by quantitative PCR. D7 eos were also analyzed for their expression of Th1, Th2 and Th17 signature genes. The role of eos was examined in iPHIL mice that experience selective ablation of eos after Diphtheria toxin (DT) administration. Balb/c left lungs transplanted into DT or vehicle treated iPHIL mice without immunosuppression were analyzed histologically and flow cytometrically at D4.

Results

We observed similar numbers of eos, higher Th1 polarization, lower IL33, a Th2 cytokine and less Th17 polarization of the lung microenvironment, and higher expression of the Th1 signature genes among eos, in rejecting compared to accepting lung grafts. Eos deficiency was associated with more rapid lung graft rejection, evidenced in higher CD8+ T cell proliferation, lower CD4/CD8 ratio, and enhanced T cell differentiation to effector memory phenotype.

Conclusion

Our results indicate that eos directly ameliorate lung graft rejection even in the absence of immunosuppression. This calls for a revalidation of the importance of eos in lung allograft pathology.
Type-1 interferon impairs the immunoregulatory activity of IL-10: Understanding the mechanisms of abrogation of transplant tolerance
Authors: Lozano Marcos Iglesias , Arun Anirudh , Chicco Maria , Lam Brandon , Ivanova Vera , Lee W.P. Andrew , Brandacher Gerald , Raimondi Giorgio
Published: January 2023

Long term transplant survival by costimulation blockade (CoB)-based regimens is impaired or eroded by inflammatory responses. Type-1 interferons (TI-IFN) are key mediators of this effect, however, their target population and mechanism of action remain unknown. To better understand how IFN-β interferes with the induction of transplant tolerance, we studied its impact on the recently discovered immunomodulatory properties of interleukin 10 (IL-10) in mouse T cells.

We first demonstrate that IL-10 has an unappreciated fundamental role in transplant tolerance, as its transient blockade in recipients of full mismatch skin grafts impairs the efficacy of a peri-transplant CoB regimen based on donor specific transfusion (DST) plus anti-CD154. Moreover, when T cells are exposed to IFN-β in vitro, they develop a selective dramatic defect in their response to IL-10 (but not to IL-6) both at the signaling and gene-induction level. This IL-10-specific unresponsiveness is not associated with any reduction of IL-10R expression or an increase in SOCS1 and 3, nor with a reduced STAT3 cytoplasmic availability. Instead, our gene-expression and functional analysis suggests a novel “cross-regulatory” role for the transcription factor STAT1. Encouragingly, this inhibition is slowly reversible with the removal of TI-IFN, proving the feasibility a pharmacological intervention. Overall, these results reveal a new molecular mechanism whereby IFN-β interferes with the suppressive function of IL-10 in T cells, and potentially with regulation of alloreactivity. Identifying a strategy to target this mechanism will then be a powerful tool to improve the efficacy of CoB-based strategies for transplant tolerance induction.
Clonal dynamics of adoptively transferred multi-virus specific T cells (MVST) in hematopoietic stem cell transplant patients
Authors: Davies Sarah I. , Yu Quan , Battiwalla Minoo , Chinian Fariba , Whitehill Gregory , Wong Susan , Koklanaris Eleftheria , Hauffe Sara , Stroncek David , Superata Jeanine , Barrett John , Muranski Pawel
Published: January 2023

Hematopoietic stem cell transplant (HCT) patients often risk complications from viral infection due to loss of protective antiviral immunity. One strategy to overcome this hurdle is to supplement the HCT with multi-virus specific T cells (MVST). In phase I trial (NCT02231853), MVSTs were generated using HLA-matched donor-derived dendritic cells pulsed with viral overlapping peptide libraries (cytomegalovirus (CMV), Epstein-Barr virus, Adenovirus, and BK polyomavirus) and then co-cultured with donor lymphocytes. These cells were infused into the patient in early post-SCT. Using T cell receptor CDR3 immnuosequencing, we investigated the fate of adoptively transferred donor derived MVST in four donor-patient pairs. Relative clonal frequencies were analyzed in the peripheral blood and bone marrow day 60, 100, 180, and 1 year post transplant. The top 10 most frequent clones in the MVST product did not necessarily survive in the patients, and only minor subsets of MVST clonal sequences survived at day 100 (2.92–7.75%). However, these minor MVST clones quickly expanded in all four patients to constitute over >20% of peripheral blood T cells even 6 months to 1 year later. Two patients analyzed had MVST antiviral clones peak during and after detectable viremia or viriuria, but were asymptomatic. One patient received steroids for treatment of GVHD, which was accompanied by the decline of MVST clones and subsequent CMV reactivation. These results suggest that adoptive transfer of viral-specific T cells can promote reconstitution of the anti-viral T cell repertoire and these cells persist in patients long-term post transplant. Furthermore, the fittest clones are dynamically selected to serve as long term sentinels against viral reactivation.
Role of exosomes in rejection following human lung transplantation – an in vitro analysis using human airway epithelial cells
Authors: Ravichandran Ranjithkumar , Gunasekaran Muthukumar , Bansal Sandhya , Mohanakumar Thalachallour
Published: January 2023

