bluuebunny/crossref_metadata_2025 huggingface dataset:
A high frequency of naïve PR1-specific CD8+ T cells in umbilical cord blood and fetal thymus suggest incomplete central tolerance to the hematopoietic self-antigen PR1. (46.24)
Authors: St. John Lisa , Clise-Dwyer Karen , Patenia Rebecca , Sergeeva Anna , He Hong , Bollard Catherine , Shpall Elizabeth , Lu Sijie , Ma Qing , Molldrem Jeffrey
Published: January 2023

Tolerance to self-antigens is established primarily in the thymus by negative selection. We have evidence that central tolerance to the HLA-A2 restricted self-peptide, PR1, is incomplete. The frequency of PR1-CTL in umbilical cord blood (UCB) is significantly higher than that seen in the peripheral blood of healthy adults. Investigation in fetal thymic tissue (FT) showed that PR1 is expressed on the surface of thymic dendritic cells (DC) but not thymic epithelial cells. The level of PR1 expression on FT DC is similar to that on UCB DC and healthy adult DC. Comparisons of antigen-specific cells in FT and UCB demonstrated pp65-CTL frequencies were comparable, yet PR1- and WT1-CTL percentages were lower in UCB than in FT. PR1-specific cells, in contrast to pp65- and WT1-specific cells, were found at a higher frequency within CD8 single positive (SP) thymocytes versus CD4+CD8+ double positive (DP) cells, suggesting that PR1-specific SP thymocytes have a survival advantage relative to WT1- and pp65-specific SP thymocytes. In response to PR1 peptide-loaded T2 cells, DP PR1-CTL undergo apoptosis to a greater degree than SP PR1-CTL, implying that higher frequencies of SP PR1-CTL in FT could be due, in part, to a lower susceptibility of SP PR1-CTL to apoptosis. Our data suggest that PR1 expression by DC in the thymus is insufficient for complete central tolerance to PR1 and PR1-specific SP thymocytes may have a survival advantage over other self and viral antigen-specific thymocytes.
Effect of immunomodulatory polyphenols on innate lymphocyte responses involved in anti-tumor defense. (46.34)
Authors: Ramstead Andrew , Robison Amy , Jutila Mark
Published: January 2023

The anti-tumor effects of polyphenols have received considerable attention in recent years. The majority of research in this area has focused on the direct effect of these compounds on tumor cells, as well as their antioxidant activities. However, few studies have focused on the effect of these compounds on leukocyte responses involved in anti-tumor immunity. Two polyphenol extracts, which have been shown to have immunomodulatory and antitumor properties, are apple peel polyphenols (APP) and oenothein B (OB), a compound isolated from the Epilobium angustifolium plant. We have previously shown that these compounds are capable of stimulating innate lymphocytes, such as NK cells and γδ T cells. Since these innate lymphocytes play an important role in anti-tumor responses through several mechanisms, the effects of these polyphenol extracts on these cells was investigated further. We found that both APP and OB can modulate the expression of certain surface proteins and cytokines involved in tumor cell recognition and killing by innate lymphocytes, such as NKG2D and IFNγ. The effect of these polyphenols on the anti-tumor activity of innate lymphocytes and their importance is currently under investigation in both human cells and mouse models. Funded by NIH NCCAM PO1 AT004986.
CD8 T cell responses to Braf-mutated melanoma upon regulatory T cell depletion (46.37)
Authors: Steinberg Shannon , Zhang Peisheng , Byrne Katelyn , Bosenberg Marcus , Turk Mary Jo
Published: January 2023

A model for studying Braf-mutated melanoma in immunocompetent mice has recently been generated, in which melanocyte-specific induction of Cre recombinase leads to expression of mutant BrafV600E and deletion of the tumor suppressor Pten. Using these Braf/Pten mice on a C57BL/6 background, we are able to characterize the melanoma-antigen specific CD8 T cell responses by adoptively transferring CD8 T cells from Pmel-1 transgenic mice, whose T cell receptors are specific for the melanoma differentiation antigen gp100. As previous work in our lab has demonstrated the ability of CD4 cell depletion to eliminate regulatory T cells and drive priming of CD8 T cells in mice bearing B16 melanomas, we characterized the effects of Treg depletion in mice with Braf/Pten tumors. In these studies, we have shown that CD4 depletion enhances the frequency of melanoma antigen-specific CD8 T cells in the tumor draining lymph node and systemically. We have also found that while untreated Braf/Pten tumors have a low frequency of infiltrating CD8 T cells, Treg depletion allows for robust infiltration of CD8 T cells into the tumor. These studies provide evidence that while poorly immunogenic, autochthonous Braf-mutated tumors can prime CD8 T cells responses in the absence of regulatory T cells.
A role for Bcl-xL in CD1d-mediated Natural Killer T cell responses to B cells (46.39)
Authors: Subrahmanyam Priyanka , Webb Tonya
Published: January 2023

Lymphomas are a heterogeneous group of malignancies, primarily divided into Hodgkin's and non-Hodgkin's lymphomas (NHL). Unlike other cancers, the incidence of NHL is steadily rising. Natural Killer T (NKT) cells are innate-like lymphocytes that recognize glycolipid antigens in the context of CD1d, an MHC class I-like molecule. They have strong anti-tumor activity and respond by massive cytokine production and/or cytotoxicity. It is known that pro-survival factors are upregulated in B cell lymphomas, but their role in antigen processing and presentation remains unknown. The effect of Bcl-xL on CD1d-mediated antigen presentation was examined utilizing the well-characterized WEHI-231 B cell lymphoma cell line. Bcl-xL expression was modulated via genetic, pharmacologic and biological approaches. We found a concomminant increase in CD1d-mediated NKT cell responses following the induction of Bcl-xL; however, cell surface expression of CD1d molecules remained unchanged. Induction of Bcl-xL in primary B cells led to increased CD1d-mediated activation of NKT cells in response to exogenous antigens, resulting in increased Th1 not Th2 responses. Taken together, these data suggest that aberrant expression of Bcl-xL may act as a sentry system to alert the CD1d/NKT system to neoplastic transformation.
Cell-intrinsic adenosine A2A receptor signaling is required for T cell homeostasis and control of tumor growth (47.1)
Authors: Cekic Caglar , Sag Duygu , Linden Joel
Published: January 2023

