bluuebunny/crossref_metadata_2025 huggingface dataset:
Scaffold protein Disc-Large Homolog 1 is required for T cell receptor-induced activation of regulatory T cell function (121.14)
Authors: Zanin-Zhorov Alexandra , Lin Jiqiang , Scher Jose , Kumari Sudha , Blair David , Hippen Keli , Blazar Bruce , Abramson Steven , Lafaille Juan , Dustin Michael
Published: January 2023

Foxp3+CD4+CD25high regulatory T cell (Treg) suppression of inflammation depends on T cell receptor (TCR)-mediated NFAT activation with reduced Akt activity. We investigated the role of the scaffold protein Dlgh1 in linking the TCR to this unique signaling outcome. The Treg immunological synapse (IS) recruited 4-fold more Dlgh1 than conventional CD4+ T cell IS. Tregs isolated from patients with active rheumatoid arthritis, or treated with tumor necrosis factor-α, display reduced function and diminished Dlgh1 recruitment to the IS. Furthermore, Dlgh1 silencing abrogated Treg function, impaired NFATc1 activation, reduced PTEN levels and increased Akt activation. Dlgh1 operates independently of the negative feedback pathway mediated by the related adapter protein Carma-1 and thus presents an array of novel targets to selectively manipulate Treg function.
Quiescence regulator Foxp1 restrains antigen-induced T cell activation and response by mechanisms including a novel negative feedback loop (121.17)
Authors: Wang Haikun , Feng Xiaoming , Takata Hiroshi , Wolf Amaya , Strauman Maura , Tian Lifeng , Erikson Jan , Jiang Ping , Tucker Philip , Hu Hui
Published: January 2023

Previously we have shown that transcription factor Foxp1 is an essential regulator in maintaining the quiescence of naive T cells during homeostasis. In mice, Foxp1 has four isoforms. Now we find that whereas the full-length Foxp1A is constitutively expressed in mature T cells, T cell receptor (TCR) stimulation induces the expression of a short Foxp1 isoform, Foxp1D, which in turn dampens TCR signaling and T cell proliferation, constituting a negative feedback loop. In Foxp1D conditional transgenic mice, Foxp1D transgene in T cells reduces the generation of CD8+ and CD4+ T effectors to viral infections. On the contrary, Foxp1-deficient T cells (lacking all Foxp1 isoforms) elicit a faster and stronger effector response to antigen challenge. These results suggest that quiescence gene Foxp1 plays an important role in restraining antigen-induced T cell activation and response with a novel negative feedback loop by Foxp1D.
cAMP responsive element modulator (CREM)α induces epigenetic remodeling of IL2 in T lymphocytes from SLE patients (121.2)
Authors: Hedrich Christian , Rauen Thomas , Tsokos George
Published: January 2023

IL-2 functions as an auto- and paracrine growth-factor for T lymphocytes and is essential during their proliferation and activation. The absence of IL-2 has been linked to the development of autoimmune phenotypes in mice and humans. The multi-factorial autoimmune disease Systemic lupus erythematosus (SLE) is characterized by disrupted transcription factor networks and dysregulated cytokine expression, antigen-presentation, and lymphocyte function. The failure to express IL-2 is a hallmark of SLE T lymphocytes and results in reduced numbers of regulatory T lymphocytes that prevent autoimmunity. Impaired IL-2 expression was linked to over-production of the transcription regulatory factor cAMP response element modulator (CREM)α that in turn binds to a CRE site within the IL2 promoter (-180 CRE) in SLE T lymphocytes. Here, we document the involvement of CREMα mediated gene-wide HDAC1-directed deacetylation of histone H3K18, and DNMT3a directed CpG-DNA hypermethylation in the silencing of IL2 in T lymphocytes from SLE patients. We provide evidence that CREMα mediates silencing of IL2 in SLE T cells through histone deacetylation, and CpG-DNA methylation. Our findings add further evidence to the discussion of whether epigenetic modifications display region and tissue-specific patterns in health and disease. Thus, CREMα may serve as a promising target to correct cytokine expression in patients with SLE.
New modes of ERK feedback regulation in T cell receptor signaling revealed by quantitative phosphoproteomic analysis (121.29)
Authors: Helou Ynes , Nguyen Vinh , Beik Samantha , Hung Norris , Salomon Arthur
Published: January 2023

Competing positive and negative signaling feedback pathways play a critical role in tuning the sensitivity of T cell receptor (TCR) activation by selectively enhancing responses to foreign ligands while suppressing signals from self-peptides. In response to TCR agonist engagement, ERK is activated to positively regulate TCR signaling through phosphorylation of Ser59 on Lck. To obtain a wide-scale view of the role of ERK in propagating TCR signaling, a quantitative phosphoproteomic analysis of 417 tyrosine phosphorylation sites by mass spectrometry was performed on Jurkat E6-1 cells in the presence of U0126, a MEK1/2 specific inhibitor of ERK activation. Relative to untreated cells, ERK-inhibited cells showed constitutive decreases in phosphorylation through a TCR stimulation time course on tyrosine residues found on upstream signaling proteins (CD3 chains, Lck, ZAP-70), as well as downstream signaling proteins (LAT, SLP-76, PLCγ1, Itk), consistent with ERK’s proposed role in TCR positive feedback signaling. Interestingly, ZAP-70 Tyr598 and CD3 ζ Tyr123 showed elevated phosphorylation in response to U0126 treatment, suggesting differential regulation of these sites via ERK feedback. Furthermore, elevated phosphorylation on the activation site of Fyn was observed in response to ERK inhibition. These findings shed light on novel signaling events that are regulated by this kinase which may fine tune TCR activation.
Title: PD-1 regulates T cell proliferation in a tissue and subset specific manner during normal mouse pregnancy (122.11)
Authors: Bonney Elizabeth , Shepard Michelle
Published: January 2023

