bluuebunny/crossref_metadata_2025 huggingface dataset:
Morphine exacerbates experimental sepsis by modulating IL17/IL22 response (IRC8P.488)
Authors: Meng Jingjing , Li Dan , Roy Sabita , Wang Fuyuan
Published: January 2023

Sepsis ranks as the top 10 cause of mortality in the intensive care units (ICU) in USA and the standard management of pain in these patients are opioids. However, very few studies have investigated the influence of opioids on development and progression of sepsis. In the present study, we determined the effects of morphine on sepsis progression using the murine cecal ligation and puncture model, which resembles many aspects of the human pathology. Our studies demonstrate that morphine treatment increases bacterial dissemination at the early stage of sepsis (24 hours) and significantly attenuates pathogen clearance at late stage of sepsis (72 hours). In addition, IL17a and IL22 responses to pathogen stimulation are impaired following morphine treatment, resulting in delayed neutrophil recruitment to peritoneal cavity, thereby compromising pathogen clearance. This is the first study to demonstrate that IL17a and IL22 responses play important roles in host defense against polymicrobial sepsis, and that this crucial response is impaired following opioid treatment. These studies imply that IL17a and IL22 may be potential therapeutic targets to treat or prevent sepsis, especially in opioid use and abuse population.
IL-33 promotes IFN-γ production by group 1 innate lymphoid cells in viral hepatitis (IRC8P.490)
Authors: Liang Yuejin , Jie Zuliang , Sun Jiaren
Published: January 2023

Innate immune response is the first line of the defense against the viral pathogens. The damage associated molecular patterns (DAMP) molecule IL-33 has been proved to be critical for T cell response in viral persistent infection. However, its role in modulating the innate immune response in viral infection is not clear. Herein, we injected the mice with lymphocytic choriomeningitis virus (LCMV) Clone 13 and found that the hepatic gene expression of IL-33 was upregulated at 1 day post-infection (dpi) and reached the peak at 6 dpi. Deficiency of IL-33 not only led to impaired anti-viral T cell responses (low IFN-γ and TNF-α), but also resulted in less numbers of IFN-γ+ innate immune cells, such as NK and γδT cells at 16h postinfection. Treatment of recombinant IL-33 (rIL-33) significantly increased the percentages of IFN-γ +γδT cells. In vitro, rIL-33 inhibited proliferation of IL-17+γδ T cells, but increased IFN-γ+γδ T cells in a dose-dependent manner. rIL-33 treatment also upregulated IFN-γ and granzyme B expression in NK cells in vitro culture. More importantly, hepatic dendritic cells (DC) in IL-33 knockout mice expressed lower level of IL-12 compared with control mice, indicating that deficiency of IL-33 may impair the DC priming. Our data reveal the key role of IL-33 in promoting IFN-γ production by group 1 innate lymphoid cells and subsequently regulate the anti-viral T cell response in viral hepatitis.
Natural killer (NK) cells but not NK T cells attenuate renal fibrosis after acute kidney injury (AKI). (IRC8P.480)
Authors: Vincent Isaah , Moscalu Stefan , Chhoun Christopher , Ye Hong , Huang Liping , Sung Sun-Sang , Okusa Mark
Published: January 2023

Renal fibrosis is the common pathway of various kidney diseases that lead to progressive renal failure and end stage renal disease. NK and NK T cells have been linked to regulating fibrosis in several different organ systems. However, there is debate as to whether they are pro-fibrotic or anti-fibrotic. To determine their role in renal fibrosis, mice were administered PK136 mAb (200μg i.p), which depletes both NK and NKT cells, twice weekly for 14 days starting 5 days after 26 minutes of unilateral kidney ischemia-reperfusion (uIRI) or folic acid (250 mg/kg, i.p.). In both models, compared to IgG-treated mice, kidneys of mice treated with PK136 had increased fibrosis as assessed by Masson’s Trichrome and mRNA expression of collagen 1 and α-SMA. This suggests that NK/NKT depletion increases extracellular matrix deposition and myofibroblast number. This may be due to changes in the kidney microenvironment as kidneys of PK136-depleted mice had decreased expression of IFN-γ and increased expression of IL-10 and TGF-β compared to IgG-treated mice. We next subjected Jα18-/- and CD1d-/- mice to unilateral IRI to determine the specific role of NKT cells in renal fibrosis. Compared to wild-type mice, Jα18-/- and CD1d-/- mice subjected to uIRI demonstrated no increase in fibrosis, thus we conclude that the that NK but not NKT cells attenuate renal fibrosis after AKI. Further investigation will focus on determining the mechanism by which NK cells regulate fibrosis within the kidney.
The pro-atherosclerotic milieu promotes the conversion of murine T regulatory cells to a spectrum of Foxp3+IFNγ+ T cell phenotypes. (IRC8P.481)
Authors: Butcher Matthew , McGary Christopher , Galkina Elena
Published: January 2023

