bluuebunny/crossref_metadata_2025 huggingface dataset:
STAT1 is required for Tfh lineage maintenance through regulation of IL-21 expression
Authors: Nurieva Roza I , Sahoo Anupama , Alekseev Andrei
Published: January 2023

T follicular helper (Tfh) cells are a specialized subset of T helper cells necessary for germinal center reaction, affinity maturation, and the differentiation of germinal center B cells to antibody-producing plasma B cells and memory B cells. The differentiation of Tfh cells is a multistage, multifactorial process involving a variety of cytokines, surface molecules and transcription factors. While to date early signaling events for fate committed differentiation of Tfh cells are sufficiently studied, mechanisms allowing Tfh cells to maintain their commitment/programming are still unclear. Here we report that STAT1 deficiency in CD4+ T cells leads to diminished number of Tfh cells at day 7 and 14 days after antigen immunization, while at day 3–4, number of Tfh cell were comparable between STAT1-sufficient and STAT1-deficient mice, suggesting the role of STAT1 in Tfh lineage maintenance rather than in Tfh cell priming. Interestingly STAT 1 deficiency in both mouse and human Tfh cells leads to significant drop in IL-21 expression, while the expression of Tfh-specific genes such as Bcl6, CXCR5 and c-Maf was not affected. Functionally, STAT1 in cooperation with STAT3 triggers IL-21 production in Tfh cells by directly binding to and activation of the IL-21 gene locus. In addition, STAT1/STAT3 cooperation influences the expression of transcriptional factor Batf as well as determines Batf-driven IL-21 expression in Tfh cells. Our results thus indicate STAT1 dependent mechanism for IL-21 expression and Tfh lineage maturation and maintenance.
IL2 production predicts Tfh differentiation
Authors: DiToro Daniel F , Winstead Colleen J , Andargachew Rakieb , Zindl Carlene , Moseley Carson Edward , Pham Duy , Evavold Brian D , Weaver Casey Todd
Published: January 2023

Naïve CD4 T cells differentiate into functionally distinct subsets in response to a set of extrinsic and intrinsic cues. Effector subsets, including Th1, Th2 and Th17 CD4s, are required for optimal cellular responses to invading pathogens, while class switched antibody responses depend on the differentiation of T follicular helper cells (Tfh). Development of both Tfh and non-Tfh occurs in parallel and involves two distinct but functionally related expression bifurcations. Upon formation of T cell receptor (TCR)/major histocompatibility (MHC) synapses with antigen-presenting cells (APC), a discrete subset of activated T cells secretes the cytokine IL2. Similarly, a portion of activated cells express the transcription factor Bcl6 and differentiate into Tfh cells, while the remainder express Blimp1 and develop into effector CD4s. However, the cellular, temporal and mechanistic relationships between these two bifurcations remain unclear. Here we show that the IL2 and Bcl6/Blimp1 expression bifurcations occur simultaneously, precede cell division, predict differentiation into Tfh and non-Tfh subsets, and are mechanistically related to antigen availability TCR density.
Characterization of memory CD4+ T cells in skin following infection
Authors: Collins Nicholas , Gebhardt Thomas , Carbone Francis R
Published: January 2023

Following elimination of virus, stable populations of memory T cells that rapidly respond to secondary infection are generated. A substantial proportion of the memory T cells in the body are found within barrier tissues such as the skin. It remains unclear to what extent the cells in these compartments exchange with those in circulation versus the proportion remaining permanently resident within the tissues. We have examined this for CD4+ T cells in mouse skin. Using parabiosis, we have found that the majority of CD4+ T cells in skin at steady-state are in equilibrium with the blood. A relatively small proportion of skin CD4+ T cells persist in skin for several months without returning to the circulation. At memory following localized infection with HSV-1 or following treatment with the sensitizing agent DNFB, the number of CD4+ T cells in skin does not return to the naïve or baseline levels. Rather, the number of CD4+ T cells in the affected region remains stable over time at a level 6–8 fold higher than in unaffected skin from the same mice. This increase in CD4+T cell numbers is a consequence of enhanced recruitment from the blood and/or prolonged persistence of recruited circulating T cells once in skin before again returning to the circulation. Hair follicle-derived chemokines are involved in retaining CD4+ memory T cells in skin and the majority of CD4+ T cells that produce IFNg in response to secondary HSV-1 infection show perifollicular localization. Overall, this characterization of the CD4+ T cell content in skin following infection and contact sensitization provides an insight into how these cells are retained in skin for enhanced response on subsequent antigen encounter.
Natural killer cells modulate B-cell affinity maturation in an antigen dose-dependent manner
Authors: Rydyznski Carolyn E. , Cranert Stacey A , Waggoner Stephen N
Published: January 2023

Affinity maturation via somatic hypermutation (SHM) in germinal centers (GC) is crucial for development of protective humoral immunity. Our discovery that natural killer (NK) cells potently inhibit GC expansion highlights a novel target for improving humoral responses during vaccination. We hypothesized that enhanced GCs in the absence of NK cells would promote increased affinity maturation via SHM. Using the immunogen, NP-KLH, we show that depletion of NK cells (ΔNK) prior to immunization results in higher numbers of follicular helper T (Tfh), GC B, and NP-specific IgG1+ B cells. Sera titers of NP-specific IgG and IgG1 were also elevated in ΔNK mice compared to controls. Unexpectedly, B-cell receptor (BCR) sequencing of NP-specific GC B-cell clones revealed a marked reduction in the total number of BCR mutations in B cells from ΔNK mice at 2 to 4 weeks post-immunization. This appeared to be independent of the generation of the affinity-increasing W33L mutation. We speculated that the increase in Tfh and GC B cell numbers may exceed antigen availability to fuel continued SHM. Thus, we gave a second dose of NP-KLH a week after the first injection. Tfh and GC B cells were still increased in ΔNK mice following a second dose. Yet in striking contrast to the extent of SHM following one dose, re-injection of antigen promoted a roughly two-fold increase in both the total number of mutations and the proportion of clones bearing the W33L mutation when NK cells were absent. These exciting results suggest that NK cells restrict the magnitude of GCs and qualitatively shape the humoral response via antigen dose-dependent effects on SHM. Thus, NK cells are a previously overlooked means of humoral regulation that might be modulated to improve vaccine responses.
Stimulation through innate immune receptors selectively upregulates co-receptor expression on B cells
Authors: Dhoonmoon Ashna , Horton Christopher G
Published: January 2023

