bluuebunny/crossref_metadata_2025 huggingface dataset:
Use of small molecule inhibitors of metabolism to enhance T cell proliferation and phenotype for adoptive T cell therapies
Authors: Waller Alexandra Lauren , Funk Christopher Ronald , Ravindranathan Sruthi , Waller Edmund K
Published: January 2023

Adoptive T cell therapies including CAR T therapy are limited by poor in-vivo persistence and moderate anti-tumor efficacy. According to literature, metabolism plays a critical role in the phenotypic state and fate of T cells post-expansion. For instance, glycolytic T cells are more active and exhibit a terminal effector phenotype, while quiescent T cells (naïve and memory T cells) rely more on oxidative phosphorylation with better proliferative capacity. Therefore, this study aims to determine whether metabolic inhibitors used during ex vivo T cell expansion promote a more quiescent phenotype leading to T cells with increased in vivo persistence and efficacy. A screening was conducted using inhibitors of T cell metabolism to alter the balance between T cell activation and senescence, thereby enhancing T cells proliferative capacity. The two inhibitors screened were: idelalisib, an inhibitor of PI3K δ and ibrutinib, an inhibitor of Bruton’s tyrosine kinase.

The study involved culturing human T cells with the above-mentioned compounds for 15 days with anti- CD3/CD28 beads and 30 U/mL of IL-2. T cells cultured with idelalisib and ibrutinib had increased expansion (50- fold: idelalisib and 21-fold ibrutinib) when compared to control (cells with beads alone) with only 6-fold expansion. Phenotypic analysis performed using flow cytometry showed an increased percentage of CD27+ CD28+ CD8+ T cells in the idelalisib treated group and decreased number of senescent T cells that are double negative for CD27 and CD28.

Future experiments focus on other modulators of metabolism that may benefit T cell expansion. They will also focus on metabolic analysis of T cells using the Seahorse assay and in-depth analysis of the PI3K pathway using western blot.
Improving T cell functionality for adoptive T cell therapy in metastatic colorectal cancer
Authors: Ravindranathan Sruthi , Jajja Mohammad Raheel , Waller Alexandra , Bommireddy Ramireddy , Selvaraj Periasamy , Yang Lily , Waller Edmund K
Published: January 2023

Adoptive T cell therapy in solid tumors such as colorectal cancers are challenging due the low frequency of tumor infiltrating T cells and high molecular tumor heterogeneity. In this study, we are proposing to address this issue by developing a novel technology to expand patient tumor specific T cells ex-vivo, using patient’s matched tumor and PBMCs. Published data from our group shows T cells isolated from heavily treated lymphoma patients when treated with inhibitors of PI3K delta (idelalisib) and vasoactive intestinal peptide (VIPhyb) signaling results in decreased levels of senescent T cells and increased persistence in-vivo in NSG mice. Additionally, we have demonstrated that tumor membrane vesicles (TMVs) prepared from human tumor cells decorated with IL-12 and B7-1 (decorated TMVs), stimulate expansion of tumor antigen specific T cells with increased IFN gamma release. This study thus combines both strategies to obtain tumor antigen specific T cells with enhanced anti-tumor activity and in-vivo persistence.

Preliminary data was generated with PBMCs obtained from consented colon cancer patients expanded with decorated TMVs with/without VIPhyb and idelalisib. Results showed that on day 14 of T cell expansion, T cells expanded with decorated TMVs + VIPhyb + idelalisib resulted in significantly increased number of IFN gamma secreting CD8+ T cells (25% of T cells), when compared to T cells treated with decorated TMVs without VIPhyb and idelalisib (13% of T cells). On the other hand, in the absence of decorated TMVs, there were less IFN gamma secreting T cells, both in the CD4 and CD8 compartment. Further experiments are underway to test the persistence and efficacy of the expanded T cells in PDX mice with matched human colon cancer.
A novel agonist of CD137 immune checkpoint stimulator serves as a cancer immunoprevention agent with efficacy against various tumor types
Authors: Batra Lalit , Barsoumian Hampartsoum B. , Shrestha Pradeep , Hawthorne Jenci L. , Bowen William S. , Zhao Hong , Egilmez Nejat K. , Gomez-Gutierrez Jorge G. , Shirwan Haval , Yolcu Esma S.
Published: January 2023

Background

CD137 immune checkpoint stimulator plays an important role in T cell responses and has been targeted for cancer immunotherapy using agonistic Abs with shown toxicity. We herein report that a novel oligomeric form of the CD137 ligand, SA-4-1BBL, as a single agent has robust cancer immunoprevention efficacy against various tumors in mice without detectable toxicity, whereas an agonistic Ab to CD137 did not protect mice against tumor challenge.

Study Design

Mice (C57BL/6 or BALB/c) were pretreated s.c. with SA-4-1BBL protein or an agonistic Ab (3H3) to CD137 and subsequently challenged s.c. at various times (0 to 12 weeks) with different tumor cell lines. SA-4- 1BBL-treated mice without tumor challenge were euthanized 7 days later to harvest lymphoid tissues for immunophenotyping.

Results

Pretreatment with SA-4-1BBL protected mice against 4 different tumor types; 3LL lung, TC-1 cervical, B16-F10 melanoma, and A20 lymphoma. The conferred protection against tumors was dose-dependent and evolved within two weeks of SA-4-1BBL pretreatment and lasted for > 8 weeks. In marked contrast, pretreatment with the agonistic Ab had no impact on the tumor growth. CD4+CD44+CD137+ T cells and NK cells producing IFN- γ were expanded by SA-4-1BBL. Depletion of CD4+ T and NK cells or blockade of IFN-γ totally abrogated the protective effect. Depletion of CD8+ T or B cells had no significant impact on tumor growth. Importantly, SA-4- 1BBL as a single agent also prevented post-surgical tumor recurrences.