The regulation of T cell homeostasis during pregnancy has important implications for maternal tolerance and immunity. Evidence suggests that Programmed Death-1 (PD-1) participates in regulation of T cell homeostasis and peripheral tolerance. To examine the contribution of PD-1 signaling on T cell homeostasis during normal mouse pregnancy, we examined T cell number or proportion, PD-1 expression, proliferation, and apoptosis by flow cytometry, BrdU incorporation, and TUNEL assay in pregnant mice given anti-PD-1 blocking antibody or control on days 10, 12, and 14 of gestation. We observed tissue, treatment, and T cell-specific differences in PD-1 expression. Both pregnancy and PD-1 blockade increased T cell proliferation in the spleen while this effect was limited to CD4 T cells in the uterine- draining nodes. In the uterus, PD-1 blockade markedly altered the composition of the T cell pool. These studies support the idea that pregnancy is a state of dynamic T cell homeostasis and suggest that this state is partially supported by PD-1 signaling.
Imaging the initial cellular events in EAE. (123.26)
Authors: Barkauskas Deborah , Myers Jay , Othman Youmna , Huang Alex
Published: January 2023

Limitations in current techniques to capture rare immune cell events have hindered the characterization of the initiation and progression of experimental autoimmune encephalomyelitis (EAE). With the cranial window and intravital two-photon microscopy techniques, we followed the initial dynamic and sequential cellular processes preceding EAE development. Disease progression was imaged daily in the first 12 days post EAE induction in either CX3CR1-GFP or CD11c-GFP transgenic mice containing adoptively transferred naïve ubiquitin-CFP myelin oligodendrocyte glycoprotein (MOG) specific T cells. Blood-brain barrier (BBB) permeability was assessed by i.v. injection of fluorescent dextran or quantum dots. We hypothesize that lymphocyte accumulation follows an initial transient BBB leak caused by pertussis toxin-initiated inflammation, with subsequent accumulation and activation of microglia and perivascular antigen presenting cells (APCs) facilitating the tissue-specific infiltration of MOG-specific T cells into the brain parenchyma via intra-luminal contact. Indeed, our sequential imaging experiments demonstrated transient leaks in BBB at the pia surface within the first 4 days post EAE induction, local APC accumulation from days 2 to 6, followed by T cell infiltration adjacent to local CNS vessel leak. Future work involves further defining the cellular and molecular regulation of CNS peri-vascular microenvironment responsible for cross-BBB attraction of MOG-specific cells.
Intrinsic Regulation of Autoimmune CD4+ Memory T Cells by Subtle Variations in CTLA4 levels (123.38)
Authors: Devarajan Priyadharshini , Miska Jason , Torres Juan , Chen Zhibin
Published: January 2023

Cytotoxic T lymphocyte antigen 4 (CTLA4) is a negative regulator of immune function. Genetic polymorphisms in the CTLA4 locus leading to subtle variations in CTLA4 expression have been linked to various autoimmune diseases including type 1 diabetes. We hypothesize that CTLA4 regulates the immune response in these disease settings through memory T cells. To test this hypothesis, a unique transgenic model of CTLA4 RNAi has been used to mimic subtle quantitative variations of CTLA4 in humans. BDC2.5 transgenic mice with the CTLA4 RNAi had a higher incidence of diabetes. An analysis of different T cell populations in the lymph nodes and pancreatic tissue revealed an increased number of CD4+ effector memory T cells. To investigate if the increase in CD4+ effector memory T cells is a direct effect of CTLA4 reduction on the conventional T cell compartment (cell-intrinsic) or if it is mediated through reduced CTLA4 on regulatory T cells (cell-extrinsic), a series of adoptive transfer experiments were done with CD4+ naïve, effector, effector memory and regulatory T cells. These experiments suggested a cell-intrinsic mechanism where reduced CTLA4 in the CD4+ effector memory T cell itself causes increased activation and survival/proliferation. These results indicate that subtle reductions in CTLA4 levels have a selective impact on CD4+ effector memory T cells, which may play a critical role in CTLA4-determined risk of autoimmune diabetes.
Metabolic regulation of CD4+ T cells by Estrogen Related Receptor-α modulates autoimmune and allergic disease (123.48)
Authors: Michalek Ryan , Gerriets Valerie , Nichols Amanda , Inoue Makoto , Theriot Barbara , Giguere Vincent , McDonnell Donald , Shinohara Mari , Walker Julia , Rathmell Jeffrey
Published: January 2023

Upon activation, naïve CD4+ T cells differentiate into effector (Teff) subsets that provide protective immunity or inducible regulatory (Treg) subsets which balance inappropriate inflammation and autoimmunity. Consistent with distinct immunological roles, Teff and Treg utilize specialized metabolic programs for specification and function with Treg increasing lipid oxidation and Teff dependent on glucose metabolism. We have identified the nuclear receptor estrogen related receptor-α (ERRα) as a critical regulator of metabolic gene expression required for Teff. Here, we demonstrate through the use of an experimental autoimmune encephalomyelitis model that ERRα contributes to the severity of autoimmune disease as limited glucose metabolism in the absence of ERRα selectively diminished Th17, but not Treg generation. ERRα contributed to inappropriate inflammation as Teff glucose metabolism, expansion, and function were reduced in ERRα-/- mice and in siRNA treated human CD4+ T cells. Additionally, elevated ERRα expression and glycolytic metabolism were also observed in a murine model of asthma and from lavage fluid of asthmatic patients. Importantly, pharmacological inhibition of ERRα following an aerosol response alleviated airway inflammation, demonstrating a metabolic requirement for ERRα in Teff maintenance. Thus, ERRα is a selective transcriptional regulator of Teff metabolism that may provide a metabolic means to modulate immunity.
Oral tolerance in patients with food protein induced enterocolitis syndrome (FPIES); Evidence of impaired induction of oral tolerance in FPIES patients with persistent reaction to multiple foods (123.9)
Authors: Jyonouchi Harumi , Geng Lee , Streck Deanna , Toruner Gokce
Published: January 2023

