bluuebunny/crossref_metadata_2025 huggingface dataset:
Modulation of immune function occurs within hours of therapy initiation for multiple sclerosis (P5189)
Authors: Sinha Sushmita , Ayers Chris , Mendoza Jason , Cunnusamy Khrishen , Greenberg Benjamin , Frohman Elliot , Karandikar Nitin
Published: January 2023

Daily subcutaneous administration of FDA-approved glatiramer acetate (GA) has beneficial effects on clinical course of relapsing remitting multiple sclerosis (RRMS). Although mechanism of GA-action has been widely investigated and partially understood, immediate immune dynamics following GA-therapy and their significance are unknown. In the present study we characterized immediate effects of GA on phenotype, quantity and function of immune cell subsets in MS patients. PBMC from RRMS patients, either untreated or initiating GA, and healthy donors were obtained at time of admission and subsequently at 4h, 12h, 24h and 72h after first GA-injection. Prominent changes in immune cells were detected within 4-12h post first GA-injection. T-cell modulation included significantly decreased CD4/CD8 ratio, perturbation in homeostasis of predominantly CD8+ T-cells, significant enhancement in CD8+ T-cell mediated suppression and inhibitory potential of induced CD4-suppressors. Changes in APCs were restricted to monocytes and included reduced stimulatory capacity in MLR and significantly increased IL-10 and TNF-α. Our study provides first evidence that GA induced rapid immunologic changes even after first dose. Interestingly, these responses are not restricted to APCs but also include complex modulation of T-cell functionality. Long-term studies will evaluate sustenance of these effects over time and their significance in clinical efficacy of therapy.
Apoptotic cell-based therapy to treat collagen-induced experimental arthritis (P5191)
Authors: Perruche Sylvain , Clauzon Amandine , Bonnefoy Francis , Saas Philippe
Published: January 2023

Biotherapies are emerging to treat rheumatoid arthritis (RA). Most of them target a cellular or soluble factor involved in the physiopathology of RA. Here we evaluated whether immunomodulatory properties of apoptotic cells can treat RA. Apoptotic cell approach has been successfully assessed to prevent arthritis development in experimental rat model, as well as in other inflammatory experimental settings. Thus we investigated apoptotic cell injection to treat RA in a mouse model of collagen-induced arthritis (CIA). Four to 8 days after CIA development in susceptible DBA/1, mice presenting arthritic symptoms were treated with apoptotic cells (5.10e6 cell/mouse). As control, methotrexate was used (MTX, 15 mg/kg, twice) alone or with apoptotic cells. Regulatory T cells (Treg) were assessed by FACS at time of sacrifice (day 40/45). Apoptotic cell injection demonstrated a significant decrease of arthritis score (p>0.01; 4 independent experiments), associated with an increased percentage of Treg in the spleen at time of sacrifice. MTX was sufficient to decrease arthritis as well and apoptotic cell injection did not influence MTX efficiency and vice-et-versa. Whereas MTX did not affect the percentage of Treg, MTX + apoptotic cell treatment induced a strong increase of Treg in the spleen and draining lymph nodes. Apoptotic cell injection is an effective treatment to reduce RA severity and should be consider in clinic as an alternative method for biotherapy refractory patients.
Laquinimod suppression of lupus nephritis in BWF1 mice associates in renal tissue with increase of myeloid suppressor cells and switch of macrophages to anti-inflammatory type II (P5193)
Authors: Lourenco Elaine , Wong Maida , Skaggs Brian , Matsuura Isao , Hahn Bevra
Published: January 2023

Background: Laquinimod (LAQ) is a novel drug that has been in clinical trial in systemic lupus erythematosus (SLE), an autoimmune disease characterized by nephritis mediated in part by autoantibodies. Aim: We investigated the immunomodulatory properties of LAQ on lupus nephritis in NZB x NZW F1 (BWF1) mice, a murine model for SLE. Methods: Six-month old BWF1 female mice were treated orally 3 times a week with LAQ 25 mg/kg or water. Results: All LAQ-treated animals survived up to 10 weeks after treatment vs 0% survival in controls. No LAQ-treated mouse had proteinuria by week 10 while it was significantly high (3 g/L) in 100% of controls by week 7 after treatment. Examination of kidney specimens showed significantly decreased histologic inflammation, damage, immunoglobulin and C3 deposition in LAQ-treated vs controls. LAQ significantly reduced macrophages (CD11b+) and their expression of MHC, and these cells produced higher levels of IL-10 and lower TNF-alfa vs controls, a phenotype characteristic of anti-inflammatory type II macrophages. In addition, increased frequency of CD11b+Ly6C+Ly6G+ and CD11b+Ly6C+Ly6G- cells was found in LAQ-treated vs controls and these cells suppressed CD4+ T cell proliferation, what characterizes them as myeloid suppressor cells. Conclusion: LAQ suppressed lupus nephritis, improved animals’ survival and increased both anti-inflammatory macrophages and myeloid suppressor cells. Thus, LAQ is a promising therapeutic for human lupus nephritis.
Therapeutic efficacy of suppressing the JAK/STAT pathway in models of neuroinflammation (P5199)
Authors: Benveniste Etty (Tika) , Qin Hongwei , Liu Yudong
Published: January 2023

