bluuebunny/crossref_metadata_2025 huggingface dataset:
“The Great College Escape”-the most difficult choice offered to COVID-19 generation of global students.
Authors: Fazal Nadeem , Cebiyev Nihat , Alakbarova Gulgaz , Huseynbayli Ayshan , Garajayev Azer , Safarova Arzu , Meher Satish , De Herde Laura
Published: January 2023

The much hyped “Great Resignation” in the world of economic forecasting has opened a big door for the students to take an easy and timely exit from education. Is it a revolving door or a one-way exit with no easy return. There are more job openings for unskilled workers, luring students to leave their studies incomplete and take over odd jobs. Remote education experience changed hearts and minds of the global students and created a survival mindset. Pandemic without question has caused significant loss of lives with profound disruption to lives and livelihoods. As the pandemic is lingering on with uncertainty and ambiguity, students are tempted to leave studies in search of more money, more flexibility, and more happiness. With the fallout of COVID-19 yet to be fully realized and fixed, the predictive-enrollment models that traditionally used to plan classes won’t hold up in the post-pandemic world. We surveyed the current student population across different continents, Asia, Europe, Russia, US and tried to figure out the mindset of student enrollment and attrition. The perception and trends data from 300 respondents across the globe showed a significant economic impact on international students failing to register for higher education abroad in western hemisphere. Majority of students surveyed believe that the education system may have oversold the value of remote education and classroom technology. Free and open-source technologies available to both teachers and students have created a monster of virtual global education. For students in Eastern part of the world, the pandemic has offered new perks. Most students reported that they are rethinking their education needs and reimagining how knowledge and learning could best shape their careers.
Novel insight into the role of the vitamin D receptor in the development and function of the immune system
Authors: Arora Juhi , Wang Jinpeng , Weaver Veronika , Cantorna Margherita T
Published: January 2023

Immune cells express the vitamin D receptor (VDR) and are therefore vitamin D targets. The VDR protein can be readily measured in the kidney using antibodies to the VDR and western blot. It is much more difficult to measure VDR protein in the spleen because of the low level of VDR expression in resting immune cells. In order to more sensitively measure VDR expression, the Cre enzyme was inserted in the 3rd exon of the VDR gene and a reporter mouse that irreversibly expresses tdTomato was made. Mice that express one copy of the VDRCre gene were confirmed to be VDR +/− and mice that express two copies were confirmed to be VDR −/−. Initial characterization of the immune cells from the VDR +/− VDRtdTomato+ compared to VDR+/+ wildtype (WT) littermates showed no effect of being hemizygous for the VDR on immune cell frequencies. Immune precursors from bone marrow and thymus showed high tdtomato expression. In the periphery, monocytes, neutrophils and macrophages had very high tdTomato+ (88–98%) expression while lymphocytes ranged from 60–70% tdTomato+. Tissue resident innate lymphoid cell (ILC) 1 and 3 cells were about 60–80% tdTomoto+, while ILC2 cells had very low tdTomato expression. Stimulation of VDRtdTomato+ splenocytes showed that the tdTomato− CD4+ and CD8+ T cells proliferated more than their tdTomato+ counterparts. Sorted tdTomato+ T cells expressed the VDR protein only after 72h post-activation and proliferated less than the tdtomato− T cells. Interestingly, activation of the tdTomato− T cells failed to induce new tdTomato expression. The data suggest that an early immune precursor expresses the VDR. In the periphery, neutrophils and monocytes are almost all tdTomato+, while some immune cells (ILC2 and some T cells) may never express the VDR.

Supported by NIH R01AT005378 to MTC and T32GM108563 to JA, USDA NIFA award PEN04771 to MTC.
Retention of epigenetic and transcriptional memory among reprogrammed T cells (T-iPSCs) imparts selective advantage for T cell re-differentiation.
Authors: Williams Robin L , Morrow Alyssa , Allred Jeremy , Tolar Jakub , Haining W Nicholas , Yosef Nir , Blazar Bruce R
Published: January 2023

Various somatic cell types reprogramed into induced pluripotent stem cells (iPSCs) can be differentiated into T cells. It remains unclear what impact the input cells reprogramed into iPSCs have on T cell differentiation. We hypothesized that preservation of T cell genetic and/or epigenetic memory would bias iPSC clones toward T cell differentiation. CD8 naïve T cells (TN), CD8 stem memory T cells (TSM), and fibroblasts (FB) were reprogramed into iPSCs using nonintegrating Sendai virus to deliver Yamanaka factors. Input cells and iPSC clones were subjected to RNA-seq and ATAC-seq to assess whether epigenetic and transcriptomic memory are retained after reprogramming. Pluripotent associated genes (NANOG, LIN28A/B, SOX2) had increased expression in both T cell− and FB-iPSCs compared to their cells of origin. Of interest, gene enrichment analysis of fibroblasts and FB-iPSCs identified fibroblast related annotations, including migration and growth factors, in reprogramed iPSCs. Among CD8 TN− and TSM-iPSCs as a group, expression of only 41 genes and 6 genomic regions were retained, but these included known mature T cell genes, CD3ɛ, ZBTB7B, and CD95 as well as a region crucial for T cell signaling. Therefore, although much of the transcriptome and accessible genome is overridden in TN/TSM-iPSCs, there exists maintenance of both the transcriptome and chromatin accessibility after reprogramming. CD8 T-iPSCs were superior to FB-iPSCs in producing T progenitors in vitro. Highlighted here is the importance of starting populations for reprogramming and differentiation into T lineage cells. Leveraging the benefits of epigenetic and transcriptomic memory of input T cells could provide a more robust source of T cells for adoptive cell therapy.

