bluuebunny/crossref_metadata_2025 huggingface dataset:
Characterization of functional antiviral CD8 T cell immune responses of HIV elite controllers by high dimensional phenotyping using mass cytometry (VIR6P.1169)
Authors: Kleinsteuber Katja , Rashidi Narges , Soghoian Damien , Nchinda Nzuekoh , Mavrothalassitis Orestes , Buus Soren , George Tad , Ornatsky Olga , Walker Bruce
Published: January 2023

Elite controllers (EC) are individuals who control HIV infection without therapy through CD8 T cells. Chronic infections can lead to T cell exhaustion characterized by reduced proliferation and cytokine expression as well as upregulation of co-inhibitory molecules like PD-1. CD8 T cells from EC are more effective at killing HIV-infected targets than those from progressors. The underlying mechanism is not completely understood, but is affected by HLA type, T-cell receptor clonotype, viral epitopes targeted, and likely also by a complex regulatory network of activation and inhibition. However, the particular phenotype of activation and exhaustion markers associated with the increased killing of HIV-specific CD8 T cells from EC has remained unclear. To elucidate these regulatory networks, we performed high-dimensional phenotyping of HIV-specific CD8 T cells using mass cytometry (CyTOF), a cutting edge technology combining flow cytometry and mass spectrometry that enables analysis of up to 40 markers with minimal signal overlap. We established and validated a CyTOF panel of 33 markers to identify up to 6 HIV-specific CD8 T-cell subsets using tetramer technology and characterize memory, activation and exhaustion status of those cells. Use of our panel enables the in-depth characterization of HIV-specific CD8 T cells at a level of unprecedented granularity and may reveal signatures of activation and exhaustion networks that underlie the enhanced lytic capacity of these cells in EC.
A framework for unbiased, robust and system-wide characterization of MHC-bound peptides and epitopes (APP5P.111)
Authors: Foster Leonard , Chan Queenie , Kuan Charlie , Yu Hong Bing
Published: January 2023

Vaccines are the most inexpensive medicine in the long run, yet developing them remains a slow process. A major challenge is to identify proteins efficiently processed by the host and presented on major histocompatibility complexes (MHCs). Furthermore, the extreme polymorphism of the MHCs means that candidate antigens must be tested across many genotypes. Immunopeptidomics, the study of all peptides presented by the host’s immune system, holds promise but is limited by informatics:standard approaches result in high error rates for these samples. We describe here an experimentally validated informatic treatment of mass spectrometric data from peptides eluted from the surface of antigen-presenting cells that can identify epitopes bound by specific MHCs, predict their core binding regions, and reveal consensus binding motifs of MHC alleles. Our approach avoids the biases inherent with MHC immunopurification or prior consensus motifs and enables rational vaccine design by allowing rapid and sensitive screening of individual immunopeptidomes.
Determinants of immunogenicity: flogging the old ovalbumin, again (APP5P.116)
Authors: Karandikar Sukrut , Srivastava Pramod
Published: January 2023

During antigen presentation, the products of intracellular proteolysis capable of binding MHC I are presented to T cells on the cell surface in context of MHC I. We queried the sequence of ovalbumin (OVA) with the NetMHC 3.0 algorithm and based on a suitable NetMHC Matrix score or predicted IC50 values, identified 19 putative epitopes with potential to bind H-2Kb or H-2Db. Generally, IC50 of 500 nM is used as a threshold to distinguish between candidate and non-candidate epitopes; sequences with an IC50 <=500 nM are considered to be good binders and candidate epitopes (Assarsson et al. J Immunol. 2007). We observe that 10 of the 19 putative epitopes of OVA have measured IC50 <= 500 (range: 1.5 nM - 248 nM). The remaining nine putative epitopes are weak binders (IC50 > 500; range: 928 nM- 13219 nM). Interestingly, three of the nine weak binders elicit antigen specific responses in C57BL/6 mice. Six of the ten strong binders also elicit such responses, while four do not. Altogether, we have identified nine novel MHC class I restricted epitopes of ovalbumin, one of the most exhaustively used model antigens. Finally, we observe that synthetic peptides corresponding to two epitopes of OVA (one weak binder and one strong binder) do not elicit specific responses upon immunization; at the same time, responses against these epitopes can be observed upon immunization with OVA. We are exploring the molecular mechanisms of these observations.
The effect of HLA-DM on cooperativity of peptide binding to MHC class II (APP5P.107)
Authors: Templeton Megan , Ferrante Andrea
Published: January 2023

HLA-DM (DM) plays a critical role in generating the peptide-MHC class II (MHCII) repertoire expressed on the surface of Antigen Presenting Cells and recognized by CD4+ T Cells. The action of DM is pH-dependent, and its effect increases at acid conditions. Previous studies performed at pH 7.4 in the absence of DM have shown that peptide binding to and release from MHCII is a flexible process as evidenced by cooperativity. Cooperativity indicates that peptide binding does not rely on independent contributions of single-point interactions, instead multiple interactions along the peptide-binding groove determine binding in a non-additive fashion To resolve whether cooperativity of binding, thus system flexibility, is a function of DM activity, we measured the affinity of peptides derived from influenza H3 HA306-319 via cycle-mutation for the human MHCII allele HLA-DR1 (DR1) at pH 5.4 and 6.4. Through competitive binding assays and peptide release studies we have determined that DM reduces cooperativity in peptide binding and peptide-MHCII kinetic stability, and the effect is a function of pH conditions. These results suggest that one component of DM action during peptide exchange is a restraining of complex structural flexibility, which imposes a higher energetic threshold for stable binding.
A critical role of Th17 cells in the development of murine Sjogeren’s syndrome (BA11P.132)
Authors: Lin Xiang
Published: January 2023

