bluuebunny/crossref_metadata_2025 huggingface dataset:
Radiotherapy-elicited cGAMP transfer by LRRC8 VRAC promotes anticancer T cell response
Authors: Xiao Hui , Cao Limin , Wang Li , Jia Xin-Ming
Published: August 2023

In addition to conducting Cl− efflux in response to cell swelling, LRRC8 VRACs have been implicated in transporting cGAMP to the bystander cells reinforcing the IFN-I response to combat HSV-1 infection. However, a plausible role of LRRC8 VRAC-mediated cGAMP transfer in cancer immunity and autoimmunity has not been explored. Here, we demonstrate that the radiation-triggered regression of implanted tumors largely depends on the host expression of Lrrc8a, especially in the T and dendritic cell compartments. Inducibly ablating Lrrc8a expression through tamoxifen or T cell-specific depletion of Lrrc8a through CD4-Cre led to diminished CD8 T cell infiltration. Moreover, the loss of Lrrc8a also impaired the expression of granzymes and IFN-γ in the tumor-infiltrated CD8 T cells. Mechanistically, our data implicate that tumor-derived cGAMP and ATP work together to trigger STING activation and IRF3/IRF7-mediated IFN-β expression in CD8 T cells. Remarkably, T cell-derived IFN-β feeds back to empower CD8 T cell’s cytolytic capacity through the induction of a subset of ISGs. In addition to radiotherapy, our observation that LRRC8 VRAC regulates anticancer immunity also extends to chemotherapy. Together, our study supports the emerging notion that both radio- and chemotherapies have the ability to boost anticancer immunity and reveal a crucial role for LRRC8 VRAC-mediated cGAMP transport in this process.

Supported by grants from National Natural Science Foundation of China.
T cell expressed microRNA-155 promotes antitumor immunity and immune checkpoint blockade responses in colon cancer through repression of Ship1
Authors: Tang William W , Ekiz H Atakan , Voth Warren P , Bauer Kaylyn M , Barba Cindy , Ghazaryan Arevik , Nelson Morgan , Lee Soh-Hyun , Weiss Allison , Thompson Jacob , Round June L , O’Connell Ryan M
Published: August 2023

Colorectal cancer (CRC) is the 2nd deadliest cancer globally and has the 3rd highest incidence and mortality in the United States. Advanced-stage disease is incurable by current clinical standards. Thus, alternative treatments are being explored, including immune checkpoint blockade (ICB) to prevent T cell inactivation by tumors. ICB induces a robust response in patients with microsatellite instability-high (MSI-H) metastatic CRC. However, most CRC patients are not responsive to ICB. Our analysis revealed MSI-H patients exhibit an immune cell-mediated inflammatory tumor microenvironment (TME) that is associated with the expression of microRNAs (miRs) linked to immune cell regulation. By studying T cell-associated miRs in MSI-H colon cancer patients, and through Crispr-Cas9 based in vivo screening, we identified T cell miR-155 as the miR most highly associated with ICB responsive patients. T cell miR-155 was necessary for ICB responses in multiple colon cancer preclinical models and promoted Th1-helper T cell and cytotoxic T lymphocyte enrichment in the TME, reflecting a robust immune response. T cell miR-155 controls ICB response and cancer immunity by repressing a network of target genes, most notably SHIP1. Loss of T cell miR-155 derepressed SHIP-1 gene expression in our study. Additionally, SHIP-1 was expressed in human tumor-associated T cells, with lower expression correlating to better patient outcomes. In vivo, we demonstrated that SHIP-1 inhibition partially restored tumor immunity in mice specifically lacking T cell miR-155. These findings suggest a dependence on T cell miR-155 and its targets for effective cancer immunity and ICB efficacy in colon cancer, identifying miR-155 as a candidate biomarker and therapeutic.

Supported by grants from NIH (F30 CA260977)
Identifying mechanisms driving macrophage differentiation and function in colorectal cancer
Authors: Saunders Ian T , Samakai Elsie , Brodeur Scott
Published: August 2023

The tumor microenvironment (TME) plays a critical role in cancer proliferation, metastasis, & responses of patients to treatment. Within the TME, tumor-initiated signaling results in aberrant activation of stromal & immune cell populations to suppress immune surveillance, induce angiogenesis, & establish premetastatic niches. Macrophages, a subset of antigen presenting myeloid cells, are the most abundant immune cell in the TME & participate in immune regulation during tumorigenesis. They functionally exhibit both anti & pro-tumorigenic properties as well as demonstrate the plasticity to repolarize between phenotypes based on environmental cues. Additionally, macrophage subset predominance within the TME & tumors have been correlated to favorable prognoses in some cancers. However, there is a lack of consensus for their role in colorectal cancer (CRC) progression. In this study, we investigated CRCs with distinct mutations, grade, & origin for their ability to drive macrophage differentiation & function in vitro. Human CRC cells were used to create multicellular tumor spheroids to better recapitulate in vivo tumor biology. CD14+ monocytes were polarized into macrophage subsets (M0/M1/M2) by cytokine/growth-factor exposure (GMCSF/MCSF/IFNγ/IL4/IL6/IL10) & co-cultured with each model. Spheroid proliferation & inflammatory profiles alone/in co-culture were assessed by live-cell microscopy, viability, & multiplex assays. Macrophage polarization, spheroid infiltration, & anti-tumor activity were examined by flow-cytometry, live-cell microscopy, & functional assessments. Overall, this study demonstrates that it may be useful to direct targeted therapies to influence macrophage polarization in CRC patients.