Adenosine generated as a result of normal metabolic activity is elevated in response to tissue stress, damage, or hypoxia. By stimulating adenosine A2A receptors (A2ARs) adenosine links metabolic activity to regulation of normal physiological functions such as sleep, blood flow, and inflammation. Although stimulation of A2ARs on activated T cells and APCs acutely suppresses inflammation and immune killing of tumor cells, global A2AR deletion has variable effects on the growth rate of syngeneic tumors, suggesting that A2AR signaling has opposing effects on immune function. Here we show that A2ARs are required for homeostatic maintenance of naïve T cells. In global A2AR-deficient mice naïve T cells undergo apoptosis, which drastically lower their number in blood and in peripheral lymph nodes. Lymphoid deletion of A2ARs, adoptive co-transfer and mixed bone-marrow reconstitution experiments showed that cell intrinsic A2AR signaling is required for T cell homeostasis. A2AR signaling is also required for interleukin 7 to optimally facilitate T cell survival ex vivo. In mice injected with syngeneic melanoma cells, lymphoid-specific A2AR deletion decreases T cell numbers in the periphery and in the tumor, reduces the proportion of tumor infiltrating T cells with an activated/memory phenotype, and markedly accelerates tumor growth. The results indicate that homeostatic adenosine production and A2AR signaling are required in naïve T cell maintenance and immune defense against tumors.
Polyfunctional capacity of T cell function in older adults (47.10)
Authors: Canaday David , Burant Christopher , Banks Richard , Aung Htin , Hornick Thomas , Higgins Patricia
Published: January 2023

The precise polyfunctional capabilities of T cells from older persons are not well known. We determined the polyfunctionality of memory CD8+ and CD4+ T cell subsets using a 13-color flow cytometric assay that simultaneously analyzes IFN-gamma, TNF, MIP1-alpha, IL-2, and perforin expression after stimulation. PBMC from 37 older (mean age 82) and 30 younger (mean age 34) individuals were stimulated with a sub-maximal concentration of the mitogen SEB. Older adults were also assessed by the Fried frailty test. The proportion of highly polyfunctional cells that could perform 3, 4, or 5 functions was similar in the memory T cell subsets from older and younger individuals. The percentage of cells capable of performing each individual function had only small differences between the age groups. Within the older group however, polyfunctionality of central memory CD4+ and CD8+ T cells and CD8+ effector cells had in inverse correlation with frailty while not having a relationship with age. In conclusion, as a whole the frequency and polyfunctionality and mitogen-activated CD4+ and CD8+ memory T cell subsets in older individuals is preserved compared to the younger group. Therefore, the phenomenon of immunosenescence particularly in the non-frail elderly may not include the loss of capability and breadth of function of T cells if stimulated sufficiently. This may inform specific strategies to boost the older immune system.
Inducible costimulator (ICOS) negatively regulate programmed death-1 (PD-1) induced apoptosis of effector memory CD4 T cells under homeostatic condition (47.11)
Authors: Suvas Susmit , Channappanavar Rudragouda , Twardy Brandon
Published: January 2023

ICOS, a T cell specific costimulator, promote the survival of effector memory CD4 T cells whereas PD-1, an inhibitory molecule, can induce apoptosis of memory CD4 T cells. However, the role of these cell-surface associated accessory molecules in regulating each other function when co-expressed on a single cell is not well known. In this report, we demonstrate that naturally occurring effector memory like CD4 T cells in unmanipulated young (12 weeks) C57BL/6 mice either do not express PD-1 and ICOS (PD-1-ICOS-) or express PD-1 (PD-1+) or ICOS (ICOS+) or both molecules (PD-1+ICOS+) when measured by flow cytometery in the spleen. A similar trend was noted in the splenocytes obtained from aged (24 months) B6 mice but the proportion and number of PD-1+ICOS+ effector memory CD4 T cell pool was significantly higher in the aged mice in comparison to young B6 mice. While comparing the ability of these four subsets of effector memory CD4 T cells to undergo spontaneous apoptosis in vivo by Annexin V staining, we determined that PD-1+ICOS+ subset was least susceptible to spontaneous apoptosis. In vivo administration of anti-ICOS antibody given to unmanipulated aged B6 mice resulted in significantly increased proportion of apoptotic PD-1 expressing memory CD4 T cells when compared to isotype treated control group. Taken together, our results suggest a novel mechanism by which PD-1 expressing antigen specific effector memory CD4 T cells might survive under steady state condition.
PI3K/Akt pathway contributes to development of apoptosis resistance during differentiation of human macrophages by maintaining antiapoptotic Bcl-xL protein expression (48.7)
Authors: Kumar Ashok , Busca Aurelia , Saxena Mansi
Published: January 2023

