bluuebunny/crossref_metadata_2025 huggingface dataset:
Gene conversion contributes significantly to IgHV somatic hypermutation in mice and humans
Authors: Dale Gordon A , Bohannon Caitlin D , Wilkins Daniel J , Dilernia Dario , Hunter Eric , Bedford Trevor , Antia Rustom , Sanz Ignacio , Jacob Joshy
Published: January 2023

During antigen-driven affinity maturation germinal center B lymphocytes undergo multiple rounds of somatic mutagenesis in the rearranged IgHV gene segment. A direct consequence of this process is the generation of multiple antibody mutants that are, in general, specific for antigen. Survival of these mutants is contingent on preserving or improving antigen-specificity in the complementarity-determining regions (CDRs) as well as retaining a functional framework region (FWR). Broadly, somatic mutations in germinal center B cells can be classified as templated or untemplated. In animals such as rabbits and chickens, somatic mutations are mostly templated. Conversely, in mice and humans it is widely accepted that mutations are untemplated. Here, we argue in favor of the alternative, that a predominant number of somatic mutations in IgHV gene segments in mice and humans can be traced to germline IgHV genes and occur through gene conversion. Using the NP hapten model, we demonstrate that mutations in germinal center B cells and plasma cells are traceable to other germline IgHV genes. Subsequently we also demonstrate using the novel V-LAIR1-DJ antibodies described by Tan et al (Nature 2016) that mutations in human antibody IgHV rearrangements, including those in non-immunoglobulin sequences (LAIR1), are traceable to the human IgHV germline repertoire. Together, this suggests that the plethora of diversity generated in antigen specific B cells is primarily directed by the germline repertoire of IgHV genes.
CD8+ tissue resident memory T cells are associated with good prognosis in breast cancer patients
Authors: Egelston Colt , Srinivasan Gayathri , Avalos Christian , Huang Yinghui , Rosario Anthony , Wang Roger , Jimenez Grecia , Simons Diana L , Yost Susan , Yuan Yuan , Lee Peter P
Published: January 2023

CD8+ Tumor Infiltrating Lymphocytes (TILs) have been shown to correlate with patient prognosis in breast cancer. Recently, resident memory T cells (TRM) have been established as a potent functional subset of memory T cells that exist in peripheral tissue without recirculation. We assayed CD8+ T cells from fresh breast tumors to phenotype memory T cells and found them to be almost exclusively made up of effector memory T cells. Further profiling by examining expression of CD103 and CD69 showed that on average 40% of CD8+ TILs are composed of TRM. Functional analysis of these T cells showed robust production of IFNγ and TNFα by these TRMs suggesting that despite existence in the tumor microenvironment TRMs maintain functional potency. To examine if CD8+ TRM have prognostic value in breast cancer patients, we examined a set of primary tumors from ‘good’ and ‘bad’ outcome patients, which we defined as having a relapse in more than 5 years after diagnosis or in less than 3 years after diagnosis respectively. In good outcome patients TRMs represented 60% of CD8+ T cells found in cancer islands, while only 20% of such in bad outcome patients. Similarly, CD8+ TRMs were observed in higher numbers in both the cancer islands and stroma of good outcome patient tumors in comparison to bad outcome patient tumors. This observed significance was greater than that found for CD8+ T cells in general, highlighting the importance of TRM in affecting patient outcome. Finally we showed that TRMs also exist in non-cancerous breast tissue and are found within the mammary ducts associated with epithelial cells perhaps in surveillance of potential pathogens. We suggest that TRMs are a potential vaccine target for preventing recurrence and metastasis in cancer patients.
TWEAK/Fn14 activation contributes to the pathogenesis of bullous pemphigoid
Authors: Liu Yale , Peng Lingling , Li Liang , Liu Chengfei , Hu Xiao , Xiao Shengxiang , Xia Yumin
Published: January 2023

Background

Tumor necrosis factor-like weak inducer of apoptosis (TWEAK) participates in various cellular effects by engaging its receptor of Fn14. Increased levels of soluble TWEAK are associated with systemic autoimmunity in patients with lupus erythematosus, rheumatoid arthritis or dermatomyositis. However, the role of TWEAK in bullous pemphigoid (BP) remains unknown.

Objectives

To explore the potential role of TWEAK/Fn14 activation in the pathogenesis of BP.

Methods

Twenty-three patients with BP and twenty healthy donors were recruited. The levels of TWEAK were determined in sera and BP fluids. Immunohistochemistry was performed with skin tissues for the expression of TWEAK, Fn14, and BP180. HaCaT cells were cultured in vitro, and received TWEAK stimulation. Some cells were pretreated with siRNA transfection or specific inhibitors before such stimuli.

Results

Serum levels of TWEAK increased in sera from BP patients, and correlated positively with anti-BP180 IgG. By immunohistochemistry, both TWEAK and Fn14 were overexpressed in BP lesion. In vitro experiments showed that TWEAK stimuli reduced BP180 expression in HaCaT cells and inhibited adhesion of cells to the culture dish. Consistently, the transfection of Fn14 siRNA preserved BP180 and protected cells from losing adherence. Moreover, specific inhibitors of the ERK and NF-κB pathways partially abrogated effect of TWEAK on cells. Finally, By silencing ADAM17 with siRNA, we found that ADAM17 mediated TWEAK-induced BP180 suppression.

Conclusion

TWEAK contributes to the pathogenesis of BP by reducing BP180 expression and cellular adherence, involving the activation of ERK and NF-κB pathways. TWEAK may serve as a biomarker or therapeutic target of BP.
Immune cell phenotype and function in different compartments of the gut-liver-axis in chronic liver disease
Authors: Bolte Fabian J , O’Keefe Ashley C. , Etzion Ohad , Ali Rabab , Serti Elisavet , Liang Jake , Heller Theo , Rehermann Barbara
Published: January 2023

BACKGROUND

The liver is exposed to gut-derived products via the portal vein and bacterial translocation is associated with chronic liver disease. Here we analyzed immune cell activation and function in three compartments of the gut-liver axis: systemic blood, portal vein blood and liver.