Supported by funding from Janssen Pharmaceutical Companies of Johnson & Johnson, Inc. under the direction of the DPDS Postdoctoral Fellowship Program and Biologics Discovery Division - Exploratory Biology Department.
Therapeutic CAR T cells differentiate into diverse memory and exhausted T cell subsets in vivo to provide long-term protection from relapse and metastasis of solid tumors
Authors: Reed Steven James , Tower Samantha J , Khanna Asheema , Kalia Vandana , Sarkar Surojit
Published: August 2023

Canonical memory T cell differentiation is programmed during the early stages of priming by a multitude of variables such as TCR signal strength, duration of antigenic exposure, and the milieu of cytokines. A combination of such stochastic variables in vivo, drive a heterogeneity in T cell priming that leads to the generation of distinct subsets of T cell effector and memory lineages. Contrarily, CAR T cell priming is largely homogenous and the diversity of the ensuing memory CAR T cells following adoptive immunotherapy remains largely unknown. In this study, we show that following adoptive immunotherapy into solid tumor bearing mice, syngeneic CAR T cells differentiate into distinct memory T cell subsets. In the peripheral tissues and lymphoid organs, CAR T cells subsets of central memory (TCM), effector memory (TEM) and resident memory (TRM) were fully represented. Furthermore, within the tumor microenvironment, distinct lineages of exhausted T cells such as progenitor exhausted (TPEX) and terminally exhausted (TEX) CAR T cells were found. When benchmarked against gold-standard T cells programmed in vivo by viral infections, the subsets of CAR memory and exhausted T cells demonstrated the hallmark qualities of protection from metastasis, and responsiveness to checkpoint blockade, respectively. These findings are among the first to demonstrate that CAR T cells differentiate into a full-spectrum of memory and exhausted T cell subsets in therapeutic applications against solid tumors. Comprehensive understandings of CAR T cell memory and exhaustion programs would be critical in developing therapeutic CAR T cells that provide long-term protection from tumor relapse, tumor metastasis and responsiveness to checkpoint blockade.

Research reported in this publication was supported by the National Cancer Institute of the National Institutes of Health under Award Number F32CA265056. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health
Gm2a as a novel regulator of CD8+ T cell threshold of activation in transplantation
Authors: Bazzano Julia Miranda R , Baecher Kirsten , Liu Danya , Tong Katherine , Fribourg Miguel L , Cravedi Paolo , Ford Mandy L
Published: August 2023

Transplant patients must remain on toxic immunosuppression for life, underscoring the need to identify immunotherapeutic targets that may facilitate transplant tolerance. We recently showed that kidney transplant recipients that remained stable following withdrawal of mainstay immunosuppression exhibited increased levels of the glycosphingolipid-catabolizing protein Gm2a, specifically in CD8+ T cells, compared to patients who went on to reject their allografts. While the role of Gm2a in lysosomal glycosphingolipid degradation in neurons is well known, little is known about Gm2a function in the immune system. To investigate this, we performed skin graft surgery in WT vs Gm2a−/− mice. Results show that Gm2a deficiency significantly increased allograft rejection relative to WT counterparts. Moreover, adoptive transfer of donor-reactive WT vs. Gm2a−/− CD8+ T cells into WT hosts resulted in increased accumulation of donor-reactive T cells and accelerated allograft rejection in recipients of Gm2a−/− CD8+ T cells as compared to recipients of WT CD8+ T cells. This increased accumulation was likely due to increased proliferation, as in vitro studies revealed enhanced proliferation of CTV-labeled Gm2a−/− vs. WT CD8+ T cells. Interestingly, Gm2a−/− CD8+ T cells exhibited increased Kb/SIINFEKL tetramer staining and sustained TCR expression following antigen stimulation compared to WT CD8+ T cells. Finally, Gm2a−/− CD8+ T cells exhibited increased responsiveness to low dose and low-affinity peptide compared to WT cells. In conclusion, these results identify Gm2a as a novel, CD8+ T cell-intrinsic regulator of the T cell activation threshold that directly impacts CD8+ T cell-mediated allograft rejection.

Supported by grants from NIH (R01AI164716)
Variegated levels of alloreactive T cell dysfunction in transplantation tolerance determine graft vulnerability to infection-triggered rejection
Authors: Allocco Jennifer Brianne , McIntosh Christine , Wang Peter , McKeague Michelle , Wang Ying , Cassano Alexandra , Xie Stephen Z , Mora-Cartin Ricardo , Chen Luqiu , Sattar Hussain , Yin Dengpin , Zhang Zheng J , Chong Anita S F , Alegre Maria-Luisa
Published: August 2023

While advances have been made in therapies that achieve tolerance in mouse models of transplantation, successful tolerance remains vulnerable to inflammatory insults, which can trigger graft rejection. To identify strategies for improving the robustness of tolerance, we aim to better understand the mechanisms by which grafts are rejected after donor-specific tolerance is established. In a mouse model of cardiac allograft tolerance in which late infection with Listeria monocytogenes (Lm) can trigger graft rejection, we previously showed that host CD4+ T cells reactive to host MHC-II presenting a donor MHC-I-derived peptide (TCR75 cells) became intrinsically dysfunctional if the alloantigen persisted for 3 weeks or greater. Intriguingly, infection-dependent transplant rejection was not associated with transcriptional or functional reinvigoration of these cells. We hypothesized that there might be heterogeneity in the level of dysfunction of alloreactive T cells depending on duration of their cognate alloantigen persistence. Unlike TCR75 cells, CD4+ T cells specific for host MHC-II presenting a peptide derived from donor MHC Class II (TEa cells), a graft antigen that declines post-transplantation, retained functionality during tolerance induction. Consequently, TEa but not TCR75 cells expanded following Lm-infection, supporting a role for TEa-like cells in driving infection-triggered rejection. Prolonging T cell exposure to cognate alloantigens aggravated TEa dysfunction and rendered grafts resistant to Lm-dependent rejection. These findings demonstrate that inducing dysfunction in a broader array of allospecific T cells, by prolonging exposure to more alloantigens, lessens graft vulnerability to infections.