Resistance to apoptosis is an important characteristic that human macrophages acquire during differentiation from monocytes. However, the intracellular mechanisms that mediate the development of resistance remain mostly unknown. We have used PMA-treated THP1 cells and MCSF-stimulated primary monocytes to generate human macrophages. By using chemical inhibitors for the PI3K and MAPK pathways, our results show that only pretreatment of cells before differentiation with the PI3K/Akt inhibitor LY294002 induced caspase dependent apoptosis that was associated with decreased expression of antiapoptotic Bcl-xL protein. These results were confirmed by inactivation of this pathway by Akt specific siRNAs. Moreover, this effect was specific if Akt was knocked down before inducing differentiation. Furthermore, Bcl-xL expression was found to be dependent on NFkB in differentiating macrophages. However, NFkB activation was prevented by LY294002 pretreatment, indicating that NFkB is responsible for Bcl-xL expression via PI3K/Akt signaling. Taken together, these results suggest that survival of macrophages is distinctly regulated during and after differentiation. A signaling pathway encompassing PI3K/Akt-mediated activation of NFkB plays a key role in survival of differentiating macrophages by specifically sustaining antiapoptotic Bcl-xL expression.
CD4+ T cells are necessary and sufficient to confer protection against C. trachomatis infection in the murine upper genital tract. (49.18)
Authors: Gondek David , Olive Andrew , Starry Georg , Starnbach Michael
Published: January 2023

Chlamydia trachomatis infection is the most common bacterial sexually transmitted disease in the United States. Chlamydia infections that ascend to the upper genital tract can persist, trigger inflammation, and result in serious sequelae such as infertility. However, current mouse models where the vaginal vault is infected with C. trachomatis do not recapitulate the course of human disease. These intravaginal infections of the mouse do not cause significant infection of the upper genital tract, do not cause persistent infection, do not induce significant inflammation, and do not induce CD4+ T cell infiltration. Here we describe a non-invasive transcervical infection model where we bypass the cervix and directly inoculate C. trachomatis into the uterus. We show that direct C. trachomatis infection of the murine upper genital tract stimulates a robust Chlamydia-specific CD4+ T cell response that is both necessary and sufficient to clear infection and provide protection against re-infection.
Conditional deletion of RANK in intestinal epithelial cells of mice results in loss of Peyer’s patch M cells and impaired acquisition of orally delivered antigen (49.2)
Authors: Rios Daniel , Knoop Kathryn , Kumar Nachiket , Tawde Suprita , Chablani Lipika , D'Souza Martin , Williams Ifor
Published: January 2023

Microfold (M) cells are antigen-sampling intestinal epithelial cells found in the follicle-associated epithelium (FAE) of Peyer’s patches (PPs). M cells initiate mucosal immune responses by transcytosis of particulate antigens for delivery to antigen-presenting cells that traffic through the intraepithelial pocket of M cells. We previously showed that RANKL from stromal cells in the subepithelial dome of PPs is necessary and sufficient for inducing M cell differentiation. To determine how selective loss of intestinal M cells affects host immune responses to antigens encountered in the gut lumen, we generated mice with a floxed allele of RANK deleted only in intestinal epithelial cells (IEC) by a villin-cre transgene (RANKΔIEC mice). The FAE of PP in RANKΔIEC mice lacked any cells expressing the M-cell marker GP2, proving that RANKL promotes M cell differentiation from intestinal stem cells via a direct action on RANK-expressing IEC. An OT-II T cell adoptive transfer model was used to analyze ovalbumin (OVA)-specific T cell responses 2 to 3 days after gavage with various forms of OVA. RANKΔIEC mice had compromised T cell responses to both soluble OVA and particulate OVA (OVA encapsulated in acid-stable microparticles prepared by spray drying). The RANKΔIEC mouse provides a first-of-its-kind mouse model for the specific assessment of the contribution of intestinal M cells to uptake of protein antigens and pathogenic microorganisms encountered in the intestinal lumen.
Anti-TBGL IgA antibody suggest mucosal immunity involvement in latent tuberculosis infection, which is frequently positive for anti-TBGL-IgG antibody. (49.28)
Authors: Hattori Toshio , Siddiqi Umme , Leano Susan , Chagan-Yasutan Haorile , Telan Elizabeth Freda
Published: January 2023

Anti-tubercular-glycolipis-IgG (TBGL-IgG) and -IgA (TBGL-IgA) elisa and interferon gamma release assay (IGRA) were done in healthcare workers (HCW, n=31) in Manila to study the immune responses in latent TB infection (LTBI). Forty eight percent (15/31) of the HCWs showed positive reactions in the IGRA indicating high incidences of LTBI in Manila. Eleven of 15 IGRA-positive HCWs had positive TBGL-IgG responses (categorical agreement 73%), whereas 5 of 16 QFT-negative subjects had positive TBGL-IgG responses (categorical agreement 68.7%). The overall κ value was 0.42, indicating a moderate association between the two assays (overall agreement: 71%; 95% CI: 0.10 ~ 0.73). The TBGL-IgG-positive proportions were also significantly different between the IGRA-positive and -negative groups of HCWs (P = 0.02). The TBGL-IgG+IgA double-positive response was shown only by IGRA-positive HCWs. A significant, positive correlation was observed between the IFN-γ levels and TBGL IgA titers in the IGRA-positive group (r = 0.74, P = 0.006) but not in the IGRA-negative group. There was no such association between the levels and TBGL-IgG titers in HCWs, although a tendency for a positive correlation was observed in the IGRA-positive HCWs (r = 0.43, P = 0.11). No association was observed between the IFN-γ concentrations in antigen stimulated IGRA-plasma and thr TBGL-IgG or -IgA titers. These findings suggest the involvement of mucosal immunity in LTBI.
Characterization of a DC Presenting a Microbial Polysaccharide to Regulatory T cells. (49.9)
Authors: Dasgupta Suryasarathi , Erturk-Hasdemir Deniz , Kasper Dennis
Published: January 2023