T lymphocytes require two signals for appropriate activation – signal one is obtained via antigen presentation to the T cell receptor and signal two is provided by a co-receptor. In the absence of signal two, T cells remain unresponsive, thus these accessory proteins serve an essential purpose in an immune response. Co-receptors include two primary classes: co-stimulatory molecules and co-inhibitory molecules. Co-stimulatory molecules including CD80 and CD86, interact with CD28 to promote T cell proliferation and survival, while co-inhibitory molecules such as B7-DC, B7-H3 and B7-H4 limit the strength and duration of immune responses. In addition to orchestrating an immune response against pathogens, co-receptors have also been implicated in several diseases. Numerous studies have revealed abundant expression of inhibitory co-receptors in tumor microenvironments, contributing to tumor immunosuppression. Furthermore, lack of inhibitory signals in mouse models leads to lupus-like autoimmunity. While the rudimentary functions of these receptors are somewhat clear, the contexts in which they are expressed are incompletely understood. In this study, we aimed to fill this knowledge gap by investigating the effects of innate immune stimuli on expression of B7-family co-receptors. Our data demonstrate that stimulation of mouse splenocytes with synthetic Toll-like receptor agonists leads to selective alterations in co-receptor expression, most notably among B lymphocytes. These data suggest an avenue by which detection of conserved microbial motifs may modulate adaptive immunity through regulation of co-receptor expression on antigen presenting cells.
Dissecting T follicular helper cell development in vivo using CRISPR
Authors: Huang Bonnie , Phelan James D. , Webster Dan E. , Shaffer Arthur L. , Schwartzberg Pamela L.
Published: January 2023

T follicular helper (Tfh) cells are specialized CD4 helper T cells that signal B cells to produce high affinity antibodies and provide long-term humoral immunity. Identifying genes that govern Tfh differentiation and function could provide targets for improving vaccines and therapies for autoimmunity. However, Tfh differentiation is only partially understood, and not fully recapitulated in vitro. To discover novel Tfh-regulating genes, we developed a CRISPR-based functional genetic system to mutate genes in primary mouse T cells, and measured the effects on Tfh cells in vivo.

Transgenic Cas9-expressing mouse CD4 T cells were transduced in vitro with an optimized retroviral vector encoding an sgRNA and GFP. With a guide targeting Tcf7 (encoding a Tfh-essential transcription factor Tcf1), >90% cells could be transduced; up to >80% of GFP+ cells were Tcf1neg by flow and mutational analyses. To evaluate the functional consequence of CRISPR editing, we adoptively transferred virus-specific CD4 T cells that were transduced with either a control or Tcf7 sgRNA construct into wild-type hosts. After viral challenge, control T cells differentiated into both Tfh and non-Tfh cells, while very few Tcf7-edited T cells became Tfh cells. Similarly, guides against the Tfh master transcription factor Bcl6 abrogated Tfh differentiation in vivo. Next, we constructed sgRNA libraries of up to 400 guides, targeting up to 80 genes, including ones implicated in primary immunodeficiencies and druggable targets. NGS analysis of T cells post-viral challenge revealed depletion of multiple guides targeting known Tfh-required genes in the Tfh vs non-Tfh populations, validating this approach. We are currently following up on potential hits from these screens.
The importance of the NFAT5/TonEBP-mediated osmotic stress response in B cells
Authors: Rokvic Ljiljana , Titze Jens , Schuh Wolfgang , Jäck Hans-Martin
Published: January 2023

Introduction

Hyperosmotic stress is well investigated within renal borders, many non-renal tissues are facing fluctuations of osmolality as part of normal, physiological behavior. For example, compared to blood, osmolality of lymphoid tissues, like spleen and thymus, is substantially hypertonic. Therefore, the ability of immune cells to respond and adapt to osmotic stress is crucial for normal immune responses. The osmotic stress response is driven by nuclear factor of activated T cells (NFAT5)/tonicity enhancer binding protein (TonEBP), the only known mammalian osmosensitive transcription factor. It has been shown that mice with a heterozygous deletion of NFAT5 display impaired adaptive immune responses. To specifically analyze the functions of NFAT5 in B lymphocytes, we generated conditional B cell specific knock-out mice.

Objectives

Using a conditional B cell-specific NFAT5 knock-out mouse model, we want to assess the importance of NFAT5 for B cell development, B cell activation and antibody production.

Results

NFAT5-deficient mice fed with high salt diet (HSD; 4% NaCl in food and 0.9% NaCl in water) and immunized with TNP-KLH showed impaired IgM and IgG responses compared to wildtype controls. Accordingly, frequencies and numbers of PC and GC B cells were reduced in immunized NFAT5-deficient mice. In support of these findings, we found lower cell numbers and diminished viability of LPS-stimulated NFAT5-deficient B cells cultured in hypertonic medium (+40mM NaCl).