T cell subsets play diverse roles in inflammation but may also display limited plasticity. The existence of a population of Foxp3+ T regulatory (Treg) cells that co-express Interferon-γ (IFNγ), has been demonstrated in several models of inflammation. In atherosclerosis, circulating Treg content inversely corresponds with atherogenesis, suggesting that inflammation affects peripheral Treg (pTregs) maintenance. Thus, we sought to examine the fate of pTregs within atherosclerotic Apolipoprotein E-deficient (Apoe-/-) mice. Foxp3+IFNγ+ T cells were elevated within the aorta, spleen, and peripheral lymph nodes (PLNs) of atherosclerotic Apoe-/- mice in comparison to C57BL/6 mice. Further phenotyping of Foxp3+IFNγ+ T cells revealed variable expression of both Treg and Th1 markers, including PD1, GITR, CD25, CD73, Tbet, CXCR3, and CCR5. To determine whether Foxp3+IFNγ+ T cells arise from de novo differentiation or peripheral conversion, we performed fate tracking experiments with co-adoptively transferred naïve T cells and pTregs within Apoe-/- mice. In this system, naïve T cells failed to generate Foxp3+IFNγ+ T cells. In contrast, adoptively transferred Tregs lost Foxp3 expression (50%), maintained Foxp3 expression (40-45%), or adopted a Foxp3+IFNγ+ phenotype (5-10%) within all of the tissues examined. Together, these results demonstrate that inflammation during atherogenesis affects the maintenance of pTregs, resulting in the generation of phenotypically diverse Foxp3+IFNγ+ T cells.
The co-stimulatory molecule GITR intrinsically enhances type 1 and follicular helper CD4 T cell responses to establish early control of a persistent viral infection and potentiate the late humoral response (IRC9P.700)
Authors: Clouthier Derek , Wortzman Michael , Zhou Angela , Luft Olga , Levy Gary , Watts Tania
Published: January 2023

During persistent infection, the immune system must be tightly regulated to balance immune control against pathology. The glucocorticoid-induced TNFR (GITR) is an important co-stimulatory molecule that plays a critical CD4 T cell-intrinsic role in the control of a mouse model of chronic viral infection, LCMV clone 13. 8 days post-infection (dpi) of wild-type (WT) and GITR-/- mice revealed that GITR-/- mice have 2-fold fewer LCMV-specific CD8 T cells, with higher PD-1 and Tim-3 levels. These defects became more striking at 45 dpi, when GITR-/- mice have a ~9-fold deficit in IFNγ+CD107+TNF+ CD8 T cells. GITR-/- mice had impaired viral control, with 35- and 10- fold higher viral load in the kidney at 8 and 45 dpi, respectively. Depletion of CD4 cells largely abrogated the differences between WT and GITR-/- mice at 8 dpi, suggesting that CD4 T cells underscore the impaired T cell responses of GITR-/- mice. GITR-/- mice have 3-4-fold fewer IFNγ+ and IL-2+ Th1 cells at 8 dpi. Using DEREG mice, we found that the compromised immunity and viral control in GITR-/- mice are independent of Foxp3+ Tregs. GITR-/- mice also had 2-fold fewer follicular helper (Tfh) cells at 21-45 dpi, with concomitant impairment in LCMV-specific IgG titres. Mixed bone marrow chimeras revealed a CD4 T cell-intrinsic role for GITR in potentiating Th1 and Tfh responses. Our findings reveal a critical role for GITR in co-stimulation of CD4 helper T cell subsets for optimal cell-mediated and humoral immunity.
A role for interleukin-15Rα signaling in the homeostasis of early T cell progenitors (LYM7P.716)
Authors: Colpitts Sara , Lefrancois Leo , Puddington Lynn
Published: January 2023

A deficiency in interleukin-15 receptor α (IL-15Rα) expression is associated with an overall decrease in thymic cellularity. To further investigate this defect, we examined T cell progenitors in the thymus of wild-type (WT) and IL-15Rα KO mice. We identified a significant reduction specifically within the subset of CD27high cells in the CD44highCD25- double negative (DN1) population of T cell progenitors in the absence of IL-15Rα. Since T cell development begins early in ontogeny, we also examined the thymus of WT and IL-15Rα KO neonates (<24hrs post-birth). Surprisingly, we found no differences in the total numbers of neothymocytes or CD27high DN1 cells suggesting that the aforementioned deficiencies are acquired over time. Finally, while IL-15Rα functions largely through the transpresentation of IL-15, it also has the capacity to signal directly. To determine which of these two roles might be involved in the long-term homeostasis of thymic progenitors, we used a transgenic mouse strain that expresses a chimeric receptor composed of the extracellular domain of IL-15Rα and the intracellular domain of the IL-2Rα, thus efficiently disrupting IL-15Rα signaling while maintaining transpresentation. We found that, similar to IL-15Rα KO mice, IL-15Rα signaling transgenics also exhibited a significant decrease in the subset of CD27high DN1 cells suggesting that direct signaling through IL-15Rα is required for optimal homeostasis of early T cell progenitors in the thymus.
Limited frequency of cytokine-producing T cells in tuberculosis lung granulomas (MPF6P.736)
Authors: Gideon Hannah , Myers Amy , Phuah JiaYao , Rutledge Tara , Coleman Teresa , Maiello Pauline , Lin Philana , Flynn JoAnne
Published: January 2023

Lung granulomas are the pathologic hallmark of Tuberculosis (TB). T cells are a major cellular component of tuberculosis lung granulomas and are known to play an important role in containment and progression of Mtb infection. We used cynomolgus macaques, a non-human primate model that recapitulates human TB, with clinically active disease and latent infection, to understand the functional characteristics and dynamics of T cells in individual granulomas and to correlate T cell cytokine response of granulomas to its bacterial burden and its systemic response. Our results suggest that each granuloma within an individual host is independent with respect to total cell numbers, proportion of T cells, pattern of cytokine response, and bacterial burden. The spectrum of these components overlaps greatly amongst various clinical status of these animals, indicating that a diversity of granulomas exist in individual hosts. Multi-parametric flow cytometry analysis of functional T cells in granulomas suggests that on average only ~6% of T cells respond to Mtb antigens with production of Th1/Th17 or IL-10 cytokines. An inverse correlation was observed between bacterial burden and Th1/Th17 response in individual granulomas. The systemic responses do not accurately reflect responses in granulomas, and suggests that, especially in active TB, the blood is a negative reflection of granuloma responses. Overall, these data provide insight into the local and systemic responses in tuberculosis.
Vanilloid receptor-1 as a regulator of gut homeostasis (MUC8P.800)
Authors: Acharya Nandini , Penukonda Sasi , Basu Sreyashi
Published: January 2023

