bluuebunny/crossref_metadata_2025 huggingface dataset:
Long-lived plasma cells in the spleen (P1472)
Authors: Bohannon Caitlin , Joshy Jacob
Published: January 2023

Immunological memory can last a lifetime, in large part due to the antigen-specific antibodies continuously secreted by long-lived plasma cells. Though a great deal of attention has been given to long-lived, IgG-expressing plasma cells within the bone marrow microenvironment, other long-lived plasma cell populations are also found within the secondary lymphoid organs. We sought to characterize the splenic plasma cell populations in comparison to those found in the bone marrow and explore to what extent they contribute to serological memory, hypothesizing that this niche may play a significant role in immune memory. Surprisingly, our initial findings showed a large population of IgM-secreting, long-lived plasma cells dominating the splenic niche in murine models. These plasma cells persisted for the lifetime of the animals. Here we demonstrate how these splenic IgM plasma cells compare to bone marrow resident IgG PCs and explored the factors that contribute to their homing.
Constitutive AKT activation impairs B cell homeostasis and class switch recombination (P1443)
Authors: Hoevelmeyer Nadine , Cox Eva Maria , Wunderlich Frank Thomas , Waisman Ari
Published: January 2023

The serine/threonine kinase AKT controls not only cellular metabolism, cell survival and proliferation but is also an important regulator for peripheral B cell maturation especially representing a critical check point in phases of proliferation and differentiation. In this study we investigated the role of constitutive AKT activation in B cell development and maturation through the use of a mouse strain allowing for the conditional expression of a N-terminally myristoylated AKT (ROSA-AKT-C) in cell types that express the Cre recombinase. The myristoylation signal recruits AKT-C to the plasma membrane where AKT becomes constantly phospho-activated. This mouse was crossed to CD19 Cre mice leading to the B cell specific overexpression of AKT-C (AKTBOE). AKTBOE mice displayed splenomegaly compared to control mice as a result of increased absolute numbers of not only B cells but also T cells, neutrophiles, monocytes and mature macrophages. Interestingly, in these spleens, a B cell specific loss of CD23 expression was observed. Of note, B cells isolated from AKTBOE mice did not form germinal centers and were not able to class switch in vitro and in vivo. The investigation of immunoglobulin titers by ELISA revealed reduced IgM, and almost no IgG1, IgG2a, IgG3 and IgA serum levels. Thus, the B cell specific overexpression of AKT-C underlines its importance in B cell maturation, homeostasis and class switch recombination.
PR1-CTL directly sort-purified from umbilical cord blood lymphocytes reduce human AML in vivo. (P4360)
Authors: St. John Lisa , He Hong , Ruisaard Kathryn , Ma Qing , Shpall Elizabeth , Karen Clise-Dwyer , Molldrem Jeffrey
Published: January 2023

Adoptive transfer of leukemia specific CTL could prove to be a valuable tool in the prevention and/or treatment of relapse after SCT. In line with this, we chose to examine the use of PR1-specific CTL derived from UCB as a means of adoptive immunotherapy for leukemia. PR1, a 9 amino acid, HLA-A2-restricted self-peptide is well established as a dominant LAA. While cells specific for PR1 are present in the peripheral blood of healthy adults at extremely low frequencies (0.0005 to 0.05% of CD8+ T cells), we have found that the frequency of PR1-CTL in UCB is significantly higher ranging from 0.007 to 0.345% (mean 0.117%; n=57) of CD8+ cells. We hypothesized that these cells could be isolated directly from UCB via cell sorting and used in the prevention or treatment of leukemia. CD8 enriched cells were sorted and then activated ex vivo with anti-CD3/anti-CD28/IL-2. Sorted, activated cells were infused into a cohort of NSG mice with established leukemia. In three separate, but comparable, experiments the frequency of leukemia cells in the bone marrow of mice receiving PR1-specific CTL was reduced by 27% (range 21%-32%) compared to those receiving no treatment, and 23% (range 4%-47%) compared to those receiving non-specific CD8+ cells. The reduction of leukemia cells in the blood was even more pronounced with a 47% (range 45%-61%) decrease compared to the level seen in mice receiving PBS alone, and 58% (range 48%-68%) compared to mice receiving non-specific CD8+ cells.
Assessing natural telomerase activators (P4342)
Authors: Valenzuela Hector , Burguez Clarissa , Chikami Kristin , Cruz Manuel , Rinaldi Domenico
Published: January 2023

Telomeres are DNA-protein complexes at the ends of chromosomes that shorten during cell division, an occurrence that causes cellular senescence. Telomere shortening is offset by the action of a reverse transcriptase enzyme called telomerase, which can extend the DNA strands at the ends of chromosomes and delay cellular senescence. These observations have lead investigators to search for methods of stimulating telomerase activity in the hopes of delaying cellular senescence and extending cellular function. Currently, there are natural compounds proven to activate telomerase activity, i.e., TA-65 and TAT2. Both compounds are extracts from the plant Astragalus membranaceus, which is commercially available. Different formulations for Astragalus membranaceus can be obtained. The objective of this research was to compare two products, TA-65 and HTA (both extract of Astragalus membranaceus) for their ability to activate telomerase activity and their affect on cellular growth on both CD4 and CD8 T cell cultures. The results of our study indicate that TA-65 was able to induce telomerase activity 1.4-3.3x fold relative to controls, whereas HTA showed increases in telomerase activity up to 1.3x-fold only in two donors but did not reach statistical significance. In addition we demonstrate that T cells treated with TA-65 had increase cell growth within 7 days of culturing, again HTA also showed increases in cell growth but did not reach statistical significance.
Transcriptome analysis of N. meningitidis PorB as an adjuvant in a multi-injection vaccination schedule (P4494)
Authors: Platt Andrew , Mosaheb Munir , Gower Adam , Wetzler Lee
Published: January 2023

