bluuebunny/crossref_metadata_2025 huggingface dataset:
Cow’s milk specific IgE and IgA as a predictor of outcome in oral immunotherapy (VAC9P.1067)
Authors: Rahman Tanvir , Lejtenyi Duncan , De Schryver Sarah , Fiter Ryan , Piccirillo Ciriaco , Ben-Shoshan Moshe , Mazer Bruce
Published: January 2023

Cow’s milk allergy (CMA) is defined as an immunologic adverse reaction to cow’s milk proteins. It affects 2-3% of infants, and 85% of CMA resolve spontaneously within the first 5 years of life. Those with persistent CMA have a lifelong threat of anaphylaxis. 20 patients were recruited for a trail of CM oral immunotherapy. Blood samples were collected at baseline and at multiple timepoints during a trial. Specific IgE, IgA to casein, b-lactoglobulin (BLG) and alpha-lactalbumin (ALA) were measured by Enzyme-Linked Immunosorbent Assay(ELISA). CM-specific IgE responses decreased by 5 to 10 times baseline. Importantly, in one patient there was no detectable sIgE after 6 months post OIT. As the role of specific IgA is poorly understood we measured specific IgA to the three CMA components in a subset of subjects. sIgA was increased significantly over time in 4 of 6 patients, unaltered in 1 patient, and was variable in one patient. At the end of oral immunotherapy, sIgA returned towards baseline. In summary, as patients underwent oral immunotherapy to milk, sIgA increased in parallel to the decrease in IgE in most subjects tested. CM specific IgA may help predict responses to oral immunotherapy for milk.
Immune modulation of IgE and T regulatory cells in horses vaccinated with viral vaccines. (VET1P.1117)
Authors: Behrens Nicole , Gershwin Laurel
Published: January 2023

West Nile virus (WNV) a neurotropic flavivirus is the leading cause of mosquito-borne encephalitis in the United States. While there is no current WNV vaccine for humans there are several vaccines for horses. These vaccines are designed to protect against this deadly virus; however, many are causing type-1 hypersensitivity IgE-mediated allergic responses that include local and severe systemic anaphylaxis. Many viral vaccines share contaminating non-target proteins that can act as antigen to elicit IgE. We compared IgE levels to those of T regulatory cell populations’ pre and post vaccination, which showed a significant inverse correlation. We hypothesize the addition of CpG oligonucleotides administered with vaccines will modulate the immune system, driving it towards protective immunity, through up-regulation of T helper 1 cells. Thirty horses were evaluated pre and 7 days post vaccination for T regulatory cell production and specific IgE. Peripheral blood mononuclear cells isolated pre and post vaccination were analyzed for expression of CD4, CD25 and intracellular Foxp3 with multicolor flow cytometry. Antigen specific IgE and cytokine supernatants were evaluated by ELISA. The addition of the CpG oligonucleotide significantly increased the production of T regulatory cells in response to antigen in-vitro and in-vivo; with this we can hope to provide a safer alternate vaccination strategy, particularly for horses that have demonstrated a pro-allergic phenotype.
Characterization of major histocompatibility complex class I genes and alleles of the California sea lion (Zalophus californianus) in relation to development of sea lion urogenital carcinoma (VET2P.1130)
Authors: Duckworth Ellen , Gulland Frances , Cohen Noah , Criscitiello Michael
Published: January 2023

Decades of global environmental change have impacted the overall health of many wildlife species, and emerging infectious diseases, biotoxicoses, and cancers are being increasingly recognized. California sea lions have an unusually high incidence of an aggressive, widely metastatic urogenital carcinoma that has affected 26% of the adult animals that died following stranding on the central California coast in the past decade. A gamma herpesvirus, environmental pollutants, major histocompatibility complex (MHC) class II alleles, and a variant of a heparanase gene have all been implicated in the multifactorial cause. The locus most commonly associated with immunogenetic susceptibility to neoplastic transformation and viral infections, MHC class I, has not been studied, however. Genomic DNA was extracted from skin samples of 20 cancer-positive and 40 cancer-negative sea lions. Using these samples, the MHC I gene sequence will be published, the MHC class I polymorphism will be assessed, and the association of specific MHC class I alleles with cancer-positive and cancer-negative sea lions will be determined. We hypothesize that particular MHC class I alleles that successfully present immunodominant peptides of the otarine herpesvirus-1 will be present in cancer-negative animals but not cancer-positive animals and that there will be increased homozygosity of MHC alleles in sea lions with carcinomas as compared to those without.
Differential mucins gene expression between respiratory human paramyxovirus infections (VIR5P.1142)
Authors: Banos-Lara Rocio , Guerrero-Plata Maria
Published: January 2023

Mucin (MUC) proteins are the main component of mucus and constitute part of the innate immune response. High production of mucins and mucus is characteristic in inflammatory lung diseases. Respiratory Syncytial Virus (RSV) and Human Metapneunovirus (hMPV) are RNA viruses belonging to the Paramyxoviridae family and represent the most important cause of severe lung diseases such as pneumonia and bronchiolitis in young children, the elderly and immunocompromised individuals. Although these viruses are close-related, the host immune response elicited by them is widely different. In this work we evaluated the lung gene expression of secreted (MUC2, MUC5AC, MUC5B, MUC8 and MUC19) and cell surface (MUC1, MUC3, MUC4, MUC13, MUC15, MUC16, MUC20, MUC21 and MUC22) mucins by qRTPCR in human epithelial cells infected with RSV or hMPV. Our data indicate that the MUC expression by RSV and hMPV differ significantly, as we observed a stronger induction of MUC8, MUC15, MUC20, MUC21 and MUC22 by RSV infection at 48 and 72 h than by hMPV. On the other hand, the expression of MUC2, MUC5B and MUC1 was dominated by the infection with hMPV at the same time points. No significant MUC5AC, MUC19, MUC3, or MUC13 expression was observed after any of the infections. Together, these data indicate that RSV appears to be a stronger inducer of MUC response than hMPV and suggest that MUC expression contribute to the differential immune response induced by these two major human respiratory viruses
FasL-dependent regulation of the magnitude of the CD8 T cell response during lethal dose influenza virus infections (VIR5P.1144)
Authors: Zacharias Zeb , Legge Kevin
Published: January 2023