Sjögren’s syndrome (SS) is characterized by progressive inflammation and tissue damage of salivary glands (SG) and lacrimal glands, leading to dry-mouth and dry-eye symptoms as a result of reduced saliva and tears secretion. Histopathologically, CD4+ T cells were the major population in the infiltration and mediated tissue destruction. However, a role of IL-17-producing CD4+ T cell (Th17 cell) has not yet been elucidated in SS pathogenesis. In the present study, experimental SS was induced in normal mice by immunization with proteins extracted from SG. A rapid increase of Th17 cells was observed in the spleen and draining cervical lymph node at acute stage. Interestingly, this pattern also emerged in the draining lymph node of non-obese diabetic mice, another murine model of SS. Further, SG-infiltrating Th17 cells were detected and increased along disease progression. In IL-17 deficient (IL-17-/-) mice, no apparent salivary dysfunction and autoantibodies were observed upon immunization. However, upon adoptive transfer of Th17 cells from wild type mice, high titer of autoantibodies, salivary dysfunction with lymphocytic infiltration were fully restored in IL-17-/- mice. Together, our findings have demonstrated a critical role of Th17 cells in SS pathogenesis.
Specifying the autoantibody-inducing CD4 T cell causing systemic lupus erythematosus out of PD-1+CD45RBlo122lo CD4 subpopulation using mass spectrometry (BA11P.135)
Authors: Shiozawa Shunichi , Miyazaki Yumi , Tsumiyama Ken
Published: January 2023

Objective: We have shown that repeated immunization of mice with antigen reproducibly led to development of experimental systemic lupus erythematosus (SLE), where a novel T cell type which we term an autoantibody-inducing CD4 T (aiCD4 T) cell was generated via TCR revision at periphery. The aiCD4 T cell induces varieties of autoantibodies and also helps full maturation of CTL to induce lupus tissue injuries. We have previously identified that aiCD4 T cell belongs to PD1+CD45RBlo122lo CD4 T subpopulation. Here we tried to further specify the aiCD4 T cell by membrane surface markers. Methods: BALB/c mice were repeatedly 12x immunized with OVA. To isolate aiCD4 T cell, we extracted cytoplasmic and membrane fractions of PD1+CD45RBlo122lo CD4 T subpopulation. They were electrophoresed and protein fractions unique to the PD1+CD45RBlo122lo CD4 T subpopulation were identified thru mass spectrometry. Results: Candidate membrane markers as suggested by mass spectrometry were DNA topoisomerase 2-beta (181 kDa), DOCK 8 (238 kDa), VWA5A (87 kDa), STAT5b (89 kDa), epidermal growth factor receptor substrate 15-like 1 (99 kDa) and FLAP (18 kDa) for aiCD4 T cell, among which we found that DOCK 8 was significantly increased in the membrane fraction of PD1+CD45RBlo122lo CD4 T subpopulation. Conclusion: We identified DOCK 8 as a possible candidate membrane marker characterizing the aiCD4 T cell as found within PD-1+CD45RBlo122lo CD4 T subpopulation.
Expression of GM-CSF in T cells is increased in multiple sclerosis and is suppressed by IFN-β therapy (BA11P.136)
Authors: Rasouli Javad , Ciric Bogoljub , Imitola Jaime , Gonnella Patricia , Hwang Daniel , Mahajan Kedar , Mari Elisabeth , Safavi Farinaz , Leist Thomas , Zhang Guang-Xian , Rostami A
Published: January 2023

Multiple sclerosis (MS) is an autoimmune disease of the central nervous system (CNS). Studies in animal models of MS have shown that granulocyte-macrophage colony-stimulating factor (GM-CSF) produced by T cells is necessary for development of autoimmune CNS inflammation. This suggests that GM-CSF may have a pathogenic role in MS as well. The objective of this study was to characterize GM-CSF production by T cells of MS patients, and to determine the effect of interferon-beta (IFN-β) therapy on its production. GM-CSF production by peripheral blood (PB) T cells and the effects of IFN-β were characterized in samples of untreated and IFN-β-treated MS patients vs. healthy subjects. GM-CSF production by T cells in MS brain lesions was analyzed by immunofluorescence. Untreated MS patients had significantly greater numbers of GM-CSF+ CD4+ and CD8+ T cells in PB compared to healthy controls and IFN-β-treated MS patients. IFN-β strongly suppressed GM-CSF production by T cells in vitro. A number of CD4+ and CD8+ T cells in MS brain lesions expressed GM-CSF. Elevated GM-CSF production by PB T cells in MS is indicative of aberrant hyperactivity of the immune system. Given its essential role in animal models, abundant GM-CSF production at the sites of CNS inflammation suggests that GM-CSF contributes to MS pathogenesis. Our findings also reveal a potential mechanism of IFN-β therapy, namely suppression of GM-CSF production.
Age-related thymic involution perturbs negative selection leading to autoreactive T Cells that induce chronic inflammation (inflammaging) (BA11P.141)
Authors: Coder Brandon , Wang Hongjun , Ruan Linhui , Su Dong-ming
Published: January 2023

