bluuebunny/crossref_metadata_2025 huggingface dataset:
Transplantation and CMV promote premature aging of CD8 T cells
Authors: Higdon Lauren E , Gustafson Claire E , Ji Xuhuai , Maecker Holden T , Maltzman Jonathan S
Published: February 2023

Cytomegalovirus (CMV) infection is a significant contributor to both immune aging and complications after solid organ transplantation. A major mediator of both effects is viral reactivation, in which latent CMV in the host undergoes new replication requiring immune control. Immunocompetent CMV seropositive individuals display hallmarks of CD8 T cell aging earlier than CMV seronegative individuals, including shortened telomeres, defects in proliferation and signaling, and altered cytokine production. In the context of transplantation, immunosuppression impairs immune control, leading to negative impacts including late-term graft loss. We hypothesized that T cell aging is accelerated after transplant in CMV+ individuals, regardless of detectable viral reactivation. To address this hypothesis, we measured telomere length and conducted phenotyping through flow cytometry and targeted single cell RNA sequencing. We found a statistical increase in CD57 expression which correlated with a trend towards decreased telomere length in the first year after transplantation. We found that CD57+ CD8 T cells expanded in the first year after transplantation specifically in CMV+ recipients. We also found that clonal expansion was associated with a terminally differentiated phenotype, defined as CD45RA+ CD27− CCR7−. Overall, our data demonstrate an enhanced aging phenotype in the first year post-transplant in CMV seropositive individuals, based on CD57+ expression and terminal differentiation. These findings suggest that CD8 T cell aging may be an important factor to understanding immunity in transplant recipients.
DNA-PKcs Inhibition Extends Allogeneic Skin Graft Survival
Authors: Burdine Marie , Waldrip Zachary , Burdine Lyle
Published: February 2023

Background:

Organ transplantation is life-saving and continued investigations into immunological mechanisms that drive organ rejection are needed to improve immunosuppression therapies and prevent graft failure. DNA-dependent protein kinase catalytic subunit, DNA-PKcs, is a critical component of both the cellular and humoral immune responses. In this study, we investigate the contribution of DNA-PKcs to allogeneic skin graft rejection to potentially highlight a novel strategy for inhibiting transplant rejection.

Methods:

Fully MHC mismatched murine allogeneic skin graft studies were performed by transplanting skin from BalbC mice to C57bl6 mice and treating with either vehicle or the DNA-PKcs inhibitor NU7441. Graft rejection, cytokine production, immune cell infiltration, and donor-specific antibody (DSA) formation were analyzed.

Results:

DNA-PKcs inhibition significantly reduced necrosis and extended graft survival compared to controls (mean survival 14 days vs 9 days respectively). Inhibition reduced the production of the cytokines Interleukin (IL)2, IL4, IL6, IL10, TNFα, and IFNγ and the infiltration of CD3+ lymphocytes into grafts. Furthermore, DNA-PKcs inhibition reduced the number of CD19+ B cells and CD19+ CD138+ plasma cells coinciding with a significant reduction in DSAs. At a molecular level, we determined that the immunosuppressive effects of DNA-PKcs inhibition were mediated, in part, via inhibition of NFκB signaling through reduced expression of the p65 subunit.

Conclusion:

Our data confirm that DNA-PKcs contributes to allogeneic graft rejection and highlight a novel immunological function for DNA-PKcs in the regulation of NFκB and concomitant cytokine production.
Cell death stage dictates the immunogenicity of ferroptotic cancer cells
Authors: Efimova Iuliia , Catanzaro Elena , Demuynck Robin , Van der Meeren Louis , Coppieters Frauke , Bachert Claus , Skirtach Andre , Krysko Olga , Krysko Dmitri
Published: February 2023

Immunotherapies hold great promise for the future treatment of cancer. It is also becoming clear that type of cancer cell death determines the antitumor immune response and, therefore, contributes to the efficiency of anti-cancer therapy and long-term survival of patients. Since tumors often develop resistance to apoptosis and necroptosis, triggering these processes is not always the optimal strategy. That is why it is crucial to select the proper cell death type potentially leading to tumor eradication. Ferroptosis, an iron-dependent form of cell death leading to lipid peroxidation in cells, is currently actively studied but whether ferroptotic cancer cells are immunogenic is unknown. We demonstrate for the first time that ferroptosis is immunogenic in vitro and in vivo. Early, but not late, ferroptotic cells promote the phenotypic maturation of BMDCs and elicit a vaccination-like effect in immune-competent mice but not in Rag-2−/− mice, suggesting that the mechanism of immunogenicity is very tightly regulated by the adaptive immune system and is time dependent. Also, ATP and HMGB1, the best-characterized damage-associated molecular patterns involved in immunogenic cell death, have proven to be passively released along the timeline of ferroptosis and act as immunogenic signal associated with the immunogenicity of early ferroptotic cancer cells. The work on approaches to increase the immunogenicity of late ferroptotic cells is underway, which will open up novel ferroptotic cell-based experimental strategies for cancer immunotherapy. In conclusion, these results indicate that induction of ferroptosis in cancer might be another option to overcome cell death resistance and enhance the efficacy of anti-cancer therapy.
Characterization, biodistribution and functional Studies of immune-therapeutic Exosomes: implications for inflammatory lung diseases and COVID 19
Authors: Elashiry Mahmoud , Elsayed Ranya , Cutler Christopher
Published: February 2023

