bluuebunny/crossref_metadata_2025 huggingface dataset:
Class II MHC-Binding Peptides are Active Over a Broad Range of Affinities for MHC (49.29)
Authors: Mage Michael , Levin Ditza , Revilleza Maria J , Boyd Lisa , Natarajan Kannan , Shevach Ethan , Margulies David
Published: January 2023

To stimulate T cells, antigenic peptides must not only bind the presenting MHC molecule, but must form a complex of sufficient stability to interact with the T cell receptor (TCR). In a murine autoimmune gastritis model based on the transgenic expression of MHC-II restricted T cell receptors (TCR) directed against peptides from the alpha chain of the gastric parietal cell proton pump, the H/K ATPase, previous studies mapped residues 630-641 (PITAKAIAASVG), and residues 889-900 (PLLCVGLRPQWE) as the epitopes recognized by TXA23 and TXA51 transgenic T cells respectively. We studied the dissociation kinetics of peptides with various amino acid substitutions (including alanine-substitution at each non-alanine position). Binding to IAd was compared to T cell stimulatory activity of the same peptides in MHC-peptide complexes. Dissociation half lives at pH 5.3 (similar to endosomes) varied from ~20 hours to over 600 hrs. Peptide/MHC stability is greatest at neutral pH, where a nonstimulatory peptide (CLIP) and a highly stimulatory peptide (GHNLAVGLRPQWHE) had half lives approximately tenfold longer than at pH5.3. We find that a half-life of >20 hours at pH 5.3 is sufficient for a peptide with the necessary epitopic residues to retain immunogenicity. These results can be explained in the context of the X-ray structure of the IAd/PLLCVGLRPQWE complex. Supported by the NIAID intramural research program.
A GMCSF-neuroantigen (NAg) fusion protein targets NAg to dendritic cells (DC) and is an effective therapy for experimental autoimmune encephalomyelitis (EAE) (50.10)
Authors: Blanchfield J. Lori , Mannie Mark D.
Published: January 2023

The GM-CSF and M-CSF cytokines were tested as domains in cytokine-NAg fusion proteins to assess targeting of NAg to different APC subsets. Fusion proteins were designed with a cytokine N-terminal domain and the encephalitogenic peptide 69-88 of guinea pig myelin basic protein (MBP) as the C-terminal domain. Studies measuring T-cell activation in vitro, prevention of EAE, and treatment of EAE showed that GMCSF-NAg was the most potent fusion protein, represented by the following rank order of activity (GMCSF-NAg > MCSF-NAg > GP69-88). GMCSF-NAg was 1000-fold more potent than GP69-88 in stimulating MBP-specific T-cell proliferation. The mechanism by which GMCSF-NAg promoted T-cell activation involved cytokine receptor-mediated uptake of NAg by antigen presenting cells (APC), because free GM-CSF inhibited the GMCSF-NAg potentiated response. GMCSF-NAg potently targeted NAg to dendritic cells and macrophages in vitro, but not to B or T cell APC. Covalent linkage between GM-CSF and NAg was required for enhanced potency of GMCSF-NAg in vitro and for the prevention and treatment of EAE in vivo. In conclusion, GMCSF-NAg potently targeted self-antigen to myeloid APC subsets and caused profound antigen-specific tolerance in EAE.

This study was supported by research grants from the National Multiple Sclerosis Society and the Brody Brothers Endowment Fund.
Gene therapy promoting inflammation-triggered IL-10 production ameliorates collagen induced arthritis (50.21)
Authors: Henningsson Louise , Eneljung Tove , Lidberg Ulf , van de Loo Fons , van den Berg Wim , Gjertsson Inger
Published: January 2023

Current treatments of rheumatoid arthritis are focused on suppression of the immune response irrespective of the inflammatory status. Interleukin (IL) -10 is a cytokine with anti-inflammatory properties. Lentiviral vectors has been used to insert an additional IL-10 gene, driven by an inflammation-inducible promoter, into the mouse genome. This enhanced endogenous production of IL-10 would allow an arthritic host to inhibit episodes of flares whilst protecting form joint destruction. Bone marrow transplants were performed to introduce the virus into DBA/1 mice. The mice were immunized with collagen type II and arthritis was evaluated throughout the experiment. The mice treated with the IL-10 construct showed a delayed onset of disease and a significantly less severe arthritis compared to controls. The frequency of arthritis in the control group increased to 70% while only 30% of the IL-10 treated mice were affected. The use of viral vectors expressing IL-10 significantly ameliorates the severity of collagen induced arthritis. Further experiments are ongoing to elucidate the mechanism of protection.

This study was supported by grants from the Swedish Medical Research Council, Göteborgs Läkaresällskap, King Gustav V's 80 Year Foundation, Reumatikerförbundet i Göteborg, Professor Nanna Svartz Foundation, Thölen and Kristler Foundation, the Swedish Foundation for Strategic Research, the 6th Framework Program of the European Union, NeuroproMiSe, LSHM-CT-2005-01863, AUTOCURE, LSHM-CT-2005-018661
Role of TLRs in the induction of tolerogenic CD8a- dendritic cells by GM-CSF (50.34)
Authors: Prabhakar Bellur S , Ganesh Balaji B , Pilli Tania , Bhattacharya Palash , Gopisetty Anupama , Vasu Chenthamarakshan
Published: January 2023