T helper cells and myeloid cells are involved in the pathogenesis of Multiple Sclerosis (MS) and Experimental Autoimmune Encephalomyelitis (EAE), an animal model of MS. The JAK/STAT pathway is used by cytokines for signaling, and is critical for development and regulation of immune responses. Many cytokines involved in MS and EAE, including IL-6, IL-12, IL-23, IFN-γ and GM-CSF, use the JAK/STAT pathway to induce biological responses. As such, targeting JAKs has implications for treating autoimmune inflammation of the brain. We used a JAK1/2 inhibitor, AZD1480, to test the potential of inhibiting the JAK/STAT pathway in EAE. AZD1480 treatment effectively inhibits disease severity in classical, atypical and relapsing-remitting EAE models by preventing entry of immune cells into the brain, suppressing differentiation of Th1 and Th17 cells, deactivating macrophages and dendritic cells, inhibiting STAT activation in the brain, and reducing expression of pro-inflammatory cytokines/chemokines. Suppression of the JAK/STAT pathway was also effective in reducing paralysis induced by transfer of pathogenic Th1 or Th17 cells. In vivo AZD1480 treatment impairs both the priming and expansion of T-cells and attenuates antigen-presentation function of myeloid cells. These results suggest the feasibility of the JAK/STAT pathway as a target for immunomodulatory therapy in MS.
Combinations of inhibitory receptors are expressed on CD8+ T cells in pediatric subjects following bone marrow or umbilical cord blood transplantation. (P2162)
Authors: Soudeyns Hugo , Gravel Catherine , Caty Martine , Mezziani Samira , Le Campion Armelle , Duval Michel , Salem Fourati Insaf
Published: January 2023

Umbilical cord blood transplantation (UCBT) is commonly used to treat a variety of hematologic or neoplastic disorders in children. Compared to bone marrow transplantation (BMT), UCBT is associated with slow engraftment and a higher incidence of graft failure and opportunistic infections. The aim of the present study was to identify immunologic biomarkers expressed by CD8+ T cells during the post-transplant period that predispose patients to develop complications following UCBT or BMT. Pediatric patients who underwent UCBT (n=16) or BMT (n=7) were enrolled at CHU Sainte-Justine. Samples were obtained from graft inoculums and from transplanted subjects during 24 months of follow-up. Expression of inhibitory receptors associated with CD8+ T cell exhaustion (PD-1, CTLA-4, 2B4, TIM-3, BTLA-4, LAG-3) was studied ex vivo using multiparameter flow cytometry. Results showed that inhibitory receptors were expressed at very low levels in UCB graft inoculums. However, in the first 100 days after UCBT or BMT, a notable increase in the frequency PD-1+ CD8+ T cells and 2B4+ CD8+ T cells was observed, and frequencies of PD-1+ and 2B4+ T cells were inversely correlated with absolute CD8+ T cell counts. A low number of CD8+ T cells also co-expressed two or more inhibitory receptors. These results suggest that expression of exhaustion markers by CD8+ T cells in graft recipients during the early post-transplant period may modulate their activity and lead to functional impairment.
Molecular analysis of human HLA-G+ trophoblasts reveals their strong immune regulatory potential (P2172)
Authors: Tilburgs Tamara , Strominger Jack
Published: January 2023

INTRODUCTION: During pregnancy HLA-G+ extravillous trophoblasts (EVTs) play a key role in the prevention of a maternal immune attack to allogeneic fetal tissue. Human EVTs are extremely difficult to study due to their low frequency and lack of proliferative capacity. In this study we obtained robust villous trophoblast (VT) and EVT preparations and performed microarray analysis on RNA isolates. RESULTS: Comparison of the expression profiles revealed >4000 differentially expressed genes based on a 2-fold difference. To identify functional differences a Gene Set Enrichment Analysis (GSEA) was performed. In EVTs, 14 functional gene sets out of the 20 most significantly enriched gene sets were associated with direct immune activation. The core genes of these immune pathways include cytokines (e.g. EBi3, TGFβ and IL8) and cell surface molecules (e.g. B7-H3 and CRTAM) which can all directly influence lymphocyte binding and activation. To evaluate the implications of this immune activating profile, co-cultures of EVT with decidual NK (dNK) cells and decidual T (dT) cells were established. Low percentages of dNK and dT cells form immune synapses with HLA-G+ EVTs. Preliminary analysis of dNK-EVT and dT-EVT co-cultures demonstrates that EVTs influence the expression of activation markers on decidual lymphocytes. CONCLUSION: Our data supports the hypothesis that proper immune activation, rather than general immune suppression, facilitates the establishment of fetal-maternal tolerance.
Skin-specific protection of bortezomib and anti-IL-6 therapy in an acute GVHD model. (P2182)
Authors: Pai Chien-Chun , Tellez Joseph , Hui-Hua Hui-Hua , Sun Kai , Chen Mingyi , Abedi Mehrdad , Murphy William
Published: January 2023

Allogeneic hematopoietic stem cell transplantation (HSCT) is currently viewed as a standard treatment for highly aggressive hematologic malignancies. However, graft versus host disease (GvHD) is a major complication that limits its therapeutic application. Although current therapies suppress GvHD symptoms, they also hinder graft versus tumor effects (GvT). Therefore, there is an urgent need to develop a novel therapy that can dissociate graft versus host disease from graft versus tumor effects. Bortezomib, a proteasome inhibitor, specifically depletes alloreactive T-cells and decreases GvHD symptoms while preserving GvT responses. However, the paradoxical treatment responses, ranging from beneficial in early administration to detrimental in late administration, hinder its clinical application. We hypothesize that serum IL-6 could be a potential biomarker to monitor the treatment response of bortezomib and as an indicator of GvHD pathology. We demonstrated serum IL-6 levels specifically correlated with skin GvHD responses but not liver or gut GvHD. By applying bortezomib or anti-IL-6 therapy, we observed skin specific protection in our GvHD model. This study demonstrated the importance of IL-6 regulation by bortezomib as well as its role in tissue specific GvHD pathology. These findings have clinical implications in selecting the appropriate patient population for bortezomib and/or anti-IL6 clinical trials for skin GvHD patients.
T cell responses to Epstein Barr Virus infections in pediatric organ transplant recipients (P2196)
Authors: Lin Dongxia , Piard-Ruster Karine , Esquivel Carlos , Martinez Olivia , Maecker Holden
Published: January 2023

