bluuebunny/crossref_metadata_2025 huggingface dataset:
Mechanism underlying why donor splenocyte transfusion helps costimulation blockade drive transplantation tolerance
Authors: Mora Ricardo , Xie Stephen , Stewart Emma , Cassano Alexandra , McIntosh Christine , Wang Peter , Wang Ying , Chen Luqiu , Chong Anita , Alegre Maria-Luisa
Published: November 2024

Solid organ transplantation is the preferred treatment for end-stage organ failure. Achieving stable donor-specific transplantation tolerance holds the potential to vastly improve the quality of life for transplant recipients by eliminating lifelong exposure to drug toxicity, a higher risk of malignancy and infection, and the development of chronic rejection associated with conventional immunosuppression. In mice, treatment with anti-CD154 (aCD154) prolongs graft survival, but the addition of donor splenocyte transfusion (DST) to aCD154 produces a robust state of graft-specific tolerance to fully MHC-mismatched cardiac allografts. In this study, we investigated parameters that dictate the efficacy of DST treatment. Our results support an antigen dispersal hypothesis whereby DST enables alloantigen to circulate to all secondary lymphoid organs where it can prime alloreactive T cells under cover of aCD154, and induce widespread tolerance in a larger repertoire of T cells than enabled by an allograft alone.
Modulating alloreactive immune responses by controlled dual delivery of Fas and IL-2R agonists using a lipocoacervate platform
Authors: Tedla Mebrahtu , Wright Nathaniel , Tarique Mohammad , Ulker Vahap , Yee-Flores Kathleen , Yolcu Esma , Wang Yadong , Shirwan Haval
Published: November 2024

Allogeneic islet transplantation is an effective treatment for type 1 diabetes (T1D). Graft rejection is controlled by immunosuppressive drugs that have adverse effects. An imbalance between Teffs and Tregs is the primary cause of T1D and graft rejection. Activated Teff cells express Fas and are sensitive to FasL-mediated apoptosis. Treg cells are relatively refractive to apoptosis and expand in response to IL-2. Thus, a combination of Fas and IL-2R agonists has the potential to modulate alloreactive responses for sustained graft survival. Given the off-target effects of systemic delivery of biologics, we established a lipocoacervate formulation consisting of a biodegradable polycation, PEAD, and polyanion heparin to form coacervate for controlled delivery of a novel Fas agonist, SA-FasL, and IL-2. Release kinetics and activities of proteins were assessed in vitro. The immunomodulatory efficacy of protein-loaded lipocoacervate was assessed in vivo. IL-2 and SA-FasL proteins showed a steady release over 30 days in vitro. IL-2 released on day 14 had minimal activity loss, whereas FasL showed ~20% activity loss on day 9. Treatment with SA-FasL/IL-2-loaded lipocoacervate modulated in vivo alloreactive T cell responses, resulting in an increased Treg/Teff cell ratio. In conclusion, lipocoacervate is an effective platform for controlled and sustained release of SA-FasL and IL-2 biologics to modulate allo and autoreactive T cells with significant therapeutic potential for T1D.
Understanding the role of neutrophils in Staphylococcus aureus-induced skin inflammation
Authors: Matsumoto Masanori , Nunez Gabriel
Published: November 2024

Staphylococcus aureus, a common pathogen in the skin, triggers skin diseases including atopic dermatitis and cutaneous abscesses. We recently found that neutrophils play differential roles in epicutaneous and intradermal S. aureus inoculation models. To understand the role of neutrophils in S. aureus-induced skin inflammation, neutrophil gene expression profiles were analyzed by single cell RNA-sequencing (scRNA-seq) analysis using those two infection models.

Methods: We infected C57BL6/J mice intradermally or epicutaneously with S. aureus USA300 strain and isolated neutrophils from the infected skin tissues followed by scRNA-seq analysis. To identify neutrophil genes that regulate skin inflammation, we generated specific gene deletion mice to investigate the underlying mechanisms.

Results: In the intradermal S. aureus infection model, we found that inducible nitrogen oxide synthetase (iNOS) gene expression was upregulated in neutrophils and that skin lesion size was significantly increased in neutrophil-specific iNOS-deficient mice. In the epicutaneous S. aureus inoculation model, however, the expressions of genes (padi4, mmp8/9, s100a8/9) required for neutrophil extracellular trap (NETs) were upregulated, and skin inflammation was suppressed in padi4-deficient mice.

Conclusion: These data suggest that neutrophil NO production is critical for dermal S. aureus killing, while NETs formation induces skin inflammation by epidermal S. aureus colonization.
Neutrophils Disrupt B-1a Cell Homeostasis by Targeting Siglec-G to Exacerbate Sepsis
Authors: Tan Chuyi , Aziz Monowar , Reilly Bridgette , Ma Gaifeng , Wang Ping
Published: November 2024

In sepsis, B-1a cells diminish in the peritoneal cavity (PerC) as they relocate to the spleen via CXCL12/CXCR4. In the spleen, these cells differentiate into plasma cells, skewing from innate to adaptive phenotype. The mechanism governing the PerC B-1a cell relocation in sepsis remains elusive. Sialic-acid-binding immunoglobulin-like lectin (Siglec)-G is a B-1a cell-specific receptor. We found that Siglec-G-/- PerC B-1a cells had a higher migration rate than WT B-1a cells. Siglec-G negatively regulated B-1a cell migration by interacting with CXCR4, as Siglec-G-/- B-1a cells showed increased activation of the CXCR4’s downstream pathway. LPS-activated neutrophils decreased Siglec-G expression on B-1a cells and promoted B-1a cell migration, while NE inhibitor reversed these. Recombinant NE treatment decreased Siglec-G expression by cleavage on B-1a cells. A small Siglec-G-derived decoy peptide (SDP) was developed to prevent Siglec-G cleavage based on the interaction site of NE at the Siglec-G protein in silico. Treatment of SDP significantly reduced the decrease in PerC B-1a cell numbers and Siglec-G expression on B-1a cells in septic mice. SDP treatment significantly reduced serum IL-6 and TNFα levels and improved survival rates in septic mice. In conclusion, we discover Siglec-G to be a new regulator of B-1a cell migration. Preventing NE-mediated Siglec-G cleavage upholds a promising strategy for maintaining peritoneal B-1a cell homeostasis to mitigate sepsis.
The Role of Textile Dyes in the Immune System Dysregulation
Authors: Lorenz -Cortes Lizette , Vinueza-Benitez Nelson
Published: November 2024