Conclusion

This study demonstrates that SA-4-1BBL is a novel CD137 agonist with desired features for cancer immunoprevention and immunotherapy.
Teriflunomide prevents the activation-induced CD25 expression on canine CD4+ T cells
Authors: Jasiecka-Mikolajczyk Agnieszka , Socha Piotr , Jaroszewski Jerzy J. , Maślanka Tomasz
Published: January 2023

Teriflunomide (TER) is an immunomodulatory agent. First reports on the use of TER in dogs have already appeared. Immune mechanisms underlying the immunosuppressive and anti-inflammatory properties of TER are not completely understood. The study attempts to shed light on the involvement of CD4+ T cells in inducing these properties in dogs. This study investigates the effect of TER on CD25 and Foxp3 expression by canine CD4+T cells in the course of in vitro activation. CD25 is T cell activation marker, whereas Foxp3 is critical for the development and function of CD4+ regulatory T (Treg) cells. Peripheral blood mononuclear cells collected from clinically healthy dogs (n = 15) were exposed to TER at concentrations reflecting its plasma levels obtained at a typical dose and at a ten-fold lower concentration with PMA/ionomycin stimulation. The percentage of CD25-expressing cells within CD4+ T cell subset in the culture treated with TER 10−4 M did not differ significantly from the value of this parameter in untreated non-stimulated cells but was significantly lower than that in untreated stimulated cells. The exposure to TER 10−4 M led to a significant increase in the percentage of Foxp3-expressing cells within the CD4+ T cell subset. However, the exposure to the lower concentration of TER did not affect the evaluated parameters. The results indicate that TER at the concentration achieved at a therapeutic dose prevented the activation-induced CD25 expression and induced Foxp3 expression by canine CD4+ T cells. These results suggest that the generation of inducible Foxp3-expressing CD4+ T cells and inhibition of CD4+ T cell activation may constitute one of the mechanisms responsible for the immunosuppressive and anti-inflammatory properties of TER.
On The Evolutionary Origins Of Cd1d And The Type I, Semi-Invariant Natural Killer T Cells
Authors: Suryadevara Naveen Chandra , Kumar Amrendra , Chimski Paul , Oh Karin , Caster Courtney , Truman Richard R , Ren Liming , Criscitiello Mike , Joyce Sebastian
Published: January 2023

The evolutionary origins of CD1d molecules and the T cell receptors (TCR) that recognize them currently remain unknown. CD1d molecules control the functions of a specialized subset of lymphocytes called semi-invariant natural killer T (type I NKT) cells. Type I NKT cells recognize both the self and non-self agonist —α-galactosylceramide (αGC) and related glycolipids— when presented by CD1d molecules. NKT cells express an invariant TCR α-chain (iTCRα) resulting from the mouse Vα14 or human Vα24 to Jα18 rearrangement. iTCRα pairs with the mouse Vβ8 (Vβ7, Vβ6, Vβ2) or the orthologous human Vβ11 TCR β-chain to create a functional NKTCR. Our phylogenetic analyses revealed that whilst the mouse Cd1 homologs have Mesozoic origin in they they evolved as far back asin the anole lizards, the orthologues of Cd1d and that of Vα14/Vα24 and Jα18 gene segments emerged with the decent of mammals. Moreover, the known avian CD1 orthologs lack the α3 domain and both the reptilian and the avian CD1 lack a functional endo/lysosomal-recycling motif critical for NKT cell development and function. As well, monotreme genomes lack Cd1d, Vα14/Vα24 and Jα18 orthologs and the marsupial Cd1d, Vα14/Vα24 and Jα18 orthologs harbor indels that would render the encoded proteins non-functional. Consistent with these findings, one of the most divergent Cd1d gene isolated from the nine-banded armadillo Dasypus novemcinctus and ectopically expressed in a cell line presented αGC and activated mouse NKT cell hybridomas. Hence, we conclude that the CD1d-restricted αGC-presentation in the immune system evolved more recently as an eutherian innovation.
The melanomacrophage center is a potential evolutionary precursor to the mammalian germinal center
Authors: Steinel Natalie C
Published: January 2023

The melanomacrophage center (MMC), aggregates of highly pigmented melanomacrophages found within the lymphoid tissues of ectotherm vertebrates, is thought to be the evolutionary precursor to the mammalian germinal center (GC). Studies of cold-blooded vertebrates suggest that the MMC is the site of the humoral adaptive immune response, where B cells proliferate, generate high affinity antibody, and undergo affinity maturation. MMCs transiently increase in size/number following immunization or bacterial infection, supporting the hypothesis that the MMC response is likely linked to the adaptive immune response. However, while morphological studies and shared expression of a GC specific gene further support the idea that the MMC is a proto-GC, melanomacrophages perform many functions not associated with the humoral adaptive immune response. Therefore, to determine if the MMC is serving in a GC-like capacity or is a more generalist feature of ectotherm biology, we turned to the fish model system, three spine stickleback. Fish MMCs increase in size in response to immunization with NP-CGG in alum, but not control injection with adjuvant alone. Fish MMCs are also found in close proximity to lymphocytes and cells expressing the B/T cell activation marker GL7. These preliminary studies not only give further support to the notion of the MMC as a GC homolog, but also provide important tools to broaden the study of adaptive immunity in other fish, reptilian, and amphibian systems.
Sphingosine 1-phosphate lyase promotes the type I interferon-mediated innate immune response to influenza but is subjected to degradation by influenza A virus NS1
Authors: Wolf Jennifer J , Xia Chuan , Vijayan Madhuvanthi , Song Yuleum , Studstill Caleb J , Ngo Hanh , Johnson Marc C , Saba Julie D , Alexander Stephen , Hahm Bumsuk
Published: January 2023