Dendritic cell (DC)-derived exosomes (DC EXO), natural nanoparticles of endosomal origin, are under intense scrutiny in clinical trials for various inflammatory diseases. DC EXO are well-tolerated by the host and can be custom-tailored for immunomodulatory functions. Previously we documented the efficacy of immunoregulatory DCs EXO (regDCs EXO) as immunotherapy for inflammatory bone disease in-vivo. However, the on- and off-target effects of these therapeutic regDC EXO and how target signaling in acceptor cells is activated is unclear. In the present report, therapeutic regDC EXO were analyzed by high throughput proteomics, with non-therapeutic EXO from immature DCs and mature DCs as controls, to identify shared and distinct proteins and potential off-target proteins. The predominant expression in regDC EXO of immunoregulatory proteins as well as proteins involved in trafficking from the circulation to peripheral tissues, prompted us to investigate how these EXO are biodistributed to major organs after intravenous injection. Live animal imaging showed preferential accumulation of regDCs EXO in the lungs. In addition, TGFB1 in regDCs EXO sustained downstream regulatory signaling in acceptor DCs. Blocking experiments suggested that sustaining TGFB1 signaling require initial interaction of regDCs EXO with TGFB1R followed by internalization of regDCs EXO with TGFB1-TGFB1R complex. Finally, these regDCs EXO that contain immunoregulatory cargo and showed biodistribution to lungs could downregulate the main SARS-CoV-2 target receptor, ACE2 on recipient lung parenchymal cells via TGFB1 in-vitro. In conclusion, these results may have important immunotherapeutic implications for lung inflammatory disorders and COVID19.
Designing a bispecific antibody targeting IL-6Rα and NKG2A receptors via in silico affinity maturation as a strategy to combat severe COVID-19
Authors: Agarwal Manusmriti , Saini Samvedna , Kaushik Spandan , Prasad Aakanksha , Kumar Yatender
Published: February 2023

High mortality in COVID-19 patients has been encountered with acute respiratory distress syndrome (ARDS). Cytokine storm has been observed as a reason for this severity in critical cases showcasing high levels of circulating cytokines along with a pronounced decrease in cytotoxic T lymphocytes and Natural Killer Cell populations. The Interleukin-6 amplifier, the activation of IL-6-signal transducer and activator of transcription 3 (STAT3) and NF-κB signaling in non-immune cells has been widely discussed as an initiator of this cytokine release syndrome. The anti-IL-6α receptor antibody tocilizumab has shown success in recovery when administered to critical COVID-19 patients. Additionally, increased release of IL-6 is shown to elicit immune cell disproportionation by upregulating inhibitory NKG2A receptors on NK cell surface, causing blunting of the host’s antiviral response. This study proposes a bispecific immuno-modulatory antibody with one Fab blocking IL-6α-receptor and the other blocking NKG2A receptor to counter hyper-inflammatory cascades and recruit NK cells to the site of viral infection, respectively. Computational site directed mutagenesis was performed to design a library of increased binding affinity mutants of tocilizumab and NKG2A inhibitory monalizumab, followed by docking studies, molecular dynamics simulations, MM-PBSA analysis to study stability of the bispecific assembly in physiologically relevant conditions. This approach utilizing immunomodulation for simultaneous recruitment of antiviral immunity and regulation of hyper-inflammatory host responses may present a potent strategy for combatting critical stage COVID-19 and thereby help in curbing COVID-19 related mortality.
The Role of Aire in the selection of Regulatory T cells in Type 1 Diabetes
Authors: Bridge Jennifer Anne , Balolong Jared , Belk Julia , Dong Shen , Cramer Nathan , Mowery Cody , Crawford Frances , Ye Jimmy , Satpathy Ansuman , Kappler John W , Bluestone Jeffrey A , Anderson Mark S
Published: February 2023

In autoimmune diseases like T1D, it is proposed that failure in central tolerance mechanisms towards pancreatic islet self-antigens leads to the escape of pathogenic clones and loss of critical immunosuppressive antigen-specific Tregs. The importance of Aire in maintaining central tolerance against self-reactive antigens is evidenced by the multiorgan autoimmune disease seen in individuals with mutation the Aire gene. When Aire-expression is specifically ablated in mouse mTECs, there is a dramatic increase in the number of self-reactive T cells that escape negative selection, in addition, a dramatic loss of Tregs. Utilizing novel high-throughput platforms and single-cell DNA barcoding technology has allowed us to assess several thousand CD4+ T-cell clones. The use of FoxP3-GFP reporter mice permits us to identify individual FoxP3+ Tregs that have undergone Aire-dependent selection in the thymus and compare the TCR repertoire to mice that have had Aire specifically ablated from the mTECs. Identification of unique clones and gene signatures within our large datasets has allowed us to start to qualitatively assess the functional role of the identified TCRs within the model of T1D. GLIPH2 clustering using our extensive insulin-specific TCRs have identified a number of motifs shared within our TCR dataset, as well as a unique V-gene usage that may predict TCR affinity. Our proposed study aims to expand our knowledge on TCR repertoire of Aire-dependent FoxP3+ Tregs and the antigen-specificity in T1D, improving our understanding of the respective function of thymic-derived antigen-specific Treg suppression in peripheral tissues associated with autoimmune T1D.
Dysregulated follicular regulatory T cells and antibody responses exacerbate CNS autoimmunity
Authors: Leavenworth Jianmei Wu , Luo Lin , Hu Xianzhen , Dixon Michael L. , Pope Brandon J. , Leavenworth Jonathan D. , Raman Chander , Meador William R.
Published: February 2023

