bluuebunny/crossref_metadata_2025 huggingface dataset:
Obesity impairs lymphocyte homeostasis and function in epithelial tissues via inflammatory mechanisms (MUC2P.932)
Authors: Jameson Julie , Cheung Kitty , Costanzo Anne , Limon Natalie , Yee Christine , Park Christa
Published: January 2023

Barrier tissues such as the skin and intestine provide vital protection to the organism from pathogens and exposure to the environment. Epithelia-resident γδ T lymphocytes maintain this protective barrier via the production of growth factors and cytokines. Obesity and type 2 diabetes disrupt normal homeostasis within the epithelium and result in complications that include delayed tissue repair. We have previously identified a reduction in the number of T cells within the epithelium of the skin and intestine of mice as obesity progresses. Here we find that obesity impairs the ability of lymphocytes to maintain homeostatic proliferation within epithelial tissues. Our studies show that this is particularly devastating in young mice that are still establishing lymphocyte populations in their epithelia. Using blocking studies and gene chip analyses we have identified inflammatory mediators that are involved in the negative impact that obesity has on intraepithelial lymphocytes. The mechanism involved is independent of diet as both mice on a high fat diet and genetically obese mice on a normal chow diet exhibit reduced and dysfunctional epithelial lymphocytes. Reducing chronic inflammation by weight loss or blocking inflammatory mediators begins to reestablish T cell homeostasis and improve tissue repair. This data identifies new therapeutic targets for improving tissue repair during obesity and type 2 diabetes via the restoration of normal cellular homeostasis and gene expression.
Resolvin E1 regulates contact hypersensitivity responses by inhibiting dendritic cell functions through leukotriene B4-BLT1 signaling blockade (MUC2P.936)
Authors: Sawada Yu , Honda Tetsuya , Nakamura Motonobu , Miyachi Yoshiki , Kabashima Kenji
Published: January 2023

Resolvin E1 (RvE1) is a lipid mediator derived from ω3 polyunsaturated fatty acids that exert potent anti-inflammatory roles in several disease models. However, the precise underlying mechanism how RvE1 exerts its action in vivo remains largely unknown. In this study, we investigated the mechanisms of the anti-inflammatory effect of RvE1 using a contact hypersensitivity model. We found that administration of RvE1 significantly suppressed skin inflammation through a blockade of BLT1, a leukotriene B4 (LTB4) receptor, on dendritic cells (DCs). Live imaging of cutaneous DCs by two-photon microscopy revealed that RvE1 significantly impaired DC motility in the skin and migration to draining lymph nodes as well as antigen presentation ability in the skin. LTB4-BLT1 signaling induced actin formation in DCs and increased DC motility by activating Cdc42 and Rac1 through the PI3 kinase pathway, which was canceled by the administration of RvE1. Taken together, our results indicate that RvE1 attenuates cutaneous immune responses by inhibiting cutaneous DC functions through LTB4-BLT1 signaling blockade.
Robust Th17 response to intranasal group A streptococcal infection requires IL-6 produced by CD301b+CD11b+CD103- migratory dendritic cells (MUC2P.937)
Authors: Dileepan Thamotharampillai , Linehan Jonathan , Cleary P. , Jenkins Marc
Published: January 2023

Intranasal (i.n.) Group A Streptococcal (Streptococcus pyogenes - Sp) infections generate a vigorous Th17 response. We tested the hypothesis that a specific dendritic cell (DC) subset in the nasal associated lymphoid tissue and cervical lymph nodes (CLN), preferential sites of colonization and lymphatic drainage site for i.n. Sp, are responsible for this skewing of the T cell response. The hypothesis was tested by tracking polyclonal Sp peptide:MHCII-specific CD4+ T cells mice lacking specific DC subsets. We found that Sp peptide:MHCII-specific Th17 formation depended on an IL-6 producing CD301b+ CD11b+ CD103- migratory DC population in the CLN. The results contrast with other findings implicating CD11b+ CD103+ DC in Th17 induction in the intestinal mucosa. Therefore, different DC subsets are responsible for Th17 generation through different mucosal surfaces.
Recirculating HIV-target T lymphocytes at the mucosal surface of the female genital tract are associated with progesterone increases during the menstrual cycle (MUC2P.938)
Authors: Swaims Alison , Haaland Richard , Evans-Strickfaden Tammy , Haadad Lisa , Sheth Anadi , Lupo L. , Cordes Sarah , Aguirre Alfredo , Lupoli Kathryn , Chen Cheng-Yen , Ofotokun Igho , Kohlmeier Jacob , Hart Clyde
Published: January 2023

The Female Genital Tract (FGT) provides a means of entry to opportunistic infections, including HIV, yet immune cell populations at this mucosal surface are poorly defined. We initiated a study of 20 healthy women to characterize resident T cell populations in the lower FGT from lavage and patient-matched blood samples. Surprisingly, we report FGT T cells were primarily CCR7hi memory CD4 T cells (P=<0.0001), consistent with a central memory phenotype and known HIV reservoir. FGT CCR7hi T cells exhibited more frequent expression of HIV susceptibility markers CCR5 and CD38 compared to blood (0.6% vs. 13.6%, P=0.0017), yet retained the ability to migrate to CCR7 ligands, suggesting these cells could migrate from the genital tract into afferent lymphatics. Finally, we demonstrate the frequency of FGT CCR7hi target cells increased during the luteal phase of the menstrual cycle (51.7-72.7), and correlated to progesterone from patient-matched blood (r=0.5176, P=0.0024). These data show that a majority of CD4 T cells at the surface of the FGT in humans do not express canonical resident memory T cell markers, but are primarily a central memory T cell phenotype with the ability to traffic across the mucosal epithelium into underlying tissue. Our findings suggest HIV may directly infect recirculating memory CD4 T cells that traffic virus across the FGT mucosal barrier and progesterone changes throughout the menstrual cycle may predict availability of these cells HIV target cells.
Functional subsets of intestinal NKT cells (MUC3P.942)
Authors: Aguiar Cristhiane , Takahashi Daisuke , Câmara Niels , Wingender Gerhard , Engel Isaac , Kronenberg Mitchell
Published: January 2023

