bluuebunny/crossref_metadata_2025 huggingface dataset:
Suppression of IL-12 production by TTP through blocking NF-kappaB nuclear translocation (P3008)
Authors: Liu Jianguo , Gu Ling , Ning Huan , Hou Rong
Published: January 2023

Tristetraprolin (TTP) plays a significant role in regulating the expression of AREs containing mRNAs. TTP-/- Mice develop a systemic autoimmune inflammatory syndrome characterized by cachexia, conjunctivitis and dermatitis. IL-12 plays a crucial role in immune defense against infectious and malignant diseases. In the present study, we found increased production of IL-12 during endotoxic shock and enhanced Th1 cells in TTP knockout mice. The levels of IL-12 p70 and p40 protein as well as p40 and p35 mRNA were also increased in activated macrophages deficient of TTP. In line with these findings, overexpression of TTP suppressed IL-12 p35 and p40 expression at the mRNA and promoter level, while surprisingly had no effects on their mRNA stability. Our data showed that the inhibitory effects of TTP on p35 gene transcription were completely rescued by overexpression of NF-kappaB p65 and c-Rel but not the p50 in activated macrophages. Our data further indicated that TTP acquired its inhibition on IL-12 expression through blocking nuclear translocation of NF-kappaB p65 and c-Rel while enhancing p50 upon stimulation. In summary, our study reveals a novel pathway through which TTP suppresses IL-12 production in macrophages, resulting in suppression of Th1 cell differentiation. This study may provide us with therapeutic targets for treatment of inflammatory and autoimmune disorders
Fli-1 drives transcription from the MCP-1 gene promoter, which may provide a novel mechanism for cytokine and chemokine activation. (P6236)
Authors: Lennard Richard Mara , Nowling Tamara , Zhang Xian
Published: January 2023

Regulation of proinflammatory cytokines and chemokines is a primary role of the innate immune response. MCP-1 is a chemokine that recruits immune cells to sites of inflammation. Expression of MCP-1 is reduced in primary kidney endothelial cells from mice with a heterozygous knockout of the Fli-1 transcription factor. Fli-1 is a member of the Ets family of transcription factors, which are evolutionarily conserved across several organisms including Drosophilla, Xenopus, mouse and human. Ets family members bind DNA through a consensus sequence GGAA/T, or Ets binding site (EBS). We have demonstrated that Fli-1 binds to three EBSs within the endogenous MCP-1 promoter by ChIP assay. Transient transfection assays of the murine MCP-1 promoter indicate that the Fli-1 gene actively drives transcription from the MCP-1 gene promoter in a dose-dependent manner. The Ets-1 transcription factor was also tested, but failed to drive transcription. Studies to determine the relationship between Fli-1 and Ets-1 in the transcriptional control of MCP-1 will be presented. Deletion studies to determine the most important regions of the MCP-1 promoter for Fli-1 gene regulation are being investigated. Together, these results demonstrate a novel pathway, separate from NFκB, for the transcriptional activation of MCP-1. Evidence suggests that Fli-1 may interact with the promoters of other chemokines and cytokines, providing a novel mechanism for activation of the inflammatory response.
Pharmacokinetics and immunological effects of human IL-15/IL-15Rα heterodimeric complexes in macaques and comparison to single-chain IL-15 (P6242)
Authors: Pavlakis George , Bergamaschi Cristina , Valentin Antonio , Kulkarni Viraj , Bear Jenifer , Rosati Margherita , Alicea Candido , Sowder Raymond , Chertova Elena , Lifson Jeffrey , Felber Barbara
Published: January 2023

