bluuebunny/crossref_metadata_2025 huggingface dataset:
CD4 T cell-mediated induction of alopecia areata is dose dependent and requires CD8 T cells
Authors: Connell Samuel J , Crotts Sydney , Christy Payton , Ortolan Luana , Henderson Nicholas , Jabbari Ali
Published: February 2023

Alopecia Areata (AA) is a common autoimmune disease characterized by infiltration of the hair follicle by T cells, resulting in nonscarring hair loss. Our previous work suggested an increased representation of IFN-γ-producing, activated CD4 T cells in the skin-draining lymph nodes of AA mice when compared to unaffected (UA) controls. Our objective was to determine the contribution of CD4 T cells to AA pathogenesis. We adapted a recently described model of mouse AA induction whereby adoptive transfer of in vitro expanded bulk lymph node (LN) cells from previously affected AA mice induced disease in previously unaffected mice. To address the role CD4 T cells play in AA pathogenesis, we first sorted CD4 T cells and assessed their ability to induce AA. Mouse recipients of in vitro expanded CD4 T cells isolated from the LNs of AA mice developed AA at a substantially increased rate compared to mouse recipients of in vitro-expanded CD4 T cells from LNs of UA mice. CD4-mediated AA induction was found to be dose-dependent, with larger numbers of CD4 T cells inducing disease in recipient mice at a higher rate. Using congenic markers, we found that the transferred CD4 T cells were present in the skin draining LNs of recipient mice at three weeks following transfer but largely absent at 16 weeks, suggesting these cells may be conferring their effect early during disease development. Additionally, we found that the CD4 T cell population is critically dependent on the presence of endogenous CD8 T cells in order to transfer disease. Our data suggests that CD4 T cells are working upstream of CD8 T cells in the pathomechanism of AA. Further studies are needed to dissect how CD4 T cells are enabling CD8 T cell-mediated autoimmune attack on the hair follicle.
CD8 follicular T cells provide helper and cytolytic function in the B cell follicule during antibody-mediated autoimmunity
Authors: Hoyer Katrina K , Valentine Kristen M , Mullins Genevieve , Davalos Oscar A , Seow Lek Wei
Published: February 2023

CXCR5+ CD8 T cells arise during situations of chronic antigen and inflammation, including chronic viral infection, cancer, and antibody-mediated autoimmunity. In some conditions CXCR5 expression facilitates CD8 T cell homing to B cell follicles, while some studies find persistent CCR7 expression prevents follicle entry. Despite robust research within this fairly new field variance in tissue localization, expression profiles, inflammatory conditions and developmental stages limit comparisons between existing CXCR5+CD8 T cell studies. We demonstrate that CXCR5+PD-1+ CD8 T follicular (Tfc) cells comprise a functionally non-redundant subset of CXCR5+ CD8 T cells in systemic autoimmune disease. CD8 Tfc develop in the absence of functional Tregs and expand during lymphoproliferation. CD8 Tfc acquire the capacity to produce helper cytokines including co-expression of IL-21 and IL-4, and maintain capacity for cytolytic function expresing granzyme B and TNFα, suggesting the potential for functional mechanisms that diverge from CD4 Tfh cells. IL-4+IL-21+ CD8 Tfc localization in the follicle and germinal center suggest that CD8 Tfc may promote B cell activities in either location during autoimmune disease. B cell activities influenced by CD8 Tfc cells are not fully defined such as regulating somatic mutation, memory and plasma cell differentiation, and germinal center movement. However CD8 T cells promote plasma cell differentiation and antibody class switch, but do not alter germinal center B cell numbers. CD8 Tfc position coupled with cytokine production in the germinal center and follicle, and induction of activation-induced cytidine deaminase by B cells suggest a dominant helper-like role with cytolytic potential.
Evaluation of Anti-nuclear Antibodies (ANA) Patterns for aging population in Hong Kong
Authors: Lau Kwun Hung , Do Yan Tung , Chan Yat Hung , Fong Wing Long , Chau Sui Ching , Cheng Wai Wan , Chan Pui Yu , Yau Kin Nam
Published: February 2023

Objective

Our aim was to investigate the association among aging population, sex and Antinuclear Antibody (ANA) patterns expressed in Hong Kong population for improving the autoimmune diagnosis by using ANA HEp-2 slides under fluorescent microscope.

Methods

A total of 50 blood samples from aging population in both males and females were collected and examined under fluorescent microscope. In this research, a HEp-2 complete Kits (Bio-Rad Kallestad) with the methodology of indirect fluorescent antibody (IFA). Positive ANA patterns classified in International Consensus on ANA Patterns (ICAP) are distinguished.

Results

In this experiment, we obtained that the number of positive samples was 43 (86%), the number of suspected (weak) positive samples was 9 (21%) and the number of negative samples was 7 (14%). In 43 positive samples, 24 out of 27 females (88.9%) and 19 out of 23 males (82.6%) were aged between 70 – 91. 21 in 43 positive samples in the result were within age group 70–78 (48.8%). 17 in 43 positive samples in the result were within age group 80–88 (39.5%). 5 in 43 positive samples in the result were within age group 90–92 (11.6%). The most frequent positive ANA patterns included AC10 – (7 patients, median age = 81, 20%), AC19 – (6 patients, median age = 82, 17%), and AC7 – Few Nuclear Dots (5 patients, median age = 84, 15%). We found that ANA positivity rate was significantly higher in female than the male in Hong Kong (6.3%).