Vaccines play an integral role in the fight against infectious diseases. Most current vaccines, however, were developed empirically, both in their formulation and schedule. A deeper understanding of the regulatory pathways involved in both adjuvanted vaccines and multi-injection schedules would contribute greatly to the rational design of future vaccines. Here we report a microarray analysis study of a 3 injection vaccination using Neisseria meningitidis PorB as a vaccine adjuvant and an Ova antigen in a murine model. We observe that the kinetics of the response in a number of gene sets is accelerated, with maximal expression after the second vaccination, rather than the third, in the adjuvanted vaccine. We also observe a recruitment of a number of innate inflammatory pathways, with adjuvanted vaccines eliciting more robust gene expression. Main effects and interactions between the antigen and adjuvant were determined, in particular as related and antibody production. In this report we demonstrate that the inclusion of PorB as an adjuvant into the formulation of a vaccine significantly alters the kinetics and nature of the immune response at a deep transcriptional level. This may provide meaningful implications for the rational design of future adjuvanted vaccines.
Comprehensive mapping of SIVmac239 envelope antigenic determinants recognized by specific polyclonal antibodies in vaccinated and/or infected macaques (P4503)
Authors: Guo Jia , Zuo Teng , Cheng Lin , Sun Caijun , Chen Ling , Zhang Linqi , Chen Zhiwei
Published: January 2023

The RV144 trial in Thailand has demonstrated a modest level of protection against HIV infection, which was likely related to V2-binding antibodies. Along with several HIV bnAbs identified recently, it is of great importance to understand the major differences between vaccination and infection in eliciting humoral immune responses. We adopted a robust mapping technique for quantitative measurement of antigenic domains of entire SIV env displayed on the yeast. The finding of reactive determinants ranges from 30 to 240 amino acids, which allows some conformational domains being evaluated as a major technical improvement. The antigen profile of all the tested sera shared two dominant domains: V1V2 stem of gp120 and ecto-gp41 region. A major distinct domain, however, was identified near the V3 loop and the main CD4 binding region which was only recognized by the effective immunized serum but not of the non-effective immunized one. Notably, binding titer of V1V2 antibodies is significantly different between the two vaccination groups, suggested a likely role of these antibodies in controlling viral replication. Unexpectedly, the anti-V1V2 antibody responses were shifted to the ecto-gp41 region when infected macaques developed AIDS. In conclusion, our findings have significant implications to help understanding the humoral immunity against neutralization-resistant SIVmac239 infection and simian AIDS, and to guide rational vaccine immunogen identification and design.
Inflammatory responses in mice exposed to various doses of TiO2 nanoparticles (P5084)
Authors: Strecker Ronald , Sharma Nilesh
Published: January 2023

Application of TiO2 nanoparticles has increased tremendously in recent years. Consumer products containing a significant amount of these nanoparticles range from paints, plastics, papers, tooth paste, food colorants, cosmetics, sunscreens to medicine pills and beverages. Ti is generally considered biologically inert, but Ti-nanoparticles were observed to cause significant toxicity including DNA damage in recent studies. In this study, C57-BL6/J mice were administered intraperitoneally TiO2 rutile crystals (30-50nm) at the rate of 333 and 1332 mg/kg body weight. Serum levels of proinflammatory cytokines, chemokines and angiogenesis growth factors (IL-1a, IL-1b, IL-6, TNFα, IFN-γ, IL-2, Rantes, G-CSF, GMCSF, MCP-1 VEGF, EGF, IGF-1and Leptin) were determined. The majority of cytokines or growth factors in exposed mice were not significantly different than in vehicle control, but VEGF level was, approximately, 50% reduced in mice exposed to the high dose, relative to control. Mice belonging to this group also demonstrated a significant decrease in the body weight, and an increase in their liver weight. The study on transcriptional activation of the related cytokine genes is in progress.
Anti-obesity effect of Sargassum muticum (Yendo) Fensholt in high fat diet-induced obese mice (P5092)
Authors: Ahn Eun-Kyung , Lee Jung A , Ko Hye-Jin , Hong Seong Su , Oh Joa Sub
Published: January 2023

In this study, we investigated the ethanol extract of Sargassum muticum (Yendo) Fensholt (ESMF) attenuate high-fat-diet-induced obesity in 57BL/6 mice. The mice were maintained on a normal diet (ND), high-fat-die (HFD) and Oral administration of ESMF with HFD for 6 weeks. ESMF administration with HFD significantly decreased the body weight gain, fat tissue content and serum total cholesterol and triglyceride levels in comparison with the HFD group. The food intakes among the groups did not show significant changes but ESMF administration groups observed significant reduction energy efficiency ratio (EER) values in comparison with the HFD group. ESMF administration with HFD induced a significant decrease in lipid droplet accumulation in the liver tissue. We demonstrated the ESMF elevated AMPK phosphoryation and activation of ACC phosphorylation in a dose-dependent manner. Our findings suggest that ESMF may be anti-obesity effect on HFD-induced mice through activating AMPK pathway.
Crucial role for non-tissue macrophages in the development of TSLP-mediated airway inflammation (P6243)
Authors: Lai Jen-Feng , Thompson Lucas , Ziegler Steven
Published: January 2023