GM-CSF is a potent dendritic cell (DC) growth factor that plays an essential role in DC differentiation. We examined the potential of GM-CSF to impart tolerogenic potential to CD8a+ and CD8a- sub-populations of DCs. We show that GM-CSF selectively modulates myeloid CD8a-, and not plasmacytoid or CD8a+, DCs in vivo. GM-CSF treatment rendered CD8a- DCs tolerogenic which was characterized by the diminished production of inflammatory cytokines IL-12 and IL1β by these DCs. Self antigen, mouse thyroglobulin (mTg), presentation by GM-CSF exposed CD8a- DCs, relative to control CD8a- or other DCs, caused a significant increase in the frequency of Foxp3+ T cells among CD4+ T cells from mTg-primed mice. The ability of GM-CD8a- DCs to induce Foxp3+ T cells was severely abrogated upon addition of IL-12, and not IL1β, into the culture and indicated that reduced IL-12 production rendered them tolerogenic. Further, we studied the effects of treatment with various TLR ligands to either augment or abolish the effects of GM-CSF on CD8a- DCs. Interestingly, addition of TLR ligands such as LPS and CpG further augmented the ability of GM-CSF exposed CD8a- DCs to induce Foxp3+Tregs. These results indicated that exposure of GM-CD8a- DCs to TLR4 and TLR9 ligands can enhance their tolerogenic potential and strongly suggested that pro-inflammatory immune stimuli may confer an enhanced antigen specific tolerogenic effect on GM-CSF exposed CD8a- DCs. NIH 1RO1AI058190
Protection from diabetes after subcutaneous insulin B:9-23/IFA immunization is mediated by Tregs that require IL-10, IFNg and NO but not IL-4 (50.39)
Authors: Fousteri Georgia , Dave Amy , Juntti Therese , Bot Adrian , von Herrath Matthias
Published: January 2023

Induction of antigen-specific tolerance in type 1 diabetes (T1D) to auto-antigens such as the insulin B chain peptide 9-23 (B:9-23) has been proven beneficial when conducted in conjunction with incomplete Freud's adjuvant (IFA) in the NOD (non-obese diabetic) mice. Due to the B:9-23/IFA ongoing clinical trials, the ill-defined mechanisms involved in achieving tolerance after B:9-23/IFA immunization could become a setback for this otherwise attractive T1D prevention strategy. Here we report that a single subcutaneous (s.c.) B:9-23/IFA immunization is as effective at later stages of the disease as at a younger stage, and loses its protective effect after IFNg or IL-10 neutralization but not in the absence of IL-4. Interestingly, when CD4+CD25+ or IFNg-producing cells from B:9-23/IFA protected mice were transferred into pre-diabetic NOD recipients, protection from T1D was conveyed, indicating that a dominant regulatory Treg-mediated effect was operational that depended on IFNg as well as NO production. In agreement, a significant increase in Treg numbers in the spleen and pancreatic draining lymph nodes (PLN) after s.c. B:9-23/IFA immunization was observed. Thus, protection from T1D after s.c. B:9-23/IFA immunization occurs through IFNg/NO and IL-10-mediated suppressive mechanisms.
Characterization of Critical Residues of the Granzyme B Inhibitor, Serpina3n (50.43)
Authors: Marcet Palacios Marcelo , Bleackley Chris
Published: January 2023

Granzyme B (GrB) is crucial for the immune system in eliminating tumor and virus-infected cells by caspase-mediated apoptosis. GrB also has detrimental effects in allograft tissue rejection and autoimmune diseases. Our aim was to design effective GrB inhibitors as unique immunosuppressive drugs in treating these disorders.

Our lab has defined a novel murine inhibitor of GrB named serpina3n (ser3n). Ser3n is a superior candidate for drug design since it is the only known GrB inhibitor that is secreted extracellularly, making it more plasma stable. We performed scanning mutagenesis of the reactive centre loop (RCL) domain, which confers serpins with their protease specificity, to test which residues have inhibitory properties. Using an in vitro transcribed/translated (IVTT) ser3n protein, we found that the putative P1 residue (Met) and the P3' residue (Lys) are important for ser3n binding to GrB.

Human antichymotrypsin (ACT) is highly homologous to ser3n, but is not a GrB inhibitor. Our goal was to generate a human secretory protein that inhibits GrB. We created an ACT-ser3n chimera by replacing the RCL of ACT with that of ser3n. We then created a mutant ACT-ser3n chimera with a Met-to-Asp mutation that resulted in greater GrB binding than the original chimera, to a degree equal to ser3n binding. This inhibitor will work on pathogenic states when secreted GrB is important. This reveals a potential drug for blocking GrB in autoimmune diseases.
Cordyceps militaris modulates presentation of exogenous antigen associated with MHC class I and MHC class II pathway in dendritic cells. (78.28)
Authors: Shin Seulmee , Kwon Jeonghak , Lee Sungwon , Moon Sunhee , Lee Seungjeong , Lee Chongkil , Cho Kyunghea , Kim Kyungjae
Published: January 2023

Cordyceps militaris has been used in traditional medicine to treat numerous diseases and reported to have antitumor and immunomodulatory activities in vivo and in vitro. The precise mechanism of its immune response activities is unclear. The water extract of Cordyceps militaris (CME) and cordycepin (3'-deoxyadenosine) were prepared. Cordycepin, nucleoside analogue, is a metabolite of C. militaris, whereas CME is included the cordycepin 1.3 ng/mg. How CME or cordycepin modulated immune reaction mechanism in antigen presenting cells (APCs) by ability to present exogenous antigens in association with the major histocompatibility complexes (MHC) in vitro and ex-vivo were examined. Microencapsulated ovalbumin (OVA) was efficiently captured, processed and presented on both MHC class I as well as MHC class II molecules. DC 2.4 cells (H-2Kb) or bone marrow- drived DCs (BM-DCs) generated from the BM cells of C57BL/6 mouse (H-2Kb) were cultured in the presence of CME or codycepin with OVA-microspheres, and the amount of OVA peptide - MHC class I complexes was measured by T cell hybridoma, CD8 OVA 1.3 T cell, that recognizes the OVA-H-2Kb complex and IL-2 production. The increment of the phagocytotic activity and the expression of MHC molecules on DCs by CME (12.5 ~ 200 μg/ml) were shown, while suppression of its activity had shown by cordycepin. CME also increased IL-2 production when we examined the effects of CME on CD4+ T cells. On the other hand, cordycepin inhibited CD4+ T cell responses in a concentration of between 5μg to 40μg. The present study demonstrates the modulation of cross priming capability could be a target of therapeutic immunoregulation.