Conclusion

Our data indicated that female aged 70–78 is more likely to result higher positivity rate of ANA than male. According to data of our study, this significantly supports the further study of the trend in the aging population who are easier to have an ANA related autoimmune disease to take better care for elderly in Hong Kong.
BTLA signaling is needed in newly generated T cells to block autoimmunity
Authors: Adegoke Adeolu , Thangavelu Govindarajan , Anderson Colin C
Published: February 2023

Radiotherapy and/or chemotherapy in cancer treatment causes lymphopenia, which favors autoimmunity. Also, coinhibitory receptor blockade triggers immune-related adverse events in more patients than it cures of cancer. Having shown that PD-1 coinhibitory receptor is required in newly generated T cells (NGTC) to prevent lymphopenia-induced autoimmunity, our objective was to determine if the coinhibitory receptor BTLA is also required to establish tolerance in NGTC during lymphopenia. NGTC from thymocytes or established T cells from splenocytes of wildtype (WT) or BTLA−/− B6 mice were adoptively transferred to lymphopenic Rag−/− B6 mice. Sorted WT or BTLA−/− CD8 or CD4 single-positive (SP) thymocytes were adoptively transferred to Rag−/− mice not expressing MHC II (CiiTA−/−) or lacking MHC I (KbDb−/−), respectively. Disease was assessed by macroscopic appearance and weight loss. About 65% (5/8) recipients of BTLA−/− NGTC developed overt autoimmunity, while 4/4 recipients of BTLA−/− splenocytes were disease-free. 10/10 recipients of BTLA−/− CD8 SP T cells showed progressive weight gain. In contrast, 11/11 BTLA−/− CD4 SP T cell recipients had diarrhea, piloerection, hunched appearance, and progressive weight loss from 3–8 weeks post thymocyte transfer (p<0.0001). 3/11 BTLA−/− CD4 SP T cell recipients developed dermatitis post thymocyte transfer. Overt autoimmunity was evident in all WT Rag−/− (n=11) and Rag−/− KbDb−/− (n=4) recipients of thymocytes, but not in any of the CiiTA−/− recipients (n=4). There were no signs of disease in the recipients of WT NGTC. Our study suggests that BTLA is required to establish tolerance in NGTC. Disease in BTLA−/− thymocyte recipients was mediated by the CD4+ T cells and was dependent on MHC II.
IL-4 and IL-13 act on immune and epithelial cells to promote gastric metaplasia during autoimmune gastritis
Authors: Noto Christine N , Bockerstett Kevin A , Jackson Nicholas M , Hoft Stella G , Mollman Johnathan , Ford Eric L , DiPaolo Richard J
Published: February 2023

Chronic inflammation in the stomach can lead to peptic ulcers and is the highest independent risk factor for developing gastric cancer. Autoimmune gastritis (AIG) and infection with Helicobacter pylori are the two major causes of chronic gastritis. Cytokines released during chronic inflammation likely regulate the degree of tissue damage and the emergence of metaplasia, a precancerous condition believed to be the origin of gastric cancer. We recently discovered that mice with AIG that lack a receptor for IL-4/IL-13 (Il4ra−/−) developed mild gastritis but failed to develop gastric metaplasia. The objective of this study was to determine how IL-4 and/or IL-13 regulate disease development and severity. Immune assays revealed that autoantibody titers were high in control mice with AIG yet were nearly absent in Il4ra−/− mice with AIG. Epithelial cell assays revealed that both IL-4 and IL-13 also act on the gastric epithelium to promote growth and induce transcriptional changes associated with metaplasia. Finally, neutralizing antibodies to IL-4 and IL-13 were effective at ameliorating disease and preventing gastric metaplasia in mice with AIG. Together these data demonstrate that IL-4 and IL-13 play critical and complex roles in exacerbating gastric disease severity and metaplasia development by increasing production of tissue-damaging autoantibodies and inducing gastric metaplasia. These novel findings identify IL-4 and IL-13 as potential therapeutic targets for treatment of diseases induced by chronic gastritis.
RNA m6A methylation guides IL-17-driven autoimmunity through IMP2-dependent regulation of C/EBP transcription factors
Authors: Bechara Rami , Amatya Nilesh , Bailey Rachel D. , Li Yang , Aggor Felix EY , Li De-Dong , Jawale Chetan V. , Jawale Chetan V. , Coleman Bianca M. , Dai Ning , Gokhale Nandan S. , Taylor Tiffany C. , Horner Stacy M. , Poholek Amanda C. , Biswas Partha S. , Gaffen Sarah L.
Published: February 2023

Dysregulated activity of IL-17 underlies many autoimmune conditions, but the molecular mechanisms by which IL-17 mediates pathogenic inflammation remain poorly understood. IL-17 regulates pathogenic inflammatory genes by two key transcription factor classes, NF-κB and CCAAT/Enhancer Binding (C/EBP) proteins. Surprisingly little is known about mechanisms that activate C/EBPs. In seeking to understand how IL-17 upregulates C/EBPs, we found that IL-17 signaling enhanced Cebpd mRNA stability, concomitant with increased levels of C/EBPδ translation. In contrast, IL-17 had only a marginal inductive effect on Cebpb mRNA, yet C/EBPβ protein was strongly upregulated. Examination of Cebpb and Cebpd noncoding sequences identified consensus sites for N6-methyladenosine (m6A) modification, an epitranscriptomic mark that influences mRNA fate. knockdown of the m6A ‘writer’ METTL3 decreased C/EBP expression, which was reversed by the ‘eraser’ FTO. Moreover, we found that loss of an unusual m6A ‘reader’ IGF2BP2 (IMP2), an RNA binding protein known to control mRNA stability, impaired IL-17 induction of C/EBPs. IMP2 bound directly to Cebps transcripts, leading to enhanced Cebpd half-life and enhanced translation of both C/EBPs. Transcriptomic analysis revealed that IMP2 regulates C/EBP-dependent genes, including IL-6 and Lcn2. Lcn2 is a biomarker of autoantibody-induced glomerulonephritis (AGN), a setting of IL-17-driven inflammatory nephritis. Imp2−/− mice were resistant to AGN, which was linked to impaired upregulation of C/EPBs and Lcn2 in kidney. Thus, IL-17-induced autoimmunity is mediated through m6A-dependent post-transcriptional regulation of C/EBP transcription factors.
Adipocyte-secreted Interleukin-9 Promotes Leukemia Development and Chemoresistance to Front-line Chemotherapies
Authors: Hamilton Jamie A.G. , Pillsbury Claire E. , Lee Miyoung , Henry Curtis J.
Published: February 2023