NKT cells constitute a distinct population of lymphocytes characterized by their reactivity to glycolipids presented by CD1d. Although they are found at a relatively low frequency, because of their clonal specificity and rapid cytokine production, they have been reported to influence many types of immune responses. Recently NKT cells have been divided into functional subsets analogous to T helper (Th) Th1, Th2 and Th17 cell subsets, called NKT1, NKT2 and NKT17. We have examined if similar functional subsets are found in the mouse intestine. NKT cells are found at a low frequency in intraepithelial lymphocytes (IEL) of the small and large intestine and a higher prevalence in lamina propria lymphocytes (LPL). We analyzed intestinal NKT cells using flow cytometry to explore the transcription factors and surface markers expressed by these cells. We found out that all the functional subsets are represented in intestinal NKT cells, however, the percentages of each subset in the intestine are different from those found in spleen. We also evaluated the response of intestinal iNKT cells to the agonist α-Galactosylceramide. We observed that, in response to in vivo exposure to the potent antigen α-Galactosylceramide, intestinal NKT cells produced IFN-γ, TNF-α and GM-CSF and increased expression of activation markers, as CD69 and CD40L, suggesting that cells with an NKT1 function are most prevalent. We are now exploring how the microbiota affects the functions of intestinal NKT cells.
Identification of highly potent and selective Tyk2 inhibitors for the treatment of autoimmune diseases through structure-based drug design (THER2P.961)
Authors: Masse Craig , Miao Wenyan , Greenwood Jeremy , Shelley Mee , Kennedy-Smith Joshua , Kapeller Rosana
Published: January 2023

The JAK family kinase Tyk2 is essential for IL-12 and IL-23 signaling, which are associated with Th1 and Th17 cell differentiation and activation. The Th1 and Th17 pathways have been implicated in the pathogenesis of psoriasis and inflammatory bowel diseases (IBD) thereby making Tyk2 a highly attractive target for the treatment of these disorders. However, given the high degree of sequence homology between the JAK family kinases, designing potent and selective Tyk2 inhibitors remains a challenge. Using an innovative structure-based approach, we have designed, synthesized and characterized small molecule inhibitors optimized for JAK family selectivity using computational free energy perturbation (FEP) methods and medicinal chemistry SAR. We have identified selective Tyk2 inhibitors with pM activity against Tyk2 (Ki=140-520 pM) and >100 fold selectivity over JAK2 and JAK1 with more moderate selectivity over JAK3. These analogs are orally bioavailable (85%F) with suitable drug-like properties. NDI-031232 was determined to be highly selective across 359 kinases, and is a potent inhibitor of IL-12 induced pSTAT4 in hPBMCs (IC50=17 nM) and IL-12 induced IFNg in human whole blood (IC50=520 nM). NDI-031232 also demonstrated robust efficacy in blocking IL-12-mediated IFNg production in an ex vivo mouse model. Therefore, selective inhibitors of Tyk2 retain the anti-inflammatory activity while reducing potential for dose-limiting side effects observed with non-selective JAK inhibitors.
RGC-32 enhances suboptimal CD8 cytotoxic T cell effector function in acute graft versus host disease and promotes lupus-like disease in chronic graft versus host disease (THER2P.965)
Authors: Rus Violeta , Nguyen Vinh , Badea Tudor , Cudrici Cornelia , Tegla Cosmin , Mekala Armugam , Rus Horea
Published: January 2023

Response Gene to Complement (RGC)-32 is an intracytoplasmatic protein expressed in a variety of cells in response to sublytic complement activation, TGFb and other cytokines. It plays a role in cell growth, fibroblast activation and renal fibrosis, Th17 and plasma cell differentiation. To assess whether RGC-32 expression promotes lupus-like disease, we used WT and RGC-32 KO mice to investigate whether lack of RGC-32 in host cells affects parameters of chronic (c)GVHD in the Bm12→B6 model and whether lack of RGC-32 in donor T cells affects parameters of acute (a)GVHD in the P→F1 model. In F1 mice injected with RGC-32 KO splenocytes, an attenuated phenotype of aGVHD was observed at suboptimal (30 x106) but not optimal (50 x106) donor cell doses as indicated by a significant decrease in donor CD8 engraftment, proliferation and host B cell elimination. Bm12→B6 cGVHD induced in RGC-32 KO hosts displayed an attenuated phenotype as indicated by decreased number of germinal center B cells and plasmablasts, mRNA expression of Prdm1, IRF-4 and Aicda and anti-dsDNA Ab production. These results suggest that RGC-32 expression in host B cells promotes autoimmune parameters in cGVHD while its expression in T cells enhances suboptimal CD8 cytotoxic T cell effector function thus preventing humoral autoimmunity in aGVHD. These data support the idea that RGC-32 blockade has the potential to reverse abnormalities in the CD8 T cell and B cell pathways that contribute to lupus pathogenesis.
Impact of human mutant TGF-β1/Fc protein on memory and regulatory CD4+T cells following lymphodepletion in rhesus monkeys (THER2P.953)
Authors: Guo Hao , Lu Lien , Wang Rijian , Abdulkerim Hayat , Reimann Keith , Thomson Angus , Ezzelarab Mohamed
Published: January 2023