Allergic asthma is a common chronic Th2-driven airway inflammatory disease. TSLP is an epithelium-derived cytokine recognized as a potent factor in the development of asthma. TSLP is known to influence the function of dendritic cells to enhance Th2 T cell responses, however many of the mechanisms underlying TSLP-induced airway inflammation remain undefined. In addition to dendritic cells, the lung tissues include other MHC class II-expressing phagocytic populations which may influence T cell responses. Among these potential antigen-presenting cells are macrophage subsets (including CD11c+ SiglecF+ F4/80+ resident tissue macrophages and CD11c-lo CD11b+ F4/80+ non-tissue macrophages) which can comprise up to 10% of hematopoietic cells in the lung during airway inflammation. Here we demonstrate that these pulmonary macrophage populations express the TSLP receptor and non-tissue macrophages are rapidly recruited to the lung tissue in response airway priming in the context of TSLP. Blockade of CCR2- and/or CX3CR1-dependent recruitment of non-tissue macrophages diminishes eosinophilic allergic airway inflammation and expression of type II inflammatory genes. These finding suggest a crucial role for non-tissue macrophages in the development of TSLP-mediated airway inflammation. By elucidating the cellular mechanisms of airway inflammation, this data will contribute to more effective therapeutic approaches for treatment of asthmatic disease.
P300, but not PCAF, collaborates with IRF-1 in stimulating TRIM22 expression, that is independent on its histone acetyltransferase activity (P6336)
Authors: Xiong Sidong , Gao Bo , Xu Wei
Published: January 2023

TRIM 22 plays an important role in IFNs-mediated antiviral activity. We previously demonstrated that IRF-1 was crucial for constitutive and IFNs-induced TRIM22 expression. Here, we further investigated the molecular mechanisms of IRF-1 in promoting TRIM22 expression. Using in vitro DNA affinity binding assay and in vivo chromatin immunoprecipitation assay, we found that IFN-γ stimulation significantly enhanced the binding of p300 and PCAF, but not other co-activators such as GCN-5, SRC-1 and ACTR, to the 5’ extended IFN-stimulating response element-containing region of TRIM22 promoter together with IRF-1. Overexpression and knockdown analysis demonstrated that it was p300, but not PCAF, that functioned as a transcriptional co-activator of IRF-1 in IFN-γ-mediated TRIM22 production. Further study revealed that p300 mainly participated in the IRF-1-mediated TRIM22 expression by recruiting RNA polymerase II to TRIM22 promoter, and this effect was independent on its histone acetyltransferases activity. Our study indicated that p300 might serve as a bridge between IRF-1 and the basal transcriptional apparatus in TRIM22 induction.
TLR2 is essential for immunity against lethal herpes simplex virus encephalitis (P6346)
Authors: Kollias Christina , Jennings Stephen
Published: January 2023

HSV-1 infection of the CNS is a serious condition. Both virus-mediated neurotoxicity and bystander inflammation contribute to pathology. Understanding the mechanisms responsible for limiting invasion and controlling overactive CNS inflammation are essential for disease prevention/treatment. Multiple cell types express TLR2, which recognizes HSV-1 glycoproteins and leads to inflammatory cytokine production. We assessed the role of TLR2 in a natural lip infection model and showed that KO mice are more susceptible to CNS infection and mortality. The KO mice displayed faster invasion, elevated viral spread, and overactive inflammatory responses in the nervous system. HSV-1 infection of the CNS drove migration of peripheral WBCs to both the WT and KO brains, therefore we were unable to discern their contribution toward mortality. Analysis of DC, NK and T cell activation and function showed these cell types were not responsible for the phenotype. These data suggested a nervous system TLR2+ cell type was essential for protection. To assess the role of TLR2 specifically within the CNS, we infected mice intracranially, however, both groups succumbed to a low dose of the McKrae strain of virus. In summary, TLR2 is critically important for controlling the level of HSV-1 that spreads to the CNS, and once the virus accesses the brain, TLR2-independent responses contribute to the heightened inflammation observed in the KO animals. We aim to identify the responsible TLR2+ cell type.
Antigen-dependent and independent NKT cell responses to microbes. (P4379)
Authors: Tyznik Aaron , Verma Shilpi , Holzapfel Keli , Hogquist Kristin , Benedict Chris , Kronenberg Mitchell
Published: January 2023

We have studied the antigen-dependent and independent roles of natural killer T cells (NKT cells) in the protective response to microbes. For mouse cytomegalovirus (MCMV), a β herpes virus infection, we establish that NKT cells are important for host protection. Using Nur77-GFP mice, a sensitive reporter for TCR stimulation, and other methods, we observe that rapid induction of IFNγ secretion by NKT cells induced by viral infection is independent of TCR triggering, in vitro or in vivo. Furthermore, compared to natural killer (NK) cells, NKT cells have a different requirement for APC-derived cytokines for optimal induction of IFNγ, being more dependent on IL-12. The different requirements for APC-derived cytokines suggest that there are nonredundant roles for NK and NKT cells in the innate response to viral infections. In contrast, during Streptococcus pneumoniae infection, a microbe that expresses known NKT cell lipid Ags, NKT cells rapidly expressed GFP as well as IFNγ following infection in Nur77-GFP reporter mice, suggesting a TCR-foreign lipid mediated activation. In conclusion, our data show that NKT cells mediate host protection against diverse infections by antigen-dependent and independent means.
Ets-1 interacts with NRON complex and facilitates the nuclear entry of NFAT (P6335)
Authors: Tsao Hsiao-Wei , Tai Tzong-Shyuan , Tseng William , Ho I-Cheng , Miaw Shi-Chuen
Published: January 2023