Background

Obese pediatric patients with B-cell acute lymphoblastic leukemia (B-ALL) have inferior overall survival outcomes compared to lean patients, with obese patients having a 2.5-fold increased risk of an adverse event and an almost 4-fold greater risk of death. Emerging data suggest that the presence of factors in the obese microenvironment may directly promote chemoresistance in B-ALL cells.

Objective

Given the increasing prevalence of obesity and negative impact of obesity on cancer, in this study, we sought to determine how adipocytes impact B-ALL progression and responses to front-line chemotherapies.

Results

We found that circulating IL-9 levels were significantly elevated in obese mice fed high-fat diets compared to mice fed control diets. Furthermore, treating human B-ALL cells with recombinant IL-9 (rIL-9) promoted cell cycle progression and proliferation. Interestingly, rIL-9 stimulation of human B-ALL cells activated survival pathways, which coincide with the induction of chemoresistance to methotrexate and doxorubicin.

Conclusions

Our findings demonstrate that IL-9 levels are elevated in obese microenvironments and that this cytokine promotes B-ALL pathogenesis. In ongoing studies, we are determining how antibody-mediated neutralization of IL-9 impacts disease progression and chemosensitivity in B-ALL cells.
Distinct spatial profile of inflammatory gene expression in the brain of a mouse model of neuropsychiatric lupus
Authors: Aw Ernest , Zhang Yingying , Carroll Michael
Published: February 2023

Systemic Lupus Erythematosus (SLE) is an incurable autoimmune disease that results in central nervous system (CNS) involvement in up to 80% of patients, with clinical manifestations ranging from anxiety and fatigue to overt psychosis. However, the mechanisms and cellular components underlying these neuropsychiatric symptoms (NPSLE) remain largely unknown. An elevated type 1 interferon (IFN) signature has been commonly observed in SLE patients, particularly within the CNS of NPSLE patients (Crow et. al., 2014, Shiozawa et. al., 1992). Given the diversity of clinical CNS manifestations, we hypothesized that type 1 interferon-mediated inflammation occurs in spatially distinct regions within the CNS, resulting in differential behavioral outcomes depending on the impacted brain region. To test this hypothesis, we first characterized behavioral phenotypes in the Sle1, Yaa mouse model, and show that these mice exhibit anxiety-like, and fatigue-like behaviors consistent with the major clinical manifestations of NPSLE. To assess the spatial distribution of inflammatory gene expression, we utilized MERFISH (Moffitt et. al., 2016), a multiplexed spatial transcriptomics platform, and observed strikingly distinct patches of interferon stimulated gene (ISG) expression within the subcortical regions of Sle1, Yaa mouse brains. Preliminary single nucleus sequencing (sNuc-Seq) and in situ hybridization results implicate astrocytes and oligodendrocytes as the major cell classes enriched in these ISG patches. In summary, our results validate a mouse behavioral model of NPSLE, and show spatially distinct regions of ISG expression within the CNS, opening up a new avenue of investigation into the fundamental mechanisms of NPSLE.
Loss of isoprenylation significantly reduces IgE mediated mast cell activation
Authors: Dailey Jordan , Ballance William , Kee Sydney Ann , Jackson Kaitlyn G. , Sebti Said , Ryan John J
Published: February 2023

Allergic disease a leading cause of chronic illness in the US and accounts for billions of dollars in healthcare costs annually. Mast cells are tissue resident innate immune cells linked to allergic disease and activated by IgE and other ligands. Upon activation they release histamine, cytokines, chemokines, proteases, and lipid mediators evoking allergic symptoms. New ways of targeting mast cells could greatly benefit allergic disease therapy. Repurposing statin drugs such as Fluvastatin as a therapeutic treatment of allergic disease showed reduced allergic symptoms using in vitro and in vivo mouse models. We found the Fluvastatin suppresses IgE mediated mast cell activation by inhibiting protein isoprenylation. However, statin suppression is dependent on genetic background. To avert genetic background dependency, we tested the hypothesis that a dual farnesyl and geranylgeranyl transferase inhibitor (FGTI 2734) would consistently suppress mast cell function. We show that FGTI 2734 reduced IgE-mediated degranulation and cytokine production similar to Fluvastatin effects, but maintained efficacy on fluvastatin-resistant genetic backgrounds. FGTI 2734 also suppressed IgE-mediated anaphylaxis in vivo on multiple genetic backgrounds. Finally, inhibition of Ras, a known target of prenylation, can mimic statin effects on IgE-mediated mast cell function. These data support the hypothesis that dual farnesyl and geranylgeranyl transferase inhibitors may be an effective therapeutic treatment for mast cell-associated diseases.
Na+/H+ exchanger regulatory factor 1 (NHERF1) enhances MRGPRX2-mediated mast cell anaphylaxis
Authors: Subramanian Hariharan , Kammala Ananth Kumar , Das Rupali
Published: February 2023

