bluuebunny/crossref_metadata_2025 huggingface dataset:
Single cell sequencing reveals mouse MAIT cell diversity
Authors: Chandra Shilpi , Seumois Gregory , Ramirez Ciro , Seo Gooyoung , Vijayanand Pandurangan , Kronenberg Mitchell
Published: January 2023

Mucosal-associated invariant T (MAIT) cells are a novel subpopulation of innate-like T lymphocytes that recognize vitamin B metabolites and express an invariant T cell receptor (TCR) α chain. MAIT cells are less abundant in mouse blood (~0.1% of T cells) as compared to human blood (~5%). MAIT cells play an important role in various infectious non-infectious diseases. Given their small number in mouse, these cells have been not been fully characterized. To unravel MAIT cell heterogeneity in thymus and peripheral tissues, we have performed single-cell sequencing of MAIT cells from various organs using 10× single-cell genomics, where we sequenced more than 7,000 cells and identified 10 clusters of MAIT cells by unbiased clustering. Cells from different organs mostly are represented in the different clusters, except one cluster was lung MAIT cell specific and another cluster consisted of cells exclusively from thymus. The lung specific cluster also reveals a tissue residency gene signature. We compared the gene expression profile of MAIT cells with iNKT cells, another T lymphocyte population that recognizes non-peptide antigens with invariant α chains. Our data reveal that most mouse MAIT cells have a Th17/NKT17–like gene signature, although there are some with Th1/NKT1 like transcriptomes, particularly in liver and spleen. A Th2/NKT2 gene expression profile was not observed. The thymus specific cluster showed a gene expression profile similar to the most immature or progenitor iNKT cells (NKT0), consistent with other data suggesting a unique thymus differentiation pathway. Therefore, our study reveals that although MAIT cells are predominantly Th17 and Th1 cells, there is an unexpected degree of heterogeneity.

Supported by R01 AI71922
Effects of TNFα signaling on iNKT cell development, activation and maturation
Authors: Zhang Hui , Modrak Samantha , Zhang Faya , Little Alexander Armstrong
Published: January 2023

iNKT cells are CD1d-restricted T cells that recognize lipid antigens. Upon activation these cells rapidly produce large amount and a broad spectrum of cytokines. They are important immunoregulatory cells involved in autoimmune disease, liver injury and antitumor immunity. The mechanisms regarding iNKT cell development, activation and maturation have been studied extensively. It is well known that many cytokines and transcription factors are involved in these processes. TNFα, an inflammatory cytokine that can be produced by iNKT cells, co-occurs with iNKT cells in steady state and many disease conditions. How TNFα affects iNKT cell development, activation and maturation is not known. Herein we used a TNFα KO mouse model to address this issue. We found that depletion of TNFα did not affect the frequency of iNKT cells in thymus and spleen, but decreased iNKT cells in liver, and increased iNKT cell apoptosis in liver. The portion of stage-2 (NK1.1loCD44hi) iNKT cells increased, stage-3 (NK1.1hiCD44hi) iNKT cells decreased in the thymus of TNFα KO mice, suggesting that depletion of TNFα impairs iNKT cell development and maturation. The percentage of CD69+ iNKT cells was significantly lower in the thymus, spleen and liver of TNFα KO mice compared to their wild-type littermates, suggesting that depletion of TNFα inhibits iNKT cell activation. Moreover, upon activation, the percentage of splenic IL-4- and IFN-g-producing iNKT cells was significantly lower in TNFα KO mice than in their wild-type littermates. Depletion of TNFα increased PLZF+ iNKT cells in thymus and downregulated the expression of CD122 on iNKT cells. Taken together, TNFα signaling plays important roles in regulation of iNKT cell development, activation and maturation.
Suppression of IL-17 producing type 3 innate lymphoid cells and increased susceptibility to Klebsiella pneumoniae sepsis in neonatal mice exposed to extended empiric antibiotics
Authors: Mirpuri Julie , Babu Sarah Thomas , Niu Xinying , Kumar Dharmendra , savani Rashmin
Published: January 2023

Background/Objective

Over 75% of newborns admitted to the neonatal intensive care unit (NICU) receive antibiotics with no proven infection (empiric) at birth. Empiric antibiotic use in the NICU increases susceptibility to late onset sepsis, but the mechanisms are poorly understood.

Methods

Neonatal mice were exposed to broad-spectrum antibiotics through their dams from birth for either 3-(short exposure, SE) or 7-days (long exposure, LE). At 14 days, qRT-PCR was used for bacterial 16S rRNA (stool), antimicrobial proteins (AMPs) and cytokines (small intestine, SI). Blood, liver and spleen cultures was utilized to detect bacterial translocation. Histology of SI was performed for Paneth cells. Flow cytometric analysis of lamina propria (LP) cells determined the presence of type 3 innate lymphoid cells (ILC3), neutrophils, macrophages, T- and B-cells. Susceptibility to sepsis was determined by IP injection of K. pneumoniae in 12-day-old pups.

Results

Mice exposed to SE and LE showed significant expansion of Enterobacteriaceae (2 log difference). LE mice showed baseline translocation of Enterobacteriaceae into the liver and spleen and were more susceptible to sepsis. By flow cytometry, LE mice showed a persistent and significant 50% decrease in IL-17 producing ILC3 in the LP, with other cell types unaffected. Further, the ILC3 present were skewed to the IL-22 producing phenotype. AMPs and Paneth cells were also increased in the SI, likely mediated by the increase in IL-22.