Supported in part by funds from NIH P01 CA065493 and R37 AI34495 from the National Cancer Institute and National Institute of Allergy and Infectious disease, respectively, the Childrens’ Cancer Research Fund, and Chan-Zuckerberg Biohub.
Differential therapeutic modulation of exhaustion among autoreactive and global CD8 T cells in type 1 diabetes (T1D)
Authors: Wiedeman Alice E , Vega Carolina Acosta , Serti Elisavet , Nepom Jerry , Long Alice
Published: January 2023

Islet-specific CD8 T cell exhaustion (TEX) is linked to slower progression of type 1 diabetes (T1D). However, in the therapeutic setting, only global TEX has been studied. Here we address whether TEX among autoreactive cells in clinical trials relates to better outcome. We assessed PBMCs from select T1D subjects who had variable preservation of pancreatic function two years after treatment with teplizumab (anti-CD3, n=7) or alefacept (LFA3-Ig, n=12). We applied a CyTOF panel incorporating pooled pancreatic islet autoantigen peptide-loaded MHC Class I tetramer and 18 phenotyping markers. CD8 T cell subsets were identified by DISCOV-R multidimensional clustering analysis and were applied to samples with >5 autoreactive cells. Better clinical outcome was linked to greater frequencies of global CD8 TEX (subsets expressing PD1, EOMES, TIGIT, and KLRG1): a PD1hi subset 2 months after teplizumab (p=0.049) and a CD57hi subset 2 years following alefacept (p=0.044) treatment. Overall, increases in islet-specific TEX were rarely detected in poor responders (1 of 8 subjects with detectable islet-specific cells) consistent with a lack of change in global TEX, However, in subjects with better preservation of beta cell function, islet-specific TEX increased in fewer subjects (2 of 6) than global CD8 T cells (4 of 8). Together these results indicate that islet-specific CD8 T cells only partially mirror global CD8 responses, and further research is needed to clarify the role of exhaustion and its relation to autoreactive T cells in determining outcome in therapeutic settings.

Supported by grants from NIH (R01 AI141952) and Immune Tolerance Network (UM1AI109565).
Sensory neurons shape allergic Type 2 inflammation in the sinonasal tract
Authors: Ortiz-Carpena Jorge F , Pastore Christopher , Hung Li-Yin , Kohanski Michael A , Vaughan Andrew E , Cohen Noam A , Herbert De’Broski R
Published: January 2023

Whether neuro-immune interactions regulate homeostasis vs. inflammation within the sinonasal tract is entirely unknown. Transient receptor potential vanilloid 1 ion channel (TRPV1+)-expressing sensory neurons of the trigeminal ganglion (TG) innervate the upper airway near sinonasal tuft cells (STC), a rare lineage of epithelial cells that initiate inflammation in the intestine by secreting pro-Type 2 cytokines. Given the vicinity of STC and TG neurons, we hypothesized that STC function(s) may be at least partially controlled by neuronal inputs. A mouse model of sinonasal allergic inflammation was developed using intranasal administration of a fungal allergen mix (FAM) that mimics key aspects of chronic rhinosinusitis pathophysiology, including increased numbers of sneezing bouts, STC, eosinophils, and levels of interleukin (IL)-25 in the sinonasal fluid. Our data show that FAM also evokes the release of neuropeptides substance P and neuromedin U from TG neurons. Also, chemical ablation of TRPV1+ neurons significantly reduces STC expansion and eosinophil accumulation, which collectively suggests that TRPV1+ sensory neurons produce factors that promote STC to release Type 2 cytokines driving allergic disease pathophysiology. Ongoing studies are designed to uncover important insight(s) into how STC/sensory neuron interactions are involved in the pathogenesis of allergic diseases in the sinonasal tract.

Supported by grants from NIH (U01 AI163062, R01 AI164715, and R01 AI123173) awarded to DRH.
Inhibiting glycolysis by targeting the enzyme PFKFB3 restricts macrophage anti-mycobacterial activity and neutrophil phagocytosis of Mycobacterium tuberculosis.
Authors: Lane Daniel Scot , Talukdar Priyanka , Junecko Beth Fallert , Mattila Joshua T
Published: January 2023

Mycobacterium tuberculosis (Mtb) causes tuberculosis (TB), a leading cause of infectious disease-related mortality around the world. Myeloid cells are important in TB as Mtb host cells and anti-Mtb effector cells but the factors that differentiate these roles are not clearly defined. Immunometabolism is a driver of immune function and may be an important determinant of this interaction and represents a potential target for host-directed therapies against TB. To better understand how immunometabolism relates to TB, we incubated macrophages and neutrophils from Mtb-infected macaques with a panel of metabolic inhibitors and measured how this affected cellular responses to Mtb infection. Specifically, we targeted glycolysis, oxidative phosphorylation, and fatty acid oxidation and measured how this affected macrophage and neutrophil phagocytosis, macrophage control over Mtb viability and replication, and neutrophil production of extracellular traps. We found that targeting glycolysis by inhibiting PFKFB3, a rate-limiting enzyme in glycolysis, reduced macrophage anti-Mtb activity and neutrophil phagocytosis, while inhibition with the glucose analog 2-DG did not. In contrast, incubating cell-free mycobacteria with PFKFB3 inhibitors severely inhibited their growth, suggesting that these bacteria use different metabolic pathways in cells and culture to survive. Inhibiting oxidative phosphorylation with metformin or fatty acid oxidation with etomoxir did not change how macrophages or neutrophils responded to Mtb or affect mycobacterial growth in culture. These results indicate a role for immunometabolism in myeloid responses to Mtb and may inform efforts for development of metabolism-targeted therapies for TB.

