bluuebunny/crossref_metadata_2025 huggingface dataset:
Regulation of expression of SLAMF4 on the surface of mucosal lymphocytes (120.13)
Authors: Liao Gongxian , Song Joo-Hye , O’Keeffe Micahel , Calpe Silvia , Castro Wilson , Magelky Erica , Wang Ninghai , Mora Rodrigo , Reinecker Hans-Christian , Terhorst Cox
Published: January 2023

SLAMF4 is a cell surface receptor, which is primarily known as a regulator of cytotoxicity and cytokine secretion by mouse NK cells upon engagement with its counter-structure CD48. Here, we found SLAMF4 is highly expressed on the surface of most mucosal CD8+ T cells, whereas it is only expressed on some CD44+ CD8+ splenic cells. Upon transfer of naïve SLAMF4− CD8+ T cells derived from Thy1.2+ B6 mice into Thy1.2− B6, donor cells were found to express SLAMF4 after entry into the mucosa. The increase in SLAMF4+ CD8+ cells was due to upregulation of the receptor gene rather than to expansion of a small subset of memory CD8+ T cells. Consistent with the high level of IL-15 in the intestinal mucosa and its requirement for the maintenance of memory CD8+ T cells, we found that recombinant IL-15 induces SLAMF4, particularly the short isoform of SLAMF4, expression on CD8+ T cells. Costimulation with αSLAMF4 significantly increased the αCD3ϵ-induced infiltration of CD8+ T cells in small intestine and exacerbated the inflammation in the small intestine of IL-10-IRES-GFP reporter mouse. As control, αSLAMF4 alone did not cause inflammation in the intestine. In conclusion, the differential expression on mucosal CD8+ T cells as well as the dual nature of SLAMF4 function suggests that SLAMF4 might be involved in regulating the fine balance between the maintenance of tolerance and immune surveillance in the intestinal mucosa.
The role of Platelet Factor 4 in local and remote tissue damage in a mouse model of mesenteric ischemia/reperfusion injury (120.18)
Authors: Ioannou Antonis , Lapchak Peter , Rani Poonam , Lieberman Linda , Yoshiya Kazuhisa , Kannan Lakshmi , Dalle Lucca Jurandir , Kowalska Anna , Tsokos George
Published: January 2023

The robust inflammatory response that takes place during ischemia reperfusion injury (IR/I) recruits factors from both the innate and adaptive immune system. However the contribution of platelets and their products such as Platelet Factor 4 (PF4; CXCL4), during the pathogenesis of IR/I has not been thoroughly investigated. We show that a deficiency in PF4 protects mice from local and remote tissue damage after 30 minutes of mesenteric ischemia and 3 hours of reperfusion in PF4-/- mice compared to control B6 mice. This protection was independent from Ig or complement deposition in the tissues. However, neutrophil and monocyte infiltration was decreased in the lungs of PF4-/- mice compared with B6 control mice. Platelet-depleted B6 mice transfused with platelets from PF4-/- mice displayed reduced tissue damage compared with controls. In contrast, transfusion of B6 platelets into platelet depleted PF4-/- mice reconstituted damage in both intestine and lung tissue. We also show that PF4 may modulate the release of IgA. Interestingly, we show that PF4 expression on intestinal epithelial cells is increased after ischemia/reperfusion at both the mRNA and protein levels. In conclusion, these findings demonstrate that PF4 represents an important mediator of local and remote tissue damage.
Knockout of mitogen-activated protein kinase phosphatase (Mkp)-1 exacerbates colitis development in interleukin-10-deficient mice (120.21)
Authors: Liu Yusen , Matta Ranyia , Barnard John , Wancket Lyn , Yan Jing , Grieves Jessica , Frazier W. Joshua , Nelin Leif , Cato Andrew
Published: January 2023

Il-10-/- mice develop colitis associated with exaggerated Th1/Th17 responses. Mkp-1 is a negative regulator of MAPKs, and its expression is enhanced by IL-10. To understand the role of Mkp-1 in the regulation of mucosal immune responses, we studied the effect of Mkp-1 deletion on the pathogenesis of colitis in Il-10-/- mice. We found that deletion of Mkp-1 on an Il-10-/- background accelerated the development of colitis. Il-10-/- mice exhibited a mild intestinal inflammation in the SPF environment, and rectal prolapse rarely appeared before 6 months. In contrast, majority of the Mkp-1-/-/Il-10-/- mice developed severe colitis rapidly and presented with rectal prolapse after 2-3 months. Colons of Mkp-1-/-/Il-10-/- mice showed diffuse transmural chronic inflammation and mucosal hyperplasia, with significant more proliferating crypt epithelial cells than those of Il-10-/- mice. Moreover, Mkp-1-/-/Il-10-/- mice also developed conjunctivitis and blepharitis. Large intestines of Mkp-1-/-/Il-10-/- mice contained significantly higher levels of pro-inflammatory cytokines, and exhibited greater p38 and ERK activities than did colons of Il-10-/- mice. Splenocytes and lymphocytes from Mkp-1-/-/Il-10-/- mice produced higher levels of Th1 cytokines ex vivo than did cells from Il-10-/- mice upon activation. Our studies support a pivotal role of Mkp-1 as a negative regulator in the mucosal immune responses and highlight its protective function against the development of IBD.
The role of the Rho-associated kinase ROCK in the differential regulation of CXCL8 and CCL2 production by Caco-2 epithelial cells. (120.23)
Authors: Perey Aaron , McGee Dennis
Published: January 2023