Conclusions

Extended empiric antibiotic exposure resulted in persistent expansion of Enterobacteriaceae and increased susceptibility to neonatal sepsis, likely mediated by a microbiota-dependent decrease in IL-17-producing ILC3.
B7-H4 expression in donor T cells and host cells negatively regulates acute graftversus- host disease lethality
Authors: Saha Asim , Taylor Patricia A. , Lees Christopher J. , Panoskaltsis-Mortari Angela , Osborn Mark J. , Feser Colby J. , Thangavelu Govindarajan , Melchinger Wolfgang , Refaeli Yosef , Hill Geoffrey R. , Munn David H. , Serody Jonathan S. , Maillard Ivan , Kreymborg Katharina , van den Brink Marcel , Dong Chen , Huang Shuyu , Zang Xingxing , Allison James P , Zeiser Robert , Blazar Bruce R.
Published: January 2023

The B7 family members are critical in positive and negative regulation of immune responses by engaging various lymphocyte receptors. B7-H4 is a member of the B7 family that can negatively regulate T cell function. We investigated the role of host and donor B7-H4 in regulating acute graft-versus-host disease (GVHD). Allogeneic donor T cells infused into B7-H4−/− versus wild type (WT) recipients markedly accelerated GVHD-induced lethality in a C57BL/6 to BALB/c GVHD model. Chimera studies pointed toward B7-H4 expression on host hematopoietic cells as more critical than parenchymal cells in controlling GVHD. B7-H4−/− recipients had rapid mortality associated with increased donor T cell expansion, gut T cell homing and loss of intestinal epithelial integrity, increased Teffector function (proliferation, pro-inflammatory cytokines, cytolytic molecules) and reduced apoptosis. Higher metabolic demands of rapidly proliferating donor T cells in B7-H4−/− versus WT recipients required multiple metabolic pathways, increased extra-cellular acidification rates and oxygen consumption rates, and increased expression of fuel substrate transporters. Interestingly, during GVHD, B7-H4 expression was upregulated on allogeneic WT donor T cells. Consistent with these data, donor B7-H4−/− T cells given to WT recipients increased GVHD mortality and functioned similarly to WT T cells from allogeneic B7-H4−/− recipients. Graft-versus-leukemia responses were intact regardless as to whether B7-H4−/− mice were used as hosts or donors. Taken together, these data provide new insights into the negative regulatory processes that control GVHD and provide support for developing therapeutic strategies directed toward the B7-H4 pathway.
Alloreactive B cells acquire suppressive function in recipients tolerant to allografts
Authors: Khiew Stella , Yin Dengping , Jain Dharmendra , Wang Qiang , Alegre Maria-Luisa , Chong Anita S F
Published: January 2023

Establishing a state of transplantation tolerance has been achieved in limited numbers of transplant recipients in the clinic. Clinical observations suggest that donor-specific antibodies can mediate allograft rejection, leading us to hypothesize that stable transplantation tolerance requires donor-specific B cell responses to also be suppressed. We hypothesize that this can be achieved through the induction of B cell-intrinsic tolerance. Using a well-established experimental model of transplantation tolerance induced to allogeneic B/c hearts with anti-CD154+donor spleen cell transfusion, we observed that donor-specific B cells are intrinsically tolerant. B cells from tolerant recipients did not produce anti-B/c IgG when adoptively transferred into naïve MD4 host and challenged with B/c splenocytes or heart grafts, and were blocked in their ability to differentiate into germinal center (GC) B cells. To test whether tolerant B cells could be rescued by ongoing GC responses, we adoptively transferred tolerant B cells into congenic hosts, followed by B/c spleen cell immunization. Tolerant B cells did not recover their ability to differentiate into antibody secreting cells, but instead they are able to suppress IgG production by naïve host B cells. Finally, we show that donor-specific tolerant B cells are not able to suppress third-party antibody responses nor exhibit linked suppression. Taken together, our data demonstrate that tolerant donor-specific B cells are profoundly altered compared to naïve B cells: they have significantly diminished ability to differentiate into antibody secreting cells, but instead acquired the ability to suppress donor-specific, but not third-party, antibody responses by naïve B cells.
C3 expression by lung transplants reduces alloimmune-mediated injury
Authors: Kulkarni Hrishikesh Satish , Liao Fuyi , Ma Lina , Wu Xiaobo , Kreisel Daniel , Atkinson John P , Gelman Andrew E.
Published: January 2023

Allograft injury is a major risk factor for death in lung transplant recipients. Complement protein C3 is primarily synthesized in the liver. However, a recent study by our group has demonstrated that C3 present in pulmonary epithelial cells can be augmented to facilitate their own ability to withstand acute environmental stress. Here, we hypothesized that pulmonary C3 expression reduces the severity of allograft rejection. To address this question, C3−/−left lungs [on a C57BL/6 (B6) background] were orthotopically transplanted into wildtype (WT) fully MHC mismatched (CBA/J) recipients. The right lung, native to the recipient, served as an internal control. CBA/J lungs transplanted into C3−/− recipients, C3−/− lungs transplanted into C3−/− recipients and CBA/J lungs transplanted into WT B6 recipients were also employed as controls. One week after transplantation, recipients were euthanized and evaluated for C3 levels and graft injury. C3 protein in C3−/− recipients of WT B6 and CBA/J lungs was not detected in the peripheral circulation but was expressed within graft tissues suggesting locally expressed C3 is sequestered within the lung. Signs of graft tissue injury were only observed in allogeneic recipients. However, acute rejection severity was significantly higher in C3−/− allografts when compared to WT allografts. Additionally, C3−/− allografts had a distinct pattern of acute alveolar damage and increased cellularity not evident in WT allografts. Taken collectively, our data indicate that expression of C3 by lung allografts attenuates alloimmunedependent responses that are associated with reduced survival in humans.
The prior exposure of CD4+ T cells to alloantigen blunts their secretion of the reparative growth factor amphiregulin
Authors: Zhang Xue , Mathews Lisa , Turnquist Heth R
Published: January 2023

Objective

Chronic rejection (CR) manifests after transplantation as function-limiting allograft fibrosis and vasculopathy. Insights into the mechanisms leading to CR are needed to develop effective therapies. The aggressive development of fibrosis during CR is similar to that observed in unproductive injury repair. Thus, we investigated how alloantigen (AlloAg) shapes novel mechanisms of immune-mediated tissue repair. Amphiregulin (Areg) is a growth factor secreted by innate and adaptive immune cells in response to IL-33 released during tissue injury. As IL-33 and Areg are implicated in muscle, epithelium, and nerve repair, we defined how AlloAg recognition by CD4+ T cells modulates IL-33-induced Areg secretion.