Bacteroides fragilis Polysaccharide A (PSA) is a unique commensal microflora molecule which has shown potent CD4+T cell mediated IL-10 dependent anti-inflammatory properties in various murine models. Little is known about how these immunoregulatory T cells are induced in order to control inflammation. We first observed that PSA dependent augmentation of regulatory T cells correlated with frequency of B220+CD11cint cells in MLN. We focused majorly on Plasmacytoid dendritic cells (PDCs), which have been implicated as tolerogenic DCs in other inflammatory situations. We observed higher expression of TLR2, a previously reported PSA receptor, in PSA treated PDCs compared to media control in vitro. Isolated PDCs from wild type compared to TLR2 deficient PDCs significantly augmented the liberation of IL-10 from CD4T cells in presence of PSA (p=0.0046), a process mediated by co-stimulators ICOSL and CD86. In a TNBS induced colonic inflammation model to study PSA mediated protection, the frequency of PDCs correlated significantly but inversely with cumulative colitis score (R2=0.4377, p=0.0002) suggesting an immunoregulatory role of these cells. Interestingly, neither protection nor change in PDCs was observed in TLR2-/-mice. In addition, PSA failed to protect mice when either PDCs were depleted or co-stimulators inhibited in vivo with monoclonal antibodies. This work suggests a tolerogenic or immunoregulatory role for PDCs most probably by generating regulatory features in T cells.
STAT4 isoforms are biomarkers in patients with inflammatory bowel disease (51.2)
Authors: Jabeen Rukhsana , Steiner Steven , Miller Lucy , Kaplan Mark
Published: January 2023

Crohn’s disease (CD) and ulcerative colitis (UC), the major forms of inflammatory bowel disease (IBD) in humans are characterized by chronic relapsing inflammation of the gastrointestinal tract (GI). Th1 and Th17 cells have been implicated as important mediators of IBD. Signal transducer and activator of transcription (STAT4) is critical for the development of Th1 cells and promotes IL-17 production from Th17 cells. STAT4 is expressed as two isoforms, a full-length STAT4α, and STAT4β that lacks the C-terminal transactivation domain. In a previous study we found that although both isoforms mediate inflammation in a mouse model of colitis, STAT4β seems more potent in generating tissue inflammation. With this we hypothesized that if STAT4β is more pro-inflammatory, ratios of the isoforms might change in disease. Compared to healthy controls, we observed increased STAT4β/STAT4α ratios in the peripheral blood mononuclear cells of UC and CD patients, correlating with the disease and ratios were normalized after patient treatment. Increased STAT4β/STAT4α ratios in GI biopsies of CD patients further confirmed that ratios correlate with the location and grade of inflammation. In contrast, we did not observe changes in STAT4β/STAT4α ratios in biopsy specimens from eosinophilic esophagitis (EE) patients. Collectively, these data indicate that STAT4 isoforms could be an important non-invasive biomarker in the diagnosis and treatment of IBD, and potentially other inflammatory diseases.
Adenosine Receptor 2A Agonist-Induced Tolerogenic Dendritic Cells Protect Mouse Kidney from Ischemia-Reperfusion Injury (52.2)
Authors: Li Li , Huang Liping , Ye Hong , Song Steven , Bajwa Amandeep , Lee Sang Ju , Jaworska Katarzyna J , Kinsey Gilbert , Day Yuan , Linden Joel , Lobo Peter , Rosin Diane , Okusa Mark
Published: January 2023

Dendritic cell (DC)-mediated nature killer T (NKT) cell activation is critical in initiating the immune response following kidney ischemia-reperfusion injury (IRI). Adenosine 2A receptor (A2AR) agonists protect kidneys from IRI through their actions on bone marrow (BM)-derived leukocytes. We hypothesized that A2AR agonists attenuate IRI by tolerizing DCs and preventing NKT cell activation. ATL313 attenuated kidney IRI in WT mice but not in mice deficient of DC A2ARs (CD11c-CreAdora2a-/-). BM-derived DCs were activated by ex vivo incubation with alpha-galactosylceramide (aGC)±ATL313 (1nM), washed and adoptively transferred into naïve WT mice 2 days before kidney IRI. ATL313-treated aGC-loaded WT DCs (DC-aGC-ATL313) but neither Adora2a-/- DC-aGC-ATL313 nor DC-aGC protected kidney function following IRI. Co-culture of NKT cells with DC-aGC-ATL313 caused less IFN-g production than with DC-aGC. ATL313 down-regulated OX40L and CD40 and up-regulated B7-DC expression but had no effect on DC surface and intracellular CD1d/aGC complexes. DC-aGC-ATL313-mediated kidney protection was not due to Treg cell expansion, but was in part due to IL-10, as IL-10mRNA level increased and IL-10 neutralization reversed the protective effect of DC-aGC-ATL313; DC-aGC-ATL313 also up-regulated PD-1 expression on co-cultured Tregs. We conclude that A2AR-induced tolerized DCs, which suppressed NKT cell activation in vivo, provide a unique and potent cell-based strategy to attenuate organ IRI.
Higher antioxidant capacity determines the survival of class I restricted antigen specific CD4 T cells in comparison to CD8 T cells (52.20)
Authors: Kesarwani Pravin , Mehrotra Shikhar
Published: January 2023