Pediatric organ transplant recipients are at highest risk of developing morbidity and mortality from EBV infection. There is broad consensus that T cell immunity is important in controlling viral replication and expansion of infected B cells. In this study, we simultaneously analyze virus-specific T cells’ phenotypes, functions, and epitope specificity using 37-parameter mass cytometry in the setting of pediatric organ transplant recipients, followed over the first two years post-transplant. We compared T cell responses to peptide pools derived from lytic and latent EBV antigens in both healthy EBV+ donors and pediatric transplant patients. The results suggest that healthy EBV+ donors can mount CD8+ T cell responses to both lytic and latent EBV antigens, with production of multiple cytokines. In contrast, pediatric patients have much weaker T cell responses, especially to lytic antigens. Based on current data, we hypothesize a polyfunctional T cell response to lytic antigens may preferentially protect against EBV viremia. We also use the SPADE algorithm for unsupervised clustering to compare the distribution of different T cell subsets in cytokine-producing cells. In addition, we analyze these T cells’ functional capacity for cytokine production by visualization in a heatmap. By employing highly multi-parameter mass cytometry and longitudinal sampling, we have created a more detailed picture of the T cell responses to EBV in the setting of pediatric transplant recipients.
miR-338-5p indirectly regulate BAFF signal by targeting TRAF3 during renal allograft antibody-mediated rejection (P2200)
Authors: Xu Haiyan , He Xizhou , Xu Renfang , Xu Xianlin , Jiang Jingting , Wu Cangping , Zhang Yanyun , Zhang Xueguang
Published: January 2023

Objective: To investigate the regulation mechanism of B cell activating factor (BAFF) signal during antibody-mediated renal allograft rejection (ABMR), microRNAs chip assay and further studies were carried out. Methods: The renal allograft tissues were diagnosed according to Banff'2005 criteria. MicroRNA chip assay was done (tested group: 4 ABMR tissues with high expression of BAFF and C4d; controlled group: 4 IF-TA tissues with no expression of BAFF and C4d). 20 other renal allograft tissues were chosen for differentially expression microRNAs testification, and TRAF3, BAFF and BAFF-R detection by IHC. Dual-lucifirase assay and further functional experiments were taken. Results: microRNA chip assay indicated that miR-200c, miR-30c, let-7b, miR-30b, and 7 other microRNAs were differentially up-regulated and only miR-338-5p was significantly down-regulated. Combing with research background, bioinformatics analysis suggested that TRAF3 was the target gene of miR-338-5p and only regulated by miR-338-5p, which was testified by luciferase assay. The real-time PCR data indicated that miR-338-5p was significantly down-regulated in ABMR tissues, and inversely correlated with BAFF, BAFF-R and TRAF3 (these three molecules high expressed in ABMR tissues). And miR-338-5p mimics and inhibitors can seperately stimulate and inhibit IgG secretion by tonsil B cells in vitro. Conclusions: miR-338-5p participate in ABMR by indirectly regulating BAFF signal via targeting TRAF3.
Ex-vivo expansion and characterization of naturally occurring CD4+CD25+Tregs in rhesus monkey (P2204)
Authors: Kim Jung-Sik , Lim Hyun Ju , Nam Hae Young , Kong Na Rae , Park Chung Gyu
Published: January 2023

Regulatory T cells (Treg) offer potential for improving long-term outcomes in cell and organ transplantation. The non-human primate (NHP) model has been a valuable resource to bridge Treg therapy to the clinic. Here we address the highly effective Treg expansion protocols and phenotypic and functional characteristics of rhesus monkey. Rhesus monkey CD4+CD25+FoxP3+Treg cells exists 2-5% in CD4+T cells in blood. Around 1-2% of CD4+CD25high population in CD4+T cells were enriched by FACSAria III and cultured, simultaneously stimulated with antiCD3/antiCD28 antibody, in the presence of hIL-2, TGF-beta and irradiated autologous CD4-T cells for 14 days. The CD4+CD25+FoxP3+Treg cells expanded from 400 to 1200 times during two round stimulations. Expansion was far retarded during third round stimulation. Expansion rates were greatly affected by the initial ratio of Treg cell to autologous CD4-T cells. Phenotypically expanded Treg are CD25+CD95+CD27+CD47+FoxP+Treg cells. CCR7, LN-homing receptor, was expressed by 70%. In contrast, inflammatory cytokines including IL-17 and IFN-r were minimally expressed by 0.4% and 1.8%, respectively. Expanded Tregs were able to suppress the monkey CD4+CD25-T cell proliferation at even 1: 32 ratio of T regs to T effector cells. These finding suggests that ex-vivo expanded Treg cells may be promising modalities to overcome cell-mediated graft rejection in preclinical transplantation model.
Apoptotic cell injection-induced tolerance preserved graft versus leukemia effect (P2206)
Authors: Perruche Sylvain , Chauchet Adrien , Vallion Romain , Bonnefoy Francis , Saas Philippe
Published: January 2023