There has been an increase in the incidence of atopic diseases and cancer in the last decades. There is an urgent need to identify the environmental triggers that fuel these conditions. In recent years, the use of synthetic textile dyes has increased. The effects of these chemicals or their metabolites on our skin and our immune system have not been well studied. There have been reports on the effects of irritants to the skin on their ability to break the dermal barrier to start a chain of immune reactions that dysregulate the immune system. The purpose of this study was to investigate the effects of the most common textile dyes in the viability, IFNg production and mitochondrial function of epidermal skin, and intestinal epithelial cells using swine as an animal model. Cultures of MPEK-BL6 and IPEC cells were cultured with disperse textile dyes Red 11, Orange 37, Blue 1, Blue 124, and Brown 1, at low and high concentrations for 3-6 hours and 1-3 days. Viability was measured with LUNA cell counter and by flow cytometry. IFNg production by flow cytometry and ELISPOT and Oxygen consumption rate (OCR) by Seahorse Agilent Technology kit. Disperse Blue 124 and Brown 1 induced IFNg production, and impaired cell viability and mitochondrial function as early as 3 hours after exposure with IPEC and MPEK cells. These data suggest that textile dyes can influence cell viability and possibly cause inflammation as a starting point to dysregulate the immune system. Supported by NCSU-DSA
Non-tumor cell IDO1 non-enzymatically decreases radiation + PD-1 mAb treatment efficacy in older adults with glioblastoma
Authors: Zhai Lijie , Bell April , Asimakidou Evridiki , Lauing Kristen , Wainwright Derek
Published: November 2024

To understand the role of indoleamine 2,3 dioxygenase 1 (IDO1) in the age-dependent immunotherapeutic effect, we compared 20-26 month-old C57BL/6 background mice that were IDO1-T2A-GFP [(WT); n=16], IDO1 enzyme null [H350A; (n=16)], IDO1KO (n=4), Ido1fl/fl(n=14), Lysm-Cre+→Ido1fl/fl (n=20), CD11c-Cre+→Ido1fl/fl (n=23), Tie2-Cre+→Ido1fl/fl (n=14) treated with RT + PD-1 mAb. Mice were intracranially engrafted (ic.) with GL261 cells followed by RT + PD-1 mAb beginning at 14-days post-ic. IDO1 WT and IDO1 enzyme null (H350A) mice had a survival rate of 12.5% and 18.75% at 90-days post ic., respectively, which was significantly decreased as compared to IDO1KO mice with a 75% survival rate (p<0.05). Tie2-Cre+→Ido1fl/fl mice, CD11c-Cre+→Ido1fl/fl, and Lysm-Cre+→Ido1fl/fl mice had a 100%, 96%, and 70% survival rate at 90-days post ic., respectively, as compared to uncrossed Ido1fl/fl mice with a 42% survival rate (p<0.05). Significantly reduced kynurenine (KYN) levels were found in IDO1 enzyme null (H350A) and IDO1KO mice as compared to WT mice (p<0.001). Unexpectedly, KYN levels were also decreased in Tie2-Cre+→Ido1fl/fl but not in CD11c-Cre+→Ido1fl/fl, Lysm-Cre+→Ido1fl/fl or Ido1fl/fl mice (p<0.05). Tryptophan (TRP) levels did not change between mouse groups. These data support the hypothesis that non-tumor cell IDO1 reduces immunotherapeutic efficacy in older adults with GBM through a TRP-KYN pathway-independent mechanism.
Targeting RAS/RAF/PI3K pathway for CD40 induction in melanoma cells to overcome resistance to anti-PD1 immunotherapies
Authors: Yan Chi , Luo Weifeng , Chen Sheau-Chiann , Johnson Douglas , Ayers Gregory , Richmond Ann
Published: November 2024

The first-line treatment for metastatic melanoma is immune checkpoint blockade (ICB), but it fails in many patients and may have serious adverse events. We showed that rigosertib (RGS), a RAS-pathway inhibitor, promotes CD40 upregulation on melanoma cells and synergizes with ICB in preclinical melanoma models. However, residual disease still occurred with uncontrolled ERK activation. Our present study explores the efficacy of RGS plus trametinib (T), a MEK1/2 inhibitor, to overcome ICB resistance. The concurrent RGS+T+αPD1 resulted in an additive effect to suppress ICB-resistant NRASmut 1014 tumor growth, and 44.4% (4/9) tumors completely regressed after 2 weeks of treatment. The RGS+T regimen promoted CD8+ T cell responses in the tumor microenvironment (TME) and extended the time to αPD1 resistance (p<0.0001). Combining RGS+T with agonist CD40 (aCD40) resulted in increased CD8+ T cells, natural killer cells, and M1 macrophages in the TME, with a reduction of myeloid-like CD11b+PD-L1+ regulatory B cells in the tumors (~70%, p<0.0001) and tumor-draining lymph nodes (~40%, p<0.0001). CRISPR/dCas9-based overexpression of CD40 (CD40-OE) in 1014 melanoma cells reduced in vivo tumor growth and successfully turned the ICB-resistant tumors into responders to αPD1 (p=0.025), which further regressed with aCD40+αPD1 (p<0.001). Our preclinical data support the therapeutic use of RAS/RAF/PI3K inhibition plus CD40 agonism for metastatic melanoma patients who do not respond to ICB.
T cell immune landscape in melanoma patients before and post immune checkpoint blockade treatment
Authors: Luo Ying , Yao Chen , Wu Tuoqi
Published: November 2024