Studies have demonstrated that exosomes expressing donor human leukocyte antigen (HLA) and lung self-antigens (SAg), Collagen V (Col-V) and Kα1 Tubulin (Kα1T), can be detected in the circulation of human lung transplant recipients (LTxR) who developed antibodies (Ab) to donor HLA and diagnosed with rejection However, the mechanisms by which Ab mediate induction of exosomes into the circulation from airway epithelial cells (AEC) remains unclear. Ab ligation to HLA could enhance PERK-mediated pathways involved in endoplasmic reticular (ER) stress-dependent exosome release. The goal of this study is to determine the mechanism by which Ab induce exosomes from AEC. Towards this we employed human AEC, ligated antigens by Ab specific to HLA (HiPRA) and lung SAg (rabbit anti-Col-V or Kα1T) and the role of ER stress signaling molecule in exosomes release were determined. Effect of exosome inhibition by GW4869 was also analyzed. AEC were incubated with HiPRA or Ab to SAg for 24hr in both normoxia and hypoxia (0.1% O2) conditions. AEC incubated with HiPRA or Ab to SAg demonstrated induction of exosomes containing lung SAg, Col-V and Kα1T. Incubation with HiPRA and Ab (20μg/ml) to SAg also demonstrated induction of exosomes containing lung SAg, MHC class II, CD80, CD86. Treatment with GW4869, exosome inhibitor (20μM) reduced the exosomes and SAg. Effect of HiPRA and Ab to SAg on stress kinase in both normoxic and hypoxic conditions and influence of GW4869 in these pathways will be delineated. Identification of stress kinase and exosome inhibition will provide novel results for Ab mediated upregulation of stress leading to exosome release from the transplanted organ resulting in immune responses thus increasing the risk for chronic rejection following LTx.
Identification of extracellular vesicle proteins in circulating exosomes from human lung transplant recipients
Authors: Bansal Sandhya , Pirrotte Patrick , McGilvrey Marissa , Garcia-Mansfield Krystine , Sharma Monal , Gunasekaran Muthukumar , Smith Michael , Bremner Ross , Mohanakumar Thalachallour
Published: January 2023

Molecular mechanisms involved in rejection following human lung transplantation (LTx) are not well understood. We identified proteomic signatures of clinical outcomes in circulating extracellular vesicles (EVs) isolated from human lung transplant recipients (LTxRs) who was diagnosed with chronic rejection (Bronchiolitis Obliterans Syndrome (BOS)), acute rejection (AR), respiratory viral infection requiring intervention (RVI) or stable following transplantation. Differential analysis revealed one protein unique to AR (Skin-specific protein 32), four unique to RVI (Guanine nucleotide-binding protein G(I)/G(S)/G(T) subunit beta-2, Transmembrane 9 superfamily member 2, Ras-related C3 botulinum toxin substrate 2, and EH domain-containing protein) and two unique to stable (Coagulation factor X, and N-acetylmuramoyl-L-alanine amidase). Comparison of each rejection group to stable identified 128, 180, and 216 significantly differentially expressed proteins (p-value<0.05, fold-change>2) in BOS, AR, and RVI respectively. Although no unique signatures were found in BOS, an increased enrichment of immune processes such as antigen processing and presentation of exogenous peptide antigen via MHC class I and Fc receptor signaling pathway were observed. Functional enrichment analysis (Gene Ontology) associated differentially expressed EV proteins in AR and RVI conditions with wound repair processes. These diverse signatures detected in each condition highlight complex immune mechanisms underlying the pathophysiology of rejection following LTx. Future studies are needed to validate these signatures in larger LTxR cohorts in order to promote early clinical diagnosis and predict outcome.
Urine levels of CCL2 and CXCL10 chemokines as potential biomarkers of kidney allograft function: a pilot study.
Authors: Czerwinska Magdalena , Gniewkiewicz Michał , Gozdowska Jolanta , Czerwińska Katarzyna , Sadowska Anna , Perkowska-Ptasińska Agnieszka , Kosieradzki Maciej , Durlik Magdalena
Published: January 2023

Introduction

Early prognostic markers that identify high-risk patients could lead to increased surveillance, personalized immunosuppression, and improved outcomes. The vigilance and rapid detection of allograft injury is what would result in the identification of patients who will require more intensive monitoring to ensure that their new allografts function well for the longest possible time.

The aim of the study was to assess whether urine levels of CCL2 and CXCL10 can be valuable, noninvasive source of information about inflammation, tubular injury, progression of fibrosis and other damage features in the kidney allograft.

Materials and methods

In this prospective study 40 patients who underwent a protocolar biopsy within 1 year post kidney transplant were included. The levels of chemokines CCL2 and CXCL10 with reference to creatinine in urine were assayed in all patients. Based on biopsy results research group (N=25) with diagnosis of BKV nephropathy, IFTA II–III, peritubular capillaritisis, C4d or rejection was selected. Patients with normal biopsy results were included as control group (N=15).