Two chemokines found at elevated levels in Inflammatory Bowel Disease (IBD), CXCL8 and CCL2, may play important roles in perpetuating the chronic inflammation that characterizes this condition. Interleukin-1 (IL-1) and Tumor Necrosis Factor-α (TNF-α), which have both been found at elevated levels in IBD, can stimulate the production of CXCL8 and CCL2 by Caco-2 epithelial cells. Previous results from our laboratory have shown that ROCK may be essential for optimal production of CXCL8, as suppression of ROCK activity with Y27632 suppressed IL-1 induced CXCL8 production. We now show that inhibiting ROCK also suppresses TNF-α induced CXCL8 mRNA and protein secretion similar to previously seen with IL-1. Furthermore, inhibiting ROCK suppressed TNF-induced phosphorylation of JNK, but had no effect on IκBα phosphorylation or degradation. Yet inhibition of ROCK actually resulted in significantly elevated levels of CCL2 mRNA with IL-1 and TNF stimulation, and TNF-induced CCL2 secretion was significantly increased while IL-1-induced CCL2 secretion was unchanged. These results suggest that ROCK does play a role in regulating chemokine production in intestinal epithelial cells, but in a very different manner for CXCL8 compared to CCL2. While the purpose of this differential regulation is unclear, it may be an important mechanism for modulating the influx of specific groups of inflammatory cells into affected tissues.
Small afferent nerve fibers control skin inflammation. (120.27)
Authors: Riol Lorena
Published: January 2023

Recent advances in the realm of the skin inflammation have made clear that diseases such as psoriasis or atopic dermatitis cannot be considered as purely immunological because cutaneous peripheral nerve fibers have been shown to play a role in both skin diseases. We have generated some exciting preliminary data in which we observed that local peripheral nerves regulate skin inflammation in animal models of psoriasis and atopic dermatitis (CHS). We have elucidated that in particular, small efferent nerve fibers play an important role in controlling the initiation and determining the magnitude of skin inflammation. Moreover, this effect occurs very promptly even before resident dendritic cells are activated and migrate to the skin-draining lymph nodes. We are currently elucidating the mechanisms by which small nerve fibers interact with the skin associated immune system which may lead to new therapeutic targets to treat skin inflammatory diseases such as psoriasis or atopic dermatitis.
Full-length IL-33 acts differently from mature IL-33 in vivo partially in an ST2-independent fashion (120.30)
Authors: Atamas Sergei , Pickering Edward , Kopach Pavel , Kang Phillip , Lockatell Virginia , Papadimitriou John , McKenzie Andrew , Luzina Irina
Published: January 2023

IL-33 is a key regulator of inflammation and immunity. It remains controversial whether protease-mediated activation of IL-33 is needed for functional effects. We constructed and validated recombinant adenoviruses for gene delivery of full-length mouse (flm) and mature mouse (mm) (aa 109-266) IL-33 to mouse lungs in vivo. Gene expression was confirmed by RT-Q-PCR and ELISA. Analyses of BAL samples and lung tissues revealed substantial differences between flmIL-33 and mmIL-33. Both isoforms caused pulmonary infiltration and BAL influx of T and B lymphocytes and neutrophils, whereas mmIL-33 also caused significant pulmonary eosinophilia (~47-55% of BAL cell count) and hyperplasia of mucus-producing goblet cells. Multiplex analyses showed that mmIL-33 caused significant increases in IL-4, IL-5, IL-13, IL-17, and KC; flmIL-33 tended to stimulate IFN-γ; and both isoforms induced MCP-1 with a greater increase induced by mmIL-33. Subsequent analyses were performed in mice deficient of ST2 (IL-33 receptor). ST2 deficiency completely abrogated mmIL-33-induced pulmonary eosinophilia, goblet cell hyperplasia, and elevations in IL-4 and IL-5. However, lymphocytic infiltration induced by flmIL-33 or mmIL-33 was attenuated but persistent in the absence of ST2. These data suggest that full-length IL-33 is independently functionally active in vivo, in part in an ST2-independent fashion, and that Th2 effects but not lymphocytosis induced by mature IL-33 are ST2-dependent.
Resolvins attenuate cigarette smoke-induced lung inflammation and injury (120.31)
Authors: Phipps Richard , Hsiao HsiMin , Thatcher Thomas , Vickery Thadeus , Serhan Charles
Published: January 2023

Cigarette smoke induced chronic obstructive pulmonary disease (COPD) is a major global health issue with an increasing incidence worldwide. Inflammation plays an integral role in the development of chronic lung diseases such as COPD. Unresolved inflammatory responses in COPD can lead to tissue damage and dysfunction. Current therapies for COPD only treat symptoms and do not alter the course of the disease. Resolution of inflammatory responses was once believed to be passive, however, recent studies have shown that resolution is a biosynthetically active process involving endogenous pro-resolving lipid mediators, derived from omega-3 and omega-6 polyunsaturated fatty acids that control and limit inflammatory responses. Resolvin D1 (RvD1) is an endogenous lipid mediator that displays potent anti-inflammatory and pro-resolving actions without being immunosuppressive. We hypothesized that RvD1 would be beneficial in a smoke-induced lung injury model. Cigarette smoke-exposed mice treated with RvD1 exhibited a greater than 50% reduction in neutrophil accumulation in the lung lavage fluid, coupled with significant reductions in the neutrophil chemoattractants KC and MIP-2. IL-6 and IL-1β were also reduced. RvD1 treated mice also had enhanced phagocytosis of apoptotic neutrophils by macrophages. Interestingly, RvD1 drives polarization of alternatively activated macrophages (M2) that may facilitate resolution of inflammation and attenuate tissue damage.
Activated Adventitial Pulmonary Artery Fibroblasts Drive Macrophages Toward a Distinct M2-like Phenotype through STAT3 Signaling in Pulmonary Hypertension (120.36)
Authors: El Kasmi Karim , Riddle Suzette , Anderson Aimee , Flockton Amanda , Noe Megan , McKeon Alexandre , Frid Maria , Sokol Ronald , Stenmark Kurt
Published: January 2023