Immunotherapy based on immune checkpoint blockade (ICB) has revolutionized cancer treatment. Immune cells, especially T cells, play a crucial role in ICB. However, ICB-induced T cell response remains incompletely investigated. Revealing T cell landscape changes caused by ICB could promote the discovery of novel biomarkers with predictive efficacy and toxicity and the potential strategy to improve the therapeutic efficacy while reducing side effects. We used single cell-based sequencing approaches to analyze transcriptome and surface protein of T cells in peripheral blood collected before and post ICB treatment from melanoma patients with immune-related adverse events. A total of 17 cell clusters were identified and annotated according to the expression of RNA and protein. Collectively, variation existed among the recruited participants. ICB treatment decreased the proportion of memory CD4 T cells while increasing the proportion of T follicular helper cells. In summary, these findings provide insight into how ICB modulates T cell responses in melanoma patients.
Investigating MAIT cell phenotype using MR1 and TCR Dextramer™ reagents
Authors: Blicher Thomas , Hansen Bjarke , Jacobsen Kivin , Brix Liselotte
Published: November 2024

Objective

Here, we demonstrate a workflow, which enables the study of mucosal-associated invariant T (MAIT) cells using MR1 reagents. The workflow covers initial detection of MAIT cells with TCR sequencing as well as cloning, production, and validation of selected MAIT TCR molecules.

Methods

Human PBMC samples were stained with relevant antibodies and MR1/5-OP-RU dCODE Dextramer™ reagents. Following staining, sorting, and partitioning on a BD RhapsodyTM Single-Cell Analysis System, the cells underwent sequencing for characterization of gene and surface marker expression as well as paired alpha-beta TCR sequences. Selected TCR sequences were produced as TCR molecules and subsequently used to detect MR1/5-OP-RU on the surface of antigen-presenting cells.

Results

The majority (70%) of the MAIT cells express typical TCRs containing TRAV1-2 and TRAJ33 gene segments. The remaining MR1/5-OP-RU-reactive MAIT cells express highly diverse, non-classical MAIT TCR sequences. The three most frequent TCR clonotypes were produced and their specificity validated using in vitro assays and staining of MR1/5-OP-RU-presenting cells.

Summary

Our workflow allows identification of MAIT cells and elucidation of their TCR sequences. The high sensitivity of MR1 dCODE Dextramer reagents enables detection of both weak interactions and rare cell types. Subsequent production of MAIT TCR molecules allows deeper characterization of individual MAIT cell specificity and its role in health and disease.
Divergent coactivator usage and CREB1 activity controls tuft lineage commitment
Authors: Eskiocak Onur , Subhash Santhilal , Shah Vyom , Moresco Phill , Beyaz Semir
Published: November 2024

The precise coordination of co-activators and transcription factors in shaping distinct intestinal epithelial cell types during intestinal stem cell (ISC) differentiation, especially the roles of CBP and P300, remains poorly understood despite extensive gene regulation studies. Here using conditional and inducible genetic models, we identify P300 but not CBP as a specific repressor of tuft and goblet lineages. Both ISC- and IEC-specific deletion of P300 elicited selective expansion of tuft and goblet cells with upregulation of gene modules that orchestrate their differentiation without affecting Paneth and enteroendocrine linages. Intestinal organoids that lack P300 more robustly upregulated tuft cell marker genes in response to IL13, a cytokine that drives type-II immunity and tuft cell expansion. Because P300 often acts as co-activator of TFs that regulate lineage specific gene expression, we hypothesized that lack of P300 repressed a repressor of tuft cell lineage. Among TFs that associate with P300, we tested whether CREB1 regulates P300 mediated restriction of tuft lineage. Surprisingly, we found that CREB1 is necessary for tuft cell commitment in vivo and expansion in response to IL13 treatment in organoids. Overall, these results contribute to our understanding of the complex regulatory mechanisms underlying the ISC differentiation to form specialized cell types in the intestinal epithelium.
IL-22 is required for preserving tissue integrity and host-microbe homeostasis in the peridontium
Authors: Saha Gundappa , Li Xiaofei , Li Lu , Diaz Patricia , Hajishengallis George
Published: November 2024

Periodontal Disease (PD) is a dysbiotic inflammatory condition of the periodontium (tooth-supporting tissues) that exerts an adverse impact on systemic health. IL-22 mediates unidirectional communication from immune cells to tissue stromal cells and plays a key role in promoting homeostatic immunity with some implications in certain inflammatory disorders. In this study, we investigated whether IL-22 is required for periodontal tissue homeostasis at steady state using IL-22-deficient (IL-22–/–) mice and IL-22+/+ littermate controls. The mice were analyzed in terms of disease phenotype, gingival transcriptomic profile (bulk RNA sequencing and quantitative real-time PCR) and microbiota composition (16S rRNA sequencing). Analysis of the bone levels in 10-week-old IL-22–/– mice revealed significantly increased naturally occurring bone loss vs controls. The IL-22 deficiency-induced bone loss was associated with significantly increased gingival tissue expression of the pro-inflammatorycytokine IL-17 and several molecules with pro-inflammatory and antimicrobial properties (e.g., S100 and Reg3 proteins). Moreover, IL-22 deficiency was associated with a significant increase in the periodontal microbial burden and alterations to the microbiota composition consistent with dysbiosis. Therefore, IL-22 appears to be required in setting the homeostatic tone of the periodontal tissue, suggesting that loss-of-function genetic polymorphisms in IL-22 may affect periodontal health.
Elucidating inflammation-induced cell fate decisions in primary human Tregs
Authors: Ho Patrick , Bluestone Jeffrey , Tang Qizhi
Published: November 2024