Transforming growth factor beta (TGFβ) is critical for regulatory T cells (Treg) homeostasis and regulation of peripheral tolerance. In rodents, combined human TGFβ1 fusion protein (TGFβ1/Fc) and rapamycin treatment prevents allograft rejection. We evaluated the influence of human TGFβ1/Fc on CD4+T cells in vitro, and in vivo following lymphodepletion in rhesus monkeys. In vitro, TGFβ1/Fc and rapamycin synergistically reduced ki67 expression and proliferation of rhesus memory T cells (Tmem), following CD3/CD28 activation or allo-stimulation. However, increased percentage of Treg and Treg/Th17 ratio were only observed in the presence of interleukin-2 (IL-2). Four monkeys received immunosuppression in the form of Thymoglobulin and rapamycin, without (n=2) or with (n=2) TGFβ1/Fc infusion. In monkeys receiving TGFβ1/Fc, trough levels were maintained at 2-7 ug/mL, and were undetectable by day-45. With no TGFβ1/Fc infusion, rapid recovery of Tmem to pre-depletion levels was observed by day-21, and continued to increase. In contrast, with TGFβ1/Fc infusion, the recovery of Tmem was delayed until day-56. In addition, increased Treg and Treg/Tmem ratio were observed in peripheral blood and lymph nodes. However, Treg/Th17 ratios were reduced in all monkeys. In lymphodepleted monkeys, infusion of human TGFβ1/Fc delays CD4+Tmem recovery, and is associated with increased Treg/Tmem ratio. Combined IL-2 and TGFβ1/Fc infusion may be essential to improve Treg/Th17 ratio after lymphodepletion.
TWEAK receptor-Fc suppresses germinal center formation and pathogenic B cells in lupus mice model via inhibition of TWEAK/Fn14 pathway (THER3P.1002)
Authors: Kim Sung-Min , Min Hong-Ki , Park Jin-Sil , Byun Jae-Kyeong , Lee Jennifer , Kwok Seung-Ki , Park Young-Woo , Cho Mi-La , Park Sung-Hwan
Published: January 2023

Systemic lupus erythematosus (SLE) is autoimmune mediated chronic inflammatory diseases. Half of SLE patients suffer from lupus nephritis, and lupus nephritis is major cause of death in SLE. TWEAK/Fn14 interactions have been identified as pathologic factor in SLE pathogenesis. We conducted TWEAK/Fn14 pathway blocking by TWEAK receptor-Fc in SLE mice model and searched for the probable therapeutic mechanisms. CD19+ B cells were harvested from sanroque mice and TWEAK was administered. Sanroque mice were treated with TWEAK receptor-Fc or control-Fc for 3 weeks. Immunoglobulin (Ig) G, IgG1, IgG2a were measured in the sera of each group. Spleens from each group were stained with antibodies against CD4, B220, GL-7, CD138, and PD-1. Expression levels of mRNAs were determined by real-time PCR. Kidneys were stained with H&E and PAS. Administration of TWEAK promoted B cell differentiation. Treatment with TWEAK receptor-Fc suppressed the levels of IgG, IgG1, and IgG2a in sera and reduced B cell, plasma cell, follicular helper T cell (Tfh) in spleen of sanroque mice. Additionally, renal protective effects of TWEAK receptor-Fc were demonstrated. In conclusion, TWEAK receptor-Fc had beneficial effects on SLE mice model by repressing auto-reactive B cell, plasma cell, Tfh, and renal damage. In futher, TWEAK receptor-Fc would be a potential therapeutic agent in SLE patient.
Soluble RAGE overexpressed mesenchymal stem cell attenuates autoimmune arthritis via induction of Th17/Treg balance and immunomodulatory effects (THER3P.971)
Authors: Lee Seung Hoon , Park Min-Jung , Lee Jung-Ah , Lee Eun-Jung , Kim Eun-Kyung , Cho Mi-La
Published: January 2023

Receptor for advanced glycation end-products (RAGE) plays the essential role in the pathogenesis of autoimmune rheumatoid arthritis (RA). Mesenchymal stem cells (MSCs) have recently shown promise as a therapeutic tool in various types of autoimmune disease models. In the persent study, we constructed stable MSC with sRAGE overexpression by DNA transfection. We hypothesized that sRAGE overexpression MSC may enhance the self-renewing and modulatory effects on RA therapy. We found that MSCs in inflammatory condition increase expression of High Mobility Group Box1 Protein (HMGB1) and IL-1beta, IL-6, VEGF. However, MSCs that overexpressed sRAGE significantly decrease these inflammatory factors. In addition, sRAGE overexpressing MSCs produced higher amounts of immune modulatory factors. MSCs that overexpressed sRAGE displayed enhanced migration activity and autophagy vesicle. Using a co-culture model of MSC and T cell, we showed that sRAGE overexpressing MSCs potently suppressed Th1, Th17 cell differentiation. Systemic infusion of sRAGE overexpressing MSCs inhibited development of arthritis and reduced bone erosion and cartilage destruction. These therapeutic effects were associated with an increase in CD4+FoxP3+ Treg cells and inhibition of Th17 cell formation in the spleen. These data suggest that enhancing the immunomodulatory activity of MSCs and modulating T cell-mediated immunity using gene-modified MSCs may be a gateway for new therapeutic approaches to clinical RA.
The upregulation of HtrA2 ameliorates autoimmune arthritis via inhibition of Th17 formation and STAT3 expression (THER3P.974)
Authors: Lee Seung Hoon , Young-Mee Moon , Seo Hyeon-Beom , Kim Eun-Kyung
Published: January 2023