Treatment for FPIES is the avoidance of offending food, i.e., a restricted diet (RD). While most FPIES children are good responders to the RD (FPIES-GR), some are not (FPIES-poor responders: FPIES-PR) with persistent non-IgE mediated reactivity to multiple foods. This study assessed production of counter-regulatory cytokines crucial for oral tolerance by peripheral blood mononuclear cells (PBMCs) in FPIES-PR (N=18, 0.6 -4.8 yr), FPIES-GR (N=22, 1.1-5.9 yr), and normal control (N=19, 1.0-5.7 yr) children after FPIES children were on the RD. PBMCs were stimulated with agonists of toll like receptors (TLRs) and representative luminal antigens (Ags) (soy, cow’s milk, and wheat proteins, and candida Ag). Microarray analysis of PB moncytes was also performed on samples from some of these children. FPIES-PR children revealed more severe clinical features including failure to thrive, intolerance to probiotics, and anaphylaxis like reaction to rice than FPIES-GR children (p<0.005). FPIES-PR PBMCs produced less sTNFRII and IL-10 both with TLR agonists and without a stimulus than the control groups. FPIRS-PR cells also produced less IL-6 with a TLR4 agonist and without a stimulus than normal controls (p<0.05). Such changes were not observed in FPIES-GR cells. FPIES-PR PBMCs produced less TGF-β with and without luminal Ag than PFIES-GR PBMCs. Taken together, FPIES-PR children may have impaired production of counter-regulatory cytokines that are crucial for oral tolerance.
Optimized buffer system for detection of transcription factors and surface proteins in T-cells validated in high throughput screening of activated T-regulatory cells. (124.10)
Authors: Hingorani Ravi , Vidal Jason , Le Hai , Waterman Paul , Rohrer Jurg , Boyce Christopher
Published: January 2023

Intracellular detection of antigens at the single cell level by flow cytometry requires optimized buffers and methods for detection of a given specificity and the compartment probed. Using a new buffer system tightly controlled for low non-specific staining, increased resolution, and significantly lower cell loss, we have developed an optimized system for detection of intracellular proteins by flow cytometry. This system works across multiple tissues from human and mouse models offering a one-method system for reliable antibody cross-reactivity tests. The optimized system detects proteins within the nucleus, endoplasmic reticulum, outer mitochondrial membrane, and the Golgi complex, with focus on optimal resolution of DNA-binding transcription factors. Intracellular targets detected included; FoxP3, T-bet, RORγT, GATA-3, Satb1, Bcl6, Bcl2, pH2AX, Ki-67, Themis, Nanog, Sox1, Sox2, Oct 3/4, Pax-6, Nestin, and various cytokines. In addition, fluorescent antibodies to cell surface antigens for multi-color flow were also found to be compatible with the optimized buffer system. Further verification of the buffer was done by high throughput screening of human cell surface markers with the BD LyoplateTM on activated human T-regulatory cells (Treg). GARP positive and negative Treg subsets were screened for various markers and the results are discussed in the poster.
Type I interferon inhibits acute IL-5 and IL-13 expression in human memory CD4+ T cells (125.12)
Authors: Gonzales Sarah , Huber Jonathan , Farrar J. David
Published: January 2023

Type I Interferon (IFN-α/β) is an innate cytokine important for fighting infections and is used to treat hepatitis C infection and multiple sclerosis, among other diseases. Although the innate role of this cytokine has been studied extensively, our group has shown that IFN-α/β plays a role in shaping the adaptive immune response in humans. Recently, we demonstrated that IFN-α/β inhibits Th2 development by regulating mRNA and protein levels of GATA3. Regulation of GATA3 in turn suppresses Th2 cytokine production, without effecting IFN-γ production. In addition to blocking Th2 development, we now demonstrate an acute inhibition of human memory T cell function upon treatment with IFN-α/β. Human memory CD4+ T cells acutely activated with anti-CD3 in the presence of IFN-α/β resulted in reduced mRNA levels of Th2 cytokine genes, IL5 and IL13, but did not effect IL4 expression. GATA3 levels were not affected, suggesting a GATA3-independent mechanism. Further experiments have determined that this regulatory mechanism does not require new protein synthesis. This mechanism seems to be specific to IFN-α/β, as neither IFN-γ nor IFN-λ treatment were able to repress Th2 cytokine mRNA expression to the extent of IFN-α/β. This study has uncovered an additional level of regulation that blocks early TCR signaling events that affect Th2 function. This novel pathway suggests that treatment of atopic asthmatic individuals with IFN-α/β can regulate both Th2 development and memory Th2 function.
Inhibition of Tyrosine Kinase Reduces Pulmonary Inflammation in A Mouse Model of Asthma (125.14)
Authors: Kim Jiyoun , Yang Beom seok , Remick Daniel
Published: January 2023