Method

C57BL/6 mice transgenic for a TCR recognizing a BALB/c-derived alloAg (Eα52–68) presented on I-Ab were administered IL-33 or Eα52–68 alone, or combined, 10 days before CD4+ T cell isolation. Areg secretion by isolated CD4+ during 4 days of culture with IL-33 or Eα52–68 was measured by ELISA. The signaling mediating Areg secretion was defined using inhibitors.

Results

CD4+ T cell exposure to IL-33 prior to alloAg secreted Areg upon de novo AlloAg exposure or secondary IL-33 stimulation. Exposure to AlloAg before IL-33, however, limited the capacity of alloreactive T cells to secrete Areg in response to either IL-33 or alloAg. Using the P38 inhibitor SB203580 and NF-κB inhibitors MG132 and TCPA-1 we established that both NF-κB and p38 are critical CD4+ T secretion of Areg.

Conclusion

Our studies suggest that recognition of AlloAg disrupts a reparative response mediated by CD4+ T cells. Limiting the reparative capacity of non-self T cells may aid pathogen clearance, but support development of CR in after transplant.
CD4+ T Cells are both necessary and sufficient to induce acute graft vs. host disease in lymphopenic recipients
Authors: McDaniel Brianyell T , Enriquez Josue , Furr Kathryn , Grisham Matthew
Published: January 2023

Allogeneic hematopoietic stem cell transplantation (HSCT) is a potentially life-saving treatment for refractory/relapsing hematological malignancies, blood disorders or autoimmune diseases. However, approximately 40–50% of patients undergoing allogeneic HSCT will develop a multi-organ, inflammatory disorder called acute graft vs. host disease (aGVHD). Experimental and clinical studies suggest that intestinal injury due to toxic, pre-transplant conditioning protocols (e.g. lethal irradiation and/or chemotherapy) may play a major role in the development of aGVHD. However, recent studies from our laboratory as well as others, suggest that this may not be the case.

Objective

We wished to quantify and compare the onset and severity of aGVHD induced by the adoptive transfer of allogeneic T cells into untreated lymphopenic mice.

Methods

Four million allogeneic or syngeneic CD4+CD62L+CD25− T cells were transferred (i.p.) into NK cell-depleted RAG1−/− mice or RAG2−/−IL2rγ−/− double knock-out (DKO) mice and assessed daily for signs of aGVHD.

Results

We found that adoptive transfer of allogeneic but not syngeneic T cells into NK cell-depleted RAG1−/− or DKO mice induced many of the clinical and histological features of aGVHD including weight loss, inflammatory cytokine production and tissue inflammation. In addition, adoptive transfer of allogeneic T cells into each recipient induced severe anemia as well as dramatic reductions in bone marrow and spleen cellularity.

Conclusions

We conclude that naïve allogeneic CD4+T cells are both necessary and sufficient to induce aGVHD in lymphopenic recipients in the absence of toxic, pre-transfer conditioning.
RNA origami nanostructures for potent and safe anti-cancer immunotherapy
Authors: Matiski Lawrence , Qi Xiaodong , Liu Xiaowei , del Villar Ryan Rodriguez , Zhang Fei , Yan Hao , Chang Yung
Published: January 2023

Nucleic acids released from microorganisms or damaged cells are perceived by the host cells as danger signals. They can be recognized by various types of pattern recognition receptors (PRRs), leading to activation of the innate immunity. Recently, we have identified a novel self-assembled RNA-origami nanostructure that shows high structural stability, feasibility of manufacture in large quantities, and demonstrates potent innate stimulatory activity. We further test its activity in murine tumor models both in vitro and in vivo. Although this RNA-origami nanostructure exerts no direct cytotoxicity on the tumor cells, it induces significant delay or regression of tumor growth in tumor-bearing mice with minimal toxicity. The mice showing tumor regression developed systemic long-term immunity and became resistant to further challenges of tumor cells. This anti-tumor immunity is dependent on both the innate and adaptive immune system. Thus, RNA-origami nanostructures represent a new line of platforms for rational design and construction of effective, safe and affordable immunotherapeutics.
A novel vaccination platform for epicutaneous allergen-specific immunotherapy based on beta-glucan neoglycoconjugates
Authors: Korotchenko Evgeniia , Machado Yoan , Strandt Helen , Joubert Isabella , Schiessl Viktoria , Bauer Renate , Sarajlic Muamera , Neuper Theresa , Wildner Sabrina , Horejs-Hoeck Utta , Gadermaier Gabriele , Scheiblhofer Sandra , Thalhamer Josef , Weiss Richard
Published: January 2023

Coupling of long chain carbohydrates to allergens can mask IgE epitopes and thus reduce the allergenicity of the neoglycoconjugates. The resulting molecules can target and activate dendritic cells via C-type lectin receptors. Neoglycoconjugates therefore have a high potential for epicutaneous immunotherapy (EPIT) by delivering a hypoallergenic vaccine to a tissue rich in dendritic cells. In the current study we test the therapeutic efficiency of the beta-glucan laminarin conjugated to ovalbumin (LamOVA) in a mouse model of allergic asthma.