METHODS

Natural killer (NK) cells, mucosal associated invariant T (MAIT) cells and conventional CD8 T cells were studied in liver and blood of 29 patients with chronic hepatitis C prior to and after Sofosbuvir/Velpatasvir-induced viral clearance.

RESULTS

NK, MAIT and conventional CD8 T cells were more activated (CD69+, HLA-DR+) and more cytotoxic (CD107a+) in the liver than in systemic and portal blood. Monocytes were also more activated in the liver and plasma levels of monocyte-derived IL-18 correlated with liver inflammation. Consistent with the activation of intrahepatic immune cells, levels of sCD14, sCD163 and sCD27 were higher in systemic than in portal plasma. However, immune cell activation and degranulation were not different in systemic and portal vein blood. Likewise, the in vitro functional responses of monocytes to lipopolysaccharide and of MAIT cells to riboflavin-synthesizing bacteria did not differ between systemic and portal blood. Intrahepatic immune cell activation declined rapidly within four weeks of antiviral therapy and remained stable for up to 36 weeks post treatment.

CONCLUSIONS

Immune cell activation and inflammation are compartmentalized to the liver in patients with compensated HCV-related liver disease. Plasma markers of immune cell activation are higher in systemic than in portal plasma. The rapid decline of intrahepatic immune cell activation suggests that it is primarily driven by viral infection.
A Regulatory Role for “MFehi” Macrophages In Adipose Tissue Iron Homeostasis
Authors: Hubler Merla , Hasty Alyssa , Moestrup Soren , Etzerodt Anders
Published: January 2023

Macrophages are the predominant immune cell in adipose tissue (AT) and exhibit phenotypic variability depending on the obesity state of the tissue. Lean AT contains a population of anti-inflammatory “M2” AT macrophages (ATMs), whereas, obesity is associated with an influx of inflammatory “M1” ATMs. The role of M2 ATMs in maintenance of a healthy lean state is relatively undefined. Our lab has previously published the discovery of the “MFehi” population of M2 ATMs, which has a two-fold increase in intracellular iron content and exhibits increased expression of genes involved in iron uptake (e.g. CD163), storage and release. This population composes 25% of M2 ATMs and exists in both lean and obese mouse and human AT. It remains unknown whether MFehi contribute to AT iron metabolism. To better understand how MFehi are regulating iron in AT, we provided iron through diet and by IP injection and predicted that MFehi compensate for excess iron to protect adipocytes from iron overload. We found that only MFehi take up the iron, and their gene expression corresponds with this uptake. Furthermore, there is no change in iron content or gene expression in adipocytes. Even though the MFehi change their gene expression in obese AT, we still observed that they are able to compensate for excess iron in the tissue. To determine the impact of MFehi on the systemic metabolic state, we depleted M2 ATMs in mice with cytotoxic CD163-targeted liposomes. Our preliminary studies have revealed CD163-targeted liposomes as a novel technique to target CD163-expressing M2 ATMs. These studies will allow us to characterize the role of the MFehi population in maintaining healthy adipocyte iron concentrations and, thereby, insulin sensitivity.
Selective oral ROCK2 inhibitor reduces clinical scores in patients with Psoriasis Vulgaris and normalizes skin pathology via concurrent regulation of IL-17 and IL-10 levels
Authors: Zanin-Zhorov Alexandra , Weiss Jonathan M , Trzeciak Alyssa , Chen Wei , Zhang Jingya , Nyuydzefe Melanie , Arencibia Carmen , Polimera Seetharam , Fuentes-Duculan Judilyn , Bonifacio Kathleen , Kunjravia Norma , Cueto Inna , Berger Mark , Krueger James , Ryan John L , Waksal Samuel D
Published: January 2023

Targeted inhibition of Rho-associated kinase 2 (ROCK2) down-regulates pro-inflammatory T-cell response, while increasing the regulatory arm of immune response in animals models of autoimmunity and T helper 17 (Th17)-skewing human cell culture in vitro. We conducted a Phase 2, open-label, dose-finding study to evaluate the safety, tolerability, and activity of selective ROCK2 inhibitor, KD025, in subjects with Psoriasis Vulgaris who failed first-line therapy (NCT02317627 at ClinicalTrials.gov). Here, we report that oral administration of KD025 reduces Psoriasis Area and Severity Index (PASI) scores in 85% of patients completing the study, with minimal side effects. In the 400 mg QD and 200 mg BID cohorts 42% and 71% of patients respectively achieved at least a 50% decrease in PASI score (PASI 50) after three month of treatment. KD025 treatment reduced levels of both IL-17 and IL-23, but no IL-6 and TNF-a in the peripheral blood of clinical responders, whereas IL-10 levels were increased at the end of the study. The clinical improvement and changes in cytokine levels were associated with decreased epidermal thickness, T-cell infiltration and down-regulation of key molecules implicated in the regulation of the Th17 pathway, such as ROCK2, pSTAT3, RORgt and IRF4 in the skin. Collectively, these data demonstrate that orally available selective ROCK2 inhibitor down-regulates Th17-driven autoimmune response and improved clinical symptoms in psoriatic patients via defined molecular mechanism that involves concurrent modulation of cytokines without deleterious impact on the rest of the immune system.
Interplay between keratinocyte STAT1 and STAT3 signaling controls skin inflammation and T-cell development in a mouse model of psoriasis
Authors: Archer Nate , Lee Steven K , Ortines Roger V , Wang Yu , Liu Haiyun , Miller Robert J , Dillen Carly A , Marchitto Mark , Ashbaugh Alyssa G , Uppal Angad , Cai Sarah , Miller Lloyd S
Published: January 2023