Key words: Cordyceps militaris, cross presentation, immunomodulator
Chlamydial respiratory infection predisposes to neutrophil dominated allergic airways disease (AAD) (79.5)
Authors: Horvat Jay C , Starkey Malcolm R , Beagley Kenneth W , Gibson Peter G , Foster Paul S , Hansbro Philip M
Published: January 2023

Chlamydial lung infections are associated with asthma but it is unknown how these Th1-inducing infections influence Th2-mediated asthma. We investigated the effect of current and resolved chlamydial infection on the development of hallmark features of asthma using a murine model of ovalbumin (Ova)-induced AAD.

BALB/c mice were infected with Chlamydia and had either resolved or current respiratory infection at the time of sensitization to Ova. The effect of infection on AAD was compared with un-infected or non-allergic controls.

Current, but not resolved, infection attenuated hallmark features of eosinophilic AAD; pulmonary eosinophil influx, T cell production of IL-5, mucus secreting cell (MSC) hyperplasia and airways hyper-responsiveness (AHR). Importantly, while current infection suppressed Th2-mediated AAD, robust Ova-specific IFN-γ release from T cells and allergen-driven neutrophilic inflammation were induced. This neutrophilic phenotype correlated with increased pulmonary expression of IL-12 and IL-17 and suppression of thymus and activation-related chemokine (TARC). Inhibition of neutrophil and macrophage chemotaxis into the lungs during current infection reversed neutrophilic inflammation and IFN-γ production in AAD and removed the attenuating effect of infection on MSC hyperplasia and AHR. These changes correlated with decreased IL-12 and IL-17 expression, increased TARC and altered antigen presenting cell function.

Ongoing chlamydial respiratory infections modify key allergen-specific immune responses in AAD with the composition of cellular inflammatory responses to infection crucial in determining the outcome of allergic phenotype.

Supported by the NHMRC
Preliminary characterization of chicken hemaglutination-enhancing factor (81.20)
Authors: Koppenheffer Thomas L. , Hampton Stephen
Published: January 2023

Hemagglutination-enhancing factor (HEF) is a chicken serum macroglobulin first described on the basis of the ability of normal chicken serum to cause pronounced aggregation of allogeneic erythrocytes (RBC) sensitized with non-agglutinating levels of alloantisera. Subsequent work showed that HEF was distinct from the first component of complement, but the identity of HEF and its ligand(s) have not been defined. We found that chicken serum agglutinated homologous RBC and sheep RBC lightly sensitized with specific IgY but not sheep RBC sensitized with rabbit IgG. Thus, the sole ligand of HEF appears to be antigen-bound IgY. HEF activity was found to be heat resistant (56o for 30 min.), unaffected by chelation with 10mM EDTA, and was not inhibited by monosaccharide ligands of C-type lectins. An isolate was obtained from chicken serum following salt precipitation, gel filtration and ion exchange chromatography that contained potent HEF activity, and consisted almost exclusively of IgM. These findings suggest that HEF is a naturally occurring IgM antibody that has the capacity to interact with antigen-bound IgY.

Supported in part by the Ralph & Louise Bricker Endowed Fund
The CD8+ T cell memory state of readiness is actively maintained and reversible (83.18)
Authors: Allam Atef , Giardino Torchia Maria Letizia , Conze Dietrich , Yagita Hideo , Munitic Ivana , Sowell Ryan T , Marzo Amanda L , Ashwell Jonathan D
Published: January 2023

The ability of the adaptive immune system to respond rapidly and robustly upon repeated antigen exposure is known as immunologic memory, and it is thought that T cell progression from the naïve to the memory state is an irreversible differentiation event. Here we report that unlike naïve T cells, which are in the G0 phase of the cell cycle, CD8+ memory T cells exist in a state of readiness in G1. This was maintained by occupancy of members the TNF receptor superfamily, CD27 and 4-1BB, which when disrupted caused memory T cells to revert to G0 and respond like naïve T cells to stimulation. Furthermore, maintenance of the G1 state was compromised in antigen-specific memory T cells of VSV-infected CD27-deficient mice. Manipulating the memory T cell state of readiness might benefit disorders involving chronic ongoing immune responses.

This work was supported by the Intramural Research Program of the National Institutes of Health, Center for Cancer Research,

National Cancer Institute
A joint role for IL-2 and IL-15 in the differentiation and survival of short-lived effector and effector memory CD8+ T cells (83.2)
Authors: Williams Matthew A , Mitchell Diana M. , Ravkov Eugene V
Published: January 2023

Interleukin-2 (IL-2) plays a central role in the differentiation of functionally competent CD8+ memory T cells. In this study, we found that in the absence of IL-2 signals, the differentiation of short-lived CD8+ effector T cells and tissue-residing effector memory CD8+ T cells in response to acute infection was greatly impaired. Due to their shared signaling apparatus, we explored the hypothesis that IL-15 signals might also contribute to the differentiation and survival of these populations. In line with this hypothesis, we observed an additional impairment in the differentiation and persistence of short-lived effector/effector memory cells in the spleen when both IL-2 and IL-15 signals were absent. Furthermore, in the absence of IL-2 and IL-15 signals this population disappeared completely by day 15. In contrast, joint inhibition of IL-2 and IL-15 signals had no further impact on the differentiation of tissue-residing CD8+ effector memory T cells. While IL-2 and IL-15 contributed to the differentiation of short-lived effector cells during the primary response, IL-15 continued to play a mandatory role in the survival of these cells during contraction and memory. These findings delineate a joint role for IL-15 and IL-2 in the differentiation of short-lived CD8+ effector cells but point to unique roles for IL-15 and IL-2 in promoting the survival of short-lived effector/effector memory cells in the spleen and the differentiation of tissue-residing effector memory cells, respectively.
Exploring Myths of CD8+ T Cell Contraction (83.22)
Authors: Prlic Martin , Bevan Michael J
Published: January 2023