Sensitized Balb/C mice were treated epicutaneously via micropores created by fractional laser ablation. Humoral and cellular responses were evaluated by ELISA, RBL assay and BAT. Lung function was analyzed by whole body plethysmography.

Laminarin remained biologically active after coupling to ovalbumin as indicated by binding to Dectin-1 receptor. IgE epitopes were successfully masked according to in vitro IgE binding aasay and skin reaction scores in therapeutic setup.LamOVA was more potent in stimulating humoral immune responses in comparison to OVA and induced the highest IgG1 and IgG2a production. Epicutaneous treatment with OVA and LamOVA reduced lung inflammation and improved lung function of sensitized mice more efficiently compared to s.c. injection with OVA/alum.

Epicutaneous immunotherapy can alleviate lung inflammation and reduce airway hyperresponsiveness in sensitized mice more efficiently than s.c. injection with alum. Coupling of allergen to laminarin reduces local side effects and increases the immunogenicity, resulting in a safer therapy.
Natural Adjuvants for in situ Vaccination Lymphoma Immunotherapy
Authors: Aleynick Mark , Peng Paul , Upadhyay Ranjan , Hammerich Linda , Roudko Vladimir , Yellin Michael , Brody Joshua
Published: January 2023

BACKGROUND

In situ vaccination (ISV) against lymphoma can be achieved with Flt3-ligand treatment to recruit dendritic cells (DC), radiotherapy to load DC with tumor antigens, and intratumoral injection of a pattern-recognition- receptor agonist (PRRa) to activate antigen-loaded DC. While clinical trials of ISV using synthetic PRRa have yielded remissions, the optimal approach to activate DC is unknown. We hypothesize that ‘natural’ PRRa, such as the attenuated pathogens in common prophylactic vaccines, could target multiple PRR, leading to more robust activation of DC as compared to synthetic PRRa.

METHODS

20 FDA-approved vaccines (BCG, MMR, etc.) were screened in vitro, for their effect on DC phenotype and function. DC tumor antigen cross-presentation was assessed using CRISPR gene-edited β2m−/−GFP-lymphoma cells and a GFP-specific CD8 T cell system. Mechanisms of immune activation were interrogated using MyD88, TRIF, MAVS, and IFNAR knockout mice, as well as a library of CRISPR-edited PRR-null Raw264 macrophages. Potent vaccines functioning through distinct mechanisms were evaluated in vivo in an ISV using the A20 murine lymphoma model.

RESULTS

Several vaccines induced robust DC activation, cross-presentation and increases in T cell activation, proliferation, and tumor killing. Some vaccines were more effective than synthetic PRRa in activating DCs to induce a T cell response. Distinct PRR and signaling pathways were engaged by different vaccines. In vivo, vaccine combination therapy induced tumor regression in a majority of animals.

CONCLUSIONS

Prophylactic vaccines, used alone or in combination, are effective DC activators and can be repurposed for use in ISV, with immediate translation into the clinic.
Optimization of human T cell activation and expansion protocols improves efficiency of genetic modification and overall cell yield
Authors: Yu Jessie , Chen Priscilla , Watson Ashley , Truong Danielle , Antonchuk Jennifer , Kokaji Andy I , Woodside Steven M. , Eaves Allen C. , Thomas Terry E.
Published: January 2023

Cancer immunotherapy using CAR T-cells is a rapidly progressing field and manufacturing these cells is a complex process that requires multiple optimization steps. We have developed reagents for the isolation, activation and expansion of human T cells that will be available for clinical cell therapy manufacturing. Soluble ImmunoCult™ Human T Cell Activators induce T cell activation via cross-linking CD3 and co-stimulatory molecules on the surface of cells. Activated T cells then can be genetically modified and subsequently expanded in ImmunoCult™-XF, a serum- and xeno-free T cell expansion medium. Here, we present several optimization strategies with ImmunoCult™ products in order to obtain high transfection efficiency and maximum cell yield. By evaluating activation dynamics of T cells and determining the optimal transfection time points, the transfection efficiency can be substantially improved in both CRISPR/Cas9- and lentiviral-mediated gene-modification methods. Our study also suggests that maintaining T cells at lower cell density after the third day following activation greatly improves cell viability and cumulative cell growth, resulting in an >1000-fold expansion of total human T cells with >85% viability over 10–12 days of culture. Expanded T cells co-express CD45RO+CD62L+ with low expression of PD-1. As an example, we applied the workflow described here to generate TCRαβ KO T cells from healthy donors with up to 90% knockout efficiency. The purity of TCRαβ KO T cells can be further increased with the use of an EasySep™ Human TCRαβ depletion kit. Taken together, the processes outlined in this study can be easily and rapidly implemented to improve T cell manufacturing efficacy.
IMPACT OF GUT MICROBIOTA ON VACCINE RESPONSE IN HIV EXPOSED INFANTS
Authors: Nyangahu Donald David , Plumlee Courtney , Wendoh Jerome , Urdahl Kevin B , Jaspan Heather
Published: January 2023

Maternal HIV infection has been shown to alter the composition and diversity of gut microbiota in their infants. We hypothesized that inheritance of an altered microbiota in HIV-exposed uninfected (HEU) infants is associated with poor vaccine responses. Here, using a germ free mouse model, we investigate the causative relationship between stool microbiota in HEU infants and BCG vaccine response.