Allogeneic hematopoietic cell transplantation (AHCT) is a curative therapy for hematological malignancies unfortunately limited by graft-versus-host-disease (GvHD) occurrence. We have demonstrated that apoptotic cell injection can favor tolerance due to their immunomodulatory properties and thus increase engraftment and limit GvHD, through TGF-β and regulatory T cell dependent mechanisms. However, since apoptotic cell injection favors tolerance we evaluated whether graft-versus-leukemia (GvL) was preserved. In an experimental mouse model of T cell-depleted bone marrow transplantation (TCD BMT), leukemic A20 luciferase+ cells were injected at d0 and allogeneic donor T cell infusion was given on d6. Apoptotic cells were injected either on d0 or d6. Eradication of leukemic cells was observed in all mice receiving T cells, as attested by tumor growth evaluated by luminescence. Mice not receiving T cells died from leukemia within 27 days. Mice demonstrating GvL also developed GvHD according to Ferrara's score, and only 27% survived at d27. In contrast, 50% of mice receiving apoptotic cells on d0 or d6 survived at d27 and demonstrated long-term GvL with the complete absence of leukemic cell growth. Our data clearly demonstrated that GvHD suppression by apoptotic cell injection does not abrogate GvL activity. Thus, apoptotic cell injection should be considered in clinic to favor engraftment, prevent GvHD, conserving high GvL therapeutic activity.
iPS cells but not their hematopoietic derivatives are immunogenic in the autologous recipient (P2210)
Authors: Zavazava Nicholas , Kim Eun-Mi
Published: January 2023

Induced pluripotent stem (iPS) cells are derived by reprogramming somatic cells, mostly fibroblasts. Since these cells can be generated from patients, the asuumption is that when the cells are transplanted back to the donor, immunological rejection can be ruled out. However, recently a manuscript showed that in fact iPS cells are rejected by "self". This result was unexpected and might significantly impact the use of iPS cells clinically. Here, we hypothesized that iPS cells are immunogenic to "self" because they express carcinogenic/embryonic antigens that are not expressed on somatic cells. Consequently, we argued that iPS cell derivatives lose the ability to express these antigens and should therefore be non-immunogenic to "self". To address this question, 129SvJ iPS cells or 129SvJ ES cells were subcutaneously injected in 129 SvJ mice, respectively. After 4 weeks, teratomas were visible in mice that had been injected with ES cells but not in mice injected with iPS cells, suggesting that iPS cells had been rejected. iPS cells were then used to derive hematopoietic progenitor cells. When these cells were infused intravenously, they easily induced long-term mixed chimerism, suggesting that non-differentiated iPS cells but not their derivatives are immunogenic to"self". Interestingly, ES cells were also rejected in" iPS-sensitized" but not in naive mice. Rejection was mediated by T cells.
Enhanced pro-inflammatory profile and Th17 alloresponse by targeting mTORC2 in DC (P2138)
Authors: Raich-Regue Dalia , Rosborough Brian , Lee Keunwook , Boothby Mark , Turnquist Heth , Thomson Angus
Published: January 2023

Mammalian target of rapamycin (mTOR) is an important integrative kinase that regulates immune cell function. mTOR functions in two independent complexes: mTOR complex (mTORC) 1 and 2. The immunosuppressant Rapamycin inhibits mTORC1 but not mTORC2. Our aim was to study the immune regulatory role and underlying mechanistic properties of mTORC2 in DC. We generated a conditional mTORC2 knockout, targeting Rictor. Bone marrow cells were obtained from KO and WT B6 controls, and DC propagated in the presence of IL-4 and GM-CSF, with or without LPS. The phenotype, cytokine production, STAT3 activation and ability of DC to induce allogeneic T cell proliferation were evaluated, and also the responder T cell phenotype and cytokine production. DC differentiation was not affected, while DC yield was slightly reduced in mTORC2-deficient BM cultures compared with WT controls. DC lacking mTORC2 activity displayed similar levels of MHC and co-stimulatory molecules, but diminished B7-H1 expression, increased levels of IL-12p40, diminished STAT3 activation and higher T cell stimulatory ability following LPS stimulation, compared with WT. Staining of allogeneic T cells stimulated by Rictor-KO DC revealed more CD4+IL-17+ cells than those stimulated with WT DC. These novel data indicate that DC lacking mTORC2 activity exhibit an enhanced pro-inflammatory profile with ability to promote Th17 responses. Thus, targeting mTORC2 provides new insight into molecular regulation of DC function.
T-bet is critical for development of acute graft-versus-host disease through controlling T cell differentiation and function (P2142)
Authors: Yu Xue-Zhong , Fu Jianing
Published: January 2023

T-bet is a master regulator for the differentiation into Th1 cells that produce IFN-γ. We evaluated the roles of T-bet and IFN-γ in acute graft-versus-host disease (GVHD) using murine model of allogeneic bone marrow transplantation (BMT). IFN-γ-/- CD4 T cells caused similar GVHD compared with WT cells, whereas T-bet-/- CD4 T cells induced significantly ameliorated GVHD. Recipients of T-bet-/- donor T cells had markedly reduced T cell infiltration and tissue damage in liver and gut, and they produced significantly lower levels of IFN-γ, TNF-α, and IL-2 but higher IL-10, IL-6 and IL-4 in serum. T-bet-/- CD4 T cells expressed significantly less IFN-γ, CXCR3 and CD122, but more IL-17 and CCR6 compared with WT CD4 T cells, indicating that T-bet-/- T cells impaired in differentiating into Th1 but not Th17 cells. IFN-γ-/- Th17 cells had a comparable ability to cause GVHD compared with WT Th17 cells, while T-bet-/- Th17 cells had a significantly reduced pathogenicity. Microarray analysis identified genes that are regulated by T-bet but independent of IFN-γ, including Cxcr3, Ccr5, Ccl3, Ccl4, Klrc1, Klrd1, Nkg7 and Pdcd1, which may explain the compromised ability of T-bet-/- not IFN-γ-/- T cells in the induction of GVHD. We conclude that T-bet is required for Th1 differentiation and migration, and contributes to optimal function of Th17 cells. Targeting T-bet or regulating its downstream effectors independent of IFN-γ may be a promising strategy to control GVHD in clinic.
JNK2 has distinct roles in CD1d-dependent and CD1d-independent activation of NKT cells (APP3P.112)
Authors: Liu Jianyun , Gallo Richard , Khan Masood , Iyer Abhirami , Kratzke Ian , Brutkiewicz Randy
Published: January 2023

