bluuebunny/crossref_metadata_2025 huggingface dataset:
Engineered Type-1 Regulatory T Cells as Cellular Therapy for Treatment of Immune Mediated Diseases
Authors: Liu Jeffrey M , Chen Pauline , Cieniewicz Brandon , Cepika Alma-Martina , Bacchetta Rosa , Roncarolo Maria Grazia
Published: January 2023

Type 1 regulatory cells (Tr1) are a promising cellular product for suppression of effector T cells in immune mediated diseases, including graft-versus-host-disease (GvHD) in allogeneic hematopoietic stem cell transplantation (allo-HSCT) (Roncarolo et al. Immunity 2018). We have developed an in vitro protocol to produce Tr1 cells by lentiviral transduction of the human IL10 with a constitutive promoter into human CD4+ T cells (Locafaro et al. Molecular Therapy 2017). These engineered Tr1 cells, called LV-10, acquire a characteristic Tr1 cytokine profile (IL-10 and IFN-g high, IL-4 low and expression of intracellular perforin and granzyme B). In vitro, LV-10 cells suppress the proliferation of responder CD4+ T cells upon activation by allogeneic dendritic cells. LV-10 cells also degranulate in response to and kill myeloid cells, including myeloid blasts from patients with acute myeloid leukemia, through a granzyme B- and perforin-dependent mechanism. Interestingly, the ability to degranulate and kill myeloid cells is not present when LV-10 are activated and expanded with anti-CD3 and anti-CD28 coated beads, suggesting that signals beyond TCR, CD28, and IL-10 receptor pathway activation are necessary to reprogram LV-10 cells into cytotoxic cells. In vivo, LV-10 cells injected into NSG mice do not induce xeno-GvHD, in contrast to control CD4+ T cells. In addition, LV-10 cells suppress CD4-induced xeno-GvHD and prevent expansion of myeloid leukemic cells. Experiments are ongoing to compare the potency and in vivo survival of allogeneic vs autologous LV-10 cells. These findings demonstrate the promise of using LV-10 to treat immune mediated diseases, including GvHD in AML patients receiving allo-HSCT.
Deubiquitinase BRCC3 ameliorates acute graft-versus-host disease and promotes graft-versus leukemia effects via promoting type I-IFN secretion
Authors: Liu Yin , Cheng Qiao , Zhang Wenli , Wang Dong , Xu Yang , Wu Depei
Published: January 2023

Acute graft-versus-host disease (aGVHD) remains the leading cause of transplant related deaths after allogeneic hematopoietic stem cell transplantation (allo-HSCT). aGVHD is mediated by the activation of donor lymphocytes in response to the host antigens, and involves a variety of immune cells, inflammatory cytokines and multiple other cytokines. Previous studies have shown that type I-IFN plays an important role in balancing aGVHD and graft-versus leukemia (GVL) responses. Ubiquitination plays essential roles in the production of type I-IFN, but deubiqutinases (DUBs) were reported rarely. Here, we showed that BRCC3 overexpression enhanced IFNβ-luciferase activity, and knockdown of BRCC3 decreased the expression of IFNβ. Interestingly, BRCC3 was down-regulated in allo-HSCT mice compared to syngeneic HSCT (syn-HSCT) mice. We further found that overexpression of BRCC3 ameliorated aGVHD, while promoting GVL effects. Relevant mechanistic studies exhibited that BRCC3 promoted IFNβ production in aGVHD mice, and reduced the absolute numbers of CD4+ T cells and CD8+ T cells in aGVHD target organs. Furthermore, we found that BRCC3 physically interacts with the E3 ligase Trim25, which is involved in RIG-I signaling pathway. In addition, BRCC3 de-ubiquitinates and up-regulates Trim25. These findings suggest that BRCC3-mediated type I-IFN production may be a strategy to control aGVHD and promotes GVL responses.
Supplementation of dietary zinc promotes T cell reconstitution after hematopoietic stem cell transplant
Authors: Iovino Lorenzo , Kinsella Sinéad , Cooper Kirsten , Jain Reema , deRoos Paul , Gagnon Alex , Ugrai Tamas M , Galimberti Sara , Petrini Mario , Dudakov Jarrod A
Published: January 2023

Hematopoietic stem cell transplant (HSCT) is a well-established therapy with curative potential for a variety of malignant and non-malignant diseases. However, delayed T cell reconstitution is an important contributor to transplant-related morbidity and mortality due to infections and malignant relapse. Optimal T cell development requires a functional thymus and many patients who could benefit from enhancing immune regeneration after HSCT have poor thymic function due to their age or their exposure to chemo- and radiation therapy. Here, we show that zinc, which is the second most abundant trace metal in the body, is crucial for thymic function and can be used to promote T cell reconstitution after HSCT. Mice that had been fed a zinc deficient diet for 3 weeks showed reduced thymic cellularity as zinc was required for the expansion and transition from DN to DP thymocytes. Conversely, supplementation of zinc in drinking water could significantly enhance T cell reconstitution after HSCT. Mechanistically, we found a switch in the bioavailability of zinc following damage, likely due to the death of thymocytes; and its release from thymocytes was able to directly stimulate the zinc-gated receptor GPR39 on endothelial cells to directly induce the production of BMP4; which we have previously identified as a key mediator of endogenous thymic regeneration. Consistent with this hypothesis, mice that had been given a pharmacologic inhibitor of BMP4 signaling did not demonstrate any improved thymic regeneration after zinc supplementation. In conclusion, we have demonstrated that zinc is important for steady-state T cell development and zinc supplementation offers a simple but innovative therapeutic strategy to improve T cell reconstitution.
Susceptibility of genetically diverse outbred mice to acute graft vs. host disease (aGVHD)
Authors: Enriquez Josue , McDaniel Brianyell T , Furr Kathryn , Grisham Matthew B
Published: January 2023

Mice are the primary animal of choice for modeling aGVHD. One aspect of these mouse models that may limit translation of promising therapeutic strategies to patient treatment is the use inbred mice as surrogates for genetically diverse human populations.