Follicular regulatory T (TFR) cells are essential for the regulation of germinal center (GC) response and humoral self-tolerance. Dysregulated follicular helper T (TFH) cell-GC-antibody (Ab) response secondary to dysfunctional TFR cells is the root of an array of autoimmune disorders. The contribution of TFR cells to the pathogenesis of multiple sclerosis (MS) and murine experimental autoimmune encephalomyelitis (EAE) remains largely unclear. Here we show that mice with a FoxP3-specific ablation of the transcription factor Blimp1 developed severe EAE and failed to recover compared to control mice, reflecting conversion of unstable regulatory T cells (Tregs) into IL-17A/GM-CSF-producing effector T-cells associated with increased TFH-Ab responses, more IgE deposition in the central nervous system (CNS) and inability to regulate CNS CD11b+ myeloid cells. Notably, serum IgE titers were positively correlated with EAE scores, and culture of CNS CD11b+ cells with sera from these EAE mice enhanced their activation, while transfer of Blimp1-deficient TFR cells promoted Ab production, activation of CNS CD11b+ cells and EAE. Moreover, compared to healthy controls, a small group of MS patients had fewer circulating Tregs and TFR cells that expressed reduced levels of Blimp1, associated with elevated serum IgE. These findings suggest that Blimp1 is essential for the maintenance of TFR cells and Ab responses in EAE, and that dysregulated TFR cells and Ab responses promote CNS autoimmunity.
T cell dysfunction in Activated PI3K δ Syndrome explained by cell intrinsic and extrinsic components
Authors: Bier Julia , Lau Anthony , Brink Robert Adrian , Ruiz-Diaz Stephanie , Lenthall Helen , Tangye Stuart , Deenick Elissa
Published: February 2023

Activated PI3K Delta Syndrome (APDS) is a rare condition caused by heterozygous gain of function mutations in PIK3CD, which encodes the leukocyte-restricted p110δ catalytic subunit of phosphoinositol 3-kinase (PI3K). PI3K is activated downstream of many T cell receptors and has been implicated in the control of CD4+ T cell helper differentiation, activation and proliferation. APDS patients have increased PI3K activity that leads to several immune manifestations including lymphoproliferation, Th2-related pathologies and impaired Ab responses.

Analysis of a CRISPR/Cas9 mouse model (Pik3cdE1020K) showed a decreased frequency of naïve and a concurrent increase in memory T cells and Tfh cells, indicating T cell hyperactivation, as well as changes in cytokine expression and T cell help. Our studies investigated CD4+ T cell intrinsic and extrinsic alterations contributing to the dysregulation of the immune response.

We identified cell intrinsic features resulting in increased IL-4 and IL-5, explaining the Th2 mediated disease in these patients. Additionally, cell intrinsic changes in Tfh generation and function were shown in the in vivo mouse model, contributing to an impaired ability to sustain GCs and alter isotype switching. Furthermore, additional studies have shown that the overactive T cells result from both intrinsic and extrinsic factors, with contributions from both dysregulated T cells and B cells exerting effects on T cell activation.

Together, these studies reveals an important role of PI3K in modulating T cell differentiation and function through both T cell-intrinsic and extrinsic mechanisms. This provides new insights into the APDS pathophysiology with potential to improve the clinical management of these patients.
γHV68 infection exacerbates arthritis and requires age-associated B cells
Authors: Mouat Isobel , Morse Zachary J. , Shanina Iryna , Brown Kelly L , Horwitz Marc S
Published: February 2023

Epstein-Barr virus (EBV) infection is associated with rheumatoid arthritis (RA), though the mechanism of contribution remains unknown and there does not exist a sufficient in vivo model to examine the relationship. Here, we utilize and expand in vivo models of EBV and RA to examine mechanisms of immune contribution. We find that infection with latent gamma-herpesvirus 68 (γHV68), a murine analogue of EBV, leads to an enhanced clinical and immunological course of collagen-induced arthritis (CIA). γHV68-infected mice display earlier and more severe CIA clinical symptoms and a Th1-skewed immune profile, compared to uninfected CIA mice. Using a latency-free strain of γHV68 we demonstrate that CIA exacerbation is not due to innate immune stimulation during acute infection but, rather, is dependent upon viral latency. Age-associated B cells (ABCs) are increased in RA patients and during viral infection, though if they act as mediators between the infection and disease remains unknown. We find that ABCs (CD19+CD11c+Tbet+) in γHV68-infected CIA mice are increased and display a proinflammatory phenotype compared to uninfected CIA. Using ABC knockout mice, we demonstrate that ABCs are critical for γHV68-enhancement of CIA, though are dispensable in uninfected CIA. This project establishes that latent γHV68 infection enhances CIA and is a viable model for examining mechanisms of EBV’s contribution to RA. Additionally, we demonstrate that ABCs mediate the viral enhancement of disease.
Inherited human PD-1 deficiency underlies tuberculosis and autoimmunity
Authors: Ogishi Masato , Yang Rui , Aytekin Caner , Boisson-Dupuis Stéphanie , Casanova Jean-Laurent
Published: February 2023