Supported by grants from NIH (R01AI134183)
CD200 is required to control LPS-induced lung inflammation.
Authors: Patoine Dany , Bouchard Karine , Bissonnette Elyse , Lauzon-Joset Jean-Francois
Published: January 2023

INTRODUCTION

Acute respiratory distress syndrome (ARDS) is a severe lung inflammatory disease caused by a variety of precipitants, including SARS-CoV-2 (COVID-19). In addition to excessive inflammation, ARDS is characterised by the dysregulation of anti-inflammatory pathways, including CD200/CD200R pathway.

OBJECTIVE

To investigate the role of CD200/CD200R pathway in lung ARDS inflammatory response.

METHODS

LPS was administered intratracheally to induce ARDS in Sprague-Dawley CD200 KO and wild type (WT) rats. Inflammation was evaluated using bronchoalveolar lavage (BAL) cellularity. Lung injury was measured by total protein level in BAL fluid, and levels of proinflammatory cytokines (TNF, IL-6) and chemokines (CXCL2, CCL2) were determined in BAL supernatants. In a second experiment, recombinant CD200Fc was administered to KO rats to restore the anti-inflammatory response.

RESULTS

Although there was no difference in total BAL cell counts at 3 h, cell recruitment was greater in CD200 KO rats given the low cell number in naïve KO rats. BAL of KO rats had higher levels of TNF, IL-6, CXCL2, and CCL2 compared to WT rats. Total protein level in BAL was higher in CD200 KO rats, implying more pronounced pulmonary edema. CD200Fc administration in KO rats significantly decreased levels of TNF and CCL2 in BAL, suggesting an attenuation of the inflammatory response.

CONCLUSION

This study shows that ARDS inflammatory response is exacerbated in absence of CD200 in an experimental model of ARDS in rats and that CD200 supplementation alleviates this phenotype. Further analyses will be needed to better understand the contribution of different cell types expressing CD200 to control lung inflammatory response resulting from ARDS.

Supported by grant by CIHR and IUCPQ fondation.
Modulating levels of cell surface CD6 is a novel mechanism for regulating T cell activity.
Authors: Chu Dalena Ngoc , Tiet Phoi , Marrocco Valeria , Ampudia Jeanette , Connelly Stephen , Ng Cherie
Published: January 2023

CD6 is a co-stimulatory receptor expressed on the surface of T cells that promotes immune synapse formation, T cell activation, and migration into tissues by engaging its ligand, ALCAM. CD6high cells have been described to be more pathogenic relative to CD6low cells. Furthermore, T cells in CD6 knockout mice have decreased proliferation and inflammatory responses. Hence, a therapeutic that can modulate surface levels of CD6 may have favorable outcomes for autoimmune/inflammatory conditions. We recently demonstrated that the anti-CD6 mAb, itolizumab, induces proteolytic cleavage of cell surface CD6. The goal of the work shown here is to characterize the mechanism of cleavage and the downstream functional consequences of reduced cell surface CD6. Treatment of PBMCs with itolizumab induces CD6 cleavage from the cell surface and a concomitant increase in the soluble form detected in the supernatant. Itolizumab-induced loss of CD6 was not observed with isolated T cells, however CD6 levels were reduced by 87% when T cells and monocytes were cocultured with itolizumab. The cleavage of CD6 is initiated through rapid cell-to-cell contact between T cells and monocytes via FcγRI. The dose-dependent reduction in surface levels of CD6 positively correlates with decreases in T cell activation markers such as CD25, PD-1, and CD71 (Pearson, p<0.001) and cytokine production including IL-2 (p<0.01) and TNFα (p<0.05). Itolizumab-treated cells are also less alloreactive as shown by a reduction in proliferation and cytokine production of responder cells in a mixed-lymphocyte reaction. This data further supports targeting CD6 as an effective means to inhibit pathogenic T cell activity in the treatment of autoimmune and inflammatory diseases.
BATF controls T cell differentiation during chronic antigen stimulation
Authors: Titcombe Philip J , Morales Milagros Silva , Zhang Na , Mueller Daniel L
Published: January 2023

CD4+ T cells preserve self-tolerance in the periphery by exerting active suppression as regulatory T cells (Treg) or entering an inactive state known as anergy. Recent evidence suggests that anergy occurs naturally within a subpopulation of polyclonal CD4+ T cells and can generate precursors to Foxp3-expressing Treg cells. To identify transcription factors (TFs) underlying T cell anergy induction and its potential plasticity, we sorted polyclonal CD4+ T cell subsets from mice for downstream sequencing-based interrogation. Anergic CD44high Foxp3− CD73high FR4high Nrp1+ T cells were compared to effector-memory, Treg, and naïve T cell populations. Integrated ATAC-seq and RNA-seq analysis revealed a distinct program in the anergic T cell subpopulation. The anergy signature was driven by bZIP/AP-1 subfamily (Batf, Maf) and NFAT family (Nfatc1) transcription factors. Anergy-enriched TFs were then investigated in antigen-specific T cell tolerization systems where Batf knockdown led to altered expansion and minimal Treg cell differentiation. BATF was furthermore required for anergy-derived Foxp3+ Treg cell generation. Our data indicate that BATF orchestrates the transition to chronically-stimulated T cell differentiation states.

Supported by grants from the NIH (T32 AI007313, P01 AI035296) and RRF (Future Physician Scientist Award)
Comprehensive comparison of adaptive immune responses to inactivated SARS-CoV-2 vaccine between young and old
Authors: Xiao Chanchan , Ren Zhiyao , Zhang Bei , Mao Lipeng , Wang Pengcheng , Liang Xiaofeng , Luo Oscar Junhong , Chen Guobing
Published: January 2023

Aging is a critical risk factor for SARS-CoV-2 vaccine efficacy. Although low humoral immunity in the elderly was observed in mRNA and recombinant protein vaccination, it is still unclear how inactivated SARS-CoV-2 vaccine stimulates humoral and cellular immunity in elderly in the real world. Here, combining neutralizing antibody, tetramer stained CD8 T cells, transcriptome, and single cell TCR-seq examinations, we comprehensively compared immune responses before and after inactivated SARS-CoV-2 vaccination between 121 young and 48 old donors. Our data demonstrated that the inactivated SARS-CoV-2 vaccine can successfully induce antibody and T cells responses in the old people, although the responses were slower and weaker than young adults. The mechanism of impaired immune responses in elderly is likely due to altered immune cell function and decreased BCR and TCR repertoire diversity. In addition, impaired immune response due to mutant epitopes from SARS-CoV-2 variants raises concerns for future vaccine design.