HtrA serine peptidase 2 (HtrA2) performs pivotal role in maintaining of the mitochondria homeostasis and involves in cellular stress response such as inflammation. Rheumatoid arthritis (RA) appears to have a basis of mitochondrial dysfunction. To identify the role of HtrA2 in RA, we used a mnd2 mutant mice. We hypothesized that HtrA2 overexpression may reduce immune inflammation and reveal therapeutic effects on RA. We observed that inhibitor and siRNA of HtrA2 in T helper cell (Th) 17 condition increase the mRNA expression of interleukin (IL)-17. On the other hand, the overexpression HtrA2 significantly reduces mRNA level of IL-17 and induces cleavage of STAT3. Moreover, p-STAT3 and lymphocyte activation is promoted in mnd2 mutant mice compared to wild type. HtrA2 overexpression attenuates collagen-induced arthritis (CIA) in mice model. Th17 and osteoclastogenesis are suppressed in CIA mice treated with HtrA2 overexpression. Additionally, HtrA2 overexpression inhibited development of plaque formation and activation of Th17 cell and STAT3 in APOE knock-out mice with proteoglycan immunization for animal model of hyperlipidemia based RA. The therapeutic function of HtrA2 in inflammatory diseases is related with the targeting of Th17 development and STAT3 pathway in splenocytes. These results suggest that HtrA2 takes part in immunomodulatory activity and upregulation of HtrA2 may shed light on therapeutic approaches to inflammatory disorder such as RA and hyperlipidemia.
PD-1 blockade does not enhance T cell reactivity to autologous tumor cells ex vivo in breast cancer. (TUM2P.1013)
Authors: Egelston Colt
Published: January 2023

Recent efforts to identify the antigens tumor specific T cells target have suggested PD-1+ T cells target epitopes made up from mutated proteins. To investigate this possibility in patients with breast carcinoma, a cancer with few mutated antigens, we sought out T cells that specifically react and kill autologous tumor cells. We found that PD-1 was highly expressed on both CD4 and CD8 T cells in the tumor and tumor draining lymph nodes (TDLNs) of breast cancer patients and to a lower extent on circulating T cells. To investigate tumor reactivity, we co-cultured CD8 T cells from tumor, TDLN, or PBMCs with autologous cancer cells overnight. Very low numbers of T cells were found to react to autologous tumor cells by measurement of either CD137 expression or IFNy secretion. Performing the same experiment in the presence of PD-1 blockade did not increase reactive T cell numbers. We have also found that there is a correlation of reduced T cell functionality and PD-1 expression in T cell clones even in the absence of PD-1 ligands. This data suggests that PD-1+ T cells in cancers with a high mutation rate may be able to be restored via PD-1 blockade, while PD-1+ T cells in cancers with few mutations may be self-antigen specific T cells that may have undergone irreversible tolerance. Such data has strong implications for checkpoint blockade therapy.
Activating self-tolerant T cells with altered structure of MHC class I (TUM2P.1024)
Authors: Parks Christopher , Hansen Michael , Ju Josephine , Reed Brendan , Kranz David , Bordner Andrew , Pease Larry
Published: January 2023

Self-tolerant T cells can cross-react weakly on self, but are usually incapable of activation. However, when activated these T cells can cross-react effectively on cells expressing self antigens. We have observed that mutants of the MHC class I molecule H2-Kb have structural differences resulting in altered antigen presentation. We hypothesize that structural manipulation of MHC class I can increase stabile amino acid contacts with the TCR. Increasing stabile contacts may decrease the energy barrier required to overcome the threshold for activation, breaking tolerance for self antigens. Using computer modeling techniques, amino acid substitutions at specific sites of MHC class I were predicted to result in stabilization of the TCR:pMHC complex. These altered MHC molecules were made and expressed in cell lines. We have shown that altered MHC enhanced TCR binding. Altered MHC enhanced activation and proliferation of naïve T cells in response to a non-stimulatory, weak antigen. Altered MHC also induced CTL killing against self-antigens. In an in vivo transplant model, protective immunity was induced against a native tumor after an altered MHC expressing tumor challenge. By altering the structure of MHC class I we have activated normally tolerant T cells that are capable of cross-reacting on self.
The quantity of CD26 on helper T cells correlates with the efficacy of adoptive cancer immunotherapy (TUM2P.1008)
Authors: Bailey Stefanie , Nelson Michelle , Bowers Jacob , Majchrzak Kinga , Stefanik Leah , Paulos Chrystal
Published: January 2023

Although Th17 cells cause striking tumor eradication upon transfer into mice bearing established melanoma, their translation into the clinic is barred by a lack of FDA-approved cytokines. Consequently, we sought to determine if IL-17-producing CD4+ cells could be enriched from peripheral blood. We found that functional Th17 cells could be isolated from blood, without the need for polarization, by using the surface marker CD26. Excitingly, very few CD26-rich cells, termed CD26high T cells, displayed striking antitumor activity and survival upon transfer into melanoma-bearing mice compared to CD26negative or bulk CD4+ T cells. Of translational significance, we found that human CD26high cells had heightened cytotoxicity and caused greater cancer cell death in vitro. Interestingly, human CD26high T cells have enhanced expression of cytokines (IL-17, IFN-γ, IL-2, TNFα), transcription factors (RORγ, Tbet), integrins (LFA-1, VLA-1) and chemokines (RANTES). Given the unique phenotype of CD26high T cells compared to cells with decreased CD26 expression, the question of this marker's importance is raised. Our preliminary studies reveal that the enzymatic activity of CD26 may be important for the enhanced function, cytotoxicity and migration of these cells and could possibly be targeted to enhance future therapies. Not only do CD26high T cells give hope for Th17 clinical translation, but also provide a potential mechanism by which to improve the T cells used for cancer immunotherapies.
Quantifying the lower limit of antibody-dependent cell cytotoxicity using a TCRm against a WT1 peptide/MHC epitope (VAC3P.1058)
Authors: Casey Emily , Scheinberg David
Published: January 2023