CD1d-mediated activation of NKT cells plays critical roles in auto-immune, anti-tumor and anti-microbial immune responses. Our previous work has shown that activation of the p38 mitogen-activated protein kinase (MAPK) pathway impairs, whereas the ERK MAPK pathway enhances, CD1d-mediated Ag presentation to NKT cells. In the current study, we found that, like p38, the JNK MAPK pathway is a negative regulator of CD1d-mediated Ag presentation. Moreover, activation of the JNK2 (but not JNK1) isoform was responsible for the reduction in CD1d-mediated Ag presentation observed. Unexpectedly, a deficiency in JNK2 (but not JNK1) in mice resulted in fewer NKT cells. We have previously shown that viral infections result in a CD1d-independent loss of NKT cells. When JNK2-deficient (KO) mice were infected with vaccinia virus, the virus-induced loss of NKT cells was substantially lower than that observed in JNK1 KO or WT mice. Furthermore, NKT cells from JNK2 KO mice expressed a slightly lower level of IL-12 receptor (IL-12R) expression on the surface. In vivo administration of IL-12 (which can induce apoptosis of WT NKT cells) to JNK2 KO mice resulted in less NKT cell loss than that observed in IL-12-treated WT littermates. Overall, our work strongly suggests that JNK2 (but not JNK1) plays an important role as a negative regulator of CD1d-mediated Ag presentation and also contributes to the virus-induced loss of NKT cells.
Tapasin-related protein TAPBPR interacts directly with peptide-free MHC-I/β2-microgolbulin complexes (APP3P.101)
Authors: Morozov Giora , Zhao Huaying , Mage Michael , Boyd Lisa , Schuck Peter , Natarajan Kannan , Margulies David
Published: January 2023

The molecular mechanism by which MHC-I molecules are loaded with peptides is crucial to fundamental aspects of antigen presentation relating to self-tolerance and CD8 T cell activation. To gain insight into the mechanism of MHC-I peptide loading we have studied human TAPBPR (TAP binding protein-related) protein, which is 22% identical to tapasin in amino acid sequence, widely expressed and IFN-γ inducible, but is not part of the peptide-loading complex (PLC). To understand the interaction of TAPBPR with MHC-I, we produced recombinant soluble TAPBPR and evaluated its interactions with MHC-I/β2m complexes in vitro. Using recombinant MHC-I molecules refolded with photolabile peptides, we show, by gel-shift analysis, size exclusion chromatography, and analytical ultracentrifugation that TAPBPR binds MHC-I/β2m after photolysis of the bound peptide, in a 1:1 molar ratio. MHC-I molecules loaded with low-affinity peptides also bind TAPBPR. The TAPBPR/MHC interaction is reversed by exposure to high-affinity peptides known to bind the MHC-I molecule, indicating a role of TAPBPR in stabilizing a peptide-receptive form of the MHC-I/β2m complex. Because of the predicted structural similarity of TAPBPR to tapasin, this system not only provides insight into the molecular details of TAPBPR/MHC-I binding, but may also elucidate tapasin interactions in the PLC that have previously eluded experimental interrogation.
LAMP-2C regulation of MHC class II antigen presentation (APP3P.105)
Authors: Pérez Liliana , Deffit Sarah , Blum Janice
Published: January 2023

In autophagy cells divert cytoplasmic components and organelles to lysosomes for degradation impacting innate and adaptive immunity. A selective form of autophagy, known as chaperone-mediated autophagy (CMA), is implicated in adaptive immunity. CMA relies on the lysosome-associated membrane protein (LAMP)-2A to translocate proteins from the cytoplasm into lysosomes. Alternative splicing of the LAMP-2 gene generates 3 highly conserved isoforms LAMP-2A, LAMP-2B and LAMP-2C. The function and regulation of these genes is not well understood. Here we identified a novel role for LAMP-2C in the regulation of CMA. Studies were undertaken here to understand the role of LAMP-2C in MHC class II presentation of cytoplasmic antigens. Ectopic expression of LAMP-2C did not alter the maturation or the enzymatic activity of lysosomal enzymes. Furthermore, LAMP-2C ectopic expression failed to perturb other degradation pathways involved in antigen presentation like macroautophagy and proteasome degradation. Thus, LAMP-2C appears to have a selective regulatory role with respect to the presentation of cytoplasmic proteins. Future studies are underway to determine mechanistically how LAMP-2C controls antigen presentation. This work was supported by NIH 5T32AI060519 and AI079065.
Protein deprivation alters trafficking of the B cell receptor and endogenous antigens in B cells (APP3P.106)
Authors: Deffit Sarah , Blum Janice
Published: January 2023

Protein malnutrition in children has been linked to increased instance of infection and higher fatality likely due to limited T and B cell-mediated host immunity. These findings suggest protein malnourishment may alter antigen presentation and the cognate interaction between T and B cells. Studies here show serum deprivation induced macroauophagy and MHC class II presentation of select antigens which utilize this pathway in B cells. Further studies established serum deprivation inhibited MHC class II presentation of cytoplasmic autoantigen, glutamic acid decarboxylase and the presentation of epitopes derived from the B cell receptor. Serum deprivation did not alter the expression MHC class II proteins but did upregulate proteasome and lysosomal protease activity. Interestingly, B cell receptor accumulation on the cell surface was detected likely due to a reduction in endocytosis. An altered subcellular distribution of glutamic acid decarboxylase protein was observed, suggesting changes in the trafficking of this antigen. Studies are currently underway to examine how protein deprivation regulates antigen trafficking and presentation. This work was supported by the T32DK007519 and NIH RO1AI079065.
Sec61 plays an essential role in facilitating antigen cross-presentation to induce immune glomerular injury: a novel ‘self-organized criticality theory’ explaining cause of SLE (BA8P.129)
Authors: Tsumiyama Ken , Shiozawa Shunichi
Published: January 2023