The presence of chronic low-level pro-inflammatory factors in elderly individuals (termed inflammaging) is a significant risk factor for morbidity and mortality. Recently, inflammaging has been partially attributed to the persistent activation of immune cells thought to arise from latent viral infection, but the contribution of activated autoreactive T cells towards the development of inflammaging remains unclear. To determine how age-related thymic involution leads to the persistent release and activation of autoreactive T cells capable of inducing inflammaging, we used a FoxN1 conditional knock-out (FoxN1-cKO) mouse model that mimics natural thymic involution while maintaining a young periphery. We found that thymic involution leads to T cell activation shortly after thymic egress, which is accompanied by cellular infiltration into non-lymphoid tissues and elevated IL-6 and TNFα levels. Autoreactive T cell clones were detected in the periphery of FoxN1-cKO mice. A failure of negative selection, facilitated by decreased expression of Aire rather than impaired regulatory T cell (Treg) generation, led to autoreactive T cell generation. Furthermore, the young environment can reverse age-related Treg accumulation but not inflammatory infiltration. Together, these findings identify thymic involution and the persistent activation of autoreactive T cells as a source of chronic age-related inflammation (inflammaging).
Autoreactive T cell trafficking into pancreatic islets during type 1 diabetes is more permissive as infiltration increases and requires CD11c+ APCs. (CAM4P.153)
Authors: Lindsay Robin , Gebert Matthew , Wigton Eric , Bradley Brenda , Haskins Kathryn , Jacobelli Jordan , Friedman Rachel
Published: January 2023

Type 1 diabetes (T1D) is a largely T cell mediated autoimmune disease that destroys the β-cells of the pancreatic islets. While initiation of disease requires T cell activation in the pancreatic lymph node, T cells must enter the islets to destroy β-cells. To investigate mechanisms required for T cell entry into the islets we established an intra-vital 2-photon pancreas imaging technique in the non-obese diabetic mouse. We determined that extravasation into the pancreatic islets is an extended process compared to lymph node entry, averaging 47 minutes from T cell arrest to completion of extravasation in islets with advanced T cell infiltration. Entry was more restricted at earlier stages of infiltration, with a duration of extravasation longer than our observation period of 2 hours. T cell entry into the islets was dependent on CD11c+ antigen presenting cells (APCs) in the islets. T cells transferred prior to CD11c+ APC depletion were able to enter the islets; while CD11c+ APC depletion prevented further entry. However, antigen was not required for entry of either activated or naïve T cells, suggesting a role for CD11c+ APCs other than antigen presentation. These studies demonstrate that T cell entry requires the presence of CD11c+ APCs within the islets, and as islet infiltration increases T cell extravasation becomes less restricted. Overall, CD11c+ APCs may be a therapeutic target for treatment of T1D by preventing T cell entry into remaining or transplanted islets.
Absence in T1/ST2 receptor results in a more severe experimental nephropathy (CCR5P.208)
Authors: Reis Vanessa , Osugui Lika , Andrade David , Martins Beatriz , Keller Alexandre
Published: January 2023

Focal segmental glomerulosclerosis (FSGS) is a major cause of chronic kidney disease. Several studies suggest an association between Th2 and the pathogenesis of FSGS. Immune system molecules are widely associated with Th1/Th2 balance. In light of this, we chose the shaft IL-33/ST2, Th2 immune responses and kidney damage as the object of study, both related to. We used BALB/c WT and ST2-/- animals, intravenously injected with a single dose of 10mg/kg of adriamycin. Since ST2-deficient animals exhibit more severe kidney damage when induced by adriamycin, the absence of this receptor signaling is intimately related to induced loss of renal function. Observing the changes of these animals, as increase in serum creatinine levels and elevated protein/creatinine curve, more severe tissue damage and decreased expression of nephrin protein, STAT3 and Smad2/3. In addition, there was an increase in PAI-1 and nephrin phosphorylation, Prha, pTyr. Serum levels of IL-33 are also higher in ST2-/- animals. In the cellular context, we observed an increase in the percentage of TCD4pos splenic cells in these ST2-/- animals. However, CD4pos / FOXp3pos cells are less present when compared to wild type animals and similar to control animals. We also observed that the effect found in ST2-/- animals was mitigated by the transfer of TCD4pos wildtype lymphocytes. Considering all results, we can conclude so far that cytokine IL-33 plays an important role in the development and control of FSGS.
IL-23 Controls CD4+ T cell pathogenicity and IL-17A production in divergent patterns during experimental autoimmune myocarditis (CCR5P.207)
Authors: Wu Lei , Diny Nicola , Ong SuFey , Barin Jobert , Hou Xuezhou , Talor Monica , Rose Noel , Cihakova Daniela
Published: January 2023

Using a model of experimental autoimmune myocarditis (EAM), we established the requirement of IL-23 for the induction of cardiac autoimmunity, as Il23a-/- mice failed to mount an efficient Th17 response after immunization and were protected from myocarditis. Furthermore, we discovered that IL-23 has divergent effects on CD4+ T cell pathogenicity and IL-17A production. First, IL-23 switches on pathogenicity in CD4+ T cells, but becomes dispensable once autoreactivity is already established: CD4+ T cells raised from WT donors were able to induce EAM in Il23a-/- recipients. Moreover, even transient IL-23 stimulation ex vivo was able to fully restore pathogenicity in otherwise nonpathogenic T cells raised from Il23a-/- donors. Second, continued IL-23 signaling is required to maintain IL-17A production. Despite of inducing same level of inflammation in Il23a-/- recipients, autoreactive CD4+ T cells downregulated IL-17A production without sustained IL-23 stimulation. These divergent effects of IL-23 on T cell pathogenicity and IL-17A production may contribute to the discrepant efficacies of αIL-23 therapy in different diseases. Finally, we demonstrated that GM-CSF mediates the IL-23-driven pathogenicity of CD4+ T cells, and neutralization of GM-CSF abrogated inflammation in EAM. Combined with our finding that GM-CSF directs disease outcome by regulating macrophage polarization during later stages of EAM, GM-CSF is decisive in both the initiation and the progression of autoimmunity.
Targeted deletion of Eed arrests thymus development (HEM7P.225)
Authors: Singarapu Nandini , Xie Huafeng , Orkin Stuart , Richie Ellen
Published: January 2023