Objective

Quantify and compare tissue inflammation and injury in inbred vs. genetically diverse outbred mice using a well-characterized model of aGVHD-mediated bone marrow (BM) failure and spleen hypoplasia.

Methods

C57Bl6 (Bl6) CD4+ T cells (20,000 T cells/g b.w.) were injected (i.p.) into sub-lethally irradiated Bl6-H2-Ab1bm12(BM12) recipients (Bl6→BM12) or into Collaborative Cross (CC) or CD1 outbred recipients.

Results

Adoptive transfer of allogeneic Bl6 T cells into BM12 recipients induced marked weight loss (25%) at 20 days post T cell transfer that was associated with dramatic reductions in hematocrit as well as large and significant decreases in circulating granulocytes, platelets and erythrocytes when compared to their syngeneic controls (BM12→BM12). Total cell numbers in BM and spleen were also found to be dramatically reduced in these mice. Histopathological inspection confirmed severe hypocellularity in BM and spleen whereas no aGVHD was present in the lungs, liver, skin and colon. In contrast, adoptive transfer of Bl6 T cells into sub-lethally irradiated CC or CD1 or CC mice induced little or no weight loss at 25–30 days post T cell transfer with no significant reductions in hematocrit, circulating leukocytes, erythrocytes or platelets. BM- and spleen-associated cell numbers were not reduced when compared their CC controls.

Conclusions

Genetically diverse outbred mice are resistant to aGVHD-mediated BM failure and spleen hypoplasia.
Diverse IL-6 signalling modalities drive pathogenic T cell differentiation and graft-versus-host-disease after allotransplantation
Authors: Gartlan Kate H , Wilkinson Andrew , Chang Karshing , Kuns Rachel D , Henden Andrea , Minnie Simone A , Ensbey Kathleen S , Clouston Andrew , Zhang Ping , Koyama Motoko , Hidalgo Juan , Rose-John Stefan , Varelias Antiopi , Vuckovic Slavica , Hill Geoffrey R
Published: January 2023

Allogeneic stem cell transplantation (alloSCT) and graft-versus-host disease (GVHD) are characterized by systemic interleukin 6 (IL-6) dysregulation, which plays a significant role in shaping donor immune responses and T cell polarization. GVHD is a T cell-mediated disease and the severity and tissue distribution is heavily influenced by T cell-derived cytokines, therefore it is critical to understand the factors that drive T cell polarization in this context to inform therapeutic strategies. IL-6 has a unique receptor system composed of IL-6Ra and the signal transducing molecule gp130, in which signaling occurs via multiple pathways either directly (classical), indirectly via a soluble IL-6 receptor (trans), or presented via antigen presenting cells (cluster). We examined the influence of IL-6 signaling modalities on T cell polarization following allotransplantation, where we found specific targeting of these pathways modulates GVHD outcomes. Using donor grafts composed of IL-6Ra deficient T cells resulted in a profound loss of pathogenic Th17/Th22 differentiation and increased GVHD survival, demonstrating these populations are highly dependent upon classical IL-6 signaling post-transplant. Whilst targeting cluster signaling through IL-6Ra deficient DC had no effect on T cell cytokine responses, trans-signaling inhibition via soluble gp130-Fc resulted in severe skin GVHD. This effect was due to significant expansion of pathogenic donor Th22 and was prevented by donor IL-22 deficiency. These data demonstrate an important role for IL-6 trans signaling in regulating pathogenic T cell polarization pathways following allotransplantation and support IL-6 classical signaling as an important target for GVHD prevention.
Treg suppressor function within allografts is required for tolerance
Authors: Ossart Jason , Pena Andressa , Dai Hehua , Bauer Lynne , Williams Amanda , Camirand Geoffrey
Published: January 2023

The role of regulatory CD4+Foxp3+ T cells (Treg) suppressor function within allografts in tolerance remain unclear. To directly address this, we first used a model where immune reactions are restrained to the graft only. Effector T cells (Teff) alone, or with Treg (2–3×106 each) were transferred to B6 mice lacking secondary lymphoid organs (SLO−; splenectomized LTβR−/−) 5 days after islet allograft (Balb/c). Adoptive transfer of Teff alone induced acute rejection (10d MST). In contrast, Teff + Treg co-transfers significantly prolonged graft survival, with 65% of recipients surviving long-term (>100d MST). Interestingly, Treg did not affect Teff proliferation or accumulation within allografts, but significantly reduced MHC-II expression on DCs and IFN-γ in Teff.

Secondly, given that S1PR1 signaling is necessary for lymphocyte exit from SLO, we prevented Treg migration to allografts during tolerance by removing S1PR1 expression in endogenous Treg specifically. Tamoxifen-fed B6.Foxp3EGFP-Cre/Ert2.S1PR1fl/fl (S1PR1KO-Treg) or B6.S1PR1fl/fl (WT-Treg) mice received Balb/c islets. Untreated mice rejected allografts acutely (18d MST). Interestingly, while anti-CD45RB treatment induced tolerance in WT-Treg control mice (95d MST), all S1PR1KO-Treg mice rejected their graft (28d MST; p<0.05 vs WT-Treg). Also, these results correlated with a 25% decrease in Treg infiltrating islet allografts and with a 22% increase in Treg in lymph nodes on day 10 in S1PR1KO-Treg + anti-CD45RB compared to WT-Treg + anti-CD45RB recipients.