The intestinal mucosa is exposed to myriads of foreign antigens. Hence the immune responses elicited to innocuous antigens in the gut are highly regulated to avoid overt inflammation. CX3CR1hi macrophages , also known as regulatory macrophages, play a critical role in maintenance of immune homeostasis in the gut. Thus therapeutic strategies leading to increase in CX3CR1hi macrophages would be beneficial for treatment of inflammatory diseases. Our present study demonstrates that Vanilloid receptor 1 (VR1), an ion channel previously identified on sensory neurons, is specifically expressed in the CD64+CX3CR1hi macrophages in intestinal lamina propria (LP). VR1-/- mice under normal steady state conditions have significantly reduced number of CX3CR1hi macrophages as compared to wild type counterpart. This data indicates a homeostatic role of VR1 and its endogenous ligands in regulating inflammation and maintaining immune tolerance in the gut. Interestingly, engagement of VR1 by orally administering its exogenous ligand Capsaicin (CP), the pungent component of chili peppers, increases the frequency of CD64+CX3CR1hi macrophages in LP. Furthermore we have shown that CP administration plays a key role in promotion of tolerance via the induction of immune-suppressive CD4+IL10+ IFN- γ+FoxP3- Tr1 cells in IL27 dependent manner. The therapeutic relevance of this phenomenon is highlighted by our study that oral administration of CP to NOD mice provides protection from Type 1 diabetes.
Enteric antigens are delivered to the infant colonic immune system to promote enhance and long-lasting tolerance (MUC8P.801)
Authors: Knoop Kathryn , McDonald Keely , Newberry Rodney
Published: January 2023

Epidemiological studies indicate early exposure to microbial and dietary antigens via the gastrointestinal (GI) tract is associated with a decreased susceptibility for food allergy and inflammatory bowel disease later in life. However, the properties of the infant GI tract conferring this benefit are largely unknown. We found introduction of a dietary antigen during infancy, as opposed to early adulthood, promoted enhanced and lasting tolerance. Using in vivo imaging and ex vivo analysis of lamina propria antigen presenting cells following intraluminal antigen administration, we observed, in contrast to adults, enteric antigens were delivered to the colonic immune system in infants, which had an increased tolerogenic capacity. Antigen delivery to the infant colon immune system was mediated by goblet cell-associated antigen passages (GAPs) and the formation of colonic GAPs was regulated by goblet cell intrinsic sensing of the luminal microbiota, which inhibited antigen delivery and GAP formation in the colon as the microbiota expanded after weaning. Additionally, inhibition of colonic GAPs during enteric antigen exposure during infancy abrogated the tolerance induction. We propose antigen delivery to the infant colon allows the immune system to become ‘educated’ to dietary and microbial antigens, preventing responses to innocuous antigens encountered in the adult colon where the environment contains abundant and complex microbial stimuli.
A role for hormones, gut microbiota and tolerogenic CD103DC in protection of male (NZBxNZW)F1 (BWF1) mice from lupus (MUC8P.805)
Authors: Chhabra Anita , Alard Pascale , Jala Venkatakrishna , Haribabu Bodduluri , Kosiewicz Michele
Published: January 2023

Mechanisms leading to gut homeostasis affect both mucosal and systemic immune function, and involve gut microbiota. Tolerogenic dendritic cells (CD103DC) play a role in gut homeostasis, including generation of induced regulatory T cells (iTregs). Previously, we found that treatment of male BWF1 mice with anti-CD103 antibody significantly increases disease incidence/mortality. Male CD103DC exhibit significantly greater tolerogenic function than either female or castrated male CD103DC in vitro. This study examines the mechanism of sex differences in CD103DC function, and the role of microbiota in lupus. Sex differences in CD103DC function correlated with increased expression of genes associated with tolerogenic/anti-inflammatory function in males, and pro-inflammatory function in females, and changes in iTreg frequency and function in vivo. Male BWF1 CD103DC expressed more Raldh2, an enzyme required for retinoic acid (RA) synthesis and CD103DC function, and supplementation with RA restored tolerogenic function in female CD103DC. Further, we found that male gut microbiota differed significantly from adult, but not immature, female microbiota, suggesting hormones influence the gut microbiota. Importantly, transfer of male gut microbiota to female BWF1 mice significantly reduced anti-dsDNA antibody production. These data suggest that an interplay between hormones, gut microbiota and tolerogenic CD103DC may play a critical role in providing protection to male BWF1 mice.
A novel intestinal intraepithelial CD8α+ population involved in innate immunity (MUC8P.807)
Authors: Olivares-Villagómez Danyvid , Algood Holly , Singh Kshipra , Parekh Vrajesh , Piazuelo Maria , Chaturvedi Rupesh , Weitkamp Jörn-Hendrik , Wilson Keith , Van Kaer Luc
Published: January 2023

Innate immune responses play a critical role during the early stages of defense against infection and other immunological processes. Here, we describe a novel lymphoid population, referred to as innate CD8α+ (iCD8α+) cells. iCD8α+ cells display a lymphoid-like morphology and are present in the intestinal mucosa, primarily residing between intestinal epithelial cells (IEC). iCD8α+ cells are a population characterized by the expression of CD45, CD103, and CD69, and by lacking expression of TCR and markers associated with intraepithelial ILC1, such as NKp46 and NK1.1. Our results indicate that iCD8α+ cells are lymphoid-derived, but contrary to ILC, they do not depend on Id2 for their development, suggesting that iCD8α+ cells belong to a new class of innate lymphoid cells. Interestingly, iCD8α+ cell numbers are considerably reduced in thymus leukemia (TL) antigen-deficient mice, indicating that expression of TL by IEC is important for the development and/or maintenance of iCD8α+ cells. iCD8α+ cells produce cytokines and chemokines associated with innate immune responses such as MCP-1, MIP-1α, MIP1-β, IFN-γ and RANTES. Moreover, iCD8α+ cells can phagocytose and kill bacteria, and are important during Citrobacter rodentium infection. In summary, here we present the description of a novel innate immune cell involved in mucosal immune responses of the intestine.
Effects of exercise and anxiety on salivary IgA production in female college athletes (MUC9P.820)
Authors: King Debra , Lamb Ashley , Carrillo Andres , Appasamy Pierette
Published: January 2023