Influenza viruses cause seasonal and pandemic influenza, remaining a threat to human health. The type I interferon (IFN)-mediated innate immune response is one of the central obstacles influenza A virus (IAV) must overcome to successfully replicate within the host. Here, we demonstrate that sphingosine 1-phosphate (S1P) lyase (SPL) enhances the type I IFN response, but this antiviral innate immunity is counteracted by IAV infection. Although SPL is an enzyme that metabolizes S1P, SPL was found to interact with IKKɛ and promote IKKɛ-mediated type I IFN responses. Thus, when SPL was knocked out of host cells, IAV replication increased and the elements of IKKɛ-induced type I IFN response decreased. However, IAV infection destabilized the SPL-mediated type I IFN response by inducing the degradation of SPL. Importantly, nonstructural protein 1 (NS1) of IAV triggered the depletion of SPL. SPL was ubiquitinated upon IAV infection or NS1 expression, whereas NS1-deficient IAV failed to elicit ubiquitination or downregulation of SPL. Transiently overexpressed SPL increased the levels of auto-phosphorylation of IKKɛ, resulting in enhanced expression of type I IFN and IFN-stimulated genes. However, this induction was markedly inhibited by IAV NS1. Collectively, this study reveals a pro-IFN function of SPL as well as a novel strategy employed by IAV to subvert the type I IFN response, providing new insights into the interplay between IAV and host innate immunity.
Influenza A virus (IAV) infection in humans leads to expansion of highly diverse CD8 T cell repertoires cross-reactive with Epstein Barr virus (EBV)
Authors: Gil Anna , Mishra Rabi , Song InYoung , Aslan Nuray , Ghersi Dario , Selin Liisa K.
Published: January 2023

The competence of T cell responses predominantly depends on how efficient T cell receptors (TCRs) are at recognizing antigenic epitopes. We show here that during acute severely symptomatic IAV infection there was an expansion of IAV-M1/EBV-BRLF1 and IAV-M1/EBV-BMLF1 double-tetramer+ cells directly ex vivo in 5 HLA-A2+ patients. We questioned whether this expansion specific to these two different cross-reactive responses would lead to alterations in the IAV-M158, EBV-BRLF1109 and -BMLF1280 TCR repertoires from before and during acute IAV infection. Using staining with VB mAb we found that T cell responses generated to these epitopes became surprisingly more polyclonal, with the sharing of Vb between M1, BMLF1 and BRLF1 populations which is not seen in healthy donor. Furthermore, by using next generation sequencing and single-cell analysis of TCRα and TCRβ repertoire of tetramer sorted IAV-M1 cells we showed dramatic changes in specific clonotype usage during acute IAV infection compared to before infection. In summary, these changes in TCR repertoire during acute symptomatic IAV infection suggest that during severe infection there is a preferential expansion of highly diverse cross-reactive responses between IAV and the persistent virus, EBV, which leads to permanent changes in TCR repertoires to both of these two viruses (NIH AI049320 and NIH AI109858).
Differential effect of GITR on lung effector T cell subpopulations during influenza infection
Authors: Chu Kuan Lun , Batista Nathalia V , Watts Tania H
Published: January 2023

Influenza remains an important global threat. Tissue resident memory T cells (Trm) are critical in protection against influenza, and it is important to identify key mechanisms regulating their formation and persistence. GITR, an NF-κB activating TNFR family member, is required on CD8 T cells for maximal responses against influenza. Our recent work (Mucosal Immunology, doi:10.1038/s41385-018-0105-5) provided evidence that GITRL on monocyte-derived inflammatory APCs provides crucial signals through GITR on T cells in the lung tissue (termed Signal 4), allowing effector T cell accumulation and optimal Trm formation during influenza infection in mice. During influenza infection, responding lung T cells are heterogeneous, some will give rise to terminally differentiated effector cells, while others will become Trm. It remains unknown how GITR affects these subpopulations. To address this issue, we characterized these CD8 T cell subsets using markers Ly6C, KLRG1, T-bet, and the chemokine receptor CX3CR1. By staining with Ly6C and CX3CR1, we identified three different CD8 T cell subsets with differential expression of Ly6C and CX3CR1 (Ly6ChiCX3CR1 hi, Ly6ChiCX3CR1 lo, Ly6CloCX3CR1 lo). Ly6ChiCX3CR1 hi CD8 T cell subset likely represents the most terminally differentiated effector cells as they have the highest expression of KLRG1 and T-bet. Using the adoptive transfer of transgenic OT-I cells, we discovered that there is a larger loss of the Ly6CloCX3CR1 loCD8 T cell in the absence of GITR when compared to the other two subsets. We are currently investigating which of the three subsets give rise to the lung Trm population after influenza infection and the timing and nature of the GITR-dependent signals involved.