Polycomb group proteins regulate cell fate and differentiation by modifying histones to maintain chromatin in a compacted state that silences expression of inappropriate genes. EED is an essential core component of the Polycomb repressive complex 2 which trimethylates lysine 27 on histone H3. To determine the role of Eed in thymus organogenesis, we excised eed specifically from thymic epithelial cells (TECs) by crossing Eedfl/fl mice with a FoxN1Cre strain. We found a hypoplastic and highly cystic thymus in four week FoxN1Cre/+;Eedfl/fl mice. Cortical and medullary TECs were depleted in the mutants; however, the cortical compartment was more profoundly affected. TEC depletion was coupled with decreased thymocyte cellularity, which is not surprising given that TECs provide indispensable maturation and survival signals to thymocytes. The thymus and parathyroid glands arise respectively from ventral (Foxn1+) and dorsal (Gcm2+) endodermal progenitors in the 3rd pharyngeal pouch. We did not observe an alteration in the allocation of endodermal progenitors to a thymus versus parathyroid fate indicating that altered pouch patterning is not responsible for the TEC phenotype. At E15.5, mutant and wildtype fetal thymic lobes were comparable in size and TEC subset composition. However, by E16.5 defects in TEC differentiation and survival are apparent and this phenotype progresses rapidly with age. Current work focuses on the role of eed in maintaining TEC progenitors.
Homeostatic proliferation of thymocytes is controlled through dynamic regulation of delta like 4 (HEM7P.229)
Authors: Serwold Thomas , Vroegindeweij Eric , Kim MiJeong , Vazquez Sara
Published: January 2023

The thymus has a remarkable ability to sustain T cell development and T cell output from thymuses with quantitative and qualitative deficiencies in T cell progenitors, suggesting a role for homeostatic pathways in controlling T cell development. However, these homeostatic pathways are currently uncharacterized. We find that Delta-like 4 (Dll4), which is expressed on thymic epithelial cells (TECs) and is essential for driving T cell lineage commitment, is dynamically regulated in response to thymocyte deficiencies, and plays a major role in thymocyte homeostasis. We observe that TECs express elevated Dll4 levels in multiple lines of mice that have defective T cell development, as well as in mice subjected to antibody or irradiation-induced thymocyte depletion. Recovery of thymus cellularity after thymic injury is accompanied by re-establishment of normal levels of Dll4. In vitro proliferation of thymocytes increases proportionally in response to elevated levels of Dll4 expressed on stromal cells. Furthermore, Dll4 levels on stromal cells diminish in response to interactions with thymocytes themselves. These findings implicate dynamic control of Dll4 levels as a major homeostatic mechanism that regulates thymocyte proliferation.
Expression analyses revealed thymic stromal co-tansporter/Slc46A2 is in stem cell populations and is a putative tumor suppressor (HEM8P.237)
Authors: Kim Moon , Kim Ki , Yoon Min
Published: January 2023

By combining flow cytometry and database searches, we extended the expression profiling of TSCOT/Slc46A2/Ly110, that was shown to be expressed in the bipotent precursor and cortical thymic epithelial cells. Genome scale analysis verified that TSCOT is tissue- and cell type- specific in thymus and is also expressed in other epithelial tissues. Coexpression profiling with genes, Foxn1 and Hoxa3, revealed the role of TSCOT during the tissue formation including early thymic organogenesis. TSCOT expression was detected in all thymic epithelial (TEC), but not in the CD31+ endothelial lineage of fetal thymus. In addition, ABC transporter-dependent side population and Sca-1+ of fetal TEC population contain TSCOT-expressing cells, indicating TEC stem cells express TSCOT. TSCOT expression was identified as early as in differentiating embryonic stem cells. TSCOT expression is not under the control of Foxn1 since TSCOT is present in the thymic rudiment of nude mice. By the searches for expression variation, TSCOT is positively associated with GRHL3 and IRF6. Cytokines such as IL1b, IL22 and IL24 are the potential regulators of the TSCOT expression. Surprisingly, we found TSCOT expression in the lung was diminished in the lung cancers suggesting TSCOT may be involved in the suppression of lung tumor development. From these results, a model for TEC differentiation from the stem cells was proposed in the context of multiple epithelial organ formation.
In vitro evaluation of B cell function using activation-induced cytidine deaminase in HIV-infected patients on antiretroviral therapy (HUM6P.244)
Authors: Gonzalez Ivan , Correa Adriana , arheart Kristopher , Frasca Daniela , Blomberg Bonnie
Published: January 2023

Background: B cell populations and function depend on the stage of HIV-1 infection. Good virologic control can normalize B cell populations, but data regarding the effect on B cell function is lacking. Method: We investigated B cell populations and intrinsic function among a perinatally and behaviorally infected HIV-1 patients based on virologic control and correlated both. Peripheral blood monocytes were collected from a HIV-negative (HN), behaviorally infected HIV patients with undetectable viral load of <40 copies/mL (BU) and two perinatally infected groups. These groups were categorized by viral load: undetectable group (PU) of <40 copies/mL and a low level viremia (PLLV) of <200 copies/mL. Activation-induced cytidine deaminase mRNA quantification was used after in vitro stimulation with CpG oligodeoxynucleotides to measure intrinsic B cell function. Results: B cell populations were similar in the HN and BU groups, which differed from the PU and PLLV groups. The PU and PLLV groups demonstrated higher percentages of naïve B cells and lower percentages of IgM and switch memory B cells, demonstrating patterns of cellular aging. However, the exhausted B cell populations were not significantly different between groups. Virologic control did not affect B cell function in the infected groups when compared to the uninfected group. Conclusion: Good virologic control, defined as < 200 viral copies/mL, normalizes B cell populations and more importantly intrinsic B cell function.
The role of Epstein-Barr virus in cervical cancer: exploring the immunological mechanisms for production of anti-Estein-Barr virus IgA in high risk women with dysplasia (HUM6P.250)
Authors: Fisher Rebecca , Melton Scott , Ferguson Amelia , Oddo Hope , Velasco-Gonzales Cruz , Hagensee Michael
Published: January 2023