Conclusion

NFAT5 in B lymphocytes controls antibody responses by controlling B cell activation and survival. Gene expression profiling will now elucidate transcriptional programs and signaling pathways involved in NFAT5-controlled B cell responses.
LVS vaccination allows BALB/c mice to survive virulent F. tularensis pulmonary challenge but does not induce sterilizing immunity
Authors: Soucy Alicia , Metzger Dennis W
Published: January 2023

There is no licensed vaccine for protection against virulent strains of Francisella tularensis, an intracellular pathogen that replicates within lung macrophages and causes lethal acute infection in mice at an intranasal challenge dose of ~10 CFU. Immunization with the attenuated F. tularensis LVS strain provides some level of protection against virulent F. tularensis SchuS4 infection; however, the basis for this induced resistance remains unknown. We found that LVS vaccination allowed complete survival of BALB/c mice (but not C57Bl/6 mice) from lethal SchuS4 pulmonary challenge for 30 days, and 50% survival for at least 100 days, yet the surviving mice contained large numbers of SchuS4 organisms in their lungs and spleens (~104 CFU/organ). Early after challenge, resistant BALB/c mice produced greater amounts of Th17-associated cytokines compared to susceptible C57Bl/6 mice. However, IL-17 and IL-22 did not appear to be important for restricting chronic infection. On the other hand, depletion of either CD4+ or CD8+ cells three weeks after SchuS4 challenge caused resistant BALB/c mice to succumb to infection within one week. Similarly, treatment of vaccinated BALB/c mice with anti-IFNγ monoclonal antibodies three weeks after SchuS4 led to fatal acute tularemia. We conclude that LVS vaccination of BALB/c mice induces an immune response that allows the animals to constrain and tolerate a chronic SchuS4 infection. Long-term resistance is dependent on CD4+ cells, CD8+ cells, and IFNγ.
CD4+ T cells prevent abacavir hypersensitivity reactions in HLA-B*57:01 transgenic mice
Authors: Cardone Marco , Garcia Karla , Yano Masahide , Mattson Elliot , Juengst Leslie , Roderiquez Gregory , Tilahun Mulualem E , Boyd Lisa F , Natarajan Kannan , Puig Montserrat , Margulies David H , Norcross Michael A
Published: January 2023

Adverse drug reactions (ADR) are a major obstacle to drug development. Genome-wide association studies identified several human leukocyte antigens (HLA)-class I alleles as risk factors for ADR. The HLA-B*57:01 allele has been found to be associated with the development of abacavir (ABC) hypersensitivity syndrome (AHS) and with the induction of liver injury by the β-lactam antibiotic flucloxacillin. The nucleoside analog ABC, an inhibitor of the HIV reverse transcriptase, can induce severe multi-organ toxicity in >50% of HLA-B*57:01+ patients with HIV infection. In a previous study we showed that ABC induced binding to the HLA-B*57:01 of altered self-peptides containing predominantly isoleucine or leucine residues at the carboxyl terminus. Recognition of these self-peptides drives in vitro activation of cytotoxic CD8+ T cells. However, the early immune events/danger signals required to overcome the immune tolerance that otherwise suppress ADR are still unknown. In order to study HLA-linked drug hypersensitivity in vivo and to understand better drug immune tolerance, HLA-B transgenic mice were generated. Here, we show that ABC activates HLA-B*57:01 transgenic CD8+ T cells in vitro via CD8 and HLA. A systemic, but partial, early response to the drug is also observed in vivo, however, ABC fails to promote skin hypersensitivity in immunocompetent transgenic mice. Instead, depletion of CD4+ T cells breaks immune tolerance to ABC and promotes ADR in HLA-B*57:01 transgenic mice.

Supported by the Intramural Research Program of the NIAID, NIH, and CDER, FDA.
Functions of the ‘alarmin’ cytokine IL-25 in house dust mites-induced allergic asthma-like chronic pulmonary inflammation
Authors: Etienne Estefania , Wang Hongshan , Tang Wanhu , Ha Hye-Lin , Siebenlist Ulrich
Published: January 2023

Allergic inflammatory lung disease is a complex immune response orchestrated by many cytokines, chemokines and other factors signaling danger. One of these is the alarmin IL-25, an epithelial cell-derived cytokine secreted at mucosal sites in response to various allergens. Although IL-25 is a member of the IL-17 cytokine family and it is the only member to promote type-2 responses. IL-25 activities overlap with those of IL-33 and TSLP, two other cytokines secreted by mucosal epithelial cells; all three activate innate lymphoid cells type 2 (ILC2s).

We set out to elucidate unique/significant roles of IL-25 in the chronic asthma-like lung inflammation model induced by repeated exposures to house dust mites. If unique/significant contributions could be identified in this physiologically relevant model, IL-25 could be a potential therapeutic target in allergic asthma. To carry out these studies we generated mice with global or conditional deletion of IL-17RB and CIKS (a.k.a. Act-1, Traf3ip2). Surprisingly we found that blocking IL-25 signaling in lung CD11c+ cells reduced expression of IL-9 and IL-13, cytokines that are critical in asthma and that derive primarily from Th9 and Th2 cells, respectively, during the chronic stages. IL-25 signaling in CD11c+ cells also contributed significantly to mast cell accumulation and ultimately lung remodeling. We will present investigations of potential mechanisms underlying the effects of IL-25 signaling in CD11c+ lung cells. Our data demonstrate that IL-25 signaling in CD11c+ cells makes physiologically important contributions in the chronic HDM-induced model of allergic asthma, findings that should encourage future studies of this cytokine as a therapeutic target.
Cognate CD40-CD40L interaction maintains thymic B cells in absence of TCR-MHC interaction between thymic T and B cells
Authors: Alkhaleel Farrah , Fujihara Chiharu , Rivera Hiraldo Luis E. , Williams Joy A. , Hodes Richard J.
Published: January 2023