The common γ-chain cytokine IL-15 plays a significant role in CD8+ T and NK cell survival, proliferation and activation, and may find clinical applications in cancer. We have previously showed that co-expression of IL-15 and IL-15Rα in the same cell allows for efficient production of bioactive IL-15 heterodimer in vivo, whereas the single-chain IL-15 is unstable and poorly secreted. In addition, the identification of soluble IL-15/IL-15Rα complexes in human and mice sera suggests that the IL-15 heterodimer is the natural form of the cytokine in vivo. We have developed stable cell lines overproducing IL-15/IL-15Rα heterodimers. We compared pharmacokinetics and biological effects of purified IL-15 heterodimers with E.coli-derived single-chain IL-15 in macaques. Upon i.v. injection, the plasma half-life of IL-15 heterodimers was increased 6x compared to single-chain IL-15. Injection s.c. of IL-15 heterodimers resulted in persistent bioactive level of plasma IL-15 for up to 72 hrs. Repeated s.c. administration of IL-15 heterodimers resulted in a massive expansion of NK, γδ and CD8 T cells. Therefore, in comparison to single-chain IL-15, IL-15 heterodimer is more stable in vivo, has longer plasma half-life and is more bioactive in macaques. This favorable pharmacokinetics/pharmacodynamics profile allows for lower dose, simple s.c. delivery and lowers the possibility of toxicity due to cytokine spike, therefore it is the most favorable form for clinical applications of IL-15.
Air-pollution mediated susceptibility to inflammation and insulin resistance via CCR2 dependent and independent effects (P6246)
Authors: Liu Cuiqing , Xu Xiaohua , Bai Yuntao , Wang Tse-yao , Rao Xiaoquan , Sun Lixian , Ying Zhekang , Maiseyeu Andrei , Sun Qinghua , Rajagopalan Sanjay
Published: January 2023

Epidemiologic and experimental studies support an association between fine ambient particulate matter <2.5µm (PM2.5) exposure and insulin resistance/type 2 diabetes mellitus. A role for innate immune cell activation has been suggested in the pathogenesis of these effects. We evaluated the role of CCR2 in PM2.5-mediated inflammation and insulin resistance. Wild-type C57BL/6 and CCR2-/- male mice were fed a high-fat diet and assigned to concentrated ambient PM2.5 or filtered air for 17 weeks via a whole body exposure system. PM2.5 exposure resulted in whole body insulin resistance and increased hepatic lipid accumulation in the liver which was attenuated in CCR2-/- mice by inhibiting SREBP1c mediated transcriptional programming, decreasing fatty acid uptake and p38 (but not JNK/ERK) MAPK activity. CCR2-/- restored abnormal phosphorylation levels of AKT, AMPK in visceral adipose tissue and adipose tissue macrophage content. However, the impaired whole body glucose tolerance and reduced GLUT4 in skeletal muscle in response to PM2.5 was not corrected by CCR2 deficiency. CCR2 modulates PM2.5-mediated insulin resistance by regulating visceral adipose tissue inflammation and hepatic lipid metabolism. These findings provide new mechanistic links between air pollution mediated insulin resistance and associated abnormalities in lipid metabolism and adipose inflammation.
Characterization of CXCR3 expression on B cell subsets in chronic Hepatitis C patients and its relation to autoimmunity. (P6250)
Authors: Reyes Elane , Sugalski Julia , Khasnis Atul , Antony Don
Published: January 2023

Hepatitis C virus (HCV) patients have extrahepatic manifestations, such as cryoglobulins. Cryoglubilins are immunoglubulins that precipitate in the cold and contain IgM rheumatoid factor. Vasculitis is an autoimmune manifestation detected in a subset of HCV patients with cryoglobulins (1-5%). HCV patients with mixed cryoglobulinemia have enhanced levels of CXCL10 (a chemotactic ligand of CXCR3) in their plasma (Antonelli.2008) and CXCR3+ B cells are detected in the liver compartment (Mizouchi. 2010). We analyzed by flow cytometric analysis a defined cohort of HCV patients (Cryoglobulin and Rheumatoid factor state) and healthy controls for CXCR3 and CXCR4 expression on B cell subsets. The chemokine expression was compared with parameters of B cell phenotype such as activation state (CD86), and cell cycling(ki-67) . We found that mature activated B cells of HCV patients had a high expression of CXCR3 (75% in HCV vs. 20% in healthy controls).. The goal of this project is to define the potential role that CXCR3 plays in the progression of HCV towards autoimmunity by comparison with alteration in B cell parameters such as alterations of cycling, apoptosis and survival.
Direct actions of TSLP on CD4+ T cells are important for in vivo Th2-type responses (P6252)
Authors: West Erin , Rochman Yrina , Kashyap Mohit , Li Peng , Spolski Rosanne , Leonard Warren
Published: January 2023