This work was supported by grants from the National Key Research and Development Program of China (2018YFC2002003 to G.C., 2021YFC2009400 to G.Z.), the Natural Science Foundation of China (U1801285 to G.C., 81971301 and 32050410285 to O.J.L.)
BCAP plays a central role in regulating canonical M2 macrophage polarization upon various cell extrinsic signals
Authors: Kawarizadeh Kiana , Warrick Kathy , Pasare Chandrashekhar
Published: January 2023

Macrophages acquire either pro-inflammatory or anti-inflammatory states following cell extrinsic stimuli. Upon microbial recognition, macrophages differentiate into a pro-inflammatory state referred to as M1 phenotype. Subsequently, clearance of the insult facilitates the transition of M1 macrophages into an anti-inflammatory M2 phenotype to repair damaged tissue and maintain homeostasis. Recent work has identified B-cell adaptor for PI3K (BCAP), acting downstream of TLRs, as a key player in facilitating this transition to switch macrophages from expression of pro-inflammatory genes to repair genes. Whether BCAP also plays a role in macrophage differentiation upon sensing other extrinsic signals remains currently unknown. For example, the anti-inflammatory cytokine Interlekin-4 (IL-4) can drive macrophage differentiation into a M2 phenotype. We found that, following IL-4 stimulation, BCAP deficient macrophages exhibited a defective M2 gene program as revealed by reduced expression of Arg1, Klf4, and Retnla. Furthermore, we found that IL-33 mediated M2 macrophage generation was also hampered in the absence of BCAP. These findings reveal that in addition to acting as a regulator of M1 to M2 transition, BCAP is a critical node for detecting cell extrinsic signals that drive canonical M2 polarization. The mechanisms by which BCAP regulates IL-4R and IL-33R mediated M2 gene program as well as the impact of BCAP on the phenotype of tissue resident macrophages is currently being investigated.

Supported by grants from NIH (R01 AI113125)
Lipin-1 integrates lipid metabolism with macrophage function to promote inflammation resolution
Authors: Woolard Matthew D , Blackburn Cassidy , Schilke Robert , Bamgbose Temitayo
Published: January 2023

Free fatty acid accumulation in macrophages alters cellular metabolism, leading to failed inflammation resolution that contributes to cardiometabolic pathologies such as atherosclerotic cardiovascular disease. Free fatty acids are either broken down by β-oxidation, stored in glycerolipids, or incorporated into sphingolipids (e.g., ceramides). Ceramide synthesis inhibits several pro-resolving macrophage functions, such as β-oxidation and efferocytosis, needed for inflammation resolution. The regulatory signals that control free fatty acid incorporation into lipids (e.g., lipid channeling) for proper macrophage function are not well understood. Lipin-1 is a phosphatidic acid phosphatase with an independent transcriptional coregulatory activity that controls cellular lipid homeostasis. Using genetically engineered mice and bone marrow-derived macrophages, we investigated the contribution of lipin-1 on macrophage pro-resolving functions. Mice lacking myeloid-specific lipin-1 had defects in the clearance of apoptotic cells in a zymosan model of inflammation resolution, and these mice had increased atherosclerotic plaques and necrotic cores in a model of atherosclerosis and a delay excisional wound closure. Bone marrow-derived macrophages lacking lipin-1 showed a striking pattern of dysregulated lipid metabolism in which il-4 stimulation promoted ceramide synthesis over β-oxidation. Additionally, lipin-1 deficient macrophages had reduced phagocytosis of apoptotic cells. Our work provides evidence that lipin-1 promotes β-oxidation while inhibiting ceramide synthesis during free fatty acid accumulation in macrophages to allow for responses that promote inflammation resolution.

Supported by grants from NIH (1 R01HL131844-04) and NIH (1P20GM134974-01A1)
Modulation of myeloid cell functions by long noncoding RNAs RN7SK and HCG11
Authors: Naqvi Raza Ali , Ahmad Imran , Estepa Araceli Maria Valverde , Naqvi Afsar
Published: January 2023

Objective

Expressional dynamics of long noncoding RNAs (lncRNA)” regulates a gamut of biological processes including immunity. Role of lncRNA in response to oral pathogens and their regulatory impact on the innate immune functions of myeloid cells remain poorly explored. Therefore, we hypothesize that periodontal pathogens may cause the alteration of lncRNA profiles in myeloid cells and modulate their innate immune functions.

Method

CD14+ monocytes sorted from human PBMCs were differentiated into macrophages (MΦ) or dendritic cells (DC), and challenged with periopathic bacteria (A. actinomycetemcomitans [Aa] and P. gingivalis [Pg] for 4, 12 or 24 h. lncRNA expression was done at these time points by RT-qPCR array. Differentially expressed lncRNAs were assessed for: 1) cell migration, 2) phagocytosis, and 3) antigen uptake/processing in . Cell surface markers were analysed by flow-cytometry.