The pathogenesis of adverse reactions to PD-1 blockade immunotherapy, including tuberculosis (TB) and autoimmunity, remains poorly characterized. Here, we study a patient with inherited PD-1 deficiency who suffered from TB and died of pulmonary autoimmunity. The patient’s leukocytes displayed no PD-1 expression and did not respond to PD-1-mediated suppression. Similar to various inborn errors of immunity impairing interferon (IFN)-γ production and underlying TB, the patient’s lymphocytes poorly produced IFN-γ in response to mycobacterial stimuli owing to both the depletion of multiple IFN-γ-producing T and NK lymphocyte subsets, including Vδ2+ γδ T, MAIT, and CD56bright NK lymphocytes, and dysfunction of residual T lymphocytes. Another set of inborn errors, including STAT3 gain-of-function (GOF) mutations, trigger an expansion of CD4−CD8− double-negative (DN) αβ T cells and underlie lymphoproliferative autoimmunity. Remarkably, the patient displayed hepatosplenomegaly and expansion of total, activated, and RORγT+ DN αβ T cells, as observed in PD-1-deficient mice and cancer patients with PD-1 blockade. Mechanistically, the patient’s myeloid cells produced excessive amounts of STAT3-activating cytokines IL-6 and IL-23 upon stimulation with lipopolysaccharide. Overall, inherited human PD-1 deficiency underlies TB through undermining IFN-γ production by T and NK lymphocytes and lymphoproliferative autoimmunity through over-activating STAT3.
RGC-32 Facilitates Reactive Astrocytosis in Experimental Autoimmune Encephalomyelitis (EAE) by Maintaining a Mature Phenotype
Authors: Rus Horea G , Tatomir Alexandru , Beltrand Austin , Cudrici Cornelia , Courneya Jean Paul , Badea Tudor Constantin , Rus Violeta
Published: February 2023

We previously showed that lack of Response Gene to Complement 32 (RGC-32) decreases the severity of clinical scores of mice with EAE and renders spinal cord astrocytes a progenitor phenotype reminiscent of radial glia. In this study, we used RNA-sequencing to investigate whether RGC-32 deferentially regulates biological processes related to astrocyte maturation and activation. We purified neonatal astrocytes from WT and RGC-32 knock-out (KO) mice, stimulated them with TGF-β and then performed RNA-seq (Genewiz, S. Plainfield, NJ). Spinal cords from WT and RGC-32 KO mice with EAE were harvested at day 0 and at the peak of disease (day 14) and subjected to immunohistochemistry for astrocytes lineage marker GFAP, as well as radial glial marker HOPX, neural stem cell marker CD133, and proliferation marker Ki-67. Pathway enrichment analysis revealed that the genes deferentially expressed in WT but not RGC-32 KO astrocytes belonged to GO categories related to brain development, axonal guidance, cell migration and extracellular matrix (ECM) regulation. Real-Time PCR confirmed that Ephrin A7, bone morphogenetic protein 1 and polycystin 1, other genes related to brain development and ECM were significantly up-regulated in WT astrocytes after TGF-β stimulation. We found that spinal cords of RGC-32 KO mice displayed a higher number of HOPX+ cells at day 14. Similarly, the number of CD133+ cells with a radial glial morphology was higher in RGC-32 KO mice. In addition, we found that RGC-32 KO astrocytes had a higher proliferative index at the peak of EAE, as shown by their increased expression of Ki-67. These data suggest that RGC-32 is an important regulator of astrocyte maturation and reactive astrocyte formation during EAE
A survey of current research trends and potential future directions in reproductive immunology clinical trials
Authors: Rossavik Eevar B , Rossavik Carolin Haeusler , Evans Michael Blake
Published: February 2023

Reproductive immunology is a relatively recent field with a wide spectrum of research opportunities. Within the reproductive system, immune system dysfunction has been suggested to contribute to a variety of conditions - such as endometriosis, infertility, recurrent miscarriage, preeclampsia, and preterm delivery. Although there is preliminary research supporting these possible connections, further studies are necessary. A survey was conducted of current clinical trials to show the current state of reproductive immunology research to assess needs in less researched conditions.

The survey was performed by accessing the ClinicalTrials.gov registry. “Reproductive immunology” was entered into the database, and the following filters were applied: “recruiting,” “active, not recruiting,” and “enrolling by invitation.” 153 studies were obtained, and the associated conditions were counted for how many times they were listed. Similar conditions were combined into one category – e.g. different types of leukemias were categorized as “Leukemia.”

Although the rate of new female breast cancer cases is significantly higher than the rate of new leukemias in the general population, this survey suggests reproductive immunology research is more focused on leukemia than breast cancer. Male and female reproductive/breast cancer/endometrial cancer/endometriosis (6.17%), autoimmune diseases (4.43%), and pregnancy/contraception (0.58%) are less frequently listed compared to leukemia (45.28%) and hematologic diseases (15.80%) in the clinical trials registry as associated conditions. This information can potentially guide future research towards less studied conditions in the relatively new field of reproductive immunology.
DOCK8-expressing Tfh cells cause SLE: autoimmunity via repeated TCR stimulation by antigen to levels that surpass system’s self-organized criticality
Authors: Shiozawa Shunichi , Tsumiyama Ken , Miyazaki Yumi , Uto Kenichi , Sakurai Keiichi , Nakashima Toshie , Matsuyama Hiroko , Doi Ai , Tarui Miho , Izumikawa Manabu , Kimura Mai , Satonaka Chisato , Fujita Yoko , Horiuchi Takahiko , Matsubara Tsukasa , Oribe Motohiro , Yamane Takashi , Kagawa Hidetoshi , Li Quan-Zhen , Mizuno Keiko , Mukai Yohei , Murakami Kazuhiro , Enya Takuji , Tsukimoto Shota , Hakata Yoshiyuki , Miyazawa Masaaki , Shiozawa Kazuko
Published: February 2023

INTRODUCTION: The cause of autoimmunity remains unclear. Candidate pathogens including autoantigens all failed to induce, exist in, or heal upon their removal the prototypical autoimmune disease, systemic lupus erythematosus (SLE). We here investigated, instead of antigen, the immune response of host that recognized antigen.

METHODS:

We test the integrity of host’s immune response by stimulating TCR maximally to levels that surpass the limit of host’s steady-state immune response, self-organized criticality, by an antigen.