Supported by the UChicago Growth Development and Disabilities Training Program (T32 HD007009). AHA predoctoral fellowships (20PRE35210946, 3PRE14550022 and 15PRE22180007). NIH T32-AI007090. UChicago Cardiovascular Pathophysiology and Biochemistry Training Grant (T32 HL07237). HHMI Med-into-Grad Program training grant (56006772). NIAID Grant P01AI-97113.
Exploiting Fas-mediated autocrine apoptosis of donor alloreactive T cells as an effective approach to prevent acute graft-versus-host disease in a humanized mouse model
Authors: Yolcu Esma S , Shrestha Pradeep , Turan Ali , Shirwan Haval
Published: August 2023

Acute GVHD initiated by donor T cells is a barrier to allogeneic bone marrow transplantation. Pan T cell elimination from the graft is associated with failure of stem cell engraftment and delayed immune reconstitution. T cells upregulate the Fas receptor on their surface following activation and become susceptible to FasL-mediated apoptosis. The objective of this study was to target Fas-mediated apoptosis for the preferential elimination of alloreactive T cells.

We previously reported a novel form of FasL protein chimeric with streptavidin (SA-FasL). Mouse and human lymphocytes were surface modified with biotin, followed by engineering with the SA-FasL protein taking advantage of biotin’s high affinity to SA. SA-FasL-engineered cells were assessed for autocrine apoptosis of alloreactive T cells in vitro and in vivo. NSG mice transplanted with 107 human PBMC were used as an acute GVHD xenogeneic model.

SA-FasL induced autocrine apoptosis in alloreactive T cells in vitro and in vivo. Mice transplanted with SA-FasL-engineered PBMC had significantly lower numbers of human T cells in the spleen and liver analyzed five days post-transplantation compared to the recipients of SA-engineered cells. Recipients of SA-engineered PBMC developed acute GVHD with a median survival time of 15 days. In marked contrast, sixty percent of SA-FasL-engineered PBMCs recipients survived for a 60-day observation period and showed minimal clinical GVHD scores.

Transient display of SA-FasL protein on the surface of donor cells provides a practical and clinically applicable effective approach to prevent acute GVHD with significant translational potential.
CRISPR/Cas9 Gene Editing of MIR155HG in Primary Human T Cells to Prevent Acute Graft-Versus-Host Disease
Authors: Neidemire-Colley Lotus M , Khanal Shrijan , Braunreiter Kara M , Snyder Katiri Jenna , Gao Yandi , Kalyan Sonu , Kararoud Meisam Naeimi , Duszynsk Molly , Chi Mengna , Malik Punam , Choe Hannah , Garzon Ramiro , Ranganathan Parvathi
Published: August 2023

Introduction

Acute graft-versus-host disease (GVHD) mediated by donor alloreactive T cells is the leading cause of non-relapse mortality in patients post-allogeneic hematopoietic cell transplantation. Here we genetically engineered donor T cells by genomic deletion of microRNA-155 host gene (MIR155HG) to prevent GVHD.

Methods

Using CRISPR/Cas9 genome editing, we targeted exon 3 (Ex3) of MIR155HG. Genomic deletion was evaluated by genomic PCR and RNA expression of mature miR-155 by qRT-PCR. In xenogeneic acute GVHD experiments, NSG mice were irradiated and transplanted with miR-155 Ex3 edited or control T cells to assess functionality in vivo. Histological analysis of target tissues (skin and liver) was performed at end of study. Weekly flow cytometric evaluation of human donor T cells in peripheral blood was performed. MOLM-13 leukemic target cells were cultured with miR-155 Ex3 edited or control CD8+ effector T cells for 72 hours and cytotoxic function was assessed by CD107a degranulation, intracellular IFN-γ production and leukemic cell death by flow cytometry.

Results

Ex3 deletion was confirmed by genomic PCR resulting in downregulation of mature miR-155 expression. Recipients of Ex3 deleted human donor T cells display lower GVHD clinical scores, lower average histopathological scores and significantly improved survival compared to mice receiving control T cells. Ex3 T cells displayed potent CTL function comparable to control T cells resulting in leukemic cell death.

Conclusion

CRISPR/Cas9 deletion of miR-155 in donor T cells protects against development of lethal GVHD while maintaining beneficial anti-leukemic effect. Our studies present genomic editing of donor T cells as a novel strategy to prevent GVHD.