Result

Challenge of DCs with Pg, Pg LPS, Aa, and Aa LPS separately result in the differential expression of 21 lncRNAs (19 up, 2 down). Knockdown of LncRNA, SNHG11, NUTM2A-AS1, MCM3AP-AS1, JPX, and HCG11 (upregulated) and RN7SK (downregulated) were used to evaluate myleoid cell functions. HCG11 knockdown revealed attenuation of cell migration, while RN7SK significantly enhanced antigen uptake/processing in APCs . Importantly, RN7SK knockdown results in downregulation of M2 Mϕ surface markers (CD163, CD206 or Dectin) and concomitant increase in M1 Mϕ markers (MHC II or CD32) suggesting its critical role in macrophage polarization.

Conclusion

Our results show that periodontal pathogens alter lncRNA profiles and impair innate immune functions in myeloid cells, thereby suggesting critical roles of lncRNAs in periodontopathogenesis.

Supported by NIH/NIDCR R03 DE027147, R01DE027980, and R21DE026259 to AN.
Role of Grail in intestinal inflammation
Authors: Nurieva Roza I , Zhao Yang-Zhi , Alekseev Andrei , Galkina Elena , Kim Sang T
Published: January 2023

Intestinal homeostasis depends on interaction between the microbiota and host immune system, and disruption of this balance results in predominance of inflammatory responses leading to inflammatory bowel disease. However, to date, precise cell-intrinsic regulatory mechanisms that controls intestinal inflammation are poorly understood. Here we show that expression of the E3 ubiquitin ligase Grail is diminished in colon tissues of mice with colitis and Grail deficiency leads to increased susceptibility to colitis. Importantly, Grail deficiency disrupted pro-/anti-inflammatory balance towards inflammatory immune responses at the steady state and during colitis. Mechanistically, loss of Grail intrinsically diminished the expression of IL-10 and IL-10 receptor (IL-10R) by adaptive and innate immune cells in colonic mucosa. Besides the intestinal immunity, Grail deficiency leads to a decrease in microbial diversity and an enrichment of pro-inflammatory microbiota. Thus, Grail plays a protective role during intestinal inflammation by supporting regulatory immune responses and shaping the microbiota.
Epigenetic Modulation of Class-switch DNA Recombination to IgA by miR- 146a through Downregulation of Smad2, Smad3 and Smad4
Authors: Li Shili , Morales Grecia , Daw Cassidy C , Chupp Daniel Phillip , Fisher Amanda Dee , Zan Hong , Casali Paolo
Published: January 2023

IgA is the predominant isotype at intestinal mucosae, where it plays a critical role in homeostasis and first line of immune protection. Class-switch DNA recombination (CSR) to IgA is directed by TGF-b which induces activation of Smad2 and Smad3 transcription factors. Activated Smad2/Smad3 dimers are recruited with Smad4 to IgH α locus Iα promoter to activate germline Iα-Cα transcription, the first step in CSR to IgA. Epigenetic factors, such as microRNAs, regulate immune cells and, as we have shown, modulate the antibody response in a B cell- intrinsic fashion. We found that miR-146a targets Smad2, Smad3 and Smad4 mRNA 3’UTRs, and keeps CSR to IgA in check in resting B cells. Enforced miR-146a expression in B cells aborted CSR to IgA by decreasing Smad levels. Induction of CSR to IgA, as directed by TGF-b, downregulated B cell miR-146a, thereby reversing the silencing of Smad2, Smad3 and Smad4, which, once expressed, led to recruitment of Smad2, Smad3 and Smad4 to Iα promoter for activation of germline Iα-Cα transcription. miR- 146a−/− mice significantly increased circulating levels of total IgA, but not IgM, IgG or IgE, and heightened the specific IgA antibody response to OVA. In miR-146a−/− mice, this was associated with increased IgA+ B cells in intestinal mucosae, increased amounts of fecal free and bacteria-bound IgA, kidney IgA deposition and IgA nephropathy. The B cell-intrinsic role of miR-146a in repression of Smad2, Smad3 and Smad4 and CSR to IgA was confirmed in miR-146a −/− B cells in vitro and in vivo in mixed bone marrow μMT/miR-146a−/− chimeric mice. Thus, by specific inhibition of Smad2, Smad3 and Smad4 expression, miR-146a plays an important and B cell intrinsic role in modulation of CSR to IgA and the IgA antibody response.

Supported by NIH grants AI 079705, AI 105813, AAI 167416 and LRA grant 641363 to PC.
Proteasome inhibition induces p53 driven apoptosis in early activated B-cells
Authors: Ochoa Trini , Allman David
Published: January 2023

Proteasome inhibition is a useful therapeutic strategy against plasma cell disorders in autoimmunity, neoplasias, and transplant rejection. However, how bortezomib induces cell death and the transcription factors that are involved in its execution are not well characterized. In this study, we aim to address the mechanism of action of proteasome inhibitors in plasma cells and in early precursor activated B-cells. We immunized mice with the hapten-protein conjugate nitrophenyl chicken-gamma-globulin (NP-CGG) and validated depletion of plasma cells and germinal center B-cells in vivo. At steady-state, we utilized B220 expression as a surface marker for newly-formed plasma cells and determined that bortezomib primarily depletes newly-formed plasma cells. To gain more mechanistic insight on how bortezomib affects newly-formed plasma cells, we employed an in vitro culture system of murine-derived primary follicular B-cells that were stimulated with CPG, IL-4, and IL5, to promote differentiation into the plasma cell fate. We find that deletion of the ER-stress induced transcription factor CHOP rescued in vitro generated plasma cells from low-dose bortezomib treatment. In addition, we identify the transcription factor p53 as a key inducer of apoptosis in in vitro generated early activated B-cells. Deletion of p53 rescued cells from apoptosis and reduced the number of cells expressing cleaved caspase-3 in response to bortezomib treatment. In conclusion, we have identified two transcription factors that are involved in bortezomib-induced cell death in B-lineage cells.