RESULTS:

After repeated stimulation by any immunogenic antigen, SLE was induced in mice normally not prone to autoimmune disease, wherein a novel T follicular helper (Tfh) cell type expressing the guanine nucleotide exchange factor DOCK8 on the cell surface was newly generated via resuscitation from antigen-induced anergy. DOCK8+ Tfh cells passed through TCR revision at the periphery, and induced a variety of autoantibodies and the lesion characteristic of SLE. They existed in splenic red pulp and in the circulation of active patients with SLE, which subsequently declined after conventional therapy. Lupus lesions of mice and the classical (NZBxNZW) F1 model mice were mostly cured by anti-DOCK8 antibody.

CONCLUSION:

Thus, disruption of host’s steady-state immune response by repeated TCR stimulation by immunogenic pathogen, either exogenous or endogenous, results in generation of DOCK8-expressing Tfh cells that cause autoantibodies and SLE.
GPR65, a critical regulator of Th17 pathogenicity, is regulated by the CREB/CRTC2 pathway
Authors: Hernandez Jeniffer B , Cave Si , Yang Qiyuan , Decker Carl , Swanson Jack , Wiater Ezra , Zheng Ye , Montminy Marc
Published: February 2023

GPR65 has been shown to be a critical regulator of Th17 pathogenicity. Loss of GPR65 in mice results in a decrease in Th17 cells and reduced susceptibility to a mouse model of multiple sclerosis. The CREB/CRTC2 pathway has emerged as an important regulator of immune function. We have previously shown that the CREB/CRTC2 pathway modulates autoimmune disease by promoting differentiation of Th17 cells. In this study we performed RNA-seq to identify Th17 genes regulated by the CREB/CRTC2 pathway. Our RNA-seq analysis led us to uncover the first mechanism of regulation of the orphan receptor GPR65 by the CREB/CRTC2 pathway. We show that GPR65 is a target of the CREB/CRTC2 pathway through expression studies and chromatin immunoprecipitation. In addition, we show that targeting of GPR65 with small molecules alters the expression of IL-17A. Understanding the regulation of GPR65 will be crucial in developing small molecules to treat patients with Th17-mediated diseases.
Emerging role of the mitochondrial branched-chain aminotransferase (BCATm) as an immunosuppressive enzyme during CD4+ T cell activation
Authors: Ananieva-Stoyanova Elitsa A , Adam Christie , Betar Rebekah , Martin Alexander , Hutson Susan , Boyer Michael
Published: February 2023

The mitochondrial branched-chain aminotransferase (BCATm) catalyzes the transamination of leucine, an amino acid that is essential for the upregulation of complex 1 of the mammalian target of rapamycin (mTORC1) during T cell activation. Because BCATm is responsible for the mitochondrial degradation of leucine, we hypothesized that BCATm limits leucine availability for mTORC1 signaling and subsequently suppresses T cell activation. To test the hypothesis, we isolated CD4+T cells from the global BCATmKO mouse and measured the rates of leucine transamination/oxidation, the activity of the mTORC1 downstream targets S6 and 4EBP-1, and the release of IFNγ after 24–72h of T cell activation. To elucidate the role of BCATm in T cells in vivo, we used the Cre-LoxP strategy to create a T cell specific deletion of BCATm in C57BL/6 mice. We completed the initial characterization of the mice, which included a validation of absence of BCATm in T cells by using qRT-PCR and Western blotting. Results demonstrated that BCATm-deficient T cells experienced significant reductions in the transamination and oxidation of leucine, which led to increased intracellular leucine concentrations. This correlated with an increased phosphorylation of S6, but not 4EBP-1, suggesting that BCATm regulates mTORC1 signaling in S6 dependent manner. The T cells released more IFNγ in the absence of BCATm, which was highest after 72h of T cell activation. Thus, BCATm appears to play an immunosuppressive role by reducing CD4+ T cell activation. The newly designed T cell conditional BCATmKO mice, which do not express BCATm in CD4+ and CD8+ T cells will be engaged in preclinical studies aiming at deciphering the therapeutic potential of BCATm in T cell-driven immunological responses.
Exploring Mechanisms of Human PD-1 Gene Regulation in T Follicular Helper Cells
Authors: Powell Michael Duane , Neeld Dennis Kyle , Rose James , Scharer Christopher , Boss Jeremy M
Published: February 2023

Programed Cell death (PD-1), encoded by Pdcd1, is an immune inhibitory receptor expressed on the surface of lymphocytes. Surface PD-1 expression is amongst its highest in a subset of CD4+ T cells called T follicular helper cells (TFH). TFH cells are pivotal for optimal germinal center formation and subsequent generation of pathogen- and vaccine-induced antibodies by B cells. Despite the clear connection to human health, the majority of previous work has centered on elucidating the mechanisms surrounding murine Pdcd1 regulation. Surprisingly, there is almost nothing known about how human Pdcd1 (hPdcd1) is regulated! Here we seek to close this gap in knowledge by identifying the cis-, trans-, and epigenetic pathways that regulate hPdcd1 in TFH cells. To this end, human TFH cells were sourced from both discarded tonsil tissue as well as from a recently described model to generate ex vivo human TFH cells through treatment with the cytokines Activin A, IL-12 and TGFβ. RNA-sequencing demonstrated that ex vivo TFH cells exhibit characteristics of bona-fide tonsillar TFH cells, including increased expression of PD-1. Furthermore, ATAC-seq and H3K27Ac Hi-ChIP experiments revealed the presence of 7 conserved accessible regions within the hPdcd1 locus that exhibit unique looping characteristics and potential enhancer properties. Functionality of each regulatory element was further defined through promoter reporter assays. Additionally, PAGE-RANK analysis elucidated transcription factors that may play a role in the induction of hPdcd1 expression. Collectively, these findings begin to unearth the mechanisms that control hPdcd1, informing the design of future therapeutics aimed at precisely manipulating human PD-1 expression.
A genome-wide CRISPR screen reveals a role for the BRD9-containing non-canonical BAF complex in Foxp3 expression and regulatory T cell function
Authors: Zheng Ye , Loo Chin-San , Gatchalian Jovylyn , Hargreaves Diana
Published: February 2023