Objective: Our novel ‘self-organized criticality theory’ which may explain the cause of SLE shows that immune glomerular injury is caused by CTL generated via antigen cross-presentation. Here we show essential role of Sec61 in antigen cross-presentation and immune glomerular injury. Methods: Bone marrow-derived dendritic cell (BMDC) was cultured with fluorescent-labeled OVA. Early endosome antigen 1 (EEA1), calnexin and Sec61 were detected using immunofluorescent staining. Sec61 was immunoprecipitated from OVA-pulsed BMDC lysate, followed by immunoblotting of OVA. Mice were repeatedly immunized with OVA to induce SLE. Splenic DC (spDC) was cultured with fluorescent-labeled OVA to examine localization of engulfed antigen. Further, exotoxin A was co-immunized with OVA to inhibit Sec61. Results: In BMDC, OVA was co-localized with an endosomal marker EEA1, and subsequently separated from EEA1. OVA was co-localized with Sec61, but not the ER marker calnexin. The result was similar when spDC was used. Engulfed OVA was co-precipitated with Sec61, and the amount of Sec61 in endosome was increased after repeated immunization with OVA. Instead, inhibition of Sec61 did not induce CTL nor renal injury, suggesting that OVA interacts with Sec61 hence exported from endosome to cytoplasm via Sec61. Conclusion: Increase of Sec61 in endosome and the export of antigen from endosome to cytoplasm via Sec61 are important for antigen cross-presentation and as the cause of immune glomerular injury.
Self-tolerance in the polyclonal CD4+ T cell repertoire (BA8P.132)
Authors: Malhotra Deepali , Linehan Jon , Jenkins Marc
Published: January 2023

Autoimmunity affects millions of people throughout the world. CD4+ T cells express T cell receptors (TCRs) that recognize peptides loaded on major histocompatibility complex II molecules (p:MHCII). Recognition of self-p:MHCII ligands may promote autoimmunity, necessitating the existence of tolerance mechanisms that regulate self-reactive CD4+ T cells. Study of TCR transgenic cells that recognize model self-antigens has identified several potential mechanisms of tolerance including clonal deletion, regulatory T cell induction, and anergy. However, TCR transgenic systems are unable to determine how each mechanism contributes to tolerance within a polyclonal repertoire comprised of a diverse set of TCRs. Thus, we have produced p:MHCII tetramers to track and study how tolerance is maintained in a polyclonal pool of self-reactive CD4+ T cells. We find that 50% of self-reactive CD4+ T cells in this polyclonal population are deleted, and following priming the remaining non-deleted self-reactive CD4+ T cells exhibit diminished proliferation and altered differentiation patterns. Ongoing work is focused on understanding if and how these tolerance pathways differentially regulate individual CD4+ T cell clones in the polyclonal repertoire and whether TCR affinity is the guiding factor for this decision.
Real time visualization of the differential mTEC and DC contributions to T cell central tolerance (BA8P.119)
Authors: Jones Jessica , Rey-Villamizar Nicolas , Sasiponganan Chayanit , Selden Hilary , Roysam Badrinath , Ehrlich Lauren
Published: January 2023

T cell central tolerance for peripherally expressed tissues is established by thymocyte:stromal cell interactions that occur within the thymic medulla. However, the roles of the medullary thymic epithelial cell (mTEC) and dendritic cell (DC) subsets in mediating negative selection against tissue-restricted antigens are unclear. While genetic models provide evidence regarding the selecting potential of either stromal cell subset in the absence of the other, these models interfere with the normal thymocyte:stromal cell crosstalk necessary to establish a properly differentiated thymic microenvironment; this leaves open the relative contribution of either stromal cell type in mediating negative selection when both subsets are intact. Here, using two-photon fluorescence microscopy, we observe thymocyte:stromal cell interactions at the onset of TCR signaling as they occur in real time in the thymic medulla. We have quantitated CD8+ and CD4+ thymocyte contacts with either Aire+ mTECs or DCs in the presence of membrane-bound or secreted soluble forms of antigen, and find that both thymocyte MHC-restriction and subcellular localization of the antigen modulate the contributions of mTECs and DCs to negative selection. These experiments provide insight into the cellular mechanisms of central tolerance that deter autoimmunity, as they occur in a physiological environment.
Generation and characterization of immune-complex specific T cells (BA8P.121)
Authors: Giles Josephine , Pinilla Clemencia , Shlomchik Mark
Published: January 2023

There is ample evidence that, in systemic autoimmune diseases such as systemic lupus erythematosus, T cells contribute to disease by both providing help for B cells and directly attacking target tissues. However, there is relatively little information on the nature and specificity of such autoreactive T cells. Moreover, there are few if any systems in which they can be identified and studied in vivo. To this end, we have generated a series of autoreactive T cell hybridomas using rheumatoid factor (RF) B cells fed with immune complexes (ICs). We are using this cognate autoreactive T and B cell system to investigate two major questions. 1) What are the T cells specific for within the immune complex? We have used a positional scanning strategy using synthetic combinatorial libraries. Using peptide reactivity patterns to search proteome databases, we identified several candidate peptides, some of which have been synthesized and validated, leading to putative implication of several DNA and RNA interacting proteins. 2) How do autoreactive T cells function in vivo? We have generated retrogenic mice expressing the autoreactive TCR cloned from one of the hybridomas. These cells are positively selected into the CD4 compartment but do not become nTregs. These mice will be used to characterize the autoreactive T cells in vivo, including their interactions with cognate RF B cells as well as their potential to promote disease.
Polyfunctionality of naive T cells is enhanced in older age (CCR3P.223)
Authors: Van Eps Puja , Banks Richard , Aung Htin , Betts Michael , Canaday David
Published: January 2023