The WT1 protein is an ideal cancer antigen, but therapeutically targeting this transcription factor has proven difficult. We have discovered a therapeutic TCR mimic antibody that specifically binds to a WT1-derived peptide (RMFPNAPYL) in the context of the MHC class I haplotype HLA-A2. This high affinity mAb, called ESK, has demonstrated significant anti-cancer activity in animal models of human cancers and leukemias. Studies in vitro and in vivo have shown the dominant role of ADCC in ESK activity. While commercially available mAbs target tens to hundreds of thousands of sites on a cell, ESK binds as few as ~500 epitopes and still promotes cancer cell clearance in vivo. Therefore, we believe ADCC can be a more sensitive mode of killing than currently estimated. To probe this question, we developed a biotinylated RMF peptide that can be pulsed onto HLA-A2 on T2 cells at various concentrations and detected using labeled streptavidin probes. By using 3D confocal microscopy photon scanning, we can detect single molecules of the streptavidin probe on a cell surface. Therefore, using a CFP-DEVD-YFP FRET reporter expressed in the pulsed T2 cells, we can measure the number of peptides bound by ESK in the immunological synapse between a target T2 cell and a human NK cell required to induce T2 cell death. Identifying this number will impact patient selection for ESK clinical trials and will influence the development of therapeutic mAbs against other low-density epitopes.
Dietary fibre-mediated effects on the primary antibody response to keyhole limpet hemocyanin differs between sexes (VAC3P.1059)
Authors: Shastri Padmaja , Brooks Stephen , Kalmokoff Martin , Green Judy , Mathias Fernando , Green-Johnson Julia
Published: January 2023

Dietary fibre can mediate effects on mucosal immunity and the gut microbial community. The aim of this study was to examine the impact of dietary fibre intake on immunization efficacy, and to assess potential sex-based differences. Following a 6 week feeding trial, male and female rats fed purified diets containing single sources of fermentable material (cellulose; C, wheat bran; WB, oat bran; OB, resistant starch; RS or oligofructan; OF) were parenterally immunized with 0.3 mg/kg Keyhole Limpet Hemocyanin (KLH). Anti-KLH and total antibody concentrations were evaluated at intervals from tail vein blood, and splenic lymphocyte percentages and cytokine levels were measured at the end of the trial. Circulating anti-KLH IgG, IgG2a and IgG2b levels decreased significantly in females fed OB, while anti-KLH IgG2a levels decreased in males fed either RS or OF-supplemented diets compared to control-fed rats. In contrast to females, males fed OB had significantly lower splenic TGF-b1/IFN-g ratios and higher total IgG antibody levels. Additionally, females fed RS or OF had significantly reduced splenic CD3+ and CD3+4+ percentages while only CD3+ percentages were significantly reduced in males fed OF, relative to their control-fed counterparts. Collectively these results suggest dietary fibre can impact systemic immune responses to immunization, and that these effects can differ between sexes.
Flagellin has differential and route-dependent adjuvant effects when encoded in heterologous systemic or mucosal gene-based prime-boost vaccines (VAC4P.1107)
Authors: Rady Hamada , Dai Guixiang , Huang Weitao , Shellito Judd , Ramsay Alistair
Published: January 2023

Flagellin has been tested as a protein-based adjuvant, with most studies focused on antibody responses. In this study, we have evaluated the adjuvant activity of flagellin for cellular and humoral immune responses in gene-based immunizations. DNA and adenovirus (Ad) vectors were constructed to encode mycobacterial Ag85B and/or flagellin (FliC) of S. typhimurium. DNA-encoded flagellin given to mice via the IM route enhanced antigen-specific splenic CD4+ and CD8+ T cell responses. Boosting either IM or intranasally (IN) with Ad vectors expressing Ag85B without flagellin enhanced antigen-specific antibody and CD4+ and CD8+ T cell responses in both spleen and pulmonary tissues, correlating with improved protection against pulmonary challenge with Mtb. However, inclusion of flagellin in both DNA prime and Ad boosting vaccines induced localized pulmonary inflammation and transient weight loss, along with route-dependent effects on vaccine-induced T cell immunity. The latter included marked reductions in mucosal CD4+ and CD8+ T cell responses following IM DNA / IN Ad prime-boosting, although antibody responses were not diminished. These findings suggest that flagellin has differential and route-dependent adjuvant activity when included in systemic or mucosal gene-based prime-boost immunization. Adjuvant activity for both T and B cell responses and safety were optimal when flagellin was included only in the DNA vaccine priming phase of systemic or mucosal DNA/Ad prime-boosting.
Lipopolysaccharide induced type I interferon is essential for effector CD4 T cell expansion and migration to liver (VAC4P.1108)
Authors: Shinde Paurvi
Published: January 2023

Vaccines that provide long-term protection against a pathogenic infection often use adjuvants to boost the immune response to less immunogenic antigens. Lipopolysaccharide (LPS) is such an adjuvant that enhances CD4 T cell clonal expansion, survival and IFN-γ production. Similarly, engagement of OX40 receptor (CD134) on antigen activated CD4 T cells increases CD4 T cell activation and clonal expansion. Administration of LPS and CD134 costimulation together profoundly increases the Ag-specific CD4 T clonal expansion, survival and memory. It is known that LPS induced TRIF signaling is needed CD4 and CD8 T migration to liver, lung and spleen as well as for IFNγ production. TRIF downstream activates Type I interferon which can upregulate co-stimulatory molecules on APC and mediate CD4 T cell priming and expansion. Hence our goal is to study how LPS induced Type I Interferon together with CD134 costimulation impacts antigen specific CD4 T cell expansion, migration and effector potential in lymphoid and non-lymphoid organs. Our results show that, IFNAR blockade reduced antigen-specific CD4 T cell expansion and migration to liver on days 6 and 12 on immunization with LPS and CD134 agonist. In pLN however, IFNAR blockade reduced T cell expansion and migration on day 6, but by day 12 T cells began to accumulate. Additionally even though Type I interferon signaling was needed for IFNγ secretion by Th1 cells in pLN, it actually reduced IFNγ and TNFα secretion of Th1 cells in liver.
Wnt signaling pathway functionally reprograms human virus-specific CD8 T cells (VAC4P.1065)
Authors: Sung Bo-Yi , Chiu Yen-Ling , Schneck Jonathan
Published: January 2023