Overall, our data demonstrate that Treg can suppress rejection by Teff within allografts without prior activation in SLO, and suggest that Treg suppressor function within allografts is required for tolerance.
Tissue-Specific NK Cell Diversity in Solid Tumors
Authors: Dunbar Zerick T , Shanker Anil
Published: January 2023

Cytotoxic innate lymphoid cells, conventionally known as natural killer (NK) cells, play major roles in cancer immunity largely due to their natural cytolytic and immune regulatory functions. However, due to the extensive heterogeneity and plasticity seen among NK cells, their functions have yet to be fully harnessed in immunotherapy. The objective of this study is to elucidate NK cell diversity maturation, recruitment and effector function profiling in solid tumor microenvironments. We hypothesize that NK cells from different tissue locations display genetic and functional profiles that can predict NK effectiveness in solid tumor microenvironments. Here, we show the biological diversity that exists among NK cells from different locations pre- and post-interaction with solid breast cancer tumors, including heterogeneity of NK cell specific cluster of differentiation markers and gene expression perspectives based on flow cytometry, qPCR, RNA, and bioinformatics analyses. Understanding NK cells in the tumor microenvironment could help answer critical NK cell research questions and lead to advancements in cancer immunotherapy applications, ultimately helping to mitigate the cancer resources-results discrepancy.
Sympathetic nervous tone influences the development and function of natural killer cells
Authors: Nevin James Timothy , Moussa Marmar , Corwin William L. , Mandoiu Ion I. , Srivastava Pramod K.
Published: January 2023

Signals derived from the nervous system inform immune development and function in a variety of ways. For example, sympathetic nervous system (SNS) activity has been demonstrated to influence the function of innate lymphoid cells (ILCs) in the context of inflammation. Natural killer (NK) cells, a subset of ILCs, are important players in the anti-tumor immune response that possess the capability to respond to signals from the SNS via expression of adrenergic receptors (ARs) on their surface. Given the critical functions of NK cells in tumor immunity and their capacity to respond to SNS signals, we hypothesized that SNS input plays a key regulatory role in the NK-mediated anti-tumor response. Here, we demonstrate that the SNS delivers a critical maturation signal that promotes NK cell development and influences their cytotoxic capacity. Mice that lack functional SNS signaling after peripheral administration of 6-hydroxydopamine exhibit a higher proportion of poorly cytotoxic NK2 cells (CD11b−CD27+) and a lower proportion of potently cytotoxic NK1 cells (CD11b+CD27−) compared to controls. Furthermore, analysis of single-cell RNA sequencing establishes that SNS ablation disrupts normal NK development and that immature NK cells accumulate in SNS-ablated tumor-bearing mice. Together, these data demonstrate that tonic sympathetic nervous activity plays an important role in NK development. Further characterization of this role will improve our understanding of the consequences of neuroimmune interactions in cancer and inform the development of cancer immunotherapy.
LncRNA AK036396 inhibits maturation and accelerates immunosuppression of polymorphonuclear myeloid-derived suppressor cells by enhancing the stability of ficolin B
Authors: Tian Xinyu , Wang Shengjun
Published: January 2023

Long non-coding RNAs (lncRNAs) are emerging as crucial regulators of cell biology. However, the role of lncRNAs in the development and function of polymorphonuclear myeloid-derived suppressor cells (PMN-MDSCs) remains unclear. Here, we identified that the lncRNA AK036396 was highly expressed in PMN-MDSCs, and lncRNA AK036396 knockdown promoted the maturation and decreased the suppressive function of PMN-MDSCs. Ficolin B (Fcnb), whose expression could be used to reflect PMN-MDSC development, was the predicted target gene of lncRNA AK036396 based on microarray results. LncRNA AK036396 knockdown attenuated Fcnb protein stability in a manner dependent on the ubiquitin-proteasome system. Moreover, Fcnb inhibition downregulated the suppressive function of PMN-MDSCs. In addition, the expression of human M-ficolin, which is an orthologue of mouse Fcnb, was increased and positively correlated with arginase1 (Arg1) level in lung cancer patients. Based on these findings, lncRNA AK036396 can inhibit maturation and accelerate immunosuppression of PMN-MDSCs by enhancing Fcnb protein stability.
CCL2/CCR2 signaling protects against bladder cancer growth in a T cell dependent manner
Authors: Mukherjee Neelam , Ji Niannian , Shu Zhen-Ju , Curiel Tyler J , Svatek Robert S
Published: January 2023