This study determined whether the fitness status of college athletes, and/or their measurable anxiety levels, had an effect on production of salivary sIgA. High-intensity physical training in elite sports has a negative effect on immune responses, while moderate-intensity training has a positive effect. In addition, psychological stress has a negative impact on immune function. Few studies have looked at the interplay between physical training and stress on the immune response. In this study, we evaluated 2 populations of female college athletes (Division III): one population participating in intense training (ice hockey), and the other group was in the “off season” (soccer). Maximal oxygen consumption (aerobic fitness) was estimated using the modified Balke sub-maximal treadmill test. Saliva samples were collected for sIgA and cortisol measurement. The anxiety levels were measured using a GAD-7 survey. We found that the team participating in their sport (hockey) had higher salivary IgA levels than the team that was “off season” (soccer). There was a wide range of anxiety levels and no correlation between anxiety and salivary IgA or sport. Higher levels of salivary IgA may confer added immune protection to the in-season athletes and these data suggest that the female college athletes were not training to the point of negatively impacting their immune responses. Strategies to maintain immune health are important to consider in designing training programs for athletes.
1-D-Methyl tryptophan treatment of lupus-prone B6.Nba2 mice reduces antinuclear autoantibody levels and B cell differentiation (THER5P.837)
Authors: Davison Laura , Carroll Thomas , Gurion Reut , Mellor Andrew , Jorgensen Trine
Published: January 2023

The enzyme indoleamine 2,3-dioxygenase (IDO) drives tryptophan degradation via the kynurenine pathway. IDO enzymatic activity is significantly elevated in lupus patients compared with controls; however the ramifications of this are unclear. The role of IDO in autoimmunity is debated, as pathogenic or protective effects have been shown in different mouse models of autoimmunity. For example, basal levels of IDO are elevated in MRL/lpr lupus-prone mice and systemic inhibition of IDO accelerates disease in this model. In contrast, systemic inhibition of IDO blocks autoreactive B cells in the K/BxN model of rheumatoid arthritis. Congenic B6.Nba2 mice spontaneously develop a lupus-like disease characterized by elevated levels of serum antinuclear autoantibodies (ANA) and glomerulonephritis. We here show that B6.Nba2 mice express significantly elevated basal levels of IDO. We therefore treated female B6.Nba2 mice with the IDO-specific inhibitor 1 methyl-D-tryptophan (1-MT) administered through the drinking water starting at 8 weeks of age. After 4 weeks of treatment, serum ANA levels were significantly and specifically reduced. This reduction was not due to differences in splenic cell subsets and treatment did not affect total IgG levels. Interestingly, cytokine-induced plasma cell differentiation was reduced in the presence of 1-MT in vitro, suggesting that inhibition of IDO may represent a novel treatment specifically targeting autoantibody production in lupus.
Targeting RhoB as a novel approach for the treatment of Rheumatoid Arthritis (THER6P.856)
Authors: Laury-Kleintop Lisa , Mulgrew Jennifer , Pigott Elizabeth , DuHadaway James , Prendergast George , Mandik-Nayak Laura
Published: January 2023

Rheumatoid arthritis (RA) is an autoimmune disease characterized by high titers of autoantibodies and increased levels of proinflammatory cytokines leading to chronic inflammation of the distal joints. Current biologic therapies, such as the B cell depleting antibody rituximab, have uncovered the important role that autoreactive B cells play in the disease process. Unfortunately, neither immunosuppressive nor biologic therapies specifically target autoreactive B cells. Here, we present data identifying a novel molecular target and newly developed therapeutic that may specifically target these cells. The small GTPase RhoB is a stress response protein that mediates tyrosine kinase receptor stimulated proliferation and migration. While much of the work on RhoB has focused on its function in cancer, we suggest a new role for this GTPase in autoimmune disease. Using the K/BxN model of RA, we show that treatment with an anti-RhoB antibody lowers autoantibody production and attenuates joint swelling. Mice genetically deficient in RhoB exhibit reduced arthritis, providing genetic confirmation of RhoB’s role in mediating autoimmunity. Interestingly, both RhoB deficient mice and mice treated with anti-RhoB Ig mounted productive antibody responses to immunization with model antigens, suggesting that RhoB may have a specific role in the autoreactive B cell. Our data suggest that RhoB antibody therapy could be a new effective therapeutic to treat antibody-mediated autoimmune diseases.
Ex vivo pre-conditioning of hematopoietic stem/progenitor cells impairs long-term hematopoietic repopulation after transplantation (TRAN3P.881)
Authors: Lee Hakmo , Park Ho Seon , Oh Ju Eun , Park Jongwoo , Chung Sung Soo , Park Kyong Soo , Jung Hye Seung
Published: January 2023