CD8+T cells are known to be effector cells, however, recent reports illustrate CD4+T cells as effectors capable of controlling tumor growth. Due to differences in specificity of these two T cell subsets, a comparison in an antigen specific manner has not been possible. Utilizing the TCR transgenic h3T (human TIL derived Tyrosinase TCR, specific for human tyrosinase epitope368-375) mice with class I restricted CD8 and CD4 T cells. We investigated the differences in class I restricted CD4 and C8 T cells in terms of differential signaling and cell death. Our results demonstrate that despite the lower cytolytic capacity class I restricted CD4 T cells were equally capable of controlling human MEL-624 cell in a xenograft model. Vβ12+CD4+ in comparison to Vβ12+CD8+ cells upon antigen specific re-stimulation showed reduced cell death and increased resistance to oxidative stress induced cell death. Our results also demonstrate that Vβ12+CD4+ cells exhibit higher cell surface thiols, higher mitochondrial membrane potential and lower ROS in comparison to Vβ12+CD8+ cells. Further we found that CD4+T cells show higher level of antioxidant enzymes such as SOD1, GPX4, CYBA and antiapoptotic molecule BCL2. In addition CD4+T cells had higher levels of micro RNA-let-7 (down regulates Myc and FasL). These results suggest that CD4+T cells have less lytic capacity they are also less susceptibility to cell death in comparison to CD8+ T cells due to innate differences at oxidative capacity.
CD11b/CD18 ligation on monocyte-derived dendritic cells dampens human Th17 cell responses (52.6)
Authors: Nowatzky Johannes , Khan Shaukat , Manches Olivier , Bhardwaj Nina
Published: January 2023

Manipulating monocyte-derived DC (moDC) to alter effector T cell responses is an important strategy for the development of suppressive immunotherapy. We have previously shown that the ligation of CD11b/CD18 on moDC decreases the secretion of the Th17-skewing inflammatory cytokines IL-1β and IL-6, but maintains “immunosuppressive” TGF-β1. Here, we hypothesized that this “tolerogenic” DC-profile would diminish Th17 responses in peripheral human T cell populations. Healthy donor PBMC were differentiated into moDC in IL-4, GM CSF-supplemented media for 5 days. CD11b/CD18 on moDC was targeted via a bead-based surrogate system developed in our lab. Irrelevant IgG, or avb5, and beads without antibodies were used as controls. DC were stimulated with LPS and MDP or PGN or not stimulated, and then co-cultured with enriched CD4+ human T memory cells. IL-17 secretion was monitored per CBA after 24 hours and/or 4 days, and Th17 frequency assessed by ICS following restimulation with PMA/Ionomycin after 2 weeks. LPS/MDP or PGN-stimulated moDC induced significant increases in co-culture supernatant IL-17 levels and CD4+ Th17 cell frequencies (up to15%). Strikingly, these changes were significantly reduced in CD11b/CD18-ligated DC/T cell conditions, showing reversal of this NOD2/TLR4 or TLR2-mediated expansion effect though ligation of CD11b/CD18 on moDC.
Involvement of Type I Interferon pathway in generating antibody responses elicited by DNA vaccination. (52.7)
Authors: Suschak John , Wang Shixia , Fitzgerald Kate , Lu Shan
Published: January 2023

Recent human study data suggests that DNA immunization can substantially enhance the protective antibody responses elicited by conventional influenza vaccines, but the mechanism of such actions is completely unknown. The current study was designed to explore the roles of innate immunity in regulating acquired immunity elicited by influenza vaccines. Innate pattern recognition receptors (PRRs), and their downstream signaling molecules, were investigated for their effect on DNA vaccination. Of particular interest was the type I interferon (IFN) pathway and its effects on the antigen-specific antibody response. Wild type C57BL/6 mice and selected Interferon Regulatory Factors (IRFs) knockout mice were immunized with DNA vaccines expressing the hemagglutinin antigen (HA) of H1 subtype influenza virus. Mouse serum anti-H1HA antibodies and non-antigen specific cytokine profile analysis showed that deletion of the IRF3 pathway alone did not result in a significantly decreased antibody response but deletion of both the IRF3 and IRF7 pathways led to a dramatic reduction of the anti-HA antibody response. Interestingly, deletion of IL-1 signaling did not significantly affect the anti-HA antibody responses. These results suggested that the IFN pathway does play an important role in shaping the antigen specific antibody response to DNA vaccines and may require the involvement of more than one key regulatory molecule.
Interruption with TNFα/TNFR1 function impairs host immune response against cutaneous vaccinia virus infection (52.8)
Authors: Tian Tian , Dubin Krista , Jin Qiushuang , Qureshi Ali , King Sandra , Liu Luzheng , Jiang Xiaodong , Murphy George , Kupper Thomas , Fuhlbrigge Robert
Published: January 2023

One strategy adopted by vaccinia virus (VV) to evade the host immune system is to encode homologs of TNF receptors (TNFR) that block TNFα function. The response to VV skin infection under conditions of TNFα deficiency, however, has not been reported. We found that TNFR1-/- developed larger skin lesions with higher virus levels after VV scarification. Following their recovery, these TNFR1-/- mice were fully protected against lethal VV intranasal challenge, suggesting their effective memory immune response. A functional systemic immune response of TNFR1-/- mice was further demonstrated by enhanced production of VV-specific IFNγ and CD8+ T cells in spleens and draining lymph nodes as well as efficient T cell skin homing. Interestingly, bone marrow (BM) reconstitution studies using WT BM →TNFR1-/- mice, but not TNFR1-/- BM → WT mice, reproduced the original results seen in TNFR1-/- mice, indicating that TNFR1 deficiency in resident skin cells, rather than hematopoietic cells, accounts for the impaired cutaneous immune response. Similar phenotype was also found in in TNFα-/- mice following VV scarification while both WT BM→ TNFα-/- mice and TNFα-/-BM →WT mice developed significantly larger skin lesions with higher virus counts than the control chimeric mice.Therefore, while the target of TNFα action is restricted to skin stromal components, the source of TNFα produced in response to VV infection would appear to include both hematopoietic cells and radioresistant skin elements.
NKp30-based chimeric antigen receptors promote T-cell effector functions and anti-tumor efficacy (53.11)
Authors: Wu Ming-Ru , Zhang Tong , Sentman Charles
Published: January 2023