Protein tyrosine kinases play a critical role in activation of inflammatory cells and expression of airways inflammatory mediators. We investigated if a discoidin domain receptor family tyrosine kinase inhibitor, LCB03-0110 will reduce pulmonary inflammation in a mouse model of asthma induced by cockroach allergen (CRA). BALB/c mice were exposed to 3 pulmonary challenges via hypopharyngeal administration of CRA on day 0, 14 and 21. Two hours after the last challenge, airway hyperresponsiveness (AHR) was measured and the mice sacrificed for bronchoalveolar lavage (BAL) fluid and lung tissue. Groups of mice were treated with LCB03-0110 or vehicle one hour prior to the 2nd and 3rd CRA challenge on day 14 and 21. AHR was significantly reduced in the LCB -treated mice when compared to vehicle -treated group (454 vs. 235% Penh increase of baseline). Inhibition of tyrosine kinase activity significantly decreased levels of BAL CXC chemokines, KC (2322 ±169 vs. 1096 ±117 pg/ml) and MIP-2 (174 ±13 vs. 64 ±8 pg/ml) and pulmonary recruitment of neutrophils (903±31 vs. 364 ±27 x1000 cell/mouse). Further, mucin production in airways was significantly reduced. Taken together, treatment with a tyrosine kinase inhibitor significantly reduces AHR and pulmonary inflammation in a mouse model of asthma induced by cockroach allergens. This suggests that inhibition of tyrosine kinase may be a novel therapeutic target in the treatment of asthma and other inflammatory airway diseases.
Rapid polyclonal desensitization with monoclonal antibodies (125.15)
Authors: Khodoun Marat , Strait Richard , Kucuk Zeynep , Krishnamurthy Durga , Finkelman Fred
Published: January 2023

Rapid desensitization, a procedure in which individuals allergic to an antigen are treated at short intervals with increasing doses of that antigen until they tolerate a large dose, is an effective, albeit risky way to induce temporary tolerance. We have adapted this procedure to suppress IgE- or IgG-mediated immediate hypersensitivity in mice by injecting them with activating mAbs to IgE, FcϵRIα, or FcγRII/RIII, starting with doses too small to induce detectable reactions and doubling doses every 90 minutes. Rapid desensitization with anti-IgE mAb suppressed IgE-mediated immediate hypersensitivity; however, some mice developed mild anaphylaxis during desensitization. Rapid desensitization with an anti-FcϵRIα mAb that only binds FcϵRI that is not occupied by IgE suppressed both active and passive IgE-mediated anaphylaxis and induced no detectable reactions, but took several days to become fully effective by eliminating nearly all mast cell FcϵRI. Rapid desensitization with this mAb also rapidly, but temporarily, desensitized mast cells by decreasing their responsiveness to FcϵRI crosslinking. Rapid desensitization with anti-FcγRII/RIII mAb induced no detectable adverse reactions and rapidly suppressed IgG-mediated anaphylaxis. Similar approaches with mAbs to human IgE or IgG receptors may be useful as prophylaxis for human immediate hypersensitivity disorders, including anaphylaxis, food allergy, atopic dermatitis and asthma.
Diversity of epitope recognition of IgG4 antibodies induced by birch-pollen specific immunotherapy (125.3)
Authors: Subbarayal Brinda , Schiller Dirk , Möbs Christian , Pfützner Wolfgang , Vieths Stephan , Bohle Barbara
Published: January 2023

Birch pollen (BP)-allergic patients sensitized to the major BP allergen, Bet v 1, often show IgE-cross-reactivity with Mal d 1 in apple and Cor a 1 in hazelnut. We recently found that specific immunotherapy (SIT) with BP induces high titers of Bet v 1-specific IgG4. In parallel, many BP-SIT-treated patients developed food-reactive IgG4. However, around 25% of patients showed no detectable food-specific IgG4 response. The diversity of epitope recognition of IgG4 antibodies (abs) in BP-SIT-treated patients developing or lacking food-reactive IgG4 were compared. Sera containing Bet v 1-, Mal d 1- and Cor a 1-specific IgE and Bet v 1-specific IgG4 with and without food-reactive IgG4 abs were included. Blocking capacity of the sera was analysed in facilitated antigen-binding and basophil activation assays. Potential epitopes recognized by allergen-specific IgE and IgG4 abs were screened by competitive immunoscreening with a random phage-displayed 12-mer peptide library. Patients showing food reactive-IgG4 with blocking capacity recognized a higher number of putative epitopes distributed over the surface of allergens. In contrast, a restricted number of putative epitopes was recognized by sera from patients who had failed to develop food-reactive IgG4. The induction of polyclonal IgG4 responses during BP-SIT may be a prerequisite for the development of abs that cross-react with Bet v 1-related food allergens and consequently, block food-reactive IgE abs.
Oral tolerance induction does not resolve allergic enteropathy due to residual IgE (125.7)
Authors: Toda Masako , Burggraf Manja , Nakajima-Adachi Haruyo , Hachimura Satoshi , Krause Maren , Kiyono Hiroshi , Hermann-Josef Gröne , Vieths Stefan
Published: January 2023

Oral immunotherapy (OIT) has gained attention as a therapy for food allergies. To investigate efficacy and safety of OIT pre-clinically, we established a murine model of allergic enteropathy. BALB/c mice were sensitised with ovalbumin (OVA, a major egg white allergen) plus ALUM and fed egg-white diet (EW-diet). The mice developed gastrointestinal symptoms (e.g. weight loss) and inflammation in the jejunum accompanied by a strong induction of IgE antibodies during the first 7 days of the allergenic diet. Importantly, the mice presented resolution of the symptoms and remarkable reduction of OVA-specific IgE levels in the sera if they were fed EW-diet more than 7 days. Reduced proliferation of T-cells from spleens and mesenteric lymph nodes of the mice on day 28 of EW-diet indicated that the continuous feeding of the allergenic diet induced oral tolerance. However, the inflammation in the jejunum of the mice was aggravated at this time point. We hypothesized that residual OVA-specific IgE antibodies promoted the intestinal inflammation, although oral tolerance against OVA was induced by the long-term EW diet. To verify it, we used mice deficient of FcϵRI, a high affinity IgE receptor. FcϵRI deficient mice presented intestinal inflammation on day 7 of EW-diet after OVA sensitisation. However, if EW-diet was continued, their intestinal inflammation was resolved. Our results suggest that residual IgE could be a poor prognostic factor for recovery from allergic enteropathy in OIT.
Anticipitated and Surprising effect of IL-17-producing CD4+ T Cells on chronic Graft-Versus-Host Disease after Unmanipulated Allogeneic Blood and Marrow Transplantation (126.17)
Authors: Zhao Xiang-Yu , Xu Ling-Ling , Lv Meng , Xu Lan-Ping , Liu Dai-Hong , Liu Kai-Yan , Huang Xiao-Jun
Published: January 2023