Human papillomavirus (HPV) alone is a necessary but insufficient cause of cervical cancer due to the high number of infections that spontaneously resolve. We have evidence supporting Epstein-Barr Virus (EBV), a known oncovirus implicated in several cancer types, as a co-factor for cervical disease in HIV+ women. Longitudinal analysis of ELISA data from 453 serum samples, collected over a five year time period from 89 HIV+ women, indicated IgA, but not IgG, against EBV viral capsid antigen (VCA) predicted cervical dysplasia. Sera were also tested for IgG and IgA against HPV16,18 L1 protein and EBV nuclear antigen-1, but no predictive value was observed for these additional targets. Measures of HIV disease control, HIV viral load and CD4 T cell count, were also not predictive of dysplasia. Multivariate models indicated that HPV IgG status did not alter the significance of the EBV VCA IgA prediction. Cervical EBV DNA detection suggests local EBV infection; however, cervical Ab responses are generally IgG. The repetitive structure of the EBV capsid may trigger T cell independent Abs, but the longevity of IgA detection opposes this notion. We hypothesize EBV in the cervix may result in formation of lymphoid-like follicles, as seen in cervicitis, for Ab production. We are collecting dysplastic cervical tissue to establish the existence and functionality of these structures as well as assessing for molecules influencing IgA class switching, such as CXCL-13, BAFF and APRIL.
Interleukin-2/anti-interleukin-2 antibody complexes expand T regulatory cells and protect against allergen-induced airway hyperreactivity (HYP7P.256)
Authors: Neunkirchner Alina , Wojta-Stremayr Daniela , Schmetterer Klaus , Mager Lukas , Reichl Victoria , Rosloniec Edward , Naumann Ronald , Dekan Gerhard , Jahn-Schmid Beatrice , Bohle Barbara , Pickl Winfried
Published: January 2023

T-cells play a key role in the development and maintenance of allergic diseases and thus represent a promising target for therapeutic interventions. Evidence from SIT studies suggest that regulatory T-cells (Treg) might counterbalance the Th2-biased immune response. We here created double transgenic (tg) allergy mice expressing a human TCR specific for the major mugwort (Artemisia vulgaris) pollen allergen Art v 1 and HLA-DR1 to study the response to the aero-allergen in vivo. After allergen-specific sensitization, only double tg allergy mice, but not single tg or wt control mice showed enhanced airway-hyperreactivity (AHR) upon challenge, which was associated with airway and lung inflammation. Significantly, treatment of allergy mice with three daily i.p. injections of IL-2/anti-IL-2 mAb complexes induced a 6-fold increase of CD25+Foxp3+ Treg among CD3+CD4+T-cells in peripheral blood (17.9±6.2% versus 3.1±1.4%) compared to a 8-fold increase of CD39+ and KLRG1+ Treg subsets, respectively (17.2±7.9% versus 2.1±1.5%; 1.6±0.2% versus 0.2±0.2%). Furthermore, expansion of Treg by IL-2/anti-IL-2 mAb complexes before sensitization prevented mice from allergen-induced AHR upon challenge and significantly reduced Art v 1-specific serum IgE, when compared to sham-treated mice. Such in vivo expanded Treg might have similar effects in therapeutic settings. In summary, expansion of Treg by IL-2/anti-IL-2 mAb complexes seems to be a promising strategy to interfere with allergic diseases.
Osteopontin regulates Cxcr2 expression and neutrophil recruitment in endodontic infection (INC6P.302)
Authors: Singh Rani , Hui Tommy , Matsui Aritsune , Rittling Susan
Published: January 2023

The integrin binding protein osteopontin (OPN) protects against bone destruction in a mouse model of endodontic infection. Here we have tested the hypothesis that this effect of OPN is mediated by effects on migration of innate immune cells to the site of infection. We used the subcutaneous chamber and air pouch as models of endodontic infection in mice. In vitro analysis of CXCR2 expression was determined by FACS and migration was assessed using transwell assay. Using both the chamber and a modified air-pouch infection model, we showed that neutrophil accumulation at the site of infection is significantly reduced in OPN KO mice, but there are very few macrophages and monocytes, and there is little effect of OPN on their accumulation. OPN does not affect neutrophil survival. Reduced neutrophil accumulation is accompanied by lower levels of myeloperoxidase activity and an increase in bacterial load in the absence of osteopontin. In vitro, OPN increases the ability of these cells to migrate in transwell assays when added in either to the upper or the lower chambers. Furthermore, OPN increases expression of CXCR2 on bone marrow neutrophils, in an alphav-integrin dependent manner. Biotinylation assays show that OPN suppresses the internalization of CXCR2. Together, these results support a model whereby OPN binding to the alphav integrin regulates CXCR2 internalization on neutrophils, increasing the ability of these cells to migrate in response to CXCR2 ligands.
Glycolysis regulates macrophage differentiation into IL-10 producing phenotype (INM6P.342)
Authors: Suzuki Hiroaki , Hisamatsu Tadakazu , Naganuma Makoto , Ebinuma Hirotoshi , Nakamoto Nobuhiro , Mori Kiyoto , Kitazume Mina , Shimamura Katsuyoshi , Chiba Sayako , Kanai Takanori
Published: January 2023