We have previously documented that in the adventitia of distal pulmonary arteries (dPA) in calves with pulmonary hypertension (PH) macrophages that express CD163 (marker of alternative activation, AAM) accumulate in proximity to adventitial fibroblasts (adFibs) in areas of remodeling. AAMs have been implicated in promoting remodeling in PH in response to STAT6 signaling. Our recently published data suggest that in the dPA, adFibs express an epigenetically stable pro-inflammatory phenotype capable of activating macrophages. We hypothesized that adFibs activate AAMs through STAT3 but not STAT6. adFibs were isolated and cultured from the dPA of hypertensive (PH-fibs) and control (CO-fibs) calves and used to generate conditioned media (CM). Bovine, murine and rat bone marrow derived macrophages (BMDMs) were incubated in CM for 16hrs followed by qRT-PCR analysis. In response to PH-fibs CM, bovine BMDMs expressed Cd163, Cd206, and IL4ra consistent with AAMs. Furthermore, in response to PH-Fib but not CO-Fib CM both murine and rat BMDMs expressed Cd206, Cd204, Irf4, Klf, jmjd3; STAT3-dependent genes Socs3, Cebp/b, Pim1, Nfatc2, Il4ra, Arg1, but not STAT6-dependent genes Chi3I3 and Retnla1; phospho-STAT3 but not phospho-STAT6; and mRNA for Arg1 (previously thought to be STAT6 dependent) in murine Stat6-/- BMDMs. These results define the activated dPA adFibs as a key locus of paracrine signaling that triggers STAT3 in macrophages, which then express a distinct AAM phenotype.
Modulatory effects of the tumor suppressor p53 in T cell activation and function (121.13)
Authors: Guo Gang , Nguyen Doan , Zhang Shu-Zhong , Huang Yunping , Ramsay Alistair , Ochoa Augusto , Cui Yan
Published: January 2023

Compelling evidence demonstrates that the tumor suppressor p53 plays important roles in regulating physiological functions, such as stem cell state, development, tissue homeostasis, inflammation, metabolism, and aging. Early immunological studies showed that p53 is rapidly activated upon TCR stimulation. Nevertheless, the mechanism that underlies the TCR-mediated p53 activation is unclear. Recently, we reported that p53 negatively regulates Th17 differentiation. Here, we further examined the function of p53 during non-polarizing T cell activation. Upon TCR stimulation (α-CD3+ α-CD28), especially under suboptimal conditions (low level of α-CD3), p53null T cells proliferated more aggressively with a higher effector function than their WT counterparts. To investigate the molecular mechanism by which p53 modulates T helper differentiation, we examined their gene expression via microarray. Among the 45,000 probe sets hybridized, 4025 genes within the Gene Ontology (GO) pathways of immune responses (innate immunity, IL-1β, IL-6, and IL-17 pathways), DNA damage responses, Cell cycle regulation, apoptosis, and survival showed significant differences (p < 0.05) between activated WT and p53null CD4 cells. We are in the process of confirming the differential expression among some of these crucial factors at the protein level and validating their cellular manifestation during T cell activation. Overall, these results support our hypothesis that p53 negatively modulates T cell responses.
The role of TNF receptor superfamily members in thymic Treg development (121.23)
Authors: Mahmud Shawn , Hogquist Kristin , Farrar Michael
Published: January 2023

CD4+FOXP3+ regulatory T cells (Tregs) express several TNF receptor superfamily (TNFRSF) members. However, it is unclear what role these molecules play in Treg development in the thymus. We found that CD4+CD25+FOXP3- Treg progenitors and CD4+CD25+FOXP3+ thymic Tregs, but not CD4SP conventional thymocytes, express high levels of OX40, GITR, and TNFR2. Utilizing a novel Nur77-GFP Bac reporter mouse in which thymocyte GFP level is proportional to TCR signal strength, we observed that OX40 and GITR expression by Tregs positively correlates with Nur77-GFP expression; this suggests that those thymocytes perceiving the strongest TCR signals during development are engendered with the highest level of expression of these TNFRSF members. Importantly, the ligands for these receptors are expressed on APCs in the thymus. To identify the functional role these TNFRSF members play in thymic Treg development, we made mixed bone marrow chimeras with WT and Gitr-/- bone marrow. Gitr-/- donor cells gave rise to normal numbers of CD4+CD25+FOXP3- Treg progenitors but exhibited an ~30% decrease in FOXP3+ mature thymic Tregs relative to WT donor cells. Furthermore, in vitro stimulation of Treg progenitors with GITRL or OX40L leads to a marked enhancement of Treg differentiation. We are presently investigating whether GITR, OX40 and TNFR2 play partially redundant roles in driving thymic Treg development by generating Ox40-/- x Gitr-/- mice in conjunction with neutralizing antibodies to TNFR2.
A Kinase/Phosphatase RNAi screen to identify genes required for PD-1-mediated inhibition of TCR activation (121.3)
Authors: Fonseca Catia , Imam Sabrina , Alexe Gabriela , Piccioni Federica , Root David , Haining William
Published: January 2023

