bluuebunny/crossref_metadata_2025 huggingface dataset:
Gut commensal flora influence the sterile inflammatory response to ventilated lung ischemia reperfusion injury by altering the activation state of alveolar macrophages and recruitment of differentially-activated neutrophils (P4029)
Authors: Prakash Arun , Sundar Shirin , Hellman Judith
Published: January 2023

Background: Ischemia reperfusion (IR) injury is a sterile inflammatory process occurring in diverse and potentially life-threatening clinical scenarios. Acute lung injury is a major cause of morbidity and mortality in severely-ill trauma patients in part due to IR injury. We previously demonstrated a role for TLR4 and alveolar macrophages in lung IR injury and hypothesize that the gut microbiome contributes to this process. Methods: A surgical model of left pulmonary artery occlusion in mice was used and the nature of the inflammatory response examined. Mice were also pretreated with antibiotics to deplete gut commensal flora in order to investigate their influence on the inflammatory response to lung IR. Results: We demonstrate that the activation state of neutrophils recruited in our lung IR injury model is altered as compared to LPS models. Interestingly, the depletion of gut commensal flora results in a significant reduction in inflammatory cell recruitment to the lung following IR. Alveolar macrophages from these antibiotic-treated mice produce lower levels of inflammatory cytokines when stimulated ex vivo with TLR agonists. This suggests that circulating endotoxin from commensal flora maintain alveolar macrophages in a primed state to respond to lung IR. However, the neutrophils recruited appear to be quiescent. This may represent an evolutionary mechanism to mobilize neutrophils to sites of lung IR, where they are ready to respond only if pathogen is present.
Pancreatic β-cells undergo apoptosis in a TLR4 and macrophage cell-dependent manner (P4031)
Authors: Rosendahl Alexander , Cucak Helena , Mayer Chistopher , Tonnensen Morten
Published: January 2023

To elucidate the expression and effect of TLR4 on pancreatic β-cells, we studied mouse islets and the murine β-cell line MIN6 in the presence of macrophages (MΦ). Diabetic islets had 40% less TLR4+ β-cells, while twice as many TLR4+ MΦ compared to healthy islets. TLR4 activation with LPS induced a 5-10 fold induction of cytokines in healthy and diabetic islets. MIN6 cells were only weakly TLR4+ and did not produce cytokines in response to LPS. LPS stimulated diabetic systemic MΦ released 3-10 fold more cytokines per cell compared to healthy MΦ. Interestingly, diabetic MΦ co-cultured with MIN6 cells secreted higher levels of cytokines compared to MΦ alone. We also measured apoptosis in healthy and diabetic islets treated with LPS and found no effect. However, a 3-fold induction of MIN6 apoptosis was obtained when co-cultured with diabetic MΦ and 2-fold when cultured with media from LPS activated MΦ. Taken together, our data suggest that the majority of the TLR4 response in the islet appears to be mediated through MΦ. Moreover a cell-dependent MΦ- β-cell interaction appears to contribute to the enhanced local immunity in the islet. Thus targeting either TLR4 or macrophages may provide a novel treatment regime for type 2 diabetes.
IL-12 regulates psoriatic plaque formation by inhibiting γδ T cells (P4033)
Authors: Kulig Paulina , Haak Stefan , Pantelyushin Stanislav , Gyülveszi Gabor , Sallusto Federica , Becher Burkhard
Published: January 2023

Cytokines are key mediators of immune responses and IL-23 is a critical player in psoriasis pathogenesis. New effective monoclonal antibody directed against common subunit p40 of both IL-12 and IL-23 is currently used in clinics to treat plaque type psoriasis. Recently was shown that Aldara cream, a drug commonly used in the treatment of benign skin disorders, is a potent stimulator of psoriasis-like plaque formation and implicates proinflammatory cytokines like IL-17A, IL-17F and IL-22 in the disease pathogenicity. Using this model we found that mice deficient in p40 as well as mice treated with neutralizing anti-p40 mAbs have a substantially reduced skin inflammation upon Aldara treatment. As anti-p40 Abs target both IL-12 and IL-23, we hypothesed, that IL-12 might have a protective role in psoriatic plaque formation. We found that skin inflammation was highly increased in mice lacking IL-12 (IL-12p35-/-) or when IL-12 signalling (IL-12Rb2-/-) was disrupted. Local injection of IL-12Fc significantly reduced skin inflammation that was corroborated by reduced leucocytes infiltrations as well diminished IL-17A expression in the skin. IL-12 stimulated CD27- γδ T cells secrete lower levels of IL-17A and F compared to their unstimulated controls suggesting direct effect of IL-12 on γδ T cells. Taken together, our findings suggest a protective role of IL-12 in psoriatic inflammation and support the need of introducing more specific treatment targeting the IL-23/IL-17 axis.
TLR2 initiates intestinal ischemia/reperfusion induced tissue damage (P4003)
Authors: Fleming Sherry , Pope Michael
Published: January 2023

Reperfusion (the return of blood flow) significantly magnifies ischemia (lack of blood flow) induced tissue damage in multiple clinical conditions including trauma and hemorrhage. During reperfusion, naturally occurring antibodies recognize endogenous damage-associated molecular patterns (DAMP), followed by an excessive complement activation, neutrophil infiltration and inflammatory response which frequently results in an excessive systemic inflammatory response which may lead to death. TLRs recognize DAMP and TLR4 is required for intestinal ischemia-reperfusion (IR). As TLR2 has been proposed to be the receptor for at least one DAMP, the serum protein, β2-glycoprotein I (β2-GPI) which is critical in intestinal ischemia reperfusion (IR), we hypothesized that IR-induced intestinal damage requires TLR2 for subsequent inflammation and tissue damage. Using TLR2-/- mice, we demonstrate that TLR2 is required for excessive IR-induced complement activation and pro-inflammatory cytokine production. TLR2-/- mice not only lacked the appropriate antibody repertoire to induce intestinal IR but treatment with wildtype antibodies was not sufficient to induce tissue damage or inflammation. Compared to wildtype mice, TLR2-/- mice have increased serum β2-GPI but β2-GPI is not deposited on ischemic intestinal tissue. Our data suggest that TLR2 interacts with β2-GPI and is required for neo-antigen expression and initiation of the systemic inflammatory response during intestinal IR.
Protective role of anti-viral human defensins during acute HIV infection (P4049)
Authors: Corleis Bjorn , Gostic William , Kwon Douglas
Published: January 2023