Funded by CIHR:FDN143250
Plasmacytoid dendritic cells contribute to HIV diseases and viral persistence via IDO-dependent mechanisms
Authors: Li Guangming , Ma Jianping , Yu Haisheng , Bi Wenwen , Wu Yaxu , Cheng Liang , Su Lishan
Published: January 2023

The combination antiretroviral therapy (cART) effectively suppresses HIV replication and enables HIV-infected individuals to live long-productive lives. However, the persistence of HIV reservoir cells with latent or lowreplicating HIV in patients under cART make HIV infection an incurable disease. Plasmacytoid dendritic cells (pDCs) suppress HIV replication through secreting type I interferon (IFN-I). We have reported that pDCs play a major pathogenic role in chronic HIV infection in vivo. In this study, we discovered that pDCs-depletion during cART completely resolved HIV immune pathogenesis and rescued anti-HIV T cell response in humanized mice. Importantly, HIV reservoirs (total cell-associated HIV DNA as well as cells with replication-competent HIV virus) are reduced by pDCs-depletion. We showed that HIV infection induced IDO expression in monocytes and DC via pDC-dependent mechanism. Interestingly, IDO inhibition during cART similarly resolved HIV immune pathogenesis and rescued anti-HIV T cell response, correlated with reduced HIV reservoirs in humanized mice in vivo. Finally, we proved that pDCs depletion and IDO inhibition acted through the same pathway to reverse HIV pathogenesis. We conclude that pDCs persistently activated by HIV lead to IDO expression that induces host immune exhaustion, contributing to HIV reservoirs persistence during effective cART. Strategies to disrupt the pDC-IDO axis will provide novel opportunities to achieve HIV-1 functional cure therapy.
Δ42PD-1 enhances HIV replication and suppresses maturation of dendritic cells
Authors: Zhou Runhong , Mo Yufei , Zhou Dongyan , Chen Zhiwei
Published: January 2023

Previously, we discovered a new PD-1 isoform, which consists of a 42-base pairs loss from the start of exon 2 that is equivalent to a 14 amino acid in-frame deletion. Therefore, we named this new PD-1 isoform as Δ42PD-1. Here, we found that Δ42PD-1 was highly expressed on monocyte-derived dendritic cell (MoDC) in healthy donors, while significantly decreased in HIV patients with or without antiretroviral therapy. Consistently, in vitro assay, HIV infection suppressed Δ42PD-1 expression in total MoDCs. However, upregulation of Δ42PD-1 was observed in mature MoDC (high CD83+CD86+) after HIV infection, although the mature MoDC population was unraised, which consistent with previous report that HIV infection do not mature MoDC. Therefore, to investigate the role of Δ42PD-1 on HIV replication, Δ42PD-1 positive and negative MoDC were sorted out by FACS and infected with R5-tropic HIV (JR-CSF) respectively. Interestingly, Δ42PD-1 positive MoDC was higher susceptible to HIV than Δ42PD-1 negative MoDC. Maturation is important for dendritic cell to perform innate and adaptive immune response. Further investigation revealed that Δ42PD-1 negative MoDC had higher maturation level than Δ42PD-1 positive MoDC after LPS stimulation, which means that Δ42PD-1 may suppress DC maturation. So far, we conclude that Δ42PD-1 not only enhances HIV replication, but also suppresses dendritic cell maturation. It warrants to further study the role of Δ42PD-1 on DC-mediated immune responses during HIV infection in vivo.
Not just acute: IRF-7 plays a key role in restriction of chronic gammaherpesvirus infection
Authors: Johnson Kaitlin E , Jondle Christopher Ned , Lange Philip T , Aurubin Carlie A , Tarakanova Vera L
Published: January 2023

Gammaherpesviruses (GHVs) are ubiquitous pathogens that establish lifelong infections and are associated with a variety of malignancies, including lymphomas. While risk factors for these virus-driven cancers are poorly understood, it is clear that increased reactivation of these viruses from latency contributes to viral oncogenesis.

Therefore, it is important to identify host factors that restrict GHV reactivation. Interferon regulatory factor 7(IRF-7) is one of two transcription factors that are primarily responsible for driving type I interferon (IFN) expression and subsequent innate immune responses. Unlike previous studies which have focused on IRF-7 in the context of acute viral infections, we found that GHV reactivation is increased in chronically infected IRF-7−/− mice. Interestingly, the virus-specific T cell response that is typically critical for control of viral reactivation during chronic infection was not decreased in IRF-7−/− mice. However, we observed a decrease in expression of select IFN induced genes (ISGs) in IRF-7−/− GHV-infected mice. Importantly, these differentially affected ISGs have been shown to inhibit GHV replication. Ongoing studies will determine the contribution of IRF- 7 to the expression of IFN, ISGs, and IFN-dependent antiviral activity in the context of chronic GHV infection. Importantly, successful completion of these studies will provide insight into how the host innate immune system controls chronic GHV infections and viral pathogenesis.
Investigating the immunological role of galectin-9 on natural killer (NK) cells in human immunodeficiency virus (HIV)
Authors: Motamedi Melika , Elahi Shokrollah
Published: January 2023

NK cells play an integral role in mediating immune responses to viral infections; however, their function is compromised during chronic diseases such as HIV, partly due to an increase expression of inhibitory receptors. Thus, the reduced immune responses elicited by NK cells in HIV can be the result of the engagement of inhibitory immune checkpoint molecules with their respective ligands. Galectin-9 (Gal-9) is a tandem-repeat type galectin that possess a wide range of immunomodulatory properties such as interacting with Tim-3, which is upregulated on T cells during chronic conditions. Previously, our group has shown that interaction of Gal-9 on regulatory T cells with Tim-3 on CD8+T cells, renders these T cells more permissible to immunosuppression and subsequently leads to their exhaustion. We aimed to further study the role of Gal-9 on NK cell function in HIV patients. As a comparison, we also analyzed another inhibitory receptor known as TIGIT. Our preliminary data indicates upregulation of surface Gal-9 on NK cells in HIV infected individuals when compared to healthy controls. We observed two effector NK cell populations with dichotomous functionality. TIGIT+NK cells expressed more cytotoxic molecules (GzmB and perforin) and less IFNγ. Conversely, Gal-9+NK cells expressed less cytotoxic molecules, but more IFNγ. Additionally, Gal-9+NK cells, in contrast to Gal-9−NK cells, co-express negligible amounts of GzmB and perforin, which is detrimental to their cytotoxic abilities. We believe that understanding the underlying biochemical pathway associated with Gal-9+NK cell dysfunction can pave the way for potential therapeutic strategies.
The Ly49G2 inhibitory receptor is essential to MHC I-dependent NK cell control of murine cytomegalovirus infection
Authors: Cronk John Michael , Gamache Awndre , Gillespie Alyssa , Brown Michael G
Published: January 2023