Adoptive regulatory T-cell (Treg) therapy has remarkable efficacy in promoting immune tolerance in preclinical models of transplantation and autoimmune disease. However, lineage-tracing studies have revealed that Tregs can undergo epigenetic reprogramming in chronically inflamed tissue environments, resulting in the acquisition of proinflammatory functions and the capacity to exacerbate autoimmunity. Despite intense interests in developing Treg therapy for humans, inflammation-induced human Treg lineage-decommitment remains poorly understood. Here, we present a robust in vitro model of IL6, IL1β, and IL23-driven human Treg instability characterized by progressive FOXP3 and HELIOS downregulation, FOXP3 conserved non-coding sequence (CNS)-2 enhancer re-methylation, diminished in vitro suppressive function, and elevated proinflammatory cytokine expression. Single-cell transcriptomic and chromatin accessibility analyses of human Tregs undergoing destabilization revealed a gradual closing of a Treg-specific element (A) and a reciprocal opening of a competing element (B) at the IRF4 locus. CRISPR-mediated excision of the B element (IRF4ΔB) preserved Treg identity and enhanced in vitro suppressive activity following prolonged inflammatory exposure. Ongoing experiments aim to evaluate the in vivo function and stability of IRF4ΔB Tregs in a xenogeneic graft-versus-host model. These findings may facilitate the design of Treg therapeutics with greater potency and improved safety.
Bi-functional IL2/CD25/IL10 fusion protein promotes Tregs, reduces inflammation, and is highly effective in limiting diabetes in NOD mice
Authors: Santos Savio Alicia , Welford Sarah , Malek Thomas
Published: November 2024

Clinical trials indicate that low-dose IL-2 is a promising approach for patients with autoimmunity. To improve upon IL-2, the mouse (m) IL-2/CD25 fusion protein was developed, which has a long half-life, is selective to the high-affinity IL-2R and expands Tregs to limit diabetes in NOD mice. However, after an extended time off-therapy, many NOD mice exhibited T1D. To improve efficacy, we developed a bifunctional fusion protein, consisting of IL-2/CD25 covalently linked to IL-10 to increase Tregs and limit inflammation. With respect to IL-2 activity, IL2/CD25/IL10 supported IL-2-dependent proliferation of CTLL and STAT5 activation by Tregs. With respect to IL-10 activity, it supported IL-10-dependent STAT3 activation by RAW264.7 cells and limited IFNγ and IL-6 production by activated T and B cells, respectively. In vivo, IL2/CD25/IL10 supported expansion of Tregs as well as activation of pSTAT3 in macrophages and reduced LPS-dependent production of IL-6 and TNFα, indicative of bifunctional activity. When 12-week-old female NOD mice received IL2/CD25/IL10 for 5-weeks, IL2/CD25/IL10 (mean survival >50 week) was more effective than IL-2/CD25 (mean survival 26 weeks) in preventing progressive diabetes. Collectively, these data support the notion that a combination approach, where Tregs are increased and autoreactive T cells are limited, in this case through IL-10, will enhance the efficacy of a Treg-targeted immunotherapy of autoimmunity.
Comparative assessment of selective Janus Kinase inhibitors in rheumatoid arthritis mouse model: Insights into immune modulation and therapeutic implications
Authors: Xue Chengfeng , Wang Jingjing , Xu Na , Pei Yaqiong , Chen Donghai , Sun Jiaping , Gu Qingyang , Zhang Qiyao
Published: November 2024

Janus kinase inhibitors (JAKi) are a class of orally available drugs for treating rheumatoid arthritis (RA). With a focus on enhancing safety profiles and therapeutic efficacy, the development of JAKi has transitioned from first-generation non-selective inhibitors to second-generation selective inhibitors. To better understand the roles of distinct JAK isoforms in the pathogenesis and progression of RA, we employed the Collagen-Induced Arthritis (CIA) mouse model to evaluate the effects of four selective JAK inhibitors: Upadacitinib (JAK1 inhibitor), CEP-33779 (JAK2 inhibitor), Ritlecitinib (JAK3 inhibitor), and Deucravacitinib (TYK2 inhibitor).

All four JAK inhibitors showed varying degrees of alleviation in paw swelling and histological severity. We utilized flow cytometry to characterize the lymphoid and myeloid compartments within the spleen and Olink technology to analyze cytokines in the plasma.

Our findings suggested that JAK1 and JAK3 play an important role in the differentiation and proliferation of T cells, while JAK2 is crucial in the development of myeloid cells. Notably, the TYK2 inhibitor exhibited significant effect in suppressing the expression of inflammatory cytokines.

In summary, this study represents a comprehensive comparison of four selective JAKi in the CIA mouse model, offering valuable insights into immune modulation and potential implications for optimizing the benefit-risk profile of selective JAK inhibitors in the treatment of RA.
Hydroxychloroquine and its derivative enhance efferocytosis and modulate the gut microbiome in pristane-induced lupus mice
Authors: Tsay Gregory , Xue Ting-Yin , Hsieh Fei-Hung , Tsay Jun-Chieh , Peng Hsin-Yi , Yeh Yung-Ju , Tsao Yen-Chi , Yin Mei-Chin , Chao Che-Yi , Tsai Shu-Yao , Lin Chung-Ping , Chen Jiunn-Horng , Huang Chung-Ming , Chang Hen-Hong
Published: November 2024

Background Impaired clearance of apoptotic cells (efferocytosis) and microbiota dysbiosis is related to pathogenesis of systemic lupus erythematosus (SLE). Hydroxychloroquine (HCQ) is frequently prescribed for the treatment of SLE. Here, we evaluate changes in efferocytosis and the gut microbiome in mice with pristane-induced lupus (PIL) by HCQ administration.

Methods PIL mice were studied with or without HCQ, the HCQ derivatives were modified from HCQ by molecular modification. Efferocytosis was determined in RAW 264.7 cells and peritoneal macrophages from mouse ascites fluid. The gut microbiome was analyzed using Illumina sequencing.