Background Human defensins are small cationic peptides with anti-bacterial and anti-viral activity. Beta defensins are expressed by epithelial cells and innate immune cells in tissue and circulating leukocytes, respectively. They are part of the first line of innate immune defense against invading pathogens at mucosal sites. Methods We investigated anti-HIV activity and production of beta defensins in intestinal mucosal biopsies and PBMCs from individuals with chronic untreated HIV, immunologically controlled HIV, acute HIV and HIV uninfected individuals using ELISA, immunohistochemistry, quantitative PCR and viral inhibition assays. Results Human beta defensin expression in PBMCs was found exclusively in classical monocytes whereas epithelial cells were found to be the main source in the intestinal tract. Expression of human beta defensins was not altered in gut tissue or PBMCs from HIV progressors and controller compared to HIV negative control sample. However, we observed increased expression in PBMCs from HIV acute patient samples. Further, recombinant human beta defensins showed anti-HIV activity in vitro. Conclusions Our data suggest that beta defensins have a direct effect on HIV and might act as an intrinsic antiviral factor early during HIV infection. Specific delivery of recombinant defensins might be a powerful tool to reduce viral burden during acute infection.
Severe pulmonary damages and high mortality rates shown in mice with combined burn and radiation injuries (P4005)
Authors: Nakamura Kiwamu , Cox Robert , Cornforth Michael , Herndon David , Kobayashi Makiko , Suzuki Fujio
Published: January 2023

Non-clinical human radiation exposure events are often coupled with burns, and victims with combined injuries are extremely susceptible to sepsis stemming from various local infections, as compared with that of patients either exposed to burns or radiation alone. In patients with combined injuries, infectious complications result in a high mortality rate, and Pseudomonas aeruginosa is a pathogen frequently isolated from these patients. In this study, the severities of burn wound infections with P. aeruginosa were studied in mice with combined radiation and burn injuries (CI-mice). All of the CI-mice infected with 10 CFU/mouse of P. aeruginosa died within 2 days of the infection, while mice with either injury alone survived more than 4 weeks after the same infection. Five CFU/mouse of P. aeruginosa was shown to be 1 LD50 in CI-mice, while 3 - 9 x 103 CFU/mouse of the pathogen were required to kill all of the mice with either injury alone. Severe pulmonary inflammation and decreased alveolar spaces were specifically observed in CI-mice. Lineage−CD45+CD34+CD44+CD49b+ cells appeared in association with combined injuries inhibited bactericidal activities of alveolar macrophages isolated from normal mice. Lineage−CD45+CD34+CD44+CD49b+ cells may play a role on the high susceptibility of CI-mice to P. aeruginosa burn wound infection.
IL17A receptor (IL17RA) signaling is important for protection against oral infection with Listeria monocytogenes (P4011)
Authors: Alam Mohammad , Kuo Jennifer , Babu Uma , Ganies Dennis , Pereira Marion , Williams Kristina
Published: January 2023

Foodborne Listeria monocytogenes (LM) causes serious illness & death every year in the US. Ingestion of LM can lead to septicemia, meningitis, & abortion. IL17 is secreted by T cells and regulates the production of CXC chemokines for monocyte and neutrophil recruitment to infected tissue. Foodborne LM infection occurs through the oral route, but mice are highly resistant to oral infection. Here, using a murinized LM strain, we explored the role of IL17RA signaling following oral LM infection. Mortality & body weight loss were compared after LM infection of C57BL/6 & IL17RA-KO mice. LM-specific proliferative responses of splenocytes isolated from infected mice were performed. Viable LM from liver & spleen tissue were enumerated. Neutrophil infiltration & inflammation were evaluated histologically. RT-PCR arrays were used to quantitate cytokine/chemokine mRNA expression in infected tissues. IL17RA-KO mice lost more body weight and died earlier following oral infection than infected WT mice. LM-Ag treated splenocytes from KO mice proliferated less vigorously than WT splenocytes. Tissues from infected IL17RA-KO mice had lower mRNA expression for IFN-γ, IL1-β & iNOS. Liver, spleen & gastric tissues from infected IL17RAKO mice showed increased necrotic tissue damage & increased LM burden. These results indicate that IL17RA signaling is important in host resistance to oral LM infection.
Forcing NF-κB in T cells promotes tumor rejection (P2042)
Authors: Evaristo Cesar , Molinero Luciana , Gajewski Thomas , Alegre Maria-Luisa
Published: January 2023