A number of investigators have tried to facilitate engraftment with limited numbers of available HSPCs by enhancing responsiveness of HSPCs to CXCL12. Recently, granulocyte derived cationic peptide, LL-37, has been suggested as a useful pre-conditioning component to overcome the shortage of available HSPCs. In this study, we first evaluated the character of LL-37 pre-conditioned HSPCs and found that LL-37 pre-conditioning effects are specific only to the clonogenic stem cells which are considered relatively dormant. This raised our concern regarding the fate of LL-37 pre-conditioned cells after transplantation and led us to evaluate their repopulation capacity as HSPCs. Not betraying our expectations, hematopoietic recovery of LL-37 pre-conditioned cells gradually decreased from 4 weeks after transplantation in competitive repopulation experiments. Further, in vitro studies revealed that LL-37 lead HSPCs to be more vulnerable to extracellular stimuli such as extracellular glucose followed by increased calcium influx from extracellular sources, which dampen self-renewal capacity by activating mammalian target of rapamycin and its downstream signaling. These results suggest that a future strategy must focus on maintaining HSPCs in dormant and protecting them from various stimuli during their long journey to the bone marrow niches rather than activating them before transplantation for successful transplantation outcome.
Low levels of SDF-1α constrain DC recovery and diminish homeostatic proliferation of naïve CD4+ T cells during graft-versus-host disease. (TRAN3P.892)
Authors: Gauthier Simon David , Leboeuf Dominique , Guimond Martin
Published: January 2023

Graft-versus-host disease (GVHD) is the major cause of morbidity and mortality after allogeneic hematopoietic stem cell transplantation (SCT) and its effect on T cell regeneration greatly increases the immunodeficiency normally associated with SCT. As a result, GVHD patients experience profound lymphopenia and lymphocyte reconstitution takes generally several months or years. The goal of this work was to assess the impact of GVHD on homeostatic proliferation (HP) of non-alloreactive naïve CD4+ T lymphocytes with the hypothesis that regeneration of the peripheral niche permissive to CD4 HP is constrained by GVHD. To study the impact of GVHD on the peripheral niche regulating CD4 HP, we used a Parent→F1 mouse model and observed a complete abrogation of CD4 HP in GVHD hosts. Loss of HP was related to depletion of all DC subsets as well as low levels of IL-7. While Flt3-ligand (FL) levels were normal, Stromal Derived Factor-1α (SDF-1α) was significantly diminished in GVHD mice. Administration of FL or SDF-1α significantly increased DC counts but CD4 HP was restored only when FL or SDF-1α was combined to IL-7. Importantly, while FL treatment aggravated GVHD, GVHD was improved by SDF-1α therapy. Our study shows that lack of immune reconstitution of CD4+ T cells is primarily due to failure of DC recovery owing to diminished SDF-1α production and diminished IL-7 and that the combination of SDF-1α and IL-7 could be used to enhance CD4 HP in GVHD hosts.
Engineering an ectopic thymus in the lymph node to induce central tolerance and long-term allograft acceptance (TRAN3P.896)
Authors: Rao Aparna , Gupta Avni , Lagasse Eric
Published: January 2023

Background: Allogeneic transplant rejection is one of the major problems plaguing the field of organ transplants. Our previous studies have established the lymph node as a functional site for thymus transplant. In this study, we hypothesized that conditioning transplant recipients with thymic cells immune-matched to the donor will induce long-term acceptance of a subsequent allograft. Methods: We transplanted Balb/c-GFP thymuses into the lymph nodes of FAH-/- mice on a 129sv background. After 6 weeks, the animals were challenged with donor-matched skin grafts or hepatocyte transfer and monitored for long-term allograft acceptance. We also assessed tolerance induction by performing mixed lymphocyte reaction assays (MLR), immunostaining of native and ectopic thymus, as well as thymic dendritic cell (tDC)-T cell co-cultures. Results: Mice receiving thymus transplants successfully accepted subsequent allografts long-term. MLR reactions demonstrated Balb/c-specific tolerance in these mice. We also observed migration of cells (mostly tDCs) from the ectopic to the native thymus. tDC-T cell co-cultures revealed that migrating DCs from the ectopic as well as native thymus were instrumental in generating the increased numbers of Tregs observed in recipient mice. Conclusion: We were able to achieve long-term allograft acceptance in mice receiving thymic transplants, which was possibly mediated by the increased Treg induction associated with cross-talk between the two thymuses.
Intrinsic MyD88-activation in B cells is necessary for development of alloreactive memory T cells (TRAN3P.902)
Authors: Singh Tripti , Ng Yue-Harn , Ramaswami Balathiripurasundari , Ippolito Renee , Chalasani Geetha
Published: January 2023

B cells help alloreactive T cells to differentiate into memory T cells. Since B cells express TLRs, we asked whether MyD88-mediated activation of B cells is required for T cell activation and differentiation to memory T cells. Methods: Irradiated μMTCD45.1 mice were transplanted with bone marrow cells from μMTCD45.1 and wtCD45.2 (μMT+wt) or μMTCD45.1 and MyD88-/-CD45.2 (μMT+MyD88-/-). Differences in congenic background allowed us to confirm that following reconstitution, MyD88 deficiency was essentially restricted to B cells. μMT+MyD88-/- and μMT+wt received Balb/c skin transplants. Results: Rejection of allografts was comparable between μMT+MyD88-/- and μMT+wt chimeras (MST=18 days). μMT+MyD88-/- recipients showed 3-fold more alloreactive IFNγ+ CD4 and CD8 effector T cells when compared to μMT+wt mice at day 14 (p<0.05). IL-10+ B cells were diminished (p<0.005) in μMT+MyD88-/- than in μMT+wt chimeras. CD8 memory precursor T cells (CD44hi CD62Llo CD127hi) in μMT+MyD88-/- showed predominantly Bcl2lo expression in both KLRG1lo memory precursor effector cell (MPEC) and KLRG1hi short-lived effector cell (SLEC) populations. Consistent with these findings, fewer alloreactive CD4 and CD8 IFNγ+ memory T cells were seen in μMT+MyD88-/- (p<0.005) resulting in impaired memory recall (MST = 27 vs. 15 days, p < 0.005) than in μMT+wt chimeras. Conclusions: Intrinsic MyD88-dependent B cell functions are important for alloreactive T cell differentiation into long-lived memory T cells.
Neddylation regulates T cell responses. (TRAN3P.914)
Authors: Mathewson Nathan , Toubai Tomomi , Sun Yaping , Wu Julia , Oravecz-Wilson Katherine , Wang Ying , Hou Guoqing , Reddy Pavan
Published: January 2023