The aim of this study was to investigate the effects of Th17 cells on chronic graft-versus-host disease (cGVHD) in patients who had undergone allogeneic transplantation. Allograft from 41 patients were analyzed for Th17 with respect to cGVHD. Seven patients with cGVHD onset and after those achieved complete remission (CR) were monitored for the Th17 in the peripheral blood (PB). RNA was isolated from PB mononuclear cells to detect the transcript levels of STAT5, T-bet, Foxp3 and RORγ. There were significantly differences in Th17 dose in allograft between patients with and without cGVHD (p=0.012), especially with local or pure cGVHD (p=0.001). Patients received a higher Th17 dose in allograft (>1.67×106/kg, p=0.018,n=20) exhibited a higher incidence of cGVHD compared to those received lower Th17 dose (n=21). Th17 cells were highly increased when cGVHD onset and decreased drastically following CR (p=0.018). Patients received a higher Th17 dose exhibited a trend to have the lower incidence of transplantation related mortality (p=0.084) and relapse (p=0.181), and achieved higher incidence of overall survival (p=0.082) and leukemia free survival (p=0.026). No differences in the transcript levels of STAT, T-bet, Foxp3 and RORγwere found between patients with cGVHD onset and after CR. These results suggested that Th17 cells contributed to cGVHD, especially skin cGVHD, and might account for the anti-leukemia effect, therefore increasing the leukemia free survival.
IL-22 protects intestinal stem cells from immune-mediated tissue damage and regulates sensitivity to graft vs. host disease (126.31)
Authors: Hanash Alan , Dudakov Jarrod , Hua Guoqiang , Liu Chen , Fouser Lynette , Kolesnick Richard , Blazar Bruce , van den Brink Marcel
Published: January 2023

Intestinal graft vs. host disease (GVHD) is a major complication of allogeneic bone marrow transplantation (allo-BMT). Strategies to limit GVHD by selective promotion of epithelial regeneration in the absence of immunosuppression are largely unknown. We investigated the role of IL-22 in allo-BMT and found that IL-22 levels in small and large intestine were increased after BMT after radiation injury (RI) without BMT. IL-22 upregulation after RI was dependent on the presence of IL-23p40. Although intestinal IL-22 levels were increased after T cell-depleted (TCD) BMT, intestinal IL-22 was reduced by GVHD, as IL-22 production was mediated by host-derived innate lymphoid cells (ILC) that were eliminated by GVHD. Furthermore, host-derived IL-22 was critical for reduction of GVHD morbidity, mortality, and intestinal pathology. GVHD in IL-22 KO mice led to increased apoptosis in epithelial crypts where the intestinal epithelial stem/progenitor cell niche is located. Immunohistochemistry and immunofluorescence demonstrated IL-22 receptor expression on intestinal stem cells (ISC) and progenitors. Allo-BMT in Lgr5-LacZ reporter mice indicated that ISC were targeted by GVHD, and GVHD in IL-22 KO mice led to dramatic ISC depletion. IL-22 is thus critical for protection of host epithelium during GVHD and critical for protection of ISC. These findings may have broad relevance for protection of ISC and intestinal epithelium in clinical GVHD and other inflammatory intestinal diseases.
Pre-transplant administration of antithymocyte globulin increases its efficacy in inhibiting anti-donor memory T cell responses (126.9)
Authors: Ayasoufi Katayoun , Fan Ran , Yu Hong , Williams John , Valujskikh Anna
Published: January 2023

Antithymocyte globulin (ATG) is a lymphoablative agent used in transplant patients. In this study, we tested how murine analog of ATG (mATG)affects pre-existent donor-reactive memory T cells. mATG treatment of heart allograft recipients on d.-7 and -4 (pre-TP)led to better outcome than treatment on d.0 and d.4 (peri-TP). Pre-TP mATG resulted in lower frequencies of donor-specific IFNγ-secreting T cells than peri-TP mATG(72000 vs 12500spots/million CD44hi cells). Studies in recipients injected with tracer subsets of donor-reactive T cells showed that while both mATG regimens depleted naïve T cells equally well, pre-TP mATG was four times more efficient in depleting pre-existing memory T cells. IFNγ production by donor-specific memory T cell was diminished by 99% with pre-TP mATG and only by 70% with peri-TP mATG. Transfer of TCR transgenic Ld-reactive 2C memory CD8 cells into recipients of Ld+ or Ld- heart allografts showed that memory T cells resistance to peri-TP mATG depletion depended on the systemic inflammation after transplantation but not on their antigen specificity. In summary,pre-TP ATG is more efficient in targeting pre-existing alloreactive memory T cells and anti-donor T cell responses. Our findings may guide the future use of ATG in sensitized transplant patients.
Expression of Germinal Center B Cell Markers in Bortezomib-Resistant Multiple Myeloma Cells (127.11)
Authors: Baughn Linda , Stessman Holly , Mansoor Aatif , Van Ness Brian
Published: January 2023