Our recent work has detailed a novel regulation of the IAV-specific CD8 T cell response during lethal dose or infection with high pathogenic IAV strains. During such infections, FasL+ plasmacytoid dendritic cells (pDC) within the regional lymph nodes (LN) target and eliminate the developing Fas+ effector T cell response, therein limiting immunity and driving severe disease. While our studies have shown that pDC are sufficient to drive this mechanism (i.e. transfer of wild type pDC into gld mice results in elimination of activated CD8 T cells), antibody targeted elimination of pDC in wild type mice does not completely halt CD8 T cell apoptosis. This suggested that an additional cell type might contribute to the overall loss of CD8 T cells during lethal/high path IAV infections. Herein we describe a novel cell population which shares some common markers with both pDC and B cells, but can be distinguished from both based on unique marker expression. These FasL+ cells are found at 4.5x greater numbers in the LN of lethal vs sublethal dose IAV mice and are 8.5x more prevalent than pDC with those LN. Further our results suggest that the FasL+ phenotype within this cell population segregates to BST2+ cells. Our current studies are focused on determining the factors regulating this novel cell population and it’s relative contribution to the overall FasL-dependent control of the magnitude of the CD8 T cell response during IAV infections.
Characterization of crossreactive T cell receptors that recognize both Epstein Barr virus and influenza A virus during acute influenza A virus infection (VIR5P.1135)
Authors: Song InYoung , Gil Anna , Stern Lawrence , Selin Liisa
Published: January 2023

Crossreactive CD8+T cells that recognize both influenza A (IAV) epitope M158, and Epstein Barr virus (EBV) epitope BRLF1109, are frequently found in HLA-A*02 populations particularly during acute infection. However, whether this crossreactivity is mediated by a single crossreactive T cell receptor (TCR) is unclear. To address this, M1- and BRLF1-MHCI oligomer positive CD8+T cells from peripheral blood of an acute IAV-infected patient who was EBV seropositive were single cell sorted and the TCR genes were sequenced and cloned. Thirty-three of 96 single cells yielded one TCRα and one TCRβ gene with 13 unique reproducible TCRα/β pairs. These 13 pairs of TCR genes were characterized in TCR deficient Jurkat cells expressing CD8α (J76) for antigen recognition and TCR signaling. In each case the transfected J76 cells bound strongly to M1-MHCI tetramer and weakly to BRLF1-MHCI tetramer consistent with the original staining in the peripheral blood. These crossreactive TCR-transfected J76 cells responded functionally to both peptides as they upregulated CD69 in response to BRLF1 as well as M1 peptide stimulation with four orders of magnitude difference. Interestingly, one TCR similar to JM22 which is a public TCR recognizing M1 epitope, was also crossreactive to BRLF1, which implies that many HLA-A*02 populations may be crossreactive to EBV. Our data clearly shows that crossreactivity between M1 and BRLF1 epitopes can be mediated by a single TCR and may be ubiquitous.
Age-related dysregulation in the lipidome compromises the immune response to the severe acute respiratory coronavirus (VIR5P.1140)
Authors: VIJAY RAHUL , Gelb Michael , Murakami Makoto , Perlman Stanley
Published: January 2023

Age-associated changes have long been known to cause dysregulation in antiviral immune responses. The SARS epidemic of 2002-03 was characteristic in this respect that it exhibited an age-associated increase in mortality. We recently reported that an age-related increase in an eicosanoid, prostaglandin D2 (PGD2) contributed to the observed higher mortality in SARS-CoV infected aged compared to young mice. To examine the factors that resulted in elevated levels of PGD2 in aged mouse lungs, we analyzed the gene expression levels of several enzymes in the eicosanoid pathway. Remarkably, secretory phospholipase A2 group 2D (Pla2g2d), which is preferentially expressed in lung CD11c+ cells, was significantly higher in aged compared to young mice. Lipid profiling of aged and young SARS-CoV-infected mice revealed that unlike young mice, aged mice failed to upregulate many pro-inflammatory lipid mediators although both groups upregulated the pro-resolving ones. Further, aged mice lacking PLA2G2D expression (Pla2g2d-/-) mice exhibited enhanced virus clearance, increased lung DC migration to the draining lymph nodes and increased virus specific T cell responses. Infection of aged Pla2g2d-/- mice with SARS-CoV increased survival from 20% to 80-90%, compared to Pla2g2d+/+ mice. Altogether our data identify a novel role for Pla2g2d in the lungs of aged mice in skewing the lipidome to an anti-inflammatory state and thus predisposing the animal to a worse outcome following SARS-CoV infection.
CX3CR1+ CD8 T cells in treated HIV infection are functionally impaired by platelet interactions (VIR6P.1171)
Authors: Freeman Michael , Mudd Joseph , Panigrahi Soumya , Lederman Michael
Published: January 2023