BCG vaccine Th1 responses were assessed using a whole blood assay and an intracellular cytokine flow cytometry panel. Stool samples from time of BCG vaccination were collected, and those from high or low BCG responder infants transferred by oral gavage to germ free pups at day 5 after delivery. Five days after gavage, pups were sacrificed to analyze inherent immunity or vaccinated with 10^6 CFUs of BCG. Vaccinated animals were sacrificed 28 days post vaccination. BCG specific T cells were quantified using TB10.4 (class I) tetramers by flow cytometry.

At d10 post-delivery, pups gavaged with high responder stool (Phr) had significantly lower spleen cell count compared to those gavaged with low responder stool (Plr). Furthermore, Phr pups had significantly higher proportions but not numbers of effector memory CD4 T cells (CD4+CD44hiCD62Llo) relative to Plr. Total number of B cells and neutrophils were significantly reduced in Phr vs Plr. When vaccinated with BCG, Phr displayed significantly higher number of TB10.4-specific CD8 T cells compared to Plr.

Our data shows a direct role of the microbiota in inherent immunity in infants. Furthermore, the microbiota in low responder HEU infants caused reduced number of vaccine specific CD8 T cells. Together, we show for the first time that intestinal microbiota in human infants at birth impacts vaccine response.
Replicating Ad-SIV recombinant vaccines elicit mucosal humoral immunity in rhesus macaques at both rectal and vaginal sites with potential protective efficacy
Authors: Hogge Christopher James , Hait Sabrina Helmold , Enyindah-Asonye Gospel , Mushtaq Zuena , Hoang Tanya , Robert-Guroff Marjorie
Published: January 2023

Humoral immunity, especially at mucosal sites, is important for preventing HIV acquisition. Replicating adenovirus (Ad)-SIV priming/gp120 boosting regimens have elicited mucosal immunity and protection against intrarectal SIV challenge in Rhesus macaques. Here we investigated mucosal responses in female macaques primed intranasally/orally, then intratracheally with Ad5hr-SIV(Env/gag/nef), boosted twice intramuscularly with SIV gp120, and challenged vaginally with repeated low-dose SIVmac251. Vaginal washes and rectal swabs and biopsies were obtained 3-weeks post-immunizations. gp120-specific antibodies were assayed by ELISA and memory B-cells by flow cytometry. Gp120-specific rectal memory B-cells increased post-Ad priming but waned post-boosting, whereas rectal and vaginal antibodies increased post-boosting. Compared to pre levels, rectal gp120-specific IgA (p = 0.011) and IgG (p< 0.0001) were elevated post-2nd boost; vaginal IgA/IgG antibodies differed post-1st boost (both p<0.0001) and IgG levels > IgA post-2nd boost (p=0.001). Rectal memory B cells post- 1st and 2nd Ads positively correlated with gp120-specific rectal IgA (p = 0.015, p = 0.0013, respectively) post-1st Ad, indicating vaccine targeting to mucosal effector sites. Significantly delayed SIV acquisition was not seen, but vaginal IgG post-1st Ad correlated with number of challenges (p=0.029). Vaccinated animals had lower acute viremia than controls (p = 0.054), consistent with higher vaginal gp120-specific IgG 2 weeks post-infection (p < 0.0001). Our data show that upper respiratory tract immunization with replicating Ad vectors elicits strong mucosal immunity with potential protective efficacy against rectal/genital transmission.
Newly isolated porcine epidemic diarrhea virus resistance to interferon and neutralizing antibody
Authors: Shin Hyun-Jin , Park Jung-Eun
Published: January 2023

Porcine epidemic diarrhea virus (PEDV) infects pigs and causes enteric disease characterized by watery diarrhea and dehydration. Variant PEDV belonging to genogroup G2 has higher pathogenicity and morbidity than conventional PEDV belonging to genogroup G1. To understand the pathogenesis of the variant PEDV, we examined the susceptibility of the variant PEDV to interferon and PEDV specific neutralizing antibodies, which are known to important for the control of PEDV infection. We found that variant PEDV (WG strain) showed higher resistance to interferon than conventional PEDV (KPEDV-9 strain). Similarly, KPEDV-9 strain grew well in interferon-deficient Vero cells but did not grow in interferon-releasing porcine alveolar macrophages, whereas WG strain showed similar infectivity in both cells. We also found that WG strain is not blocked by the neutralizing antibodies against KPEDV-9, suggesting the differences in the antigenicity of two strains. To elucidate the possible mechanism of resistance to interferon and neutralizing antibody in variant PEDV, sequencing analysis was performed. We found huge variations in spike and ORF3 proteins. To test the impacts of these mutations on the acquisition of interferon and neutralizing antibody resistance, we are currently generating pseudotyped PEDV and recombinant PEDVs with mutations. Our results indicate that the recently introduced variant PEDV exhibits higher resistance to the interferon than the existing PEDV and avoids the neutralizing antibody against the existing PEDV. These results also provide understanding of the occurrence of variant PEDV and its pathogenesis.
Non-therapeutic administration of antimicrobial growth promoters to interrogate suppression mechanisms of innate and adaptive immune responses using an ovine model of stress
Authors: Clarke Sandra T , Inglis G. Douglas
Published: January 2023