Multiple myeloma (MM), the second most common hematopoietic malignancy, is an incurable plasma cell neoplasm. The proteasome inhibitor, bortezomib (Bz), has been widely used to treat MM. However, patients eventually relapse due to drug resistance. Identifying signatures that distinguish Bz sensitive from resistant cells is critical for the development of novel approaches to reverse resistance. We have utilized isogenic pairs of Bz sensitive and resistant lines derived from the iMycCα/Bcl-xL mouse model of MM. This highly robust model resembles human MM based on gene expression, chromosomal abnormalities and progression of disease and response to therapy. In populations with an acquired resistance to Bz, we observed increased expression of Pax5 and AID and reduced expression of CD93 compared to parental Bz sensitive populations. Although most parental cells are CD93+, we have isolated a subpopulation of CD93- cells that expresses 20x more AID and display an innate resistance to Bz, while the CD93+ population remains sensitive. Time course analysis following low-dose Bz treatment indicates that Bz increases the expression and promotes the stabilization of Pax5 and AID demonstrating drug-induced changes in markers associated with B cell differentiation relating to the ultimate emergence of the resistance phenotype. Targeting B cell differentiation may represent a rational therapeutic approach to overcoming drug resistance.
In vivo three dimensional MRI of GL261 syngeneic gliomas concurrently with analysis of CNS infiltrating tumor-specific killer T cell responses (127.15)
Authors: Johnson Aaron , Litterman Adam , Ohlfest John , Jin Fang , Hanson Lisa , Gamez Jeff , Chae Michael , Carlson Brett , Sarkaria Jann , Parney Ian , Pirko Istvan
Published: January 2023

Glioblastoma multiforme (GBM) is among the most lethal of cancers. Enhancing anti-tumor killer T cell responses via dendritic cell vaccines has correlated with a positive outcome in selected GBM patients. Nevertheless, the mechanisms by which killer T cell responses to GBM are inhibited or enhanced remain poorly defined. We therefore developed the GL261 “Quad Cassette” glioma cell line that expresses model T cell epitopes in the immunocompetent C57BL/6 mouse. Tumor size and inflammatory profiles observed in these animals was then compared to C57BL/6 mice administered the parent GL261 glioma cell line. Resulting tumors present with a tumor mass surrounded by considerable edema visible by gadolinium enhanced T1 and T2 weighted MRI. Both edema and tumor mass visible by MRI were quantified using Analyze 10.0 software which enables 3D volumetric analysis of MRI images. C57BL/6 mice with GL261 “Quad Cassette”, but not parent GL261 gliomas, presented with smaller tumor mass and significant brain infiltrating tumor specific Kb:ova specific CD8 T cells. We therefore conclude that the GL261 Quag Cassette system is suitable for studying tumor epitope specific CD8 T cell responses using the vast genetic and immunologic resources available for the C57BL/6 mouse background. Furthermore, incorporation of the first true 3D volumetric analysis of GL261 glioma size with small animal MRI will enable investigation of immunotherapeutic treatments in vivo without euthanizing the animal.
Activated mouse CD4+Foxp3- T cells facilitate melanoma metastasis via Qa-1-dependent suppression of NK cell cytotoxicity (127.17)
Authors: Gao Wenda , Wang Xiaojuan , Hu Jie , Cui Yanyan , Wang Qinghong , Luo Gaoxing , He Weifeng , Yuan Jun , Wu Yan , Sun Xiaofeng , Robson Simon , Li Xianchang , Tan Jiangling , Peng Yanmeng , Zhou Junyi , Xue Gang , Lu Linrong , Wu Jun
Published: January 2023

The regulatory activities of mouse CD4+Foxp3+ T cells on various immune cells, including NK cells, have been well documented. Under some conditions, conventional CD4+Foxp3- T cells are able to acquire inhibitory function on other T cells, but their roles in controlling innate immune cells are poorly defined. As a potential cellular therapy for cancer, ex vivo activated CD4+Foxp3- effector T cells are often infused back in vivo to suppress tumor growth and metastasis. Whether such activated T cells could affect NK cell anti-tumor function is unclear. We found that mitogen-activated CD4+Foxp3- T cells exhibited potent suppressor function on NK cell proliferation and cytotoxicity in vitro, and notably facilitated B16 melanoma metastasis in vivo. Suppression of NK cells by activated CD4+Foxp3- T cells is cell-cell contact dependent and is mediated by Qa-1:NKG2A interaction, as Qa-1-blocking antibody could completely reverse this suppression, and significantly inhibited otherwise facilitated melanoma metastasis. Moreover, activated CD4+Foxp3- cells from Qa-1-/- mice completely lost the suppressor activity on NK cells, and failed to facilitate melanoma metastasis when transferred in vivo. Taken together, our findings indicate that innate anti-tumor response is counter-regulated by the activation of adaptive immunity, and thus the regulatory role of activated CD4+Foxp3- T cells in NK cell activity must be taken into consideration in the future design of anti-tumor biotherapies.
Tumor-derived factors promote differentiation of immunosuppressive Gr-1+cDC through TLR2. (127.25)
Authors: Tang Michael , Diao Jun , Zhao Jun , Cattral Mark
Published: January 2023