In the era of highly effective antiretroviral therapy (ART), HIV-infected patients have a better long-term prognosis than ever; however, increased inflammation and coagulation in HIV infection is associated with an increased risk of cardiovascular and other morbidities. CD8 T cell expansion is characteristic of HIV infection and has been linked to increased morbidities especially cardiovascular disease in treated HIV infection. Here we have identified a population of circulating memory CD8 T cells that express the vascular endothelium homing receptor CX3CR1 (fractalkine receptor) and is enriched in ART-treated HIV+ patients. CX3CR1+ CD8 T cells have elevated expression of the platelet-binding receptor PSGL-1 and the thrombin-activated receptor PAR-1, suggesting a possible role in cardiovascular disease via endothelial localization and interactions with platelets and mediators of coagulation. We found that even though thrombin stimulation via PAR-1 could directly enhance CD8 T cell responses in vitro, CD8 T cell cytokine production was diminished in the presence of thrombin-activated platelets through mechanisms mediated in part by TGF-β. Thus, we have identified a population of circulating memory CD8 T cells in HIV infection that are activated by thrombin, are susceptible to platelet-mediated regulation, and could contribute to cardiovascular disease risk in ART-treated HIV-infected patients.
Mouse polyomavirus-specific CD8+ T cells establish a brain-resident memory population during persistent infection (VIR6P.1174)
Authors: Frost Elizabeth
Published: January 2023

Human JC polyomavirus (JCV) causes progressive multifocal leukoencephalopathy (PML), a rare but life-threatening demyelinating disease in patients under profound immunosuppression and those receiving therapeutic immunomodulation for autoimmune and inflammatory disorders. Using persistent mouse polyomavirus (MPyV) infection in mice, we are developing a model of PyV-induced CNS disease to elucidate the role of immunosuppression in PML pathogenesis. Histology revealed corpus callosum demyelination in 100% of persistently infected mice. Following intracerebral injection with MPyV, immunocompetent C57BL/6 mice showed robust infiltration by MPyV-specific CD8 T cells with minimal contraction. During persistent CNS infection, a significant population of CD8 T cells exhibited a tissue-resident memory (Trm) phenotype (CD103+CD69+CD62Llo), as reported for acute VSV and latent HSV-1 infections, and incorporated BrdU in situ, suggesting capacity for self-renewal. Furthermore, these Trm cells develop and survive independently of replenishment by circulating antiviral CD8 T cells or CD4 T cell help. Unlike in the spleen, MPyV-specific CD8 T cells are PD-1hi upon infiltration of the brain without evidence of exhaustion. Interestingly, levels of CD8 and T cell receptor also increase on cells in the brain over time. Whether brain-infiltrating T cells exhibit higher sensitivity to antigen due to higher affinity and/or functional avidity is currently under investigation.
Strain differences in CD8 T cell expansion revealed in vivo during LCMV infection and ex vivo by TCR stimulation (VIR6P.1176)
Authors: Tarrio Margarite , Ploquin Mickael , Suarez-Ramirez Jenny , Biron Christine
Published: January 2023

Lymphocytic choriomeningitis virus (LCMV) infection of C57BL6 (B6) mice induces dramatic virus-specific CD8 T cell expansion. Comparison of B6 and 129-Sve (129) mouse strain responses demonstrated that the magnitude of total LCMV-specific CD8 T cell expansion was much lower in the 129 mice. The difference occurred even though viral titers and proportions of CD8 T cells specific for the three immunodominant epitopes of LCMV were similar. A higher type 1 IFN response was associated with the enhanced expansion in the B6 mice, and the responding CD8 T cells in B6 as compared to 129 mice had elevated levels of the signal transducer and activator of transcription (STAT) 4 but lower levels of the STAT1 molecule used to mediate inhibition of proliferation. The magnitude of the response was significantly diminished in B6 but not 129 mice genetically altered to block in IFN responsiveness, i.e. IFN receptor or STAT1 deficient, such that overall CD8 T cell expansion was now similar in both strains. Culture studies with purified cells demonstrated that in comparison to B6, 129 CD8 T cells had poor proliferative potential when stimulated through the TCR. Thus, intrinsic mechanisms linked to genetic differences regulate the magnitude of CD8 T cell proliferation. The data suggest that these differences have consequences for type 1 IFN requirements in shaping the magnitude of the response and overall immunity to viral infections. Supported by the National Institutes of Health, USA.
The effect of staphylococcal enterotoxin B on antiviral CD8+ T cell responses (VIR6P.1177)
Authors: Meilleur Courtney , Memarnejadian Arash , Haeryfar S. M.
Published: January 2023

Staphylococcal enterotoxin B (SEB) is a bacterial superantigen capable of activating a large percentage of the T cell repertoire. Contact with SEB causes T cells to proliferate and secrete inflammatory cytokines, ultimately resulting in deletion or anergy of the activated T cell. This may leave holes in the T cell repertoire, altering the ability of the host to respond to viral infection. We have used a mouse model of influenza (IAV) vaccination and infection to gauge the effect of SEB administration on the quality of the antiviral CD8+ T cell response. Our results show that SEB alters the immunodominance hierarchy of epitope-specific CD8+ T cells during primary, memory and recall responses. We also found that these changes can be explained by the presence or absence of SEB-reactive Vβ regions in the TCRs of epitope-specific CD8+ T cell populations. Intriguingly, SEB exposure also augments the ability of one epitope-specific CD8+ T cell population to kill their target cells. Contrary to previous reports, these results show that contact with SEB may enhance CD8+ T cell responses in the context of IAV infection. In summary, our work uncovers a novel effect of SEB on the immune system, one that has potentially useful implications for the treatment of viral diseases.
Paradigm-violating HLA class II-restricted CD8 T cells exist in HIV infected individuals (VIR6P.1164)
Authors: Ranasinghe Srinika , Lamothe Pedro , Damien Soghioan , Jones Brad , Sidney John , Sette Alessandro , Streeck Hendrik , Picker Louis , Kappler John , Kaufmann Daniel , Walker Bruce
Published: January 2023