There are a lot of reports indicating that cellular metabolic state or some metabolites regulate the phenotype of immune cells. In case of macrophages (Mφ), it is believed that regulatory and inflammatory Mφ shifts ATP production to each oxidative phosphorylation and glycolysis, respectively. However, it is not clear how metabolic pathway affects Mφ in the acquisition of their specific phenotypes. In this report, we tried to reveal how glycolysis affects Mφ during the acquisition of IL-10 producing phenotype. We used in vitro differentiated human monocyte derived Mφ cultured in the presence of M-CSF or M-CSF plus IFNγ as IL-10 producing-Mφ (M-Mφ) or inflammatory Mφ (Mγ-Mφ), respectively. M-Mφ showed regulatory Mφ-like phenotypes and produced more IL-10 when it was stimulated with LPS compared to Mγ-Mφ. Interestingly, M-Mφ differentiated with glycolysis inhibitors (2-deoxy-D-glucose or DCA) produced significantly lower amount of IL-10 and lager amount of IL-6 compared to M-Mφ differentiated without glycolysis inhibitors. Glycolysis inhibited M-Mφ reduced the expression of regulatory Mφ marker genes (MRC1 and CCL22) and exhibited distinct pattern of cell surface antigens (CD163, CD206, CD209 and HLA-DR) compared to M-Mφ. DNA microarray analysis revealed that inhibition of glycolysis affects gene expression profiles during Mφ differentiation. Our finding suggests that glycolysis plays key roles in the course of differentiation of Mφ into IL-10 producing phenotype.
Identifying biomarkers from an in vitro carrageenan model of inflammation in swine (INM8P.359)
Authors: Myers Michael , Deaver Christine , Peters Sharla , Swaim Heidi , Yancy Haile
Published: January 2023

Carrageenan-induced inflammation, though extensively used as a model of local inflammation, has not been fully characterized on the genomic and proteomic level. The goal of this study was to develop an in vitro carrageenan model of local inflammation for use in studies of novel non-steroidal anti-inflammatory drug efficacy. We evaluated lambda carrageenan (l-CGN) in vitro and its capacity to affect previously identified inflammatory biomarkers in swine, using swine whole blood cultures. Blood was collected from 9 male Yorkshire barrows, cultured in vitro. The blood was divided into two groups (stimulated and unstimulated) per each time point. Unstimulated blood was used as control to compare samples treated with l-CGN. Blood was then separated into two fractions, serum collection for ELISA analysis and white blood cells for RNA extraction per time point. We found that l-CGN increased the production MCP-1 in vitro, both on the proteomics level and the genomic level. Conversely, TNF-a and IL-6 demonstrated a decreased or no change in cytokine production when measured via ELISA. Phosphokinase-3, Thrombospondin-1 and Alveolar Macrophage Chemotactic -II showed increased gene expression when normalized to GAPDH. These results also demonstrate that carrageenan initiates an inflammatory response that differs from a typical endotoxin effect, such as LPS stimulation in terms of the pathways and gene products altered.
Fibrocytes secrete periostin to promote myofibroblast differentiation in lung fibrosis (IRC10P.407)
Authors: Ashley Shanna , Wilke Carol , Moore Bethany
Published: January 2023

Fibrocytes are bone-marrow-derived circulating mesenchymal precursors (CD45+, col 1+) that are recruited to fibrotic areas. Fibrocytes secrete a number of profibrotic mediators including periostin; a matricellular protein that regulates cellular interactions with extracellular matrix components (ECM). In bleomycin-induced fibrosis,loss of periostin by either structural or hematopoietic cells can limit the development of pulmonary fibrosis.To test the hypothesis that hematopoietic-derived fibrocytes might secrete soluble factors to signal structural myofibroblast differentiation, wild-type (WT) fibroblasts were treated with conditioned medium from fibrocytes isolated from bleomycin-treated WT or periostin-/- mice. After 48hrs we observed an increase in α-smooth muscle actin expression in cells treated with the conditioned medium from WT fibrocytes. Adoptive transfer of WT fibrocytes augmented lung fibrosis to a greater extent than transfer of fibrocytes from periostin-/- mice. We performed in vitro analyses of lung mesenchymal cells isolated from WT and periostin-/- mice by sorting fibroblasts and fibrocytes then treating with TGFβ1 or periostin. We demonstrate that these treatments increase the expression of X-linked inhibitor of apoptosis protein and collagen 1 mRNA. Taken together these data suggest that fibrocytes may augment bleomycin-induced fibrosis via secretion of periostin and TGFβ and which then promote the accumulation of apoptotic resistant myofibroblasts.
Synergistic effects of lipopolysaccharide on Treg cell generation is through an IDO-dependent pathway (IRC11P.431)
Authors: Goldberg Michael , Appel Moshe , Elizur Arnon , Katz Yitzhak
Published: January 2023

Over the past several years there has been increased awareness of the importance of early-life exposures, to long term immune disposition. Using lipopolysaccharide (LPS) activation as a surrogate marker for environmental stimulation, we previously noted that a high proliferative response to LPS in cord blood mononuclear cells (CBMC) was impaired in infants born to atopic parents (Ped. Res. 2013 Aug;74(2):211-6). Furthermore, LPS treatment can result in hypomethylation of the FOXP3 promoter in CBMC. To explore the role of LPS in promoting an immunoregulatory phenotype in CBMC, its effects on the generation of Treg cells was explored. LPS minimally induced Foxp3 expression on FACS analysis. However, in a subset of CBMC (7/14), it synergistically increased the generation of Foxp3+ Tregs induced by T cell stimulation by anti-CD3 antibody and IL-2. Priming by LPS was a necessary first step for this synergy. LPS treatment of CBMC increased indoleamine pyrrole 2,3-dioxygenase (IDO)-1 and IDO-2 expression, characteristically expressed in tolerogenic dendritic cells. Importantly, the addition of 1-Methyltryptophan, an IDO inhibitor, completely blocked FoxP3 expression. These results suggest that LPS priming, working through an IDO-dependent pathway may have early effects on the cellular milieu, resulting in the generation of Treg cells. Future experimentation will address whether early life environmental induction of Tregs is indicative of one's longer term immune disposition.
Repository corticotropin injection (H.P. Acthar® Gel) exerts direct effects on human B cells to suppress IL4/CD40L-stimulated proliferation and immunoglobulin G production in vitro (IRC11P.437)
Authors: Olsen Nancy , McAloose Carl , Benko Ann , Kovacs William , Decker Dima , Becker Patrice
Published: January 2023