Antimicrobial growth promoters (AGPs) have been administered in non-therapeutic concentrations in livestock feed to increase weight gain per unit of feed consumed. Daily low-dose antibiotic administration is capable of modulating immunity by suppressing inflammation, potentially increasing metabolic resources that can be allocated to muscle development. It is unknown how the AGPs tylosin (TYL) and chlortetracycline (CTC) influence acquired immunity. The objective of this study was to measure acquired immune responses, assess impacts on innate immunity and to elucidate mechanisms of immunosuppression by immunizing 10-week old lambs (male Suffolk cross) with keyhole limpet hemocyanin (KLH) during AGP administration (TYL, CTC or Control diets) while enduring intermittent physical stress. Over a 4-week period, 18 lambs (n=3 per diet/stress group) were monitored. Between the third-fourth experimental week, stressed lambs gained less weight (1.50 ± 0.49 kg) than non-stressed lambs (3.06 ± 0.37 kg, P < 0.03). The weight of lambs fed AGPs did not surpass those on the control diet. Diet and stress did not impact serum concentrations of total IgG and IgM, or anti-KLH IgG and IgM specific titres. Endpoint hematological analysis revealed that stressed lambs had decreased red cell distribution width (RDW%, 29% vs. 31%, P<0.01) and peripheral lymphocyte populations (5.72 × 103/μL vs. 4.69 × 103/μL, P<0.02). A diet × stress interaction was observed for peripheral granulocyte population (P=0.02). The impending restriction of AGP usage in North America has generated interest in identifying AGP alternatives, and elucidation of the mechanisms of AGP function is needed to develop effective non-antibiotic alternatives that mimic their action.
T cells responses during resolving Hepacivirus A infection, a surrogate animal model for human Hepatitis C
Authors: Gimenez Fernada , Mealey Robert H. , Ramsay Joshua D.
Published: January 2023

Hepacivirus A (HcV A) is a hepatotropic equine virus and the closest known relative of hepatitis C virus (HCV). HcV A is also highly similar to HCV in its replication kinetics and severity of associated acute hepatitis, but, unlike HCV, HcV A is typically cleared by the host before causing significant disease. As such, HcV A is considered a tenable model for defining the correlates of resolving hepaciviral infection and vaccine development. The pathogenesis of acute HcV A-hepatitis and the mechanism(s) that lead to viral clearance are unknown. In this study we aimed to characterize T cell responses in horses (n=2) following inoculation with plasma containing HcV A. Viremia and hepatic injury were monitored by RT-PCR and plasma liver enzyme activity [gamma glutamyl transferase (GGT) and sorbitol dehydrogenase (SDH)], respectively. HcV A-specific T-cell responses were evaluated every two weeks by interferon gamma Elispot following ex-vivo PBMC stimulation with peptides pools spanning the HcV A polyprotein. Liver biopsies were collected prior to infection, at the peak of liver enzyme activity, and following viral clearance to quantify CD4, CD8 and proinflammatory cytokine mRNA levels, and characterize CD3+ T cell infiltration by immunohistochemistry (IHC). No distinct HcV A specific peripheral T cell response was detected during infection, but when liver enzymes peaked, IHC revealed a mean 15 fold increase in CD3+ T cell infiltration. CD4, CD8 and proinflammatory cytokine mRNA levels were also significantly increased at the same time point. In conclusion, our findings suggest that acute HcV A-hepatitis is mediated by CD3+ T cells and that HcV A-specific T cell response are likely sequestered in the liver.
Functional comparison of human and non-human primate neutrophil responses
Authors: Brinkworth Jessica F , Van Etten Kathrine , Bhatt Priya , McClure Keaton , Valizadegan Negin , Woo Minkyu , Gunasekera Suvanthee , Suarez Yaravi , Aldridge Brian
Published: January 2023

Humans and apes are neutrophilic, maintaining high levels (50–70% of circulating leukocytes) of this short-lived, highly reactive cell type (neutrophils), while most primates maintain significantly lower proportions(20–40% circulating leukocytes). Neutrophils are key limiters of bacterial and parasitic infection, amplifying inflammation, phagocytosing and presenting, and trapping invading foreign material, as well as committing bystander tissue damage in these efforts. The comparative neutrophilia of humans, therefore, suggests alterations in neutrophil activities such that higher proportions are both required and tolerated in this species. To address this possibility, we analyzed transcriptomic, and cell physiological responses of neutrophils from humans, and two biomedically important primates, rhesus macaque and common marmoset. Cells were isolated via a density gradient and stimulated with 1ng-1ug of lipopolysaccarhide (LPS) from Escherichia coli K12 or the bacteria itself (MOI 10:1) for 1–3 hours. Transcriptomic responses were captured by RNAseq, and physiological responses [phagocytosis, apoptosis, extracellular trapping (NETing)] by fluorescent microscopy. The species differed strongly the expression of genes in innate immune and cell trafficking pathways. Overall, primates exhibited NETing and apoptosis sensitivity to even the lowest dose of LPS. Humans, however, displayed a unique complement of neutrophil antimicrobial responses - phagocytosing and apoptosising more, while NETing less than the other species. These results suggest inter-species differences in important neutrophil antimicrobial strategies that may impact interpretation of primate biomedical models.
IFN-lambda modulates dendritic cells to facilitate protective T cell immunity during influenza A virus infection
Authors: Hemann Emily A , Green Richard , Turnbull Bryan , Langlois Ryan A , Savan Ram , Gale Michael
Published: January 2023