Funding for this project was provided through The Ohio State University Leukemia Research Program to PR and RG, NIH R01CA252469 to PR, and NIH T32CA090223 fellowship to KB.
Sentinel flaps correlate with pro-inflammatory and immunoregulatory mediators of human face transplantation rejection
Authors: Crisler William J , Win Thet Su , Barrera Victor , Ho-Sui Shannan , Clark Rachael A
Published: August 2023

Early diagnosis and treatment of immune rejection is critical for face transplants (vascularized composite allografts, VCA). VCA rejection is diagnosed by histologic evaluation of skin biopsies, yet repeated biopsies are a considerable aesthetic problem. Sentinel flaps, fasciocutaneous radial forearm flaps, have been used for remote-site rejection monitoring, but the concordance between tissues during rejection remains unclear. With access to the world’s largest cohort of VCA patients at a single center worldwide, we studied 14 paired skin biopsies from sentinel flaps (SF) and facial allografts (FA) during rejection and non-rejection. We previously identified 15 pro-inflammatory genes (including GZMB, HLA-DPB1, IFNG) and 15 immunoregulatory genes (including SOCS1, CTLA4, IDO1) upregulated in VCA rejection. SF and FA had an average correlation coefficient of 0.88 and 0.76 for pro- and anti-inflammatory gene sets, respectively. During rejection, there was no significant difference in expression of the 15 pro-inflammatory genes between tissue type (SF versus FA), and only one immunoregulatory gene (PDCD1) was significantly different. Across all samples, there were 64 differentially expressed genes (DEG) between rejection and non-rejection, 13 DEG between tissue types, and only 2 DEG when statistically accounting for differences in tissue type driven by rejection status (log2FC>1, p<0.05). High throughput sequencing (HTS) of the CDR3 of TCR-β genes from 4 matched pairs during rejection identified a mean of 9.5 expanded shared T cell clones, indicating that shared antigens drove rejection in both tissues. Together, these data indicate that SF could serve as effective remote-site monitoring for face transplant rejection.

NIH T32 Training Grant 2T32AR007098-47 Dept. of Dermatology, Harvard Medical School RT170025 (P.I. Rachael A. Clark) Department of Defense - congressionally Directed Medical Research Programs; "T cells and Rejection in Vascularized Composite Allografts).
Interleukin 18 (IL-18) Suppresses Thymus Regeneration
Authors: Granadier David , Cooper Kirsten , Acenas Dante , Kinsella Sinéad , Evandy Cindy , Iovino Lorenzo , deRoos Paul , Hernandez Vanessa , Shannon-Sevillano Steve , Kousa Anastasia , Dudakov Jarrod
Published: August 2023

The thymus is highly sensitive to acute injury, particularly to cytoreductive conditioning given pre-hematopoietic stem cell transplant (HCT). The thymus is capable of a remarkable degree of regeneration, however its reparative capacity and T cell productivity decline with age. This leaves HCT recipients vulnerable to relapse of malignancy and opportunistic infection – the leading causes of post-HCT mortality – during a prolonged period of lymphopenia. Better understanding the endogenous mechanisms by which thymus regeneration is regulated may inform therapeutic interventions to improve T cell reconstitution in these patients. Here, we identify the inflammatory cytokine interleukin 18 (IL-18) as a suppressant of thymic function and propose its targeting to improve immune reconstitution.

We posit that distinct forms of acute injury (i.e., irradiation, chemotherapy, infection and stress) lead to caspase-1 activation within the thymus and subsequently increased active IL-18. Our preliminary data suggests that IL-18 receptor (IL-18R) expressing natural killer (NK) cells are activated following acute damage in an IL-18 dependent fashion. Importantly, depletion of this NK1.1+ IL-18R+ population improves thymus regeneration, suggesting that IL-18 induces an inflammatory response in organ-resident NK cells capable of impairing thymus repair.

Our findings implicate IL-18 as a novel regulator of thymic regeneration and identify NK cells as likely mediators in its pathway. Furthermore, we suggest that IL-18 can be therapeutically targeted to improve T cell reconstitution following cytoreductive therapy and HCT.

Supported by grants from NIH (R01 HL145276, P01 AG052359)
Expanded T regulatory cells fail to control germinal center responses in the lung lymph nodes of pulmonary fibrosis patients
Authors: Velez Tania Estefania , Yoon Young me , Hollinger Maile K , Blaine Kelly M , Lee Cathryn T , Strek Mary E , Guzy Robert , Sperling Anne I
Published: August 2023

Idiopathic pulmonary fibrosis (IPF) is the most common form of interstitial lung diseases (ILD), which is characterized by progressive scarring of the lung. While immune suppression is a viable therapeutic option for many ILDs, it is detrimental in IPF. Nevertheless, enlargement of lung-draining lymph nodes (LLN) is prevalent and predicts survival outcomes in all ILD patients, including IPF, suggesting that there is also an immune component to IPF pathogenesis. To test this hypothesis, we investigated the cellular composition of the LLN in IPF and non-IPF ILD patients. All ILD patients’ LLN had increased germinal centers (GC), implicating that immune responses are not being resolved. As T regulatory cells (Tregs) are key in the control and resolution of immune responses, we investigated the phenotype of Tregs in the LLN of both IPF and non-IPF ILD patients to reveal their role in lung fibrosis. Tregs were expanded in LLN of all ILD patients compared to controls. Interestingly, there was a predominance of GATA3-expressing Tregs in ILD patients’ LLN. However, Tregs of ILD patients expressed significantly elevated levels of ICOS and CD137 compared to controls, indicating recent activation. Surprisingly, CD25 expression was significantly lower on Tregs from ILD patients’ LLN. Sc-RNAseq confirms similar Treg phenotypes in the lungs of IPF patients. In a mouse model of bleomycin-induced pulmonary fibrosis, Tregs expand and display a similar activated phenotype. Preliminary experiments suggest that GATA3 expression in Tregs is detrimental in this model. Together, these data indicate that there are unresolved GC responses in the LLN of all ILD patients, including IPF, and suggest that Tregs are ineffectual at controlling the GC response.