The T cell paradigm dictates that CD8 T cells recognize peptides presented by MHC class I. Yet, class II-restricted CD8 T cells were recently observed in rhesus macaques vaccinated with a RhCMV/SIV vector (reviewed in Ranasinghe and Walker 2013 Nat Biotech). Restriction of CD8 T cells by class II is rare, with less than a dozen published studies in two decades. Our study aimed to assess whether class II-restricted CD8 T cell responses exist in HIV infection. We detected class II-restricted CD8 T cells in 3 HIV infected individuals. The responses were identified using a novel 'HLA-DRB1 ELISpot' where patient CD8 T cells were co-cultured with HIV peptide-pulsed mouse LCL expressing human recombinant DRB1 (N=2/30 DRB1*01 and N=1/22 DRB1*11 subjects). Antibody blocking of class I and II, and class II tetramer binding confirmed the DRB1 restriction. Strikingly, up to 12% of total CD8 T cells bound class II tetramer directly ex vivo. The class II-restricted CD8αβ T cells expressed a TEMRA phenotype with a distinct polyfunctional profile and single TCR vBeta clonotype when compared intra-patient to class I-restricted CD8 TEM responses. Class II-restricted CD8 T cells exhibited high levels of Perforin and Granzyme B and efficiently lysed peptide-pulsed autologous BCL and CD4 T cells, yet showed impaired killing of autologous HIV-infected CD4 T cells in vitro. In our study, we have demonstrated the first proof-of-principle detection of class II-restricted HIV-specific CD8 T cells.
A virus-encoded miRNA in mouse polyomavirus regulates viral replication in the kidneys in a CD8 T cell dependent manner. (VIR6P.1166)
Authors: Maru Saumya , Lukacher Aron
Published: January 2023

The polyomaviruses JCV and BKV are ubiquitous DNA viruses in the human population which persist as low-level infections in host kidneys and give rise to disease upon immunosuppression, resulting in significant morbidity and mortality. The polyomaviruses encode one endogenous miRNA, with both the 5p and 3p mature miRNAs functioning to reduce levels of target transcripts. The mouse polyomavirus (MPyV) also encodes a miRNA within the sequence for the early nonstructural proteins large (LT) and middle (MT) T-antigen, both of which have been established as miRNA targets. Other targets of the MPyV miRNA and their physiologic consequences have been poorly defined. The dl1013 virus is a deletion mutant of MPyV which eliminates expression of the endogenous miRNA while preserving LT and MT functions vital to the viral life cycle. We have shown that infecting immunocompetent C57BL/6 mice with dl1013 results in lower viral levels in the kidneys than infection with parental virus, but not in other organs. No difference in kidney viral levels between parental and dl1013 virus is seen in CD8α-/- mice, suggesting that the effect of the miRNA on viral replication is dependent on CD8 T cells. We are currently investigating whether this MPyV miRNA affects recruitment of myeloid and lymphoid cells into the kidney and their role in controlling MPyV in the dominant reservoir of viral persistence of both human and mouse polyomaviruses.
Identification of ribosomal proteins as targets for protective CD4+ T cell responses against mycobacteria (APP5P.114)
Authors: Johnson Alison , Kennedy Steven , Lindestam Arlehamn Cecilia , Hegde Subray , Seidel Ronald , Blanchard John , Sette Alessandro , Chan John , Jacobs William , Porcelli Steven
Published: January 2023

Mycobacterium tuberculosis (Mtb) infects one-third of the global population, and a more efficacious vaccine is urgently needed for control and prevention of disease caused by Mtb. We previously reported a genetically modified strain of Mycobacterium smegmatis called IKEPLUS, which is a promising vaccine candidate due to its ability to generate an unprecedented level of bactericidal immunity against Mtb in mice. Protective immunity induced by IKEPLUS is dependent on antigen-specific CD4+ T cell memory, and we hypothesized that specificity of the CD4+ T cell response was a critical feature of this protection. Using in vitro assays of IFNγ production by splenocytes from IKEPLUS-immunized mice, we identified an immunogenic peptide within the mycobacterial RplJ protein encoded by the Rv0651 gene. In a complementary approach, screening T cell hybridomas against Mtb ribosomal proteins identified as the RplA protein, a product of the Rv0641 gene in Mtb, as an additional specific target. Both RplJ and RplA are structural proteins of the 50S large subunit of the mycobacterial ribosome. Our results demonstrate that the CD4+ T cell response in IKEPLUS-immunized mice has multiple targets including components of the mycobacterial ribosome, among which RplA and RplJ represent novel specificities for protective responses against Mtb. Further studies to more fully define the CD4+ T cell responses to ribosomal targets should help to inform the design of effective vaccines against Mtb.
HLA-DO inhibition of HLA-DM catalytic function is regulated by acid-promoted HLA-DO inactivation (APP5P.106)
Authors: Jiang Wei , Strohman Michael , Somasundaram Sriram , Ayyangar Sashi , Wang Nan , Hou Tieying , Macmillan Henriette , Mellins Elizabeth
Published: January 2023