Post-translational modifications (PTM) of proteins regulate cellular processes by proteasomal degradation. PTM of Cullin-RING ligase (CRL) protein via neddylation and the presence of its critical adapter element, sensitive to apoptosis gene (SAG) protein, modulate the process of neddylation-dependent ubiquitination. The role of neddylated proteins in regulating T cells is not known. We explored the role of neddylation in T cells by utilizing two different, but complementary approaches. We generated a T-cell specific knockout (SLCK-KO) of SAG (critical for the neddylation process) and utilized a small molecule inhibitor specific for neddylation (MLN4924). Phenotypic analysis of thymic and splenic T cells from SLCK-KO showed no differences when compared to WT. In vitro analysis of SLCK-KO T cells, with either allo-splenocytes or non-specific CD3/CD28 antibodies demonstrated reduced activation (CD69, CD44), proliferation, and secretion of T helper signature cytokines (IFNγ, IL-4, IL-17) (P<0.003). In vivo studies utilizing multiple MHC disparate models of BMT (B6→BALB/c, B6→B6D2F1), demonstrated that SLCK-KO T cells in the recipient animals caused reduced severity of GVHD and increased survival (P<0.01). Similar results were obtained in vitro and in vivo with small molecule, MLN4924. Our results demonstrate a novel biological role for neddylation in regulating T cell functions in vitro and in vivo.
Andrographolide-triggered mitophagy-mediated NLRP3 inflammasome inhibition accounts for the prevention of colitis-associated cancer (TUM7P.919)
Authors: Sun Yang , Guo Wenjie , Xu Qiang
Published: January 2023

Nonresolving inflammation in the intestine predisposes individuals to the development of colitis-associated cancer (CAC). Inflammasomes are believed to mediate intestinial homeostasis, and their dysregulation contributes to inflammatory bowel diseases and CAC. However, few agents targeting inflammasomes to reduce CAC have been reported thus far. Here we show that small molecule andrographolide (Andro) protects mice against azoxymethane/dextran sulfate sodium-induced colon carcinogenesis through inhibiting NLRP3 inflammasome. Andro significantly attenuated colitis progression and tumor burden. Further study showed that Andro inhibited NLRP3 inflammasome activation in macrophages in vivo and in vitro. Furthermore, Andro triggered mitophagy in macrophage, which in turn inactivated the NLRP3 inflammasome. Moreover, downregulation of the PIK3CA-AKT1-MTOR-RPS6KB1 pathway accounted for Andro-induced autophagy. Finally, inhibition of IL-1β production and amelioration of murine experimental colitis induced by Andro were almost completely blocked by the knockdown of BECN1 expression, or by autophagy inhibitors 3-methyladenine, chloroquine, bafilomycin A1 and ammonium chloride in vitro and in vivo. Taken together, our findings demonstrate that Andro-driven mitophagy-mediated NLRP3 inflammasome inhibition is responsible for the prevention of CAC. Our data may help guide decisions regarding the use of Andro in patients with colitis, which ultimately reduces the risk of CAC.
Plasma-derived exosomes mediate immune suppression and serve as a biomarker of response to chemotherapy in Acute Myeloid Leukaemia (AML) (TUM7P.945)
Authors: Hong Chang-Sook , Muller Laurent , Boyiadzis Michael , Whiteside Theresa
Published: January 2023

Exosomes are virus-sized (30-100nm) vesicles formed within endocytic compartment and released into extracellular space. While all cells are capable of releasing exosomes under physiologic conditions, tumor cells are avid exosome producers. We have previously shown that plasma of AML patients at diagnosis contain high levels of exosomes relative to normal controls’ plasma (NC). AML exosomes carry various immunosuppressive molecules, including TGF-β1 related to NK-cell dysfunction in AML. Here we show that molecular content of exosomes isolated from patients’ plasma before, during and after chemotherapy (CT) reflects disease status. Thus, exosomes present in patients’ circulation carry biomarkers that can be useful in evaluating responses to therapy. AML exosomes carry different forms of TGF-β1 (pro-peptide, latency-associated peptide or active form) in different proportions. Only TGF-β1 pro-peptide was seen in exosomes obtained from NC or patients in remission. While TGF-β1 levels tracked protein levels in exosomes, elevated TGF-β1 levels discriminated better between patients receiving CT whose leukemia was likely to recur. In addition, co-incubation of normal NK cells with exosomes carrying active TGF-β1 resulted in down-regulation of NKG2D expression. In conclusion, elevations in exosomal protein and/or TGF-β1 levels during CT may reflect an incomplete response to CT and suggest the presence of residual disease in patients considered to have achieved complete remission.
An in vitro screen employing human neonatal dendritic cells identifies distinct synergy between Toll-like receptor and C-type lectin receptor agonists (VAC10P.966)
Authors: van Haren Simon , Foppen Willemina , Dowling David , Levy Ofer
Published: January 2023