T cells from tumor-bearing hosts have reduced NF-κB activity. Our previous results indicate that reduced NF-κB activation results in impaired T cell survival, decreased Th1 differentiation and increased iTreg differentiation. We hypothesized that forced activation of NF-κB in T cells should have the opposite effect, which would be beneficial for tumor rejection. We generated mice with constitutively active IKKβ in T cells (CA-IKKβ mice). T cell numbers were comparable to littermate controls, but CA-IKKβ mice had fewer Tregs and increased frequency of activated T cells that produced IFNγ and IL-4 upon re-stimulation. When B16-SIY melanoma cells were injected subcutaneously, tumors grew progressively in control littermates, but were rejected by CA-IKKβ mice. Tumor control was associated with a massive increase in the number of tumor-specific IFNγ-producing CD8+ T cells and IFNγ blockade restored tumor growth. CA-IKKβ+ CD8+ T cells were able to control tumor growth in the absence of CD4+ T cells. Interestingly, CA-IKKβ+ CD4+ T cell help was sufficient to induce tumor control by WT CD8+ T cells. Finally, enhanced tumor control was observed in immune-competent mice when fewer than 5% of T cells expressed CA-IKKβ. Our results suggest that NF-κB is at the cross-roads of major T cell fate decisions that uniquely synergize for control of tumor growth and identify a therapeutic pathway with potential clinical applicability.
Polyanhydride nanoparticle vaccine platform delays tumor growth (P2050)
Authors: Darling Ross , Huntimer Lucas , Ross Kathleen , Ramer-Tait Amanda , Narasimhan Balaji , Wannemuehler Michael
Published: January 2023

Immunotherapies designed to direct the immune system to elimate malignancies is a growing area of research aimed at providing a safe and effective alternative and/or adjunct therapy to chemotherapeutic and surgical interventions. These approaches require the development of immunization regimens that induce efficacious cell-mediated immunity directed at antigens expressed by tumor cells. Particles consisting of polyanhydride copolymers based on 1,6-bis(p-carboxyphenoxy) hexane (CPH), and 1,8-bis(p-carboxyphenoxy)-3,6-dioxaoctante (CPTEG) have been shown to enhance dendritic cell activation, which in turn induces activation and proliferation of antigen-specific CD4+ and CD8+ T cells in vitro. We demonstrate that immunization of mice with a polyanhydride nanoparticle-based vaccine platform induced a potent immune response to a specific tumor-associated model antigen in order to control tumor progression in vivo. Specifically, we challenge immunized mice with a transgenic tumor cell line, expressing ovalbumin (Ova) as a target antigen, to determine if our vaccination strategy is effective. Mice immunized once with a regimen consisting of soluble Ova and Ova encapsulated in 20:80 CPTEG:CPH nanoparticles exhibited delayed tumor growth during the course of our study when compared to mice immunized with soluble Ova adjuvanted with Alum, soluble Ova alone, or PBS. Mice that receive the nanoparticle formulation also have improved survival rates when compared other formulations.
Acute TLR5 stimulation with a flagellin-derived agonist induces cytotoxic lymphocyte-mediated tumor immunity (P2064)
Authors: Leigh Nicholas , Bian Guanglin , Ding Xilai , Liu Hong , Aygun-Sunar Semra , Burdelya Lyudmila , Gudkov Andrei , Cao Xuefang
Published: January 2023

Toll-like receptor (TLR) mediated recognition of pathogen associated molecular patterns allows the immune system to rapidly respond to a pathogenic insult. This feature makes TLR agonists attractive agents that can stimulate a favorable immune response. CBLB502 is a pharmacologically optimized TLR5 agonist derived from Salmonella enterica flagellin. In this study, we examined the effect of CBLB502 on tumor immunity using two lymphoma models, both of which do not express TLR5, and thus do not directly respond to CBLB502. Upon challenge with the T cell lymphoma RMAS, CBLB502 treatment after tumor inoculation protects approximately 50% of C57BL/6 mice from death caused by tumor growth. This protective effect is both natural killer (NK) cell and perforin dependent. In addition, CBLB502 stimulates clearance of the B cell lymphoma A20 in BALB/c mice in a CD8+ T cell dependent fashion. ImageStream flow cytometry analyses reveal that CD11b+ and CD11c+ cells, but neither NK nor T cells, directly respond to CBLB502 as determined by nuclear translocation of NFκB. NFκB can regulate a variety of pro-inflammatory cytokines; accordingly, CBLB502 increases serum levels of IL-6, IL-15, TNFα, and IFN-γ. Furthermore, CBLB502 promotes antigen-presenting cell maturation as evidenced by CD86 upregulation. These findings demonstrate that CBLB502 can stimulate a robust antitumor immune response by indirectly activating cytotoxic lymphocytes through TLR5-expressing accessory immune cells.
Improving the identification of beneficial mimotopes using a representative tumor-specific T cell receptor (P2066)
Authors: Buhrman Jonathan , Jordan Kimberly , Slansky Jill
Published: January 2023

Vaccination with peptide mimotopes is a promising strategy to improve antitumor immunity. Mimotopes function by eliciting increased numbers of T cells that cross-react with the native tumor antigen. Unfortunately, mimotope vaccines often elicit T cells that do not cross-react or have low affinity for the tumor Ag. Therefore, determining which mimotopes are most effective upon vaccination is a challenge. Using a high affinity tumor-specific T cell clone, we identified a panel of mimotopes for the dominant Ag (AH1) of a mouse colon tumor cell-line (CT26) that elicits a range of tumor protection when used in vaccines. Ex vivo evaluation of the TCRs of T cells initiated by mimotopes that protect against tumor challenge revealed increased representation of a Vβ8.3/Jβ2.6 CDR3 motif. The original high affinity clone was not representative of the tumor-specific TCRs identified. We identified a ‘representative’ clone that made up nearly 8.5% of sequences following immunization with mimotopes. We tested the hypothesis that using a representative TCR to screen peptide libraries would improve the frequency of discovery of efficacious mimotopes. We demonstrated that the representative T cell bound to more protective mimotopes than the high affinity clone by screening genetically encoded peptide libraries and comparing mimotope vaccines identified by each TCR. These results have important implications when determining the optimal mimotopes for cancer immunotherapy.
Targeting miR-23a in CD8+ cytotoxic T lymphocytes for tumor immunotherapy (P2072)
Authors: Lin Regina , Chen Ling , Hu Chunyan , Chen Gang , Jiang Shan , Sevilla Jose , Wan Ying , Zhu Bo , Li Qi-Jing
Published: January 2023