Supported by NIAID Diversity Supplement: R01AI139123
Flagella and indole produced by commensal bacteria protect the intestinal barrier to prevent food allergy
Authors: Kemter Andrea Maria , Patry Robert T , Arnold Jack , Campbell Evelyn , Hesser Lauren A , Ionescu Edward , Nagler Cathryn
Published: January 2023

Lifestyle-induced changes to the diversity of the commensal microbiota have been causally linked to the increasing prevalence of food allergies and other non-communicable chronic diseases. We have shown that bacteria from the Clostridia class prevent an allergic response to food by eliciting an IL-22 dependent barrier protective response that limits allergen access to the systemic circulation. We have now examined the mechanisms by which commensal Clostridia induce this allergy-protective effect. We identified taxa in a consortium of Clostridia that possess flagella and produce indole, which are ligands for TLR5 and AhR, respectively. Lysates and flagella isolated from this consortium induced IL-22 in mouse intestinal explants. IL-22 was not induced in explants from mice in which TLR5 or MyD88 was knocked out globally or conditionally in CD11c+ cells. Treatment with the commensal flagellar isolate also reduced detection of intragastrically administered FITC dextran in the serum of antibiotic-treated mice. Similarly, indole exposure induced IL-22 in intestinal explants and reduced intestinal permeability to FITC dextran. Importantly, AhR signaling in RORγt+ cells was necessary for IL-22 induction by flagella. These results suggest that flagella and indole act synergistically to prevent an allergic response. Finally, we have isolated and characterized two Clostridial taxa which bear flagella and produce indole. We hypothesize that germ-free mice colonized with these two taxa will exhibit improved IL-22 dependent barrier function and be protected against an allergic response. Our work reveals novel features of Clostridia key to their allergy-protective capability which may be further exploited to develop therapeutics.

Supported by NIH AI106302 and the Bunning Professorship Endowment Fund
Genetic manipulations to improve intratumoral trafficking of adoptively transferred T cells
Authors: Vianzon Vianca , Garg Ishita , Rogers Laura M
Published: January 2023

T cell–mediated cancer immunotherapies, including anti–PD-1 and CAR-T cells, are becoming standard treatments for many cancer types. One potential strategy to overcome resistance is to enhance the ability of T cells to traffic into tumors. We recently reported results from an innovative, genome-wide screen to identify endogenous T cell genes that enhance intratumoral accumulation using the Sleeping Beauty (SB) transposon system. We successfully identified many promising candidates with this in vivo approach using immunocompetent models of solid tumors.

Others have performed similar independent whole-genome and targeted screens in T cells assessing tissue localization using adoptive T cell transfer approaches. To determine how our gene candidates compared with significant candidates identified in these independent screens, we performed a formal rank aggregation to generate a single, integrated ranked list in descending order of selection strength. Aggregated results yielded approximately 1500 T cell gene candidates whose biological functions are enriched in chemotaxis. We hypothesized that these candidates represent novel therapeutic targets to improve T cell migration into solid tumors.

Here, we present in vivo validation of two of the most strongly selected candidates, including AP2-associated protein kinase 1 (Aak1) and Small Proline-Rich Protein 1B (SPRR1B). Importantly, in vivo migration of adoptively transferred T cells into tumors was significantly increased when the T cells overexpressed AAK1DN125 or SPRR1B. Our observations strongly support the strength of genomic screens to identify truly novel candidates for further development as immunotherapies to enhance T cell homing to the tumor microenvironment.

Supported by Mayo Clinic Graduate School of Biomedical Sciences, K22 (K22CA225786, PI: LM Rogers), and Cancer Immunology and Immunotherapy Program Pilot Award (PI: LM Rogers)
The PARP inhibitor ABT-888 impairs the suppressive capacity of regulatory T cells through modulation of FoxP3 and CTLA-4.
Authors: Falcon Daniel M , Adams Sarah F
Published: January 2023

Immune checkpoint blockade (ICB) has achieved long-term survival in melanoma but results remain limited in other solid malignancies including ovarian cancer. Combination treatments are a promising strategy to optimize responses to ICB. We have previously shown that the PARP inhibitor (PARPi) ABT-888 synergizes with CTLA-4 ICB to achieve long-term survival in a preclinical model of ovarian cancer. While the role of PARPi on DNA repair is well established, the impact on T cells is unclear. Published data using PARP1-KO mice suggests that PARP impairs regulatory T cell (Treg) function through modulation of FoxP3. Tregs suppress conventional T cell responses using multiple mechanisms including consumption of co-stimulatory molecules via the inhibitory receptor CTLA-4. Here we sought to define the impact of ABT-888 on the suppressive function of Tregs using FoxP3-eGFP transgenic mice. Using flow cytometry, we examined the levels of FoxP3 and CTLA-4 in Tregs stimulated in the presence of increasing doses of ABT-888 (0 – 50μM). Independent of stimulation, the GMFI of FoxP3 and CTLA-4 were significantly reduced by ABT-888 in a dose-dependent manner (P < 0.05). To test suppressive function, we pre-treated Tregs with increasing doses of ABT-888 prior to use in a standard proliferation assay. Similarly, ABT-888 pre-treatment significantly reduced the suppressive capacity of Tregs in a dose-dependent manner (P < 0.05). Taken together, these data identify a potentially novel area of synergy between PARPi and ICB. Furthermore, these data highlight the immunomodulatory effects of PARPi which are independent of their role in DNA repair. Future studies will focus on the impact of ABT-888 on tumor-associated Tregs and optimization of ICB therapy.