Anaphylaxis is a severe life-threatening allergic reaction that is caused by mast cell hyperactivity. Two different mast cell receptors play critical roles in mediating anaphylaxis: 1) the IgE receptor that is stimulated by IgE-bound allergen and 2) the MRGPRX2 receptor that can bind to several cationic molecules and FDA-approved drugs resulting in mast cell activation. Activated mast cells degranulate to release proinflammatory mediators. We have recently identified that Na+/H+ exchanger regulatory factor 1 (NHERF1) regulates IgE-dependent mast cell activation in vitro and promotes passive cutaneous anaphylaxis (PCA) and passive systemic anaphylaxis (PSA) in vivo. In the current study, we investigated the role of this adapter molecule in affecting IgE-independent mast cell activation via the MrgprB2 (mouse orthologue of human MRGPRX2) receptor using a novel mouse strain, Cpa3-Cre+ NHERF1fl/fl that lacks NHERF1 in mast cells. PCA and PSA to compound 48/80 (a known MrgprB2 agonist) were significantly attenuated in the Cpa3-Cre+ NHERF1fl/fl mice as compared to control Cpa3-Cre− NHERF1fl/fl mice. In agreement with our in vivo data, peritoneal mast cells isolated from Cpa3-Cre+ NHERF1fl/fl mice showed a significant reduction in mast cell degranulation to the MrgprB2 ligands, compound 48/80 and substance P. Additionally, NHERF1-silenced human LAD2 mast cells showed decreased degranulation to these ligands as well. Collectively, our data suggests that NHERF1 promotes mast cell activation though the MRGPRX2/MrgprB2 receptor and determining the mechanisms through which NHERF1 modulates MRGPRX2 responses will lend insights into the development of new therapeutics for anaphylaxis and other allergic diseases.
A Basophil-Neuronal Axis Promotes Itch Flares
Authors: wang Fang , Trier Anna M. , Li Fengxian , Kim Seonyoung , Chen Zhen , Chai Jiani N. , Mack Madison R. , Morrison Stephanie A. , Hamilton Jennifer D. , Baek Jinok , Yang Ting-Lin B. , Ver Heul Aaron M. , Xu Amy Z. , Xie Zili , Dong Xintong , Kubo Masato , Hu Hongzhen , Hsieh Chyi-Song , Dong Xinzhong , Liu Qin , Margolis David J. , Ardeleanu Marius , Miller Mark J. , Kim Brian S.
Published: February 2023

Itch is an evolutionarily conserved sensation that facilitates expulsion of pathogens and noxious stimuli from the skin. However, in organ failure, cancer, and chronic inflammatory disorders like atopic dermatitis (AD), itch becomes chronic, intractable, and debilitating. Recent discoveries have implicated type 2 cell-associated cytokines such as IL-4, IL-13, and IL-31 in directly stimulating sensory neurons to promote chronic itch in AD. However, in addition to chronic itch, patients often experience intense acute itch exacerbations. Whether this form of itch represents amplification of known itch pathways or a different mechanism remains overlooked. Herein, we show that a large proportion of patients with AD harbor allergen-specific IgE and exhibit a propensity for acute itch flares. In mice, we found that while acute itch is mediated by the classical mast cell-histamine axis in steady-state, AD-associated inflammation renders this pathway dispensable. Instead, a previously unrecognized basophil-leukotriene receptor (CysLTR2) axis emerges as critical for acute itch flares. By probing fundamental itch mechanisms, our study highlights a novel basophil-neuronal circuit that may underlie a variety of pathological neuroimmune processes.
Dual role of CCL3 and follicular regulatory T cells in the control of germinal center response
Authors: Ke Fang , Benet Zachary , Grigorova Irina L
Published: February 2023

Follicular regulatory T cells (Tfrs) have been reported to play multiple roles in the control of B cells response. They repress foreign antigen-specific germinal center (GC) B cells at the peak of GC response. At the same time, under some conditions they promote GC B cell cycling in IL-10 dependent fashion and ensure optimal affinity maturation. Our previous studies suggested that CCL3 produced by GC centrocytes (CCs) promoted their direct contacts with Tfr cells and negative regulation of GC B cells at the peak of GC response. However, which fraction of CCs upregulates CCL3 expression, which receptors on Tfrs may respond to CCL3, and the long-term effect of CCL3 on GCs has been unclear. Based on the single cell and bulk qPCR analysis we found that CCL3 is upregulated in about 10% of CCs that express Myc and are undergoing positive selection. qPCR and transwell analysis revealed expression and synergistic involvement of CCR5 and CCR1 chemokine receptors on Tfr cells in their chemotaxis to CCL3. Interestingly, both an adoptive transfer and mixed bone marrow chimeras models suggest that after the peak of GC response B cell-intrinsic production of CCL3 promotes their prolonged participation in GCs and B cell affinity maturation, as well as better memory and plasmablast response. We also showed that both the negative and positive regulation of GC B cells by CCL3 was dependent on the presence of Tregs. To summarize, our studies suggest that in addition to the role of CCL3 and Tregs in the modest repression of foreign antigen-specific B cells at the peak of GC response, it is also important for optimal selection of B cells in GCs. Further studies are underway to determine the role of Tfr cells specifically in the negative and positive CCL3-mediated regulation of GCs.
Redefining the targets of the vitamin D receptor (VDR) utilizing a novel VDR reporter mouse.
Authors: Arora Juhi , Cantorna Margherita T
Published: February 2023