American Heart Association Postdoctoral Fellowship, Award number (835938)
1 Liver + 4 Diseases = n Drugs
Authors: Dell Garrison Jeffrey , Steele Hope , Cheng Jason Liang , Breckenridge Joey Emery , Willicut Ashley , Ghastine Andrew , Culberson Erica , Herro Rana
Published: August 2023

The burden placed on the global population by the instance of liver diseases is significant and there exists a great need for accurate diagnostic method alternatives to invasive biopsy. Here we conduct a comparative histopathological and transcriptomic analyses of 4 models of liver diseases to identify distinct biological pathways which aide in understanding the mechanisms of each disease and provide insight into biomarkers for diagnosing disease, monitoring disease progression, and for evaluating future therapeutic efficacy. The NASH model is metabolically induced by high fat/high fructose diet, whereas the three other models are hepatotoxicants, and involve Allyl Alcohol (AA), 4,4′-Methylenedianiline (MDA), or Bromobenzene (BB). Analysis of liver sections stained with Hematoxylin and Eosin and Masson’s trichrome revealed distinct pathological differences between each of the models. These differences were further confirmed by gene set enrichments and pathway analyses. This transcriptomic comparative study outlines distinct pathways important for inflammation, steatosis and fibrosis of the liver that are unique to each model in addition to common pathways contributing to disease pathogenesis. It further validates the histopathological features characteristic of each disorder.
Negative regulation of intestinal IgA by inflammatory cues in absence of infection
Authors: Sung Chi Chang , Barbet Gaetan
Published: August 2023

Pathogen-specific IgA elicited at mucosal site is associated with a better immune protection upon vaccination. Although some effective mucosal vaccines are available and approved, the majority of current vaccines are administered intramuscularly or subcutaneously and were developed irrespectively of a particular site of infection. Vaccine adjuvants are selected in their capacity to elicit a strong inflammatory response. However, few studies show the effectiveness of FDA-approved adjuvants to mucosal vaccines. Here, we unveil the role of inflammatory pathways in their negative regulation of IgA production in murine intestines. We are currently studying and identifying the molecular and cellular pathways involved in the production of protective IgA antibodies in the intestines in absence of particular inflammatory cues. Indeed, in absence of some inflammatory signals, we observed an increase of free IgAs and IgA-bound bacteria in the intestines of mice in steady state. IgA levels were normal in the serum and the concentration of IgG antibodies were also normal in both the intestines and serum suggesting a skewed toward IgAs restricted to the intestines. Furthermore, we could show that this IgA production is for the most part T-dependent and involves the formation of germinal centers localized in the Peyer’s Patches. We are now developing vaccine models to elicit potent IgA-mediated protection in the aim to use these finding at our advantage to promote pathogen-specific intestinal responses through new designs of mucosal vaccines. We believe that developing mucosal vaccines that elicit a protective immunity through natural routes of infection hold the promise to act better, faster and safer against worldwide pathogenic threat.

RWJF Grant Project # 826399 Moderna Research Fellowship
TFR cell-mediated regulation of IgA and gut microbiota in food allergy
Authors: Abdi Abdullahi M , Dent Alexander L
Published: August 2023

About two grams of secretory Immunoglobulin A (sIgA) are secreted daily into the intestine, where it serves as the first line of defense against pathogens and protects the host’s commensals by coating them, ensuring mucosal immune system homeostasis. Gut microbiota (Mb)-specific IgA can be generated via T cell-dependent (TD) or T cell-independent (TI) pathways. However, the TD route is required to generate high-affinity, pathogen-specific antibodies (Abs). T follicular helper (Tfh) cells are essential for germinal center (GC) development, promoting B cells expansion. Nevertheless, the role of T follicular regulatory (Tfr) cells in Mb-reactive IgA production is unclear. Unpublished results and a prior work demonstrated that deleting Tregs or Tfr cells in mice increased antigen-specific IgA titers, indicating that these cells negatively control IgA response. Thus, we hypothesized that TFR cells regulate gut Mb-reactive IgA via the regulation of high-affinity IgA production against gut Mb. To test this hypothesis, TFR cell-deficient Bcl6FC (Foxp3 cre-Bcl6 fl/fl) and WT mice were primed with peanut plus cholera toxin through oral gavage in a food allergy model. Then, we stained fecal Mb with anti-mouse IgA, sorted commensals into IgA +and IgA −populations and sequence 16S rRNA gene of the sorted bacteria (IgA-seq) to evaluate the Mb-reactive IgA response. Flow cytometry data showed significantly higher IgA-coated bacteria in Bcl6FC than in WT mice, showing TFR cells adversely modulate IgA response to gut Mb. Our current experiments will dissect if the higher Mb-reactive IgA secreted in the absence of TFR cells were non-specific and polyreactive or were specific towards certain members of gut commensals.

R21 AI172087 NIH
Synergy between anti-commensal antibodies and IL-10 in gut homeostasis and disease
Authors: Hsu Jung-Shan , Klocke Barbara J , Landuyt Ashley E , Muir Rachel Q , Jennings Melissa S , Molina Patrick A , Randall Troy D , Elson Charles O , Lee Goo , Maynard Craig L
Published: August 2023