Polyfunctionality, a single T cell producing a set of different effector molecules, is a hallmark of protective immunity against infection and tumor, but the molecular pathways governing the polyfunction T cells were not well known. In our lab, we found that memory CD8 T cells treated with TWS119, a Wnt pathway activator, increased highly polyfunctional memory T cells formation during expansion. The treatment of TWS119 enhanced CD62L+, CD28+ and KLRG1- central memory T cell phenotype. We further analyzed the impact of Wnt activation on influenza virus or CMV(cytomegalovirus) antigen-specific stimulation, and found TWS119 also improved the polyfunctionality. Especially in CMV-specific T cells, terminal differentiated non-polyfunctional population, Wnt signaling reversed their non polyfunctional state. Wnt-activated T cells were capable of superior homeostatic proliferation and expressed more anti-apoptotic molecules. Mechanistically, RNA microarray analysis revealed that Wnt activation led the gene expression profile correlated with stem-cell specific gene expression pattern. Our findings provided the evidence that non-polyfunctional antigen-specific human CD8 T cells response can be pharmacologically reprogrammed and have important translational implications for immunotherapy against chronic viral infections and malignancies.
Skin dendritic cells induce follicular helper T cells and protective humoral immune responses (VAC4P.1101)
Authors: Yao Chen , Zurawski Sandra , Jarrett Elizabeth , chicoine brian , Crabtree Juliet , Peterson Erik , Zurawski Gerard , Kaplan Daniel , Igyarto Botond
Published: January 2023

Skin-resident dendritic cells (DC) play a crucial role in initiation of adaptive immune responses against cutaneous pathogens as well as in the maintenance of peripheral tolerance. Recent studies have suggested that steady-state Langerhans cells (LC) induce regulatory T cells against self-antigens. However, the immune response induced by skin DC against foreign antigen in the absence of adjuvants has not been addressed. Here we report that, using anti-huLangerin/ muLangerin antibodies, we could specifically target antigens to LC or CD103+ dermal DC. Presentation of the foreign peptide 2W1S by either LC or CD103+ dDC was sufficient for expansion of naïve CD4+ T cells and induction of T follicular helper cell (Tfh) differentiation. Notably, topical application of peptide or protein also efficiently induced Tfh. The expansion of Tfh specific to foreign peptide was accompanied by activation and expansion of antigen-specific B cells and the development of a robust antibody response that provided systemic protection against influenza infection. This study not only reveals a major unappreciated function of skin DC in humoral response, but also provides insight into DC-targeted vaccine design.
Measuring the properties of different forms of keyhole limpet hemocyan reveals important differences (VAC5P.1116)
Authors: Bi Shuguang , Milbrandt Laura , Brisson Catherine
Published: January 2023

KLH can exist as cylinders composed of 20 copies (didecamer) of subunits, or as separate subunits. It is a well-established immune stimulant and vaccine component. However, literature reports cite dramatically different immune responses to different preparations of KLH. We are developing functional assays to investigate the immunogenicity of different forms and preparations of KLH with the goal of improving the predictive outcome of its use as vaccine carrier or diagnostic agent for both research and clinical studies. Dendritic cells (DCs) derived from mouse bone marrow mononuclear cells, human PBMC, mouse DC line JAWSII, as well as human leukemia lines THP-1 and KG-1, were treated with different forms of KLH. To compare the efficiency of DCs to stimulate KLH-specific adaptive immune responses, KLH treated DCs were used to activate T cells extracted from spleens of mice that were immunized with KLH. DC antigen uptake efficiency, DC maturation, T cell proliferation and cytokine secretion were compared. Ovalbumin was conjugated to different forms of KLH. DCs derived from mouse bone marrow mononuclear cells were treated with the KLH-Ovalbumin conjugates for antigen uptake efficiency and ovalbumin-specific T cell activation studies. We found that didecameric KLH and subunit KLH have significantly different properties. We report here the results of these studies which suggest the need for methods to identify appropriate forms of KLH when used as hapten carrier or immunogen.
Immunostimulatory activity of indium phosphide quantum dots in vitro and in vivo (VAC5P.1128)
Authors: Lyle Christopher , Byrne Kristen , Falcon Daniel , Dienglewicz Robert , Jang Hyeonmin , Aguilar Zoraida , Erf Gisela
Published: January 2023