Psoriasis is an autoimmune skin disease affecting 2–3% of the population that is driven by IL-17 responses. Although STAT3 promotes the development of IL-17-producing T cells, STAT3 overexpression in keratinocytes is sufficient to induce psoriasiform dermatitis in mice. To study the role of STAT3 in keratinocytes versus T cells in psoriasis, the imiquimod mouse model of psoriasiform dermatitis was performed on mice with specific deletion of STAT3 in either keratinocytes (K5-creERT2×STAT3fl/fl [K5-STAT3]) or T-cells (Lck-cre×STAT3fl/fl [Lck-STAT3]). Unexpectedly, K5-STAT3 mice but not Lck-STAT3 mice had reduced skin inflammation and inflammatory cell influx compared to wt mice, suggesting that STAT3 signaling in keratinocytes rather than T cells was more important determinant for mediating the skin inflammation. K5-STAT3 mice also had increased IFN-γ+ T cells but less IL-17+ T-cells compared to wt mice, indicating that loss of STAT3 signaling in keratinocytes dampened inflammation by inhibiting IL-17 responses while promoting IFN-γ responses. Since interferon/STAT1 signaling responses can be inhibited by STAT3, we hypothesized that loss of STAT3 signaling in keratinocytes would lead to enhanced interferon/STAT1-signaling and responses. Consistent with this possibility, histologic sections from the inflamed skin of K5-STAT3 mice had increased epidermal STAT1 phosphorylation and increased mRNA expression of interferon-regulated chemokines CXCL10 and CXCL11 compared with wt mice. Taken together, these findings indicate that keratinocytes can regulate T cell responses in the skin and STAT3 signaling in keratinocytes could represent a therapeutic target in psoriasis.
Map3K14 signaling attenuates the development of colorectal cancer through activation of the non-canonical NF-κB signaling cascade
Authors: Allen Irving C , Eden Kristin , Heid Bettina , Holl Eda K
Published: January 2023

Dysregulation of NF-κB signaling is a common feature in Colorectal Cancer (CRC). While the canonical signaling pathway is well characterized, the non-canonical NF-κB signaling cascade is understudied. Thus, we evaluated the hypothesis that non-canonical NF-κB signaling significantly modulates CRC pathogenesis. To evaluate this hypothesis, we initially conducted a meta-analysis of human gene expression data. Using this approach, we found that non-canonical NF-κB signaling is significantly down-regulated in CRC patients and the extent of pathway attenuation is correlated with disease severity. These findings were confirmed in patient biopsy specimens, where we found significantly reduced expression of ligands, receptors, regulators, and chemokines associated with non-canonical NF-κB signaling. Notably, we observed significantly reduced CCL19, CCL21, CXCL12, and CXCL13 expression in CRC patients compared to healthy controls. To gain mechanistic insight, we utilized genetically modified mice lacking the kinase Map3K14 (NIK), which is essential for non-canonical NF-κB signaling. Nik−/− mice exhibited ablation of non-canonical NF-κB signaling and significantly increased epithelial cell proliferation in the GI tract compared to wild type mice in both in vivo and in vitro studies. Together, our data defines a protective role for non-canonical NF-κB signaling in the gut during CRC and identifies a highly protective role for Map3K14 in attenuating epithelial cell proliferation.
The innate immune receptor, NLRX1, functions as a tumor suppressor by reducing colon tumorigenesis and key tumor-promoting signals
Authors: Koblansky A Alicia , Truax Agnieszka D. , Liu Rongrong , Montgomery Stephanie A , Ding Shengli , Wilson Justin E , Brickey W June , Hu Peizhen , Li Zengshan , Lund Pauline Kay , Ting Jenny P.Y.
Published: January 2023

NLR (NOD-like receptor) proteins are intracellular innate immune sensors/receptors that regulate immunity. This work shows that NLRX1 serves as a tumor suppressor in colitis-associated cancer (CAC) and sporadic colon cancer by keeping key tumor promoting pathways in check. Nlrx1−/− mice were highly susceptible to CAC, showing increases in key cancer-promoting pathways including NF-kB, MAPK, STAT3 and IL-6. The tumor-suppressive function of NLRX1 originated primarily from the non-hematopoietic compartment. This prompted an analysis of NLRX1 function in the Apcmin/+genetic model of sporadic gastrointestinal cancer. NLRX1 attenuated Apcmin/+colon tumorigenesis, cellular proliferation, NF-kB, MAPK, STAT3 activation and IL-6 levels. Application of anti-IL6R antibody therapy reduced tumor burden, increased survival and reduced STAT3 activation in Nlrx1−/−Apcmin/+mice. As an important clinical correlate, human colon cancer samples expressed lower levels of NLRX1 than healthy controls in multiple patient cohorts. These data implicate anti-IL6R as a personalized therapeutics for colon cancers with reduced NLRX1.
Merkel Cell Carcinoma viral oncoprotein-specific polyclonal T cells can be generated from patients and from healthy donors
Authors: Davies Sarah I , Whitehill Gregory , Yu Quan , Chang Mark Jesse , Keyvanfar Keyvan , Brownell Isaac , Buck Christopher , Barrett John , Muranski Pawel
Published: January 2023