The chemokine CCL2 (C-C motif ligand 2) is an important signaling axis underlying recruitment of pro-tumorigenic myeloid cells and is associated with worse outcomes in several tumor models. Currently, anti-CCL2 antibodies are being tested in cancer trials for solid tumors. To test CCL2 signaling in bladder carcinogenesis, wild type (WT) mice and mice deficient in CCL2 (CCL2KO) were given the urothelial carcinogen N-butyl-N-(4-hydroxybutyl) nitrosamine (BBN). Surprisingly, tumor incidence was higher in CCL2KO mice demonstrating an unanticipated protective role for CCL2 signaling in bladder cancer (BC). We then orthotopically challenged WT, CCL2KO, and CCR2KO mice (lacking the major CCL2 receptor) with MB49 BC and confirmed the protective effect of CCL2/CCR2. WT mice had significantly more intratumoral T cells compared to CCL2KO mice suggesting that CCL2 is involved in recruiting T cells to the bladder. Further, depletion of T cells abolished this protective effect of CCL2. Adoptive transfer of CCR2+ T cells into CCR2KO mice restored protection against MB49. CCR2+ T cells were also more activated, functional and tumor specific compared to their CCR2− counterparts. We found that anti-CCL2 promotes BC growth highlighting a concern for use of anti-CCL2 in BC. Moreover, intravesical recombinant CCL2 (rCCL2) either alone or in combination with intravesical gemcitabine reduced bladder tumor and improved survival of mice with MB49 BC. We further developed a slow-release encapsulated nanoparticle formulation of rCCL2 which successfully treated MB49, C3H mice with MBT-2 BC and reduced urine hematuria in our first ever humanized model of orthotopic BC. Our results are rapidly translatable and identify a novel treatment strategy in BC.
Bifidobacterium improves the outcome of immune checkpoint blockade by modulating Treg cell function
Authors: Wang Feng
Published: January 2023

Immune checkpoint-blocking antibodies that attenuate immune tolerance have been used to effectively treat cancer, but they can also trigger severe immune-related adverse events. Previously, we found that Bifidobacterium could mitigate intestinal immunopathology in the context of CTLA-4 blockade in mice. Here, we examined the mechanism underlying this process. We found that Bifidobacterium altered the composition of the gut microbiota systematically in a regulatory T cell (Treg)-dependent manner. Moreover, this altered commensal community enhanced both the metabolic and suppressive functions of intestinal Treg cells, contributing to the amelioration of colitis during CTLA-4 blockade. Importantly, Bifidobacterium ameliorated gut immunopathology and boosted anti-melanoma immunity with combination therapy of anti-CTLA-4 and anti-PD-L1.
Emerging roles of Cxcl12/Cxcr4 signaling axis in breast cancer metastasis
Authors: Karagiannis George S , Rivera-Sanchez Luis , Duran Camille L , Oktay Maja H , Condeelis John S
Published: January 2023

The Cxcl12/Cxcr4 axis promotes breast cancer metastasis in mice and humans. Prior studies have linked Cxcl12/Cxcr4 to cancer cell homing, survival and proliferation at distant sites secreting Cxcl12, such as lungs and liver. However, the mechanism via which Cxcr4+ cancer cells escape primary tumors in the first place (which also highly express Cxcl12), remains elusive. Here, by measuring chemotactic gradients using immunofluorescence, we show that Cxcl12 gradients are concentrically expressed around cancer cell intravasation sites, and spatially associate with Cxcr4+ tumor cells. Without affecting the gradient itself, pharmacological inhibition of Cxcl12 suppresses the translocation of Cxcr4+ cancer cells to intravasation sites. Paradoxically, tumor cell-specific deletion of Cxcr4 results in a suboptimal prometastatic response. We theorized that Cxcr4+ tumor-associated macrophages (TAMs), previously shown to accompany migratory tumor cells, could instead read the Cxcl12 chemotactic gradient, thus partially rescuing the prometastatic phenotype. Indeed, clodronate-mediated TAM depletion abrogates Cxcr4+ cancer cell translocation to intravasation sites and subsequent dissemination to secondary sites. We also found that breast cancer endothelium is lined by Cxcl12+Pdgfrb+ stromal cells; however, Cxcl12 gradients originating from intravasation sites are specifically tied to a subset of Cxcl12High perivascular TAMs. As such, pharmacological inhibition of Pdgfrb depletes Pdgfrb+Cxcl12+ stromal cells, but does not affect Cxcl12+ TAM-dependent translocation of Cxcr4+ tumor cells to intravasation sites. Overall, our data support a new model for the implication of Cxcl12/Cxcr4 in the early stages of metastasis.
Preclinical evaluation of efficacy and cytokine release of bispecific T cell engagers in humanized NSG mice
Authors: Yao Li-Chin , Cai Danying , Aliperta Roberta , Colthart Allison , Meier Werner , Cheng Mingshan , Keck James G
Published: January 2023

Bispecific T cell engagers (BiTEs) are a growing class of cancer therapy that redirects effector T cells against target-expressing tumors. Mouse models to assess efficacy and cytokine release associated with T cell engagement are needed. JAX humanized NOD scid gamma (Hu-NSG™) mice are engrafted with cord blood-derived CD34+ HSC and have shown robust T cell reconstitution. In this report we evaluated two novel BiTEs, EGFR-CD3 and BCMA-CD3, in Hu-NSG™ mice implanted with MDA-MB-231 human breast cancer cells and NCI-H929 multiple myeloma, respectively. Following the initial dosing, we measured human cytokines and analyzed T cell activation by flow cytometry. Clinical observations and body weight change were also monitored. Treatment with EGFR-CD3 for 2 weeks resulted in complete tumor regression when the dosing was initiated around 100 mm3. 50% of the treated-tumors remained nonpalpable for 10 weeks after dosing ceased. EGFR-CD3 also inhibited tumor growth in mice bearing tumors of ~400 mm3 average in volume. In both cases human IFN-γ, IL-6, and IL-10 levels were elevated from the first dose before returning to the baseline and the frequency of CD69+ cells in T cell subsets increased after the treatment. BCMA-CD3 treatment also induced T cell activation and elevated levels of human IFN-γ, TNF-a, IL-6, and IL-10. NCI-H929 tumor growth inhibition by BCMA-CD3 showed donor to donor variability. We observed that significant growth inhibition in one donor while the other donor did not respond. Together, our Hu-NSG™ platform enables preclinical evaluation of drug efficacy and cytokine release induced by BiTEs and has the potential to be applied to other immunotherapies.
Therapeutic targeting of CD38 enhances temozolomide activity in glioblastoma cells and modulates their tumor microenvironment by eliciting an enhanced CD8+ T cell response
Authors: Manna Alak , Aulakh Sonikpreet , Chanan-Khan Asher , Paulus Aneel
Published: January 2023