To limit immune responses to gut-resident microbes, mucus and the epithelial barrier physically separate the host immune system from microbial communities. Immunoglobulin A (IgA) antibodies are known to help to regulate the colonization and functions of mucus-associated commensals. However, the mechanisms whereby other isotypes, including IgG, interface with other components of the immune system to promote tolerance of MA microbes are still being unraveled. Our lab previously showed that, in mice with impaired production of T-dependent (TD) anti-commensal antibodies, IgA- and IgG-mediated recognition of mucus-derived bacterial antigens is significantly reduced. Moreover, transient depletion of IL-10-producing cells from mice devoid of TD antibodies resulted in rapid onset colitis. Therefore, we hypothesized that anti-commensal antibodies synergize with IL-10 to promote immune homeostasis in the colon. Here, we show that, relative to wild type mice, there are increased levels of colonic IL-10-producing CD4 T cells in mice lacking both IgG and IgA (Aicda−/−) but not in mice lacking only IgA (Iga−/−). Additionally, transient depletion of IL-10-producing cells resulted in severe colitis in Aicda−/− but not Iga−/− mice. Importantly, this colitis developed despite elevated levels of IgM in the colon mucus layer of Aicda−/− mice. Furthermore, we observed elevated levels of IgG in the colon mucus layer of Iga−/− mice suggesting a potentially compensatory role for IgG in mice devoid of IgA. Collectively, our data suggest an important contribution for anti-commensal IgG, including in combination with IL-10, in promoting immune tolerance of border-dwelling microbes.

R01 AI162736, R21 DK118386
Endogenous retroviruses act as a tonic signal for Treg induction and oral tolerance development
Authors: Rivera Claudia A , Krishnamurthy Siddharth R , Belkaid Yasmine
Published: August 2023

Endogenous retrovirus (ERVs) result from accumulated germline infections by ancient exogenous retroviruses and constitute a sizable proportion of the mammalian genome. Recent findings support the idea that these elements control tissue immune threshold of activation and inflammation. Within this context, our lab has recently described that, in the skin, the beneficial immunity to the microbiota depends on endogenous retrovirus expression. How the crosstalk between ERVs and the microbiota controls tissue immunity at other barrier sites and how dysregulation of this dialogue impairs tolerance responses remain unknown. Our results reveal that antiretroviral (anti-RT) treatment (blocking the conversion of ERV into cDNA) impairs gut regulatory T cell homeostasis and differentiation and compromises the induction of oral tolerance to food antigens. Consistently, our data demonstrate that antiretroviral treatment impairs type I IFN signaling in mucosal dendritic cells involved in the induction of regulatory T cells. Further, our results show that epithelial ERV expression is highly conserved across tissues and seems to be only partially affected by the microbiota in the small intestine. Altogether, our results propose that ERV control immunoregulation within the gastrointestinal tract by acting as a tonic signal required for Treg induction and differentiation thereby promoting oral tolerance and preventing allergic responses.

This work was supported in part by intramural funds of NIAID, NIH. C.A.R is supported by Damon Runyon Fellowship program (DRG-2496-23).
Homeostatic intestinal immunity is dominated by microbiota-independent cytotoxic Th1 cells
Authors: Gine Eduardo Ansaldo , McFadden Taryn , Yong Daniel , Bouladoux Nicolas , Link Verena M , Corral Dan , Belkaid Yasmine
Published: August 2023

Barrier tissues such as the skin and gut form the first line of defense against pathogens and establish an immune dialogue with the resident microbiota. To that end, they harbor immune populations geared towards barrier function, tolerance, and repair. Of particular interest are the large numbers of T cells. Some are specific towards commensal microbes or recognize previously encountered pathogens. However, the origin, antigen specificity, and function of most barrier tissue-resident T cells remains elusive. We have identified a dominant CD4 T cell population in the small intestine lamina propria characterized by the expression of T-bet. Surprisingly, this population is present in both germ-free and conventionally reared (SPF) mice at homeostasis, and accounts for almost half of T cells at this site. Bulk and single-cell transcriptome analysis revealed that this population has cytotoxic potential. Furthermore, single-cell TCR sequencing showed parallel T cell repertoires in germ-free and SPF mice, suggesting shared antigen specificity. To explore the ontogeny of this population, we have identified clonally expanded TCRs and are generating low frequency retrogenic mice. Additionally, we have generated several T cell hybridomas from small intestine Th1 cells. Interestingly, preliminary results suggest that small intestinal Th1 TCRs exhibit some degree of self-reactivity, which may determine their development and differentiation in a foreign antigen-independent manner. Collectively, these studies will shed light on the function of a dominant intestinal T cell population and reveal novel insights into homeostatic tissue immune networks.

This research was supported by the Intramural Research Program of NIAID, NIH.
In neonatal mice, intestinal CD64+ CD206+ MHCII− macrophages do not recover one-week post-anti-CSF1 antibody injection, which affects key pro-angiogenic proteins
Authors: Managlia Elizabeth , Yan Xiaocai , De Plaen Isabelle
Published: August 2023

The small intestine of neonatal mice has abundant embryonically derived macrophages (MΦ) but only a small subset remains into adulthood. These MΦ play a role in fetal and neonatal development. In the intestine, they are juxtaposed to endothelial cells where they facilitate postnatal vascular expansion. Postnatal recruitment of bone-marrow-derived blood monocytes complicates the identification and study of the embryonically derived compartment since adult monocytes differentiate into intestinal MΦ of similar phenotype. Whether the different lineages have unique roles in neonates needs further investigation. To explore the role of intestinal MΦ during the neonatal period, we depleted neonatal MΦ by injecting 1-day old pups with anti-CSF1 antibodies and allowed the intestine to repopulate with monocyte-derived MΦ for a week.