Truncated large T (LT-T) and small T (ST) antigens of Merkel Cell Polyomavirus (MCV) are viral oncoproteins critical for pathogenesis of Merkel Cell Carcinoma (MCC), making this malignancy an attractive target for immunotherapy. We explored feasibility of generating LT-T and ST specific T cells from 12 healthy donors and 4 patients with MCC. Manufactured overlapping peptide libraries (pepmixes) spanning entire LT-T and ST proteins were pulsed onto autologous dendritic cells or peripheral blood mononuclear cells and co-cultured with lymphocytes in presence of IL-2, IL-7 and IL-15. Analogous cultures were generated using BK and JC polyomavirus LT pepmixes and PRAME (MCC patients). On day 14, cells were tested for recognition of cognate pepmixes. T cells from 4/12 (33.3%) healthy donors released IFN-γ and/or TNFα (average 0.48 ± 0.144 %) in response to MCV T antigens, predominantly in CD4± cells. 2/4 patients with MCC had a robust T cell response against MCV LT (2.23 ± 0.65%), 3/4 patients responded to JCV LT (3.56 ± 1.92%), and 2/4 responded to PRAME (1.18 ± 0.707%). T cells specific for JC or BK LT antigens demonstrated mutual cross-reactivity, but negligible cross reactivity with MCV LT and vice versa, suggesting that the more abundant JC and BK-specific cells do not confer protection against MCC. Our data indicate that while MCC subjects have strong responses healthy donors harbor only rare memory T cells specific for MCC oncoproteins. Based on these findings we are developing highly-active clinical-grade MCC-specific T cells for adoptive immunotherapy.
Engineering adoptive T cell therapy for efficacy in ovarian cancer
Authors: Anderson Kristin G , Bates Breanna M. , Chiu Edison Y. , Greenberg Philip D.
Published: January 2023

Over 20,000 women are diagnosed with ovarian cancer annually; more than half will die within 5 years, which has changed little over the last 20 years. One promising new treatment strategy employs immune T cells engineered to target proteins uniquely overexpressed in tumors, with the potential to control tumor growth without toxicity to healthy tissues. Recently, mesothelin (MSLN) has been identified as a valid antigen target in ovarian cancer; minimally expressed in healthy cells, MSLN contributes to malignant and invasive phenotypes. In preclinical studies using patient-derived cell lines or the mouse ID8 ovarian tumor model, we found that T cells engineered to express a high-affinity MSLN-specific T cell receptor (TCR) can kill ovarian tumor cells in vitro. In vivo, adoptively transferred TCR-engineered T cells preferentially accumulate within disseminated ID8 tumors, delay tumor growth and prolong mouse survival, but our data also show the tumor microenvironment (TME) can limit engineered T cell persistence and function. Other analyses show human therapy will face similar TME-mediated obstacles. The ovarian cancer TME is a nutrient- and oxygen-deprived milieu, and adaptive metabolic responses by infiltrating T cells have protean effects on T cell function. Thus, strategies that modulate T cell metabolic pathways, and thereby influence activity in the TME, might enhance T cell function and improve anti-tumor efficacy by overcoming a critical component of immune evasion by solid tumors. Ongoing studies will be discussed that are exploring strategies to overcome elements common to the human and murine TME, including direct modulation of the environment and T cell engineering to promote T cell survival and function.
Dissection of CD8+ T cell immunodominance hierarchy in human influenza virus infection
Authors: Sant Sneha Ashok , Parra Sergio Quinones , Rossjohn Jamie , Gras Stephanie , Loh Liyen , Kedzierska Katherine
Published: January 2023

Based on magnitude, virus-specific CD8+ cytotoxic T lymphocyte (CTL) responses can be grouped as immunodominant and subdominant. Such immunodominance hierarchies are governed by multiple factors, including naïve T cell precursor frequency, proliferative capacity and clonotypic diversity of Ag-specific CTLs. However, how these factors determine together inter/intra-individual immunodominance remains elusive. In this study, we dissected CTL immunodominance hierarchy for universally conserved influenza (IAV)-specific epitopes. Our data from 59 donors covering the HLAI types presenting universally conserved IAV-specific peptides, HLA-A* 0201, A*0301, B*2705, B*5701, B*1801 and B*0801, revealed that CTLs directed against HLA-A* 0201:M158 were immunodominant, except in A*0201+B*2705+ heterozygous individuals, where the HLA-B* 2705-NP383 recalls higher magnitude CTL responses. Subsequently, we further dissected the basis of B*2705 immundomination over A*0201 by analyses of T cell Receptor (TCRαβ) repertoire, TCR avidity and kinetics of killing and proliferation. Our data show that following in vitro peptide stimulation, B*2705:NP383 CTLs proliferate rapidly acquiring higher levels of polyfunctionality than A*02:M158 specific CTLs. Furthermore, ex vivo single-cell paired CDR3αβ analysis revealed that public A*0201:M158 TCRs characterized by Vβ19/Vα27 profiles in A*0201 individuals are significantly reduced/absent in A*0201+B*2705+ individuals. Clonal analysis of M158 dominant public TCRs suggested B*2705-mediated removal of self-reactive TCRs and is under investigation. Our study provides insights into the importance of human IAV-specific immunodominance at quantitative and qualitative levels.
Skin homing and highly suppressive (CD45RA−CD27−) Tregs associated with ultraviolet radiation exposure
Authors: Hesterberg Rebecca , Akuffo Afua Adutwumwa , Hampras Shalaka S , Messina Jane L , Fenske Neil A , Cherpelis Basil , Schell Michael J , Reed Rhianna , Balliu Juliana , Rollison Dana E , Epling-Burnette Pearlie K
Published: January 2023