Regulatory T cells (Tregs) play a pivotal role in suppressing auto-reactive T cells and maintaining immune homeostasis. Treg development and function are dependent on the transcription factor Foxp3. Although a large body of work has been devoted to dissecting the molecular mechanisms regulating Foxp3 expression, a systematic, genome-wide approach has not been reported. Here we performed a genome-wide CRISPR/Cas9 knockout screen to identify the regulators of Foxp3 in mouse primary natural Tregs. The screen results not only confirmed a number of known Foxp3 regulators, but also revealed many novel factors that control Foxp3 expression. Gene ontology analysis showed that Foxp3 regulators are highly enriched in genes encoding SWI/SNF and SAGA complex subunits. Among the three SWI/SNF-related complexes, the non-canonical or ncBAF (also called GBAF or BRD9-containing BAF) complex promoted the expression of Foxp3, whereas the PBAF complex repressed its expression. Gene ablation of BRD9 led to reduced Foxp3 expression and compromised Treg function in inflammatory disease and anti-tumor immunity. BRD9 co-localized with Foxp3 in genome-wide binding studies, including at the CNS0 and CNS2 enhancers at the Foxp3 locus. Functional genomics revealed that BRD9 is required for Foxp3 binding and expression of a subset of Foxp3 target genes. Thus, we provide an unbiased analysis of genes and networks regulating Foxp3, and reveal the ncBAF complex as a novel target that could be exploited to manipulate Treg function.
Factor VIII-Fc activates natural killer cells via Fc-mediated interactions with CD16
Authors: Lagasse H.A. Daniel , Hopkins Louis B. , Jankowski Wojciech , Sauna Zuben E. , Golding Basil
Published: February 2023

FactorVIII Fc-fusion (rFVIIIFc) is an enhanced half-life antihemophilic factor usedin replacement therapy of hemophilia A. Fc-fusion is a successful protein bio-engineering platform technology. In addition to enhancement of plasmahalf-life via neonatal Fc receptor (FcRn) binding, other Fc-mediatedinteractions, including engagement with Fc gamma receptors (FcγR), may haveimmunological consequences. Previouslywe and others demonstrated rFVIIIFc interactions with activatingFcγRIIIA/CD16. Here, we investigated ifrFVIIIFc activates natural killer (NK) cells via CD16. We demonstrated rFVIIIFc signaling via CD16independent of Von Willebrand Factor (VWF):FVIII complex formation. We established that rFVIIIFc potentlyactivated NK cells in a CD16-dependent fashion resulting in IFNγ secretion andcytolytic perforin and granzyme B release. We also demonstrated an association between rFVIIIFc-mediated NK cellIFNγ secretion levels and the high affinity (158V) CD16 genotype. Furthermore, we show that rFVIIIFc-activatedCD16+ NK cells were able to lyse a B-cell clone bearing ananti-FVIII BCR (BO2C11) in an antibody-dependent cellular cytotoxicity (ADCC)assay. These findings provide anunderlying molecular mechanism that may help explain clinical case reports andretrospective studies that suggest rFVIIIFc may be more effective in tolerizinghemophilia A patients with anti-FVIII inhibitory antibodies compared to FVIIInot linked to Fc. Our findings suggest apotential use of Fc-fusion proteins acting via NK cells to target B-cells, inthe management of unwanted immune responses directed against immunogenicself-antigens or therapeutic protein products.
Curricular Framing of the Undergraduate Immunology Classroom
Authors: Bruns Heather A. , Taylor Rebekah , Wisenden Brian , Kleinschmit Adam , Liepkalns Justine , Lal Archana , Vanniasinkam Thiru , Mixter Philip F , Paustian Timothy , Sletten Sarah , Sparks-Thissen Rebecca , Condry Danielle , Pritchard Rachel E , Elliott Samantha L , Justement Louis B , Pandey Sumali
Published: February 2023

Immunology education has historically been delivered at the graduate level. Calls for reform in biology education and increased workforce demand for individuals trained in immunology have spurred efforts to examine immunology education at the undergraduate level. The Undergraduate Immunology Education Consortium, a newly formed international group of immunology educators representing diverse institutions, is contributing to this effort through the development of curricular resources and student learning outcomes. We have identified a set of fundamental concepts that are critical to the understanding of immunology for undergraduate students and align with the 2011 AAAS Vision and Change Report. To identify consensus among immunology educators this group distributed an initial online survey to immunology educators to evaluate the identified fundamental concepts. Following survey feedback, the concepts were revised, submitted for further evaluation by immunology educators in focus groups, and then distributed in a final survey. Analyses of the data from the initial survey demonstrated that the respondent pool represented a varied and diverse range of expertise. Although teaching experience and current role in teaching influenced the proportion of time allotted to some immunology subtopics, topics of greatest emphasis included innate immunity, adaptive immunity, host-pathogen interactions, and molecular mechanisms. An understanding of current curricular practices by immunology educators will further aid in the refinement of the fundamental concepts, their interdisciplinary use in a diverse range of undergraduate science curricula, and future collection of assessment data to analyze effective best practices.
Learning outcomes with flipped and traditional classrooms in medical immunology.
Authors: Jafferjee Mufaddal , Abdullah Ikram , Brown Teresa , Geis Abby L
Published: February 2023

Education literature reports the benefits of flipped classrooms (FC), but medical educators still typically rely on traditional didactic lectures (TL) as a primary teaching modality. In fact, when given a choice of delivery modalities, learners often prefer to engage in the easier and comfortable route to knowledge, including listening to lectures. However, learning through passive techniques is less effective because it fails to facilitate application and recall of concepts. High order learning and retention is significantly improved when the learning process is challenging and thought-provoking.