Flow cytometry on small intestinal tissue revealed that at D8, 2.77% (± 0.32) of intestinal MΦ were CD64+ CD206+ MHCII− in IgG injected pups compared to 0.18% (± 0.05) in anti-CSF1 injected pups (p=0.001). These data show that postnatal bone-marrow derived MΦ do not reconstitute the intestinal CD64+ CD206+ MHCII− MΦ, which are abundant at birth but not detected in adults. We determined by western blot that intestinal Vegfr2 and Igf1r protein expression, which we have found to be critical for neonatal intestinal microvascular development, were decreased in 8-day old pups injected with anti-CSF1 2.1-fold (p=0.047) and 1.5-fold (p=0.15) respectively when compared to IgG injected littermates. Taken together, these results suggest that CD64+ CD206+ MHCII− MΦ may be embryonically derived and their transient presence may be important for neonatal microvascular development.

NIH DK116568 (IDP)
Inactivation of GSK3β by Ser389 phosphorylation prevents thymocyte necroptosis and impacts Tcrβ and Tcrα repertoire diversity
Authors: Pereira Felipe , Sheridan Ryan , Riemondy Kent , Thorton Tina , Fang Qian , Barret Brad , Paludo Gabriela , Thompson Claudia , Collins Patrick L , Santiago Mario , Oltz Eugene M , Rincon Mercedes
Published: August 2023

Double negative (DN) thymocytes go through consecutive rounds of Tcrβ and Tcrα gene recombination that involves the generation of DNA double-strand breaks (DSB), the addition of N-nucleotides between coding ends, and DNA repair with the ultimate goal of enhancing TCR repertoire diversity. How DN thymocytes can overcome such genomic instability in order to generate functional Tcrβ and Tcrα genes remains unclear. We performed scRNA-seq analyses of isolated DN3 and DN4 thymocytes to demonstrate a clear trajectory from DN3 through the DN4 stage that is delineated by G1 and G2M cell cycle checkpoints to facilitate DNA repair during recombination of Tcrβ and Tcrα prior to cell proliferation. Inactivation of GSK3β by phosphorylation on Ser389 in response to V(D)J recombination DSB is required to prevent thymocyte death by necroptosis. Failure to inactivate GSK3β through this pathway alters the Tcrβ and Tcrα V segments usage. We also show that inactivation of GSK3β by phosphorylation on Ser389 is required for expression of the cytidine deaminase Apobec3 in DN3 thymocytes to increase N-nucleotide diversity within the Tcrβ N2-region. Thus, G1 and G2M cell cycle checkpoints to ensure proper DNA repair during V(D)J recombination and unique GSK3β-dependent survival pathways to prevent necroptosis during this process are critical to generate a large diversity of pre-selection TCR repertoire.

Double negative (DN) thymocytes go through consecutive rounds of Tcrβ and Tcrα gene recombination that involves the generation of DNA double-strand breaks (DSB), the addition of N-nucleotides between coding ends, and DNA repair with the ultimate goal of enhancing TCR repertoire diversity. How DN thymocytes can overcome such genomic instability in order to generate functional Tcrβ and Tcrα genes remains unclear. We performed scRNA-seq analyses of isolated DN3 and DN4 thymocytes to demonstrate a clear trajectory from DN3 through the DN4 stage that is delineated by G1 and G2M cell cycle checkpoints to facilitate DNA repair during recombination of Tcrβ and Tcrα prior to cell proliferation. Inactivation of GSK3β by phosphorylation on Ser389 in response to V(D)J recombination DSB is required to prevent thymocyte death by necroptosis. Failure to inactivate GSK3β through this pathway alters the Tcrβ and Tcrα V segments usage. We also show that inactivation of GSK3β by phosphorylation on Ser389 is required for expression of the cytidine deaminase Apobec3 in DN3 thymocytes to increase N-nucleotide diversity within the Tcrβ N2-region. Thus, G1 and G2M cell cycle checkpoints to ensure proper DNA repair during V(D)J recombination and unique GSK3β-dependent survival pathways to prevent necroptosis during this process are critical to generate a large diversity of pre-selection TCR repertoire. Supported by NIH R01 AI051454
Defining the Role of HDAC3 Deacetylation and Decrotonylation in T Cells
Authors: Martinez Kodi , Shapiro Michael , Rajcula Matthew , Lee Hyun Se Kim , Hammer Brady , Mai Elissa , Shapiro Virginia
Published: August 2023

Histone acetyltransferases (HATs) and histone deacetylases (HDACs) facilitate addition or removal of various acyl groups from lysine residues, including acetylation (KAc) and crotonylation (KCr). Recently, mutations in p300 and CBP were identified that abrogate their function as an acetyltransferase but not the ability to mediate crotonylation. These mutants can activate transcription independent of their ability to acetylate histones, demonstrating that crotonylation can also lead to transcriptional activation. The Class I deacetylases HDAC1, HDAC2, and HDAC3 have been shown to have both decrotonylase and deacetylase activity, but the relative importance of each function in T cells is not known. Genetic knockout of HDAC3 using CD2 icre mice results in a severe block in T cell development and spontaneous development of severe colitis. Examination of HDAC3 cKO mice has shown an aberrant ratio of Treg:Th17 cells within the gut, driving inflammation and loss of mucosal barrier integrity. Our lab has also determined that HDAC3 is required for at least three aspects of T cell development: CD4+ CD8+ (DP) cell survival, positive selection, and CD4+ lineage development. Recently, a mutation was identified in either HDAC1 or HDAC3 that eradicated deacetylase activity but retained decrotonylase activity (“VRPP”). Through this mutation we were able to generate a novel mouse model with Cre-mediated deletion of endogenous HDAC3 and linked re-expression of HDAC3 VRPP. Strikingly, this mouse does not develop colitis, although T cell development is still impaired. Thus, HDAC3 decrotonylase activity alone is sufficient to impede the IBD phenotype.