Immune suppression is a critical risk factor for the development of cancer. Understanding how carcinogens impact the immune system in immunocompetent individuals may give rise to relevant information about the biological processes that occur in precancerous lesions and how these contribute to cancer progression. UV radiation (UVR) is a known risk factor for keratinocyte cancer, but its association with immunosupperssive CD4+FoxP3+ regulatory T (Treg) cells is not established. To evaluate the association between UVR and Tregs, an epidemiological study (n=350) was conducted among a cohort undergoing skin cancer screening. Patients were recruited at the University of South Florida Dermatology clinic and UVR exposure was quantified by spectrophotometric readings from the axilla (sun-unexposed) and two sun exposed regions including the forehead and upper forearm. Populations of T cells were determined by flow cytometry using peripheral blood samples collected at the same clinic visit. Subsets of CD8+ T cell, CD4+ T cells, and total Treg numbers showed no significant correlation to UVR exposure. Interestingly, the proportion of Tregs that lacked expression of CD45RA and CD27 (ie, effector Tregs) were positively associated with UVR exposure (r=0.127, p=0.020 and r=0.197, p=0.0002, respectively). This population of Tregs has been shown to be more suppressive and expanded in a pre-leukemic patient population. Skin-associated CLA+ (r=0.167, p=0.002) and CCR4+ (r=0.19, p=0.0004) Tregs were also positively correlated with UVR exposure. These data demonstrate the association between UVR exposure and the expansion of immunosuppressive skin-associated Treg subsets that may contribute to the process of skin cancer progression.
Eradication of hepatocellular carcinoma by augmenting PD-L1 blockade efficacy with suppression of cell-cycle related kinase (CCRK)-induced myeloid-derived suppressor cells
Authors: Zhou Jingying , Liu Man , Sun Hangyong , Feng Yu , Wong John , Chan Anthony W.H. , Chow King-Lau , Chen Zhiwei , Cheng Alfred S.L.
Published: January 2023

Myeloid-derived suppressor cells (MDSCs) constitute to tumor immunosuppression and immune checkpoint blockade therapy resistance. We have recently reported the cell-autonomous pro-tumorigenic functions of cell cycle-related kinase (CCRK) in hepatocellular carcinoma (HCC), but its role in tumor immune evasion remains unknown. Here we report that the hepatoma-intrinsic CCRK signaling renders T-cell dysfunction by induction of MDSCs. CCRK stimulated immunosuppressive CD11b+CD33+HLA-DR−MDSC generation from human PBMCs through up-regulating interleukin-6 (IL-6). Mechanistically, CCRK activated nuclear factor-kappa B (NF-κB) in an enhancer of zeste homolog 2 (EZH2)-dependent manner and facilitated NF-κB-EZH2 co-binding to IL-6 promoter. In corroboration with the CCRK-inducible transgenic mouse model, we demonstrated that hepatic CCRK induced IL-6 to specifically expand and recruit polymorphonuclear-MDSCs with potent T-cell-inhibitory function to the liver. In contrast, genome-deletion of tumoral Ccrk expression or nanoparticle-mediated hepatic IL-6 inhibition increased cytotoxic IFN-γ+TNF-α+CD8+T-cell infiltration and impaired tumorigenicity, which was rescued by restoring polymorphonuclear-MDSCs. More importantly, Ccrk deletion significantly enhanced the efficacy of anti-PD-L1 therapy to eradicate large hepatoma in an intrahepatic model. As we also showed that CCRK positively correlated with the intra-tumoral CD11b+CD33+HLA-DR−MDSCs and poor survival rates of HCC patients, our results establish an immunosuppressive function of the hepatoma-intrinsic CCRK signaling and highlight a therapeutic target whose inhibition might potentiate cancer immunotherapy.
TGFβ superfamily members regulate radiation-induced anti-tumor immunity.
Authors: Vanpouille-Box Claire I , Formenti Silvia C , Demaria Sandra
Published: January 2023

Transforming Growth Factor-beta (TGFβ) and activin A (actA) are TGFβ superfamily members with overlapping functions in many processes including regulation of inflammation and immunity. We have recently found that actA release by breast cancer cells is enhanced by radiotherapy (RT). Interestingly, prolonged exposure to TGFβ inhibitors also resulted in actA upregulation, consistent with a previously described compensatory mechanism. Here we hypothesized that actA and TGFβ regulate RT-induced anti-tumor immunity.

4T1 mouse breast cancer cell derivatives with inducible actA knockdown (KD) (4T1shActA) or non-silencing control (4T1shNS) were injected s.c. in mice (day 0). ActA KD was induced by doxycycline at day 8. TGFβ neutralizing antibody (1D11) was given i.p. every other day starting on day 12. 6Gy RT was delivered to the tumor on days 13, 14, 15, 16 and 17.

TGFβ blockade improved RT-mediated tumor control, an effect mediated by T cells. However, tumor recurred. Notably, ActA KD or 1D11 increased intratumoral Tregs (Control: 11%; 1D11: 26%, shActA: 21%) and enhanced Tregs infiltration induced by RT (RT: 15%; RT+1D11: 27%; RT+shActA: 30%). When both TGFβ and actA were blocked, Tregs significantly decreased in both untreated (1D11+shActA: 13%) and RT-treated tumors (RT+1D11+shActA: 8%). Tumor-specific IFNγ production by CD8+ T cells was higher in RT+1D11+shActA compared to RT+1D11 (*p) and RT+shActA (**p). ActA KD in mice treated with RT+1D11 reduced tumor recurrence and improved survival (RT+1D11 vs RT+1D11+shActA **p; RT+shActA vs RT+1D11+shActA ***p).

Data indicate that both TGFβ and actA impair RT-induced anti-tumor immune responses. Concomitant inhibition of actA and TGFβ is required for optimal in situ vaccination by RT.
Adjuvant Immunotherapy with Surgery Improves Survival in Murine Models of Thoracic Malignancies
Authors: Dunbar Ashley , Baldassari Michael , Connolly Courtney , Mizelle Jack , Newton Andrew , Singhal Sunil , Predina Jarrod
Published: January 2023

Objectives

Immunotherapy is an alternative therapy for thoracic malignancies, but results in humans have not been as impressive as anticipated. We hypothesized that adjuvant immunotherapy with surgery debulking can improve efficacy and long term response rates.