Signaling via PD-1 negatively regulates T cell activation and plays a central role in the state of T cell dysfunction known as exhaustion. However the signaling mechanisms by which PD-1 inhibits TCR activation are not fully understood. Because protein phosphorylation/dephosphorylation regulates TCR signaling, we screened an shRNA library targeting all human kinases and phosphatases to determine which were required for PD-1 inhibition. To model PD-1 inhibition of TCR stimulation we used Jurkat cells engineered to expressed high levels of PD1; ligation of PD-1 by bead-bound antibodies inhibited CD3/CD28-induced IL2 secretion in a dose-dependent fashion. We screened a lentiviral-based arrayed shRNA library of 518 kinases and 150 phosphatases for genes that could rescue PD-1-mediated inhibition of IL2 expression (measured by multiplex qPCR). We identified 25 kinases or phosphatases for which knockdown by two or more shRNAs increased IL2 expression in PD-1-inhibited cells but not in unstimulated cells. Nine of 25 hits were validated in multiple cell lines and secondary assays.Hits included known negative regulators of T cell activation, suggesting our screen recovered expected gene targets. This study uncovered new potential negative regulators of TCR signaling mediated by PD-1 and can be adapted to identify signaling intermediates downstream of other inhibitory receptors.
Molecular signature of human CD8+ T cell with different polyfunctionality in response to different antigen dose (121.8)
Authors: Chiu Yen-Ling , Mathias Oelke , Schneck Jonathan
Published: January 2023

T cell polyfunctionality has been shown to be crucial in protective immunity. However, the molecular mechanism controlling CTL polyfunctionality have not yet been determined. Specifically the genetic control linking antigen doses to polyfunctionality has not been explored. In this study, we found that human antigen specific CTL expansion induced by high dose antigenic stimulation is associated with CTL with lower polyfunctionality and higher inhibitory receptors expression, while low does antigen stimulation results in higher T cell polyfunctionality and lower level of inhibitory receptors. Notably, we found that even in an artificial APC system with only signal 1 and signal 2, antigen dose still directly mediated T cell polyfunctionality independent of any inhibitory receptor signaling. The molecular signature associated with optimal CTL polyfunctionality is identified by genomic microarray and these findings are thus important framework for further studies on how to preferentially modulate T cell polyfunctionality.
Chemokine Receptor CCR7 Does Not Play an Appreciable Role in the Trafficking of Central Memory CD4 T Cells to Lymph Nodes. (122.1)
Authors: Campbell James , Vander Lugt Bryan , Tubo Noah , Boes Marianne , Malissen Bernard , Fuhlbrigge Robert , Kupper Thomas
Published: January 2023

CCR7(-/-) mice exhibit profound anomalies in the architecture of their secondary lymphoid organs, which complicates the study of CCR7-mediated T cell trafficking in these mice. To circumvent this confounding variable, we established in vivo models that force WT and CCR7(-/-) populations to compete for access to various tissues within mice possessing otherwise normal lymphoid organs. We found CCR7(-/-) naïve T cells to be profoundly reduced within lymph nodes with respect to their WT counterparts, a reduction unrelated to effects of CCR7 on thymic development. Strikingly, we found the entry of memory CD4 T cells into lymph nodes from the blood to be entirely autonomous of CCR7, calling into question the function of CCR7 molecules expressed on the surface of these cells. Contrary to previous studies, we found that CCR7 deficiency had no effect on the exit of CD4 T cells from inflamed skin. CCR7(-/-) CD4 T cells were inadequately imprinted for development into skin-selective E-lig(+) cells during primary cutaneous responses, most likely due to the inability of CCR7(-/-) naïve T cells to enter peripheral lymph nodes.
Human Th17 cells have stem cell-like features and promote long-term tumor immunity (122.10)
Authors: Kryczek Ilona , Zhao Ende , Liu Yan , Wang Yin , Vatan Linda , Szeliga Wojciech , Klimczak Aleksandra , Lange Andrzej , Zou Wiping
Published: January 2023

Th17 cells play a role in multiple pathological scenarios. However, their functional phenotype, self-renewal potential, survival pattern and the underlying controlling mechanisms are poorly investigated. Now we have studied Th17 cells in the pathological microenvironments of GVHD, ulcerative colitis and cancer in humans. Th17 cells are increased in the chronic phase of these diseases. Despite their phenotypic markers of terminal differentiation, in vivo adoptive transfusing experiments demonstrate that Th17 cells mediate long-term anti-tumor immunity. Th17 cells have stem cell-like features including high capacity of proliferative self-renewal, potent persistence and apoptotic resistance in vivo, and the generation of other types of T helper cells and express high levels of multiple core stem cell genes and anti-apoptotic genes. We demonstrate that their stem cell-like characters are regulated collaboratively by HIF-1α and Notch. Our data indicate that human Th17 cells may be a long-lived proliferating T cell population with stem cell characters.
Functional significance of Syntaxin 8 in Cytotoxic T Lymphocyte cytotoxicity (122.3)
Authors: Bhat Shruthi , Qu Bin , Kummerow Carsten , Rettig Jens , Schwarz Eva , Hoth Markus
Published: January 2023

Cytotoxic T Lymphocytes (CTLs) are activated CD8+ T cells which identify and kill pathogen infected, tumor and nonself cells by FAS ligand expression and by secretion of perforin and granzymes from lytic granules specifically at the immunological synapse (IS). This prevents killing of healthy bystander cells. Therefore at the time of killing these cytotoxic molecules are to be delivered to the IS. SNAREs are membrane associated proteins involved in vesicle transport, docking and fusion. Mutations of these proteins can cause diseases like Familial Hemophagocytic Lymphohistiocytosis. We have shown previously that Vti1b is required for docking of lytic granules at the IS. We also found that Vti1b and Syntaxin 8 (Stx8) colocalize with perforin in human CTLs. Further experiments showed that Stx8 partially colocalized with Vti1b. To elucidate the role of SNAREs in CTL cytotoxicity, siRNA were used to downregulate their expression. Stx8 downregulation did not impair the IS formation as CD3 accumulation at the IS was unaffected. We have previously shown that the killing capacity of CTLs is impaired by Vti1b downregulation. Here, Stx8 downregulated CTLs used in real time killing assay showed reduction in killing. Cycloheximide treatment of control and Stx8 downregulated CTLs showed that Stx8 is required for killing by perforin secretion. This conclusion is also supported by our preliminary ELISA data, which shows reduced perforin secretion in Stx8 downregulated CTLs.
The effects of age on CD4+ T cell TLR-2 expression and TLR-2 mediated direct co-stimulation (122.6)
Authors: Lancioni Christina , Sinnott Brian , Null Megan , Lewinsohn David , Lewinsohn Deborah
Published: January 2023