CD8+ cytotoxic T lymphocytes (CTLs) are ideal candidates for tumor immunotherapy. However, the potential of CTL-based cancer intervention has been limited by the challenges of in vitro expansion and the susceptibility of modified CTLs to immunesuppression in vivo. Here, we report a novel microRNA-targeting approach to augment the cytotoxic capacity of tumor-specific CTLs. We have identified miR-23a as a key inhibitor of anti-tumor CTL effector responses in vivo. miR-23a down-regulates its mRNA target Blimp-1, and simultaneously inhibits the expression of the key CTL effector molecules, Granzyme B and IFNγ. Conversely, functional suppression of miR-23a in CTLs augments CTL effector molecule expression, and enhances their cytotoxic potency on a per-cell basis. In mouse models of established B16 melanoma or Lewis lung cancer, immunotherapy with miR-23a-inhibited tumor-specific CTLs dramatically retards tumor growth to confer superior protection against tumor progression in vivo. We further establish that, by suppressing cMyc-mediated inhibition, tumor-derived TGFβ functions as a key enhancer of miR-23a expression in effector CTLs. In human lung cancer patients, elevated miR-23a expression in pleural effusion CTLs correlates with their impaired anti-tumor potential. Our findings not only demonstrate how TGFβ-induced miR-23a may impair CTL function to promote tumor immune evasion, but also provide the first proof-of-principle of a miRNA-based strategy for tumor immunotherapy.
Anti-tumor effects of adoptive transfer inactivated MHC-haploidentical tumor specific lymphocytes in mouse model (P2082)
Authors: Zhang Shuren , Shi Guilan , Liu Binlei
Published: January 2023

In this study, we evaluated effect of tumor-antigen-primed MHC-haploidentical lymphocytes in a TC-1 (H-2b) murine lung cancer model. The haploidentical effector cells prepared from splenocytes stimulated with the tumor cell lysates pulsed-DCs from F1 (C57BL/6 × Balb/C, H-2bd) mice, or immunized from F1 mice vaccinated with inactivated tumor cells. Haploidentical lymphocytes immunized with TC-1 could specifically kill the TC-1 cells, as well as up-regulate the NK activity. The effector cells inactivated with Mitomycin C in vitro kept the killing capacity, and migrated to tumor tissue. Compared with the cells from non-inactivated, or the syngeneic mice, the cells from Mitomycin C inactivated mice with haploidentical showed enhanced tumor inhibition. The survival time increased in tumor bearing mice and some showed tumor free, which developed no tumor after rechallenged with TC-1.Th1 cytokines including IL-2, IFN-γwere significantly increased. These studies demonstrate that the tumor antigen immunized MHC-haploidentical lymphocytes could specificity target to the tumor and kill them. MHC-mismatched alloantigens could be an effective adjuvant to break the immune tolerance and to activate innate and adoptive immunity in the tumor bearing host. The infusion of the inactivated MHC-haploidentical lymphocytes immunized with tumor antigen could be a safe and effective adoptive immunotherapy against tumor.
An IL-33 immunotherapy that blocks tumor growth by establishing innate lymphoid cells. (P2084)
Authors: Kim Wonyoung , Kim Juyang , Moon U-jeong , Jung Soyeon , Cho Hong , Kwon Byungsuk
Published: January 2023

Innate lymphoid cells are recently identified cell population, which produces IL-5, IL-13, and GM-CSF in response to IL-33. These cells play an important role in anti-helminth immunity and allergic diseases. However, little is known about the role of innate lymphoid cells in tumor. We report in this study that sustained intratumoral expression of IL-33 establishes a high numbers of innate lymphoid cells secreting high levels of GM-CSF which are required for inhibition of tumor growth. GM-CSF strongly inhibits proliferation of mural cells (pericytes and smooth muscle cells), thereby preventing normal angiogenesis in tumor mass. Our results identify a previously unrecognized innate immune pathway involving innate lymphoid cells which mediated type-2 innate immunity leading to antitumor activity.
Tumor-derived IL-35 promotes tumor growth by enhancing myeloid cell accumulation and angiogenesis (P2034)
Authors: Bai Xue-Feng , Wang Zhihui , Liu Jin-Qing , Liu Zhenzhen , Shen Rulong , Basu Sujit , Feng Youmei
Published: January 2023

IL-35 is a member of the IL-12 family of cytokines consisting of IL-12 p35 subunit and IL-12 p40-related protein subunit, EBV-induced gene 3 (EBI3). IL-35 functions through IL-35R and has a potent immune suppressive activity. Although IL-35 has been demonstrated to be produced by regulatory T cells, gene expression analysis has revealed that IL-35 is likely to have wider distribution including expression in cancer cells. In this study we have demonstrated that IL-35 is produced in human cancer tissues such as large B cell lymphoma, nasopharyngeal carcinoma and melanoma. In order to determine the roles of tumor-derived IL-35 in tumorigenesis and tumor immunity, we generated IL-35 producing plasmacytoma J558 and B16 melanoma cells, and observed that the expression of IL-35 in cancer cells does not affect their growth and survival in vitro, but stimulates tumorigenesis in both immune competent and Rag1/2 deficient mice. Tumor-derived IL-35 increases CD11b+Gr1+ myeloid cell homing in tumor microenvironment, and thereby promotes tumor angiogenesis. In immune competent mice, spontaneous CTL responses to tumors are diminished. IL-35 does not directly inhibit tumor antigen specific CD8+ T cell activation, differentiation and effector functions. However, IL-35-treated cancer cells had increased expression of gp130 and reduced sensitivity to CTL destruction. Thus, our study indicates novel functions of IL-35 in promoting tumor growth via enhancing myeloid cell accumulation, tumor angiogenesis and suppression of tumor immunity.
Real-time in vivo analysis of T cell migration and activation in the periphery and in the central nervous system using a genetically encoded calcium indicator (P4103)
Authors: Mues Marsilius , Bartholomäus Ingo , Thestrup Thomas , Griesbeck Oliver , Wekerle Hartmut , Kawakami Naoto , Krishnamoorthy Gurumoorthy
Published: January 2023