Reduced number of naïve T cells along with an accumulation of differentiated cell types in aging has been described but little is known about the polyfunctionality of T cells. In this study we compared the individual cytokine and polyfunctional profiles of T cell subsets, including newly described stem cell like memory T cells (TSCM) in response to stimulation with submaximal concentration of non-specific mitogen in older (median age 80, n=23) versus younger (median age 27; n=23) adults using multicolor flow cytometry. Secretion of IFN-γ, MIP-1α, TNF-α, perforin, and IL-2 were measured. Polyfunctionality was determined using FlowJo and SPICE (NIAID). Aging was associated with a marked decline in CD8+ naïve cells (11% vs. 47 %; p<0.0001) and an expansion in memory cell types including central memory (p<0.05), effector memory and effectors (p<0.001 for both) compared to younger subjects. There was also a decline in CD4+ naïve cells with age (33% vs. 45%; p=0.02). There were no differences in frequencies or cytokine profiles of TSCM between groups. CD8+ naïve cells in the older group had increased secretion of all cytokines measured compared to the younger subjects and exhibited greater polyfunctionality (p=0.03). CD4+ naïve cells among the older group also had greater polyfunctionality with a TNF-α and IL-2 dominance (p=0.001). The decline in naïve T cells in aging, rather than their polyfunctional potential maybe contributing to immunosenescence.
Initial IL7R signaling regulate the induction of effector CD8+ T cells and subsequent anti-tumor immune response in lymphopenic host (CCR3P.208)
Authors: Suzuki Toshihiro , Kishimoto Hidehiro , Abe Ryo
Published: January 2023

Induction of lymphopenia and adoptive transfer of T cells are followed by lymphopenia-induced proliferation (LIP), and generated a anti-tumor immune response. However, the distinct role of redundant IL7 in lymphopenic host and subsequent LIP of T cells for anti-tumor immune response has been unclear, since multiple mechanisms are involved in the anti-tumor immune response under lymphopenic conditions. To clarify the correlation between LIP and the anti-tumor effect, LIP was inhibited by IL7R blockade with anti-IL7Ra mAb at various stages, and the anti-tumor effect was assessed. When LIP was occurred, the growth of transplanted LLC expressing model antigen gp33, was markedly regressed. Using IL7R blockade with mAb at various stages, we found that IL7R signaling at the start of LIP is crucial for the anti-tumor immune response. Although some reports focused on the benefit of IL7 for the improvement of effector T cell function, later IL7R blockade did not arrest the expansion of CTLs, and anti-tumor effect was occurred. IFN-g production also depend on IL7R signaling during induction phase, but not later phase, and, the expression of co-inhibitory molecules, PD-1, on CTLs was unchanged in IL7R blockade hosts. In conclusion, our results demonstrated that initial IL7R signaling and subsequent LIP regulated the expansion of IL7Ra+ effector precursors mainly, but that the effector function of CTL precursors and migration into tumor did not require IL7R signaling.
Notch signaling regulates T follicular helper cell function and maintenance during allergic immunity (CCR3P.209)
Authors: DellAringa Mark , Reinhardt Richard
Published: January 2023

Success of most current vaccines relies on the production of high-affinity antibodies. The generation of high-affinity antibody producing B cells occurs within the germinal center, and CD4+ T follicular helper cells (Tfh) play an indispensable role during the maturation of these B cells. A critical step in this process is the secretion of interleukin-4 (IL-4) by Tfh cells. IL-4 signaling in germinal center B cells is required for affinity maturation of IgG1. IL-4 production by Tfh cells appears to occur independently from the classical Th2 differentiation pathway. Currently, the mechanisms regulating IL-4 production by Tfh cells are unknown. One intriguing possibility for non-canonical IL-4 production in Tfh cells is the Notch pathway. Notch signaling is required for the differentiation of Tfh cells, and has been shown to modulate IL-4 production. Here, we provide mechanistic evidence for Notch signaling in the production of IL-4 by Tfh cells. Inhibition of Notch signaling after germinal center formation resulted in a significant decrease in the percentage and number of Tfh cells actively producing IL-4. Furthermore, inhibition of Notch signaling led to decreased expression of the chemokine receptor CXCR5 in IL-4 producing CD4+ T cells, which corresponded to a repositioning of IL-4-competent cells outside of the germinal center. These findings suggest a model in which Notch signaling is not only important in Tfh differentiation, but also in Tfh function and maintenance.
Biallelic T cell receptor rearrangement enhances the efficiency of thymocyte development and positive selection. (HEM4P.234)
Authors: Schuldt Nathan , Auger Jennifer , Hogquist Kristin , Binstadt Bryce
Published: January 2023