We used immunology within a medical school curriculum to compare the effectiveness of and student preferences for two distinct teaching modalities: FC and TL. Data was collected from 170 first year medical students at our institution. In the FC modality, prerecorded lectures were provided to students prior to class. In class, students completed a quiz with low-level questions, the quiz was reviewed, and the remaining class time was spent doing high-level application problems. For the TL, students were provided with the low-level quiz and high-level application problems to do at their own discretion. No aspect of the learning activities was required or graded. Our data show that students who performed poorly on exam items linked to the traditional lecture benefitted disproportionately from the flipped classroom modality, though there was no difference in learning outcomes between the two modalities overall. Finally, students who provided feedback about their learning preferences overwhelmingly preferred traditional lectures and denied learning gains from the flipped modality.
Enhancing engagement of graduate students in class via use of an interactive teaching platform improves learning and long-term retention
Authors: Alard Pascale , Kosiewicz Michele M
Published: February 2023

Engagement of students in the classroom has been shown to improve understanding and retention of material. However, the most common teaching format in Graduate, Medical or Dental School is lecturing via powerpoint, which minimally engages students, an issue amplified during the pandemic due to remote teaching. Assessing previous knowledge, predicting/retrieving information, and using academic games are teaching strategies that facilitate more effective learning. We used TopHat, an interactive teaching platform allowing integration of these strategies, to teach a graduate Immunology course. Two cohorts of first-year graduate students were provided with the opportunity to anonymously discuss topics learned during the current or previous sessions, hear different perspectives, and correct what they had not understood during face-to-face or virtual sessions. Only some instructors in the course used the platform. Students who perform overall at a lower level obtained higher exam scores with instructors who used the platform. However, all students received higher scores on comprehensive final exam questions provided by instructors who used the platform, suggesting that engaging graduate students in class helped with long-term retention. Lastly, a student survey about the utility of the platform indicated that a large majority of students felt more engaged, and believed that they were better able to understand the material and retrieve the information both short-term and long-term. This study suggests that using an interactive teaching platform with graduate students resulted in better engagement, improved learning and long-term material retention, and could be easily implemented at any level of Immunology education.
Generation and implications of oral resident memory T cells
Authors: Stolley J. Michael , Masopust David
Published: February 2023

CD8+ tissue resident memory T cells (TRM) represent the predominant memory T cell population in many nonlymphoid tissues (NLT) where they are major contributors to localized immunosurveillance. TRM accelerate protection against reinfection, may be associated with tumor control, and may also facilitate the persistence of certain allergic and autoimmune diseases. While extensively studied in other mucosal sites, there is presently a fundamental void in our understanding of the ontogeny, function, and therapeutic implications of oral-mucosal TRM. Given their well-documented and critical functions in mediating barrier immunosurveillance in other NLT, oral TRM are likely to play a major role in antiviral immunity and oral immune homeostasis. TRM may also perpetuate chronic immune responses observed in periodontal disease and oral lichen planus. However, addressing their role in these clinically relevant settings has been mired by a lack of animal models for generating sufficient oral TRM to manipulate and study. Here we introduce a novel oral ‘prime-pull’ strategy for generating large quantities of tractable TRM of a defined antigen specificity within the oral mucosa. Leveraging this approach, we demonstrate that oral TRM 1) are abundant within mouse gingiva, 2) are intimately associated with salivary ducts of the tongue and buccal mucosa, and 3) can be locally reactivated to potentiate oral immunity. Ongoing work will further define the tissue-specific functions of oral TRM and describe new mechanisms by which oral immunity can be attenuated or augmented to achieve the desired clinical outcome.
Tissue resident memory T cell responses to RSV infection are reduced in infant mice compared to adults
Authors: Lipp Madeline Anne , Eichinger Katherine M , Kosanovich Jessica L , Empey Kerry M
Published: February 2023

Tissue resident memory T cells (TRMs) are an important yet incompletely understood cell population that enhance the clearance of respiratory syncytial virus (RSV) infection in both humans and animal models of disease. Limited data in an influenza model of disease suggests the establishment of TRMs in infants is impaired, but the establishment of TRMs during infant RSV infection is unknown. The purpose of this study was to characterize the quantity and function of infant TRM responses to RSV infection. To address this, BALB/c mice were infected with RSV as 10-day old mice or as 6–8-week-old adults. Peripheral T cells were then depleted using FTY720 at the time of an RSV re-challenge 4–5 weeks later. TRM response was evaluated using flow cytometry; viral titers and histological staining were used to assess disease outcomes. Mice infected as infants had significantly lower total numbers of CD4+ and CD8+ TRMs compared to those infected as adults. Furthermore, TRMs established in infancy showed a Th2-biased response, with increased production of IL-4 and IL-10 compared to adults, while adult TRMs produced more granzyme B from CD8+ cells and increased IFNγ from both CD4+ and CD8+ T cells. Mice infected in infancy also showed increased %PAS positive airways compared to those infected initially as adults, suggesting more severe RSV disease. In conclusion, infants have reduced ability to establish TRMs following RSV infection and their TRM population exhibits Th2 skewing. Further studies are needed to elucidate the mechanisms that prevent effective establishment of TRMs following infant pulmonary infections.
Blocking of CA-MSC-induced desmoplasia reprograms the tumor immune microenvironment and enhances cancer immunotherapy
Authors: Cascio Sandra , Chandler Chelsea , Zhang Linan , Sinno Sarah , Bruno Tullia , Osmanbeyoglu Hatice , Vlad Anda , Coffman Lan , Buckanovich Ronald
Published: February 2023