CD4+ T cells recognize MHC-II-peptides, whose generation is controlled by HLA-DM and its inhibitor, HLA-DO. Activation of DM-DO-expressing cells alters DO:DM in favor of active DM, but the molecular mechanism(s) underlying this process are unclear. DM is known to catalyze exchange of MHC-II-associated invariant chain peptides (CLIP) for antigenic peptides. As expected, We identified the positive correlation between the total CLIP level and DO:DM, and confirmed sufficient DM inhibition in high DO:DM cells. Importantly, super-resolution microscopy shows co-localization of CLIP with only the early endsomal marker but not the lysosomal marker in these cells, indicating the pH regulation of DM inhibition by DO and the presence of active DM in lysosomes. We demonstrated that acid-expose of DM-DO complexes yields active DM, and isolated free functional DM ectodomains using size exclusion chromatography. The study of pH-effects on the interaction between soluble DM and a stabilized DO equivalent (DOaP11A) indicates that the generation of active DM from DM-DO complexes is mainly due to acid-promoted DO denaturation. It is also suggested that free DM ectodomains are subjected to acid-promoted inactivation. Therefore, association by DO may regulate the endosomal pH range of DM activity while extending DM active life span at the lysosomal pH condition. Acidification of endosomes will down-regulate DO:DM via acid-promoted DO inactivation for efficient DM catalysis.
Involvement of γδ T cells and scavenger receptors in the immunopathogenesis of Pemphigus Vulgaris (BA11P.129)
Authors: Das Dayasagar , Anand Vivek , Singh Antil Parul , Khandpur Sujay , Sharma V.K , Sharma Alpana
Published: January 2023

Pemphigus Vulgaris (PV) is an auto-immune blistering disease mostly due to formation of auto-antibodies against desmoglein3 (Dsg3) and aberrant T cell function. γδ T cells play significant role in the immune surveillance at the epithelial surface and modulate other immune cells. Scavenger receptors (CD36 and CD163) are Pattern recognizing receptor (PRR) molecules and key players in several inflammatory diseases. γδ T cells and its signature molecular markers has found to be involved in several autoimmune diseases. However the role of γδ T cells and associated Scavenger receptor molecules are not characterized in the Immunopathogenesis of PV. In our study we observed significantly higher frequency of γδ T cells (6.7% vs. 3.8%) and IL17 producing γδ T cells in circulation of PV patients (n=25) vs. controls (n=15) respectively along with Th1 polarization (p<0.010) suggesting the potential role of γδ T cells and IL17 secreting γδ T cells in the pathogenesis of PV. Cytotoxic activity of γδ T cells from PV patients (26±11%) was observed to be higher as compare to control (12±7%). The molecular expression (mRNA) of scavenger receptors CD36 and CD163 were elevated in patients as compared to controls indicating the possible involvement of these PRR along with γδ T cells. These findings may help to understand the pathogenesis of PV and find better approach for novel therapeutics.
BAFF promotes T follicular helper cell development and germinal center generation via BR3 in mice (BA11P.120)
Authors: Zheng Song , Chen Maogen , Li Qiang , Wang Julie , Olsen Nancy
Published: January 2023

BAFF (B cell activating factor belonging to the TNF family) promotes B cells maturation in germinal center. The generation of germinal center depends on T follicular helper cells (Tfh). Overexpression of BAFF and Tfh is found in many autoimmune diseases. However, whether BAFF has effects on Tfh development is still unclear. In this study, we have explored the relationship between BAFF and Tfh in mice. Mice were immunized with KLH for the induction of Tfh in vivo. Naive CD4+ T cells were used for Tfh differentiation in vitro. BAFF−/−, BAFF transgenic (Tg), April−/−, BR3−/−, TACI−/−, BCMA−/−, μMT mice, BCL-2 Tg/μMT, BAFF−/−/BCL-2 Tg and TCRβ-/- mice were used for the mechanism studies. Tfh cells, mature B cell and germinal centers were determined. KLH specific IgG levels were measured by ELISA. The expression of IL-6 and IL-21 was determined by qPCR. We showed that BAFF but not April promotes the development of Tfh cells in vivo and in vitro. Furthermore, the development of Tfh cells is dependent upon BR3, but independent on BCMA and TACI. BAFF facilitates the generation of germinal center, and promotes B cells maturation and secretion of IgG. B cells are required for BAFF-promoted Tfh cell formation. We also demonstrated that BR3 in CD4+ T cells promotes the development of Tfh cells in vivo. In summary, these findings provide evidence that BAFF promotes Tfh development and germinal center generation, and these effects depend upon, at least partially BR3 signal.
Loss of NAPDH oxidase-derived superoxide exacerbates diabetogenic CD4 T cell effector responses (BA11P.122)
Authors: Padgett Lindsey , Anderson Brian , Liu Chao , Piganelli Jon , Mathews Clayton , Tse Hubert
Published: January 2023

In Type 1 diabetes (T1D), infiltrating leukocytes secrete reactive oxygen species (ROS) and pro-inflammatory cytokines that participate in β-cell destruction and enhance T cell autoreactivity. We previously demonstrated that superoxide-deficient Non-obese diabetic (NOD.Ncf1m1J) mice were T1D-resistant due, in part, to blunted Th1 CD4 and cytotoxic CD8 T cell responses. To dissect the role of ROS on autoreactive CD4 T cells, we created the NOD.BDC-2.5.Ncf1m1J (BDC-2.5.Ncf1m1J) mouse with diabetogenic, superoxide-deficient CD4 T cells and hypothesized that reducing ROS would diminish autoreactivity. BDC-2.5.Ncf1m1J splenocytes stimulated with the BDC-2.5 mimotope or islets displayed elevated IFN-γ and pro-inflammatory chemokine ligands CXCL10 and CCL5 (p<0.05). BDC-2.5.Ncf1m1J CD4 T cells were more diabetogenic (p=0.002), with only 26% of transferred NOD.Rag (n=45) mice euglycemic at 10 days post transfer, in contrast to 56% of BDC-2.5-transferred mice (n=24). In vivo suppression assays revealed that enhanced diabetogenicity was due to defective Treg activity, as BDC-2.5.Ncf1m1J Tregs curbed T1D in only 33% of BDC-2.5-transferred NOD.Rag mice (n=9), while BDC-2.5 Tregs induced 70% protection (n=10). Our results highlight the intricate role that ROS induce and restrict T1D autoreactivity. Future studies will dissect the TCR adaptor molecule oxidation status in BDC-2.5.Ncf1m1J CD4 T cells, as these targets may reveal novel biomarkers for T1D progression and prevention.
Mesenchymal stem cell and low level laser therapies reduce T CD4+, CD8+ and PMN cells in lung on experimental model of COPD (CAM4P.144)
Authors: Vitoretti Luana , de Britto Aurélia , da Silveira Elaine , de Moraes Gabriel , Aimbire Flávio , Vieira Rodolfo de Paula , Peron Jean , Ligeiro de Oliveira Ana Paula
Published: January 2023