Effects of melanocortin peptides on human B cells were described more than two decades ago, but therapeutic applications have not been fully explored. This study examined B cell responses to a clinically available melanocortin peptide preparation, repository corticotropin gel. Peripheral blood B cells isolated from healthy donors using CD19 magnetic beads were cultured with and without Interleukin 4 (IL-4) and CD40 ligand (CD40L). Repository corticotropin or Placebo Gels were added in serial dilutions. Cultures were harvested after 7 days for measurement of secreted IgG by ELISA, B cell proliferation using CFSE and viability with trypan blue. Mean IgG in IL-4/CD40L-stimulated cultures was 241.4 ng/ml and was reduced in a dose-dependent fashion with corticotropin gel; maximal suppression was to 65.55 ng/ml (p= 0.008; n=8). Addition of Placebo Gel or dexamethasone (1μM) to IL-4/CD40L cultures resulted in no change in IgG (240.3 and 248.1 ng/ml). The effect on IgG was paralleled by inhibition of B cell proliferation. In IL4/CD40L-stimulated cultures, 61% of cells were dividing, vs 8% with corticotropin gel (p<0.0001) and 58% with Placebo Gel (ns). Inhibition of proliferation was not accompanied by increased cell death. Repository corticotropin gel exerted dose-dependent inhibition of IgG production and B cell proliferation without effects on cell viability. These data support potential efficacy of repository corticotropin gel in diseases characterized by B cell activation.
Treg suppressor function in inflamed peripheral tissue and lymphoid tissue occurs through distinct mechanisms (IRC11P.426)
Authors: Camirand Geoffrey , Rothstein David
Published: January 2023

The mechanisms by which Treg (CD4+Foxp3+) affect DC function and modulate immunity in inflamed non-lymphoid tissues remain poorly understood. Here, we used 2-photon intravital microscopy to visualize how Treg suppressor function operates within allografts. To restrain the immune reaction to the transplant site, we adoptively transferred effector T cells (Teff), wt Treg, or both (2-3x106 each) to B6 mice lacking 2° lymphoid organs (SLO; splenectomized LTβR-/-) 3-4 days after transplantation of BALB/c islets. Teff alone induced acute allograft rejection. However, the co-transfer of Teff + wt Treg led to long-term allograft survival in ~50% of recipients. Intravital microscopy of transplanted islets on day 2 showed that wt Treg infiltrated the peri-islet region, where they predominantly made contacts with DCs. Wt Treg restrained the motility parameters of Teff in the peri-islet region, reduced Teff-DC contacts, and decreased Teff intra-islet infiltration (insulitis), compared to Teff alone. Treg constitutively express the ecto-nucleotidase CD73, which generates anti-inflammatory adenosine from AMP. CD73-/- mice do not develop autoimmunity, and CD73-/- Treg are potent suppressors in vitro. In contrast, CD73-/- Tregs were unable to prevent allograft rejection by Teff, despite their presence at the graft site. These results indicate that Treg function does not require priming in SLO and that their mechanisms of action may differ from those used in SLO.
Intrinsic function of B7-H1 is linked with p38 MAPK pathway in activated CD8 T cells for T cell survival. (IRM14P.443)
Authors: Dong Haidong , Liu Xin , wu xiaosheng , Krco Christopher
Published: January 2023

B7-H1 (also named PD-L1) is up-regulated on activated CD8 T cells at the late stage of antigen stimulation, and is required for T cell survival during contraction phase. Since more anti-B7-H1 (PD-L1) antibody has been used in clinical trials of treatment of human cancers, it is necessary to know whether antibody ligation of B7-H1 expressed by T cells would affect T cell function and survival. After screening several anti-B7-H1 antibodies, we found that some antibody triggered the activation of p38 MAPK in activated CD8 T cells. In addition, the levels of activated p38 MAPK increased in T cells isolated from B7-H1 knockout mice compared to wild type mice. Inhibition of DNA-PKcs activation increased the activation of p38 MAPK in wild type T cells but not in B7-H1 KO T cells, suggesting B7-H1 may negatively regulate p38 MAPK through DNA-PKcs pathway. To that end, we identified an association of B7-H1 with DNA-PKcs in activated human T cells but not in resting T cells. Since p38 MAPK inhibition rescued activated CD8 T cells from apoptosis, the pro-survival function of T cell intrinsic B7-H1 may be mediated by suppression of p38 MAPK. However, ligation of B7-H1 by agonist antibody may disrupt B7-H1’s pro-survival function by increasing p38 MAPK activation. Our results identified a link of B7-H1 with p38 MAPK pathway and may help to define agonist function of anti-B7-H1 antibody prior to clinical application.
Smad4 plays an integral role in memory CD8 T cell differentiation (IRM14P.444)
Authors: Hu Yinghong , Cauley Linda
Published: January 2023