A small fraction of the CD8 T cell effector population that responds to an infection progresses through the contraction phase to the memory stage. The factors regulating the extent of contraction are poorly understood. In previous experiments, parameters such as inflammation, antigen presentation, etc. were modified during the priming/expansion phase and the consequences of these changes were monitored in the contraction phase. A major caveat of these studies is that the experimental setup did not allow examination of the contraction phase without altering the priming phase first. We developed a system based on bim-/- CD8 T cells that overcomes this confounding problem. Competition for limited resources has been widely postulated to be the cause of cell death during the contraction phase, but our data show that competition does not affect contraction kinetics. All effector cells present at the peak of the response have the potential to become bona fide memory cells, thus excluding selection based on functionality. We propose that cell death during contraction is the default pathway for an effector cell and identify a set of genes that may be key regulators for surviving contraction.

This work was supported by the Howard Hughes Medical Institute and National Institutes of Health Grant AI-19335 (to M.J.B.). M.P. is a fellow of The Leukemia & Lymphoma Society. The authors have no conflicting financial interests.
Suppression of CD8 T cell survival by the synthetic adjuvant poly (I:C) at low dosage (83.3)
Authors: Ngoi Soo Mun , Vella Anthony T
Published: January 2023

The synthetic double-stranded RNA (dsRNA) poly I:C is commonly used as an adjuvant to boost CD8 T cell functions through type I IFN production but is also effective at suppressing disease development in certain autoimmunity models. It is unclear how a single adjuvant achieves two distinct immunoregulatory roles. By utilizing the transgenic T cell OTI adoptive transfer model, we found that low dose poly I:C suppresses costimulation-enhanced CD8 T cell survival. Perhaps surprisingly, early expansion and effector differentiation were not significantly impacted. Reduction in CD8 T cell survival was rescued by increasing the poly I:C dosage and is dependent on the precursor frequency of antigen-specific CD8 T cells. We hypothesize that the opposing effects of poly I:C on CD8 T cells at low versus high doses depend on differential activation of multiple dsRNA receptors. Compared to the intracellular dsRNA receptors such as MDA5 and RIG-I, TLR3 may have better access to poly I:C in soluble form due to its strategic location in the endosomal compartment. Hence we postulate that TLR3 is responsible for suppression of CD8 T cell survival when low dose poly I:C is administered. Indeed, a minor subset of splenic B cells that express CD5 and CD21 surface markers produce IL-10 in response to poly I:C. Interleukin-10 synthesis was inhibited in TLR3-/- B cells, although these cells were capable of producing IL-10 in response to PMA and ionomycin. The relationship of IL-10 and reduced T cell survival is being explored, as is the anatomical location of the CD5+CD21+ B cells. Consistent with other reports on the immunoregulatory role of IL-10-producing B cells on adaptive immunity, our findings further suggest the feasibility of fine-tuning such regulatory balance by altering the dosage of TLR ligands.
Cell division predicts CD62L expression in vivo (83.6)
Authors: Davenport Miles P , Schlub Timothy , Sabel Jaime L , Harty John T , Badovinac Vladimir P
Published: January 2023

Naïve CD8+ T cells express a predominantly CD62Lhigh phenotype, but CD62L expression is rapidly lost during the effector phase of the immune response and only slowly regained during the memory phase. We used adoptive transfer of TCR-transgenic T cells followed by Listeria infection to study the dynamics of cell division and differentiation in vivo. Adoptive transfer of larger quantities (400 000) of naïve TCR-transgenic T cells leads to diminished T cell growth following infection and a higher proportion of cells remaining CD62Lhigh than populations derived from adoptive transfer populations of smaller quantities (3200 naïve TCR-tg T cells). This suggests a process of 'division-linked differentiation', where a proportion of cells change phenotype (in this case CD62Lhigh => CD62Llow) upon division. A simple model of division-linked CD62L differentiation where 20% of CD62Lhigh cells differentiate to become CD62Llow on each division accurately predicts the phenotype of cells during acute infection. By contrast, expression of CD127 did not conform to this simple pattern of differentiation. These results suggests that CD62Lhigh and CD62Llow cells arise from the same precursors, and CD62Llow cells are formed from the CD62Lhigh population by a process of progressive, division-linked differentiation.
Phosphoinositide dependent kinase 1 (PDK1) maintains T-cell metabolism during proliferative stress (84.20)
Authors: Macintyre Andrew N. , Kelly April P. , Cantrell Doreen A.
Published: January 2023

Phosphoinositide dependent kinase 1 (PDK1) is a serine/threonine kinase that phosphorylates and activates protein kinase B (PKB/Akt) and the p90 ribosomal S6 kinase (RSK). Naïve T-cells lacking PDK1 are viable in a thymic environment, show normal ex-vivo survival responses to IL7 and can migrate normally to secondary lymphoid tissue. However, when induced to proliferate by antigen receptor triggering or a lymphopenic environment PDK1 deleted naïve T-cells are unable to survive. Activated CD8+ effector T-cells lacking PDK1 suffer gross metabolic defects with reduced rates of aerobic glycolysis, decreased glucose uptake and abnormal lipid metabolism, and cannot sustain proliferation or survival. A T-cell's requirement for PDK1 as a driver of metabolism is therefore limited to periods of proliferative stress. Strikingly, in peripheral T cells the ability of PDK1 to sustain T cell metabolism is not mediated solely by Akt but includes a critical synergistic contribution by RSK kinases. PDK1 thus has a key role in controlling energy metabolism and the survival of proliferating T cells, but distinct PDK1 independent signalling pathways regulate naïve T cell survival.