The vitamin D receptor (VDR) is a ligand-activated nuclear protein that regulates gene transcription. Vitamin D target cells are identified by the expression of VDR. Kidney and colon epithelial cells constitutively express high levels of the VDR. Immune cells also express the VDR; however, at significantly lower levels. Western blots show very low expression of the VDR in spleen, and lymph nodes. Gene expression studies in macrophage, B cells and T cells showed that activation for 24–48 hours increased the VDR mRNA levels. We have developed transgenic mice that express Cre recombinase within the VDR locus (VDRcre) and crossed these with a reporter line (Tdtomatofl/fl). Cre expression in the VDR locus results in a VDR +/− mice. The frequencies of immune cells were similar between VDR−/− and VDR +/− mice. The VDRcre expressing offspring had pink skin. Kidneys, small intestine and colon were also distinctly pink suggesting high expression of the Tdtomato-VDR reporter. Splenic T cells, activated with CD3e antibody showed upregulation of VDR mRNA and protein. Regardless of activation status, naive, effector or memory T cells had similar and high levels of the VDR reporter. VDR expression was detected at various stages of T cell thymic development with a 80% in the CD4-CD8- cells and 90% of CD4+CD8+ cells, 84% CD4+ and 88% CD8+ thymocytes. High expression of the VDR reporter in multiple T cell types and precursors suggests that the VDR is expressed throughout the life of a T cell. Future work will identify immune cells negative for the VDR reporter and the kinetics of VDR expression following activation in various cell types.
The C3-like molecule CD109 controls Th1 versus Th17 induction in CD4+ T cells
Authors: Merle Nicolas S , Kolev Martin , Rahman Jubayer , West Erin , Yan Bingyu , Kazemian Majid , Afzali Behdad , Kemper Claudia
Published: February 2023

Recent workdefined an unexpected and critical role for intracellular/autocrine active complement in human Th1 biology. Specifically, autocrine stimulation of CD46via TCR-induced C3b generation is needed for IFN-γ production by human CD4+ T cells. Thus, CD46-deficient patients are unable to generate normal Th1 responses. Analysis of the CD46-driven gene signature in Th1 cells revealed CD109 as a direct target of CD46. Indeed, T cells from CD46 deficient patients and from healthy donors in which CD46 expression was ablated via CRISPR-Cas9 technology failed to upregulate CD109. CD109 is aGPI-linked surface protein, mostly expressed on non-immune cells, and belongs to the complement C3 protein family. Though CD109 has been reported as a major negative regulator of TGF-β receptor signaling, direct functional activity for of CD109 on T cells remains unexplored.

Knock-out of CD109 in human CD4+ T cells with CRISPR-Cas9 technology induced uncontrolled Th1 and Th17 activation upon in vitro stimulation under non-skewing conditions. Similarly, CD4+T cells from Cd109−/− mice displayed augmented Th1 and Th17 in vitro responses and caused significantly more tissue pathology and weight loss in a T cell-transfer colitis model. Surprisingly, on T cells, CD109 does not restrain IFN-γ and/or IL-17 induction via modulation of TGF-β receptor activity. Instead, CD109 engages with a non-canonical costimulatory molecule and controls stemness- and metabolism-related signaling pathways.

Together, these data suggest that the complement-related protein CD109 serves as an important and novel molecular switch on CD4+ T-cells, where it regulates the balance between Th1 and Th17-induction pathways.
Time- and tissue-dependent roles of regulatory T cells in the immune response to HSV-2
Authors: Traxinger Brianna , Vick Sarah , Voillet Valentin , Erickson Jami , Woodward-Davis Amanda , Prlic Martin , Lund Jennifer M
Published: February 2023

Herpes simplex virus 2 (HSV-2) is a chronic sexually transmitted infection that causes recurrent genital ulcers and increases HIV susceptibility. Efforts to design a vaccine have assumed regulatory T cells (Tregs) restrain immune activation and impede viral clearance. However, we have previously shown that in a mouse model of vaginal primary HSV-2 infection, Treg removal leads to impaired T cell priming and migration. This suggests an alternative model where Treg function is location- and context-dependent. Additionally, research suggests that tissue-resident memory T cells (Trm) localized in the vaginal tract (VT) are crucial for efficient viral control, and Trm are likely regulated by local Tregs. These findings underscore the importance of understanding the specific contributions of VT Tregs during viral infection and vaccination.

We performed RNA sequencing of VT and lymph node (LN) Tregs after HSV-2 infection to reveal a highly activated transcriptional profile in VT Tregs compared to LN Tregs. Using flow cytometry, we confirmed an elevated activation and tissue residency phenotype in VT Tregs, exacerbated after HSV-2 infection. Notably, we found that VT Tregs express Granzyme B (GzmB) by both RNA-sequencing and flow cytometry. These Tregs persist in the VT up to 90 days, suggesting that they remain poised to respond to HSV-2 reencounter by restraining memory Trm to prevent tissue damage, possibly through GzmB-mediated toxicity. Tregs from healthy human VT corroborated mouse VT Treg phenotypes. These results suggest that VT Tregs are distinct and uniquely poised to respond to viral infection. Studies are ongoing to interrogate the signals that drive Treg migration into the VT and that lead to the tissue-specific Treg phenotype.
Sexual dimorphism in the antigen-specific T cell response in S. Typhimurium infections within extra-lymphoid tissues
Authors: D’Souza Shaina J , Godwin Matthew , Immethun Vicki E , Bauer David L , McLachlan James B
Published: February 2023

Differences in the immune response between the sexes, particularly in the susceptibility to and severity of infectious diseases, are well established. Females commonly generate a more robust response that is beneficial when combatting infections but leads to an increased risk of developing autoimmune disorders. While the sex-related hormone, estradiol (E2), is known to affect CD4 T cell cytokine release in vitro, less is known about how T cells differ in non-lymphoid tissues in vivo or how lymphoid tissues might influence T cell responses to infection. To address this, we intravenously infected lymphotoxin-alpha (LTa) male and female mice that genetically lack lymphoid tissues save for a small, disorganized spleen that was either left intact or removed surgically, with attenuated Salmonella enterica sp. Typhimurium (S. Tm). In infected mice lacking all lymphoid tissues, rates of survival between the sexes were similar. In sharp contrast, females with intact spleens had a significantly lower rate of survival in comparison to their male counterparts. We show that, in mice with or without intact spleens, the number of S. Tm-specific CD4 T cells are higher in male livers on day 10 post infection; however, by day 15, female mice with intact spleens had significantly increased CD4 T cell expansion in comparison to male mice with intact spleens, correlating to lower survival. This difference in T cell expansion, and the commensurate effect on survival, in the presence or absence of lymphoid tissue indicate that females may have evolved a separate ability to regulate immune responses outside of traditional lymphoid organs. These studies will provide insights into sex-based differences seen in cellular immunity against bacterial pathogens in vivo.
Impact of early like trauma on murine multiple sclerosis phenotype and development via dysregulated β1 adrenergic signaling
Authors: Khaw Yee Ming , Inoue Makoto
Published: February 2023