This research was supported by NCI R37CA229221 (PI: Adams) and UNM Comprehensive Cancer Center Support Grant NCI P30CA11810. D.F. was supported by the Academic Science Education and Research Training program at the University of New Mexico Health Sciences Center (NIGMS Institutional Research and Academic Career Development Award; K12-GM088021).
The novel coreceptor CD8α:MyD88 enhances T-cell proliferation, activation, and antitumor response at low-dose IL-2 in B16 melanoma
Authors: Cao Phoebe , Chen Jiangli , Ciavattone Nicholas , Davila Eduardo
Published: January 2023

The gamma common chain (γc) cytokine receptor plays a central role in Type I cytokine signaling, and in the generation and expansion of antitumor T-cells. Adoptive T-cell therapies (ACT) benefit from IL-2 treatment but is limited by a short half-life and high toxicities. Reducing IL-2 mediated toxicities while retaining antitumor T-cell efficacy is needed to improve clinical outcomes in ACT-treated patients. Other γc cytokines such as IL-7 and 15 have shown benefits for ACT by increasing generation and expansion of long-lived antitumor T-cells with reduced cytokine toxicity. Our lab previously demonstrated that MyD88 (critical adaptor protein in toll-like receptor signaling) is necessary for CD8 T-cell expansion. Induced MyD88 signaling in T-cells via expression of novel coreceptor CD8a:MyD88 (CM T-cells) resulted in enhanced proliferation, activation, and durably reduced tumor size in mouse melanoma models upon ACT treatment. CM T-cells expressed higher γc receptor levels which is required for IL-2, IL-7 and IL-15. Therefore, we hypothesized that CM T-cells have increased sensitivity to γc cytokine signaling resulting in enhanced proliferation, activation, and antitumor activity. CM T cells cultured at 10–25 times lower concentrations of IL-2, -15, -7 demonstrated enhanced cell proliferation and activation and was associated with increased JAK-STAT signaling intensity and persistence. CM T-cell ACT treatment of mice with B16-F10 tumors reduced tumor growth even at the lowest IL-2 concentration (5000 IU IL-2). In conclusion, CM T-cells require lower concentrations of type I cytokines for enhanced antitumor immunity and highlights the potential of CM T-cells to improve clinical outcomes with reduced toxicities.

Supported by grants from NSF GRFP, NCI (R01CA207913, P30CA046934), Dept of VA Merit Award (BX004935-01), University of Colorado Comprehensive Cancer Center (UCCCC), Leukemia and Lymphoma Society Translational Research Program
Tumoral Expression of NAC1 Restrains Antitumor Immunity through the LDHA-mediated Immune Evasion
Authors: Song Jim Jianxun
Published: January 2023

T cell-mediated antitumor immunity has an important role in cancer prevention and treatment; however, immunosuppressive tumor microenvironment (TME) constitutes a major contributor to immune evasion that weakens antitumor immunity. We report here that tumoral expression of nucleus accumbens-associated protein-1 (NAC1), a nuclear factor of the broad complex, tramtrack, bric-a-brac/poxvirus and zinc finger (BTB/POZ) gene family, negatively impacts the CD8+ T cell - mediated antitumor immunity via through LDHA-mediated suppressive tumor microenvironment. We show that NAC1 could positively regulate the expression of lactate dehydrogenase A (LDHA) at transcriptional level, and LDHA-mediated lactic acid accumulation only inhibited cytokine production but also induced the exhaustion and apoptosis of CD8+ cytotoxic T lymphocytes (CTLs), impairing their cell-killing ability. We further show that in the immunocompetent and immunodeficient mice bearing melanoma, tumors with NAC1 depletion grew significantly slower and had elevated infiltration of tumor antigen specific CTLs following adoptive cell transfer, as compared with the control groups. These findings imply that tumor expression of NAC1 contributes substantially to immune evasion through its regulatory role in LDHA expression and lactic production. Thus, therapeutic targeting of NAC1 warrants further exploration as a potential strategy to reinforce cancer immunotherapy such as adoptive cell transfer of CTLs.
Immune characterization in platinum resistant ovarian cancer patients treated with pembrolizumab and guadecitabine
Authors: Xie Ping , Chen Siqi , Cowan Mathew , Huang Hao , Cardenas Horacio , Kocherginsky Masha , Matei Daniela , Zhang Bin
Published: January 2023

Immune checkpoint inhibitors (ICI) have limited activity in ovarian cancer (OC). Accumulating preclinical and early clinical results support that interventions targeting the epigenome could elicit an inflamed tumor milieu to augment ICI. Here we explore the clinical and biological activity of guadecitabine, a second generation Hypomethylating agents (HMA), given in low dose as a priming strategy before pembrolizumab, a humanized anti-PD1 antibody in a clinical trial for patients with recurrent, platinum-resistant OC. Among 35 evaluable patients, there were 3 partial responses (8.6%) and 8 (22.9%) patients with stable disease, resulting in clinical benefit rate (CBR) of 31.4%. Median duration of clinical benefit was 6.8 months. High dimensional immune profiling of PBMCs showed higher frequency of naive and/or central memory CD4+ T cells and of classical monocytes in patients with durable CBR. Higher baseline density of CD8+ T and CD20+ B cells and presence of tertiary lymphoid structures in tumors were associated with durable CBR. The molecular features such as expression levels of PD-L1 or A2AR linked to therapeutic benefit for resistant OC. Our study provides an in-depth view of the immune milieu of platinum resistant OC and of the effects of the combination of HMAs and pembrolizumab on the interactions between immune cell populations and tumor cells.