Due to impairments in cell-mediated immunity, newborns are markedly susceptible to infection with intracellular pathogens. Impaired newborn immunity includes a reduced response to Toll-like Receptor agonists (TLRAs) by dendritic cells (DCs), placing them at risk for infection and limiting Th1-responses to many vaccines. Our hypothesis was that dual stimulation with TLRAs and C-type Lectin Receptor agonists (CLRAs) may overcome the reduced response of newborn DCs to common vaccine formulations. We screened TLRAs, CLRAs and their combinations for their ability to induce Th1-polarizing cytokine production from neonatal dendritic cells. Human adult- and cord blood monocyte-derived DCs (MoDCs) were generated in the presence of autologous plasma and stimulated for 18 hours. Cytokines and markers for cellular toxicity were measured to assess correlates of immunogenicity and reactogenicity. Dual activation of newborn DCs with a TLR8 agonist (R848) and a Mincle agonist (TDB) or with a TLR4 agonist (MPLA or GLA) and a Dectin-1 agonist (alkali-treated Zymosan) synergistically enhanced the production of the T-cell activating and -polarizing cytokines TNF-α, IL-12p70 and IL-1β. Our data suggest that targeting vaccinal antigens to the endocytic receptors Mincle or Dectin-1 is a powerful approach to induce Th1-mediated immunity in newborns. This study has the potential to inform development of novel adjuvanted neonatal vaccines and reduce infections in infants.
Anti-tumor response in T cells with CD8 independent high affinity TCR remains unaffected with aging (VAC11P.1009)
Authors: Kesarwani Pravin , Kristina Andrijauskaite , Rubinstein Mark , Mehrotra Shikhar
Published: January 2023

Advancement in adoptive cell therapy has led to the use of T cells engineered with tumor antigen reactive high affinity T cell receptors (TCR) in clinical trials. However, T cell engineered with anti-tumor epitope reactive TCR from an aged individual could affect the clinical outcome. In order to study if aging affects the functional efficacy of a CD8 independent high affinity TCR, we compared anti-tumor T cell response from young/old TCR transgenic h3T(human TIL derived Tyrosinase TCR) mice developed in our lab using TIL1383I TCR obtained from TILs of a melanoma patient. In vitro comparison of h3T T cells from old/young mice shows that antigen specific stimulation of the T cells from old mice resulted in an increased expression of CD25 and increased cytokine(IL2,IFNy,TNFa) secretion as compared to T cells from young mice. Remarkably, an overall decreased immune response from old hgp100 reactive T cells from Pmel transgenic mice was observed. Further, adoptively transferred h3T old/young splenocytes in vivo showed comparable control of human melanoma tumor 624-MEL established in a xenograft model. Further, aged T cells engineered with TIL1383I TCR and transferred to treat established melanoma in aged HLA-A2 recipient also showed efficient tumor control. These results demonstrate that aging does not dampen the recognition and activation of CD8+T cells bearing CD8 independent high affinity TCR and thus its broad immunotherapeutic potential in treating patients across all ages.
Ad5 but not Ad26 induces an IFN-γ+IL-10+ CD4+ T cell population following immunization (VAC12P.1023)
Authors: Larocca Rafael , Provine Nicholas , Borducchi Erica , Barouch Dan
Published: January 2023

Adenovirus-based vaccines have arisen as an attractive method to elicit robust cellular and humoral immune responses. Multiple vector serotypes have been developed, but the CD4+ regulatory T cell responses generated by these vectors have not previously been compared. We therefore, compared the functionality of antigen-specific CD4+ regulatory T cells induced following Ad5 or Ad26 immunization. C57BL/6 mice were immunized intramuscularly with 1010vp of Ad5-SIVgag or Ad26-SIVgag. Spleen cells were harvest at day 10, and SIVgag-specific CD4+ regulatory T cell responses were evaluated. Immunization with 1010vp of Ad5-SIVgag induced a population of IL-10+ producing cells that comprised 30% of gag-specific IFN-γ+ CD4+ T cells. In contrast, the number of CD4+ T cells that co-produced IFN-γ and IL-10 was 8 times lower following Ad26 immunization. These cells were T-bet+ and FoxP3-. In addition, Ad5 immunization induced a substantially larger fraction of IL-10+ cells that co-express PD-1 than Ad26 immunization. Moreover, a larger fraction of IFN-γ+ CD4+ T cells were Tim-3+ following Ad5 versus Ad26 immunization. We also observed increasing frequency of IFN-γ+IL-10+ CD4+ T cells with increasing doses of Ad5. These data suggest that Ad5 vectors, but not Ad26 vectors, induce a large population of CD4+ T cells that co-produce IFN-γ and IL-10, an immunosuppressive cytokine that may potentially limit vaccine efficacy.
Development and characterization of mouse monoclonal antibodies reactive with chicken TL1A (VET2P.1029)
Authors: Lee Sung Hyen , Lillehoj Hyun , Jeong Misun , Sullivan Yvonne B.
Published: January 2023

Two mouse monoclonal antibodies (mAbs) which specifically detect chicken TL1A (chTL1A) were developed and characterized. Both mAbs identified a 32 kDa E. coli-derived, poly-histidine-tagged fusion protein by Western blot analysis. Both mAbs identified TL1A-secreting cells in the chicken thymus, cecal tonsil, and bursa of Fabricius by immunocytochemistry, and were used to measure serum TL1A levels in normal and necrotic enteritis (NE)-afflicted chickens by antigen capture ELISA. These mAbs inhibited chTL1A-induced spleen lymphocyte proliferation, nitric oxide production by HD11 chicken macrophages, and blocked the cytotoxic effect of chTL1A against LSCC-RP9 chicken B tumor cells. These new mAbs detecting chTL1A will be important immune reagents for basic and applied research in poultry immunology
Characterizing immune cells of Atlantic Bottlenose Dolphins (VET2P.1047)
Authors: Nouri-Shirazi Mahyar , Bible Brittany , Zeng Menghua , Tamjidi Saba , Bossart Gregory
Published: January 2023