Environmental triggers have important functions in multiple sclerosis (MS) susceptibility, phenotype, and trajectory. Exposure to early life trauma (ELT) has been associated with higher relapse rates in MS patients; however, the underlying mechanisms are not well-defined. Using C57BL/6J mice, we show that ELT induces mechanistic and phenotypical alterations during experimental autoimmune encephalitis (EAE). ELT induces not only increases in severity and susceptibility in EAE but also induces a phenotype shift characterized by interferon-β insensitivity, severe neuronal damage in the spinal cord, in addition to membrane-bound lymphotoxin and CXCR2 involvement. To understand how ELT induces phenotype change in EAE development, we assessed sympathetic adrenergic neuron activity in immune cells of ELT mice. Using flow cytometry, we found that immune cell adrenergic receptors are chronically downregulated in ELT mice, attributable to chronic norepinephrine circulation. ELT-mediated EAE phenotypic changes are observed in control EAE mice treated with β1 adrenergic receptor antagonist. Conversely, β1 adrenergic receptor agonist treatment to ELT mice abrogates phenotype changes via restoration of immune cell β1 adrenergic receptor function. Our results indicate that ELT alters EAE phenotype via downregulation of β1 adrenergic signaling in immune cells. These results have implications for the effect of environmental factors in provoking disease heterogeneity and may enable prediction of outcomes in MS.
Cooperative ectodomain interaction among TCRαβ, CD3δɛ and CD3γɛ
Authors: Yuan Zhou , Cong Peiwen , Ge Chenghao , Natarajan Aswin , Travaglino Stefano , Krogsgaard Michelle , Zhu Cheng
Published: February 2023

The T-cell receptor (TCR) complex comprises of the ligand-binding subunit TCRαβ, and the signaling subunits CD3δɛ, CD3γɛ and CD3ζζ, with the Cα/Cβ in proximity to both CD3δɛ and CD3γɛ extracellularly. Direct measurements of the ectodomain interactions had not been successful, although they are believed to be important for TCR stability and functionality. Mechanical force has been shown to modulate TCR–ligand interactions. The TCR mechanosensor hypothesis predicts that force-encoded information may transmit from pMHC to CD3 via TCR-CD3 interaction. Evaluating ectodomain interactions among TCRαβ, CD3δɛ and CD3γɛ can help elucidate the TCR triggering mechanism and further guide the design of TCR-based immunotherapy.

Using two mechanical based assays, we were able to measure the weak two-dimensional (2D) affinities among ectodomains of human TCRαβ (2B4-LC13), and human CD3δɛ or CD3γɛ and showed catch bond formation where lifetimes of TCRαβ–CD3δɛ and TCRαβ–CD3γɛ bonds are prolonged by forces <15 pN. Remarkably, CD3δɛ and CD3γɛ bind TCRαβ cooperatively, forming more bonds that last longer when both CD3s interact with TCRαβ as a whole than the sum of either CD3 interacting with TCRαβ individually. Interestingly, these measurements are comparable to 2D affinity and force-dependent bond lifetime of the 2B4 TCR interaction with its cognate ligand K5:I-Ek, supporting their relevance to TCR function. Using molecular dynamics simulations based on a published Cryo-EM structure, we identified the formation of long-lasting CD3δɛ–TCRαβ–CD3γɛ trimolecular bonds as the structural mechanisms of the cooperativity. Our work helps explain TCR function under force and suggests strategies for engineering of TCR for immunotherapy applications.
Inhibiting Yap for improved T cell activation and anti-tumor immunity.
Authors: Cheng Nan , Stampouloglou Eleni , Federico Anthony , Monti Stefano , Szeto Gregory Lee , Varelas Xaralabos
Published: February 2023

Immunotherapies have shown promise in the treatment of cancers, but challenges such as sustained T cell activation and recruitment into tumors have prevented their wide-spread success. We have identified Yes-associated-protein (Yap), a transcriptional regulator that plays key roles in mechanotransduction, as an important immunoinhibitory protein. Yap is rapidly induced at the mRNA and protein levels in CD4-posititve and CD8-positive T cells upon activation with mechanical stimuli impacting Yap nuclear-cytoplasmic dynamics. We found that T cell specific deletion of Yap results in enhanced T cell activation, differentiation, and function, which translates in vivo to an improved ability for Yap-deleted T cells to repress solid tumor growth. Strikingly, Yap-deleted T cells showed an enhanced ability to infiltrate solid tumors. Gene expression analyses of Yap-deleted tumor-infiltrating T cells demonstrates Yap as a mediator of T-cell responses in the local tumor microenvironment, and implicates Yap as a negative regulator of T-cell tumor infiltration and patient survival in diverse human cancers. Our observations indicate that Yap plays critical roles in T-cell biology and suggest that Yap inhibition may offer a method for improving T cell recruitment and activation in therapies for solid tumors.
CD5 regulates the heterogenous depot of NFκB available for T cell activation
Authors: Matson Courtney Alexandra , Choi Seeyoung , Livak Ferenc , Love Paul E , Singh Nevil J
Published: February 2023