Quantum dot nanoparticles (QDs) have shown increasing promise in biological applications including enhanced immunomodulatory effects that may be leveraged as vaccine adjuvants. While cadmium selenium based QDs were shown to have toxic effects in vivo, recent studies have shown that indium phosphide QDs offer a safer alternative. The objective of this study was to examine the bioactivity of 7 nm InP/ZnS QDs (670 nm emission) in vitro and in vivo in the avian model. In vitro studies on the effects of QDs on chicken macrophages revealed uptake of QDs within 45 min with minimal nitric oxide production and cytotoxicity. To monitor in vivo effects of QDs, we utilized the pulp (dermis) of growing feathers (GF) as a test-tissue. For this, 10 μL of QDs (4, 1, or 0.25 μM) were injected into the pulp of 20 GF/chicken (n = 4/treatment). GF were collected 0.25, 1, 2, 3, 5, and 7 days post-injection for cell population analysis of infiltrating leukocytes by flow cytometry and immunohistochemical staining of frozen sections for localization of leukocytes and QDs. Leukocyte infiltration was observed by 6 h. Leukocyte profiles were both time and dose dependent and inflammatory in nature. Combined immunohistochemical and fluorescence studies of frozen GF sections confirmed the presence of QDs and immune cells. Gene-expression (qRT-PCR) analyses are underway. To our knowledge this is the first study to assess in vivo immune activities in response to InP/ZnS QDs injected into a complex tissue.
Immune response induced by vaccination with non-replicative type I strain of Toxoplasma gondii (CPS-1) induces more tetramer specific CD8 T cells with central memory phenotype. (VAC5P.1117)
Authors: Dominguez Mariana , Hwang Sujin , Nixon Douglas , Moretto Magali , Khan Imtiaz
Published: January 2023

Role of CD8 T cells in immunoprotection against Toxoplasma gondii infection is well established. However, in an encephalitis model, CD8 immunity is functionally impaired (immune exhaustion), leading to reactivation of latent infection. Studies from our laboratory have reported that functional defect during chronic infection is primarily exhibited by memory subset of CD8 T cells. Thus restoration of dysfunctional CD8 response may be critical for maintaining the chronicity and preventing the reactivation of latent infection. As a first step we evaluated and compared the antigen-specific CD8 T cell response induced by vaccination with the non-replicative type I strain of Toxoplasma gondii (which efficiently controls per-oral infection with type II strain) to the one induced during chronic infection. While type II strain induced a stronger SLEC (short lived effector CD8 cell) response as compared to vaccine strain in all the tissues tested, MPEC (memory precursor effector cell) response with both the strains was similar. However, interestingly the frequency of central memory cells subset (CD127+CD62Lhigh) in the vaccinated mice was significantly higher as compared to those receiving type II infection. These findings suggest that the development of inadequate central memory CD8 response may be an important defect responsible for reactivation of infection in mice susceptible to toxoplasmic encephalitis.
Passive immunization via neutralizing scFv-Mab to specific antigen of Aggregatibacter actinomycetemcomitans ameliorates periodontal infection and destruction in vivo (VAC5P.1120)
Authors: Teng Andy , Liu Yen Chun
Published: January 2023

Aggregatibacter actinomycetemcomitans-Aa, a G(-) anaerobe, is involved in medical/dental infection, i.e., aggressive periodontitis, & systemic disorders. To explore anti-bacterial immunity & therapeutics, its genomic library was screened by expression-cloning using disease-associated CD4+T-cells of HuPBL- NOD/SCID mice. Such cloning yielded one novel gene, designated cagE-homologue, encoding a bacterial type-IV secretion system homologous to Helicobacter pylori cagE & Agrobacterium tumefaciens virB4. We showed that: i) Aa-CagE induced apoptosis of targets, based on biochemical & genetic studies; ii) anti-CagE IgG-response was significantly correlated with disease progression. By CagE immunization in HuPBL-SCID mice and the recombinatorial phage-display library, we screened to develop therapeutics for Aa. The screening yielded human sc-Fv-Mabs to CagE (CagE-scFv-Mabs), as confirmed by binding assay & sequencing. To this end, our latest studies showed that human CagE-scFv-Mabs were manifested by: i) high binding activity (>108~9M-1); ii) strong neutralizing capability by inhibiting CagE-induced caspase3-mediated apoptosis (>80%); iii) possessing anti-toxin-like Ig-CDR sequences; iv) significantly reduced Aa colonization in biofilm and reduced alveolar bone loss in mice. It is concluded that CagE-scFv-Mabs capable of inhibiting apoptosis, Aa colonization and bone destruction via immunization exhibit the potential to ameliorate periodontal & medical infections associated with Aa.
De novo detection and HLA-association of public T cell responses to Cytomegalovirus using high-throughput immune repertoire sequencing (VIR1P.1134)
Authors: Emerson Ryan , DeWitt William , Desmarais Cindy , Rieder Mark , Carlson Christopher , Gravley Jenna , Hansen John , Robins Harlan
Published: January 2023

We investigated the public T cell response to cytomegalovirus by sequencing rearranged T cell receptors (TCRs) in 640 subjects (287 with & 353 without CMV). We performed an experiment similar to a GWAS study, assessing the concordance of millions of unique TCRs with CMV serostatus, looking for significant associations. With CMV serostatus and TCR sequencing data, we identify 142 public T cell responses to CMV (each with p ≤ 1.0 x 10-4; FDR ≈ 0.20). Using this method to identify public clones, we predict CMV serostatus in a leave-one-out cross-validation experiment with a diagnostic odds ratio of 66. Using HLA typing, we then estimate the HLA-association of each public CMV clone by assessing the over-representation of particular HLA types in associated subjects. Of 142 CMV public clones, 26 were HLA-associated at p < 10-6; no clones were significantly associated with multiple HLA alleles. When comparing our results to the literature, we find substantial concordance; most previously-reported public CMV clones are seen in our data, though only 2 are significantly associated with CMV serostatus in this cohort; both were significantly HLA-associated and in both cases the associated allele confirms previous findings. We demonstrate the validity of association studies using immunosequencing for detection and HLA-association of public T cell responses to infection, and report that assessing the presence of public T cell responses can serve as a powerful diagnostic classifier.
Age-dependent defects in the draining lymph node and naïve CD4+ T cell trafficking impair adaptive immunity and control of virus infection (VIR1P.1137)
Authors: Richner Justin , Gmyrek Grzegorz , Miller Mark , Diamond Michael
Published: January 2023