Results HCQ enhanced efferocytosis in RAW 264.7 cells, the HCQ derivative AL127 also much enhanced efferocytosis. HCQ also significantly suppressed ascites production of proinflammatory cytokines interleukin (IL)-6 and tumor necrosis factor alpha (TNF-α) in PIL mice. The Firmicutes/Bacteroidetes (F/B) ratio was notably decreased in comparison to untreated controls (p<0.05). Furthermore, administration of HCQ in PIL mice resulted in an increased F/B ratio.

Conclusions In this study, both HCQ and its derivative exhibited a capacity to enhance efferocytosis in RAW 264.7 cells and peritoneal macrophages of PIL mice. Furthermore, HCQ treatment reversed the pristane-induced reduction in the Firmicutes/Bacteroidetes (F/B) ratio. These findings underscore the roles of HCQ in facilitating efferocytosis.
Tumor-associated KLRG1+ Tregs drive a dominant immunosuppressive program in Non-small Cell Lung Cancer
Authors: Oluwatola Oluwaseyi , Oseni Saheed , Bag Arup , Adeegbe Dennis , Pilon-Thomas Shari
Published: November 2024

Killer cell lectin-like receptor G1 (KLRG1) is a co-inhibitory receptor expressed on T and NK cells. While KLRG1’s role in effector T and NK cells has been extensively documented, its impact on regulatory T cells (Tregs) remains poorly understood. This study aims to characterize and compare the immunosuppressive potential of KLRG1+ Tregs in contrast to KLRG1- Tregs and their therapeutic targeting in non-small cell lung cancer (NSCLC).

CTV-labelled effector T cells were cultured with TAMs and tumor digests in the presence or absence of each Treg subpopulation for 5 days in a Treg suppression assay. For in vivo studies, T cells with either KLRG1+ or KLRG1- Tregs were adoptively transferred into lung tumor-bearing mice. Intracellular staining for GZB, IFN-γ and TNF-α was performed on the processed tumor cells. Lastly, cohorts of tumor-bearing KP mice were treated with a KLRG1-blocking antibody for 6 weeks.

Our study revealed that KLRG1+ Tregs exhibit robust suppressive functions both in vitro and in vivo, contingent upon intact KLRG1 signaling. Blockade of KLRG1 in vitro and in vivo profoundly arrested tumor growth, augmented effector T cell activity, and significantly prolonged the survival of tumor-bearing mice.

In conclusion, KLRG1+ Tregs drives immunosuppressive activity in the tumor microenvironment; this provides novel insights into the functional dynamics of KLRG1+ Tregs and underscores their therapeutic potential for boosting anti-tumor T cell responses in NSCLC.
CAR T cells against TRP1 significantly reduce melanoma growth in vitro and in vivo
Authors: Boucher Justin , Austin Anna , Kostenko Elena , Guevara-Patino Jose
Published: November 2024

CAR T cells have shown great efficacy for patients with liquid malignancies. However, CAR T cell treatments have had limited efficacy in patients with solid lesions, such as melanoma. One reason is the lack of suitable targets specific to the tumor antigen, which also do not cause toxicity. TRP1 is abundantly expressed in melanoma cells as well as in healthy melanocytes. While targeting healthy melanocytes may cause vitiligo, that condition is not life threatening. Previous work shows monoclonal antibodies against TRP1 reject melanoma tumors and lung metastases. Therefore, we designed a CAR with an scFv against human TRP1 along with 41BB and CD3z costimulation. When we examined CAR T cell cytotoxicity in vitro, we found a significant reduction in melanoma target cells after 48hrs co-culture with CAR T cells at 10:1 and 5:1 E:T ratios compared to untransduced (UT) cells. CAR T cell cytokine production after melanoma co-culture also showed significantly higher IFNg compared to UT cells. To examine CAR T cell efficacy in vivo, we challenged NSG mice intradermally with melanoma tumor cells. Four days after the tumor challenge, mice were given CAR T or UT cells by tail vein injection. We found that mice given anti-TRP1 CAR T cells had significantly reduced tumor area by day 10 and had significantly increased survival compared to mice given UT cells. This work shows that TRP1 can be effectively targeted by CAR T cells and increase the survival of mice with melanoma.
B2AR Deletion from T-Cells Enhances Tumor Immunity in a Tumor Metastasis Model
Authors: Massalee Rachel , Mustapha Habeebat , Puppala Eswar , Cao Xuefang
Published: November 2024

Previous studies suggest that Beta-2-adrenergic receptor (β2-AR) signaling plays a role in dampening anti-tumor immune response. Given that B2AR is expressed in innate and adaptive immune cells, , it remains to be determined how B2AR signaling affects the activity of specific types of immune cells in the setting of tumor immunity. Therefore, we have developed T cell-specific deletion of B2AR by crossing LCK-Cre mice with B2AR Floxed mice, and used the resultant T cell-specific B2AR knockout (LCRCre-B2ARFL) mice to study tumor metastasis. We utilized the B16-F10 melanoma model via intravenous injection and examined metastatic tumor nests in the lungs 2-3 weeks after tumor injection. Our findings indicate that (LCRCre-B2ARFL) mice showed significantly lower burden of metastatic tumor nests in the lungs compared to control groups (LCKCre mice and B2ARFL mice). These results indicate that endogenous B2AR signaling in T cells plays an immune checkpoint role, restraining their anti-tumor activity, while selective deletion of β2AR from T cells enhances their anti-tumor function, resulting in significant reduction of tumor metastasis. Therefore, this study provides an important rationale for blocking B2AR signaling to improve tumor immunity and disabling B2AR in adoptive T cell therapy for cancer patients.
Lineage tracing reveals heterogeneity within tumor-reactive CD8 T cells in mismatch repair-proficient (MMR-p) CRC
Authors: Duhen Thomas , Rosales Wesley , Fesneau Olivier , Samson Kimberly , Jones Brett , Rajamanickam Venkatesh
Published: November 2024