Biallelic rearrangement of TCR loci has been proposed to increase the likelihood of productive gene rearrangement, thereby promoting thymocyte survival. When allelic exclusion is incomplete, this process also results in dual TCR-expressing T cells, which may increase repertoire diversity, but may also increase autoreactive potential. We investigated the effects of biallelic TCR gene rearrangement by comparing wildtype (wt) T cells to those from mice hemizygous for the genes encoding TCR α, TCR β, or both. Using bone marrow chimera competition assays we found that Tcrb hemizygous thymocytes were impaired in the DN1 to DN2 transition relative to wt cells. In addition, Tcrb hemizygous mice exhibited a higher frequency of T cells committed to the γδ lineage. Bone marrow chimera competition assays revealed a dramatic increase in the ratio of wt thymocytes to Tcra hemizygous thymocytes between the pre-selection and post-selection DP compartments, demonstrating that biallelic Tcra rearrangement increases the rate of positive selection. Despite their impairments in thymocyte development, mice hemizygous for both Tcra and Tcrb generated polyclonal T cell repertoires similar to wt mice as assessed by numbers of antigen-specific naïve T cells, responses to multiple self and foreign antigens, and TCRβ sequence diversity. Thus, biallelic rearrangement of Tcra and Tcrb loci enhances the efficiency of thymocyte development, but is not necessary for normal TCR repertoire diversification.
Signaling through FcRγ receptors on dendritic cells drives IL-33 dependent Th2-type responses (HYP6P.262)
Authors: Tjota Melissa , Blaine Kelly , Barrett Nora , Sperling Anne
Published: January 2023

Dendritic cells (DCs) play a critical role in the development of Th2 responses during atopic asthma, but the mechanism by which allergens drive DCs to promote Th2 responses is still unclear. Previously, we demonstrated that allergen-specific IgG immune complexes (ICs) enhance secondary Th2 responses via an IL-33-dependent mechanism by ligating FcγRIII on antigen presenting cells. To identify if this pathway was common to other Th2 stimuli, we used house dust mite (HDM) extract. While HDM-mediated allergic inflammation was not dependent on FcγRIII, it was reliant on the common signaling component FcRγ as FcRγ-/- mice failed to develop Th2 responses in the lung. Notably, ligation of FcRγ associated receptors, FcγRIII and Dectin-2, on DCs treated with ICs or HDM respectively, upregulated IL-33 in an FcRγ and PI3-kinase dependent manner suggesting a common pathway by which Th2 stimuli can direct the development of DCs that induce Th2 differentiation. Reestablishment of Th2 responses in the FcRγ-/- mice with an adoptive transfer of HDM-sensitized FcRγ+/- DCs indicated that the lack of response in the FcRγ-/- mice was due to a deficient response from the DCs. Interestingly, addition of recombinant IL-33 during HDM sensitization was sufficient to restore airway inflammation in FcRγ-/- mice. Overall, these data identify a mechanism whereby Th2 stimuli signal through FcRγ associated receptors on DCs to upregulate IL-33 production and induce Th2-mediated allergic airway inflammation.
Dissecting mechanisms underlying the pathogenesis of eosinophilic esophagitis (HYP6P.263)
Authors: Tait Wojno Elia , Noti Mario , Kim Brian , Siracusa Mark , Nair Meera , Benitez Alain , Ruymann Kathryn , Muir Amanda , Yearley Jennifer , Menard-Katcher Paul , Kubo Masato , Obata-Ninomiya Kazushige , Karasuyama Hajime , Comeau Michael , de Waal Malefyt Rene , Sleiman Patrick , Hakonarson Hakon , Cianferoni Antonella , Falk Gary , Wang Mei-Lun , Spergel Jonathan , Artis David
Published: January 2023

Eosinophilic esophagitis (EoE) is an allergic disease characterized by esophageal eosinophilia, inflammation, and dysfunction. EoE has become increasingly common, but current management strategies are nonspecific. Thus, there is an urgent need to identify new pathways that could be targeted to treat EoE. Recently, EoE was associated with a gain-of-function polymorphism in the gene that encodes thymic stromal lymphopoietin (TSLP), a cytokine that promotes allergic inflammation and peripheral basophilia. However, how TSLP and basophils might contribute to the development of eosinophil responses during EoE remains unknown. Here, we employed a new murine model of EoE-like disease to investigate the role of TSLP and basophils in promoting esophageal eosinophil responses. Development of esophageal eosinophil responses was dependent on TSLP-elicited basophils, and antibody-mediated neutralization of TSLP or depletion of basophils ameliorated established esophageal eosinophilia. In addition, we examined how sort-purified human basophils influence eosinophil responses in vitro. Finally, elevated TSLP levels and exaggerated basophil responses observed in esophageal biopsies from EoE patients correlated with eosinophil responses. Together, these data indicate that TSLP-elicited basophil responses may play a key role in mediating eosinophil responses in EoE, suggesting that the TSLP-basophil axis could represent a new and promising therapeutic target to treat this disease.
Diesel exhaust particles potentiate allergic Th2/Th17 recall responses (HYP7P.301)
Authors: Brandt Eric , LeCras Timothy , Khurana Hershey Gurjit
Published: January 2023

Diesel exhaust particles (DEP) are a major component of traffic related air pollution and have been implicated in asthma exacerbations. In mice intra-tracheally exposed 9 times over the course of 3 weeks to either saline, DEP, house dust mite (HDM) or HDM and DEP, we assessed the rate of DEP clearance from the lung and examined its impacts on HDM recall responses. DEP alone did not induce the classic hallmarks of asthma but promoted a Th17 response resulting in long lasting chronic neutrophilia. DEP was still observed in a majority of alveolar macrophages 6 weeks after exposure. Coexposures to HDM and DEP generated a mixed Th2/Th17 response with a more severe asthma phenotype that completely resolved within 4 weeks. However, upon in vitro restimulation with HDM, Th2/Th17 responses were significantly enhanced in lung and draining lymph node cell cultures of HDM+DEP exposed mice compared to mice exposed to HDM alone. In conclusion, the prolonged presence of DEP in the lungs is associated with chronic neutrophilia. DEP-mediated exacerbation of allergen-induced asthma promotes long-term expansion of effector/memory Th17 and Th2 populations.