Interaction of T cells with cells of the surrounding milieu may result in activation of one or both partners. In particular, T cell activation following recognition of antigen presented by antigen presenting cells (APCs) triggers a swift rise of intracellular calcium levels within the T cell. Synthetic calcium indicators have been successfully used to monitor the activation status of T cells in vitro. However, these chemical dyes do not persist intracellularly for a sustained period which creates a major challenge for calcium imaging in the living animal. We now present a newly developed fluorescence resonance energy transfer (FRET)-based, genetically encoded calcium indicator (GECI) expressed in (auto)antigen-specific CD4+ T cells to study T cell activation in vivo at real time. Using two-photon microscopy, we explored the responses of retrovirally transduced, GECI-expressing T cells to antigen. In lymph nodes, the administration of exogenous antigen caused an almost immediate arrest of T cells around APCs concomitant with an instant rise of cytosolic calcium levels. In contrast, encephalitogenic T cells entering the leptomeningeal space, one main portal into the central nervous system parenchyma during experimental autoimmune encephalomyelitis, showed elevated intracellular calcium levels while still meandering through the space. This approach enables us for the first time to follow the migration and activation patterns of T cells in vivo during the course of the disease.
The effects of Trypanosoma cruzi infection and vaccination on priming T. cruzi-specific CD4+ T cells. (P3346)
Authors: Blase Jennifer , Eickhoff Christopher , Zhang Xiuli , Hoft Daniel
Published: January 2023

Trypanosoma cruzi is a protozoan parasite and the causative agent of Chagas disease. The T. cruzi protein trans-sialidase (TS) is currently under investigation as a vaccine candidate. There are defects in CD4+ T cell responses after T. cruzi infection as compared to TS vaccination. We sought to compare T cell receptor transgenic (Tg) TS-specific CD4+ T cell responses induced by infection versus vaccination through RNA microarrays. Tg TSaa57-74-specific CD4+ T cells were purified from naïve transgenic mice and activated in vitro by peptide-pulsed DCs or T. cruzi-infected and peptide-pulsed DCs, as well as in vivo by TS-DNA vaccination or T. cruzi infection. At various time points post activation, RNA was extracted from purified Tg CD4+ T cells and analyzed by microarray. In vitro, infection of DCs resulted in an increase in the expression of the signaling molecule Map3k10, the cytokine Il21, and factors involved in cell death/survival in CD4+ T cells. Following in vivo activation, proliferation was similar among Tg T cells stimulated with TS vaccination and T. cruzi infection. The microarray will reveal genes differentially expressed in response to T. cruzi infection with further analysis ongoing. These in vitro and in vivo studies using our transgenic T. cruzi-specific CD4+ T cells suggest that T. cruzi infection and TS vaccination differentially activate CD4+ T cells which may explain the defects in CD4+ T cells observed following T. cruzi infection.
A role for T-bet in regional responses required to control parasite replication. (P3302)
Authors: Pritchard Gretchen , Hall Aisling , Wagage Sagie , Dupont Christopher , Reiner Steven , Hunter Christopher
Published: January 2023

The role of T-bet, a hallmark transcription factor of Type 1 immune responses, has been examined in a variety of infection models. The results of these studies have been contradictory in that the immune response to some pathogens (e.g. Salmonella, TB) requires T-bet while its expression is dispensable for resistance to other pathogens (e.g. LCMV, Listeria monocytogenes). The intracellular parasite Toxoplasma gondii induces a rapid Type 1 response in mice, characterized by production of the cytokine interferon-γ, which is required for controlling the parasite. In the absence of T-bet, mice challenged with T. gondii develop normal NK responses and have largely intact T cell activity, including production of IFN-γ. However, despite production of IFN-γ by NK and T cells, T-bet deficient mice succumb to acute infection; this increased susceptibility is characterized by control of parasite replication in the liver and peritoneum, but higher parasite burden in other sites. These data suggest local differences in the requirement for T-bet to control parasite replication and reveal an essential function of T-bet in the regional vs. systemic immune response necessary for control of T. gondii.
Th17 cells confer long-term adaptive immunity to oropharyngeal candidiasis (P3308)
Authors: Hernandez-Santos Nydiaris , Huppler Anna , Peterson Alanna , Khader Shabaana , McKenna Kyle , Gaffen Sarah
Published: January 2023