Results

Median urinary CCL2-to-creatinine (Cr) ratio [ng/mmol] was significantly elevated in research group compared to control group (21.76 ± 18.57 vs 8.99 ±5.12, p=0.003). There was no statistical difference between groups regarding CXCL10/Cr ratio (7.96 ± 9.08 vs 5.58 ±5.7 p=0.32). Patients with BKV nephropathy (N=3) had much higher level of CCL2 [ng/l] than control group but the difference did not reach statistical significance (429.69 ± 166.49 vs 78.04 ±53.61, p=0.06).

Conclusion

CCL2/Cr may be used as a non-ivasive tool useful for monitoring graft condition and predicting recipients with high risk of transplant kidney loss.
Towards the repair of marginal liver grafts: Mesenchymal stromal cell therapy.
Authors: Bartczak Agata , Linares Ivan , Kollmann Dagmar , Zhang Xing-Hua , Ma Xue-Zhong , Echeverri Juan , Ganesh Sujani , Manuel Justin , Selzner Markus , Keating Armand , McGilvray Ian
Published: January 2023

Despite the recent inclusion of livers donated after cardiac death (DCD), more than 25% of people listed for a liver transplant in Canada die before obtaining a suitable graft. Over half of possible DCD livers undergo long periods of ischemia that sensitize the liver to reperfusion injury (HIRI) and render the liver grafts unsuitable for transplantation. Mesenchymal Stromal cells (MSC) show promise in protecting liver tissue against HIRI: MSC stimulated with inflammatory agents have enhanced anti-inflammatory activity. Kupffer Cells (KCs) are central to HIRI, are activated after reperfusion, and release various inflammatory mediators that promote hepatocyte death. We hypothesize that LPS-primed MSC-treatment can mitigate the tissue damage inflicted by HIRI by modulating KC activation. To test this, we injected LPS-primed MSCs(LPS) into rats undergoing warm ischemia to 70% of the liver. Rats that received MSCs(LPS) had significantly lower levels of AST (2288 ± 2121 IU/L) compared to sham controls (6614 ± 3495 IU/L). Histological analysis showed decreased TUNEL and Caspase-3 positive staining in the MSC(LPS) group: the necrotic area in the MSC- and sham-treated groups affected >50% of the liver parenchyma whereas, only ~20% was necrotic in the MSC(LPS) group. In this latter group, the number of infiltrating inflammatory CD68+ macrophages in the necrotic tissue and CD163+ macrophages in the viable tissue was significantly decreased. Systemic treatment of rats with activated MSCs resulted in the decreased migration of macrophages into inflamed tissue and limited parenchymal tissue damage. Primed MSCs are a viable treatment to protect marginal DCD donor liver grafts from the damaging effects of HIRI.
The adapter protein TSAd maintains alloimmune T regulatory cell stability and function
Authors: Wedel Johannes , Stack Maria P. , Seto Tatsuichiro , Sheehan Matthew M. , Liu Kaifeng , Flynn Evelyn , Briscoe David M.
Published: January 2023

TSAd is an SH2 domain containing intracellular adapter molecule that binds to Lck and functions to elicit T cell receptor-mediated signals and cytokine production. Deficient expression has been associated with autoimmunity in humans and mice, but its mechanism of function in Tregs is not known. We used TSAd KO mice to evaluate its function in CD4+ Tregs, and find that KO Tregs suppress effector proliferation in vitro similar to WT Tregs. However, TSAd KO Tregs displayed higher methylation of the TSDR region of the Foxp3 gene suggesting that TSAd may function via epigenetic regulation of Treg stability. TSAd KO Tregs also produced significantly more effector IFNγ upon mitogen stimulation (vs. WT) indicating that they possess proinflammatory function. To assess function in vivo, we transplanted MHC class II mismatched B6.C-H-2bm12 heart transplants into C57BL/6 WT or TSAd KO recipients. While allograft survival was >50 days in WT recipients, allograft rejection was markedly accelerated in TSAd KO recipients (mean survival time [MST] 22 days, P<0.01). By FACS, CD4+CD44hiCD62Llo Teffs expanded in TSAd KO recipients (vs. WT) supporting the possibility that TSAd KO Tregs are less efficient in suppression and/or possess effector function. To further evaluate these possibilities, we adoptively transferred CD4+CD25+ WT or TSAd KO Tregs into C57BL/6 Rag2γc KO recipients of fully MHC mismatched Balb/c allografts on day 2 post-transplant; subsequently WT CD4+CD25− Teffs were transferred on day 18. In this model WT Tregs (MST 68 days) were significantly more efficient than TSAd KO Tregs (MST 48, P<0.05) in suppression of effectors in vivo. Thus, TSAd provides cell-intrinsic signals to suppress proinflammatory effector function in Treg populations.
A novel function of platelets and CD40L in NAFLD-promoted HCC development
Authors: Fu Qiong , Ma Chi , Heinrich Bernd , Brown Zachary , Zhang Qianfei , Elzey Bennett , Ratliff Timothy , Greten Tim
Published: January 2023

Hepatocellular carcinoma (HCC) is the 2nd leading cause of cancer-related death worldwide. Chronic liver inflammation, such as non-alcoholic fatty liver disease (NAFLD), which is characterized by increased fat deposition in the liver, is one common cause of HCC. The prevalence of NAFLD is increasing rapidly with the growing epidemics of diabetes and obesity, but little is known about how NAFLD proceeds to HCC.