We previously demonstrated that maintenance of the thymic B cell population requires cross-talk between thymic B cells and T cells, analogous to the cross-talk that had been demonstrated between thymic epithelial cells and developing T cells. Maintenance of thymic B cells specifically requires interaction between CD40 ligand (CD40L) and cell autonomous CD40 on thymic B cells. Co-stimulatory interactions such as those mediated by CD40 commonly exert their effects in concert with pMHC-specific T cell receptor (TCR) recognition. We have therefore analyzed the requirement for MHC-TCR interaction for thymic B cell maintenance. Analysis of mixed bone marrow (BM) chimeras constructed from MHCI/MHCII double knockout (DKO) and WT donor BM revealed that there is no requirement for expression of MHCI or MHCII on thymic B cells for their maintenance. Thus, the thymic B cell population is regulated by cognate CD40-CD40L interaction in the absence of direct pMHC-TCR interaction with T cells. Experiments are currently in progress to determine whether CD40-dependent thymic B cells are sustained by direct interaction with T cells expressing membrane-bound CD40L, or by soluble CD40L in the absence of direct T cell-B cell interaction.
N-glycan branching inhibits pro-inflammatory B cell activity by controlling TLR4 and BCR signaling.
Authors: Mortales Christie-Lynn , Demetriou Michael
Published: January 2023

N-glycosylation is a critical regulator of T cell function. The branching and number of N-glycans on cell surface glycoproteins drive binding to galectins, forming a galectin-glycoprotein lattice that controls T cell growth, differentiation and development by regulating receptor clustering, distribution and endocytosis. N-glycan branching is ~1.5–2 fold higher in B cells compared to T cells, yet effects on B cell function are unknown. Here we report that branching regulates pro-inflammatory signaling via Toll Like Receptor – 4 (TLR4) and the B cell receptor (BCR). Mechanisms controlling pro-inflammatory (cell surface) versus anti-inflammatory (endosomal) TLR4 signaling in B cells is poorly understood. Loss of branching in B cells de-couples LPS induced proliferative from pro-inflammatory responses by inhibiting TLR4 endocytosis. Similar de-coupling is observed with BCR signaling, where loss of branching inhibits BCR driven proliferation and Lyn/Syk/PLCγ2/Ca2+ signaling via reduced CD19 surface expression while concurrently driving pro-inflammatory effector functions and PI3K/Akt/mTOR activation. Together, these data reveal N-glycan branching as a critical negative regulator of TLR4 and BCR triggered pro-inflammatory B cell responses.
Ezh2 is required for the division-specific epigenetic programming of plasma cell differentiation
Authors: Scharer Christopher , Guo Muyao , Haines Robert , Kania Anna , Boss Jeremy M
Published: January 2023

Following activation of naïve B cells, epigenetic remodeling is required to facilitate the induction of the plasma cell gene expression program. Ezh2 is an epigenetic repressor, catalyzing the trimetylated state of histone H3 lysine 27 (H3K27me3), and is essential for B cell development and the maintenance of a germinal center reaction. Recently, Ezh2 has been shown to directly interact with the master transcriptional regulator of plasma cell fate Blimp1. To investigate a wider role in regulating other aspects of B cell fate, a tamoxifen-inducible deletion of Ezh2 was developed, allowing deletion during distinct phases of B cell differentiation. Ezh2 was not required for mature B cell homeostasis, however; in response to differentiation signals, a cell-intrinsic reduction in antibody secreting plasma cells was observed. Additionally, Ezh2 deficient plasma cells secreted fewer antibodies, indicating a failure to fully induce the plasma cell fate. To determine the molecular processes regulated by Ezh2, chromatin accessibility and the transcriptome were profiled by ATAC-seq and RNA-seq, respectively. Ezh2 deficient plasma cells demonstrated an up regulation of inflammatory related genes and B cell lineage transcription factors. Chromatin accessibility data indicated a failure to repress B cell enhancers and promoters. Additionally, Blimp1 target genes displayed increased expression in Ezh2 deficient plasma cells providing a genetic link to the interaction of these two repressors. Together, these data indicate that Ezh2 facilitates the epigenetic repression of the B cell program and is required for full induction of plasma cell fate and function.
POLD3 Plays an Essential Role in Somatic Hypermutation
Authors: WU WENJUN , XIAO ZHIHUI , Poltoratsky Vladimir
Published: January 2023

Somatic hypermutation (SHM) increases the affinity of antibodies to antigen. While the initial steps of SHM, including development of the lesion, its excision and formation of an abasic site, are well established, the repair and mutation events are still under investigation. Multiple DNA repair pathways have been suggested for the SHM.

POLD3, as a part of a scaffold complex, connects error free or error prone DNA polymerases with the processivity factor PCNA and this interaction plays an essential role in mutagenesis and recombination.

In this study we tested the role of POLD3 in SHM. We demonstrated elevated expression of POLD3 in activated mice B cells and accumulation of this protein at DNA damaged sites. We created a Burkett’s lymphoma Raji cell clone (Raji 2C9 clone) expressing endogenous mutant POLD3 lacking C-terminus PCNA interaction site, using the CRISPR system. Functional studies shown that the Raji cells expressing mutant variant demonstrated abnormal cell cycle distribution, hyper-sensitivity to genotoxic agents and compromised SHM, compare to the control cells.

The frequency of mutations in locus encoding immunoglobulin heavy chain variable region was are five times lower in cells expressing mutant POLD3 compare to control cells.