Background: Functional Toll like receptors (TLR) are found on human T cells. Neonatal antigen presenting cells (APC) have altered responses to TLR ligands; it is unknown if neonatal CD4+ T cell TLR responses are comparable to adult. Objective: Comparison of TLR-2 expression and responsiveness to TLR-2 direct co-stimulation among total and naïve CD4+ T cell subsets from neonates and adults. Methods: T cell subsets (CD3+CD4+ or CD3+CD4+CD45RA+) were isolated from cord and adult peripheral blood mononuclear cells by flow sorting ( >99.5% purity) and cultured in an APC-free model with α-CD3 ± α -CD28 ± TLR-2 ligand Pam3CYS4. Quantitative PCR and ELISA determined TLR-2 expression and cytokine production. Results: Neonatal CD4+ T cells are predominately naïve (97.2% CD45RA+) but express 5-fold more TLR-2 mRNA than adult CD4+ T cells. Pam3CYS4 provides direct co-stimulation in the presence of α-CD3 to adult and neonatal total CD4+ T cells resulting in IL-2, TNF-α, and IFN-γ production; provision of additional co-stimulation (α-CD28) further enhances cytokine production. When comparing total CD4+ T cell populations, neonatal cells generate significantly less cytokines than adult. When naïve neonatal and adult T cells are compared, neonatal cells generate more cytokines in response to TLR-2 mediated co-stimulation. Conclusions: Naïve neonatal CD4+ T cells have enhanced TLR-2 expression and increased sensitivity to direct TLR-2 co-stimulation as compared to naïve adult cells.
Mechanisms of age-associated T cell defects: With age naïve CD4 T cells become Th1 prone, make increased OPN and acquire epigenetic changes in methylation of Type I and 2 cytokine loci consistent with that profile (122.8)
Authors: Zhang Wenliang , Kuang Yi , Browne Eva , Arcaro Kathleen , Swain Susan
Published: January 2023

As animals age, their T cells develop defects that compromise their immune responses and prevent effective vaccination to new pathogens. Our research has shown that naïve CD4 T cells become progressively more defective with age. When they respond, they produce less IL-2 and expand less. This pattern is intensified in the memory cells to which they give rise, which expand poorly and produce little if any IL-2, and more Th1, but less Th2-polarized cytokines. The molecule mechanisms that cause these shifts are largely unknown. We find that during their 10 response, aged naïve CD4 cells produce more IFNγ and more of the Th1-polarizing cytokine osteopontin (OPN). We find that the IFNγ promoter region of the aged naïve CD4 T cells, has become hypomethylated, while promoters for IL-4 and GATA3 are hypermethylated, suggesting that epigenetic changes that modify chromatin accessibility may be responsible for the altered cytokine production during initial response. We are investigating whether the increased initial levels of IFNγ and OPN are responsible for programming the aged cells to become memory cells with restricted cytokine profiles. Our hypothesis is that signals that accumulate during the increased persistence of naïve T cells that occurs during aging, cause epigenetic changes in cytokine loci that in turn contribute to the development of age-associated functional defects.
The role of ICOS co-stimulation on CD8 T cell differentiation during infection. (122.9)
Authors: Mouriès Juliette , Yagita Hideo , Lefrançois Leo
Published: January 2023

Co-stimulatory molecules give critical signals to CD8 T cells during their priming phase to allow the generation of protective cytotoxic T cells (CTLs). In this study, we explored the role of ICOS on CTL differentiation by blocking ICOSL with an antibody in vivo. Using a bacterial and a viral infection models, we showed that despite normal expansion during ICOSL blockade, the generated CD8 T cells had lower granzyme expression, they preferentially differentiated more into memory precursor effector cells (CD127hi KLRG1low) and their cytokine production (IFN-γ, TNF-α, IL-2) was enhanced. Finally, CTLs generated in the absence of ICOS signaling at initial priming accumulated more at memory stage. Interestingly, the observed phenotype is very similar to Blimp-1 deficiency, a transcription factor involved in terminal differentiation, suggesting a link between ICOS and the Blimp-1/Bcl-6 axis. In fact, after ICOSL blockade, Bcl-6 expression was increased in CTLs. Bcl6 and Blimp-1 are known reciprocal and antagonistic regulators, indicating that Blimp-1 might be down regulated following ICOSL blockade. The molecular basis of this regulation is still under investigation. Finally, using mixed bone marrow chimera with cells from WT and ICOS KO mice, we were able to recapitulate the phenotype observed with ICOSL blockade, indicating that the direct triggering of ICOS on CD8 T cells is responsible for tuning the response.
Lupus-prone (NZBxNZW)F1 mice exhibit decreased ability to induce iTregs due to defects in both antigen presenting cells and T cells (123.1)
Authors: Chhabra Anita , Parnell Sarah , Alard Pascale , Kosiewicz Michele
Published: January 2023