Tumor cells secrete endogenous danger signals that modulate the tumor inflammatory microenvironment. We have found that the intra-tumor inflammatory milieu skews differentiation of DC precursors (pre-cDC) towards a novel immunosuppressive Gr-1+ subpopulation. Inhibition of this differentiation enhanced cytotoxic T cells expansion and suppressed tumor growth. We sought to identify signaling mechanisms that regulate Gr-1+DC development. Splenic Pre-cDC from wildtype and MyD88-/- mice were incubated with tumor-conditioned medium (TCM) derived from the Lewis lung carcinoma cell line or fibroblast control medium. Supernatants were collected to measure various cytokine levels and cell surface expression of Gr-1 was monitored by flow cytometry. We found that TCM, but not control medium induced IL-6, IL-10 & IL-1β production and Gr-1+DC development. These cells produced high amounts of IL-10 after secondary stimulation with LPS. MyD88-/- Pre-cDC stimulated with TCM did not produce cytokines or differentiate into Gr-1+DC. Tumors from MyD88-/- mice contained a significantly lower frequency of Gr-1+DC compared to wildtype mice (14.1% vs. 1.1%; p=0.001, n>5). To determine whether Toll-like receptor 2 (TLR2) was involved in Gr-1+DC development, we stimulated Pre-cDC with different TLR agonists. We found that both Pam3CSK4 (TLR2/TLR1) and FSL-1 (TLR2/TLR6), but not LPS (TLR4) mimicked the activity of TCM. Together these data suggest that MyD88 and TLR2 are involved in Gr-1+DC development.
Targeting Tumor-Expressing CCR6 Inhibits Colorectal Cancer Progression in Mice (127.44)
Authors: Wang Honglin , Liu Jinlin
Published: January 2023

Background: CCR6/CCL20 expressed by colorectal cancer (CRC) cells. Although their interactions may play an essential role in proliferation and migration of CRC, the in vivo evidence is lacking. Methods: Expression of CCR6/CCL20 was evaluated in 130 primary tumor specimens from CRC patients. Mouse models was established by the injection of CMT93 transfected with luciferase in CCR6-/- mice, or by crossing CCR6-/- mice with APCmin/+ mice. Intratumor injection of anti-CCR6 was performed in CCR6-/- mice grafted luciferase-labeled CMT93. Luciferase intensity of CMT93 was monitored using the IVIS Imaging System. Intestinal tumor development in APCmin/+ mice was studied with a dissecting microscope. Migration of CMT-93 treated with anti-CCR6 was investigated by the Real-Time Cell Analyzer. Results: In contrast to paratumor tissues, CCR6/CCL20 predominantly expressed by tumor cells in CRC patients of all tumor stages. CCR6/CCL20 was consistently identified in CMT-93. Injection of anti-CCR6, significantly inhibited the growth of CRC in CCR6-/- mice. Knock-out of CCR6 resulted in a decrease in numbers and size of intestinal adenomas in APCmin/+ mice. Anti-CCR6 treatment markedly inhibited the migration of CMT-93 in vitro. Conclusion: The CCR6/CCL20 interaction plays an essential role in proliferation and migration of colorectal cancer cells via the autocrine mechanism in mice, and selectively targeting tumor-expressing CCR6 may harbor a novel therapeutic strategy for human CRC.
Circulating biomarker-IgM complexes in the serum of pediatric patients with osteosarcoma and Ewing tumor (127.9)
Authors: Savitskaya Yulia , Rico Genaro , Linares Luis , Delgado Ernesto , Martinez Elisa , Tellez Rene , Estrada Erendira , Marin Norma , Ibarra Clemente
Published: January 2023

Osteosarcoma (OS) and Ewing tumor (ET) together account for the majority of musculoskeletal sarcomas diagnosed in patients of all ages and nearly 90% of those occurring in children and adolescents. Natural IgM antibodies to tumor antigen have been reported in patients with early-stage cancer, and a panel of serum antibodies can detect cancer many years prior to imaging detection. Circulating immune complexes formed by tumor antigens and immunoglobulin M (IgM) represent a novel class of biomarkers with diagnostic value for early cancer detection. In the present study, we investigated the presence of ANG-IgM, VEGF-IgM, bFGF-IgM, PDGF-IgM in the sera of children with bone tumors. The study included 174 pediatric patients with newly diagnosed bone tumors. Express ELISA techniques have been developed to quantify immune complexes in INR. Serum samples from all patients were analyzed for the presence of biomarker-IgM immune complexes. We compared the serum level of angiogenic factors and immune complexes in children with different types of bone tumors. The highest concentration of VEGF-IgM was detected in ET patients and ANG-IgM in OS patients. The present study demonstrated increased ANG-IgM expression in the sera OS patients and a positive correlation between its expression levels and tumor vascularity evaluated by CTA. Our results have potential applications as biomarkers for controlling unwanted angiogenesis, early diagnosis and response to therapy in children with bone tumors.
Marginal zone B cells contribute to autoimmunity in the NZM2410 lupus prone mouse model. (159.13)
Authors: Zheng Ying Yi
Published: January 2023

Lupus is an autoimmune disease in which the affected individual generates antibodies (Abs) against nuclear antigens such as DNA. Our NZM2410-derived triple congenic (TC) model of lupus expresses 3 previously characterized lupus susceptibility loci. We have previously shown that the breach of follicular exclusion of TC MZB cells is associated with disease progression. The production of nuclear autoAbs occurred in tandem with a large influx of TC MZB cells in the follicles. We also found an expansion of MZB cells in TC mice as compared to non-autoimmune B6 mice that occurs prior to the production of anti-DNA autoAbs, further suggesting that MZB cells may contribute to autoimmunity. MZB cell expansion is associated with the upregulation of Notch2 signaling in these cells. Adoptive transfer of TC or B6 splenic B cells into B6.Rag1-/- recipients indicated no difference in the homeostatic expansion of MZB cells from either donor strain, whether the recipients were treated or not with CpG. However, CpG-treated recipients of TC B cells produced more anti-ssDNA IgM than recipients of B6 B cells. Our transfer results showed that although TC MZB cells do not expand more than B6 MZB cells in a lymphopenic host, TC splenic B cells produce higher level of anti-ssDNA IgM in response to CpG stimulation. Further characterization of the TC MZB cells will determine more precisely how they contribute to autoimmunity.
Genomic and functional characterization of the diverse immunoglobulin domain-containing protein (DICP) family (160.5)
Authors: Yoder Jeffrey , Haire Robert , Cannon John , O’Driscoll Marci , Ostrov David , Mueller M. , Turner Poem , Litman Ronda , Litman Gary
Published: January 2023