Funded by the Wellcome Trust.
Microarray analysis of genes induced by 2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) in thymic progenitor cell populations prior to thymic atrophy (84.3)
Authors: Silverstone Allen E , Laiosa Michael D , Lai Zhi-wei , Middleton Frank A , Mills Jeffrey H , Singh Kameshwar P , Gasiewicz Thomas A
Published: January 2023

TCDD activates the Aryl Hydrocarbon Receptor (AhR), a basic helix-loop-helix transcription factor, and causes thymic atrophy in all species examined. We have previously shown [Laiosa, et. al. J. Immunol. (2004) 171 4582-91] that the earliest detectable effect associated with TCDD induced thymic atrophy was a decline in the percentage of S phase cells in the CD3-CD4-CD8- triple negative (TN) intrathymic progenitor 3 and 4 stages within 9-12 hours of treatment, prior to any detectable reductions in thymocyte cell number. We also demonstrated that this effect was strictly by activation of the AhR in the developing lymphocytes. We now show, using microarray analysis, that 108 distinct genes are affected in the TN1-4 thymic progenitor stages 6 and 12 hours after TCDD treatment. While most of these genes have specific AhR recognition sequences, only 7 genes were altered exclusively in the TN3 and 4 stages. Interestingly, all 7 of these genes demonstrated a decrease in expression. These 7 genes represent novel targets of AhR transcriptional activity in the developing immune system. Future work will test the hypothesis that these 7 genes play a critical role for thymocyte cell cycle regulation and proliferation following TCRβ selection.

Funded by 5RO1- ES04862 (TAG) and Hendricks Fund (Syracuse University).
A haplotype of the HLA-DRA gene promoter region was associated with susceptibility to gastric cancer in a Korean population (88.1)
Authors: Kwack KyuBum , Pyun Jung-A , Kim Kyoung-Yeon , Lee Seung Ku , Oh Ah Reum , Kim Min-Ho , Kang HyunJun , Won SungSik , Lee Kwang-Jae , Cho Sung Won , Kim Jeong-Geun
Published: January 2023

Gastric cancer, the second leading cause of mortality by cancer in the world, shows particularly high incidence and mortality in Korea. To screen the causative genes or loci that control the transition from gastritis to gastric cancer, whole genome approach was applied using Illumina's 510S SNP chip technology. One of the interesting associated genes, HLA-DRA1 was investigated. HLA-DRA gene is very limited in polymorphism compared to the highly polymorphic HLA-DRB1, 3, 4, and 5 loci. HLA-DR alpha protein has roles as a non-polymorphic partner for HLA DR-beta for transport and surface expression as a functional HLA-class II protein. The expression level of the HLA-DR alpha/beta on the cell surface may be controlled by the transcriptional level of the HLA-DR alpha. To test this hypothesis, we tested susceptibility to gastric cancer compared to gastritis in terms of SNPs, haplotypes, and diplotypes covering the HLA-DRA promoter region. To find causative SNP(s), the whole linkage disequilibrium (LD) region of the significant haplotype using more than 20 Koreans were sequenced. We found that a diplotype composed of significantly associated SNPs and a haplotype was associated with susceptibility to gastric cancer significantly. These data suggested that the expression level of HLA-DRA might control the HLA-DR protein levels on the cell surface.
Immune contexture and cancer prognosis (88.28)
Authors: GALON Jerome , TOSOLINI Marie , KIRILOVSKY Amos , CAMUS Matthieu , MLECNIK Bernhard , BINDEA Gabriela , TRAJANOSKI Zlatko , BRUNEVAL Patrick , BERGER Anne , FRIDMAN Wolf-Herman , PAGES Franck
Published: January 2023

To date the anatomic extent of tumor (TNM classifications) has been by far the most important factors to predict the prognosis of colorectal cancer patients. However, the impact of immune responses and tumor escape on patient prognosis in human cancer is poorly understood.

We showed that tumors from human colorectal cancer with a high density of infiltrating memory and effector memory T-cells (TEM) are less likely to disseminate to lymphovascular and perineural structures and to regional lymph-nodes. We showed that the combination of immune parameters associating the nature, the density, the functional orientation and the location of immune cells within the tumor was essential to accurately define the impact of the local host immune reaction on patients prognosis. We proposed to define these immune criteria as "immune contexture". Investigation of the CRC primary tumor microenvironment allowed us to uncover the association of favorable outcomes with efficient coordination of the intratumoral immune response. We described four major immune coordination profiles within CRC primary tumors depending on the balance between tumor escape and immune coordination.

In conclusion, the density and the immune-cell location within tumor regions had a prognostic value that was superior of those of the TNM classifications. Tumor invasion was dependent on the host immune reaction.
The role of IL-6 and IL-23 in colitis associated cancer (88.30)
Authors: Grivennikov Sergei , Mucida Daniel , Terzic Janos , Rose-John Stefan , Eckmann Lars , Cheroutre Hilde , Karin Michael
Published: January 2023

Colitis associated cancer (CAC) is the most serious complication of inflammatory bowel diseases (IBD). Pro-inflammatory cytokines regulated by NF-B were suggested to regulate pre-neoplastic growth during CAC tumorigenesis. We tried to dissect the roles of individual cytokines in CAC tumorigenesis. Using genetic tools and a mouse model of azoxymethane/dextrane sodium sulfate induced CAC we demonstrate that IL-6 and IL-23 are critical tumor promoters during early CAC tumorigenesis. IL-6 and IL-23 act on cells of innate and adaptive immunity, particularly on T cells. Additionally, these cytokines have direct and indirect effects on intestinal epithelial (IEC) and neoplastic cells. These effects are mediated by transcription factor STAT3, whose IEC-specific ablation reduces CAC tumorigenicity. We conclude that IL-6 and IL-23 regulate multiple stages of colonic tumorigenesis, particularly tumor initiation and tumor growth. Selective inhibition of these cytokines may be beneficial for treatment and prevention of CAC.