Platelets, which are a critical component in hemostasis, have been shown to release CD40L after activation, and there is accumulating evidence for an important role of platelets in adaptive immunity. Platelet-derived CD40L, by interacting with its receptor CD40, is able to induce dendritic cell maturation, prime T cells, and stimulate B cell isotype switching.

NAFLD patients have increased mean platelet volume, which is an indicator of platelet activation, and an increased level of CD40L in their serum. However, this potential role of platelets in NAFLD-promoted HCC is completely unknown.

Here we studied the platelet function in a murine HCC model of NAFLD and HCC.

We found that inhibition of platelet activation using Aspirin and Clopidogrel greatly increased HCC incidence. Similar results were found when mice were treated with a CD40L blocking antibody, while the combination treatment did not show an additive effect. However, Aspirin and Clopidogrel treatment had no effect on HCC in the absence of NAFLD, while using only a CD40 activating antibody significantly reduced HCC incidence without NAFLD. Finally, an increased CD40L level was found in the serum from mice with NAFLD using a bioassay. In summary, our results suggest an important role of platelets in NAFLD-promoted HCC, and will provide useful information for HCC treatment.
Systemic dysregulation of antigen cross-presenting dendritic cells occurs early in preinvasive pancreatic neoplasia and is reversed by CD40 agonism
Authors: Lin Jeffrey H. , Vonderheide Robert H.
Published: January 2023

Pancreatic ductal adenocarcinoma (PDAC) ranks as the third-leading cause of cancer-related deaths in the United States with a five-year survival rate of 9%. While immune checkpoint blockade (ICB) has revolutionized treatment of many metastatic diseases, cancers like PDAC that lack tumor-infiltrating CD8+ T-cells remain refractory to ICB. To determine whether this phenotype is due to defective CD8+ T-cell priming by antigen cross-presenting XCR1hi classical dendritic cells (cDC’s), we use the KPC mouse model of PDAC driven by Pdx1-Cre KrasLSL-G12D/+Trp53LSL-R172H/+ to interrogate how XCR1hi cDC’s change over the course of cancer development. In the present study, flow cytometric analysis reveals a progressive systemic decline in XCR1hi cDC differentiation and activation that begins in mice bearing preinvasive pancreatic intraepithelial neoplasias (PanIN’s). These changes occur in the setting of intact cDC maturation, consistent with induction of a tolerogenic program, and strengthen upon progression to malignancy. Administration of CD40 agonist to tumor-bearing mice effectively reverses defects in XCR1hi cDC activation and drives CD8+ T-cell infiltration into the tumor microenvironment when combined with the chemotherapies gemcitabine and nab-paclitaxel. Our results therefore demonstrate that antigen cross-presenting cDC’s are dysregulated early and prominently in the neoplastic process and position CD40 agonism as an ideal strategy to potentiate ICB in PDAC.
The coupling of MDSCs with a computational neural network (NN) to detect solid tumors.
Authors: Dominguez George A. , Maslar Kristen , Sholevar Cyrus , Polo Alexander , Roop John , Campisi Anthony , Gabrilovich Dmitry I. , Rauscher Frank J. , Kumar Amit
Published: January 2023

The goal of this study was to create a non-invasive cancer detection assay that analyzes flow cytometry data in an objective method. By using an artificial neural network (NN), we were able to distinguish between cancer patients (CPs) and healthy donors (HDs) based upon the flow cytometry profiles of MDSCs and other leukocytes. Myeloid-derived suppressor cells (MDSCs) are known to be key contributors in supporting tumor progression and tumor escape through their ability to suppress anti-tumor responses. Several studies have demonstrated their utility as indicators for tumor development, but there is significant overlap with healthy individuals. We used standard multiparametric flow cytometry techniques to immunophenotype MDSCs and other leukocytes found in the peripheral blood of 65 biopsy-confirmed CPs with solid tumors and 84 HDs. A series of NNs utilizing pattern recognition computational algorithms were created using three data sets: 1) the training set – this ‘teaches’ the two output categories of cancer and not cancer, 2) the validation set – this uses backpropagation to improve the accuracy of the trained networks, and 3) the testing set – this is used to rank the trained networks against each other. Finally, a naïve testing set is then used to determine the overall sensitivity and specificity for the top-ranking networks. Here, we incorporated a standard 12 marker flow cytometry assay with NN technology to achieve a sensitivity of 92% and a specificity of 89%. Pairing the advanced analytical capabilities of our NN with surface biomarker based analysis of MDSCs and certain leukocytes measured in peripheral blood has enabled us the ability to objectively identify patterns indicative for the existence of a solid tumor.
Relevance of host and tumor PD-L1 in PD-L1 pathway blockade
Authors: Kryczek Ilona , Lin Heng , Wei Shuang , Green Michael , Zou Weiping
Published: January 2023