NFAT transcription factors are critical for the expression of IL-2 by Th cells. In resting Th cells, NFATs are phosphorylated and trapped in cytoplasm by NRON complex. Calcium dependent signals downstream of TCR lead to dephosphorylation of NFATs and disassembly of NROM complex, releasing NFAT into nucleus to transactivate IL-2. However, how the assembly and disassembly of NRON complex is regulated is not fully understood. Here we report that Ets-1, the prototype of the ETS family of transcription factors was critical for the expression of IL-2 in primary human Th cells. A sizable fraction of nuclear Ets-1 quickly exited nucleus in response to calcium dependent signals and interacted with protein components of NRON complex. Ets-1 deficiency resulted in impaired nuclear entry of NFAT proteins and their recruitment to the IL-2 promoter. To the best our knowledge, Ets-1 is the first transcription factor that is known to facilitate nuclear entry of NFAT proteins by interacting with NRON complex.
Cellular expression of the alarmin IL-33 in acute skin inflammation (P6337)
Authors: Sundnes Olav , Loos Tamara , Khnykin Denis , Rankin Andrew , Sponheim Jon , Pflanz Stefan , Haraldsen Guttorm
Published: January 2023

Background: IL-33, a recently described member of the IL-1-family, is an important player in a variety of inflammatory settings and is thought to be released from damaged or necrotic cells. Little work has so far been published on the cellular expression and regulation of IL-33 in inflammatory lesions. Results: In normal mouse skin IL-33 showed a nuclear expression in non-proliferating keratinocytes (Ki67-) and periglandular myoepithelial cells. After injection with Staphylococcus aureus (6h i.d.) we observed that nuclear IL-33 disappeared from keratinocytes in the center of the lesion and instead appeared in fibroblasts at the periphery. The keratinocyte layer remained intact and expressed nuclear HMGB1, revealing no evidence of overt necrosis at this time point, thus indicating possible secretion. Wound lesions of the ear showed a similar picture at the margins. At later time points IL-33 reappeared in keratinocytes, initially in a hyperproliferative, multilayer structure in which IL33 and Ki-67 remained minimally co-localized. Similarly, in cultured human keratinocytes there was constitutive expression of nuclear IL-33 in non-proliferating cells and in contrast to endothelial cells, expression levels appeared unaffected by cell density or pro-inflammatory activation (TNF-a or IL1b) Conclusion: The regulation of IL-33 in keratinocytes appears to involve molecular signals different from the regulation seen in vascular endothelial cells and deserves further investigation.
Clean-up on IL-7 (P6290)
Authors: Martin Christopher , Surh Charles
Published: January 2023

IL-7 availability determines the size and character of the resting T cell pool and therapeutic intervention to manipulate IL-7 levels represents a point at which to control T cell numbers and naïveté. However, the mechanisms that naturally regulate IL-7 abundance remain ill-defined. Using novel mouse models and the IL-7-specific proliferative response of wild type T cells as a direct measure of the IL-7 available to T cells in vivo, we demonstrate that radioresistant cells are the peripheral source of IL-7 for both CD4+ and CD8+ mature T cells. Hematopoietic lineage cells, although apparently unimportant as a source of IL-7, are primarily responsible for limiting IL-7 availability through their expression of IL-7R. The results of this study are the first to directly test and support the consumption model of IL-7 homeostasis wherein the IL-7 that is constitutively produced by stromal cells is maintained in limited supply via receptor-mediated degradation by hematopoietic cells. Collectively, these data illuminate a formidable IL-7 cytokine sink that exists in vivo and inform the design of next-generation IL-7 therapies.
Mesenchymal stem cell effect on lymphocyte apoptosis is partially contact dependent. (P1056)
Authors: Marti Luciana , Normanton Marilia , Ribeiro Andreza , Kondo Andrea , Hamerschlak Nelson , Rizzo Luiz Vicente , Marti Luciana
Published: January 2023

Although a vast evolution in mesenchymal stem cell (MSCs) knowledge and their immunomodulatory properties, still unexplored fields, such as, its effect on T cell death. This study objective was to evaluate MSCs effect on PHA activated lymphocyte (CD3+) apoptosis in vitro. CD3+ co-cultivated with MSC presented less early apoptosis (EA) and late apoptosis/necrosis (LN) determined by Annexin V and Annexin V/Propidium Iodide co-expression, compared to the ones without MSC. After 48hs of MSC co-cultivation CD3+ EA mean was lower (9.1+2.8) then without MSC (12.7+2.0), the same was observed for LN that was lower (28.0+13) then without MSC (41.9+13) p<0.05. To assess if this effect on lymphocytes were contact dependent, the co-cultivation was performed with or without 0.2µm transwell, and we observed that is partially contact, dependent since the CD3+ EA mean after 72hs of MSC co-cultivation (25.0+3.3) was lower than with transwell (32.7+4.0), but both were inferior than without MSC (45.0+7.3). Next, to evaluate cell type specificity, lymphocytes were co-cultivated with MSC, fibroblast and HUVEC. CD3+ cells presented less EA when co-cultured with MSC and fibroblast then with HUVEC. After 72hs of lymphocytes co-cultivation with MSC (n=4) (29.95+0.29) and fibroblast (30.70+1.62) EA were significant lower compared to HUVEC (38.85+1.33) p<0.05. These results have shown that the MSC effect on CD3+ is partially contact dependent, and similar for MSCs and fibroblast, but not for HUVEC.
Antigen choice determines vaccine-induced generation of immunogenic versus tolerogenic DC (P1104)
Authors: Farkas Adam , Finn Olivera
Published: January 2023