Supported by RFA from Crohn`s and Colitis Foundation of America (CCFA #1762) to S.G. and by grants from the NIH and a Jeannik M. Littlefield-AACR grant in metastatic colon cancer to M.K.
A short term assay for evaluating the functional potential of isolated human regulatory T cells (89.14)
Authors: Ruitenberg Joyce J. , Ghanekar Smita A.
Published: January 2023

Naturally occurring human regulatory T cells (Tregs) are able to actively suppress auto reactive immune responses. Phenotypically, they are characterized as CD4+, CD25++, CD127- and FoxP3+. Determining whether Tregs can suppress the antigen-specific cytokine expression of autologous PBMC would provide useful insight into the functional abilities of these cells. Long term assays such as proliferation are routinely used to analyze the functional capability of Tregs, but for practical reasons, a short term intracellular cytokine staining (ICS) assay would seem to be a more valuable tool for assessing the potency of Treg. In this study, we have demonstrated that highly purified and in vitro expanded Treg are capable of suppressing T-cell specific cytokine responses in 7 hr ICS assays. Treg were purified from healthy donor PBMC by sorting the CD4+CD25++CD127dim/- cells with a BD FACSAria(tm) flow cytometer. The functional ability of the highly purified Treg was evaluated either after activation for approximately 20 hours, or after in vitro expansion for 12 to 14 days. Activation of PBMC with SEB and/or CD3/CD28 results in the expression of inflammatory cytokines (IFNγ, and TNFα) by CD4+ and CD8+ T cells. The data from this study indicate that the addition of CD4+CD25++ cultured Treg to the ICS assay of autologous PBMC results in suppression (i. e. reduced frequency of) cytokine expressing T cells. There was no correlation between the frequency of FoxP3+ Treg and % suppression of cytokine+ effector cells. Since the intracellular T-cell cytokine expression assay has been validated extensively, this short-term assay could reduce the time needed to qualify Treg for immunotherapy.
Expansion of Tregs in Aged, but not Young Mice following Influenza Infection (89.7)
Authors: Williams-Bey Yolanda , Jiang Jiu , Murasko Donna M
Published: January 2023

The increase mortality and morbidity commonly seen with aging is believed to be in part due to a negatively altered immune response. The role of regulatory T cells (Tregs) in relation to reduced immune response in aging was investigated. In this study we compared the function and phenotype of Tregs isolated from young (4-6 mos) and aged (18-22mos) C57bl/6 mice. Both young and aged Tregs significantly reduced the activation and expansion of specific CD8 T cells, however there was no difference in the level of suppression induced by young versus aged Tregs. There was a strong negative correlation between Treg percentage and CD8 T cell activation. To further establish the negative correlation between specific CD8 T cell expansion and Tregs, mice were infected intravenously (i.v.) with influenza virus (PR8 strain) and sacrificed 5, 7 and 10 days post infection. While adult mice produce a strong response to influenza aged mice had a reduced and delayed response. Aged mice demonstrated a significant increase in the proportion of Treg over the course of infection, while young mice demonstrated no such change, maintaining a consistent percentage of Tregs. Our results suggest the increase in Tregs following influenza infection interfere with specific CD8 T cell expansion and function. These studies demonstrate the further need to assess the role of Tregs in the decreased immune response in elderly individuals.
Characterization of the in vivo function of the leukocyte-associated Ig-like receptor (LAIR)-1 (90.27)
Authors: tang xiaobin , Esteso Tornero Gloria , Coligan John E , borrego francisco
Published: January 2023

Leukocyte-associated Ig-like receptor-1 (LAIR-1) is an immunoreceptor tyrosine based inhibitory motif (ITIM) containing receptor. The ligand for LAIR-1 is the GPO repeats present in all collagens. Cross-linking of LAIR-1 in vitro with antibodies or collagens delivers an inhibitory signal that is able to down-regulate TCR mediated signals. To study the in vivo function of LAIR-1, we have generated LAIR-1-/- and LAIR-1flox/flox mice on a C57Bl/6 background. LAIR-1-/- mice appear to be healthy and fertile, and have normal longevity. LAIR-1-/- mice show a slight increase in the percentage of splenic B cells, along with a decrease in T cells. Since LAIR-1+/+ and LAIR-1-/- T cells traffic equally to peripheral lymphoid organs, this is probably not the result of abnormal T lymphocyte trafficking. When OT-II LAIR-1-/- CD4 T cells are cultured with APC loaded with OT-II OVA peptide, they proliferate significantly less than OT-II LAIR-1+/+ CD4 T cells. However, in vivo, OT-II LAIR-1-/- CD4 T cells proliferate similarly to OT-II LAIR-1+/+ CD4 T cells in response to challenge with albumin. Without any stimulation, LAIR-1-/- mice have lower levels of IgG3 and IgG1 in the serum. In response to T-dependent immunization with TNP-OVA in alum, LAIR-1-/- switch less efficiently to IgG2a and IgG2b production, while switching to IgG1 is not affected. In response to T-independent immunization with TNP-LPS and TNP-ficoll, no significant differences in immunoglobulin levels were observed between LAIR-1-/- and LAIR-1+/+ mice. Several mouse disease models, including those for experimental autoimmune encephalomyelitis (EAE), inflammatory bowel disease (IBD) and hapten induced contact hypersensitivity (CHS), were utilized to try to determine how LAIR-1 functions in vivo. No differences were observed, except that preliminary results show LAIR-1-/- mice are more susceptible to CHS.
Regulation of T cell responses by a distinct subset of resident splenic macrophages (90.3)
Authors: Kurotaki Daisuke , Morimoto Junko , Kanayama Masashi , Uede Toshimitsu
Published: January 2023