NKp30 is a natural cytotoxicity receptor (NCR) that is expressed on natural killer (NK) cells and recognizes B7-H6, which is expressed on several types of tumors but few normal cells. To target effector T cells against B7-H6-positive tumors, we have developed several chimeric antigen receptors (CAR) based on NKp30, which contain the CD28, Dap10, CD27, and/or CD3ζ signaling domains with the transmembrane domains from CD3ζ, CD28, or CD8α. The NKp30 CAR expressing T cells produced IFN-γ and killed B7-H6 ligand-expressing tumor cells. This response was dependent upon ligand expression on target cells but not on MHC expression. The inclusion of other signaling domains enhanced chimeric NKp30 CAR-mediated activity compared to CD3ζ alone. Adoptive transfer of T cells expressing a chimeric NKp30 receptor containing a CD28 signaling domain inhibited the growth of a B7-H6-expressing murine lymphoma (RMA/B7-H6) in vivo. Moreover, mice that remained tumor-free were resistant to a subsequent challenge with the wild-type RMA tumor cells, suggesting the generation of immunity against other tumor antigens. Overall, this study demonstrates the therapeutic potential of adoptive immunotherapy with NKp30 CAR-expressing T cells against B7-H6+ tumor cells.
Comprehensive, longitudinal T cell functional analyses correlated with clinical outcome of a melanoma immunotherapy trial (53.18)
Authors: Ma Chao , Cheung Ann , Chodon Thinle , Comin-Anduix Begonya , Koya Richard , Ribas Antoni , Heath James
Published: January 2023

Adoptive cell transfer (ACT) of transgenic T lymphocytes expressing tumor antigen specific T cell receptor (TCR) can mediate objective responses. However, little is known about the functional activity of the transferred cells and the dynamics of host immunity. We monitored the transgenic and endogenous immunity of 8 metastatic melanoma patients over 8-10 time points in a MART-1 TCR ACT therapy. Using 3 highly multiplex clinical immune diagnostics methods (Ma, C. et al. Nature Medicine 17, 738-743 (2011)), the abundance of 35 melanoma antigen specific cytotoxic T lymphocyte (CTL) populations, 19 functional proteins from 6 types of phenotypically defined single T cells, and 37 blood tumor and immune markers was quantified, yielding millions of readouts. In all patients there was an initial clinical antitumor response followed by disease progression, which correlated with T cell changes observed. The transgenic MART-1 T cells expanded rapidly after infusion, producing multiple cytotoxic and antitumor cytokines. However, their abundance and polyfunctionality diminished within 2-3 weeks. Around 30 days post infusion, a multi-clonal expansion of melanoma specific CTLs emerged only in patients with regressing metastatic lesions. These cells exhibited antitumor functions initially, but gained inhibitory and non-specific functions and diminished in number when tumor progressed. This study points to the need of maintaining T cell functionality to improve ACT therapy effectiveness.
Adoptive immunotherapy using liver natural killer cells for preventing recurrence of hepatocellular carcinoma in cadaveric donor liver transplantation (53.20)
Authors: Ohira Masahiro , Nishida Seigo , Levi David , Tekin Akin , Selvaggi Gennaro , Ruiz Phillip , Ricordi Camillo , Ohdan Hideki , Tzakis Andreas
Published: January 2023

Tumor recurrence is the limitation of liver transplant (LT) in patients with hepatocellular carcinoma (HCC). However, there is no prevention or treatment for HCC recurrence after LT. Here, we describe a clinical-scale method for an adoptive immunotherapy that uses natural killer (NK) cells derived from donor liver graft perfusate to prevent HCC recurrence after LT. Liver mononuclear cells (LMNC) that were extracted from the deceased donor liver graft perfusate contained a large percentage of NK cells (45.0% ± 4.0%) compared with peripheral blood mononuclear cells (PBMC) (21.8% ± 5.2%) from the same donor. Furthermore, interleukin (IL)-2-stimulated NK cells showed greater upregulation of activation markers and the tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), which is critical for NK cell-mediated anti-tumor cell. Moreover, IL-2 stimulation induced LMNC to exhibit a stronger cytotoxicity against HCC compared with PBMC (p < 0.01). After obtaining approval from the FDA and IRB of our institute, we successfully applied this approach to 10 liver cirrhotic patients (9M / 1F) with HCC (Clinicaltrial.gov #NCT01147380). The average number of administered NK cells was 175 × 106 cells/body. After infusion, cytotoxicity, TRAIL, NKp44, and CD226 expression on NK cells significantly increased in patients’ PBMC (p < 0.05). There are no study related adverse events. In conclusion, the administration of IL-2-stimulated cadaveric donor liver NK cells is well tolerated.
Viral infection induces immune responses to many self-proteins, some of which are known tumor associated antigens (53.23)
Authors: Iheagwara Uzoma , Minden Jonathan , Ross Ted , Beatty Pamela , Finn Olivera
Published: January 2023

It has been previously described that viral infection and inflammatory events induced abnormal expression and specific antibodies to tumor associated antigens (TAA). We hypothesize therefore that normal cellular molecules that undergo changes following viral infections generate an immune memory response that participates in tumor immunosurveillance. We are testing this hypothesis by infecting C57BL/6 mice with influenza virus and examining the post infection antibody repertoire for newly generated antibodies that might recognize TAA in Lewis Lung Carcinoma (3LL). We also aim to identify specific molecules most significantly associated with tumor protection. Pre- and post-infection sera were used to immunoprecipitate proteins from the whole cell 3LL tumor lysate. The immunoprecipitated proteins were be subjected to Difference In-Gel Electrophoresis (DiGE) to detect proteins uniquely precipitated by post-infection sera. The selected spots were removed from the gels and analyzed by mass spectrometry. We identified the protein content of 32 new spots precipitated by post-infection serum. Many of these proteins are involved in cytoskeletal and metabolic processes. To start examining their expression on normal, infected or transformed cells, as well as their immunogenicity, we selected the first group of molecules based on previous reports implicating them in anti-tumor immunity. They are: enolase, alpha-actinin-4, 14-3-3 protein sigma, GAPDH, Histone H4, HSP70, and HSP90.
MicroRNA-155 regulates inflammation in alcoholic liver disease via targeting SOCS1 and SHIP1 (54.15)
Authors: Bala Shashi , Petrasek Jan , Csak Timea , Catalano Donna , Kodys Karen , Mundkur Shiv , Szabo Gyongyi
Published: January 2023