A peptide derived from the tumor-associated antigen MUC1 (MUC1p) is seen as “self” by the immune system of MUC1.Tg mice. TCR.Tg CD4+ T cells specific for MUC1p proliferate less in MUC1.Tg mice relative to WT after vaccination with DC loaded with MUC1p. We hypothesized this hyporesponsiveness acted to prevent autoimmunity, and investigated the phenotype of post-immunization (i.v.) splenic DC as likely mediators of this tolerance to self. At 24 hours these DC primed naïve CD4+ T cells into Foxp3+ Treg versus IFNy+ T cells. Immunized MUC1.Tg mice also had fewer splenic DC compared to WT. This was due to a concurrent defect, relative to DC from immunized WT mice, in DC-produced myeloid cell-recruiting chemokines such as MIP-1a/b, RANTES, and CCL2. These DC were also less inflammatory as measured by lower IL-6, TNF-a and IL-1a and increased IL-10 production. Splenic DC were also phenotypically immature (CCR2+ MHCIIlo CD40lo CD86lo) and exhibited impaired motility ex vivo that was mechanistically linked to decreased NFkB pathway activation versus WT. The repressor of DC activation Zbtb46 was increased in DC exposed to “self’ antigen and these DC expressed more Aldh1/2 suggesting a novel role for retinoic acid-mediated tolerance in the spleen. Immunization of RIP.OVA mice with OVA gave similar results suggesting a general mechanism not unique to MUC1p as an antigen. Lastly, MUC1-specific Treg induced this tolerogenic DC phenotype, the mechanism by which we are investigating.
LRRK2 affects cytokine production and Ig secretion in activated B cells (P1060)
Authors: Chen Yi-Chun , Monroe John
Published: January 2023

Leucine-rich repeat kinase 2 (LRRK2), a 286 KDa multi-domain protein, is strongly associated with familial Parkinson disease, Crone’s disease, as well as Lupus in GWAS studies. However, the pathogenic mechanisms of LRRK2 are still unclear. In order to further study its functions, we developed a strain of GFP knockin knockout (KIKO) mice that replaced the exon2 of lrrk2 gene with gfp and eliminated endogenous LRRK2. Since it is highly expressed in different immune cells, especially B cells, we first determined that LRRK2 mainly expressed in T2, follicular, and marginal zone B cells, but not in pro-, pre-, immature, T1 or B1 B cells. After B cell activation by LPS or anti-IgM/ anti-CD40, there was no difference in proliferation as well as apoptosis between Wt and KIKO B cells. However, Wt and KIKO B cells showed statistically significant differences in cytokine production profiles. Re-stimulation of activated B cells by PMA and ionomycin showed KIKO B cells has differential cytokine productions. Meanwhile, activated KIKO B cells also showed impaired IgA and IgG3 secretion, though the Ig switching was normal. These indicate that LRRK2 may have a role in B cell effector functions, and further investigation is needed.
Myeloid cells regulate plasma cell responses in the lymph node (P1066)
Authors: Fooksman David , Nussenzweig Michel , Dustin Michael
Published: January 2023

Differentiation and survival of plasma cells requires the coordinated signaling from many different cell types, and these requirements vary depending on the tissue. In the lymph node, differentiating plasma cells migrate to the medullary cords where they can either arrest or exit the lymph node. Previously, we imaged and characterized plasmablast migration by intravital microscopy in the lymph node by transferring B cells expressing a YFP plasma cell reporter. Using recipient mice that express GFP in myelomonocytic cells, we observed that plasmablasts migrated along these cells in the medullary cords and arrested in contact with them. Since several cell types express GFP in this strain, we used a various ablation techniques to narrow down which subsets were engaged with plasmablasts and determine what role they perform. Ablation of one subset increased plasmablast numbers by 3 fold. This increase was largely due enhanced cell proliferation, but plasmablast apoptosis was also reduced. We have evidence to suggest that cytokine signaling and adhesion molecules play important roles. Localization of plasma cells in the lymph nodes may play an important function in controlling infection locally during early stages, while under autoimmune conditions, excessive plasma cell recruitment and survival can be detrimental. Regulating plasma cell survival and localization may provide ways to manipulating the immune response in both contexts.
Regulation of FcϵRI-mediated signaling and mast cell activation by adaptor protein Tespa1 (P1068)
Authors: Lu Linrong
Published: January 2023

Tespa1 (thymocyte-expressed, positive selection-associated 1) is a newly identified adaptor protein that is expressed in T cells, B cells and mast cells. Our previous study revealed its critical role in mediating TCR signaling and thymocyte positive selection. Tespa1-deficient mice have fewer mature CD4+ and CD8+ SP cells in thymus, reflecting the impairment of T cell development in these mice. We further revealed that Tespa1 interacts with Lat signolosome components Grb2 and PLC-γ1 and is essential for the assembly of this signolosome and mediating of downstream signals. Tespa1 is also highly expressed in Mast cells. Here we further explored the role of Tespa1 in Mast cell activation. We found that disruption of Tespa1 causes mast cells to be hyperresponsive upon the stimulation of FceRI receptor, evidenced by enhanced degranulation, cytokine production, and their ability to mediate anaphylaxis. We also found that more Grb2 and PLC-γ1 were recruited to the LAT1 signolosome rather than LAT2 signolosome, suggesting Tespa1 might regulate the balance of LAT1 and LAT2 signolosome assembly and downstream distal signaling transduction in mast cells.
Regulation of inflammatory diseases by cestode antigens: multiple receptors and a possible common pathway (P3128)
Authors: Terrazas Luis I. , Terrazas Cesar A. , Ledesma-Soto Yadira , Callejas Blanca , Espinoza-Jimenez Arlett , Bonifaz Laura , Satoskar Abhay R.
Published: January 2023