The balance between immune activation and tolerance must be regulated to maintain immune homeostasis. It has been well demonstrated that dendritic cells are critical regulators to maintain such balance. Tissue macrophages (Mφs) constitute the major cellular subsets of antigen presenting cells within body, however, it is poorly understood what kind of tissue resident macrophages is involved in the regulation of immune homeostasis in the peripheral lymphoid tissues. Here, we found that splenic Mφs could be divided into two subsets due to α9 integrin expression (F4/80intMac-1hiα9- and F4/80hiMac-1lowα9+). F4/80intMac-1hiα9- Mφ spontaneously secreted IL-6 and IL-12p40 and induced robust proliferation and differentiation of naive CD4+ T cells. These characteristics were met with the features of conventional splenic Mφ (cMφ). Mφs expressing α9 integrin (α9Mφs) were mainly localized in the red pulp. Unlike cMφs, α9Mφs expressed TGF-β and inhibited T cell proliferation by a mechanism partially dependent on TGF-β. Furthermore, α9Mφs were capable of inducing the differentiation of naive CD4+ T cells into Foxp3+ regulatory T cells via TGF-β. These results suggest that these distinct two Mφ subsets reciprocally control T cell immune responses in the spleen.
Foxp3 is expressed in LPS activated mouse microglial and modulates inflammatory responses. (92.12)
Authors: Chung Hwan-Suck , Lee Jun-Ho , Kim Hyunseong , Shin Min-Kyu , Hong Moo-Chang , Kim Yangseok , Bae Hyunsu
Published: January 2023

The starting point of this study was the serendipitous observation that forkhead box P3 (FOXP3) gene was up-regulated over 3 times in the LPS activated BV-2 microglial cells at 0.5h, 1, 3 and 6h in previous microarray. We confirmed that FOXP3 was expressed in the LPS activated mouse microglial by real time PCR and RT-PCR. Using FOXP3 mutant mouse (Scurfy), Scurfy microglial were released higher levels of CXCL10 and monocyte chemotactic protein 1 (MCP-1) by LPS treatment compared with wild type microglia. However, IL-6 and IL-1¥â in LPS activated scurfy microglia were same levels compared with wild type microglial. Scurfy microglial increased nitric oxide (NO) and Reactive oxygen species (ROS) generation by LPS treatment. These results suggest that FOXP3 may contribute to the suppression of CXCL10 and MCP-1 and to the decrease of NO and ROS in the LPS activated mouse microglial cells.
Increased aortic atherosclerotic plaques in female apolipoprotein E-null (ApoE-/-) mice is associated with increased production of thromboxane A2 (93.17)
Authors: Smith Donald  D. , Tan Xiaoyu , Tawfik Ossama , Stechschulte Daniel J. , Dileepan Kottarappat N.
Published: January 2023

Prostaglandin I2 (PGI2) and thromboxane A2 (TXA2) are products of the cyclooxygenase pathway and their production is increased in patients with atherosclerosis. In this study, we evaluated the influence of gender on PGI2 /TXA2 homeostasis and its impact on atherosclerosis progression using the ApoE-/- mouse model. ApoE-/- and C57BL/6 control mice were fed a normal diet or the high fat Western Diet for 3 months, and then blood, urine samples, and aortas were collected. The EIA analyses of 24-h urine samples for 11-dehydro TXB2 and 2,3-dinor-6-keto PGF1α indicated that female ApoE-/- mice produce 15-fold higher TXA2 (M=0.20; F=2.99 ng/mg creatinine) and approximately 50% less PGI2 (M=116; F=61 ng/mg creatinine) than males. The increase in urinary 11-dehydro TXB2 was also evident in wild type female mice maintained on normal diet. En face analyses of aortas showed marked increases in plaque formation in female ApoE-/- mice (male=9.9%; female=22.0%). Quantification of the H & E-stained cross sections of the aortic arch revealed 3 to 4-fold higher plaque thickness in female ApoE-/-mice. Interestingly, serum triglyceride and total cholesterol levels in ApoE-/- females were approximately 25% lower than males. The results suggest that the markedly elevated TXA2 production and reduced PGI2 production are gender-related proatherogenic risk factors in female ApoE-/- mice. Thus, a tightly regulated PGI2 /TXA2 homeostasis is critical for the prevention of cardiovascular disease. (Supported by NIH R01-HL070101 and the Carey Arthritis Fund)
Chemoattractant receptors and lymphocyte egress from extralymphoid tissues: changing requirements during the course of inflammation (94.21)
Authors: Debes Gudrun F , Brown Meghan N , Fintushel Sarah R , Hay Jack , Butcher Eugene C
Published: January 2023

Memory/effector T cells traffic efficiently through extralymphoid tissues entering from the blood and leaving via the afferent lymph. During inflammation, T cell traffic through the affected tissue dramatically increases, however, dynamics and mechanisms of T cell exit from inflamed tissues are poorly characterized. Here we show that, unexpectedly, large numbers of lymphocytes including T cells capable of producing IFN-γ and IL-17 leave the chronically inflamed skin, showing that memory/effector T cells also egress sites of inflammation. Whereas efficient egress from acutely inflamed skin required lymphocyte-expressed CCR7, chronic inflammation promoted CCR7-independent exit. CCR7-independent exit at late time points of inflammation was sensitive to pertussis toxin implying the contribution of alternative chemoattractant receptors. Our data suggest that CCR7 is an important receptor for lymphocyte egress from both resting and inflamed tissues, but that alternative exit receptors come into play during chronic inflammation allowing the egress of CCR7- as well as CCR7+ lymphocytes.
Enhanced Effector CD8 T Cell Development to IL-17 producing Cells in IFN-g Receptor Deficient Mice (96.11)
Authors: Kish Danielle D. , Fairchild Robert L.
Published: January 2023