Quantitative and qualitative defects in regulatory T cells (CD4+CD25+Foxp3+; Tregs) are associated with many autoimmune diseases, including systemic lupus erythematosus (SLE). SLE-prone (NZBxNZW)F1 (BWF1) mice have very low frequencies of CD4+Foxp3+ in lymphoid organs compared to non-autoimmune strains, and adoptive transfer of Tregs prevents SLE presumably by restoring the “proper” Treg:effector T cell ratios. In this study, we investigated the mechanisms that could affect Treg frequencies in BWF1 mice. There were no differences in either thymic production or peripheral proliferation of Tregs between BWF1 and control mice. However, in vitro induction of Treg conversion (iTregs) was defective using either BWF1 antigen presenting cells, i.e., TGFβ-treated bone marrow-derived dendritic cells (DC) or untreated CD103+ DC (DC able to induce conversion constitutively), or BWF1 T cells. Furthermore, BWF1 mesenteric lymph nodes contained decreased CD103+ DC concomitant with decreased Helios-CD4+Foxp3+ cells (a potential marker of iTregs) frequencies compared to controls. Taken together, these data suggest that defects in both DC and T cells in BWF1 mice lead to defective conversion of iTregs and may contribute to the decreased Treg frequencies, and consequently, to the development of disease. Identifying the mechanisms that cause these defects could lead to the development of therapeutic strategies that restore an optimal Treg compartment, and thereby treat lupus.
Oncostatin M suppress activation of IL-17/Th17 via SOCS3 regulation in CD4+ T cells of autoimmune arthritis (123.22)
Authors: Cho Mila , Lee Seon-Yeong , Son Hye-Jin , Ryu Jun-Geol , Moon Young Mee , Heo Yang Mee , Kim Ho Youn
Published: January 2023

Objective: We investigated whether the OSM was induced SOCS3 in Treg and Th17 generation in type II collagen induced arthritis (CIA) mice. Method: Isolated T cell of CIA mice was incubated with Th17 or Treg differentiation condition for 30min or 3day. The Th17 and Treg culture condition was incubated with or without OSM 5, 10, 20 and 50 ng/ml. The cell lysated was analyzed using western blot for phospho(p)-STAT3705, p-STAT3727 andp-STAT5 IL-17production was measured in 3day culture supernatant. IL-17 and SOCS3 mRNA levels were analysis by real-time PCR. Result: OSM was increased p-STAT3705 in Th17 and Treg culture condition, but not p-STAT5. However, production of IL-17 and IL-17 mRNA was decreased in Th17 and Treg culture condition. Inaddition, the OSM was increased SOCS3 mRNA in Th17 and Treg condition. Next we investigated expression of OSM and SOCS3 mRNA levels in Treg cell. P-STAT5, OSM and SOCS3 were activated inTreg, while p-STAT3 was decreased. Although OSM was induced activation of STAT3, that was suppressed differentiation from Treg to Th17. These result ssuggest that OSM was regulated expression of SOCS3, thereby tightly regulated plastically differentiation of Treg and Th17. Conclusion: SOCS3 induced by OSM is an acting negative feedback loop and since OSM-mediated signaling is suppression IL-17 production in mouse CD4 T cells, which is provide for feedback inhibition of inflammatory response in the CIA.
Insulin B:9-23 peptide-reactive γδ T cells in the non-obese diabetic (NOD) mouse recognize an oxidized dimeric form of the peptide (123.23)
Authors: Aydintug Mustafa , Zhang Li , Michels Aaron , Day Brian , Eisenbarth George , O'Brien Rebecca , Born Willi
Published: January 2023

The insulin peptide ins2 B:9-23 is recognized by diabetogenic αβ T cells in NOD mice, and by γδ T cells. Unlike the αβ T cell response to the peptide, the γδ T cell response is independent of APCs. Furthermore, the cysteine residue in position 19 of the peptide is required for the γδ but not the αβ response. Since cysteine residues can dimerize upon oxidation, we hypothesized that γδ T cells recognize oxidized dimeric peptide molecules. We therefore compared the stimulatory activities of monomeric, copper chloride-oxidized (mostly dimeric) and DMSO-oxidized, HPLC-purified dimeric insulin peptide B:9-23 preparations. We found that oxidized and purified dimeric peptide preparations stimulated γδ T cells far better than untreated or purified monomeric preparations, which still contained some oxidized peptides. In contrast, αβ T cells responded only to monomeric peptides except when live APCs were present. The γδ T cell response to the dimeric insulin peptide was TCR dependent because responsiveness could be transferred with transduced TCR-γ/δ genes derived from the originally described B:9-23-reactive γδ T cell hybridoma SP9D11. These findings provide an explanation for the differential response requirements of insulin peptide-reactive αβ and γδ T cells, and they raise the possibility that B:9-23-reactive γδ T cells function as sensors of oxidative stress.
The Role of Th17 cells in Type 1 Diabetes. (123.30)
Authors: Li Cheng-Rui , Bradley Linda
Published: January 2023

Type 1 diabetes (T1D) has been considered to be elicited primarily by Th1 cells. However, the role of Th17 cells, which have been implicated in other autoimmune diseases, is controversial. Using the NOD model, we found that CD4 T cells infiltrate the pancreas by 2 weeks of age, a significant proportion of which expressed either IFNγ or IL-17, suggesting that islet-specific Th1 and Th17 both develop very early. Although serum IL-17 levels increased at diabetes onset, Th17 cells from spontaneously diabetic mice failed to transfer disease to NOD.Scid recipients whereas Th17-depleted CD4 cells elicited diabetes as efficiently as non-depleted cells. To further address the pathogenic potential of Th17 cells, we differentiated polyclonal or BDC2.5 Th17 cells in vitro and transferred them into NOD or NOD.Scid recipients. Polyclonal Th17 cells failed to induce diabetes, and the recovered donor cells maintained their phenotype, suggesting that Th17 cells can be stable. In contrast, BDC2.5 Th17 cells readily induced diabetes in both recipients, although a majority of the donor cells became IFNγ-producers. However, neither the acquisition of IFNγ production nor IFNγ itself were required for diabetes induction. Instead, TNFα was essential for their pathogenicity. Our results raise the possibility that Th17 cells do not make a critical contribution to spontaneous T1D unless re-differentiation that is marked by IFNγ production occurs as a consequence of local inflammation in the pancreas.
PKC-θ shifts balance between inflammatory and regulatory T cells (123.42)
Authors: Ma Jian , Wang Ruiqing , Fang Xianfeng , Sun Zuoming
Published: January 2023