Although adaptive B and T cell responses decline with age, the basis for impaired immunity of the elderly against viruses remains uncertain. Herein, in the context of infection with West Nile virus (WNV), we identify new mechanisms for compromised adaptive immunity that occurs with aging. Old mice had blunted humoral immune responses to WNV, increased viral burden, and enhanced mortality. This decline in immunity and enhanced vulnerability to infection was associated with decreased trafficking capacity of naïve CD4+ T cells but not B cells from old mice, which manifested as impaired T cell diapedesis at high endothelial venules and reduced cell motility within draining lymph nodes (DLN) as judged by intravital microscopy, as well as delayed germinal center formation. Additionally, immune cell accumulation in the DLN after virus infection or antigen-adjuvant administration was diminished more generally in old mice and associated with a separate aging-related defect in local cytokine and chemokine production. Our results support a model in which age-dependent cell-intrinsic and cell-extrinsic defects result in delayed immune cell recruitment and antigen recognition in the DLN, which compromises induction of adaptive immune responses and contributes to the susceptibility of old animals to acute viral infections, and likely impaired vaccine responses.
T cell expression of the histone demethylase UTX promotes clearance of a persistent virus infection by modulating Tfh cell differentiation (VIR1P.1155)
Authors: Cook Kevin , Shpargel Karl , Starmer Joshua , Whitfield-Larry Fatima , Conley Bridget , Allard Denise , Magnuson Terry , Su Maureen , Whitmire Jason
Published: January 2023

T cell differentiation and memory formation are impacted by epigenetic changes that modulate nucleosome structure to regulate transcription factor accessibility. The enzymes and specific targets that mediate these processes within T cells are not clear. We show that UTX, a histone H3 lysine 27 (H3K27) demethylase, promotes the formation of the CD4+ T follicular helper cell (Tfh) phenotype during a chronic virus infection. Mice with T cell specific UTX deletion had defects in Tfh cell generation and function that resulted in reduced germinal center responses and virus-specific immunoglobulin production. Furthermore, clearance of the chronic virus infection was specifically dependent on T cell expression of UTX. H3K27me3-ChIP-sequencing and RNA expression analysis revealed a subset of genes that were specifically regulated by UTX via H3K27 demethylation. This group included IL-6Ra, a known regulator of Tfh responses, as well as other genes, which are potentially novel Tfh effector genes. Thus, we identify a critical link between UTX-dependent H3K27 demethylase activity in T cells and immunity to infection.
Virus-infected human mast cells enhance Natural Killer cell functions (VIR1P.1160)
Authors: Portales-Cervantes Liliana , Haidl Ian , Lee Patrick , Marshall Jean
Published: January 2023

Mast cells are widely distributed throughout vascularised tissues. At mucosal surfaces they play important roles in host defence. Human mast cells produce chemokines, such as CXCL8, that selectively recruit Natural Killer (NK) cells in models of viral infection. However, the ability of mast cells to enhance NK cell effector functions has not been previously examined. Primary human mast cells were infected with reovirus and derived mast cell products were used for stimulation of human NK cells. Mast cell products induced expression of the activation marker CD69 (n=7; p<0.001), the cytotoxicity-related genes PRF1 (n=6; p<0.01) and TIA-1 (n=5; p<0.05), and enhanced NK cell cytotoxic activity against K562 cells (n=6; p<0.01). Interferon-γ was produced by NK cells treated with virus-induced mast cell mediators in the presence of IL-18 (n=7; p<0.001). In vivo, reovirus-infected human mast cells induced a 7-fold increase in recruitment and activated murine NK cells (42.3% CD69+ vs 5.8% CD69+ cells) in comparison to uninfected mast cells in a subcutaneous matrigel model (n=9; p<0.05). Soluble products of reovirus-infected mast cells included IL-10, type I and type III interferons. Blockade of type I interferon receptors revealed that they were important for mast cell-mediated NK cell activation. Our data define a novel mast cell-NK cell immune axis in human host defense against viral infection, which could be rapidly induced prior to the generation of acquired immunity.
Immune restoration, activation and viral reservoirs in gut-associated lymphoid tissue in SIV-infected long-term nonprogressing Chinese rhesus macaques on antiretroviral therapy (VIR1P.1130)
Authors: Ling Binhua , Perez Stefanie , Johnson Ann-Marie , Doyle-Meyers lara
Published: January 2023

HIV infected long-term nonprogressors (LTNPs) and elite controllers (ECs) maintain enduring control of HIV infection without antiretroviral therapy. However, persistent tissue reservoirs and chronic inflammation still exist. We previously found that 1/3 of SIV-infected Chinese rhesus macaques mimics HIV-infected LTNPs/ECs. Here we studied whether therapy can further decrease tissue reservoirs, restore immune function and decrease inflammation in this model. Six LTNPs/ECs and 4 progressors were treated with PMPA and FTC daily for up to 24 weeks during chronic infection. Blood, lymph nodes and gut tissues were collected. Cell-associated viral DNA levels from purified CD4+ T cells in blood and gut lymphocytes were quantified by real-time PCR. The size of reservoirs was evaluated by quantitative coculture assays and real-time PCR. Immune cells restoration including IL-17+ subsets, proliferation and immune activation were monitored by flow cytometry. We found that plasma viral loads in all animals were suppressed and maintained to undetectable throughout ART. The levels of IL-17 expression in CD4 and CD8 T cells were not fully restored but slightly increased after ART. Immune activation significantly decreased. In conclusion, the ChRM nonhuman primate model is excellent for testing novel therapeutic interventions on TP and LTNPs/ECs for HIV eradication. Initiation of ART may be necessary for improvement of immune function for HIV/SIV infected LTNP/ECs.