Oropharyngeal candidiasis (OPC) is an opportunistic infection of the oral cavity caused primarily by Candida albicans. OPC particularly afflicts HIV+ patients with low CD4 counts, suggesting a role for CD4+ T cells in host defense. Experimental models of OPC have strongly suggested that immunity to OPC is IL-17-dependent, and humans with rare genetic defects that result in Th17 deficiency are associated with the development of OPC. However, other sources of IL-17 have been described, particularly in the innate compartment, but the relative contribution of adaptive and innate sources of IL-17 during OPC has not been well articulated. Here, we showed that development of adaptive immune responses in OPC results in a ~1 log decrease in fungal burden, tightly associated with an increased frequency of antigen-specific CD4+IL-17+ (Th17) cells. Importantly, adoptive transfer of Candida-primed Th17 cells into Rag1-/- mice resulted in protection from disease. Surprisingly, however, CD4-/- mice were resistant to OPC, which was linked, at least in part, with compensatory IL-17 production by both CD8+ and double-negative (DN) T cells. The adoptive transfer of CD8+IL-17+ or DN cells into Rag1-/- mice resulted in protection from OPC. Therefore, Th17 cells contribute to C. albicans clearance but their absence can be compensated by alternative sources of IL-17. These findings implicate CD8+IL-17+ and DN cells as vaccine targets in immunocompromised populations.
Microfluidic single cell array for dynamic T cell imaging (P3368)
Authors: Hung Paul , Chen Cindy
Published: January 2023

Imaging of individual live cells over time is essential for studying dynamic cellular processes within the heterogeneous cellular population. There is growing interest in using imaging technology to decipher the signaling pathway governing the stimulation of CD4 positive T cells by antigen-presenting cells in order to better understand the associated adaptive immune response. However, there are several technical challenges when conducting time-lapse live cell imaging to study the activation and the differentiation of T cells. First, primary T cells are non-adherent and become highly mobile upon activation. Second, T cell differentiation processes are relatively slow which could take over 72 hours. At last, ability to introduce different reagents as well as a second population of cells to monitor the cellular responses in real time is almost impossible with conventional cell culture substrates. Here we present a microfluidic single cell trapping array platform for dynamic T cell imaging. The platform allows the capture and growth of non-adherent immune cells, long-term monitoring and tracking of cells, separate introduction of antigen presenting cells as well as concurrent imaging with automatic liquid exchange. The protocol to execute each function was developed with a sample experiment demonstrated to screen for cytosolic calcium transients at single cell resolution.
Direct monitoring of phagocytosis and endocytosis in live cells with novel pH-sensing fluorescent dyes. (P3393)
Authors: Langsdorf Chris , Beacham Dan , Jaron Shula , Zhang Yu-Zhong , Janes Mike
Published: January 2023

The basic cellular internalization processes of endocytosis and phagocytosis are important to many areas of cell biology including receptor internalization, pathogen response, and apoptotic cell clearance. However, the ability to study these processes has historically been limited by the lack of quality tools to directly monitor the internalization and acidification of cargo. Here we present an assortment of tools to enable research into cellular internalization pathways with particular relevance to immunology. Two fluorogenic, pH-sensitive probes, one green and one red, are presented. These dyes are non-fluorescent at the neutral pH typically found in the cytosol and extracellular environment, but become brightly fluorescent in acidic cellular compartments, permitting direct monitoring of internalization and acidification processes. We present experimental data in which these dyes were used to label microorganisms including yeast and gram-positive and -negative bacteria. Live cell imaging and flow cytometry were then used to monitor the phagocytosis and acidification of these labeled particles by cultured murine macrophages over time. Additionally, we investigated the utility of these dyes to study phagocytic clearance of apoptotic cells by primary human monocyte-derived macrophages. We anticipate that these tools will enable additional research into the fields of receptor trafficking, drug delivery, host-pathogen response, and clearance of apoptotic cells.
A direct approach for measuring total TCRαβ diversity (P3354)
Authors: Schoettle Louis , Wei Xixi , Blattman Joseph
Published: January 2023

Two cardinal features of adaptive immunity are diversity and specificity: the immune system must be able to recognize virtually any pathogen while maintaining enough cells specific for each pathogen in order to mount effective responses. T cells accomplish this by expressing clonotypic αβ heterodimeric T-cell receptors (TCR) that recognize pathogen epitopes presented by major histocompatibility (MHC) proteins. Although the diversity of TCR chains have been independently measured, quantitating diversity due to pairing of a and b chains has been problematic; single cell sequencing is too costly while molecular approaches have not worked. We have developed a novel approach for directly measure diversity resulting from TCR αβ pairing employing DNA origami nanostructures to physically link mRNA encoding both TCRα and β chains, coupled with Ion Torrent deep sequencing to obtain CDR3 sequence information for both chains from the same cell. We have designed and generated such DNA origami structures for ligation of TCR mRNA, transfected with high efficiency naive primary T cells with these origami structures, and re-isolated origami with bound TCR mRNA. Thus, we are now able to directly estimate the total diversity of the T cell receptor repertoire (including TCRαβ pairing diversity) and can use this approach to assess how decreased TCR diversity (such as occurs during aging or immune reconstitution) affects the ability to mount effective responses to pathogens.
Analyzing cytotoxicity kinetics with a novel automated high-throughput real-time killing assay (P3366)
Authors: Qu Bin , Schwarz Eva , Bhat Shruthi , Lis Annette , Zhou Xiao , Lyrman Hélène , Backes Christian , Stephan Cora , Bufe Bernd , Zufall Frank , Hoth Markus , Kummerow Carsten
Published: January 2023

Cytotoxicity is an essential parameter to examine killer cell activity, to screen hits for new drugs, and to proof safety evaluation. Cytotoxicity assays are routinely used in basic research, clinical studies, and industrial applications. However, the most commonly applied standard end-point cytotoxicity assays largely suffer from the lack of kinetic information, which is indispensable to assess killing competence. Here we report a quantitative and highly sensitive calcein-based real-time high-throughput cytotoxicity assay on a single cell level and for population use. By determining fluorescence loss as a function of the fraction of lysed cells, this assay measures the kinetics of cytotoxicity over a broad dynamic range. The high time resolution of the assay also provides kinetic information with the potential to reveal neglected/immeasurable properties in the process of cytotoxicity of both purified human cytotoxic T lymphocytes and natural killer cells. To test its clinical potential, we have also applied this assay to screen the cytotoxic potential of (easy to prepare) peripheral blood mononuclear cells in healthy blood donors. In summary, this assay should substantially increase the screening efficiency for clinical and industrial purposes and offer quantitative kinetic data with a high time resolution.
Visualizing the physiological self-reactivity of epidermal γδ TCR (P3280)
Authors: Zal Tomasz , Chodaczek Grzegorz , Zal M. Anna
Published: January 2023