Background Fatty liver and inflammation characterize alcoholic liver disease (ALD). Sensitization of liver macrophages (Kupffer cells; KCs) to gut-derived LPS and increased TNFα play a central role in ALD. MicroRNA-155 (miR) is a master regulator of inflammation. In this study we evaluated the role of miR-155 in regulating inflammation after chronic alcohol feeding. Methods C57Bl/6 female mice were fed with 5% alcohol (Liber-DeCarli diet) for 4 weeks and total liver and KCs were isolated. Results MiR-155 positively regulates TNFα and we found increased TNFα mRNA and protein in KCs of alcohol-fed mice. Furthermore increased miR-155 levels correlated with TNFα production in KCs of alcohol-fed mice. To dissect the mechanism by which miR-155 regulates TNFα in ALD, we evaluated the miR-155 target genes SOCS1, SHIP1 and IRAKM that are negative regulators of LPS signaling. We found increased miR-155 and decreased expression of SOCS1, SHIP1 and IRAKM in KCs of alcohol-fed mice. Importantly inhibition of miR-155 by using a specific miR-155 inhibitor, inhibited TNFα production in KCs of alcohol-fed mice, suggesting a role of miR-155 in TNFα regulation. C/EBPβ, another miR-155 target, regulates monocyte maturation and cytokine production. We found increased miR-155 and decreased DNA binding of C/EBPβ in livers of alcohol-fed mice. Conclusion Collectively our results indicate that alcohol induced-miR-155 contributes to inflammation via targeting multiple genes of LPS/TLR4 signaling.
Differential lumbar spinal cord mononuclear cell responses among wild type, CD4 knockout, and CD40 knockout mice in spinal nerve L5 transection-induced neuropathic pain (54.17)
Authors: Cao Ling , Beaulac Holly , Eurich Adriana
Published: January 2023

We have previously demonstrated that both lumbar spinal cord-infiltrating CD4+ T cells and lumbar spinal cord microglial CD40 contribute to the maintenance of mechanical hypersensitivity in the murine model of neuropathic pain spinal nerve L5 transection (L5Tx). To further delineate the CD4 and CD40-mediated mechanisms involved in the development of L5Tx-induced neuropathic pain behaviors, we examined the lumbar spinal cord mononuclear cells of wild type (WT) BALB/c, BALB/c-CD4 knockout (KO), and BALB/c-CD40 KO mice in time course studies via flow cytometry. In WT mice, L5Tx significantly increased the total numbers of lumbar spinal cord mononuclear cells, microglial cells (CD45loCD11b+), and infiltrating leukocytes (CD45hi) in the ipsilateral side of the spinal cord on both days 3 and 7 post-surgery. Meanwhile, in CD4 KO mice, the only significant elevation in microglial cells was observed on day 7 post-L5Tx, and there were no increases in mononuclear cells and infiltrating leukocytes numbers post-L5Tx. On the other hand, in CD40 KO mice, L5Tx did not induce any of the changes observed in WT mice. Altogether, these data indicate that both CD4 and CD40 play a role in L5Tx-induced leukocyte infiltration into the lumbar spinal cord but have differential contributions to spinal cord microglial activation following peripheral nerve injury.
Inflammation Promoting Activity Of HMGB-1 In Type-II Diabetes And As a Marker Of Tissue Injury . (54.3)
Authors: Kannan Krishnaswamy , Ortmann Robert
Published: January 2023

HMGB-1 is a well characterized DNA-binding protein and a potent proinflammatory molecule when released extracellularly during tissue injury. Accelerated atherosclerosis is a major cause of morbidity in diabetes and a causative factor of coronary artery disease, stroke and vascular disease. HMGB-1 is elevated in ischemia-reperfusion injury of the heart and also highly expressed in endothelial cells, vascular smooth muscles, and macrophages present in atherosclerotic lesions. Since HMGB1 is a ligand for advanced glycation end products (RAGE) and is capable of interacting with toll-like receptors, it is likely that a vicious cycle of pro-inflammatory activity occur in patients with diabetes. In this study, the role of HMGB-1 in type II patients (T2D) was examined to better understand its role in chronic inflammation. Plasma samples from T2D patients (n=13) and age matched healthy normal controls (n=20) were analyzed for HMGB-1 and proinflammatory cytokines IL-6 and TNF-α. Our results showed that patients with T2D had significantly elevated levels of TNF-α and IL-6 as compared to healthy controls. Elevated levels of pro-inflammatory cytokines also correlate well with HMGB-1 levels found in patient sera. Collectively, our data suggests a pro-inflammatory activity in patients with T2D. Endogenous danger signals such as HMGB-1 may promote autocrine loop of inflammation and contribute to the persistence of inflammation in type II diabetes and autoimmune diseases.
Adenosine receptor involvement in both the initiation and resolution of inflammation in the central nervous system during experimental autoimmune encephalomyelitis (54.6)
Authors: Mills Jeffrey , Kim DoGeun , Chen Jiang-Fan , Bynoe Margaret
Published: January 2023