TSLP is a pleiotropic cytokine that has broad effects in multiple T helper type 2 (Th2) driven immune responses, including in asthma, intestinal immunity, and tumor progression. Initially, TSLP was shown to indirectly influence CD4+ T cells through direct actions on dendritic cells, resulting in CD4+ T cell priming towards a Th2 polarized response. Subsequently, direct actions of TSLP on CD4+ T cells were also demonstrated, but these direct actions were not analyzed in vivo. Herein, we show that TSLP induces both Th2 (IL-4, 5, 13) and Th2-associated (IL-3, 9, 10 and 31) cytokines early after TCR activation. Interestingly, TSLP can increase IL-5 production independent of IL-2 and IL-4 signaling; however, production of the other Th2 cytokines was a least partially dependent on IL-2 and IL-4. Additionally, we demonstrated that direct actions of TSLP on CD4+ T cells were important for Th2 polarization and cytokine production in an in vivo mouse lung inflammation model. Lastly, we found that TSLP plays a role in the development of IL-9 producing cells during a mouse chronic asthma model. Taken together, these data indicate a role for critical direct actions of TSLP in Th2 responses in vivo and indicate strategies for manipulating Th2 responses, including in allergic disease.
Lithium treatment reduced microglia activation and inflammation after irradiation to the immature brain (P6256)
Authors: Zhu Changlian , Xie Cuicui , Zhou Kai , Blomgren Klas
Published: January 2023

To evaluate the effects of lithium on microglia activation and inflammation after irradiation to the immature brain, male rat pups were injected 2 mmol/kg lithium chloride i.p. on postnatal day 7 (P7), additional lithium injections, 1 mmol/kg, were administered at 24 h intervals. Pups were subjected to whole brain 6Gy irradiation on P11. The pups were sacrificed at 6h and 24h after IR. Microglia scattered in the brain can be detected by counting their numbers, their size, engulfment of cell debris or by the production of cytokines and chemokines. Microglia were stained using the marker Iba-1, revealing the presence of these cells throughout the brain under normal conditions, and apparently higher numbers and bigger sizes after IR, particularly in areas where cell death occurred. Quantification of Iba-1+ cells in the GCL of DG showed a significant increase after IR, but with a lower increase in the lithium-treated brains. The concentration of MCP-1, IL-1α, IL-1β, GRO/KC in the hippocampus were increased significantly at 6h after IR compared with non-irradiation control. Lithium treatment significantly inhibited the increase. There was no significantly difference with these cytokines/chemokines at 24h after IR between lithium and vehicle treated group. These data indicate that lithium can specifically reduce inflammation through GSK3-beta inhibition or modified microglia activation.
Targeted delivery of HMGB1 siRNA to macrophages and dendritic cells for sepsis treatment in humanized mouse model (P6223)
Authors: Ye Chunting , Choi Jang-Gi , Abraham Sojan , Shankar Premlata , Manjunath N.
Published: January 2023

Over expression cytokine by innate immune cells is thought to initiate multiple organ failure in murine models of sepsis. Whether human cytokine storm also plays a similar role is not clear. Here we show that human hematopoietic cells are required to induce sepsis-induced mortality following cecal ligation and puncture (CLP) in the severely immunodeficient NOD/SCID/IL2Rγ-/- mice and siRNA treatment to inhibit HMGB1 release by human macrophages and dendritic cells dramatically reduces sepsis-induced mortality. Following CLP, as compared to immunocompetent WT mice, NOD/SCID/IL2Rγ-/- mice did not show high levels of serum HMGB1 or murine proinflammatory cytokines and were relatively resistant to sepsis-induced mortality. In contrast, NOD/SCID/IL2Rγ-/- mice transplanted with human hematopoietic stem cells (humanized BLT mice) showed high serum levels of HMGB1 as well as multiple human, but not murine proinflammatory cytokines and uniformly succumbed, suggesting human cytokines are sufficient to induce organ failure in this model. Moreover, targeted delivery of HMGB1 siRNA to human macrophages and dendritic cells using a short AchR binding peptide (RVG-9R) effectively suppressed secretion of HMGB1, reduced the human cytokine storm, human lymphocyte apoptosis and rescued humanized mice from CLP-induced mortality. SiRNA treatment was also effective when started after the appearance of sepsis symptoms. These results show that: CLP in humanized mice provides a model to study human sepsis; HMGB1 siRNA might provide a treatment strategy for human sepsis; and RVG-9R provides a tool to deliver siRNA to human macrophages and dendritic cells that could potentially be used suppress a variety of human inflammatory diseases.
Structure function analysis of NOD2, ATP and muramyl-dipeptide interaction (P1272)
Authors: Davis Beckley , Mo Jinyao , Smith Dylan , Pollock Sarah , Duncan J.
Published: January 2023