Chronic Obstructive Pulmonary Disease (COPD) is characterized by airflow limitation, mucus gland hyperplasia, fibrosis and inflammatory cells recruitment into the lung. Cellular and molecular mechanisms of COPD have not yet been elucidated, complicating the use of effective therapies. Low level laser (LLL) and/or mesenchymal stem cell (MSC) are relatively new and effective therapies. We evaluate whether the use of LLL (670 nm, during 15 days) and human tubal-derived MSCs (i.n. 15 and 7 days before euthanasia) could induce changes in polymorphonuclear (PMN) and mononuclear (MN) cells in peribronchial space and T cells in lung of COPD induced mice (submitted to cigarette smoke for 75 days, 2 times/day). We also measured the IL-8 in supernatant of human bronchial epithelial cells (BEC) incubated with 2,5% of cigarette extract, irradiated or not with LLL. Compared to COPD group, we observed less PMN and MN cells in peribronchial space of treated groups, being the association of htMSCs+LLL most effective. The frequency T CD4+ and T CD8+ cells in the lung were significantly reduced irrespective of the therapeutic approach used. For CD8+ T cells, LLL therapy alone seems less efficient than htMSCs alone or in association. We noted reduction of IL-8 levels in BEC supernatant after laser therapy. Other data indicate that the therapies used here could reduce or impair cellular transmigration, T cell activation and cytokines release, being effective to decrease the inflammation in COPD.
CCR2 promotes accumulation of conventional dendritic cells in the lung during inflammation (CAM4P.149)
Authors: Nakano Hideki , Moran Timothy , Nakano Keiko , Whitehead Gregory , Thomas Seddon , Cook Donald
Published: January 2023

Migratory dendritic cells (DCs) take up antigens in peripheral tissues and migrate to tissue-draining lymph nodes (LNs) to present antigen-derived peptides to naïve T cells. Our group recently found that although preDC-derived conventional DCs (cDCs) migrate from the lung to LNs, monocyte-derived DCs (moDCs) do not. It is well established that interactions between the chemokine receptor CCR7 and its ligands direct cDCs from peripheral tissue to LNs, but LNs also contain non-migratory ‘resident’ cDCs derived from preDCs that likely migrate directly from the blood to LNs. We found that these LN resident cDCs are present in CCR7-deficient mice, suggesting that preDC migration from the blood is directed by a different and unknown mechanism. To investigate the mechanism, we used quantitative PCR to study chemokine receptor expression in preDCs. Surprisingly, we found that preDCs express high levels of the gene encoding CCR2, which is primarily recognized as a molecule that directs monocyte trafficking. Ccr2-deficient mice had normal numbers of cDCs in the lung at steady state, but these cells were significantly decreased compared to those of wild-type mice during inflammation. These findings suggest that CCR2 regulates accumulation of preDCs during inflammation, and that a different chemokine receptor directs preDC migration at steady state.
A novel population of myeloid-derived Gr-1+ CXCR2+CD11c+Ly6C+cells expressing FasL and perforin is required for contact hypersensitivity responses (CAM4P.150)
Authors: Kish Danielle
Published: January 2023

Allergic contact hypersensitivity (CHS) is a CD8 T cell mediated response to hapten skin sensitization and challenge. Hapten-primed CD8 cell infiltration into the skin challenge site during elicitation of CHS requires prior infiltration of Gr-1+ CXCR2+ cells expressing FasL and perforin. This study further characterized these Gr-1+ cells. These cells were purified from harvested bone marrow of hapten sensitized and challenged mice by depletion of CD3+, B220+, NK1+, F4/80+, class II MHC+ and Ly6G+ cells. Flow cytometry analyses indicate >90% of the remaining bone marrow cells are FasL+, CD11c+, Ly6C+, CXCR2+, CCR2+, CX3CR1+ and express perforin and granzyme B. These cells are at low numbers in the bone marrow of naïve mice but increase to peak numbers 2 hrs. after hapten challenge of sensitized WT and Rag-/- mice and T-cell depleted WT mice. Generation of these myeloid cells is inhibited by anti-TNFa mAb. Transfer of these myeloid-derived cells from WT donors restores the absent CHS-response in sensitized gld/perforin-/- mice and induces expression of the T cell chemoattractants CCL1, CCL2, and CCL5 in the skin 3-6 hrs after challenge. These results describe a novel population of myeloid cells expressing FasL and perforin/granzyme B that are generated in the bone marrow following hapten challenge of sensitized mice and are activated to induce expression of the chemoattractants directing hapten-primed T cell infiltration into the hapten challenged skin site to mediate CHS.
CCR2 mediated inflammation and gut microbiota in promoting intestinal cancer (CCR5P.214)
Authors: Jala Venkatakrishna , Bodduluri Sobha , Chilton Paula , Bodduluri Haribabu
Published: January 2023