Glioblastoma (GBM) is an aggressive primary malignant brain tumor with poor prognosis. Surgical resection followed by chemoradiotherapy (temozolomide [TMZ] + radiation) results in a median survival of only 12-15 months. Recently, immunotherapy with targeted antibodies, therapeutic vaccination and adoptive cell therapy offers a promising option for treating GBM. Herein we observed that CD38, a catalytic receptor and regulator of NAD homeostasis, is expressed on GBM cells and their microenvironment in primary patient-derived GBM tissues. Blacher et al have shown that blocking CD38 enzymatic functions results in reduced GBM cell growth. This evidence prompted us to investigate the antitumor and immune engaging effects of a clinical grade anti CD38 therapeutic monoclonal antibody (Daratumumab/DARA) in preclinical models of GBM. We demonstrated that DARA significantly enhanced the direct apoptotic effect of TMZ and induced ADCC/CDC of GBM cells, irrespective of the presence of microglia. In an in vitro 3D simulated GBM + T cell coculture system, DARA induced activation (CD69/GITR upregulation/decreased PD1) and enhanced proliferation of CD8+ T cells/cTL activity against GBM cells. DARA also resulted in increased IFNγ and decreased IL-10; suggesting an improved antitumor cytokine milieu and ensued in downregulated inflammatory cytokine associated JAK2/STAT3 signaling pathway. Finally, we tested TMZ+DARA in a human GBM xenograft in vivo model and noted significantly prolonged survival of mice [TMZ+DARA vs. vehicle: 98.13±1.88 vs. 63.63±3.77 days, p<0.001]. Altogether, these observations led us to investigate the clinical activity of DARA+ TMZ in GBM patients in a phase I/II clinical trial.
Outlining key inflammation associated parameters during early phase of an experimental gram-negative sepsis model in rhesus macaques (Macaca mulatta)
Authors: Rosado-Franco Jose J , Ramos-Benitez Marcos J , Parodi Laura M , Rosario Derick , Compo Nicole , Giavedoni Luis D , Espino Ana M
Published: January 2023

Sepsis is a life-threatening syndrome characterized by metabolic and systemic dysregulation in the presence of an invading pathogen, such as bacteria or fungi. In the initial stages of this syndrome, the body, in an attempt to eradicate the pathogen, secretes high levels of pro-inflammatory cytokines and chemokines that leads to an anti-inflammatory state, which leads to organ dysfunction and death. Despite the efforts to find targeted therapeutics to treat septic patients, physicians rely on the administration of antibiotics and fluids. Hence, the development of novel targeted therapeutics to treat septic patients at early stages of sepsis is highly important to save the lives of these patients. The aim of this study was to identify inflammation markers during the early phase of sepsis in rhesus macaque. Four rhesus macaque were given an intravenous dose of 1010 CFU/kg of E. coli. Blood samples were collected before, 30 minutes, 2, 4, 6 and 8 hours after E. coli infusion. Physiological parameters, bacteremia, endotoxemia, C-reactive protein (CRP), procalcitonin (PCT), and cytokines and chemokines were determined for each animal at each time point. Bacteremia was present in all animals from 30 minutes to 3 hours after E. coli infusion. Endotoxin, CRP and PCT levels remained detectable during the whole experimental window. Signature cytokines and chemokines such as TNFα, MIP1α, and MIP1β peaked about 2 hours after E. coli infusion and decreased thereafter. Plasma IL6, IL12p40, IFNγ, and IL1Ra, ITAC, MIG, IP10 and MCP1, remained at detectable levels after 4 hours of E. coli infusion. This non-human primate model could be useful for the assessment of new therapeutics aiming to suppress key inflammatory markers throughout sepsis early phases.
Glucose is required for IFN-γ secretion but not IFN-γ mRNA expression by peripheral T cells
Authors: Loving Crystal L , Bond Zahra F
Published: January 2023

T cells play a key role in adaptive immunity, proliferating and producing effector molecules upon activation. T cell effector function is regulated by changes in their metabolism, which is necessary for ensuring availability of biosynthetic precursors for expansion, gene transcription, and protein translation. Impaired or altered immune T cell function often coincides with cellular metabolic impairment or shifts, and may be a cause for defective immune responses. For example, peripheral porcine T cells have increased glycolytic capacity following Arterivirus infection, but fail to produce IFN-γ protein in response to antigen stimulation. To understand the mechanism by which porcine T cell effector function is controlled by metabolic substrate availability a series of in vitro studies with varying culture conditions were performed. Specifically, peripheral blood mononuclear cells were stimulated with phorbol 12-myristate 13-acetate plus ionomycin in the presence or absence of glucose for varying amounts of time, and IFN-γ mRNA expression and IFN-γ protein secretion assessed. Glucose was required at the time of stimulation for both IFN-γ mRNA expression and translation/secretion. If glucose was removed 24h after stimulation, IFN-γ mRNA and protein were still produced, though protein levels were reduced. However, if glucose was not readily available upon stimulation, IFN-γ mRNA was transcribed, but not secreted. L-glutamine was supplied in all assays. Collectively, glucose availability to T cells can impact effector function and may be a factor in impaired responses during infection.
Cross-species gene expression analysis of human, canine and murine natural killer cells suggests convergence of activated dog and human transcriptomic profiles
Authors: Gingrich Alicia A , Reiter Taylor , Judge Sean J , Sturgill Ian , Monjazeb Arta M. , Murphy William J , Brown C Titus , Canter Robert J.
Published: January 2023