Marine mammals are ideal sentinel species for human health due to exposure to the same oceans and consumption of the same foods. There have been many studies which demonstrate that wild Atlantic Bottlenose Dolphins are exposed to high levels of contaminants which lead to a suppressed immune system and are therefore more susceptible to opportunistic infections, many of which are zoonotic diseases. However, nearly no research has been done on determining defects in the immune cell population of dolphins, especially DCs which are essential for initiating an immune response. We hypothesize phenotypic and functional differences in the PBMC, including DC precursors, of wild dolphins as compared to managed dolphins. Specifically in this study, we have used terrestrial-specific antibodies and growth factors to characterize immune cells in PBMC and to generate monocyte-derived DCs. We have identified cross-reactive terrestrial antibodies that could detect immune cell subsets within PBMC, including B cells, T cells, NK cells, monocytes and APCs. Interestingly, using these antibodies we found significant changes in immune cell subsets within PBMC of wild and managed dolphins. Finally among the terrestrial DC growth factors tested we found rat GM-CSF and IL-4 generated DCs expressing higher levels of CD11c, CD14, CD40, CD80, CD86, MHC I and MHC II. Our findings allow us to further study defects in the immune cells, especially DCs, in response to environmental contaminants.
Evaluation of Montanide™ ISA 71 VG adjuvant against coccidiosis (VET2P.1030)
Authors: Lillehoj Hyun , Lee Sung Hyen , Dupuis Laurent , Ben Arous Juliette
Published: January 2023

Chickens were immunized subcutaneously with an Eimeria recombinant profilin protein plus MontanideTM ISA 70 VG (ISA70) or MontanideTM ISA 71 VG (ISA 71) water-in-oil adjuvants, or with profilin alone. While immunization with profilin/ISA 70 vs. profilin alone altered the levels of more total transcripts compared with profilin/ISA 71 vs. profilin alone (509 vs. 296), the latter was associated with a greater number of unique biological functions, and a larger number of genes within these functions, compared with the former. Canonical pathway analysis identified 10 pathways related with genes encoding the altered transcripts in animals immunized with profilin/ISA 71 vs. profilin alone, compared with only 2 pathways in profilin/ISA 70 vs. profilin alone. Vaccination with profilin/ISA 71 was associated with greater body weight gain following E. acervulina infection, and decreased parasite fecal shedding after E. maxima infection, compared with profilin alone. Anti-profilin antibody levels were higher in sera of E. maxima- and E. tenella-infected chickens vaccinated with profilin/ISA 71 compared with profilin alone. The levels of transcripts encoding interferon-γ, interleukin (IL)-2, IL-10, and IL-17A were increased in intestinal lymphocytes from E. acervulina-, E. maxima-, and/or E. tenella-infected chickens vaccinated with profilin/ISA 71 compared with profilin alone.
Semi-quantitative analysis of T cell receptor (TCR) β and TCR γ gene expression in xenopus laevis: a novel animal model to study γδ T cells (VET2P.1036)
Authors: Kongprachaya Yuthana , Wilson Darayu , Appasamy Pierette
Published: January 2023

The polypeptide chains of heterodimeric TCRs divide T cells into two classes: αβ or γδ T cells. The functions of γδ cells overlap with cells of the innate and adaptive immune system and include cytokine production, maintaining tissue homeostasis and antigen presentation. γδ TCR ligand recognition is MHC-independent and TCRs bind not only peptides, but also other soluble, non-soluble and membrane-bound antigens. In humans and mice, γδ T cells represent only five percent of the total T cell population, although the percentage is higher in certain epithelial tissues such as the intestines and urogenital tract in adult animals. Phylogenetic analysis of the amino acid sequence of γδ TCR suggests that γδ T cells evolved prior their αβ counterparts and it is likely that γδ T cells may be more abundant in relatively primitive vertebrates like frogs and fish. We developed a semi-quantitative RT-PCR assay to compare levels of γ-TCR and β-TCR RNA in adult Xenopus laevis tissues, as markers for the presence of αβ and γδ T cells, respectively. Preliminary data indicate that γδ cells are as abundant as αβ cells in adult X. laevis spleen; ongoing experiments are evaluating γ-TCRC and β-TCRC RNA in X. laevis intestines, lungs, thymus and skin and in these tissues during metamorphosis. These studies will provide valuable information to establish Xenopus laevis as a novel animal model to study the development and function of γδ TCR-expressing cells.
A few changes in wild-type picornavirus genome lead to immunosuppression (VIR8P.1068)
Authors: Cusick Matthew , Libbey Jane , Fujinami Robert
Published: January 2023

Viruses, such as human immunodeficiency virus and poliovirus, use a variety of mechanisms to suppress the immune response and evade clearance by the host. Therefore, investigating how a few changes in the viral genome of a non-lethal virus can lead to an alteration in disease, from survivable to immunosuppression and death, would provide valuable information into viral pathogenesis. Here we investigate a picornavirus, the Theiler’s murine encephalomyelitis virus (TMEV), in its natural host, the mouse. C57BL/6 mice infected intracerebrally (i.c.) with the DA strain of TMEV develop acute encephalitis; mice survive the acute disease and clear the virus. A mutant of the DA strain of TMEV was inadvertently created, called H101, which encodes a point mutation (T101I) in VP1. Additional sequencing showed several nucleotide substitutions in the 5’ untranslated region as well as additional amino acid substitutions in the capsid protein coding region, suggesting that there are a number of perturbations in the viral genome. C57BL/6 mice infected with the H101 mutant via a peripheral route [intraperitoneal (i.p.)] become immunosuppressed through specific depletion of T cells. For C57BL/6 mice infected with the H101 mutant virus by either the i.c. or i.p. route, the immunosuppression is profound. This study provides experimental evidence that infection by a virus having just a few changes in the viral genome can now result in profound immunosuppression due to T cell depletion.