We demonstrate here that PKC-θ is a critical molecule to maintain immunological balance by shifting the ratio of inflammatory Th17 and inhibitory Treg cells. TGFβ-induced T regulatory cell (iTreg) differentiation was enhanced in naive PKC-θ -/- T cells or WT cells treated with a specific PKC-θ inhibitor. PKC-θ -/- T cells had reduced activity of the AKT kinase, and the expression of a constitutively active form of AKT in PKC-θ -/- T cells restored ability to inhibit iTreg differentiation. Furthermore, knockdown or overexpression of the AKT downstream targets FoxO1 and FoxO3a was found to inhibit or promote iTreg differentiation in PKC-θ -/- T cells, indicating that the AKT-FoxO1/3A pathway plays an inhibitory role in the differentiation of iTreg downstream of PKC-θ. In the other hand, we show that naive PKC-θ -/- T cells were defective in Th17 differentiation in vitro, whereas in vitro differentiation of Th1 and Th2 appeared normal. PKC-θ -/- T cells had notably lower levels of Stat3, a transcription factor required for Th17 differentiation. PKC-θ-mediated activation of the Stat3 promoter was inhibited by dominant negative AP-1 and IκB kinase β mutations, but stimulated by WT AP-1 and IκB kinase β, suggesting that PKC-θ stimulates Stat3 transcription via the AP-1 and NF-κB pathways. We conclude that PKC-θ is able to control T cell-mediated immune responses by shifting the balance between the differentiation of Th17 inflammatory and regulatory T cells.
Innate and adaptive tolerance to apoptotic cells is controlled by an IDO- dependent mechanism. (123.49)
Authors: Ravishankar Buvana , Liu Haiyun , Shinde Rahul , Chandler Phillip , McGaha Tracy
Published: January 2023

Marginal zone macrophages (MZMs) play a crucial role in generation of systemic tolerance to apoptotic cell (AC)-associated antigens. However, the mechanism(s) by which MZMs drive tolerogenic responses is not understood. In this report, we show that systemic administration of ACs induces splenic expression of the immunosuppressive enzyme indoleamine 2-3 dioxygenase (IDO) in Marco+SignR1+ MZMs. Moreover, we found abrogation of IDO activity altered immunity to ACs resulting in decreased TGF-β synthesis and increased pro-inflammatory cytokine production associated with a reduction in Treg function and a loss of T cell tolerance towards AC antigens. When pre-symptomatic, lupus-prone MRLlpr/lpr mice were examined we found significant IDO expression in the splenic MZ analogous to the observations in AC challenged mice. When IDO was inhibited, autoimmunity was rapidly accelerated in MRLlpr/lpr mice with a >10-fold increase in anti-dsDNA IgG titers and increased renal pathology. Finally, IDO-/- mice, which do not show signs of autoimmunity, were challenged chronically with ACs. The mice developed high titer serum IgG autoreactivity, kidney inflammation, and increased mortality compared to B6 mice, which did not develop autoimmunity. Thus, the data demonstrate that a novel IDO-dependent regulatory circuit is induced in MZMs upon apoptotic cell phagocytosis providing clues to future therapeutic targets to re-establish tolerance in systemic autoimmune disease.
Green tea epigallocatechin-3-gallate modulates differentiation of naive CD4+ T cells into specific lineage effector cells (123.6)
Authors: Wang Junpeng , Pae Munkyong , Meydani Simin , Wu Dayong
Published: January 2023

CD4+ T cells are implicated in pathogenesis of autoimmune disease: Th1, Th9, and Th17 cells are shown to induce autoimmunity whereas regulatory T cells (Treg) may be protective. We previously showed that green tea catechin epigallocatechin-3-gallate (EGCG) attenuated experimental autoimmune encephalomyelitis (EAE) through altering CD4+ T cell subset balance, but the mechanisms are yet to be elucidated. We thereby determined how EGCG impacts differentiation of naïve CD4+ T cells into different effector lineages. Results showed that EGCG impeded Th1, Th9, and Th17 but slightly promoted Treg differentiation. In the presence of TGF-β, IL-6 promoted Th17 and suppressed Treg development; this effect of IL-6 was diminished by EGCG. Further, EGCG inhibited T-bet, PU.1, and RORγt, the transcription factor for Th1, Th9, and Th17 differentiation, respectively. EGCG also induced down-regulation of p-STAT1 & p-STAT4, and p-STAT3, the key transducers for IFN-γ-induced T-bet activation and IL-12-amplfied Th1 response, and for IL-6-induced RORγt activation, respectively. In vitro EGCG inhibited soluble IL-6Rα and membrane gp130, and plasma soluble gp130 was increased in EAE mice fed EGCG, which may contribute to reduced generation of Th17. Taken together, our results suggest that EGCG modulates development of CD4+ T cell lineages through impacting their respective regulatory networks which are also interactive, as such affording a protective effect in T cell-mediated autoimmune diseases.
Application of Brilliant Violet™ fluorescent polymer-labelled antibody reagents in 15-color flow cytometric immunophenotyping (124.12)
Authors: Lundsten Kelly , O'Connor Carolyn , Wang Elena , Li Sky , Ilic Alex , Lay Gene
Published: January 2023