In control cells, mutations accumulated at the RGYW/WRCY “hot spots”, and majority of mutations were C to T or G to A transitions. In cells expressing mutant POLD3 mutations accumulated outside “hot spots” and are represented by transversions. The overall results indicate that POLD3 plays an important role in SHM.
Tetraspanin 33 (TSPAN33) regulates cytoskeleton rearrangements and the Notch signaling pathway in human B cells.
Authors: Maravillas-Montero Jose Luis , Cervantes-Diaz Rodrigo , Acevedo-Ochoa Ernesto , Romero-Ramirez Sandra , Navarro-Hernandez Itze Cecilia , Santos-Argumedo Leopoldo
Published: January 2023

TSPAN33 is the last described member of the tetraspanin protein family; it is highly expressed in malignant B cells and is one of the most controversial tetraspanins on regard of its function in leukocytes. In human B cells, we observed that the expression of TSPAN33 is rapidly induced by IL-4 and anti-CD40 stimulation and its antibody-induced cross-linking reduced the cell proliferation rate. Furthermore, by over-expressing TSPAN33 in Raji B cells, we detected dramatic alterations at different membrane features of these lymphocytes. Our results show that TSPAN33 promotes the formation and extension of microvilli and membrane protrusions generated during cell spreading by altering the cortical actin cytoskeleton and the surface expression of LFA-1, CD29 and CD44. In this way, we also demonstrate that TSPAN33 participates in adhesion events such as immune synapse formation with T cells and extracellular matrix substrate adhesion. TSPAN33 belongs to the TspanC8 tetraspanin subgroup, which is associated with the metalloprotease ADAM10 regulating its traffic and activity in different cell membranes. ADAM10 mediates the proteolytic cleavage of several proteins including the Notch receptors. We observed that TSPAN33 does not just alter the expression ADAM10 but also modifies the expression and activity (measured by proliferation and target genes activation assays) of Notch receptors in B cells. Taken together, all these findings demonstrate that TSPAN33 plays a role in the cytoskeleton dynamics and Notch signaling pathway in B cells, by altering different membrane features which in turn modify the surface ADAM10/Notch interactions, regulating its signaling capacity.
PSGL-1 is a negative regulator of effector, exhausted, and memory T cells
Authors: Tinoco Roberto , Carrette Florent , Barraza Monique L , Otero Dennis C , Bradley Linda M
Published: January 2023

Effective T cell responses depend on adhesion mechanisms that guide T cells to sites of infection and inflammation. P-selectin glycoprotein ligand-1 (PSGL-1) has been shown to impact T cell migration; however, its role in the differentiation of responding T cells has not been investigated. Since PSGL-1 is highly expressed on naïve, effector, and memory T cells with varying degrees of glycosylation that impact ligand binding, we hypothesized that PSGL-1 may regulate T cell fates. Using mice deficient in PSGL-1 and three murine models we found that PSGL-1 limits the magnitude of the ensuing T cell response. PSGL-1-deficient mice had an increased accumulation of anti-viral CD4+ and CD8+ T cells after both acute and chronic LCMV infection. Virus-specific PSGL-1-deficient T cells had enhanced survival and were not hyper proliferative. Furthermore, PSGL-1-deficient T cells had superior effector function, downregulation of PD-1 and other inhibitory receptors, and enhanced viral control. The enhanced T cell response resulted in significant mortality of PSGL-1-deficient mice infected with chronic LCMV, whereas all PSGL-1-deficient mice survived the acute LCMV infection. After viral clearance, more CD4+ and CD8+ PSGL-1-deficient T cells survived to form memory cells. Interestingly, resting memory T cells from PSGL-1-deficient mice sustained PD-1 downregulation implying that PSGL-1 may regulate inhibitory receptors in memory T cells. We found that PSGL-1-deficient T cells had superior function in a tumor model and were more effective in controlling melanoma tumors. Our findings in three separate models show a previously unrecognized role for PSGL-1 in negatively regulating T cell responses and may have high therapeutic potential.
Antigen presentation profiling reveals T-cell recognition of lymphoma immunoglobulin neoantigens
Authors: Olsson Niclas , Khodadoust Michael , Wagar Lisa , Swaminathan Kavya , Haabeth Ole Audun Werner , Chen Binbin , Rawson Keith , Liu Chih Long , Steiner David , Lund Peder J. , Rao Samhita , Zhang Lichao , Marceau Caleb , Stehr Henning , Newman Aaron M. , Czerwinski Debra K. , Carlton Victoria , Moorhead Martin , Faham Malek , Kohrt Holbrook E. , Carette Jan , Green Michael R. , Davis Mark M. , Levy Ron , Alizadeh Ash A. , Elias Joshua E.
Published: January 2023

Presentation of novel antigenic peptides (neoantigens) that distinguish malignant from normal cells by major histocompatibility complexes (MHC) can serve as potent substrates for specific anti-tumor immune responses. We sought to identify mantle cell lymphoma (MCL) neoantigens by taking an integrated genomic and proteomic strategy that interrogates antigen peptides presented by MHC-class I and class II. Peptides bound to MHC were purified via immunoprecipitation followed by identification using mass spectrometry. Mass spectra were searched against patient-specific proteome databases generated by whole exome sequencing and targeted immunoglobulin gene sequencing. This approach was applied to systematically characterize over 36,000 immunopeptides from 17 patients’ tumor specimens. Interestingly, 52 neoantigenic peptides were derived from the lymphoma immunoglobulin (Ig) heavy or light chain variable regions. Although we identified MHC presentation of private germline polymorphic alleles, no mutated peptides were recovered from non-Ig somatically mutated genes. Furthermore, somatic mutations within the immunoglobulin variable region were almost exclusively presented by MHC-II. T-cells specific for Ig-derived neoantigens were found in two patients. Following ex vivo activation and expansion, the T-cells were remarkably able to mediate killing of autologous lymphoma cells. These results demonstrate that combining MHC isolation, peptide identification and exome sequencing is an effective platform to uncover tumor neoantigens. Application of this strategy to MCL implicates immunoglobulin neoantigens as targets for lymphoma immunotherapy.
Fatty Acid Uptake Unexpectedly Supports a Th17 cytokine signature in Type 2 Diabetes (T2D) Inflammation
Authors: Nicholas Dequina , Proctor Elizabeth A. , Jones Albert , Raval Forum , Habib Chloe , Corkey Barbara , Apovian Caroline M. , Lauffenburger Douglas A. , Nikolajczyk Barbara S.
Published: January 2023