Methods

We retrospectively reviewed our group’s experience with human immunotherapy trials for NSCLC and mesothelioma. We developed murine models of NSCLC and mesothelioma surgery to evaluate clinically applicable immunotherapy approaches (tumor vaccination and checkpoint inhibitors) and optimized regimen. Outcomes were assessed for overall survival and tumor progression. Flow Cytometry, ELISA, PCR and in vivo neutralization assays were utilized to determine immune responses.

Results

The most important predictor of immunotherapy response in human trials was minimal disease at the time of immunotherapy. Surgery generates a state of minimum disease, thus we used a murine cancer surgery model to evaluate clinically available adjuvant immunotherapy approaches in a controlled setting. Adjuvant vaccination strategies were associated with decreased tumor volumes (p<0.01) and were associated with an increase in postoperative survival (42 days vs 24 days, p=0.03). In vivo lymphocyte assays demonstrated only partial cytotoxic activity of CD8+ T-cells postoperatively. The addition of checkpoint inhibitor (CTLA4i) with tumor vaccination improved potency and nearly tripled post-operative survival (54 days vs 19 days; p=0.004). Additionally, 34% of mice receiving combination immunotherapy with surgery were cured (p=0.03).

Conclusion

This evidence supports a new approach to thoracic malignancies including multi modal immunotherapy with surgical resection.
Chemotherapy-induced tumor dormancy and relapse
Authors: Aqbi Hussein F , Butler Savannah E. , Keim Rebecca , Idowu Michael O. , Manjili Masoud H
Published: January 2023

Breast cancer mortality is due to distant recurrence of the disease as a result of awakening dormant tumor cells that were established by cancer therapies. The mechanisms through which dormant tumor cells are maintained or escape from dormancy, and relapse remain elusive. One such mechanism that may play a role in tumor dormancy maintenance or escape is autophagy; a process important in all cells for the removal of damaged proteins and organelles. Although autophagy is a mechanism of tumor suppression, it also confers stress tolerance that enables tumor cells to survive under adverse conditions. Here, we wanted to determine the role of autophagy in tumor dormancy and sensitivity of dormant tumor cells to immunotherapy, i.e., IFN-gamma treatment. A mouse mammary carcinoma (MMC) cell line was established from the neu over-expressing FVBN202 transgenic mouse. We also used Adriamycin (ADR) for the establishment of chemotherapy-induced tumor dormancy, and blocked autophagy by chloroquine (CQ). We demonstrated that a transient blockade of autophagy by CQ during ADR treatment prolonged tumor dormancy, in vitro, but not in vivo. Also, we determined that dormant tumor cells established by ADR or ADR+CQ were more sensitive to IFN-gamma induced apoptosis compared with non-dormant, proliferating tumor cells. These observations suggest that autophagy could participate, in part, in tumor dormancy without affecting the sensitivity of dormant cells to IFN-gamma treatment.
Combination therapy of a PI3K p110α-isoform-selective inhibitor and anti-HER2/neu antibody enhances the anti-tumor immunity in breast cancer mouse model
Authors: Choi Jae Hyeog , Kim Ki Hyang , Roh Kug Hwan , Jung Hana , Seo Su Kil , Choi Il Whan , Yea Sung Su , Park SaeGwang
Published: January 2023

Trastuzumab (Herceptin) is a humanized recombinant monoclonal antibody against HER2 that has shown the clinical benefit in HER2+ breast cancer patients. Because non-responders to trastuzumab have been observed, one strategy to overcome the resistance is combination therapy. Combination therapies with phosphoinositide 3-kinase (PI3K) inhibitors and trastuzumab (anti-HER2/neu antibody) are effective against HER2+ breast cancer mouse model. The therapeutic effect and anti-tumor immunity of combination therapy with an anti-HER2/neu antibody and a pan-PI3K inhibitor (GDC-0941) or a p110a isoform-selective inhibitor (A66) was evaluated in vivo breast cancer models. Combined the anti-neu and PI3Ki treatment synergistically occurred in anti-tumor immunity and HER2/neu-mediated memory T cell responses. In the presence of the anti-neu antibody, A66 was more effective in increasing the T cells and IFN-γ+ CD8+ T cells in TILs. IFN-γ ELISPOT data showed that tumor-specific T cells against neu and non-neu tumor antigens increased synergistically after combination PI3Ki and anti-neu treatment, and A66 was more potent. In a TUBO (neu+) and TUBO-P2J (neu-) mixed model representing IHC2+ tumors, A66 showed greater tumor mass control and survival prolongation than GDC-0941, when combined with the anti-neu antibody. Combined with the anti-neu antibody, A66 was more effective than GDC-0941, and A66 synergized with the anti-neu antibody in terms of anti-tumor immunity. We propose A66 plus anti-HER2/neu antibody as an effective strategy in trastuzumab-resistant breast cancers.
TLR-stimulated T cells acquire resistance to MDSC mediated suppression
Authors: Joseph Ann Mary , Parker Dominique , Hawkins Tarik , Ciavattone Nicholas , Davila Eduardo
Published: January 2023