While CRC develop in the colon and rectum, little is known about the relationship between tissue resident memory (TRM) CD8 T cells and newly recruited tumor-reactive T cells. We compared the phenotype of CD8 T cells from paired adjacent normal colon (NC) and tumor by flow cytometry and observed a higher T cell infiltration in the tumor with an enrichment in CD8 T cells. Interestingly, a subset of CD103+ CD8 T cells in NC expressed CD39, a phenotype reminiscent of tumor-reactive T cells, but those cells lacked PD-1 and Ki-67. To better understand the relationship between T cells from NC and tumor and to uncover the differentiation path of tumor-reactive CD8 T cells, we performed scRNA-seq and scTCR-seq together with CITE-seq on paired NC and tumor from five patients. We confirmed that DP CD8 T cells from NC were distinct from tumor-infiltrating DP CD8 T cells (DP CD8 TILs). In contrast, the latter represented an heterogenous population with cells at different stages of differentiation as illustrated by the detection of cells sharing the same TCRs in multiple cell clusters. To further appreciate the differentiation path of neoantigen-reactive CD8 TILs, we performed neoantigen discovery on three of the five patients. We are now evaluating whether T cell differentiation and activation are linked to functional avidity and cellular location in the tumor. This work provides essential information regarding the differentiation of tumor-reactive CD8 TILs in MMR-p CRC tumors.
fluorescent tracing unveils the impact of radiation therapy on the migration and plasticity of Tregs in the tumor microenvironment
Authors: Friedman David , Blair Tiffany , Kramer Gwen , Kaur Aanchal Preet , Baird Jason , Alice Alejandro , Potuznik Megan , Crittenden Marka , Gough Michael
Published: November 2024

Radiation therapy (RT) is a pivotal component of cancer treatment, yet its impact on the immune system is not fully understood. RT induces DNA damage in cancer cells, promoting cancer cell death and RT-induced immune responses with the potential to clear surviving cancer cells. However, RT can also recruit suppressive T regulatory cells (Tregs), that hinder adaptive immunity. Following RT-mediated death of tumor Tregs, we hypothesize that immature Tregs are recruited to the post-RT tumor and converted into suppressive phenotypes. Using the Kaede mouse model, we tracked photoconverted cells from either the tumor or TdLN after RT. In photoconverted tumors, Tregs in the TdLN constituted a higher proportion of photoconverted cells compared to non-Treg CD4 or CD8 T cells. When RT was applied to tumors, photoconverted Tregs and non-Treg CD4 T cells decreased in the TdLN. When photoconverting the TdLN we observed selective migration of Tregs to the tumor rather than to NdLN. Analyzing these newly arrived photoconverted Tregs in the tumor post-RT revealed increased expression of CD69, KLRG1, and Ly6C compared to non-photoconverted Tregs, indicating limited suppressive capacity. Ongoing studies are examining the impact of the irradiated tumor environment on newly-entered Treg plasticity. These findings unveil intricate dynamics between RT, Tregs, and the use of site-specific photoconversion for tracking immune cell movement over time.
Critical role of 4-1BB:4-1BBL axis in anti-malaria immune response
Authors: Caloba Carolina , Sturtz Alexandria , John Lijo , Ramachandran Akshaya , Malkina Kateryna , Minns Allen , Lindner Scott , Vijay Rahul
Published: November 2024

Malaria is a deadly disease caused by Plasmodium and remains as a major public health threat. The effective elimination of the parasite and protection against reinfections depend on the generation of high-affinity plasma cells (PCs) and memory B cells (MBCs). Due to the emergence of drug-resistant strains and difficulty of developing an efficacious vaccine, host-directed therapies have been gaining more attention as promising approaches. The co-stimulatory receptor 4-1BB is required for an optimal humoral immune response against Plasmodium. Here, we show that exogenous stimulation of 4-1BB by 3H3 improved memory B cell response against Plasmodium. Despite having fewer polyclonal and antigen-specific MBCs, 3H3-treated mice showed enhanced protection upon heterologous reinfection. Adoptive transfer of MBCs from 3H3-treated mice was more protective than control MBCs and generated a higher number of IgM+ PCs in the bone marrow, suggestive of an extrafollicular memory response. Collectively, our data show that upon 3H3 treatment, B cells are reprogrammed to differentiate into a superior MBC population that provides enhanced protection against repeat infections.
Altered T cell phenotypes in M. tuberculosis ‘resisters’ correlated with bacterial control.
Authors: Sun Meng , Phan Jolie , Kieswetter Nathan , Yu Krystle , Smith Malisa , Huang Huang , Liu Yiran , Gupta Sanjana , Obermoser Gerlinde , Maecker Holden , Krishnan Akshaya , Suresh Sundari , Rieck Mary , Acs Peter , Khatri Purvesh , Boom W Henry , Hawn Thomas
Published: November 2024

A subset of individuals with a high probability of exposure to M. tuberculosis (M.tb) appears to exhibit ‘resist’ infection, as demonstrated by serially negative tuberculin skin test or IFN-γ release assay results. While these ‘resisters’ (RSTR) display IFN-γ-independent T-cell responses to the M.tb-specific antigens ESAT-6 and CFP-10 (PP1), it is currently unknown whether specific T-cell functions are associated with this clinical outcome. We used multi-modal scRNA- and TCR-seq to compare the phenotypes and functions of PP1- and M.tb lysate-specific CD4 T-cells between RSTRs and matched controls with ‘latent’ M.tb infection (LTBI). M.tb-specific CD4s were clonally expanded in both RSTRs and LTBIs, confirming the priming of adaptive immune responses after M.tb exposure. However, RSTR CD4s showed an early differentiation phenotype as well as enrichment of Th17-like transcriptional programs compared to LTBIs, which were characterized by Th1*-like effector programs. These results were validated in an independent set of RSTRs and LTBIs using flow cytometry and Luminex. We found that these IFN-γ-independent CD4 T-cell phenotypes correlated with bacteria control in both published human and non-human primate cohorts. Together, these data suggest that ‘resisters’ may successfully control M.tb after exposure and immune priming, establishing a set of T-cell biomarkers to facilitate further study of this important clinical phenotype.
Enteral Supplementation with Bovine Lactoferrin or Glutamine Alters Neonatal Iron Status, Intestinal Permeability and Sepsis-related Mortality in a Mouse Model
Authors: Watral Kyra , Bermick Jennifer
Published: November 2024