Licensed Ly49G2+NK cells mediate H-2Dk-dependent control of murine cytomegalovirus (MCMV) in NK gene complex (NKC)C57L mice. However, a precise role for the Ly49G2 inhibitory receptor in Dk-dependent MCMV control is not defined. Thus, we generated Ly49g2C57L mutant mice via CRISPR/Cas9 gene-editing. A single cytosine insertion resulted in Ly49G2 truncation and undetectable NK cell surface expression. Ly49G2null mice exhibited increased mortality due to abrogated MCMV control by NK cells, despite an otherwise unmodified Ly49 repertoire and normal NK cell development. We further found Dk-dependent MCMV control requires Ly49R signaling, as blocking the activation receptor prior to infection resulted in rapid MCMV spread. Coincident with MCMV control, Ly49R+Ly49G2+ (R+G2+) NK cells were more frequently Klrg1hi, CD27−CD11b+, and CD62Llo than their Ly49R+ (R+) counterparts, and this effect was Dk-dependent. R+G2+ NK cells likewise out proliferated R+ NK cells in infected Ly49G2wt mice. NK cell subset activation and proliferation differences suggested impaired virus control in Ly49G2null mice might result from inept target cell sensing or poor NK cell expansion. To verify a role for Dk-licensed NK cells, we examined donor NK cells after adoptive transfer to NK cell-deficient hosts. Licensed R+G2+ NK cells in peripheral blood and spleen robustly expanded and mediated MCMV control in contrast to R+ NK cells. Both Ly49G2 and Ly49R receptors expressed together in NK cells thus are essential to Dk-dependent MCMV control. Our data and novel mouse model suggest licensed NK cells may be better equipped to sense infected targets, which aligns with human genetics studies implicating a role for licensed NK cells in control of chronic infection.
LAG-3 modulation of natural killer cell immunoregulatory function
Authors: Ali Ayad , Moran Michael T , Tuazon Jasmine , Gyurova Ivayla , Vignali Dario AA , Waggoner Stephen N
Published: January 2023

Natural killer (NK) cells suppress antiviral immunity by killing a fraction of virus-specific CD4 T cells. This occurs during the first 3 days of infection, is perforin-dependent, and coincides with NK-cell localization in the splenic white pulp. While the receptors governing this NK-cell activity are ill-defined, it is clear that negative feedback mechanisms must exist to prevent excessive pruning of T-cell responses. Comparative transcriptome analysis of red pulp and white pulp localized NK-cells derived from late in the window of immunoregulatory activity (day 3) reveals increased expression of Lag3 in white pulp NK-cells. LAG-3 is an inhibitory receptor that binds peptide-bound major histocompatibility complex II (pMHC-II). We find a progressive increase in LAG-3 protein expression on NK-cells with time post-infection and localization to the white pulp. Consistent with its putative inhibitory role, antibody-mediated neutralization of LAG-3 enhances NK-cell killing capacity in vitro. Preliminary analysis reveals reduced virus-specific T cells in mice lacking Lag3 in NK-cells (Ncr1Cre+Lag3fl/fl) relative to control mice. We hypothesize that LAG-3 upregulation on NK-cells confers inhibitory feedback signals from surrounding MHC-II+ cells to terminate NK-cell suppression of T cells. Uncovering the role of LAG-3 in NK cell immunoregulatory function will provide a novel target to constrain this suppression and enhance vaccine-elicited T cell responses.
Latency III gene products of Epstein-Barr Virus (EBV) are associated with Systemic Lupus Erythematosus (SLE) genetic risk loci
Authors: Laurynenka Viktoryia , Chen Xiaoting , Carter Martha , Parameswaran Sreeja , Eswar Shruti , Kaufman Kenneth M. , Namjou Bahram , Weirauch Matthew T. , Kottyan Leah C. , Harley John B.
Published: January 2023

SLE affects millions with increasing diagnostic prevalence. Genetic studies have identified >600 mostly regulatory variant associations. EBV has been nominated as a causal factor for SLE from immunochemistry and epidemiologic studies. EBNA2 concentrates at SLE risk loci (Nat Genet 50:699 2018) suggesting mechanisms. Additional evidence supporting EBV as an SLE origin would advance this hypothesis of etiology.

Previous simulation using our RELI algorithm revealed an astonishing association with Epstein-Barr virus nuclear antigen 2 (EBNA2) (OR=5.96, Pc=E-25) (Nat Genet 50:699, 2018). The >100 risk alleles associated at p<5E-8 for SLE were curated and reduced to 83 loci by linkage disequilibrium pruning. We evaluated the available 53 virally encoded and 13,051 human TF ChIP-seq (chromatin immunoprecipitation with DNA sequencing) datasets. In addition to EBNA2 (now OR=3.7, Pc=1.96E-18 after Bonferroni correction), two of the 53 viral TF data sets, EBNA3C and EBNALP, each of which are EBV Latency III gene products, were also strongly associated with the SLE risk loci (OR=6.0, Pc=4.19E-18; and OR=3.3, Pc=5.53E-18). Moreover, among the human TF datasets strongly associated with SLE risk loci (Pc<E-6), those from EBV infected and transformed B cells are highly concentrated (OR≈56, P30, p<E-25). Meanwhile, genetic associations for depression, anxiety, and schizophrenia show no such relationships.