The immunosuppressive tumor microenvironment presents a significant challenge to developing effective T cell-based cancer immunotherapies. Myeloid-derived suppressor cells (MDSCs), a heterogeneous group of cells, are a major contributor to the suppressive tumor microenvironment. MDSCs are immature myeloid cells that develop in response to chronic inflammation generated by an infection or a tumor. Currently, strategies to block MDSC-mediated suppression generate modest anti-tumor responses. This is in part due to lack of specific markers to target MDSCs and inability to simultaneously inhibit the multitude of suppressive mechanisms employed by MDSCs. Generating tumor-reactive T cells with the capacity to resist MDSC-mediated suppression would help facilitate the production of potent anti-tumor T cell therapies. The activation of the Toll-like receptor-Myeloid differentiation factor 88 (TLR-MyD88) signaling pathway in CD8+ T cells enhances cell proliferation, cytotoxic function, and survival. Our studies show that TLR-stimulated T cells are resistant to MDSC-mediated suppression. MyD88-activated CD8+ T cells co-cultured with tumor-derived MDSCs displayed enhanced proliferation and cytokine production over control T cells. Our future objectives are to understand the molecular mechanisms by which TLR-activated T cells acquire resistance properties and to exploit this knowledge to improve antitumor immune responses by overcoming MDSC-mediated suppression.
Targeting Sphingosine kinase 2/S1P axis Decreases Immunosuppressive Potential of Myeloid Derived Suppressor Cells (MDSCs) and Improves Tumor Control
Authors: Chatterjee Shilpak , Chakraborty Paramita , Paneerselvam Shanmugham , Ogretmen Besim , Mehrotra Shikhar
Published: January 2023

One of the major hindrances for harnessing therapeutic potential of T cells against tumor is the presence of immunosuppressive cells (MDSC, TAMs etc.) in the tumor microenvironment. Thus, strategies to boost the anti-tumor T cell function in vivo by overcoming immunosuppression holds merit. In the present study, we observed that MDSCs isolated from the tumor site have high expression of both Sphingosine kinase 1 (SphK1) and 2 (SphK2). However, genetic ablation of SphK2, but not SphK1, completely diminished the suppressive potential of MDSCs as evident by the increased proliferation and IFN-γ secretion by T cells co-cultured with SphK2−/− MDSCs. Similar dysfunctionality in suppressive potential was observed when MDSCs from ABC294640 (SphK2 specific inhibitor) treated tumor bearing mice was compared with vehicle control treated mice. The functional impairment of SphK2−/− MDSCs also contributed to the better tumor control when melanoma epitope gp100 reactive CD8+ T cells were adoptively transferred to treat subcutaneously established B16 melanoma tumor bearing SphK2−/− mice, as compared to those transferred in WT tumor bearing host. Studies delineating the underlying mechanisms revealed that SphK2−/− MDSCs exhibit reduced expression of various suppressive molecules like arginase 1, TGF-β and STAT3. Additionally, SphK2−/− MDSCs also displayed reduced expression of HIF-1α - that in turn regulates MDSC suppressive function and metabolic commitment. Thus, intracellular level of S1P acts as an important signaling mediator regulating the suppressive potential of MDSCs. Our study unveils an important therapeutic potential of inhibiting SphK2/S1P axis in regulating MDSCs functionality to improve T cell mediated tumor control.
Potential Role of the 15kDa Selenoprotein in Colorectal Inflammation
Authors: Peters Kristin M , Carlson Bradley A , Canter Jessica E , Tobe Ryuta , Seifried Harold E , Yu Yunkai , Cao Liang , Gladyshev Vadim N , Davis Cindy D , Hatfield Dolph L , Tsuji Petra A
Published: January 2023

Previously, systemic knockout (KO) of the ER-resident 15kDa selenoprotein (Sep15, SelenoF) protected mice against the formation of chemically-induced pre-neoplastic lesions and suggested modulation of inflammatory pathways. Our objective is to elucidate the potential role of SelenoF in the early steps of colon tumorigenesis by assaying gene expression of KO mice and littermate controls (WT) that were either treated with 2% dextran sulfate sodium (DSS) to induce colitis or given water (control). Based on previous results, we hypothesize that SelenoF expression is inversely correlated with a protective interferon (IFN)-γ pro-inflammatory response. Microarray analyses of colonic mucosa suggest that knockout of SelenoF expression itself caused more significant gene expression changes than DSS treatment (inflammation). Gene pathway analyses indicated galectin-6 was the highest up-regulated gene in DSS-treated (133-fold) and untreated (30-fold) KO compared to WT mice. The top significantly changed network included genes involved in the differentiation of T-lymphocytes. Colonic gene expression changes in SelenoF-KO mice, as validated with qPCR, included up-regulation of guanylate-binding protein 1 (p=0.004), and a down-regulation of T-box transcription factor 21 (p=0.08). Interestingly, a decrease in IFN-γ levels (p=0.026) was detected in the serum of KO compared to WT mice, whereas the opposite trend was shown in colonic mucosa. Thus, our preliminary analyses indicate that SelenoF may indeed influence the expression of inflammatory genes in the colon. Its contribution to the regulation of colitis will continue to be elucidated.
Emergence of inflammatory Treg in premature fetal Rhesus macaques in a model of LPS-induced chorioamnionitis
Authors: Jackson Courtney , Rueda Cesar M. , Presicce Pietro , Jobe Alan H. , Kallapur Suhas G. , Chougnet Claire A.
Published: January 2023