Colorectal cancer is strongly associated with inflammation accompanied by increased infiltration of leukocytes. CCR2/CCL2 axis plays an important role in recruitment of several immune/inflammatory cells. Elevated expression of CCL2 in tumor cells is very well correlated with increased metastasis and poor prognosis in several types of human cancers including colon cancer. Gut microbiota plays an important role in modulating colon tumorigenesis. However, interactions between chemokines-microbiota-colon tumorigenesis are remains to be determined. Our data demonstrated that CCR2-/- mice in the ApcMin/+ (a spontaneous intestinal tumor mouse model) background showed substantial survival advantage compared to ApcMin/+ mice. CCR2-/-ApcMin/+ mice developed significantly fewer and smaller size small intestinal and colon tumors. Further, polyps in CCR2-/-ApcMin/+ displayed decreased tumor infiltrating F4/80+ cells, decreased inflammatory molecules (e.g., IL-1β, SOCS1), increased apoptosis and CD8+ T cells compared to ApcMin/+ mice. Interestingly, inflammatory cytokines IL-23 and IL-17 were significantly reduced in CCR2-/-ApcMin/+. Gut microbiota analysis revealed that tumor promoting Bacteroides genus are significantly reduced in CCR2-/-ApcMin/+ mice compared to ApcMin/+ mice. These data suggests that CCR2/CCL2 mediated recruitment of tumor-associated macrophages (TAMs) and Th17 cells modulate inflammation and microbiota in the tumor microenvironment to promote intestinal tumorigenesis.
Intracerebroventricular TWEAK (TNF-like weak inducer of apoptosis) induces depressive-like behavior and cognitive dysfunction in non-autoimmune mice (CCR5P.200)
Authors: Wen Jing , Chen Chris , Stock Ariel , Doerner Jessica , Burkly Linda , Gulinello maria , Khodakhah Kamran , Putterman Chaim
Published: January 2023

Fn14, the sole receptor for the TNF family member TWEAK, is inducibly expressed in the brain in endothelial cells, astrocytes, microglia, and neurons. Previously, we had found that Fn14 knockout lupus MRL/lpr mice display significantly attenuated neuropsychiatric manifestations. To establish whether this improvement in disease is secondary to ablation of TWEAK-Fn14 signaling within the CNS or the periphery and determine whether TWEAK-mediated neuropsychiatric effects are strain dependent, we performed intracerebraventricular injection (ICV) of Fc-TWEAK or a control protein to B6 mice. Interestingly, we found that the Fc-TWEAK injected mice developed significant depressive-like behavior and abnormal cognitive dysfunction. Inflammatory mediators including MCP-1, C3, and iNOS were significantly elevated in the brains of Fc-TWEAK treated mice. Furthermore, Fc-TWEAK directly increased BBB disruption, as demonstrated by increased fibronectin deposition in the brain and reduced aquaporin-4 expression. Finally, Fc-TWEAK increased apoptotic cell death in the cortex, hippocampus, and brain stem. In conclusion, TWEAK can contribute to lupus-associated neurobehavioral deficits by acting within the CNS to enhance production of inflammatory mediators, promote permeability of the BBB, and induce apoptosis in resident brain cells. Our study provides further support that the TWEAK/Fn14 signaling pathway may be a potential therapeutic target for inflammatory diseases involving the CNS.
Altered innate, adaptive, and TNF-superfamily soluble inflammatory mediators mark impending systemic lupus erythematosus disease flare, while regulatory mediators distinguish lack of impending disease flare in African-American lupus patients (CCR5P.201)
Authors: Munroe Melissa , Vista Evan , Merrill Joan , Guthridge Joel , Roberts Virginia , Air Gillian , Thompson Linda , James Judith
Published: January 2023

SLE is a multifaceted disease characterized by immune dysregulation and varied disease activity. We evaluated changes in plasma soluble mediators (n=52) preceding clinically-defined disease flare, in 13 African American (AA) SLE patients who developed disease flare 6 or 12 weeks after baseline assessment compared to matched SLE patients without impending flare and healthy controls. In addition, mediators within samples preceding SLE disease flare and during a clinically stable period from the same individual were compared in 18 AA SLE patients. Patients with impending flare had significant (q<0.01) alterations in 32 soluble mediators at baseline with significantly higher levels of pro-inflammatory mediators, including innate and adaptive cytokines. Baseline levels of regulatory cytokines, including IL-10 and TGF-β, were higher in non-flare SLE patients, while baseline levels of soluble TNFRI, TNFRII, TRAIL, FasL, and CD40L were significantly greater in pre-flare patients (p<0.05). A normalized and weighted combined soluble mediator score was significantly higher in pre-flare SLE patients versus those with stable disease (p<0.0001). Pro-inflammatory soluble mediators are elevated prior to disease flare, while regulatory mediators are elevated during periods of stable disease. Alterations in the balance between inflammatory and regulatory mediators may help identify AA patients at risk of disease flare and help decipher SLE pathogenic mechanisms.
Functional analysis of protective IL1RL1 variants associated with asthma risk (CCR6P.215)
Authors: Ramirez-Carrozzi Vladimir , Dressen Amy , Lupardus Patrick , Yaspan Brian , Pappu Rajita
Published: January 2023