These new results further the possible validity of the hypothesis that SLE is originally caused by the Latency III expression program of EBV.
The Effect of Dietary Fiber Intake on Systemic Lupus Erythematosus (SLE) Disease in NZB/W Lupus Mice
Authors: Panther Eric James , Ren Jingjing , Cabana-Puig Xavier , Abdelhamid Leila , Swartwout Brianna , Luo Xin M. , Reilly Christopher M.
Published: January 2023

Dysbiosis in the gut microbiota has been observed in a various autoimmune disease, including SLE, which could cause a leaky gut, triggering an immune response, and thus worsening autoimmune disease expression. In our current studies, we hypothesized that increasing dietary fiber would create a healthy microbiota environment in the gut, leading to decreased leakiness of the gut and to decreased disease expression in and NZB/NZW female mice. NZB/NZW mice were placed on standardized high fiber (HF 30%) or low fiber (LF 0.4%) for 12 weeks beginning at 10 weeks of age. Mice were assessed as they aged for various parameters of disease including proteinuria and anti-dsDNA antibody production. Alteration of the microbiota and short chain fatty acid levels were also assessed. At 36 weeks-of-age, the mice were euthanized and we assessed occlusion protein expression, splenocyte profiles, and kidney tissue. We found as the mice aged, their body weights, anti-dsDNA antibody levels, and proteinuria were not significantly different between the groups. Similarly, there were no differences in SCFA levels. In regard to the microbiota, Chlostridiales bacteria were consistently increased in the HF treated mice compared to the LF treated mice. In regard to disease progression, spleen weight, immune cell profiles, proteinuria, dsDNA levels, and kidney pathology, there was no difference between the HF and LF treated groups. Taken together, these results indicate that in the NZB/W female mouse, a HF diet may alter the microbiota but does not influence disease progression.
Single-cell RNA sequencing of pancreatic islets reveals the complexity of autoimmune diabetes throughout its initiation and development
Authors: Zakharov Pavel , Hu Hao , Wan Xiaoxiao , Unanue Emil R
Published: January 2023

Type one diabetes (T1D) is a spontaneous autoimmune disease that results in pancreatic islet beta-cell death driven by diverse immune cells. However, molecular signatures of the leukocyte populations in T1D remain poorly defined. Here, using single-cell transcriptional analysis, we examined intra-islet leukocytes at various stages of disease: from its very early start, through its control phase and finally to the final effector stage. Early stages before clinical diagnosis are not accessible for human studies.

We found that both CD4 and CD8 T cells showed heterogeneity at the initial stage of autoimmune inflammation and already contained effector cells. Moreover, the disease progression at all times was accompanied by regulatory sets establishing control mechanisms. In line with that, we also detected accumulation of anergic and exhaustion-associated phenotypes over time. Analysis of the islet macrophages showed substantial divergence, including novel population with anti-inflammatory and dead-cell clearance features. Autoimmune process resulted in polarization of islet macrophage into terminal pathogenic state via two-step activation program. While stage 1 marked by NF-kB activation was present in non-diabetic condition, stage 2 was inflammation-induced and had additional IFN-gamma activation. Interestingly, the final effector stages were not dominated by CD8 cytotoxic T cells but by pro-inflammatory macrophages.

Overall, our analysis comprehensively describes stages of T1D on the basis of lymphoid and myeloid transcriptional activation programs, providing a detailed single-cell atlas of autoimmune diabetes initiation and development. Our findings will guide future studies in staging human T1D.
Characterization and use of human insulin transgenic mouse models for type 1 diabetes
Authors: Amdare Nitin Purushottam , Shultz Leonard D , Greiner Dale L , DiLorenzo Teresa P
Published: January 2023

Type 1 diabetes (T1D) is characterized by T cell-mediated islet-specific autoimmunity leading to β cell destruction and loss of insulin production. Insulin is the primary antigen produced by β cells and due to incomplete conservation between the mouse and human insulin genes, some of the important disease-relevant T cell epitopes can be missed in the widely studied non-obese diabetic (NOD) mouse model. Thus, considering the limitations of NOD mice, we generated a novel NOD mouse model transgenically expressing human insulin (NOD.hIns mice) under the control of the human promoter. Female NOD.hIns mice spontaneously developed autoimmune diabetes as early as 15 weeks (incidence 62% by 30 weeks of age, n=21) vs NOD mice (41%, n=24), accompanied by numerous hallmarks of human T1D (including thymic human insulin expression and variable degrees of pancreatic islet infiltration by immune cells). Next, we cultured islet-infiltrating CD8 T cells from NOD.hIns mice and tested them for recognition of peptides unique to human preproinsulin. These efforts uncovered at least three T cell epitopes, validating that human insulin is a target of the autoimmune response in NOD.hIns mice. This concept was confirmed using T cells derived from NOD.hIns mice deficient in both murine insulin genes (NOD.Ins1ko.Ins2ko.hIns mice). NOD.hIns mice expressing human class I MHC molecules associated with T1D (HLA-A*24:02 and B*39:06) are currently being studied. Such well-characterized, and highly human-relevant, T1D models can be used to develop robust immune monitoring tools and to test and refine antigen-specific therapeutic strategies for T1D.
Follicular helper T cells express a metabolic and cell signaling gene signature in a lupus-prone murine model
Authors: Gong Minghao , Cornaby Caleb , Michailidis George , Morel Laurence
Published: January 2023