A murine model of E. coli sepsis results in significant mortality in pups but universal survival in adults despite equivalent immune responses, suggesting that neonatal susceptibility is due to immune-independent factors. Previous data shows that increased intraperitoneal(IP) iron in pups contributes to increased E. coli growth and subsequent mortality. Neonates are known to have decreased intestinal barrier function, so we hypothesized this iron-rich depot was due to a leaky intestinal barrier allowing iron to more freely pass from the intestinal lumen into the IP cavity. To determine if altering intestinal barrier function would influence sepsis-associated mortality, neonates were enterally gavaged with lactoferrin(LF) or glutamine(Gln) daily. LF decreased intestinal permeability when measured by FITC-dextran assay(p<0.0001) and resulted in less IP iron (p<0.0001). LF also resulted in decreased E.coli translocation following E.coli oral gavage sepsis(p=0.0011) and decreased mortality following E.coli IP sepsis(p<0.05). Gln increased intestinal permeability when measured by FITC-dextran assay(p<0.0001) but had no effect on IP iron. Gln increased E.coli translocation(p<0.05) and burden(p<0.05) following E.coli oral gavage sepsis. Gln also increased mortality following E.coli IP sepsis(p<0.05). Overall, early-life supplementation can alter intestinal permeability and iron status, and these alterations influence neonatal E.coli sepsis-associated mortality.
TRPV1+ Sensory nerves modulate antigen specific immune responses
Authors: Tynan Aisling , Tsaava Téa , Bravo Iñiguez Carlos , Chavan Sangeeta , Tracey Kevin
Published: November 2024

Sensory neurons recognize harmful or noxious stimuli and initiate a protective response by eliciting pain and defensive behavior. Nociceptors also interact with immune cells to regulate immune responses and play a key role in modulating antibody class switching to IgE. However, it is still unknown whether nociceptors participate in regulating primary IgG antibody responses to novel antigens. Here, we show that TRPV1 expressing nociceptors are required to develop an antigen specific immune response and that direct activation of TRPV1+ signals by selective optogenetic stimulation enhances primary IgG antibody responses to novel antigen. Specific ablation of TRPV1+ neurons, which include nociceptors, significantly blunts NP-specific IgG antibody responses in TRPV1-DTA mice compared to controls (anti-NP2 IgG AU, Day 28 control, 18,401 ± 3393 n=18 versus TRPV1-DTA, 9,028 ± 1,498, n=18, p< 0.001). Selective optogenetic stimulation of TRPV1+ nociceptors on the dorsum of the paw with blue light (473nm, 3Hz, 20%DC, 15min) immediately prior to a local immunization in TRPV1-ChR2 mice significantly increases KLH-specific IgG antibody responses compared to sham stimulation controls (anti-KLH IgG ng/mL Day 28, TRPV1-ChR2: 2,581 ± 476, n= 6 versus Control, 1,224 ± 332, n=8; * p<0.05). Together these studies with genetic and selective functional evidence indicates that nociceptors are required during immunization to produce primary antigen-specific IgG antibody responses to novel antigens.
Tularemia-associated hyperinflammation regulation by Natural Killer T cell subsets
Authors: Torres Cameron
Published: November 2024

Inflammation is critical for effective clearance of viral and bacterial challenges, while simultaneously a mechanism utilized by many highly infectious pathogens to cause disease. The bacterium Francisella tularensis (Ft), the causative agent of human tularemia, elicits a cytokine storm, in which the immune system over-responds to the presence of the bacterium causing a dysregulated release of proinflammatory and regulatory cytokines. Without treatment, tularemia mortality can be as high as 30%, with inhalation of as few as 10 CFUs inducing respiratory tularemia. Natural Killer T (NKT) cells are innate-like T lymphocytes with a diverse array of regulatory functions. NKT cells are activated very early following Ft infection suggesting a critical role in the innate immune response in tularemia. NKT cells are categorized into two distinct subsets, types I and II, with a yet to be defined understanding of their individual characteristics in regulating inflammation. Through a combination of in vitro and in vivo techniques, we have observed a cooperative suppression of inflammation when both subsets of NKT cells are present. This phenotypic suppression is disrupted when each subtype responds individually to Ft. The cellular mechanism controlling this interdependence of function will yield a better understanding of NKT-regulated inflammation in tularemia, and more broadly characterize their involvement and potential therapeutic application for a number of NIH priority pathogens.
IL-12p35 and IL-12p40 functionally synergize to active NK cells
Authors: Wang Jinghua , Erickson Christian , Renelt Mark , Gavino Vida , Sun Jing , Li Jun , Bi Ming , Person Anthony
Published: November 2024