Extracellular adenosine is a key regulator of inflammatory immune responses. Utilizing the experimental autoimmune encephalomyelitis (EAE) animal model of multiple sclerosis, we have shown that both the lack of CD73 (catalyzes extracellular adenosine) and antagonist blockade of the A2A adenosine receptor (AR) can protect mice from EAE development. To fully define the importance of A2AAR signaling during EAE progression, we induced disease in A2AAR-/- mice. Surprisingly, A2AAR-/- mice were not only susceptible to EAE, but developed more severe disease associated with an increased number of brain and spinal cord lymphocytes and activated microglial cells. Severe EAE was also found in radiation bone marrow chimeric mice given stem cells from A2AAR-/- donor mice. However, irradiated A2AAR-/- mice given wild type donor cells were protected from disease progression. Our data strongly suggests that A2AAR expression on lymphocytes is important in controlling the magnitude of the inflammatory response during EAE, as A2AAR-/- lymphocytes had a higher proliferative potential and produced more IFNγ following MOG stimulation compared to those from wild type mice. However, A2AAR signaling on non-hematopoietic cells also has an important role in promoting disease progression as A2AAR blockade prevented EAE development in T cell deficient mice given A2AAR-/- donor cells. Overall, our results indicate that A2AAR signaling is involved in both the progression and resolution of EAE.
Dectin-1 controls maturation of fungal phagosomes (55.1)
Authors: Mansour Michael , Tam Jenny , Khan Nida , Puranam Srav , Vyas Jatin
Published: January 2023

Dectin-1 recognizes the fungal wall carbohydrate, β-1,3-D-glucan. Defects in Dectin-1 have been isolated in patients suffering from recurrent Candidal infections demonstrating the essential role of carbohydrate recognition in eliciting protective immunity. In the following work, we sought to characterize the intracellular steps following β-1,3-D-glucan phagocytosis by macrophages. In previous studies, we described a fungal-like particle (FLP) consisting of a polystyrene bead platform covalently conjugated to β-1,3-D-glucan thus mimicking the geometry and surface of fungal pathogens. Using a RAW cell line expressing GFP-Dectin-1, we show that there is translocation of Dectin-1 from the surface to the FLP-containing phagosome which is eliminated by 2h. In order to explore the contribution of Dectin-1-specific intracellular signaling towards the off-rate, a TYR to PHE substitution was introduced within the ITAM-like motif of the cytoplasmic domain of Dectin-1 (ΔY3). GFP-Dectin-1-ΔY3 showed prolonged retention to the phagosome when compared to WT Dectin-1. We next explored subsequent phagolysosomal maturation milestones. Interestingly, phagosomes with intense Dectin-1-ΔY3 did not acidify or recruit LAMP-1 suggesting phagolysosomal maturation blockade. Using Candida albicans, similar deficits in phagosomal maturation were seen with Dectin-1-ΔY3-positive Candida containing phagosomes. These data suggest novel role for Dectin-1 as a controller of fungal phagosome maturation.
Elevated resistin levels in severe pulmonary tuberculosis weaken mycobacterium-mediated inflammasome activation (55.10)
Authors: Chao Wen-Cheng , Yen Chia-Liang , Shieh Chi-Chang
Published: January 2023

Resistin, a protein produced by immune cells in human, causes insulin resistance and impairs the reactive oxygen species (ROS) production in granulocytes. Recent studies suggested that IL-1β production in pulmonary tuberculosis (TB) correlates with inflammasome activation which may be regulated by ROS. We first investigated the serum levels of resistin in pulmonary TB patients with different clinical severity. We found that serum resistin levels were significantly higher in severe TB cases and diabetes-only (DM) group when compared with mild TB cases and healthy controls. Moreover, elevation of serum resistin correlated with impairment of ROS production of granulocytes in DMTB cases. In human macrophages, we further demonstrated that resistin inhibits ROS production and ROS regulated the mycobacterium-mediated inflammasome activation. Finally, using macrophages derived from WT and Ncf1-/- mice, we verified that ROS regulate the mycobacterium mediated inflammasome activation. We concluded that resistin, which suppresses the mycobacterium-mediated inflammasome activation through inhibition of ROS production, is a useful biomarker in severe pulmonary tuberculosis.
Role of inflammasome-dependent cytokine production and neutrophil recruitment during acute lung injury that protects from Burkholderia species infection (55.17)
Authors: Re Fabio , Sahoo Manoranjan , Barrio Laura , Ratsimandesy Rojo , Miller Mark , Ceballos-Olvera Ivonne
Published: January 2023

Burkholderia pseudomallei is a Gram-negative bacterium that infects macrophages and other cell types and causes melioidosis. We have recently shown that the Nod-like receptors (NLR) NLRP3 and NLRC4 differentially regulate pyroptosis and production of IL-1β and IL-18 in response to B. pseudomallei infection. Pyroptosis and IL-18 production were equally important for resistance to B. pseudomallei. Surprisingly, IL-1β was found to be deleterious in melioidosis. The detrimental role of IL-1β during melioidosis was due, in part, to excessive recruitment of neutrophils to the lung. We showed that neutrophils do not express NLRC4, fail to undergo pyroptosis, and, therefore, may be permissive to B. pseudomallei intracellular replication leading to increased bacterial burden and morbidity/mortality. Results will be presented to extended the analysis of the role of the inflammasome components NLRP3, NLRC4, ASC, and Caspase-1 and of IL-1β, IL-18, and pyroptosis during lung infection with other Burkholderia species like B. thailandensis and B. cepacia. Preliminary studies indicate that mice that underwent an acute lung injury (ALI) protocol showed an increased resistance to Burkholderia infection. We will show results that characterize the role of IFNγ and neutrophils recruitment in the protective effect of ALI.