Helminth parasites bias the immune response toward Th2 and/or to a regulatory environment associated with high levels of IL-4, IL-13, IL-5 and IL-10. In addition, helminth infections impair immunity against other unrelated infections. Thus, helminths can modulate the immune response in their host, and they have multiple ways to escape or modulate the immune system. One of their potential evasion strategies involves modulating the early response of macrophages and dendritic cells (DCs). These modulatory activities have been exploited to interferer development of pro-inflammatory-mediated diseases such as arthritis (AR), type 1 diabetes (T1D), experimental autoimmune encephalomyelitis (EAE) and colitis. Here we show that Taenia crassiceps, a cestode, is able to ameliorate the severity of EAE, T1D and colitis, but not AR, mainly by down-regulating IL-12, TNF-a, IL-15, IL-17, IL-1b and NO production. However, as most of the effects of helminth infections in these kinds of diseases, the mechanisms involved remain mainly unknown. Using T. crassiceps antigens we reproduced the effects of the infection. Such antigens are rich in glycoproteins that appear to be binding by multiple receptors, including MGL, MR, and TLR2, and interfered with the inflammatory-induced NFκB p65 and p38 MAPK pathways. Interestingly, blockade of just one intracellular signaling pathway was enough to avoid the effect of T. crassiceps antigens on modulating DCs and programing Th2 responses.
Temporal co-option of host cell trafficking by Yersinia pestis during bubonic plague. (P3140)
Authors: Ang Wei Xin Gladys , St. John Ashley , Kunder Christian , Chan Elizabeth , Gunn Michael , Abraham Soman
Published: January 2023

Bubonic plague, caused by the gram-negative bacteria Yersinia pestis, is characterized by pathologically swollen lymph nodes (LNs) or buboes. The mechanism by which these buboes form and the adaptive function they provide for the pathogen, if any, remains unknown. We hypothesize that bubo formation is a strategy for flea-borne Y. pestis to establish a foothold in the host for subsequent systemic infection. Our studies indicate that colonization of the draining lymph node (DLN) from the site of infection occurs through the trafficking of infected dendritic cells and monocytes in temporally distinct waves. This is effected by redundant chemotactic signals, including through the receptors CCR7 and CCR2, as evidenced by the upregulation of multiple chemoattractants in the DLN during Y. pestis infection and the improved survival of infected CCR7KO and CCR2KO mice compared to wild-type.
Identification of factors required for intracellular survival of Pseudomonas aeruginosa (P3146)
Authors: Penaranda Cristina , Hung Deborah
Published: January 2023

Antibiotic resistance is becoming a greater threat to combating bacterial infections. Pathways directly responsible for infection, such as those involved in virulence or immune evasion, are likely to be good targets for novel approaches to treating infection; however, these approaches are currently not being pursued due to the lack of understanding of the requirements for bacterial survival within the host. Pseudomonas aeruginosa (PsA) is one of the most virulent opportunistic human pathogens and is often highly resistant to antibiotic therapy. Although it is traditionally considered an extracellular pathogen, PsA has been shown to invade and survive inside host cells. We hypothesize that this ability to survive intracellularly allows bacteria to become antibiotic insensitive, leading to the formation of reservoirs for recurrent infections. We have developed an in vitro infection model where we have identified genes required for intracellular survival in epithelial cells. We have also found strain specificity among clinical isolates in the kinetics of survival and in their ability to become antibiotic insensitive, suggesting a genetic basis for these differences. We are screening a PsA mutant library to find factors involved in intracellular survival that could be the basis for the development of novel anti-infectives.
The role of regulatory T cells in the Leishmania (Viannia) panamensis mouse model: a target for immunotherapy. (P3091)
Authors: Ehrlich Allison , Castilho Tiago , Goldsmith-Pestana Karen , Sparwasser Tim , McMahon-Pratt Diane
Published: January 2023

Leishmania (Viannia) parasites are etiological agents of cutaneous leishmaniasis in the New World. Infection in humans is characterized by mixed Th1/Th2 and hyperinflammatory responses. Treg suppressive function is diminished and only restored with disease resolution. Using the mouse model of chronic L. (V.) panamensis infection, we examined the hypothesis that Treg impairment contributes to pathogenesis. Upon infection, Treg CD4+Foxp3+ cell levels were reduced and dysregulated. Treg cells produced IFNγ and had a reduced ability to suppress T cell proliferation in vitro. Ablation of Tregs in DEREG mice resulted in enlarged lesions, increased parasite load and enhanced production of IL-17 and IFNγ. Conversely, adoptive transfer of Tregs from naïve mice halted disease progression, lowered parasite burden and reduced production of IL-10, IL-13, IL-17 and IFNγ. As Treg function appeared to be important for controlling pathogenesis, we examined whether directly modulating Treg function provided a target for immunotherapy. As a proof of principle, chronically infected mice were treated with high dose CpG to induce Tregs. Immediately after treatment, increases in TGFβ and Foxp3+ cells were observed, with an ablation of Foxp3+IFNγ+ cells and reduction of IL-10, IL-13, IL-17 and IFNγ. CpG treatment led to amelioration of disease. Thus, immunotherapy targeting Tregs may provide an alternate treatment strategy for leishmaniasis caused by L. (Viannia) panamensis parasites.
EBNA-1 specific effector T cell deletion associated with holoendemic malaria exposure in the etiology of endemic Burkitt’s lymphoma (P3063)
Authors: Mulama David , Chelimo Kiprotich , Collins Ouma , Jura Walter , Otieno Juliana , Vulule John , Munz Christian , Moormann Ann
Published: January 2023