Systemic lupus erythematosus (SLE) is an autoimmune disease in which autoreactive follicular helper T (Tfh) cells license high-affinity autoantibody production. Strikingly, the frequency of circulating Tfh is correlated with disease activity in SLE patients. As such, understanding the molecular mechanisms responsible for the aberrant Tfh cell generation and activation in lupus is of fundamental significance. We previously demonstrated that expanded Tfh cells in the B6.Sle1.Sle2.Sle3 (TC for triple congenic) lupus model exhibit high glycolysis and oxidative metabolism, which can be constrained by inhibiting glycolysis with 2-deoxyglucose (2DG). We performed RNA-seq analyses of splenic Tfh and naïve CD4+ T cells (Tn) comparing between TC and B6 mice. First, data revealed a large number of shared gene signatures in Tfh and Tn comparing between TC and B6 group, implicating that the aberrant development of Tfh initiates as early as Tn state. Further alignment of the Tfh transcriptome obtained from RNA-seq and earlier microarray assays demonstrated concerted alterations in numerous gene signatures of overactivation of T cells including dysregulated tyrosine kinase signaling and MAPK signaling pathways. Gene set enrichment analyses (GSEA) further revealed altered metabolic pathways (e.g., oxidative phosphorylation and pyruvate metabolism) among splenic TC Tfh cells. Finally, we demonstrated that 2-DG partially correct the aberrant expression patterns in TC splenic Tfh cells, restoring oxidative phosphorylation and the MAPK pathways. Since 2DG eliminates the expansion of autoreactive TFh cells, these results suggest that these two pathways may be critical to lupus Tfh cell function.
T cell cooperation in the induction of autoimmunity in a murine model of multiple sclerosis
Authors: Garza Celeste , Joern Rachel Robinson , Rojano Elba Mirelle , Forsthuber Thomas
Published: January 2023

Multiple sclerosis (MS) is a T cell-driven autoimmune disease. There are many unknowns regarding the disease etiology and pathogenesis. Importantly, the mechanisms of peripheral tolerance and dysregulation of pathogenic mechanisms are poorly understood. It is known that patients with MS exhibit myelin reactive T cells in the blood; however healthy controls also show myelin reactive T cells in the blood. Additionally, T cells from patients with MS react to many different myelin peptides. Current human studies do not support that patients exhibit large populations of T cells specific for a single myelin-antigen responsible for the induction of autoimmune pathology. In contrast, a quorum of several low avidity autoreactive T cells specific for one epitope might suffice to induce autoimmunity. We hypothesized that a quorum could be established and induce autoimmunity by small numbers of antigen-specific T cells recognizing different myelin antigen peptides. We found that disease in the experimental autoimmune encephalomyelitis (EAE) model of MS can be initiated by the cooperation of small numbers of myelin peptide-specific T cells reactive against different myelin epitopes. These results support the hypothesis that a quorum of autoreactive T cells could suffice to initiate autoimmune disease. We have developed a system whereby T cells of different antigen specificities can be traced in order to track the dynamics and kinetics of those T cells in disease initiation and progression. The results of this study provide further insights into the dynamic interactions between T cells of different antigen specificities.
Noncoding RNAs modify GATA3-driven transcription, leading to the immune profiles seen in psoriasis and atopic dermatitis
Authors: Mazahery Claire , Valadkhan Saba , Levine Alan D.
Published: January 2023

Atopic dermatitis (AD) and psoriasis are clinical manifestations of immune dysregulation in the skin. Psoriasis is generally classified as a T cell-mediated, IFN-γ dependent disease, while AD is skewed toward interleukin (IL)-4 dependence. Consistent with these classifications are clinical observations that IL-4 improves psoriasis disease score and an IL-4 receptor blocking antibody is approved for treatment of severe AD. However, the immunobiology is far more complex, as IL-13, often produced by IL-4 dependent T cells, can be elevated in psoriasis. Since GATA3 controls both IL-4 and IL-13 expression, we propose that regulation of GATA3 differs between the two diseases, resulting in overlapping yet distinct cytokine profiles. Our meta-analysis of two RNA sequencing profiles for each disease identified a noncoding RNA of unknown function (NRUF) in all four datasets (p<1×10−10). Upon analyzing 48 datasets of immune responses to viral infection in epithelial cells and leukocytes, we found IL-13 and NRUF expression levels are most strongly correlated in lymphocytes. In T cell lines, we found elevated expression of the NRUF noncoding RNA associates with IL-4 and IL-13. However, IL-31, which is elevated in AD independently from the GATA3/IL-4/IL-13 axis, is not correlated to NRUF expression, supporting our hypothesis that NRUF drives disease pathology via the GATA3/IL-4/IL-13-dependent inflammatory response. Using overexpression or silencing approaches, we have characterized the ability of this NRUF to direct cytokine polarization of T cells from healthy control and donors with AD or psoriasis. We propose this NRUF modulates these two cutaneous diseases by regulating GATA3-based IL-4/IL-13 balance in T cells.
Delineating T cell autoreactivity in autoimmune diabetes by assessing the MHC-II-bound peptidome
Authors: Wan Xiaoxiao , Vomund Anthony N. , Peterson Orion J. , Lichti Cheryl , Unanue Emil R
Published: January 2023