Key species differences exist between human and murine natural killer (NK) cells. Although outbred dogs are a link for translational NK studies, better characterization of dog NK cells is needed. We used RNAseq to profile resting and activated NK cells from mice, dogs and humans. Cells were isolated based on putative NK cell populations from healthy humans (CD56+CD3−), beagles (CD5dim) and C57BL/6J mice (NK1.1+CD3−) and sequenced from steady-state or following exposure to irradiated human feeder cells (K562 clone 9) with 100 IU/mL rhIL-2 × 14 days (human and dog) or 1000 IU/mL rhIL-2 × 7 days. Purity of expanded human and dog NK cells was ~ 90%, while for mouse it was ~ 70%. We performed differential gene expression (DGE) across species and conditions using ~7000 1:1 orthologous genes. DGE revealed distinct transcriptional profiles for each species (FDR <0.05). Hierarchical clustering demonstrated mouse NK cells as an outgroup compared to dog and human. PCA showed within-species spatial clustering of resting NK cells. After activation, variance between dog and human NK cells decreased, variance between human and mouse NK cells increased (PC1 40%, PC2 28%). Species differed in expression of key genes after activation, including PRF1, GZMA, SPP1, IL2RB, CTSD and FCER1G, which showed greater increases in abundance counts in mice than dogs and humans. In this first transcriptomic sequencing of dog NK cells, we show distinct gene profiles of resting cells across the most commonly used mouse, dog, and human NK populations, with convergence of dog and human NK cells upon stimulation under these standard conditions. By defining the cross-species comparative transcriptional profiles of putative NK cells, these data fill a gap in translational NK studies.
Host Transcriptome Studies in Response to Feline Coronavirus Reveal Differences in Macrophages vs CRFK
Authors: Drechsler Yvonne , Vasconcelos Elton JR , Diniz Pedro PVP
Published: January 2023

Feline coronavirus (FCoV) is a highly contagious virus that exists as two pathotypes, with feline infectious peritonitis (FIP) caused by the pathogenic FIPV. The pathogenesis of FIPV strains remains an enigma, and several different theories are still being discussed. A predominant theory has been that the pathogenic FIPV arises from a mutation of the enteric form, so that it could replicate not only in enterocytes of the intestines but also in monocytes, which subsequently transported the virus systemically. A strong argument can also be made for a role of the immune status and genetics of affected cats in pathogenesis. While some studies have looked at ex vivo infection of macrophages with virulent and avirulent strains, most of these studies focus on viral entry or differential replication instead of host responses. Considering the importance of genetics and host immune responses in viral infections, transcriptome studies to elucidate host pathogen interactions in macrophages and CRFK at different time points were performed by next-generation sequencing of mRNA. CRFK and macrophages from healthy male cats infected with FIPV 79-1146 ex vivo displayed differential gene expression in response. While CRFK gene expression was enriched for many classical virally induced genes and pathways, gene expression in macrophages was quite different. Instead, macrophage responses were very individual and showed limited overlap among cats. Interestingly, some of the commonly downregulated genes in macrophages involved innate anti-viral factors, while upregulated genes fell within unexpected pathways, such as autophagy, cellular senescence and other cellular processes not directly linked to infection.
The emergence and evolution of the mammalian germinal center: analysis of the splenic microarchitecture of the teleost threespine stickleback (Gasterosteus aculeatus)
Authors: Steinel Natalie C , Vaidyanathan Saraswathy
Published: January 2023

Lymphoid cell-based adaptive immunity first arose in jawed fish ~500 mya and fish lymphoid cells share many conserved features with their mammalian counterparts. Both fish and mammalian B cells respond to T-dependent antigen by proliferating, differentiating into plasma cells, and generating high-affinity, antigen-specific antibody. In mammals, this response occurs in the germinal center (GC) and is highly dependent on GC architecture and cellular dynamics, which direct B cell SHM and clonal selection. Though most cellular components are conserved, little is known with regard to the immune architecture of the fish humoral adaptive response. Therefore, we sought to investigate the organization of B cell activation within the lymphoid tissues of the fish, threespine stickleback (Gasterosteus aculeatus). Stickleback splenic B cells are organized around melanomacrophages (MM), highly pigmented melanomacrophages which are proposed homologues to mammalian follicular dendritic cells (FDCs). We find that MM aggregates, known as melanomacrophage centers (MMCs), increase in size in response to immunization, but not control injection with adjuvant alone, suggesting that the MMC response is antigen driven. Analysis of lymphoid tissue architecture indicates B and T lymphocytes inhabit distinct regions of the stickleback spleen. For instance, IgM mRNA-expressing cells aggregate in discrete regions surrounding MMC clusters, comparable to the organization of mammalian B cells around FDCs. These findings support the hypothesis that MMCs are evolutionary precursors to GCs. Furthermore, it suggests that the microarchitecture necessary to sustain somatic hypermutation and clonal selection evolved early in vertebrates.
A bursa of Fabricius structural analogue in the teleost fish Atlantic salmon
Authors: Bjoergen Haavard , Løken Oskar Mongstad , Hordvik Ivar , Koppang Erling Olaf
Published: January 2023