Etiological mechanism of eBL remain largely unknown despite long-standing epidemiological link between malaria and EBV co-infection. Age-dependent deficiency in EBV-specific CD8+ T cell-mediated IFN-γ immunosurveillance has been reported in malaria holoendemic exposed children which has been associated with eBL diagnosis. We sought to investigate whether qualitative differences in EBNA-1 specific T cells existed in malaria-exposed children to explain their increased risk for eBL. We conducted a cross-sectional study of healthy children from western Kenya with divergent malaria exposure:Kisumu (holoendemic) and Nandi (hypoendemic) and eBL age-matched patients. T cell effector function was evaluated by multiparameter flow cytometry against T cell lineage markers(CD3,4,8,45RA & CCR7) and functional phenotype (IFN-γ, IL10, IL17 & PD1) to EBNA-1. EBNA-1 specific responses were compared to MSP-1, EBNA2 and EBNA3A. We found significantly higher IFN-γ responses to MSP1 in the eBL & Kisumu compared to Nandi children (p=0.03), in contrast to EBNA1, which were significantly lower in eBL and Kisumu compared to Nandi children (p=0.01). Responses to EBNA2 and EBNA3A did not differ between groups. IFN-γ was generated from CD4 TEM and a heterogeneous combination of CD8 T-cells. PD-1 expressing T-cells were less frequent in eBL patients and highest in Kisumu children.This suggests that the EBNA1-specific deficiencies are due to deletion of T-cell population due to malaria induced exhaustion.
mTORC1 inhibition pronounces CD8 T cell effector defects in aging (P1233)
Authors: Goldberg Emily , Smithey Megan , Nikolich-Zugich Janko
Published: January 2023

Mammalian target of rapamycin complex 1 (mTORC1) is a critical cell nutrient sensor and its role in CD8 T cell development, homeostasis, and fate decisions during infection are becoming increasingly evident. Seminal studies showed that direct mTORC1 inhibition with rapamcyin favored memory CD8 T cell development after infection. Simultaneously, rapamycin was shown to extend lifespan in mice. Whether or not rapamycin treatment alters the CD8 T cell immune response in old mice has not been studied. This question is particularly critical given recent data from our lab that CD8 T cell defects after Listeria monocytogenes (Lm) and West Nile Virus infection are both associated with increased infection mortality in old mice. Therefore, we tested the effect of rapamycin (75μg/kg), when given during the T cell priming phase, on CD8 T cell effector responses in adult (3 months) and old (18 months) mice infected with Lm. We found that mTORC1 inhibition with rapamycin significantly inhibited effector differentiation of antigen-specific CD8 T cells and this was especially pronounced in old mice. Additionally, contrary to previous reports, we measured no benefit on CD8 T cell memory formation in either old or adult mice. Therefore, we conclude that any beneficial effects of rapamycin on memory formation likely come at the cost of effector function, and these effects may be pathogen-specific. Furthermore, rapamycin may not be a suitable candidate for extending lifespan or “Healthspan.”
Cell-extrinsic mechanism of CTLA4-mediated regulation of dendritic cell maturation depends on STAT3 (P1202)
Authors: Zhang Li , Kowalczyk Aleksandra
Published: January 2023

The role of cytotoxic T lymphocyte antigen 4 (CTLA4) in hampering immune responses has been well documented; however, the exact mechanism by which CTLA4 exerts its effect is still under debate. Here, we investigated whether CTLA4 signal alone is sufficient to inhibit dendrtic cell (DC) maturation, stimulatory function, and the molecular mechanisms involved. We demonstrate that stimulation of DCs with CTLA4, either expressed by inducible regulatory T cells (iTregs) or by cross-linking with CTLA4Fc fusion protein, is sufficient to inhibit LPS-induced maturation of both mouse and human DCs. The CTLA4 signaling significantly decreased DC CD80 and CD86 expression as determined by flow cytometry, real-time PCR and Western blot analyses. Importantly, CTLA4Fc-treated DCs had reduced ability to stimulate T cell proliferation and cytokine production in both syngeneic and allogeneic settings. Furthermore, we identified a novel reverse signaling pathway that is dependent on direct activation of STAT3 in DCs and subsequent suppression of NFκB activity. Our data demonstrate, for the first time, that a reverse signal induced by CTLA4 is sufficient to suppress LPS-induced DC maturation and function. This work opens new windows for generation of tolerogenic DCs that could ultimately be used for treating autoimmune diseases and transplant rejection.
Raf-1 kinase inhibitor protein: an important molecule for modulating inflammatory responses? (P1188)
Authors: Wright Kyle , Vella Anthony
Published: January 2023