Development of tissue-specific autoimmune diseases is tightly linked to major histocompatibility complex class II alleles (MHC-II). In humans, the HLA-DQ2/DQ8 variants contributes the highest genetic risk to type 1 diabetes (T1D). However, it remains unclear how the MHC-II-specific peptidome defines the outcomes of T cell self-reactivity. To address this question, we performed unbiased analyses of the MHC-II immunopeptidome of the pancreatic islets of non-obese diabetic (NOD) mice at the start of the autoimmune process. NOD mice spontaneously develop autoimmune diabetes due to the expression of the I-Ag7 MHC-II molecule, which is structurally homologous to HLA-DQ8. Combining mass spectrometry analysis and immunoassays, we identified the precise MHC-II-specific epitopes derived from diverse β-cell proteins and functionally characterized the corresponding T cell responses. The major peptides derived from insulin B chain and C-peptides and consisted of sets of varying length, usually around 14–16 residues. Their presence correlated with the initial T cell autoreactivity. Other minor epitopes elicited T cell responses at a more advanced stage, the phenomenon of epitope spreading. We also identified post translational change consisting of deamidation of C peptides- see abstract of Lichti et al. Importantly, most of the immunogenic epitopes were independently identified in the MHC-II peptidome of pancreatic lymph nodes and spleen, revealing a correlation of peripheral presentation with the tissue autoreactivity. In sum, these data demonstrate the landscape of the MHC-II epitopes being presented in a target autoimmune organ and delineate the characteristic features of a self-peptide that foster pathogenic responses.
Treg-restricted deletion of TRAIL (Tnfsf10) reduces autoimmune diabetes
Authors: Dadey Rebekah , Grebinoski Stephanie J , Zhang Qianxia , Gocher Angela , Brunazzi Erin , Andrews Lawrence Peter , Burton Amanda , Workman Creg , Vignali Dario
Published: January 2023

Regulatory T cells (Tregs) are a suppressive cell population that limit the anti-tumor response. One mechanism Tregs utilize is production of suppressive cytokines. We previously asked if deletion of the two main suppressive cytokines made by Tregs would have any overall effect on the suppressive ability of the Tregs. We found that Treg-restricted deletion of the cytokines IL-10 and IL-35 led to a compensatory upregulation of Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL; Tnfsf10). In these studies, upregulation of TRAIL was dependent on deletion of IL-10 and IL-35; however, we also wondered if TRAIL could also be upregulated in inflammatory environments and if deletion would affect Treg function in these environments.

We made a Tnfsf10L/L mouse, backcrossed onto both B6 and NOD backgrounds, and crossed each with respective Foxp3Cre lines. Surprisingly, we found little effect on tumor growth in B6 mice but found a substantive decrease in autoimmune diabetes incidence in the NOD mouse model. In the latter, we found that Treg-restricted deletion of TRAIL may extrinsically effect CD8+ and CD4+ T cell phenotype by increasing expression of suppressive molecules such as CD73 and LAP-TGFβ in these cells. Future studies are necessary to understand if this phenotypic change is responsible for the decrease in diabetes incidence.
A lifestyle-educational program improves depression levels of those with autoimmune disease
Authors: Ramirez Francisco E , Nedley Neil , Hunter Jennifer Michelle , Finlayson Lance Takeshi
Published: January 2023

Background

Patients with autoimmunity may have problems with depression. We explore the severity of depression among this population.

Methods

Participants that started and finished an 8 week depression educational program were studied. From n=5,861 participants from 5 continents, n=431 suffered from autoimmune disease and were used for the study. The program consisted of 2-hour long weekly meetings which occurred for a total of eight weeks. During each meeting, a 45-minute DVD presentation by a skilled physician was shown, a small group discussion was guided by trained facilitators. The focus of the program was to educate participants about healthy lifestyle behaviors such as exercise, nutrition, sleep hygiene, etc. The participants were assessed pre-post with the Depression and Anxiety Assessment Test that measured depression and demographics. Depression scores were classified into 4 categories: none (0–6), mild (7–10), moderate (11–19) or severe (20 or more).

Results

At baseline the average age was 54.9 (SD 13.3) and 85.4% were female. At baseline, mean group depression was 14.2 (SD 7.4), the depression levels were as follows: none (17.2%), mild (18.3%), moderate (34.3%), severe (30.1%). After the lifestyle-educational program, mean group depression decreased to 7.8 (SD 6.2). Post-program depression levels were as follows: none (51.97%), mild (17.6%), moderate (24.36%), severe (6.03%). The decrease in depression levels was significant with t(431) = −21.19 p <.01.

Conclusions

The eight-week educational program was effective in improving depression levels for most of the participants with autoimmune disease. A follow up study is being planned to see if the results last long term.
Impact of age-associated decrease in thymic B cell expression of Aire and self-antigen genes on T cell tolerance
Authors: Cepeda Sergio , Xiao Yangming , Kraig Ellen , Griffith Ann
Published: January 2023

Susceptibility to many autoimmune diseases increases with age, however, mechanisms linking aging and susceptibility to autoimmunity remain incompletely understood. One hallmark of the aging immune system is atrophy of the thymus, the primary site of T lymphocyte generation and tolerance induction. Aging is also associated with declines in critical thymic functions, including expression of tissue restricted self-antigen (TRA) genes important in tolerance induction, many of which are regulated by the Autoimmune regulator (Aire). TRA expression in the thymus allows T cells to be tolerized to antigens typically expressed in peripheral tissues, and declining TRA expression is predicted to contribute to diminished T cell tolerance in aging. Until recently, medullary thymic epithelial cells (mTECs) were the only known source of thymic TRA expression, but it is now clear that a subset of thymic B (tB) cells also express Aire and mediate tolerance to Aire-dependent self-antigens. We found that expression of Aire and Aire-dependent self-antigens declines in tB from aged mice and humans, which is likewise predicted to diminish central T cell tolerance induction. To test these predictions, we used I-Ab MHCII tetramers to detect T cells recognizing a model TRA expressed in tB cells, Titin (Ttn, an auto-antigen associated with late-onset myasthenia gravis), in the thymus and secondary lymphoid organs (SLO) of young and aged mice. We find an increase in potentially auto-reactive Ttn-specific T cells in SLO of aged mice, concomitant with declining Ttn expression in tB cells. Ongoing experiments will compare the capacity of young and aged thymic B cells to tolerize T cells using the BDC2.5 TCR- and Aire-driven BDC peptide-transgenic mouse models.