PD-L1 and PD-1 pathway blockade is a promising therapy against cancer. However, the mechanistic contribution of host and tumor PD-L1 and PD-1 signaling to therapeutic efficacy of PD-L1 and PD-1 blockade remains elusive. Using three tumor bearing mouse models that differ in their sensitivity to PD-L1 blockade, MC38, ID8, and B16-F10, we found a loss of therapeutic efficacy of PD-L1 blockade in immunodeficient mice and in PD-L1 and PD-1 deficient mice. In contrast, knockout or overexpression of PD-L1 in tumor cells had no effect on PD-L1 blockade. Human and murine studies show high levels of functional PD-L1 expression in dendritic cells and macrophages in the tumor microenvironments and draining lymph nodes. Further, expression of PD-L1 on dendritic cells and macrophages in ovarian cancer and melanoma patients correlates with the efficacy of anti-PD-1 and anti-PD-1 plus anti-CTLA-4 therapy. Thus, PD-L1+ dendritic cells and macrophages may mechanistically shape and therapeutically predict clinical efficacy of PD-L1/PD-1 blockade.
Tumor-associated myeloid cells support T-ALL survival and proliferation through close contact
Authors: Lyu Aram , Triplett Todd , Godfrey Wesley , Ehrlich Lauren I.
Published: January 2023

T cell acute lymphoblastic leukemia (T-ALL) is a malignancy of developing T cells. Although intensified radiation and chemotherapy have improved survival rates to ~85% in pediatric patients, these therapies are toxic, and patients who relapse have no alternative treatments. Research on the genetic mutations driving T-ALL have been extensively characterized, but despite numerous tumor-promoting genomic alterations, primary T-ALL cells require cells/signals from the tumor microenvironment (TME) to survive. We have previously reported that myeloid cells from the TME are capable of supporting survival and proliferation of T-ALL cells in vitro. Here, we report that myeloid cells are required for T-ALL tumor growth in vivo. Treatment of a transplantable murine model of T-ALL with Clodronate liposomes, which deplete myeloid cells, resulted in a significant reduction in tumor burden in multiple tissues compared to control-treated mice. Because the myeloid compartment is heterogeneous, we assessed the capacity of purified myeloid subsets to support the survival of T-ALL cells, and identified several myeloid cell types capable of providing tumor support. To address the mechanism by which these tumor-associated myeloid cells support T-ALL growth, we performed transwell assays and found that T-ALL cells require close contact with myeloid cells to receive survival signals. We report on recent functional experiments, implicating VCAM-1 and ICAM-1 as important mediators of myeloid support of T-ALL. Future studies aim to provide further insight into the cell types and mechanisms by which stroma from the TME of distinct organs promote T-ALL survival, with the ultimate goal of developing effective therapeutics.
Tumor cell-intrinsic MHC class II expression as a determinant of immunotherapy responsiveness in an orthotopic mouse model of non-small cell lung cancer
Authors: Clambey Eric T. , Johnson Amber M. , Neuwelt Alex , Kimball Abigail K. , Kaspar Rachael , Bullock Bonnie , Kwak Jeff , Poczobutt Joanna , Li Howard , Nemenoff Raphael
Published: January 2023

Lung cancer is the leading cause of cancer-related deaths worldwide and the second most common cancer among both men and women in the United States. Immunotherapy regimens that disrupt the inhibitory PD-1/PD-L1 axis have recently emerged as promising treatments that can unleash anti-tumor immunity against a wide range of cancers, including a subset of lung cancers. How these therapies elicit potent anti-tumor immunity remains controversial, with markers for immunotherapy-sensitivity still an active area of investigation. To study determinants of lung cancer growth in vivo we studied two orthotopic mouse models of non-small cell lung cancer (NSCLC). These Kras-mutant tumor lines dramatically differ in their response to immunotherapy: CMT167 tumor cells are exquisitely susceptible to PD-1/PD-L1 targeted immunotherapy whereas LLC tumor cells are immunotherapy-resistant. Immunotherapy-sensitive cells showed induction of the MHC class II antigen processing and presentation pathway in vitro and in vivo, and were capable of stimulating CD4 T cells directly in ex vivo co-culture. To study the impact of tumor cell-intrinsic MHC class II induction, we used RNA interference to knockdown CIITA, a transcriptional inducer of the MHC class II pathway. CIITA knockdown in immunotherapy-sensitive cells impaired the induction of tumor cell-intrinsic MHC class II in vitro and abrogated CD4 T cell stimulation in co-culture. CIITA knockdown further converted tumors from immunotherapy-sensitive to immunotherapy-resistant. These studies identify tumor cell-intrinsic expression of MHC class II as a potential direct target for CD4 T cell recognition that may promote sensitivity to PD-1/PD-L1 blockade.
USP22 maintains regulatory T cells by stabilizing Foxp3 protein level and impairs anti-tumor immune response
Authors: Montauti Elena , Zhang Yusi , Fang Deyu
Published: January 2023