Dendritic epidermal T cells (DETCs) remain activated in vivo at steady state through their monomorphic Vgamma5-Vdelta1 TCR, forming phosphotyrosine-rich aggregates located on projections, called PALPs (Chodaczek et al. 2012). This recent discovery suggests the existence of beneficial self-reactivity on the part of the evolutionarily-preserved γδ TCR. Located at barrier-forming squamous keratinocyte junctions, and remarkably long-lived, the PALPs display multiple hallmarks of true immunological synapses, including TCR clustering and CD3-zeta and ZAP70 tyrosine phosphorylation. Using two independent super-resolution microscopy techniques: the structured illumination microscopy (SIM) and stimulated emission depletion (STED), we show that PALPs consist of several tens of peripherally distributed, tyrosine-phosphorylated TCR microclusters. Individual microclusters are uniformly sized at 150-160 nm and are associated with distinct signaling intermediates, suggesting that they represent supra-molecular signaling assemblies of a defined composition. Intravital fluorescence recovery after photobleaching and high speed time-lapse imaging showed that small intracellular cargo granules trafficked bi-directionally within T cell protrusions between large stationary granules and the PALPs. These studies reveal the PALPs as dynamically active TCR-activatory immunological synapses, and suggest that this novel form of physiological TCR self-reactivity mediates homeostatic secretory functions.
Over-expression of CD200 protects mice from dextran sodium sulfate induced colitis (P3286)
Authors: Chen Zhiqi , Yu Kai , Gorczynski Reginald
Published: January 2023

CD200:CD200R interactions have been shown to lead to potent immunosuppression, resulting in enhanced allograft survival; reduced collagen-induced arthritis; and increased growth of leukemic, lymphoma cells and breast cancer cells. We have investigated the effect of "knockout"of CD200R, or over-expression of CD200 in transgenic mice (CD200tg), on susceptibility to dextran sodium sulfate (DSS) induced colitis in C57BL/6 female mice. Control animals given DSS developed weight loss (~15%) and diarrhoea over 7 days, before slow recovery. These effects were more pronounced in CD200RKO mice, with ~40% mortality at day 7. In contrast CD200tg mice showed attenuation of all symptoms. Immunohistological analysis and protein expression array confirmed severe inflammation in CD200RKO and controls, with loss of villi, inflitration by macrophages, neutrophils and CD3+ cells, and high expression levels of IL-1β and TNF-α in the intestine. In CD200tg mice intestinal tissue showed enhanced inflitration by Foxp3+Tregs, with increased expression of IL-10. Taken together these data suggest for the first time that CD200:CD200R interactions play a role in regulating inflammatory colitis in mice, which may open new avenues for treatment of inflammatory diseases in man.
TRAF6 in DCs controls intestinal immune tolerance (P3292)
Authors: Han Daehee , Walsh Matthew , Cejas Pedro , Surh Charles , Choi Yongwon
Published: January 2023

The intracellular signaling mediator TRAF6 is critical for Toll-like receptor (TLR)-mediated activation of dendritic cells (DCs). We now report that DC-specific deletion of TRAF6 (TRAF6ΔDC) results, quite unexpectedly, in loss of mucosal tolerance, characterized by spontaneous rise in lamina propria Th2 cells, and development of eosinophilic enteritis and fibrosis of the small intestine. Loss of TRAF6ΔDC mucosal tolerance requires the presence of commensal microbiota in a manner that appears independent of the MyD88 pathway. Further, TRAF6ΔDC mice exhibit decreased regulatory T cell (Treg) numbers in the small intestine, and diminished induction of iTregs in response to model antigen. Evidence suggests this defect may be associated with diminished DC-expressed IL-2. Finally, we demonstrate that aberrant Th2-associated responses in TRAF6ΔDC mice can be mitigated via restoration of Treg activity. Collectively, our findings reveal a novel role for TRAF6 in directing DC maintenance of intestinal immune tolerance through balanced induction of Tregs versus Th2 immunity.
Skin-resident T cells limit UV damage through sensing the danger signal ATP (P3246)
Authors: MacLeod Amanda , Havran Wendy
Published: January 2023

Exposure to ultraviolet radiation (UVR) is a major risk factor for skin cancers and skin inflammation. However, molecular and cellular mechanisms to minimize UV damage in the skin are incompletely understood. Based on their sentinel function, we hypothesized that resident T cells in the skin, murine dendritic epidermal T cells (DETC) and human skin-resident T cells, sense UVR-induced danger signals such as extracellular adenosine triphosphate (ATP) to initiate protection and repair. Supporting this hypothesis, we find that epidermal keratinocytes release ATP upon UVR to activate DETC and human skin-resident T cells in a direct and IL-1β-mediated manner and this response could be inhibited by ATPases. Using mice deficient in DETC, we show a previously unrecognized role for DETC in repairing keratinocyte DNA damage upon UVR. Functionally, ATP enhanced TCR-mediated DETC proliferation and IL-17A production. IL-17A induced molecules relevant to DNA repair and apoptosis in keratinocytes, including TNF related weak inducer of apoptosis (TWEAK) and Growth arrest and DNA damage gene (GADD45), providing a possible link to the observed UV protective mechanisms exerted by DETC. Ongoing studies quantifying UVR-induced DNA damage and apoptosis in wildtype, TCRδ-/-, and IL-17A-/- skin cells and in human skin cultures will provide novel information on skin-resident T cell function, and may identify new targets to treat and prevent sunburn and skin cancer in humans.
Fucosylation is a hallmark of inflammatory macrophages and a novel therapeutic target in rheumatoid arthritis (P5137)
Authors: Li Jun , Hsu Hui-Chen , Yang PingAr , Wu Qi , Spalding David , Mountz John
Published: January 2023