Influenza A virus (IAV) remains a global health burden for which therapeutic and vaccination strategies to limit infection and drive robust, protective immune responses are still needed. Type III interferon (IFN), IFN-lambda (IFNλ), restricts IAV in vivo compared to type I IFN. IFNλ signaling in both epithelial cells and neutrophils contribute to this innate antiviral control of IAV. However, the contribution of IFNλ to program protective adaptive immune reposes against IAV has not been defined. To investigate the role of IFNλ in programming effective immunity against IAV, we evaluated responses in lungs and lymph nodes (LN) of WT and IFNλ receptor knock out (Ifnlr−/−) mice following IAV infection. Ifnlr−/− mice had significantly reduced pulmonary IAV-specific CD8 T cell responses during primary infection compared to WT that resulted in loss of protection against subsequent heterosubtypic IAV re-challenge. We found that this defect in the T cell response was not an intrinsic T cell defect, but instead was due to disruption of IFNλ-dependent dendritic cell (DC) function, as confirmed by infection of conditional Ifnlr−/− mice. Analysis of cell function revealed BMDCs respond to IFNλ, and that Ifnlr−/− DCs are defective in antigen uptake, processing, cell maturation, and cell migration. Transcriptomic analysis of DCs sorted from LN show that IFNλ (but not type I IFN) signaling is essential to modulate an IL-10 immunoregulatory program during IAV infection that links with the reduced function observed in Ifnlr−/− DCs. Our studies reveal a novel role for IFNλ in programming DCs for effective T cell actions to control IAV infection that may be broadly applicable to development of therapeutics against respiratory and other viral infections.
Regulation of IL-37 and its signaling co-receptor IL-1R8 in HIV infection
Authors: Ahmad Ali , Samarani Suzanne , Abulkhir Ayoub , Amre Devendra
Published: January 2023

IL-37 is a member of the IL-1 family with potent anti-inflammatory and immune deviating effects. The cytokine exerts its biological effects via IL-1R8 (also formerly known as TIR-8 and SIGIRR), the signaling component of its receptor. Little is known about the regulation of the cytokine and of its receptor in HIV infection. The objective of this study was to investigate how the production of the cytokine and the expression of the signalling component of its receptor on immune cells was regulated in HIV infection. The study was conducted using biological samples from a cross section of HIV-infected individuals. Concentrations of the cytokine and soluble (shed) IL-1R8 in serum samples were determined by ELISA. The expression of IL-1R8 on immune cells was determined by flow cytometry. The effects of exogenous IL-37 on HIV replication in human PHA blasts were determined in in vitro assays. We found that the cytokine concentrations tended to decrease in treatment-naïve HIV-infected individuals compared with the ART-treated patients. Higher concentrations of the cytokine were observed in sera from LTNP. The transcripts for the most complete isoform of the cytokine, b, disappeared from the patients’ PBMC. The expression of IL-1R8 on immune cells was decreased in HIV-infected individuals. On the other hand, concentrations of its soluble form increased in the sera from the virus-infected individuals. The trend was reversed in the patients undergoing anti-retroviral treatment. Soluble IL-1R8 attenuated anti-inflammatory effects of the cytokine. Collectively, these results suggest that the IL-37/IL-1R8 axis is functionally compromised in HIV-infected individuals.
Regulation and perturbation of Myocyte enhancer factor-2 (MEF-2) activity in Adult T-cell leukemia/lymphoma
Authors: Madugula Kiran Kumar , DeMarino Catherine , Panfil Amanda , Dobzanki Alex , Ginwala Rashida , Khan Zafar , Kashanchi Fatah , Lemasson Isabelle , Green Patrick , Harhaj Ed , Jain Pooja
Published: January 2023

HTLV-1 is a complex human retrovirus, an etiologic agent in causing malignant and intractable T-cell neoplasia. About 5% of the infected population would progress to acquiring a more aggressive form of non-Hodgkin’s lymphoma (NHL), termed as Adult T-cell leukemia/Lymphoma (ATLL). MEF-2 transcription factors are a family of genes that are involved in distinct functions in various tissues and isoforms of MEF-2 are 2A, 2B, 2C, 2D in mammals whose mutations are implicated in various cancers. Earlier studies from our lab delineated the role of MEF-2A in increased viral gene expression and inhibition of MEF-2A lead to the reduction in HTLV-1 viral replication and associated T-cell transformation. Presently, we have adopted to novel cyto-analytical techniques to quantitate gene transcripts and protein expression patterns at a single cell level for all the MEF-2 isoforms in different cell systems representing productive viral infection and/or ATLL. These studies revealed post-translationally modified, overexpression of MEF-2C and downregulated expression of MEF-2B which are known to be oncogene and tumor suppressor respectively. Similar results were obtained with a unique and specific HDAC-IIa inhibitor namely MC1568, which led to a dose-dependent cancer cell (but not normal) death in vitro while inhibiting viral proteins (Tax and HBZ) expression in vivo. In addition, exposure of HTLV-1-infected cells to MC1568 resulted in the activation of autophagy marker, LC3-II that is currently under investigation to a more in-depth mechanistic analysis.
HIV infection induces CD44+PD-L1+ tumor initiating cells in AIDS associated non-AIDS defining cancers
Authors: Gupta Harshita B. , Zhang Xinyue , Curiel Tyler J , Kulkarni Smita
Published: January 2023

The antiretroviral therapy (ART) significantly increased the number of aging HIV patients and the non-AIDS defining cancers (NADC) have become the most frequent co-morbidity and causes of death among HIV patients. The role of HIV infection in cancer initiation and progression is unclear. Tumor initiating cells (TIC) are responsible for chemo-resistance, metastasis and mortality. Our previous work demonstrated that tumor-intrinsic PD-L1 correlate with proportion (%) of TIC and virulence. Supernatants from HIV infected peripheral blood lymphocyte (PBMC) cultures (HIV-sup) significantly increased % TIC (CD44+PD-L1+) in two human tumor cell lines-SCC4 (squamous carcinoma) and HeLa (cervical cancer). HIV single stranded RNA (ssRNA) binds TLR7/8, and leads to immune activation and inflammation. The Cancer Genome Atlas database analyses showed that squamous cancer and cervical adenocarcinoma patients with TLR7/8 amplification had significantly poor overall survival. TLR7 agonist R848 led to significant increase in % TIC, whereas treatment of the cancer cells with HIV-sup in presence of TLR7/8 inhibitor ODN2088 reversed this effect. Similarly, pharmacological inhibition (by ACHP) of TLR7/8 mediated NFkB signaling pathway decreased TIC % two-fold, confirming HIV mediated TLR7/8 signaling has pro-TIC effects. High levels of inflammatory cytokine IFNα correlates with HIV load and persistent infection. Treatment with IFNα led to a significant increase in TIC whereas blocking IFNα-induced JAK signaling by ruxolitinib, in presence of HIV-sup reduced TIC significantly. Thus, our novel data gives insight into HIV induced host-pathogen interactions that lead to TIC prevalence.
Microglia secrete exosomes during persistent virus infection of the central nervous system that promote demyelinating disease
Authors: Olson Julie K , Luong Nhungoc
Published: January 2023