Ubiquitin-specific peptidase 22, a member of the deubiquitinase family, has been recently defined as a “cancer stem cell” marker in tumor development and progression. However, the physiological function of USP22, particularly in the immune system, remains unknown. Here, we identify USP22 as a specific deubiquitinase of Foxp3, a lineage-specifying transcription factor of regulatory T (Treg) cells. A genetic deletion in USP22 results in a dramatic reduction in FoxP3 expression levels and impairs the suppressive functions of Tregs without affecting the activation of conventional T cells. Consequently, a targeted deletion of USP22 in mice, either specifically in T cells or in Tregs, results in chronic T cell activation and the development of spontaneous autoimmune syndromes. Importantly, the impairment in Treg suppressive function of USP22-null Tregs boosts anti-tumor immunity. Collectively, our findings reveal a previously unappreciated physiological function of USP22 in maintaining Treg stability and identify USP22 as a potential target for cancer immunotherapy.
Molecular control of Treg-induced effector T cell DNA damage and senescence for tumor immunotherapy
Authors: Liu Xia , Mo Wei , Ye Jian , Li Lingyun , Hsueh Eddy C. , Hoft Daniel F. , Peng Guangyong
Published: January 2023

Regulatory T cells (Treg) have a critical function for the tumor immunosuppressive microenvironments. Defining the suppressive mechanisms utilized by Treg cells is critical for the development of effective strategies to treat human cancers. However, the molecular processes that occur in responder T cells that are suppressed by Treg cells remain largely unknown. We discovered that both naturally occurring Treg and tumor-derived Treg cells induce responder naïve/effector T cell senescence with potent suppressive activity. We further identified that initiation of DNA damage is critical and the main cause for the induction of responder T cell senescence and dysfunction mediated by human Treg cells. In addition, Treg-induced effector T cell DNA damage and senescence is due to the glucose competition between Treg and responder effector T cells. Furthermore, MAPK ERK1/2 and p38 signaling functionally cooperate with the transcription factors STAT1/STAT3 to control the molecular process of responder T cell senescence induced by human Treg cells. Senescent T cells have unique phenotypes, transcriptional profiles, gene regulation signatures, and active functions, distinct from that of exhausted and anergic T cells. Importantly, our in vivo studies suggest that human Treg-induced T cell senescence can be prevented via the inhibition of DNA damage response and/or STAT signaling in T-cell adoptive transfer mouse models. These studies identify novel molecular mechanisms of human Treg suppression and provide an emerging concept that targeting Treg-induced effector T cell senescence is a new checkpoint for immunotherapy against cancer and other diseases associated with Treg cells.
Unbiased testing of all single nucleotide variants specific to a tumor for anti-tumor CD8+ T cell immune response
Authors: Karandikar Sukrut Hemant , Shcheglova Tatiana V. , Hagymasi Adam T. , Brennick Cory A. , Mandoiu Ion I. , Srivastava Pramod K.
Published: January 2023

Neoepitopes presented in the context of MHC render tumors immunogenic. However, not all potential neoepitopes elicit immunity, and the attributes of immunogenic neoepitopes are yet to be fully defined. Based on the premise that regressing tumors must elicit T cells that recognize the true anti-tumor neoepitopes, we have developed and used here an ELISPOT based high-throughput assay that allows testing of the immunogenicity of a large number of candidate neoepitopes. Naïve C57BL/6 mice were challenged with the colorectal tumor line MC38 and treated, starting day 5 post-tumor challenge, with a checkpoint-blocking antibody, leading to the beginning of tumor regression. MC38-specific CD8+ T cell response was observed in tumor-bearing hosts with regressing tumors. Exome and transcriptome sequencing of MC38 and accompanying bioinformatic analyses have revealed >800 single nucleotide variations (SNVs) in MC38. To identify the SNVs contributing to the immunogenicity of MC38, reactivity of the anti-MC38 CD8+ T cells isolated from mice with regressing tumors, towards individual peptides corresponding to each identified SNV, was tested. Indeed, CD8+ T cells against a number of mutant peptides were detected. These immunogenic mutant peptides are potential MHC I-restricted neoepitopes of MC38 and their role in tumor rejection is being tested.
Complex phenotyping of PD-1+ CD39+ exhausted CD8+ T cells in human carcinomas
Authors: Egelston Colt , Avalos Christian , Huang Yinghui , Wang Roger , Solomon Shawn , Tu Travis Y. , Simons Diana L. , Lee Peter P.
Published: January 2023