Tissue resident memory CD8 T cells (TRM) are a unique subset of virus-specific CTLs which bolster local immune responses after becoming lodged in previously infected tissues. These cells provide enhanced protection by intercepting returning pathogens before a new infection gets established. In contrast, central memory CD8 T cells (TCM) circulate in the bloodstream and proliferate in secondary lymphoid organs before replenishing effector and memory CD8 T cell populations in remote parts of the body. Both populations of virus-specific memory CD8 T cells participate in immunity to influenza virus infection however the signaling pathways that instruct developing memory CD8 T cells to distribute to specific tissues are poorly defined. We show that TGFβ promotes the development of pulmonary TRM cells via a signaling pathway that does not require the downstream intermediate Smad4. In contrast, circulating memory CD8 T cells have no requirement for TGFβ but show signs of arrested development in the absence of Smad4 including aberrant CD103 expression. These signaling pathways alter the distribution of virus-specific CTLs in the lungs, but do not prevent robust cytokine responses. Our data show that Smad4 is required for normal differentiation of multiple subsets of virus-specific CTLs. In normal circumstances, Smad4 may be activated via a pathway that bypasses the TGFβ receptor. Improved understanding of these signaling pathways could be used to augment vaccine-induced immunity.
Endocytosis of cytotoxic granules in CD8 T lymphocytes is required for multiple killing of target cells (IRM14P.448)
Authors: Chang Hsin-Fang , Halimani Mahantappa , Krause Elmar , Rettig Jens , Pattu Varsha
Published: January 2023

Cytotoxic (CD8+) T lymphocytes (CTLs) eliminate tumorigenic and virus-infected target cells by releasing the contents of cytotoxic granules (CGs) at the immunological synapse. Even though CTLs have only a limited number of pre-existing fusogenic CGs, they can efficiently kill multiple target cells upon activation. We therefore hypothesized that, following CG fusion, the remaining components are recycled and subsequently refilled with cytotoxic substances to become available for the next round of fusion, thus enabling efficient multiple killing. We visualized endogenous CGs and their recycling in real-time in CTLs from synaptobrevin2-mRFP knock-in mice by fluorescently-labeled anti-RFP antibodies. We found that endocytosis (recycling) of CGs was blocked by Dynasore, an inhibitor of dynamin. Additionally, clathrin was colocalized with endocytosing CGs at the plasma membrane following dynasore pretreatment. Thus, endocytosis of CGs is clathrin- and dynamin-dependent. Importantly, although CTLs pre-treated with Dynasore showed no immediate effect on CG exocytosis, they exhibited a 50% deficiency in killing target cells after 2 hours of incubation. Our data strongly suggest that recycling of CGs plays an important role in CTLs for executing multiple killing of target cells.
Role of atypical protein kinase on Th17 differentiation and function (IRM15P.606)
Authors: Yang Jun-Qi , Guo Fukun , Zheng Yi
Published: January 2023

We have reported that PKCλ/ι, a member of atypical protein kinase family, is critical for the control of cell polarity during T cell activation, Th2 cytokine production and allergic airway inflammation. However, the effect of PKCλ/ι on Th17 cells remains unknown. By the use of mice with conditional PKCλ/ι knockout in activated T cells, we investigated the potential roles of this protein kinase on Th17 differentiation, effector cytokine secretion and allergic airway inflammation. We found that loss of PKCλ/ι in activated CD4+ T cells impaired Th17-differentiation in vitro. The secretion of Th17 effector cytokines was down-regulated from PKCλ/ι-deficient T cells either under non-polarized or Th17-polarizd conditions. Furthermore, PKCλ/ι-deficiency inhibited OVA-induced allergic airway inflammation in vivo, where Th17 effector cytokines were also reduced along with Th2 cytokines. Further studies are ongoing for underlying mechanisms whereby PKCλ/ι controls Th17 cells. Our preliminary findings indicate that PKCλ/ι might be a novel regulator for Th17 cells.
OX40 ligation recruits the E3 ubiquitin ligases TRAF6 and cIAP2 that cooperatively activate the non-canonical NF-κB pathway (IRM15P.608)
Authors: Fan Yihui , Xiao Xiang , Shi Xiaomin , Ghobrial Rafik , Minze Laurie , Li Xian
Published: January 2023

The signaling cascades triggered by OX40 in the control of diverse responses from initial T cell activation to the generation of memory and regulatory T cells remain incompletely defined. OX40 preferentially activates and sustains the non-canonical NF-κB pathway, but the underlying mechanisms remain unknown. Here, we found that in primary CD4+ T cells activated under OX40 costimulation, TRAF6 and cIAP2 are two essential molecules and both cooperatively activate the non-canonical NF-κB pathway. Mechanistically, OX40 directly recruited TRAF6 and TRAF2/TRAF3/cIAP2/NIK complexes in which TRAF6 acted as the E3 ligase to mediate Lys63-linked polyubiquitination of cIAP2. Consequently, cIAP2 used its E3 ligase activity to promote TRAF3 Lys48-linked polyubiquitination and degradation. The UBA domain of cIAP2 inhibited the E3 ligase activity of cIAP2 targeting to TRAF3 and this autoinhibition could be released by the C-terminal RING domain polyubiquitination mediated by TRAF6. The lysines 585 and 594 at cIAP2 C-terminus were two main ubiquitin acceptor sites for TRAF6-mediated polyubiquitination, as combined mutation of such sites abolished Lys48-linked TRAF3 polyubiquitination. Thus, in TRAF6 knockout T cells, OX40 failed to induce sufficient non-canonical NF-κB activation and the downstream IL-9 production due to insufficient TRAF3 degradation. Our study identifies a previously unknown mechanism of OX40 controlled non-canonical NF-κB activation in T cells.