Knowledge of the construction of immune organs of different species is important to understand the evolution of immunity. In birds, the bursa of Fabricius is a cloaca-based lymphoid organ that has been vital for our current understanding of the adaptive immune system of vertebrates. So far, this structure has not been described in other species. Here, we show a substantial lymphoepithelial tissue in a previously undescribed bursa in the Atlantic salmon, situated analogous to the placement of the avian bursa of Fabricius, also developing from the ectoderm. Investigations of three different age groups of Atlantic salmon revealed a dynamic lymphoepithelium that regressed following sexual maturation. This is similar to the situation in birds. Morphological investigations including dissection, radiography, in situ hybridization and immunohistochemistry revealed presence of T and B cells embedded within a CCL19-positive lymphoepithelium. CCL19 expression is indicative for lymphoid organs, and thus we suggest that the salmonid bursa should be regarded as such. We found however no evidence for primary lymphoid organ functions including recombination events in the lymphocytes, this contrasting the situation in birds. Our results demonstrate the presence of a salmonid lymphoepithelial compartment with anatomical and developmental similarities to early stages in the organogenesis of the avian bursa of Fabricius. Such information provides novel and exciting possibilities both for our understanding of the function of the immune system in salmon and for our understanding of the development of lymphoid structures and niches in vertebrates.
Immune genomic response associated with preference behavior: an examination in a freshwater fish
Authors: Price Sarah , Schumer Molly , Young Rebecca L. , Wang Silu , Cummings Molly
Published: January 2023

Immune activity in the brain underlies many social behaviors. This immune-behavior relationship can be viewed under a genomics scope by isolating transcriptomic correlates and molecular pathways present following various social encounters. Here we explore how immune genes correlate with different social exposures: mate-choice and social affiliation. Using an established model for sexual selection studies, we compared the immune gene responses in female brains following exposure to either a potential mate or a conspecific female. Females from the Poeciliidae family of fishes, the sailfin molly (Poecilia latipinna), were profiled for their social preference behavior and brain transcriptomes using RNA-seq. During the 30 minute behavioral assay, females were placed into one of two groups: (n=8) given the choice of a large male and small male (‘mate choice’) or a choice between a large and small female (n=8; ‘social affiliation’). The relationship between immune gene responses (categorized using Gene Ontology analysis) and behavior (preference, social affiliation, activity) will be explored using a linear model approach for differential expression (limma; Ritchie ME, et al, 2015) and WGCNA (weighted gene co-expression network analysis; Langfelder & Horvath, 2008). The RNA-seq transcriptomes from each female (n=16) will be analyzed by comparing differential expression between the two groups (‘mate-choice’ vs ‘social preference’).
Mycobacterium avium subspecies paratuberculosis (MAP) promotes expression of Th17 related cytokines in bovine CD4+ and CD8+ T cells in the presence and absence of antigen-presenting cells
Authors: Coussens Paul M.
Published: January 2023

The overall purpose of work to be presented was to further examine the potential role of Th17 immune responses and expression of Th17 related cytokines in Johne’s disease (JD). JD is a chronic inflammatory gastrointestinal disease of ruminants caused by Mycobacterium avium subspecies paratuberculosis (MAP). Recently, it has become clear that Th17 immune responses may play a protective role in early mycobacterial infections, but could also contribute to inflammation in later disease. Detection of MAP infection by current immunological methods at early stages of JD is difficult. In addition, correlates of protection are not well known in model systems of JD, complicating vaccine development efforts. Finally, the sources of inflammation in late-stage JD are not well characterized. We have proposed that robust Th17 responses to MAP may help improve upon detection, vaccine development, and control efforts. We have shown that MAP antigens induce mRNAs encoding the Th17 associated cytokines IL-17A, IL-17F, IL-22, IL-23, IL-27, and IFNγ in co-cultures of CD3+ T cells and monocyte derived macrophages (MDMs). Although not as robust as when co-cultured with MDMs, MAP is able to stimulate the upregulation of these cytokines from sorted CD3+ T cells in the absence of APCs. CD4+ and CD8+ T cells are the main contributors of IL-17A and IL-22 in the absence of APCs. However, MAP stimulated MDMs are the main contributor of IL-23. In-vivo, JD+ cows have more circulating IL-23 than JD- cows, suggesting this proinflammatory cytokine could be an important factor in clinical JD. Our data suggests that Th17-like cells and associated cytokines may indeed play an important role in immune responses to MAP infection and development or control of JD.
CD4+ T cell recognition of haemagglutinin epitope across Influenza strains
Authors: Nguyen Andrea T , Sloane Hannah , Grant Emma , Szeto Christopher , Jayasinghe Dhilshan , Gras Stephanie
Published: January 2023

In 2018 the World Health Organisation listed influenza pandemics as one of the top threats to global health. Influenza pandemics occur as a result of the emergence of antigenically distinct strains, which arise due to genomic re-assortment or through the acquisition of mutations. The resulting strains have different antigenic make-ups and implicate the ability of the immune system to respond quickly following infection. Particular HLA class II allomorphs including HLA-DRB1*07:01 have been linked with the occurrence of severe influenza infections and non-response to the influenza vaccine. However, other allomorphs such as HLA-DRB1*11:01 have been shown to be protective in viral diseases such as HIV and HCV, and might be important for influenza infection. The mechanism surrounding these associations of influenza infection and HLA class II molecules expression is unclear. As some individuals expressing particular HLA allomorphs are at a greater risk of developing severe influenza infection, further investigation into the mechanism of this association is warranted. Through stimulating T cells from donors expressing HLA-DRB1*11:01 or HLA-DRB1*07:01 with an influenza epitope (HA) derived from pandemic and epidemic influenza strains, we determined the level of cross-reactivity, phenotype and polyfunctionality of the responding CD4+ specific T cells. We have compared different allomorphs and provide the first insight into the molecular basis of influenza epitope recognition by different HLA class II allomorphs, and its functional impact.
CCR4 and CCR6 Expression Characterizes Memory Tregs Subsets During HIV Infection and Correlates with Disease Progression
Authors: Salgado Gonzalo , Del Río - Estrada Perla Mariana , Avila-Ríos Santiago , Reyes-Terán Gustavo
Published: January 2023

Chemokine receptor expression on Tregs has been associated with pathogenesis of several diseases such as cancer, as CCR4 promotes Treg recruitment into tumors. During HIV infection, continuous Treg stimulation due to chronic inflammation has negative effects such as tissue fibrosis and limited antiviral response. We studied CCR4, CCR6, CXCR3 and CXCR5 on Treg subsets to identify homing patterns during HIV disease.