Metabolism controls T cell function, but the role of cellular metabolism in regulating our published T2D-associated Th17 cytokine signature is untested. Published work showed Th17 function requires fatty acid (FA) uptake, and suggested Th17s burn fat to support inflammation. We thus hypothesized that chronic exposure of immune cells to FAs, which occurs in T2D patients, drives Th17 inflammation. We used extracellular flux (XF) and cytokine analyses to show that CD3/CD28-stimulated PBMCs from BMI-matched T2D vs. non-T2D subjects were more glycolytic despite overall equal mitochondrial respiration. Consistent with this difference, T cell stimulation in glucose-free media followed by partial least squares analysis of cytokines showed glucose impacted cytokine profiles from T2D and non-T2D cells differently. To challenge the resulting prediction that PBMCs (predominantly T cells) from the two groups used different mechanisms to fuel cytokine responses, we performed XF analysis in the presence of FA +/− glucose. Non-T2D cells utilized FA as a fuel source, but T2D cells were dependent on glucose, despite data showing diabetic vs. non-T2D PBMCs store more lipid in response to elevated exogenous FA. Etomoxir, an inhibitor of FA transport into the mitochondria, caused non-T2D cells to increase glycolysis, but had no effect on T2D cells. These data confirmed that T2D cells exclusively burn glucose, not FA, to generate energy, and that T2D cells cannot be forced to use FA as a fuel. Finally, blockade of FA transport into the mitochondria revealed that FAs were associated with Th17 cytokine production. Together, our results support the unexpected conclusion that FA uptake is uncoupled from FA oxidation to drive the Th17 inflammation in T2D.
Mouse CD19-targeted CAR T cells with an optimized 4-1BB domain enhance protection against CD19+ leukemia and retain proliferation potential after long-term rest in vivo.
Authors: Li Gongbo , Beatty Nolan , Vishwasrao Paresh , Yu Bin , Park Paul , Zhang Yongliang , Davila Marco L
Published: January 2023

Chimeric antigen receptor (CAR) T cells have mediated exciting results for patients. The CAR is a novel antigen-receptor that includes an antigen-binding domain, derived from an antibody, linked to an intracellular T cell activation domain. This 1st generation CAR design worked well in vitro but mediated poor results in vivo. Modification of the CAR to include a co-stimulatory domain, classified as a 2nd generation CAR design, achieved optimal T cell persistence and tumor killing in mice, which served as the rationale for evaluation of CAR T cells in patients. The most common clinically evaluated CARs target CD19 and include CD28 or 4-1BB co-stimulatory domains. However, there has been no extensive comparison of 2nd generation CAR T cells in mice, which could serve as a model for how these domains impact CAR T cell function in patients. Therefore, we evaluated and compared the function of mouse CD19 targeted CARs modified with a CD28 or 4-1BB domain. In contrast to clinical results, mouse CD28 CAR T cells imparted better leukemia-killing and persistence than mouse 4-1BB CAR T cells. We identified a defect in CAR T cell signaling mediated by mouse 4-1BB and mutation of this domain restored CAR signaling and in vivo leukemia killing. Also, we determined that CAR T cells with the mutation-optimized 4-1BB co-stimulatory domain proliferated in response to antigen in the bone marrow (BM) after a > 1-month period of rest, while CAR T cells with the CD28 co-stimulatory domain did not. We determined that this is likely due to the acquisition of an exhausted phenotype in the CD28 CAR T cells. Our results illustrate how co-stimulation modulates CAR T cell function in mice and suggest mechanisms for its impact on persistence and cancer-killing in patients.
Two-pronged Cell Therapy: Engineering NK cells to target CD22 and redirect bystander T cells to CD19 for the adoptive immunotherapy of B-cell malignancies
Authors: Szoor Arpad , Velasquez Mireya Paulina , Bonifant Challice Lee , Vaidya Abishek , Brunetti Lorenzo , Gundry Michael , Parihar Robin , Goodell Margaret , Gottschalk Stephen
Published: January 2023

CD19-CAR T cells have shown antitumor activity for B-cell derived malignancies, but they also have shown limitations regarding their toxicity and development of CD19- escape variants. Given their short lifespan and potent cytolytic function NK cells are attractive candidate effectors to express CARs. The goal of this project was to devise a strategy to genetically modify NKs that express a CAR to target CD22 and secrete a CD19-specific T-cell engager (CD19-ENG) to redirect T cells to B-cell leukemias. NKs were transduced with a retroviral vector encoding a CD22-CAR and/or CD19-ENG molecules. To mimic immune escape, CD19 or CD22 knockout (ko) leukemia cells (BV173) were generated. We performed coculture and cytotoxicity assays using CD22 and/or CD19+ targets with NKs +/− T cells. In the absence of T cells, CD22-CAR +/− CD19-ENG NK cells recognized tumor cells in an antigen-dependent manner as judged by cytokine production and tumor killing. Moreover, CD22 CAR +/− NK cells secreting engager molecules efficiently redirected T cells in the presence of CD19+ targets. In vivo, CD22-CAR/CD19-ENG NK cells recognized tumor cells in an antigen-dependent manner, redirected T cells to tumor cells, and induced significant regression of leukemia in xenograft models in comparison to mice treated with CD22-CAR NK cells or NK cells targeting irrelevant antigens. We have generated for the first time NK cells that kill B-cell malignancies through a CAR and simultaneously redirect bystander T cells to a 2nd B-cell antigen to enhance antitumor effects and prevent immune escape. Genetic modification of NK cells to enhance their antitumor activity and redirect bystander T cells may represent a promising addition to current cell therapies for B-cell malignancies.
S1P/PPARγ axis metabolically reprograms the T cells to modulate their anti-tumor potential
Authors: Chakraborty Paramita , Thyagarajan Krishnamurthy , Chatterjee Shilpak , Nganga Rose Nithenya , Selvam Shanmugam Panneer , Ogretmen Besim , Mehrotra Shikhar
Published: January 2023