Monitoring populations of immune cell subsets, such as B lymphocytes, T lymphocytes, NK cells, NKT cells, Treg cells, monocytes and dendritic cells, is important in many different infectious diseases, including HIV/AIDS, to assess response and recovery to treatment and therapy. In the last decade, multicolor flow cytometry has greatly increased the capabilities and consistency of immunophenotyping and immunomonitoring. However, limitations still preclude a wider adaption of >10 color flow cytometry, largely due to the limited availability of fluorophores with flexible utility in a complex panel. Although instruments with 18-20 parameters are now increasingly common, until recently, the available reagents had significant practical limitations, such as limited brightness and permeability, and undesirable cross-beam excitation, resulting in reduced sensitivity when detecting lowly abundant antigens in these assaulted channels. The Brilliant Violet™ family of fluorescent probes, released in 2011, overcomes these limitations, with significantly increased overall brightness and signal-to-noise. Here, we present a 15 color/ 17 parameter flow cytometry assay, introducing Brilliant Violet™ 605 and Brilliant Violet 650™ in our expanding family of fluorescent polymers. This family of fluorescent probes will be instrumental in making a wide array of commercially available conjugates with a broad range of brightness and no limitations for intracellular detection.
A flow cytometer with a standardized fluorescence detection system simplifies multi-color panel design. (124.13)
Authors: Roys Stacey , Rogers Clare , Dinkelmann Maria
Published: January 2023

Multi-color panel design can be difficult due to the many factors which impact signal detection including the fluorescence detection capability of the cytometer, target molecule density and the spectral characteristics of available fluorophores (fluors). We demonstrate that a flow cytometer with a fixed and standardized fluorescence measurement system simplifies design of multi-color flow experiments, using a common conundrum researchers face as illustration: several different fluor conjugates detectable by the cytometer are available for a given clone. Which is best to use? First we show that by locking down optical alignment and optimizing fluorescence detection at the time of manufacture the BD Accuri C6 gives reproducible, predictable fluorescence measurements both for individual instruments over time and between instruments. We also show that this design leads to predictable fluorescence spillover for stable fluors. Since fluorescence spillover due to bright fluors and/or highly expressed epitopes can mask dimmer signals in neighboring channels, successfully predicting spillover greatly simplifies multi-color panel design. We illustrate this by comparing the spillover of four fluors optimally detected in FL3 of the BD Accuri™ C6 (PerCP, PerCP-Cy™5.5, PE-Cy7, BD Horizon™ PE-CF594), when conjugated to antibody clones of a high (CD45) and low (CD19) epitope-number antigen. We also quantitate the impact of the spillover on detection of weak signals in neighboring channels.
A Novel Approach to Improve Immunological Animal Study Conduct Efficiency, Data Integration, Consistency and Security and the Sharing of Results (124.16)
Authors: Kumer Jeff , Ibsen Eric
Published: January 2023

The traditional management of immunological animal studies and results is challenging due to complex, varied workflows as well as the acquisition, analysis, secure storage and accessibility of disparate data types acquired and stored in multiple formats and locations. Existing approaches often poorly reflect the details of planned-versus-actual study conduct, animal welfare considerations, and integration of data types in various paper and electronic formats. Errors and non-standardized condition scoring, data acquisition, processing, analyses are common problems as is impeded access to current and historical results due to compartmentalized data silo storage. The result is a highly inefficient use of financial, labor and animal resources and the unintentional loss of historical results over time. In-house-developed and commercially-available desktop and web-based applications have been designed specifically to manage complex and variable study designs, comprehensive task workflow and information management. For the first time, disparate data types from immunology studies can be acquired consistently, linked, analyzed, graphed and securely shared with stakeholders in real time and in perpetuity saving precious research resources and minimizing historical information loss. Several non-commercial and commercially-available solutions will be described and their respective impacts on immunology study workflow efficiency and data quality, integration, security and accessibility.
A specialized tube to make enrichment of specific cell subsets faster and easier (124.3)
Authors: Fairhurst Maureen , Fadum Jodie , Woodside Steve , McQueen Karina , Thomas Terry , Peters Carrie
Published: January 2023

Many experimental protocols require the enrichment of specific cell subsets from peripheral blood. RosetteSep™ cell enrichment and standard mononuclear cell (MNC) preparation both involve density gradient centrifugation, which entails slowly layering the sample over the density gradient medium to avoid mixing, and carefully pipetting to remove the enriched cells after centrifugation. Centrifugation must be performed with the brake off to avoid disturbing the enriched cell layer, further lengthening the process. SepMate™, a centrifugation tube with a specialized insert, was developed to allow rapid layering of the sample onto the density gradient medium, and pouring off of the enriched cells after centrifugation, thus simplifying the entire process. When using SepMate™, the cocktail incubation time and centrifugation time could each be shortened to 10 min, making RosetteSep™ cell enrichment even faster. RosetteSep™ enrichments of mononuclear cell subsets using the SepMate™ tubes and protocol gave equivalent purity and recovery of desired cells compared to using the standard RosetteSep™ protocol, and desired cells could be enriched from whole blood in <30 min. Purities of specific cell types were: CD3 T Cells 96 ± 1 (n=5), CD4 T Cells 94 ± 5 (n=3), CD8 T Cells 85 ± 11 (n=4), B Cells 92 ± 6 (n=3), NK Cells 85 ± 5 (n=5), monocytes 68 ± 8 (n = 4). The protocol is easily scalable to process multiple samples simultaneously, and the SepMate™ tube can also be used to prepare MNCs.