CD4+ T cells (Tc) from 33 chronic untreated (CHR) and 52 antiretroviral treated (ART) HIV donors were characterized by FACS using memory markers. Tregs were defined as CD127lowCD25hi. Analysis was performed on Cytobank and Prism 6 using non-parametric tests.

Tregs represented 7.0% and 8.0% of total CD4+ Tc in CHR and ART, respectively. Average Treg memory subsets distribution in CHR vs. ART was: Tscm=17.2% vs. 15.3%, Tcm=30.7% vs. 36.3% (p=0.0008), Ttm=27.4% vs. 25.7%. Chemokine receptor expression on total Tregs was (CHR vs. ART): R4=46.9% vs. 55.2% (p=0.0261), R6=31.6% vs. 40.1% (p=0.0043), X3=20.8% vs. 20.9% and X5=7.6% vs. 10.3%. In CHR, R4 (p=0.0007 r=0.3161) and R6 (p=0.0054 r=0.2241) frequencies correlated with CD4+ Tc count. Both markers were preferentially found on memory Treg (Tcm: R4=70.5%, R6=51.2%; Ttm: R4=93.4%, R6=65.7%) and had a significantly lower expression on non-Treg subsets (Tcm: R4=43.2%, R6=46.9%; Ttm: R4=37.0%, R6=29.9%). A similar pattern was also observed in people on ART.

We showed that Tregs are characterized by R4 and R6 expression during treated and untreated HIV infection. Both molecules are markedly found on memory phenotypes, suggesting a role in controlling homing of effector Tregs. Accumulation of R4+R6+ Tregs may reflect a response to control chronic activation that persists after ART.
Tfh Cell Metabolism and HIV-1 Infection
Authors: Syed Fahim , Yang Jing , Xia Ying , Li Wei , Yu Qigui
Published: January 2023

HIV-1 predominantly infects and depletes CD4 T cell populations. Multiple subsets of CD4 T cells exist, one of which is the follicular helper T (Tfh) subset. This subset is primarily present in lymphoid tissues, such as tonsil tissue, where it is specialized in helping B cells form germinal centers (GCs) for generation of high-affinity class-switched antibodies. Tfh cells in HIV-1 patients are expanded and become a major active and latent HIV-1 reservoir. To explore whether Tfh cells have a unique metabolic program that is responsible for Tfh cell expansion and survival under HIV-1 infection, we used flow cytometric analysis to sort GC Tfh (CD3+CD4+PD-1highCXCR5high) versus non-Tfh (CD3+CD4+PD-1−CXCR5−) cells from tonsil mononuclear cells for RNA sequencing (RNAseq). We found that Tfh cells express significantly lower levels of glycolytic genes, but higher levels of gluconeogenic and fatty acid oxidation genes. Inhibition of glycolysis via a lack of glucose in culture medium aided in the differentiation of Tfh cells from naive CD4 T cells and promoted Tfh cell survival against HIV-1 infection. Furthermore, use of chemical inhibition of alternative metabolic pathways effected the response of T cell subsets to HIV-1 infection. Our studies into metabolism in Tfh cells suggest a relationship between their unique metabolic phenotype and HIV-1 infection, replication and latency.
PD-1hi CD8+ tissue-resident memory T cells balance immunity and fibrotic sequelae following influenza infection
Authors: Wang Zheng , Wang Shaohua , Goplen Nick P. , Sun Jie
Published: January 2023

CD8+ tissue-resident memory T (TRM) cells provide frontline immunity in mucosal tissues. The mechanisms regulating CD8+ TRM maintenance, heterogeneity, and protective and pathological functions are largely elusive. Here, we identify a population of CD8+ TRM cells that is maintained by major histocompatibility complex class I (MHC-I) signaling, and CD80 and CD86 co-stimulation after acute influenza infection. These TRM cells have both “exhausted-like” phenotypes and memory features, and provide heterologous immunity against secondary infection. Using recombinant influenza virus, we demonstrate that the development of these PD-1hi “exhausted-like” TRM cells is dependent on the doses of the antigen during primary infection. Furthermore, we showed that lung CD11c+ antigen presenting cells are required for sustaining these TRM cells at the memory stage. Our data suggest that continuous antigen presentation in situ is required for the development and maintenance of these “exhausted-like” TRM cells in the lung. Strikingly, PD-L1 blockade after the resolution of primary infection promotes the rejuvenation of these “exhausted-like” TRM cells, restoring protective immunity at the cost of promoting post-infection inflammatory and fibrotic sequelae. Thus, PD-1 serves to limit the pathogenic capacity of “exhausted-like” TRM cells at the memory phase under specific tissue environment with chronic low-levels of antigen presentation following viral clearance. Our data indicate that TRM cell “exhaustion” is the result of a tissue-specific cellular adaptation that balances fibrotic sequelae with protective immunity.