Cancer associated MSCs (CA-MSCs) are multipotent stromal cells that generate tumor desmoplasia and promote tumor growth. Here, we investigated the impact of CA-MSCs on the ovarian tumor immune microenvironment. In vitro and in vivo analyses confirmed that CA-MSCs secrete numerous cytokines and chemokines, including Ccl2, Cx3cl1 and Tgfb1, that are known to induce the recruitment of monocytes into the tumor and promote macrophage immunosuppressive phenotype. Using a syngeneic and immune responsive murine ovarian tumor model, we found that CA-MSCs restrict the intra-tumoral trafficking of CD8+ T cells and inhibit the response to immune checkpoint inhibitor therapy. Similarly, in patient samples the presence of CA-MSC is inversely correlated with CD8+ T cell infiltration into tumor islets. Single cell RNAseq analyses revealed that tumoral monocytes and macrophages of a-PD-L1 treated-CA-MSCs enriched tumors express high levels of the protein transforming growth factor beta induced (Tgfbi). In ovarian cancer patient samples TGFBI is primarily expressed in the desmoplastic stroma and correlated with poor prognosis. Importantly, we found that hedgehog signaling inhibitor (HHi) therapy reversed CA-MSC driven tumor desmoplasia, reduced the number of tumor-associated myeloid cells and decreased their expression of Tgfbi. These events restored the infiltration and activation of CD8+ T cells into the tumor islets and response to a-PDL1 therapy. We are currently evaluating the role of Tgfbi in the regulation of innate and adaptive immune response. Our findings demonstrated CA-MSCs critically regulate anti-tumor immunity and support HHi as an adjunct to anti-PDL1 therapy to increase repose to immune therapy in ovarian cancer.
M2 macrophages, B cells, and PD-L1 immune checkpoint protein exhibit sexual dimorphism in the outcomes of non-muscle invasive bladder cancer
Authors: Chenard Stephen , Jackson Chelsea , Vidotto Thiago , Chen Lina , Hardy Celine , Jamaspishvilli Tamara , Berman David , Siemens D. Robert , Koti Madhuri
Published: February 2023

Non-muscle-invasive bladder cancer (NMIBC) is more than three times as common in men as it is in women. However, female patients with NMIBC do not respond as well to immunotherapeutic treatments and experience worse clinical outcomes than their male counterparts. The underlying causes of these discrepancies have yet to be fully elucidated.

We hypothesized that sexual dimorphism in the tumor immune microenvironment (TIME) may contribute to the inferior clinical outcomes observed in female patients with NMIBC. To test this hypothesis, we investigated immune-associated gene expression in tumors from male (n=357) and female (n=103) patients. High-grade tumors from female patients exhibited significantly increased expression of B cell-associated genes CD40 and CXCL13, and the immune checkpoint genes CTLA4, PDCD1, LAG3, and ICOS.

Based on these differences, we utilized multiplexed immunofluorescence to evaluate the density and spatial distribution of 12 immune cell markers (CD79a, CD3, CD8, FoxP3, Ki67, CD103, CD163, GATA3, CK5, IDO1, PD-1, and PD-L1) in tumors from an independent cohort of 332 patients with NMIBC (n=259 males and n=73 females). Tumors from female patients showed significantly higher infiltration of PD-L1+ cells and CD163+ M2-like macrophages compared to tumors from male patients. Notably, increased abundance of CD163+ macrophages and CD79a+ B cells were independently associated with decreased recurrence-free survival.

This study has the potential to inform the rational utilization of immunomodulatory treatments for NMIBC based on the TIME of both male and female patients. Furthermore, these novel findings highlight the necessity of considering sexual dimorphism in the design of future immunotherapy trials.
The Tumor Microenvironment Enhances Treg Fitness through Upregulation of a Foxp3-specific Deubiquitinase Module
Authors: Montauti Elena , Weinberg Samuel E , Chaudhuri Shuvam , Fang Deyu
Published: February 2023

Through abnormal vascularization, cytokine production, and nutrient depletion, tumors can create a highly immunosuppressive microenvironment that favors the functionality of immune regulatory programs over anti-tumor effector responses. Particularly, the highly immunosuppressive tumor microenvironment (TME) favors T regulatory (Treg) cell stability and function, while diminishing the anti-tumor activity of effector T cells. Here, we characterized previously unknown TME-specific cellular and molecular mechanisms that promote intratumoral Treg adaptation through Foxp3. We uncovered the critical role of FOXP3-targeting deubiquitinases, ubiquitin specific peptidase 22 (Usp22) and 21 (Usp21) as environmentally sensitive factors of multiple TME-specific environmental stressors including TGF-β, hypoxia, and nutrient deprivation. Specifically, Usp22 and Usp21 maintain optimal Foxp3 expression through HIF, AMPK, and mTOR activity. The simultaneous loss of both USPs synergizes to alter Treg metabolic signatures and impair suppressive mechanisms, resulting in enhanced anti-tumor activity. Our findings unveil new mechanisms underlying the functional uniqueness of intratumoral Treg cells, and identify Usp22 and Usp21 as a antitumor therapeutic targets that inhibit Treg adaptability in the TME.