CD8+ T cell infiltration into solid tumors has been shown to correlate with patient prognosis in numerous carcinomas. Furthermore, response to checkpoint blockade therapies has been linked to an induction of T cell function and rescue of T cell exhaustion in patients. Expression of both PD-1 and CD39 on exhausted CD8+ T cells has been described in murine models of chronic disease and recently human carcinomas. Here we profile breast tumor, melanoma, and chronic lymphocytic leukemia (CLL) for the frequency and phenotype of exhausted CD8+ T cells. We find that the frequency of exhausted CD8+ T cells is highest in melanoma among these tumor types which may suggest correlation with mutational burden. Functional analysis of PD-1+ CD39+ cells in human tumors shows significant reduction of IFNγ, TNFα, and IL-2 production as assessed by intracellular flow cytometry. Further phenotyping of exhausted CD8+ T cells revealed a loss of expression of both the IL-7 receptor alpha (CD127) and KLRG1, suggesting terminal differentiation of these cells. PD-1+ CD39+ T cells also expressed CD103 suggesting potential similarities with differentiation into a peripheral resident memory phenotype. Indeed PD-1+ CD39+ exhausted T cells were only identified in peripheral tissues where cancer cells were present, such as solid tumors, tumor positive tumor draining lymph nodes, and one CLL patient, but not identified in the circulation or tumor negative tumor draining lymph nodes of patients. Finally, single cell and T cell receptor sequencing revealed distinct transcriptional and clonal signatures of PD-1+ CD39+ CD8+ T cells suggesting them to be a unique population in response to tumor antigen.
Novel T cell therapy using spherical nucleic acids
Authors: Dominguez Donye , Chernyak Natalia , Guan Monica , Long Alan , Lee Andrew , Chou Yushang , Chen Siqi , Qin Lei , Fan Jie , Mirkin Chad , Zhang Bin
Published: January 2023

Immune stimulating Spherical Nucleic Acids (SNAs) are nanoparticles comprised of a spherical, hollow, lipid core surrounded by an oligonucleotide shell, allowing for the simultaneous delivery of tumor antigens and immune stimulating oligonucleotides into immune cells. Interestingly, we have found that SNAs can readily enter primary nonphagocytes such as T lymphocytes. The uptake of SNAs increases expression of co-stimulatory molecules and presentation of antigen via MHC class I molecules by T cells. SNA treated T cells are endowed with the ability to prime tumor antigen specific CD8+T cells in a long lasting and durable manner compared to “free” oligonucleotide and antigen admix-treated T cells. Furthermore, SNA treated T cells are able to act as cellular chaperones and transfer both SNA-derived oligonucleotide and antigen to other cells. In an amplifying cascade manner, these “bystander” cells, which receive SNA material, then gain the ability to prime antigen-specific CD8+ T cells and achieve stronger antitumor effect. The cellular chaperone cascade only occurs when cells are treated with SNAs compared to the “free” material. The unique structural properties of SNAs thus confer a set of key biological properties that make SNAs particularly compelling for the efficient and specific delivery of tumor antigen and adjuvant in adoptive T cell therapy.
CPMA9, a newly immune checkpoint molecule, regulates T cell function
Authors: Jeon You-kyoung , Choi Inhak
Published: January 2023

We identified a new immune checkpoint molecule, CPMA9, belonging to the immunoglobulin superfamily (IgSF). In TCGA (The Cancer Genome Atlas) data set, CPMA9 is highly expressed and associated with poor survival in human prostate, lung, liver, breast. Also, we showed that CPMA9 differentially expressed between responder and non-responder with Pembrolizumab, PD1 blockade, treated renal cell carcinoma patients (n=49). CPMA9 inhibits TCR mediated T cell activation through suppress of proliferation, various cytokine secretion. Furthermore, hydrodynamic injection of plasmid encoding CPMA9. Ig into mice that were immunized with ovalbumin inhibits T and B cell response to chicken OVA protein. These data suggest that CPMA9 directly inhibits T cell activation as new immune checkpoint.
Neoadjuvant OX40 therapy in patients with head and neck cancer induces profound changes in tumor-infiltrating lymphocytes
Authors: Duhen Rebekka A. , Ballesteros-Merino Carmen , Bell R. Bryan , Leidner Rom , Koguchi Yoshinobu , Bifulco Carlo , Fox Bernard A. , Weinberg Andrew D.
Published: January 2023

OX40 is a member of the tumor necrosis factor (TNF) receptor family and, as a co-stimulatory molecule, enhances T-cell proliferation, survival and memory formation. We have also shown that it induces robust antitumor responses in patients with metastatic disease. To examine the effect of OX40 stimulation on the immune response in cancer patients, 16 head and neck squamous cell carcinoma (HNSCC) patients were enrolled in a neoadjuvant phase Ib trial and received anti-OX40 infusion 2 days, 1 week and 2 weeks prior to surgical resection.