GWAS studies have identified polymorphisms in both IL33 and IL1RL1, the gene encoding ST2, the high affinity chain of the IL-33 receptor, that associate with asthma susceptibility. We identified amino acid changing variants in IL1RL1 associating with asthma incidence and found these SNPs to be protective from asthma risk in our study population. These variants result in coding changes to the intracellular region of ST2, which contains the TIR domain of the receptor that is critical for signaling downstream of IL-1 cytokine family and TLRs. Mutations or deletions to this region can inhibit ligand-induced responses. IL-33-mediated dimerization of ST2 and IL-1RAcP promotes TIR-TIR domain interaction and recruitment of the adaptor molecule MyD88 leading to AP-1 and NF-kB activation. IL-33 responses were diminished in cell lines expressing all 4 IL1RL1 missense variants. To further elucidate how this haplotype could affect IL-33 activity, we compared IL-33 activity and ST2 expression between donors carrying either haplotype. We observed reduced IL-33 mediated IL-8 secretion from purified blood eosinophils derived from individuals carrying the protective haplotype. We also observed greater soluble ST2 expression in these individuals. Our results provide a link between the genetic predisposition to asthma and IL-33 mediated responses. Given IL-33 promotes Th2 immunity, perturbations that diminish this response may provide protection from asthma risk.
The effects of TNF-a on T cell homeostasis in obese and diabetic mice (CCR6P.219)
Authors: Barba Cindy , Mendoza German , Park Christa
Published: January 2023

Obesity is a growing epidemic in the United States that leads to many complications, including exacerbation of colitis and diminished immune response. Obesity increases levels of TNF-a systemically, causing chronic inflammation. This inflammation suppresses gd-T lymphocytes in epithelial tissues, which play key roles in tissue repair through the release of growth factors and activation of macrophages. Our previous research shows that blocking TNF-a for four days prior to wounding can help improve T cell function. This research focuses on how TNF-a affects intestinal epithelial T cells, splenic T lymphocyte homeostasis, and TNF-a function in obesity. Our study used mice deficient in TNFR1 and TNFR2 to investigate the changes imposed by an absence of TNF-a signaling. Data collected from splenic T cells and small intestine were compared to provide us with insight to the local and peripheral effects of mice with and without TNFR1 and TNFR2. Our current data suggest that TNFR1 and TNFR2 deficient obese mice show a decreased number of splenic T lymphocytes. Wound repair remains dysfunctional in obese mice even in the absence of TNFR1 and 2 suggesting epithelial T cells require TNFR signaling for normal function. The results thus far suggest that TNF-a is an essential cytokine in maintaining the homeostasis and function of T lymphocytes in obese mice. Further research will be conducted involving TNF-a and its receptors on the immune cells of obese and diabetic individuals.
Dual CCR2/CCR5 antagonism by Cenicriviroc efficiently inhibits both MCP-1 and RANTES induced chemokine receptor internalization in murine pro-inflammatory monocytes (CCR6P.221)
Authors: Byron Mary Margaret , D'Antoni Michelle , Premeaux Thomas , Lefebvre Eric , Ndhlovu Lishomwa
Published: January 2023

Persistent immune activation, driven in part by monocytes and macrophages (MO), has been implicated in HIV-associated comorbidities and progressive liver disease. Binding of chemokines MCP-1 and RANTES to their respective chemokine receptors (CKR), CCR2 and CCR5, selectively regulates the migration of MO. Cenicriviroc (CVC), an oral, dual CCR2 and CCR5 antagonist, has completed Phase 2b development in HIV and is undergoing Phase 2b evaluation in subjects with non-alcoholic steatohepatitis (NASH) and liver fibrosis. We assessed CVC receptor occupancy, measured by inhibition of MCP-1 and RANTES induced CKR internalization, compared to single antagonists BMS-22 (CCR2) and Maraviroc (MVC; CCR5) in pro-inflammatory (CD11c+F4/80+Ly6c+) MO in male BALB/c mice (n=6) by flow cytometry and quantified using Molecules of Equivalent Soluble Fluorochrome microspheres. CVC significantly reduced both MCP-1 induced internalization of CCR2 (p<0.01) and RANTES induced internalization of CCR5 (p<0.01). Doses of 0.25-1μM achieved >90% CCR2 and CCR5 occupancy by CVC compared to 49% CCR2 occupancy by BMS-22 and 69% CCR5 occupancy by MVC in blood pro-inflammatory MO. Doses of 0.02-1μM achieved >85% CCR2 and CCR5 occupancy by CVC compared to 72% CCR2 occupancy by BMS-22 and 73% CCR5 occupancy by MVC in spleen pro-inflammatory MO. These data suggest that CVC leads to efficient CCR2 and CCR5 target coverage in blood and tissues with effective doses lower than that of respective single CKR antagonists.
CCR4 is required for efficient thymocyte medullary entry and central tolerance induction (HEM7P.230)
Authors: Ehrlich Lauren , Hu Zicheng , Lancaster Jessica , Sasiponganan Chayanit
Published: January 2023

Clonal deletion of auto-reactive thymocytes is essential for the establishment of central tolerance. The thymic medulla is a specialized microenvironment in which post-positive selection thymocytes are screened for reactivity against a wide range of self-antigens. Autoreactive cells are tolerized via clonal deletion or differentiation into the regulatory T cell lineage. Both thymic dendritic cells (DCs) and medullary thymic epithelial cells (mTECs) present auto-antigens to thymocytes to induce central tolerance. Thus, it is critical that post-positive selection thymocytes enter the medulla and interact efficiently with antigen-presenting cells therein to avoid export of autoreactive naïve T cells. We previously demonstrated that although CCR7 is required for medullary accumulation of thymocytes, additional GPCRs must be involved in medullary entry. Here we report that CCR4 is expressed by post-positive selection thymocytes, while CCR4 ligands are expressed by thymic DCs. Using two-photon time-lapse microscopy, we find that CCR4 is required for accumulation of post-positive selection thymocytes in the medulla, as well as efficient interactions between thymocytes and medullary DCs. In the absence of CCR4, thymocytes fail to undergo clonal deletion to low avidity antigens, autoreactive naïve T cells accumulate in secondary lymphoid organs, and autoimmunity ensues. Taken together, these data demonstrate an essential role for CCR4 in the establishment of thymic central tolerance.