Placental inflammation, termed chorioamnionitis (chorio) is one of the largest contributors to premature birth, and has been associated with several complications post-birth, mainly pulmonary and cerebral. However, a full understanding of the fetal responses to the inflammatory milieu created by chorio remains limited. To address this question, we developed an experimental model of chorio in which intra-amniotic (IA) injection of LPS in pregnant Rhesus macaques lead to inflammation of the placenta similar to what is described in human chorio. In this model of fetal inflammation, fetal splenic regulatory T cells (Treg) frequency decreased, but CD4+IL-17+ cells increased. IL-17 and other pro-inflammatory cytokines (IL-2, IL-8, IFNγ) were mainly expressed by a subset of FoxP3+ T cells that also had a low expression of Helios, thus exhibiting characteristics of “inflammatory Treg”. Several pro-inflammatory cytokines, notably TNFα and IL-1 are elevated in the cord blood and amniotic fluid of LPS exposed neonates. IL-1 receptor antagonist (IL-1RA) and anti-TNFα decreased the frequency of splenic inflammatory Treg; however, they did not normalize overall Treg frequency. Collectively, our results show that in-utero exposure to inflammation triggers a rapid pro-inflammatory response in the fetus, particularly affecting Treg frequency and phenotype, and cytokines like IL-1 and TNFα are important for many of these pathways. Due to the role of Treg in maintaining immune homeostasis, altered Treg frequency and phenotype in response to in-utero inflammation potentially contributes to the detrimental outcomes of these neonates.
A rare subset of skin-tropic regulatory T cells expressing Il10/Gzmb inhibits the cutaneous immune response
Authors: Tomura Michio , Ikebuchi Ryoyo , Teraguchi Shunsuke , Vandenbon Alexis , Francis Shand H.W. , Honda Tetsuya
Published: January 2023

We reported that Foxp3+ regulatory T cells (Tregs) migrating from the skin to the draining lymph node have a strong immunosuppressive effect on contact hypersensitivity (CHS) response. However, the Treg subsets that regulate the CHS response remain poorly defined.

In this study, we used a combination of single-cell real-time PCR high-dimensional gene expression profiling data with spatiotemporal information about cellular movement from the mice expressing the photoconvertible protein KikGR to elucidate the role and characteristics of Treg subsets in CHS response. We found that although immunosuppressive genes Ctla4 and Tgfb1 were expressed in the majority of Tregs, Il10-expressing Tregs were rare and unexpectedly, the majority of Il10-expressing Tregs co-expressed Gzmb and displayed Th1-skewing. Furthermore, Gzmb-/Il10-expressing Treg subsets expressed the novel markers CD43 and CCR5. CD43+ CCR5+ CXCR3− Tregs highly expressed skin-tropic chemokine receptors CCR4 and CCR8. We also measured the retention of Treg subsets within inflamed skin using KikGR mice, and found that the subset of Tregs that was most highly retained in the skin, CD43+ CCR5+ CXCR3− Tregs, had superior in vivo inhibitory function to other Treg subsets. These results suggested that even if only present in small numbers, highly activated Tregs that co-express Gzmb and Il10 and that have the capacity to remain in inflamed tissue are likely to be clinically relevant due to the role of these molecules in the control of excessive immune responses.

Taken together, the identification of a rare Treg subset co-expressing multiple immunosuppressive molecules and having tissue-remaining capacity offers a novel strategy for the control of skin inflammatory responses.
Contribution of the immunoproteasome to the inflammatory phenotype observed in obese mice
Authors: Kimura Hiroaki , Karasawa Tadayoshi , Watanabe Sachiko , Suzuki Koichi , Caturegli Patrizio , Takahashi Masafumi
Published: January 2023

The immunoproteasome, a proteolytic machinery derived from the constitutive proteasome expressed predominantly in immune cells, plays a critical role in the regulation of T cell polarization that occurs in autoimmune and inflammatory diseases. Recently, it has been shown that inflammation contributes to the development of obesity and metabolic disorders. We found that LMP7, one of subunits in the immunoproteasome, is expressed in epididymal adipose tissue of obese mice. In this study, we assessed the role of LMP7 in the pathogenesis of murine obesity induced by high-fat diet (HFD). LMP7 deficiency conveyed resistant to obesity, and improved glucose intolerance and insulin sensitivity. Using bone marrow-transferred chimera, we found that LMP7 contributed to the development of obesity through involvement of both bone marrow- and non-bone marrow-derived cells. LMP7 deficiency decreased inflammatory responses, such as macrophage infiltration and chemokine expression, as well as increased serum adiponectin levels and decreased serum leptin levels. Overall, LMP7 deficiency in both bone marrow- and non-bone marrow-derived cells contributed to the suppression of the inflammatory responses in the HFD-induced obesity. We propose the immunoproteasome as a therapeutic target in inflammatory disorders.
Distinct Myeloid Cell Subsets Promote Angiogenesis and Damaged Tissue Clearance Following Mild Traumatic Brain Injury
Authors: Russo Matthew V , McGavern Dorian B
Published: January 2023

Mild traumatic brain injury (mTBI) leads to a robust immune response featuring resident microglia/macrophages and recruitment of peripherally-derived monocytes/macrophages. We sought novel insights into the dynamics and function of innate myeloid cells following brain injury by studying a closed-skull focal contusion model of mTBI that permits real-time imaging by intravital two-photon microscopy (TPM). TPM studies of CX3CR1GFP/+ CCR2RFP/+ reporter mice revealed an early microglia response followed by CCR2+ monocytes and CX3CR1+ macrophages that accumulated 1 to 7 days post-TBI. This transition coincided with dynamic changes in gene expression that shifted from pro-inflammatory to wound-healing. Early cell death and vascular destruction led to an impressive re-vascularization of the lesioned meninges, with 80–100% wound closure observed within 7 days of injury. We used histo-cytometry to define the anatomical position of the different myeloid subpopulations within the injured meninges during the repair phase. This revealed the presence of non-classical and resident macrophages that highly expressed the remodeling enzyme matrix metalloproteinase 2 (Mmp2) in the lesion core. Depletion of neutrophils and classical monocytes increased pathology due to inefficient dead cell clearance, but re-vascularization of the wound was not affected. Interestingly, vascular regeneration was significantly impaired only when non-classical monocytes and resident meningeal macrophages were depleted, resulting in reduced numbers of CD206+macrophages in the lesion. These studies demonstrate that neutrophils, monocytes, and resident macrophages participate in coordinated yet divergent wound-healing responses to mTBI.