Support by grants from National Institute of Allergy and Infectious Diseases (R01 AI150100)
Learning to live with the thymic roller coaster
Authors: Kang Seung Woo , Manley Nancy R
Published: August 2023

The thymus is the primary lymphoid organ responsible for naïve T-cell production, vital for adaptive immunity. Thymus size and T-cell output dynamically change throughout the lifetime. The thymus increases its size and output during the perinatal stage; hits a plateau around postnatal day 10 and maintains homeostasis during the juvenile stage; then starts to undergo involution. The perinatal thymus is unique, for it exponentially increases the T-cell export whilst generating virtual memory-like T-cells (TVM), poised to respond to foreign antigens.

Our questions are how the thymic microenvironment changes from perinatal expansion to juvenile homeostasis and how FOXN1 regulates the transition. FOXN1 is a master transcription factor that is exclusively expressed in thymic epithelial cells (TECs) and works in a highly dosage-sensitive fashion to determine TEC differentiation and proliferation as well as the total thymic microenvironment via crosstalk. In this study, we use the Foxn1-lacZ model that has a normal Foxn1 expression level during development but has 60% decreased Foxn1 expression level around postnatal day 7. Using scRNA-seq of total thymic stroma, we are studying stromal changes from perinatal expansion to juvenile homeostasis and the effect of decreased Foxn1 level on the transition. We have found changes in TEC subsets, endothelium and fibroblasts before and after the transition contributing to the different perinatal thymic microenvironments. Moreover, we have found a decrease in Foxn1 level causes accelerated transition with the premature occurrence of ageing TECs. Successful completion of the project will determine how the thymic stroma changes across perinatal to juvenile and how Foxn1 regulates the transition.

Supported by grans from NIH-NIAID 1P01AI139449-01A1.
Going oversized: Testing the limits of endogenous thymic regeneration and uncovering the mechanisms of thymic hypertrophy
Authors: Acenas Dante Dennis V , Granadier David , Iovino Lorenzo , Cooper Kirsten , deRoos Paul , Evandy Cindy , Hernandez Vanessa , Dudakov Jarrod
Published: August 2023

The thymus is the primary site of T cell development, generating a diverse repertoire of T cells that protects us from various threats. Despite its crucial role in T cell immunity, the thymus undergoes chronic involution that leads to a decline in T cell production and impaired T cell immunity. Although the thymus is highly sensitive to various forms of damage, and everyday insults like stress and infection, it also has a remarkable capacity to regenerate itself. Though this reparative capacity declines with age. We hypothesized that age-related thymic decline may be attributed to repeated damage and regeneration cycles in response to everyday insults. We expected impaired regeneration with each round of damage. However, we found that after three rounds of damage and repair cycles, overall thymic cellularity consistently returned to homeostasis. Moreover, we also found that unlike injury from ionizing radiation, Dexamethasone (Dex) mediated damage led to thymic hypertrophy.

Preliminary studies revealed that type 2 cytokines, previously implicated to promote thymic regeneration, are elevated in the early phases of Dex induced damage, but not during irradiation. Comparing thymic cellularity between damage models, we found that Dex induced damage had faster recovery kinetics and led to thymic hypertrophy 28 days after damage. We hypothesize that type 2 immunity may be a key contributor to hypertrophic thymic regeneration in Dex induced thymic damage. We will test our hypothesis through genetic mouse models, cytokine modulation, and standard immunophenotyping. The positive results of this study will augment our basic knowledge of the endogenous mechanisms of thymic regeneration and inform the creation of thymic boosting therapies.

I am supported in part by PHS NRSA T32GM007270 from NIGMS.
Nucleated red blood cells: unexpected orchestrators of fetal immunity
Authors: Clements Rebecca Lynn , Jurado Kellie Ann
Published: August 2023

Red blood cells (RBCs) are not typically considered active immune mediators. This dogma stems from the fact that RBC precursors discard organelles as they mature, thus losing the ability to alter gene expression in response to stimuli. Intriguingly, nucleated RBCs (nRBCs) circulate in human fetuses and neonates. Due to evolutionary pressure for successful reproduction, circulation of nRBCs during pregnancy is likely important. However, the role of nRBCs in utero remains unknown. To define the role of human nRBCs, we queried single cell RNA-seq data and found that transcriptomics support nRBCs as putative immune mediators.

Unexpectedly, we found that nRBCs express antigen processing & presentation machinery. nRBCs constitutively express MHC II & co-stimulatory molecules, hallmarks of specialized antigen-presenting cells. We further demonstrated that nRBCs internalize and cleave antigens. Currently, we aim to determine if nRBCs present antigens to T cells and to characterize the elicited response.

In addition, we found that nRBCs express pattern recognition receptors capable of detecting pathogens and initiating an antimicrobial response. We demonstrated that nRBCs respond to viral infection through upregulation of antiviral genes, interferons/interferon-stimulated genes, MHC I, and pro-inflammatory cytokines/chemokines. These data suggest a putative functionality for nRBCs in mediating antiviral immunity in utero. We are currently investigating antiviral functions of nRBCs.

Together, our findings shed light on an unexpected orchestrator of fetal immunity. Knowledge of the fetal immune system has potential to help us understand health and disease during pregnancy, as a neonate, and likely every stage of life after.

Supported by grants from NIH/NIDDK 5T32DK007780, NIH/NIGMS K12GM081259, Burroughs Wellcome Fund 2022 Next Gen Pregnancy Initiative Award