Contact hypersensitivity (CHS) is a CD8 T cell mediated immune response to skin sensitization and challenge with hapten. In wild-type (WT) mice, hapten-primed CD8 T cell populations producing IFN-g and IL-17 are required for elicitation of CHS. This study tested the role of the IFN-g receptor (IFN-gR) in CHS to 2,4-dinitrofluorobenzene (DNFB). The magnitude of responses in sensitized IFN-gR-/- and WT mice were similar 24 hr after challenge but were markedly extended in duration in the IFN-gR-/- mice. CD8 T cell depletion abrogated CHS in both WT and IFN-gR-/- mice. Sensitization of WT mice induced high numbers of CD8 T cells producing IFN-g and IL-17 that were detected on day 5 but were reduced to naïve levels by day 8. In contrast, sensitization of IFN-gR-/- mice induced 2.5 more IFN-g producing and 5 fold more IL-17 producing CD8 T cells on day 5 and these levels were only slightly decreased on day 8. These increases were associated with increased cellular infiltration into the hapten challenged skin site of sensitized IFN-gR-/- vs. WT mice through 48 hr post-challenge. Treatment of sensitized IFN-gR-/- mice with anti-IL-17 but not anti-IFN-g mAb during challenge attenuated the CHS response. Thus the absence of IFN-g receptor signaling dysregulates CD8 T cell priming for CHS, increasing the development of hapten-specific CD8 T cells producing IL-17 which mediate CHS without the need for IFN-g.
Characterization of neutralizing anti-mouse IL-15 antibodies (96.15)
Authors: Xia Dong , Smith Carole , Shaffer Kathy , Butz Eric
Published: January 2023

IL-15 is believed to play a central role in the pathogenesis of a wide variety of disease states. We have developed neutralizing anti-mouse IL-15 antibodies from IL-15-null mice immunized with E.coli-derived recombinant mIL-15. Monoclonal antibodies M96 and M95 blocked the mIL-15-induced proliferation of CTLL-2 cells in vitro. In a mouse acute GVHD model, M96 inhibited donor CTL expansion and limited donor anti-host cytotoxic activity. In normal mice M96 induced a profound loss of NK cells and, though the total number of CD8 T cells, CD4 T cells, and B cells was not affected by M96, there was a slight reduction in the number of memory CD8 T cells in M96-treated mice. During time course studies, mice treated with 10μg of M96 lost about 50% of NK cells from the spleens within the first day and 95% of NK cells were eliminated by day 4. By day 14 the mice had recovered about 75-80% of their splenic NK cells, and by day 28 splenic NK cell levels were indistinguishable from untreated controls. We believe that M96 will be a useful tool for dissecting the roles of IL15 in normal homeostasis and inflammatory diseases.
Modulation of human pDC function by IFN-Lambda (IL-29) (97.17)
Authors: Gallagher Grant E. , Gallagher Grant , Megjugorac Nicholas
Published: January 2023

The Type III family of IFNs comprises three members, IFN-λ1, 2 and 3 (IL-29, IL-28A&B, respectively). Each signals through the same heterodimeric receptor complex, which consists of the binding and signaling subunit (IL-28Rα) plus the IL-10Rβ chain. We have identified high levels of IL-28Rα mRNA in plasmacytoid DC (pDC) from peripheral blood, and hypothesized that IFN-λ plays in important role in pDC maturation and development. Others have treated monocyte-derived DC with IFN-λ and reported an increased ability to generate Treg cells. However, there are no data regarding its effects on additional DC subsets or in fresh, untouched populations. We have previously reported on the differential regulation of costimulatory and homing molecules on pDC and mDC by IFNλ. Here, we show, using allogeneic MLRs, that IFN-λ alters the immunostimulatory capacity of pDC, which then inhibit production of hallmark cytokines of T cell polarization, including IFN-γ and IL-13. Not only does IFN-λ exert variable effects on DC surface molecule expression, but also regulates DC activation and downstream activity.
Cell cycle progression following naïve T cell activation is independent of Jak3/gc cytokine signals (97.6)
Authors: shi min , Lin Tsung , Appell Kenneth , Berg Leslie
Published: January 2023

T cell proliferation following activation is an essential aspect of the adaptive immune response. Multiple factors, such as TCR signaling, costimulation, and signals from cytokines each contribute to determine the magnitude of T cell expansion. In this report, we examine in detail the role of Jak3/γc-dependent cytokines in promoting cell cycle progression and proliferation of naïve T cells. Using naïve CD4+ T cells from Jak3-deficient mice and wild type CD4+ T cells treated with a small molecule inhibitor of Jak3, we find that these cytokine signals are not required for proliferation; instead they are important for the survival of activated T cells. In addition, we show that the percentage of cells entering the cell cycle and the percentage of cells in each round of cell division are comparable between Jak3-deficent and wild-type T cells. Further, cell cycle progression and the regulated expression of key cell cycle proteins are independent of Jak3/γc cytokine signals. These findings hold true over a wide range of TCR signal strengths. However, when CD28 costimulatory signals, but not TCR signals, are limiting, Jak3-dependent cytokine signals become necessary for the proliferation of naïve T cells. As CD28 signaling has been found to be dispensable for autoreactive T cell responses, these data suggest the potential for interfering with autoimmune T cell responses by inhibition of Jak3 signaling.