Inflammatory macrophages (MΦ) play an important role in rheumatoid arthritis (RA). Fucosylation is a regulator of inflammation and we have observed a highly positive correlation between Fucosyltransferases (Fut)1 or Fut3 and Tnfα (p=0.0001) in RA synovial tissues. In sorted cells from human RA synovial fluid, Fut1 and Fut3 was highly expressed in M1 inflammatory MΦ (CD68+CD80+), but not in M2 MΦ (CD68+CD80-), synovial fibroblasts, Th1, Th17, and other T cells (p<0.01, n=5). Fucosylation inhibitor, 2-Deoxy-D-galactose (2-D-gal), precluded the differentiation of M1 MΦ derived by GM-CSF from mouse bone marrow or human PBMC monocytes, strongly indicating the indispensable role of fucosylation in M1 MΦ differentiation. Additionally, phalloidin staining showed 2-D-gal disrupted MΦ actin, suggesting a potential effect of fucosylation in antigen processing and presentation. Indeed, 2-D-gal treatment of fully differentiated M1 MΦ for 2 days dramatically reduced their uptake, processing and presentation of GFP-Eα (from I-Edα) and FITC-CII antigens in a dose dependent manner (p<0.01). In vivo, 2-D-gal dramatically blocked the bovine CII-induced arthritis in DBA/1J mice (scores 9.5±1.7 vs 0.5± 0.3, p<0.01) with reduced inflammatory MΦ in draining LN (1.3±0.3% vs 0.5±0.1%, p<0.05), decreased TNF-α (130 vs 39 pg/ml, p<0.05), and anti-CII in the serum. Our study indicated that M1 MΦ differentiation and function is orchestrated by fucosylation, which is a novel therapeutic target in RA.
Kv1.3 deletion induces Foxo1 expression independently of Foxp3 and biases Th cells toward an immunoregulatory phenotype (P5127)
Authors: Gocke Anne , Grishkan Inna , Calabresi Peter
Published: January 2023

Myelin-reactive Th cells in patients with multiple sclerosis (MS) express Kv1.3, a voltage-gated K+ channel essential for Th cell activation and function. Recently, we have demonstrated that Kv1.3 deletion prevents experimental autoimmune encephalomyelitis, a murine model of MS, by inducing a regulatory Th cell phenotype independently of Foxp3. However, the mechanism by which Th cells with regulatory properties develop in the absence of Kv1.3 remains unclear. Foxo1 has been shown to function in induction of tolerance and in Treg cell lineage commitment. Foxo1 nuclear exclusion is induced in effector Th cells by downstream T cell receptor (TCR) signaling. Herein, we demonstrate an increase in nuclear Foxo1, with concomitant reduction in T-bet, in Kv1.3 KO Th cells. Surprisingly, Kv1.3 deletion does not impair TCR-induced intracellular Ca2+ flux or the activation of the Ca2+-sensitive transcription factor NF-AT. Instead, the absence of Kv1.3 leads to p56lck relocalization from TCR to the IL-2R complex, thereby contributing to the increase in Foxo1 and the differentiation of Treg cells in the absence of changes in Foxp3 expression. Herein, we demonstrate that regulatory Th cells are generated in the absence of Kv1.3 as a consequence of disrupted TCR signaling during formation of the immunological synapse, rather than impaired Ca2+ flux, resulting in upregulation of Foxo1. These data highlight Foxo1 as a novel target for induction of Treg cells in autoimmune diseases such as MS.
MyD88 plays a role in deciding between apoptotic and necroptotic cell death after UV irradiation (P1238)
Authors: Harberts Erin , Fishelevich Rita , Liu Juan , Atamas Sergei , Gaspari Anthony
Published: January 2023

Ultraviolet irradiation (UV) induced cellular damage is classically associated with apoptosis and known to result in systemic immunosuppression. How the decision to undergo apoptosis is made following UV is not fully understood. We hypothesize that a central mediator of TLR signaling, MyD88, determines cell fate after UV. Survival after UV of immortalized bone marrow-derived macrophages (BMM) from MyD88 germline-deficient mice (MyD88-/-) was significantly higher and apoptotic morphology reduced, vs. wild-type (WT) BMM. UV-induced DNA laddering in peritoneal macrophages (PM) and epidermis of MyD88-/- animals, vs. WT, was decreased. In MyD88-/- PM, decreased cleavage of pro-necroptotic protein RIP1, elevated mRNA levels of RIP1 and RIP3, and significantly increased TNF-α release suggests that necroptosis rather than apoptosis has been initiated. In vivo studies after UV showed significantly less TUNEL-positive cells and more profound inflammation in MyD88-/- skin sections. MyD88-/- mice are also found to be resistant to UV-induced immunosuppression. Considering that MyD88 participates in many TLR pathways, TLR2-/-, TLR4-/-, and WT BMM were compared for evidence of UV-induced apoptosis. Only TLR4-/- BMM had a similar phenotype to MyD88-/-, suggesting that the TLR4-MyD88 axis importantly contributes to cell fate decision. Our study describes a new cellular consequence of MyD88 signaling after UV, and may provide rationale for therapies to mitigate UV-induced immunosuppression.