Adoptive T cell transfer (ACT) holds promise in cancer therapy, and strategies to further improve its efficacy are being tested. It is reported that sphingosine 1-phosphate (S1P), generated by sphingosine kinase 1 (SphK1), regulates lymphocyte egress into circulation and also plays role in the differentiation of regulatory T cells and T helper-17 cells. Here, we examined the roles of SphK1/S1P signalling in regulating antitumor potential of T cells. We found that adoptive transfer of melanoma epitope gp100 reactive CD8 T cells (pMel) with SphK1 deficiency showed improved tumor control in murine melanoma model. Our data also indicated that Sphk1−/− T cells exhibited enhanced central memory phenotype, higher mitochondrial respiration and resistance to TGF-β mediated suppression. Importantly, Sphk1−/− T cells spontaneously secreted higher levels of IL17 compared to their wild type counterparts. Mechanistically, we demonstrate that SphK1/S1P signaling regulates IL17 generation through attenuation of PPARγ, which is down-regulated in Sphk1−/− T cells. In reciprocal studies, addition of S1P in culture media enhanced PPARγ expression, resulting in decreased IL17 production in Sphk1−/− T cells. Furthermore, down-regulation of PPARγ in Sphk1−/− T cell increased lipolysis to produce free fatty acids and metabolically reprogram T cells to depend on mitochondrial metabolism for meeting their energy demand. Thus, our data reveal that SphK1/S1P signaling regulates anti-tumor functions and persistence of T cells by controlling T cell energy metabolism through induction of lipolysis via PPARγ modulation. Overall, these data highlight the clinical potential of targeting SphK/S1P axis for improving efficacy of ACT for solid tumors.
Augmenting Ag presentation at the “CD4 memory checkpoint” can overcome age-associated “defects” in CD4 helper response and enhance vaccination.
Authors: Xia Jingya , Yi Kuang , Bautista Bianca L. , Vong Allen M. , Liang Jialing , Swain Susan L
Published: January 2023

Vaccines are the most efficient approach to prevent infection and transmission of viruses such as influenza, but they are inefficient in the elderly. Age-associated functional defects in naive CD4 T cells, lead to few Tfh and poor help for B cells and thus to reduced long-lived Ab and memory CD4 T cells. Therefore, a vaccination strategy that would enhance the response of aged CD4 T cells could promote B and T cell memory generation and more effective protection.

We found CD4 effectors generated by influenza A virus (IAV) made very few memory cells if they could not make autocrine IL-2 and that such IL-2 production depended on cognate Ag recognition at the effector stage to drive transition to memory, defining what we call the ‘memory checkpoint’. Full differentiation of CD4 effectors to Tfh/GC-Tfh effectors also requires such Ag recognition at this checkpoint.

Here we ask if providing adult or aged CD4 T cells with Ag/APC at the ‘memory checkpoint’ can augment the CD4 and Ab response to inactivated IAV (IIV) vaccine. We use a reductionist in vivo model with an irrelevant TcR transgenic host, where we can add the IAV-specific naïve CD4 T cells (young or aged from TcR transgenic donor) and vaccinate with inactivated IAV as the vaccine. We introduce Ag at the memory checkpoint. When with either young adult or aged CD4+ T cells, again recognize cognate Ag at the ‘memory checkpoint’, they yield more IAV-specific IgG memory B cells (MBCs), IgG-secreting long-lived plasma cells (LLPCs) and more tissue resident memory CD4 T cells. This suggests novel strategies that augment Ag presentation at this CD4 checkpoint may improve vaccine efficacy in both young and elderly.
Cereblon ablation increases T cell anti-tumor immunity
Authors: Hesterberg Rebecca , Beatty Matthew S , Epling-Burnette Pearlie K
Published: January 2023

Druggable targets that reinvigorate T cells may improve the efficacy of immunotherapy in melanoma or other cancers. T cells exist in a hostile tumor microenvironment associated with hypoxia, high lactic acid, and poor nutrient availability which limits their anti-tumor activity. We found that the genetic deficiency of Crbn, a novel E3 ubiquitin ligase substrate receptor, improves effector T cell function. Growth of subcutaneous B16 tumors was slower in Crbn−/− mice and the adoptive transfer of Crbn−/− T cells into mice with established B16 tumors lead to a significant growth reduction. While similar total tumor infiltrating lymphocytes (TIL) numbers and normal splenic populations were present, a significant increase in the proportions of CD44+ and PD1+ CD8+ TIL but not KLRG1+CD8+ TIL were present in Crbn−/− mice. These results suggest that CRBN represses antigen-dependent anti-tumor T cell immunity and represents a novel regulator of T cells in the tumor microenvironment. In melanoma, hypoxia is a mode of T cell suppression. Activated Crbn−/− T cells demonstrated higher extracellular acidification rate, oxygen consumption and glucose uptake compared to Crbn+/+ T cells. Under hypoxic conditions (2% O2), effector cell cytotoxicity is reduced at high densities of B16 melanoma compared to normal atmospheric oxygen (~21% O2) and proliferation is suppressed due to reduction of OxPhos. Using anti-CD3ɛ/anti-CD28 stimulation in hypoxic conditions, Crbn−/− T cells retained their proliferative capacity compared to Crbn+/+ T cells indicating that deletion of Crbn restores metabolic activity of T cells in a hostile tumor microenvironment and may serve as a novel drug target in immunotherapy.
5- Fluorouracil regulation of myeloid-derived suppressor cell differentiation in vitro and in vivo
Authors: Klement John David , Paschall Amy V , Savage Natasha M , Nayak-Kapoor Asha , Liu Kebin
Published: January 2023