A heretofore-unrecognized multigene family encoding diverse immunoglobulin (Ig) domain-containing proteins (DICPs) was identified in the zebrafish genome. Twenty-nine distinct loci mapping to three chromosomal regions encode receptor-type structures possessing two classes of Ig ectodomains (D1 and D2). The sequence and number of Ig domains, transmembrane regions and signaling motifs varies between DICPs. Interindividual polymorphism and alternative RNA processing contribute to DICP diversity. Molecular models indicate that most D1 domains are of the variable (V) type; D2 domains are Ig-like. Sequence differences between D1 domains are concentrated in hypervariable regions on the front sheet strands of the Ig fold. Recombinant DICP Ig domains bind lipids, a property shared by mammalian CD300 and TREM family members. These findings suggest that novel multigene families encoding diversified immune receptors have arisen in different vertebrate lineages and effect parallel patterns of ligand recognition that potentially impact species-specific advantages.
Leukotriene B4 receptor BLT1 expression on CD8+ T cells is critical for antitumor immunity (161.5)
Authors: Sharma Rajesh , Jala Venkatakrishna , Chheda Zinal , Haribabu Bodduluri
Published: January 2023

Leukotriene B4 (LTB4) receptor, BLT1 is expressed on a verity of immune cells and has been implicated in diverse inflammatory disorders, including arthritis, atherosclerosis and asthma. In contrast, whether biological responses mediated via this receptor generate tumor promoting inflammation or anti-tumor immunity remains unexplored. Herein, we investigated the role of BLT1 in cancer progression using implantable TC-1 syngenic mouse cervical cancer model and observed accelerated tumor growth and decreased survival of BLT1−/− mice. Analysis of the tumor infiltrating leukocytes by flow cytometry and confocal microscopy revealed a significant decrease in T cells and NK cells in the tumors of the BLT1−/− mice compared to the BLT1+/+ mice. Expression profile of mRNA from the tumors of BLT1−/− mice confirmed the dramatic decrease in T and NK cell associated markers such as IFN-γ, granzyme-B and IL-2. Furthermore, depletion of CD8+ T cells enhanced the tumor growth in BLT1+/+ but not in BLT1-/- mice. Adoptive transfer of tumor educated CD8+ T cells from the BLT1+/+ but not BLT1-/- mice significantly decreased tumor growth and increased the survival in Rag2-/- mice. While the homeostatic proliferation of adoptively transferred BLT1+/+ and BLT1-/- CD8+ T- cells appears to be similar only the BLT1+/+ T-lymphocytes enter the tumors in greater numbers. These results suggest that BLT1 expression on CD8+ T cells is critical for generating effective anti-tumor immunity.
The G protein-coupled receptor mFPR2 promotes anti-tumor host defense (161.7)
Authors: Wang Ji Ming , Liu Ying , Chen Keqiang , Liu Mingyong , Gong Wanghua , Yoshimura Teizo , Ford Jill , McVicar Daniel
Published: January 2023

Abstract mFPR2 is a G protein-coupled receptor interacting with bacterial and host-derived chemotactic agonists. In vivo, mFPR2 plays a major role in innate and adaptive immune responses as shown by reduced severity of allergic airway inflammation in mFPR2-KO mice due to impaired trafficking of antigen presenting dendritic cells (DCs). The aim of this study is to examine the role of mFPR2 in anti-tumor host responses. We found that mFPR2-KO mice bearing subcutaneously implanted Lewis lung cancer (LLC) cells exhibited significantly shortened survival due to more rapidly growing tumors. In contrast, in mFPR2-transgenic (Tg) mice, subcutaneously implanted LLC tumors grew more slowly than those in wild type (WT) littermates. Mechanistic studies revealed an increased number of macrophages associated with tumors grown in mFPR2 KO mice and macrophages derived from mFPR2 KO mice showed a more potent chemotactic response to LLC-derived supernatant (LLC Sup), which could be neutralized by an anti-CCL2 antibody. In addition, macrophages from mFPR2 KO mice were more prone to assume an M2 phenotype after incubation with LLC Sup. These results suggest that mFPR2 plays an important role in host defense against tumor by sustaining macrophages in an M1 phenotype.
Anti-PDL-1 immunotherapy alters T cell anti-tumor responses and inhibits tumor progression in the lung (162.13)
Authors: Bartkowiak Todd , Zal Tomasz
Published: January 2023

Lung cancer is the second most common form of cancer-behind prostate cancer in men, and breast cancer in women-accounting for 15% of all new cases. It is also the leading cause of death among both men and women. Secondary lung cancer is also an important problem which arises from primary tumors in other tissues aggressively metastasizing to the lungs. The lung is a key site of tumor metastasis, and these secondary tumors are very difficult to treat. Treatment of lung cancer is typically invasive and ranges from chemotherapy, to radiotherapy, to surgical removal of the cancerous tissue. However, a new horizon of cancer therapy involves immunotherapy--altering and/or boosting the immune system to help fight cancer. One immune-targeted cancer therapy involves blocking the PD1/PDL1 pathway; a pathway which acts to suppress the immune system by inhibiting T cell signaling. However, little is known about how exactly this and other immunotherapies modulate anti-tumor T cell responses in vivo. We used confocal and multiphoton microscopy to track T cell motility and T cell/tumor interactions in vivo within lung tumors in PDL-1 treated mice. Blockade of PDL-1 altered the spatio-temporal patterning of T cell motility and their tumor killing power, and inhibited lung tumor progression. The knowledge of how αPDL-1 cancer immunotherapy affects the immune microenvironment of tumors can lead to the development of safer, more potent treatments for lung cancer and other forms of cancer.