T cells bearing receptors to the same antigen often differ considerably in their ability to respond. We hypothesized that variations in intracellular signaling molecules in T cells may underlie these cell-to-cell heterogeneities. We found that the expression levels of PLCγ and IκBα, varied considerably between individual peripheral T cells. The ~1000-fold variation in IκBα levels, but that not of PLCγ, strictly correlated with surface levels of CD5. CD5 is typically used as a marker for T cell self-reactivity and differences in responses between CD5-high and -low cells is attributed to the self-awareness of the TCR. Our data suggested that CD5 directly regulates the NFκB pathway to modulate T cell responses. Accordingly, ablating CD5 using germline or conditional knock-out mice resulted in a significant reduction in IκBα. This regulation was independent of SHP-1 and casein kinase II and not evident in mRNA levels, suggesting a previously unknown post-translational mechanism. Overexpression of CD5 in TCR-deficient cells also increased basal IκBα levels indicating independence from TCR signaling. Intriguingly, cells with higher CD5 and IκBα expression maintained a proportionally greater amount of NFκBp65 in a cellular reservoir. This suggests that CD5-high T cells with a greater NFκB reservoir might allow for a more robust NFκB-dependent gene expression. Consistent with this postulate, we found that CD5-high thymocytes survived ex vivo better than CD5-low ones, but only when NFκB signaling was intact. These data suggest a key role for CD5 in the control of T cell heterogeneity, independent of the self-reactivity of a TCR itself.
T cell stemness and ST6Gal-I mediated sialylation
Authors: Harrington Laurie E , Shin Boyoung , Anderson Ashlyn E , Ingram Jennifer T , Bellis Susan , Zajac Allan J
Published: February 2023

Populations of both CD4 and CD8 T cells with stem-like properties have been documented in the setting of autoimmunity, chronic infection, and cancer. These cells exhibit the capacity to self renew, as well as serve as progenitors to more differentiated T cell phenotypes, but the cell intrinsic factors that promote the development and maintenance of T cell stemness are not well known. In a mouse model of chronic intestinal inflammation, we observed that a subset of effector CD4 T cells displayed many of the attributes consistent with T cell stemness; these cells expressed the key stemness transcription factor TCF1, could persist long term in vivo, and further transitioned into terminal effector cells. Importantly, these stem-like effector CD4 T cells were able to both sustain and confer intestinal inflammation. To discern molecules that contribute to the stemness phenotype, we employed gene set enrichment analyses with multiple data sets and identified the glycosyltransferase ST6Gal-I, which facilitates the addition of a2,6-linked sialic acid moieties to surface N-glycans. We found that this sialyltransferase was preferentially expressed in stem-like CD4 T cells during colitis and CD8 T cells during chronic lymphocytic choriomeningitis virus infection, and that these cell populations displayed a2,6-linked sialic acids on the cell surface. Moreover, in the colitis model, we observed that genetic deletion of ST6Gal-I in T cells correlated with diminished TCF1 expression and reduced inflammation. Furthermore, constitutive expression of ST6Gal-I by T cells was associated with the ability to promote intestinal inflammation. Together, these data highlight ST6Gal-I as a potential regulator of the T cell stemness phenotype.
Serum- and feeder-free culture expansion of human peripheral blood NK cells
Authors: Le Fevre Tim , Ang Elaine , Wognum Albertus W , Szilvassy Stephen J , Louis Sharon A , Eaves Allen C , Tabatabaei-Zavareh Nooshin
Published: February 2023

Natural killer (NK) cells are critical effectors of innate immunity that secrete proinflammatory cytokines and kill tumor cells and virus-infected cells. The ability to expand human NK cells in culture provides a ready source of cells for disease modeling, development of immunotherapies, and basic research. We have developed a culture system that supports the expansion of primary NK cells in the absence of feeder cells and serum. NK cells were enriched from peripheral blood using EasySep™ negative selection and cultured in serum-free medium with interleukin-2 in plates coated with a NK cell ‘activation coating matrix’ for 14 days. Cells were passaged with medium changes on days 7 and 11, and were harvested on day 14 for assessment or functional characterization. The average frequency of CD56+CD3− NK cells was 92% (range 80 – 97%, n=16) with an average expansion of 121-fold (range 10 – 274). On average 79% (48–92%) of expanded NK cells expressed CD16. The ability of expanded NK cells to produce interferon-γ (IFN-γ) and to degranulate were tested after stimulation with phorbol 12-myristate 13-acetate (PMA) + ionomycin or co-culture with K562 cells. As detected by intracellular flow cytometry, the average frequency of IFN-γ+ NK cells was 56% (range 41 – 63%, n = 4) and 40% (range 12 – 55%) when stimulated by PMA/ionomycin or K562 cells, respectively. Similarly, the average frequency of degranulated NK cells as detected by surface expression of CD107a, a lysosomal-associated membrane protein, was 69% (range 35 – 84%, n = 4) and 53% (range 18 – 75%), respectively. These results show that NK cells can be expanded and stimulated under serum- and feeder-free conditions to generate large numbers of functional NK cells for basic and translational research.
A unique multiplex assay to profile growth factors and cytokines in CSF of MS and ALS patients
Authors: Sastry Amani , Madiraju Charitha , Krajewska Maryla , Krajewski Stan , Tomik Barbara , Szczudlik Andrzej , Matson Robert
Published: February 2023