Supported by USAMRMC/CDMRP (W81XWH170141)
Absence of Toll-like receptor 5 confers survival in mice bearing ovarian tumors treated with anti-PD-L1
Authors: McGinty Mitchell Thomas , Feng Tzu-Yu , Kolli Sree , Putelo Audrey , Rutkowski Melanie
Published: January 2023

Ovarian cancer accounts for more deaths than any other cancer of the female reproductive system. Patients bearing ovarian tumors infiltrated with high frequencies of T cells associate with a greater survival probability. However, therapeutic strategies targeting T cells in ovarian cancers are largely ineffective. We have observed in our models of late-stage murine ovarian cancer that in the absence of Toll-Like Receptor 5 (TLR5) signaling, anti-PD-L1 therapy promotes significant survival and subsequent protection against tumor rechallenge. Ovarian tumors from rechallenged TLR5KO mice exhibited significantly higher frequency/number of both CD4 and CD8 effector memory T cell subsets and greater numbers of antigen experienced T cells. Thus, in the absence of TLR5 signaling, T cells are better able to mount/maintain a response to ovarian tumors after inhibitory receptor blockade. Furthermore, protection against tumor progression in TLR5KO mice corresponds with a significant increase in tumor infiltrating cross presenting and IL-12 producing Dendritic cells (DCs) which could account for the differences in T cell infiltrates. We hypothesize that TLR5 signaling functionally alters DCs to become more suppressive in the tumor microenvironment, impacting T cell differentiation and anti-tumor activity, thereby reducing survival during ovarian tumor progression with aPD-L1 therapy. Clinical implications are already present, as roughly 7.5% of the general population harbor a TLR5 SNP that diminishes TLR5 signaling and is associated with increased long-term survival for ovarian cancer patients. Therefore, patients who express the TLR5 SNP may immediately benefit from anti-PDL1 therapy and those without the SNP, TLR5 antagonism.

Supported by NIH/NCI 1R01CA253285 UVA CIC Collaborative Grant
Nitration of CCL2 disrupts the tumor-protective function of CCL2 in bladder cancer
Authors: Mukherjee Neelam , Ji Niannian , Shu Zhen-Ju , Curiel Tyler J , Svatek Robert S
Published: January 2023

The chemokine CCL2 (C-C motif ligand 2) is best known for its ability to induce trafficking of immune cells by binding its primary receptor, CCR2. The recruitment of immunosuppressive monocytes by CCL2 promotes cancer in several tumor types. Surprisingly, we previously found an unanticipated protective role for CCL2 signaling in bladder cancer (BC). To determine if CCL2’s effect to suppress bladder tumor growth is dependent on T cells, we examined the effects of α-CCL2 antibody in wild-type versus T cell-deficient mice. The protective effect of CCL2 was lost in T cell-deficient mice and on T cell depletion in wild-type mice. Mixed bone marrow chimera experiments also showed that CCR2+ T cells were preferentially recruited by CCL2 to the bladder compared with CCR2− T cells suggesting that the protective effect of CCL2 in BC is through CCR2+ T cells. Most studies researching chemokines, including CCL2, in cancer assume the chemokine to be completely active and do not consider their different modified states which may be one of the critical reasons behind the failure of chemokines in clinical trials. We show for the first time that bladder tumors induce post-translational nitration of CCL2 and block T cell recruitment to the bladder which is restored by exogenous recombinant (r) CCL2 treatment. However, the chemical nitration of rCCL2 abolished this therapeutic efficacy of rCCL2 and was associated with decreased bladder T cell infiltration and more monocytes infiltration in BC. Our study analyzed the role of nitrated CCL2 in BC which explained certain paradoxes between CCL2 levels and patient outcomes in cancer. We also developed a novel BC treatment strategy using rCCL2 which should lead to effective combinations with existing BC immunotherapies.

Supported by (1) the Mays Family Cancer Center at University of Texas Health San Antonio (P30 CA054174), (2) the Roger L. And Laura D. Zeller Charitable Foundation Chair in Urologic Cancer, (3) the Glenda and Gary Woods Distinguished Chair in GU Oncology, (4) the Max & Minnie Tomerlin Voelcker Fund, (5) CDMRP CA170270/P1P2, (6) Bladder Cancer Advocacy Network (BCAN), (7) Research Training Award (RP170345) from the Cancer Prevention & Research Institute of Texas (8) MSTP Program (NIH T32GM113896)
Granzyme F production by CD8 T cells in the tumor microenvironment
Authors: Hay Zachary LeeZer , Knapp Jennifer R , Camp Faye A , O’Connor Brian P , Slansky Jill E
Published: January 2023

Granzymes are a class of cytotoxic proteases and are the primary mechanism utilized by T cells to directly eliminate cancer cells. Each granzyme acts upon a unique set of substrates in target cells to induce cytotoxicity through a range of different mechanisms. Granzymes are some of the most differentially regulated genes in CD8+ tumor infiltrating lymphocytes, relative to T cells outside of the tumor microenvironment (TME). We and others have determined by microarray and qPCR that granzyme F is highly upregulated in the TME. Granzyme F expression is restricted to a small subset of antigen-experienced and exhausted T cells, as determined by flow cytometry-based detection of granzyme F RNA transcripts and may represent a marker of a unique T cell cytotoxic function. Single cell RNA sequencing of CD8 TIL has revealed that granzyme F-high expressing cells are unique from both granzyme A and B expressing cells, and that it is therefore likely these TIL utilize a unique mechanism of cytotoxicity in their elimination of cancer cells. Recombinant granzyme F has previously been shown to induce a unique form of cell death, characterized as being caspase-independent and resulting in rupture of target cell plasma membrane. By over expressing granzyme F we are determining if this mechanism of cell death is leverageable to improve the cytotoxic capacity of TIL, and if induction of different forms of T cell-mediated cytotoxicity can modulate the immunogenicity of the TME. These experiments are designed to provide insight into how to improve adoptive T cell therapies by directly improving cytotoxicity, the terminal step of T cell interaction with tumor cells.

This work was supported by the National Institutes of Health NIAID training grant (Training Program in Immunology; T32-AI07405) award to Zachary Hay