Theiler’s murine encephalomyelitis virus (TMEV)-induced demyelinating disease is a mouse model for multiple sclerosis (MS). TMEV infection of susceptible mice leads to a persistent viral infection of microglia in the central nervous system (CNS) which contributes to the development of a chronic progressive demyelinating disease associated with an inflammatory immune response. We have previously shown that TMEV infection of microglia activates an innate immune response with expression of cytokines, chemokines, and effector molecules associated with development and progression of demyelinating disease. Recently, we have shown that TMEV-infected microglia secrete exosomes, which are membrane-bound nanovesicles (60–150 nm). Exosomes contain and facilitate intercellular transfer of a wide variety of materials, including miRNA, mRNA, and proteins. We wanted to determine whether the exosomes secreted by TMEV-infected microglia can be taken up by other CNS resident cells and contribute to the persistent virus infection and inflammatory response during demyelinating disease. Our studies showed that exosomes secreted from microglia during TMEV-induced demyelinating disease (63–98 dpi) contain viral RNA/genome and can be taken up by other CNS resident cells. These exosomes activated the CNS resident cells to express inflammatory cytokines and chemokines. More importantly, the exosomes secreted by microglia during TMEV-induced demyelinating disease could induce MS-like pathology including neuroinflammation and demyelination in naïve mice. These results suggest that exosomes secreted by infected microglia may directly contribute to the development and progression of demyelinating disease.
IL-33 promotes antiviral immunity and protects against congenital Zika disease in mice
Authors: Liang Yuejin , Escano Jerome , Sun Jiaren
Published: January 2023

Zika virus (ZIKV) infection during pregnancy can cause congenital Zika syndrome and other birth defects. The alarmin IL-33 can evoke strong antiviral immune responses during viral infection. However, whether IL-33 is involved in immune responses during ZIKV infection is not clear. In this study, we infected three-week-old wildtype (WT) and IL-33 knockout (KO) mice with ZIKV and analyzed the ZIKV-specific cytotoxic T lymphocyte (CTL) responses at 6 days post infection. We found that IL-33 deficiency resulted in decreased ZIKV-specific CTL responses as evidenced by lower numbers of IFN-g+TNF-a+ CD8+ T effectors. To determine if IL-33 contributes to anti-ZIKV immunity during pregnancy, we infected WT pregnant mice with ZIKV and found increased IL-33 in the placenta at a mid-gestational period (embryo E13.5) compared with that of uninfected ones. Likewise, elevated IL-33 expression was also observed in human trophoblast cell lines JEG-3 and HTR-8 upon ZIKV infection. To determine if IL-33 plays a protective role in congenital Zika disease, we infected pregnant WT and IL-33 KO mice by i.p. injection of ZIKV at E6.5 and found higher percentages of fetal demise and absorption in IL-33 KO mice compared with that in WT mice (16.7 % vs. 3.3 %) from necropsy on E13.5. In addition, the fetuses from IL-33 KO mice displayed smaller sizes than those of WT fetuses (55 mm2 vs. 48 mm2). Moreover, IL-33 deficient mice had lower numbers of virus-specific CD8+ T cells in the placenta, uterine and decidua. Importantly, IL-33 deficiency resulted in higher viral loads in both the placenta and fetus. In summary, our study highlights IL-33 as a key immune regulator in inducing anti-ZIKV CTL responses and protecting against congenital Zika disease.
Determining the impact of HLA type and chronic viral infection on peripheral T-cell receptor sharing between unrelated individuals
Authors: Johnson Sarah , Gittelman Rachel M , Sanders Catherine , Robins Harlan
Published: January 2023

T cells generate highly diverse T-cell receptors (TCRs) to recognize antigens, most of which are private to each individual. TCRs with low junctional complexity have a higher likelihood of being generated and shared across individuals. Additional factors that may further limit diversity and increase the likelihood that particular TCRs are public include: (a) thymus selection for maturation of TCRs that bind antigens presented by an individual’s human leukocyte antigen (HLA) proteins, and (b) T-cell proliferation upon exposure to cognate antigens, which remain in the periphery as memory T cells. Numerous public TCRs have been associated with particular HLA restrictions and antigen specificities1. Chowell et al. also showed that HLA diversity is important for mounting an effective T-cell response against solid tumors2. However, little is known about the impact of these forces on the diversity of the TCR repertoire and expected TCR sharing across individuals. Here, we leverage the TCR repertoires of 666 healthy individuals sequenced with the immunoSEQ® Assay (Adaptive Biotechnologies, Seattle, WA) to address these questions. We investigate (a) how HLA restriction affects immune repertoires by examining the effect of HLA heterozygosity and divergence on the diversity of the TCR repertoire; (b) how the similarity of HLA alleles between individuals impacts the number of TCRs shared between individuals and (c) the impact of shared antigen exposure by determining how age and CMV status impacts clone sharing between individuals. These data show that HLA type, age, and CMV exposure shape the immune repertoire and influence the sharing of clones between individuals.