Growth factors and cytokines play a critical role in the pathogenesis of multiple sclerosis (MS) and amyotrophic lateral sclerosis (ALS). The shift in the balance between pro-inflammatory and anti-inflammatory cytokines contributes toward autoimmunity. Epidermal growth factor (EGF) and fibroblast growth factor (FGF) are elevated in the cerebrospinal fluid (CSF) of MS and ALS patients. Identification of relevant biomarkers helps in the understanding of the disease pathogenesis. The goal is to establish a sandwich ELISA-based multiplex assay to assess growth factor and cytokine levels in CSF samples from MS and ALS patients. Capture antibodies against EGF, FGF, IFN-gamma, IL-6, IL-1beta and IL-4 imprinted in a multiplex array format into each well of a 96-well plate. Arrays subjected to multiplex sandwich ELISA of CSF samples. Test samples included CSF from Relapsing-Remitting MS (RRMS, N = 10), Secondary Progressive MS (SPMS, N = 10) and ALS patients [N=10]. Control samples included CSF from healthy subjects [N=10]. Colorimetric read-outs from multiplex ELISA are quantified using ImageJ analysis of standards and samples. Results suggest that multiplex array designed for ELISA is a unique, robust, and a cost-effective method for profiling growth factors and cytokines present in biological matrices.

[*In memoriam: abstract submitted as a tribute to honor Dr. Barbara Tomik for her valuable contributions towards the collection and preparation of patient samples].
Linking immune receptor sequence to transcriptional states with clonotype neighbor graph analysis (CoNGA)
Authors: Schattgen Stefan A , Guion Kate , Crawford Jeremy Chase , Souquette Aisha , Barrio Alvaro Martinez , Stubbington Michael , Thomas Paul Glyndwr , Bradley Philip
Published: February 2023

Recent advances now allow for deep simultaneous profiling of T cell clonotypes, defined by T cell receptor (TCR) sequence, and phenotype, as reflected in gene expression (GEX) profile, surface protein expression, and epitope binding at the single-cell-level. However, there currently few tools available for unsupervised discovery of relationships between TCR sequence and cell phenotype. We hypothesized that by identifying correlations between “TCR neighborhoods”, defined by shared TCR sequence and GEX features, we could move beyond simply measuring GEX variation within clonal descendants and identify novel associations between T cell specificities and states. Previously, we introduced TCRdist, a measure for assessing inter-TCR similarity capable of identifying closely related clonotypes based on shared sequence features. Using TCRdist to quantify TCR similarity, we developed a graph-theoretic approach—clonotype neighbor-graph analysis or “CoNGA”—that identifies correlations between GEX profile and TCR sequence in an unbiased and automated manner through statistical analysis of GEX and TCR similarity graphs. Applying CoNGA, we uncovered novel associations between TCR and GEX space including a previously undescribed “natural lymphocyte” population of human blood CD8+ T cells; an association between TRBV gene usage and EPHB6 expression; and TCR sequence determinants of differentiation in thymocytes. These examples demonstrate that CoNGA is able to effectively deconvolve complex relationships between TCR sequence and cellular state. Conceptually, CoNGA could be extended to other clonally-related populations (e.g. B cells, tumors), and can easily incorporate other measurable features (e.g. ATAC-Seq).
Development of a 30-parameter flow cytometry panel for functional characterization of unconventional T cells and innate lymphoid cells
Authors: Iftikhar Yaser , Liechti Thomas , Roederer Mario
Published: February 2023

Unconventional T cells, which include mucosal-associated invariant T (MAIT) cells, invariant natural killer T (iNKT) cells, and γδ T cells, are defined by their recognition of antigens and microbial products, such as vitamin B metabolites, presented on non-classical MHC molecules. These “innate-like” cells have garnered attention for their unique potential for bridging the gap between innate and adaptive immunity, although their exact roles during infection is still not well-understood.

To evaluate the phenotypic and functional characteristics of these cells, we developed and optimized a comprehensive, 30-parameter flow cytometry panel which delineates these cell subsets on the basis of surface marker expression. In addition, we simultaneously assess effector functions through the measurement of intracellular markers for cytokines, activation molecules, and cytotoxic molecules, following stimulation. We also included markers for the evaluation of natural killer (NK) cells and innate lymphoid cells (ILCs), as they play distinct roles at this junction of innate and adaptive immunity. We optimized our panel in combination with innate, pan-T cell (IL-12/IL-18) and subset-specific (e.g. HMBPP for Vγ9 T cells) stimulation reagents to evaluate responses to distinct innate-like and adaptive immune stimulation modes.

We will use this flow cytometry panel to assess the immune profiles of various cohorts, including individuals who are cytomegalovirus (CMV) positive and human immunodeficiency virus (HIV) positive, in order to further elucidate how different acute and chronic infections can alter the frequencies, phenotypes, and functional characteristics of these cells.
Analysis of the structure and dynamics of synaptic interface revealed functional anomaly of naive CD8+ T cells during HIV infection
Authors: Anikeeva Nadia , Steblyanko Maria , Kuri-Cervantes Leticia , Buggert Marcus , Betts Michael , Sykulev Yuri
Published: February 2023

While it is thought that HIV infection leads to a global defect in T cells, effect of the infection on the T cells at various differentiation stages has not been studied. We have exposed freshly isolated polyclonal CD8 T cells at various differentiation stages to planar lipid bilayers that display anti-CD3 antibody and soluble ICAM-1 and analyzed morphology and dynamics of the synaptic interface formation by the T cells, parameters that are linked to their functional activities. We have found that a large fraction of purified naïve CD8 T cells from HIV-infected people developed mature synapses exhibiting atypical degranulation capacity. These data provide evidence that even at the earlier stages of differentiation CD8 T cells derived from HIV-infected patients reveal functional anomaly causing changes in the ability to degranulate. The latter mediated by aberrant functioning of integrins and actin cytoskeleton resulting from continuous inflammation during chronic HIV infection. Functional anomaly of naïve T cells further impairs functions of CD8 T cells at later differentiation stages contributing to a global defect in T cells and failure to control the infection.