To evaluate changes in the tumor microenvironment (TME), tissue and peripheral blood samples were obtained prior to infusion and at time of surgery. Assessment of changes in tumor infiltrating lymphocyte (TIL) and peripheral blood populations were performed by flow cytometry and fluorescent multiplex immunohistochemistry (IHC).

OX40 administration was well tolerated with low toxicity. Activation and proliferation of CD4+ and CD8+ memory T-cell populations in both the TME and periphery peaked between 12 and 19 days after OX40 infusion, as demonstrated by increased levels of Ki67, CD38 and ICOS. In the TIL, expression of CD39, ICOS and PD-1 was increased on CD4+ T cells in most patients and the frequency of tumor-reactive CD39+CD103+ CD8+ T cells was increased in some patients. IHC analysis revealed striking changes in tissue integrity and increased lymphocyte infiltrates in 4/16 patients.

With this study, we have found that neoadjuvant OX40 immunotherapy is feasible and safe in patients with HNSCC and results in robust activation and proliferation of T cells within the tumor. Based on these findings, we are now comparing the transcriptome and changes in TIL repertoire and metabolism before and after treatment.
Modulation of TFH-like cells and anti-tumor activity of immune checkpoint blockade.
Authors: Zappasodi Roberta , Budhu Sadna , Hellmann Matthew D. , Postow Michael A. , Senbabaoglu Yasin , Manne Sasikanth , Gasmi Billel , Liu Cailian , Zhong Hong , Li Yanyun , Huang Alexander C. , Hirschhorn-Cymerman Daniel , Panageas Katherine S. , Wherry E. John , Merghoub Taha , Wolchok Jedd D.
Published: January 2023

A significant proportion of cancer patients do not respond to immune checkpoint blockade therapy. To deepen our understanding of the mechanisms of resistance to immunotherapy, we studied a population of CD4+Foxp3− T cells expressing PD-1 (4PD1hi), which we found to be up-regulated in B16-melanoma bearing mice after CTLA-4 blockade in association with limited response to treatment. We observed that 4PD1hi accumulate intratumorally as a function of tumor burden in untreated tumor-bearing hosts. Interestingly, CTLA-4 blockade promotes intratumoral and peripheral 4PD1hiincreases in a dose-dependent manner, while combination with PD-1 blockade mitigates this effect in both B16-bearing mice and melanoma patients and significantly improves anti-tumor activity. In addition, we found that persistence of high levels of 4PD1hi after PD-1 blockade correlates with poor prognosis in melanoma patients. Mechanistically, we show that mouse and human circulating and intra-tumor 4PD1hi inhibit T-cell functions in a PD-1/PD-L1 dependent fashion. In addition, we found that mouse and human 4PD1hi resemble follicular-helper-T-cell(TFH)-like cells and that CTLA-4 blockade activity is improved in TFH deficient mice. These findings broaden our understanding of the mechanisms that limit anti-tumor immunity, providing an additional explanation for the incremental activity of combined CTLA-4 and PD-1 blockade. Our study also defines 4PD1hi as a new prognostic and pharmacodynamic biomarker for the design of optimal checkpoint blockade combination schedules and dosage.
Bacterial and viral pathogen associated molecular patterns create different early transcriptomic landscapes in bovine macrophages
Authors: Toka Felix Ngosa , Dunaway Kiera , Smaltz Felicia , Szulc-Dabrowska Lidia , Drnevich Jenny , Mielcarska Matylda Barbara , Bossowska-Nowicka Magdalena , Schweizer Matthias
Published: January 2023

Pathogens stimulate immune functions of dendritic cells and macrophages (MΦ). There is no clear understanding of the nature of transcriptomic changes caused in cells upon stimulation through pattern recognition receptors by various pathogens. Our study investigated the early transcriptomic changes of bovine MΦ in response to stimulation with CpG or poly I:C. BMΦ were incubated with CpG or pI:C for 6 h and RNA isolated for transcriptomic analysis by RNAseq. Bioinformatics tools were used to analyze differentially expressed genes (DE). Results showed that, of the 13740 genes mapped to the bovine genome, 2245 had p-value ≤0.05, deemed as DE. At 6 h post stimulation, pI:C induced a different transcriptomic profile from that induced by CpG. CpG upregulated 347 and downregulated 210 genes. pI:C upregulated 761 and downregulated 414 genes. The highest expressed genes in both stimulations belonged to the TNF superfamily, TNFSF18 (CpG) and TNFSF10 (pI:C) and in both cases the lowest downregulated gene was CYP1A1. CpG induced canonical pathways that are unrelated to immune response in BMΦ in contrast to pI:C which induced canonical pathways directly related to antiviral immune functions dominated by IFN signalling genes. The transcriptomic profile after pI:C stimulation was consistent with TLR3 signalling. CpG influenced expression of genes involved in molecular and cellular functions in free radical scavenging. We conclude that CpG and pI:C induce different early transcriptional landscapes in BMΦ line, but each suited to a specific function of BMΦ during interaction with pathogens.