IL-12 plays critical roles in regulating the immune response toward different effector pathways. The formation of functional IL-12 requires association of the IL-12p35 subunit with the IL-12p40 subunit. IL-12p40 can be released from these hematopoietic cells as a monomer. Previous studies have reported that IL-12p40 associates with p35 extracellularly to generate IL-12 like activities on T cells, but the biological significance on NK cells remains unclear. In this study, we show that only high concentrations of IL-12p35 could activate NK cells to secrete IFN-gamma and induce cell proliferation and IL-12p40 had no effect on NK cell activation. Interestingly, IL-12p35 and p40 functionally synergized to activate NK cells by inducing IFN-gamma secretion and cell proliferation in vitro. Surprisingly, pretreatment of NK cells with IL-12p35 or p40 didn’t affect IL-12 induced IFN-gamma secretion and cell proliferation in NK cells. Furthermore, a functional ELISA binding assay revealed that IL-12p35 binds to both IL-12R beta1 and IL-12R beta2 with a low affinity, while IL-12p40 only binds to IL-12R beta1 with a high affinity. Fluorescent conjugated human IL-12, IL-12p35 and IL-12p40 proteins all significantly bound to human NK cells and IL-12p35 and p40 didn’t significantly interfere with IL-12 binding to these cells. Collectively, the data we show herein indicates that IL-12 activity can come from the collaboration between the IL-12p35 and p40 subunits and impact NK cell activation.
The CD4 T cell-intrinsic C3-like molecule CD109 prevents hyper-inflammatory T cell responses
Authors: Merle Nicolas , Kolev Martin , Ding Jienyu , West Erin , Rahman Jubayer , Bibby Jack , Bingyu Yan , Kazemian Majid , Afzali Behdad , Biertuempfel Christian , Mizuno Naoko , Kemper Claudia
Published: November 2024

Immune cell autonomous complement expression and function emerges as pivotal regulator of tissue immunity and T cell biology. Specifically, CD4 T cell intrinsic engagement of CD46 by TCR stimulation-triggered generation of the CD46 ligand C3b, is required for IFN-γ production. In consequence, CD46-deficient patients are unable to generate normal Th1 responses. We identified CD109, a GPI anchored C3-like protein and a direct CD46 signaling induced target. Despite its known ability to regulate TGF-ßR1 signaling in cancer cells, a direct functional activity for CD109 on T cells remains unexplored.

Human CD4 T cells lacking CD109 expression displayed significantly augmented IFN-γ and IL-17 production upon in vitro stimulation under non-skewing conditions. Similarly, CD4 T cells from Cd109–/– mice showed in vitro Th1 and Th17 hyperactivation and caused more severe EAE pathology in vivo. Unexpectedly, T cell CD109 does not control TGF-ß signaling but restrains IFN-γ and/or IL-17 by binding to and inhibiting the activity of a non-canonical costimulatory molecule linked to Th1/Th17 biology. Guided by modeling and Cryo-EM data, we designed a CD109/costimulatory inhibitory peptide that recapitulates the phenotype observed in CD109-deficient T cells.

Together, these data suggest that the C3-like protein CD109 serves as an important molecular brake (or switch) on CD4 T cells by fine-tuning costimulatory signals upstream of the hyper-inflammatory Th1/Th17 induction pathways.
Neutrophil specific glycogen metabolism supports antifungal function in the infected tissues
Authors: Choi Wonseok , Li De-dong , Peroumal Doureradjou , Rahimi Hossein , Wang Shuxia , Biswas Partha
Published: November 2024

Neutrophils are critical for controlling systemic candidiasis (candidiasis), a bloodstream infection caused by Candida albicans. Neutrophils rely on extracellular glucose to fuel their antifungal activities at tissues. However, C. albicans consumes glucose, making it challenging for the neutrophils to survive and function at infected tissues. Hence, encountering fungal pathogens in glucose-depleted tissues exert metabolic challenges for neutrophils. It is unknown how neutrophils dynamically reprogram their metabolic pathways to function in glucose-deprived tissues in candidiasis. We show that neutrophils activate PYGL, an enzyme essential for the breakdown of intracellular glycogen storage to glucose, when glucose supply is limited. C. albicans regulate glycogenolysis in neutrophil in a dectin-1/Syk/Ca2+/PKA-axis dependent manner. Blockade of PYGL function in neutrophils inhibits fungicidal activities only when glucose availability is low and neutrophil specific PYGL ablated mice show increased susceptibility to candidiasis due to impaired ROS formation. Finally, treatment with β2-adrenergic receptor agonist, an approved drug and activator of PYGL function, improved antifungal activities of neutrophils in candidiasis. These results provide insights on how glycogenolysis supports compensatory metabolic reprograming required for antifungal function of neutrophils in the glucose-depleted infected tissues, a knowledge that can be therapeutically exploited by an approved drug.
Macrophages restrain pathological interferon responses during viral respiratory infection
Authors: Rodriguez-Morales Patricia , Hoagland Daisy , Mann Alexander , Yu Shuang , Lai Alicia , Baez Vazquez Alan , Medzhitov Ruslan , Franklin Ruth
Published: November 2024

Uncontrolled inflammation triggered by infection can cause deleterious effects on human health. Therefore, it is important to understand the mechanisms that constrain inflammatory responses. We observed that macrophages, key regulators of inflammation, produce the cytokine Oncostatin M (OSM) in response to cellular stressors and viral stimuli. Upon challenge with influenza or the viral mimic, poly(I:C), Osm-deficient mice exhibited increased mortality and this phenotype was recapitulated in mice lacking OSM specifically in macrophages. Transcriptional and protein analyses revealed that mice lacking OSM specifically in macrophages display heightened lung type I interferon (IFN-I) responses, and excessive IFN-I production by lung epithelial cells. Acute blockade of IFN-I signaling protected mice lacking macrophage-derived OSM against morbidity and mortality, revealing that these mice succumb to viral stimuli due to heightened IFN-I responses. Moreover, reconstitution of OSM in the respiratory tract of mice lacking macrophage-derived OSM was sufficient to protect against morbidity, highlighting the importance of local OSM signaling. Altogether, these data suggest that macrophage-derived OSM restrains excessive IFN-I production by epithelial cells during exposure to viral stimuli. This work reveals a novel mechanism by which macrophages dampen inflammation in the lung through the negative regulation of IFN-I, thus providing protection against inflammation-induced pathology.