The chemotherapeutic agent 5-fluorouracil (5-FU) is the standard therapy for patients with advanced colorectal cancer (CRC). 5-FU not only targets tumor cells for apoptosis but also induces apoptosis in myeloid cells, leading to myelosuppression, which has long been thought as a side effect of 5-FU therapy. Myeloid-derived suppressive cells (MDSCs) are a heterogeneous population of immature myeloid cells that exhibit potent suppressive activity to inhibit T and NK cell function. Recent studies have found that 5-FU suppresses MDSCs in mouse models. However, we observed that MDSCs still massively accumulate in human CRC patients after multiple rounds of 5-FU therapy. We hypothesize that a subset of the heterogeneous MDSCs is resistant to 5-FU and that 5-FU therapy selectively eliminates the sensitive MDSCs, enriching the 5-FU-resistant MDSCs. To test this hypothesis, we made use of both in vitro BM-derived MDSC (BM-MDSC) and in vivo mouse tumor models. We observed that 5-FU therapy significantly decreases CD11b+Gr1+ MDSC accumulation in an orthotopic colon cancer mouse model. BM-MDSCs can be induced by cytokines, including GM-CSF, G-CSF, M-CSF and IL-6, either alone or in combination. Cytological analysis indicates that various cytokines induce BM-MDSC to differentially acquire the morphological appearance of myeloid cell subsets, including macrophage- and granulocyte-like cells. Interestingly, all of these BM-derived MDSCs display resistance to 5-FU-induced apoptosis in vitro following exposure to 5-FU at doses as high as 10 μg/mL. Our data indicates that some MDSC subsets display resistance to 5-FU, providing a rationale to explain the enrichment of MDSCs in human CRC patients following successive treatments with 5-FU.
Induction of Nrf2/HO-1 pathway suppresses neuroinflammation in ischemic stroke
Authors: Yen Jui-Hung , Kuo Ping-Chang , Yu I-Chen , Scofield Barbara A , Brown Dennis , Curfman Eric T , Paraiso Hallel C , Chang Fen-Lei
Published: January 2023

Ischemic stroke induces reactive oxygen species, causing oxidative and inflammatory responses in ischemic brain. To date, recombinant tissue plasminogen activator is the only available therapy for the treatment of ischemic stroke. However, the treatment does not prevent oxidative stress and inflammation in the ischemic brain. Nrf2, a major regulator of oxidative responses, is a key transcription factor, which induces a wide set of phase II anti-oxidant enzymes, including HO-1, Gclc, and NQO1. In addition, Nrf2 has been shown to exert an anti-inflammatory effect through induction of HO-1. D3T, a sulfur-containing dithiolethione compound, is found in cruciferous vegetables and has been reported to induce anti-oxidant genes through activation of Nrf2. In this study, we evaluated the effect of D3T on the suppression of neuroinflammation in ischemic stroke. Our results show D3T attenuates brain infarct and ameliorates neurological deficits in stroke animals. In addition, D3T reduces CNS infiltrating inflammatory immune cells including neutrophils and monocytes in the ischemic brain. Moreover, D3T-induced suppression of inflammatory cytokine production was observed in wild-type but not in Nrf2-deficient microglia. Furthermore, the protective effect of D3T on the attenuation of ischemic brain infarct was abolished in Nrf2-deficient stroke animals and in the stroke animals administered with HO-1 inhibitor. Altogether, our results demonstrate that D3T-mediated suppression of inflammation in the ischemic brain is mediated through Nr2/HO-1 pathway, suggesting that targeting Nrf2/HO-1 pathway may be a promising therapeutic strategy for the amelioration of neuroinflammation in ischemic stroke.
Inflammatory signals from fat: Visfatin promotes pro-inflammatory activation and leukocyte interaction in endothelial cells
Authors: Singh Manindra , Benencia Fabian
Published: January 2023

Obesity is a global epidemic and is associated with a cluster of metabolic diseases that lead to premature mortality. Research in the past decade suggests that chronic, low-grade inflammation – a hallmark of obesity, is a major contributing factor for the pathophysiology of several cardiovascular and metabolic diseases, including atherosclerosis and type 2 diabetes. Evidence suggests that obesity leads to abnormal levels of adipokines, which is linked to insulin resistance and inflammation of the adipose tissue. Visfatin, an adipokine predominantly secreted by visceral adipose tissue and that circulates at increased levels in obesity has been associated with several inflammatory disorders and cancer. Whether visfatin functions as a pro-inflammatory or anti-inflammatory factor is still a subject of debate. We investigated the effect of visfatin on human endothelial cells at the level of gene expression, cytokine release, and interaction with leukocytes in vitro. We found that visfatin upregulates the gene expression of the cytokines – CCL2, IL6, GM-CSF, CXCL2, and CXCL8 (p<0.05) at the transcript level. Additionally, visfatin significantly upregulated the secretion of CXC-cytokines – CXCL2 and CXCL8, in a dose and time-sensitive manner. Finally, we explored if visfatin treatment has any effect on endothelial-leukocyte interaction; we found that visfatin-treated endothelial cells exhibited pronounced adhesion to monocytes (p<0.05). Visfatin also upregulated the expression of adhesion markers – ICAM1, VCAM1, and E-Selectin (p<0.05). Overall, these results consolidate the notion that extracellular visfatin is a marker for vascular and adipose inflammation and a potential target for therapeutic intervention.