Systemic inflammatory response syndrome is a costly burden for health care systems both nationally and globally. SIRS results from the systemic release of exorbitant quantities of proinflammatory cytokines, which leads to severe complications including acute lung injury, multiple organ dysfunction syndrome, and even death. RKIP is a regulator of MAPK and NF-κB signaling cascades which are both critical for production of the cytokines responsible for SIRS initiation. Using a T cell dependent mouse model of SIRS, we have shown that RKIP is necessary for the synthesis of pathologic quantities of IFNγ from antigen specific CD8+ T cells, but not CD4+ T cells, which is due to an intrinsic signaling defect downstream of the TCR in RKIP-/- T cells. Importantly, RKIP deficient mice were able to mount a productive response to vaccination, suggesting that RKIP may be critical for cytokine production during serial TCR triggering in SIRS, but less so during primary T cell responses (data submitted for publication). Additionally, we have found that RKIP may also play a role in antigen presenting cell function. This is evidenced by a rapid, but transient, inability to detect RKIP by western blot after stimulation of splenocytes with TLR ligands. Although an exact molecular mechanism underlying RKIP’s effects is presently unclear, we are currently utilizing cutting-edge PF 2D proteomic mapping to further elucidate its role within the signaling machinery of immune cells.
Easy, column-free procedure for the enrichment of human Th1 cells from peripheral blood (P3239)
Authors: Fairhurst Maureen , Yu Jessie , de Jong Susan , Thomas Terry
Published: January 2023

Upon activation naïve T helper (Th) precursor cells are polarized toward various cell subtypes and can be distinguished from each other based on effector function and chemokine expression. Th1 cells regulate cellular immunity via IL-2 plus IFN-γ, activating macrophages, cytotoxic T, NK and B cells in an effort to eliminate intracellular pathogens. Th1 cells preferentially express the chemokine receptors CXCR3 and CCR5 and can be further characterized by intracellular staining for IFN-γ. We have developed a 2-step EasySepTM immunomagnetic column-free method for the enrichment of CD4+CXCR3+ T cells from fresh peripheral blood nucleated cells. First, non-CD4 T cells are targeted for depletion using dextran-coated magnetic particles and a cocktail of antibody complexes. Labeled cells are separated using an EasySepTM magnet, and pre-enriched CD4 T cells are poured off. CXCR3+ cells are then positively selected from the pre-enriched fraction. The procedure can be automated using RoboSepTM. With an initial frequency of 10 ± 3% CD4+CXCR3+ cells, purities of 90 ± 4% (n=12) can be obtained. When stimulated, a high proportion of these cells produce IFN-γ (68 ± 12%) compared with total CD4 T cells and pre-enriched CD4 T cells. Less than 1% of isolated cells produce cytokines associated with other Th cell subtypes (IL-4, IL-17). Enrichment of unstimulated human Th1 cells enables the investigation of chronic inflammation responses and mechanisms of regulation in human models of disease.
TGFβ down-regulates PGE2 receptor subtype 2 in differentiating naïve human CD4+ T cells, resulting in a lack of expression and function in Th17 cells (P6281)
Authors: Poloso Neil , Vu Chau , Woodward David
Published: January 2023

Prostaglandin E2 (PGE2) is an important component in inflammatory diseases. Recently, PGE2 has been recognized as playing a critical role in determining CD4+ T helper cell differentiation and expansion (Th1 and Th17, respectively). In order to better understand the role prostaglandins play in differentiated human T-helper cells we determined the expression of various PG receptors in in vitro differentiated human Th1, Th2, and Th17 cells. Surprisingly, we found that while Th1 and Th2 cells express all four PGE2 receptor subtypes, Th17 cells did not express EP2 subtype as determined by qPCR. Stimulation with specific EP receptor agonists confirmed that Th17 cells did not express a functional EP2 receptor. This is in contrast with mouse Th17 which do not show this phenotype. We further found that this down-regulation of EP2 in human Th17 cells was dependent on exposure to TGFβ, and was dose dependent. This is in contrast to TGFβ reported effects on other cell types and also in mouse CD4+ T cells. This finding could explain why differentiated Th17 cells expand in the presence of PGE2, as they down-regulate an important regulatory feedback loop through EP2. Additionally, our data suggests that PGE2 receptor expression and biology in T cells may not translate from rodents to humans.
B7x modulates the adaptive immune response and diminishes renal injury in murine antibody mediated nephritis (P5168)
Authors: Pawar Rahul , Goilav Beatrice , Xia Yumin , Herlitz Leal , Ghosh Kaya , Zang Xingxing , Putterman Chaim
Published: January 2023

B7x (B7-H4, B7S1), a member of the B7/CD28 co-stimulatory superfamily, is an inhibitor of T cell activation and proliferation. B7x is highly expressed in peripheral, non-lymphoid tissues. However, the in vivo function of B7x is largely unknown. We hypothesized that B7x may modulate the pathogenesis of antibody mediated nephritis through its effects on T cells. We induced nephritis in B7x-deficient (B7x-/-) (n=10) and B7x-wild type (WT) B6 mice (n=10) pre-immunized with rabbit IgG by intravenous injection of nephrotoxic sera (rabbit anti-mouse glomerular antibodies). Following nephritis induction we observed a significant increase in the levels of serum IgG, particularly of the IgG2b and IgG1 isotype, in B7x-/- mice as compared to B7x WT mice. At the mRNA level, IL-23, CCL2 and CCR5 were significantly upregulated in B7x-/- kidney tissue. Immunohistochemistry revealed significantly increased glomerular infiltration of CD3+ T cells and CD68+ macrophages in B7x-/- kidneys. Furthermore, kidney histopathology was more severe in B7x-/- mice, in terms of infiltrating polymorphonuclear cells in both glomerular and interstitial compartments, crescent formation, and tubular atrophy/dilatation, as compared to B7x WT mice. In conclusion, B7x plays a protective role in immune mediated nephritis, likely through exerting an inhibitory effect on T cell activation and restricting the migration of immune cells. Our results indicate a potential role for modulation of the B7x pathway for the treatment of nephritis mediated by pathogenic antibodies, including anti-glomerular basement membrane disease (Goodpasture’s syndrome), lupus nephritis, and related conditions.