Nucleotide binding and oligomerization domain containing protein 2 (NOD2) is an intracellular protein that is involved in the recognition of bacterial cell wall derived muramyl-dipeptide. Mutations in the gene encoding NOD2 are associated with inherited inflammatory disorders including Crohn’s disease and Blau syndrome. NOD2 is a member of the Nucelotide-binding domain and leucine-rich repeat- containing protein gene family (NLR). Nucleotide binding is thought to play a critical role in signaling by NLR family members. However, the molecular mechanisms underlying signal transduction by these proteins remains largely unknown. Mutations in the nucleotide-binding domain of NOD2 have been shown to alter its signal transduction properties in response to muramyl-dipeptide in cellular assays. Using purified recombinant protein, we demonstrate that NOD2 binds and hydrolyzes ATP. Additionally, we have found the purified recombinant protein is able to bind directly to muramyl-dipeptide and can associate with known NOD2 interacting proteins in vitro. Binding of NOD2 to muramyl-dipeptide and homo-oligomerization of NOD2 are enhanced by ATP binding, suggesting a model that involves binding of nucleotide, muramyl-dipeptide, and oligomerization into a signaling complex. These findings set the stage for further studies into the molecular mechanisms that underlie detection of muramyl-dipeptide and assembly of NOD2-containing signaling complexes.
STING an essential trigger of DNA-mediated innate immune signaling. (P1258)
Authors: Barber Glen
Published: January 2023

Cellular innate immune sensor pathways are essential for identifying invading pathogens and for initiating host defense responses. However, how cells recognize DNA based microbes and trigger innate immune gene activation remains to be fully resolved. Here, we demonstrate that STING, an endoplasmic reticulum associated transmembrane protein, acts as a receptor for cytosolic pathogen associated DNA. This event invoked STING trafficking from the ER through autophagy-related endosomes to promote the activation of interferon regulatory factors IRF- 3, 7 and NF-κB, which triggered the production of host defense genes. STING, predominantly found in endothelial cells, macrophages and dendritic cells, was found to be essential for protecting mice against lethal HSV-1 infection through production of type I interferon. However, while STING was found to be essential for host defense, chronic activation of STING was observed to be responsible for inflammatory disease in two murine model systems. For example, DNaseII-/- and Trex1-/- mice die prematurely due to self-DNA manifested cytokine production. Lethality was prevented in both models through loss of STING since cytosolic self-DNA failed to robustly trigger cytokine production through STING signaling pathways. Our data suggests that STING senses aberrant cytosolic DNA species and is responsible for initiating innate immune gene activation, though may play a key role in inflammation and autoimmune disease manifested through self-DNA.
Defining cellular characteristics of human IL-7Rαlow memory CD8+ T cells using a genome-wide DNA methylation analysis (P1432)
Authors: Yoo Seung-Ah , You Sungyong , Shin Min Sun , Lee Naeun , Kang Ki Soo , Hwang Daehee , Kang Insoo
Published: January 2023

DNA methylation is one of the predominant epigenetic mechanisms to propagate heritable epigenetic information. Recently, we identified two subsets of memory CD8+ T cells expressing high and low levels of IL-7Rα in humans. IL-7Rαlow memory CD8+ T cells have impaired proliferation and survival in response to TCR triggering and IL-7, respectively. Importantly, the expansion of IL-7Rαlow memory CD8+ T occurs in human aging and lupus, suggesting a potential role for this cell subset in immunosenescence and autoimmunity. However, the exact characteristics of this cell subset are yet to be defined. Of interest, IL-7Rα expression in T cells is tightly regulated by DNA methylation in the IL-7Rα gene promoter. Here, we investigated how DNA methylation contributes to determining the distinct characteristics and functions of IL-7Rαhigh and IL-7Rαlow memory CD8+ T cells using genome-wide DNA methylation profiling technique. Methylation profiling revealed genes that are differentially methylated between IL-7Rαhigh and IL-7Rαlow memory CD8+ T cells. In particular, we found that the most significantly hypomethylated genes in IL-7Rαlow memory CD8+ T cells are associated with chemotaxis (CX3CR1, CXCR1, CCL3, and CCL4). Furthermore, the hypomethylation of these genes directly correlated with increased gene expression in this cell subset. Our data suggest that DNA methylation can be an important mechanism involved in defining the characteristics of IL-7Rαlow memory CD8+ T cells.
Histone deacetylase 11 is an epigenetic regulator of cytotoxic T-lymphocyte effector function and memory formation (P1404)
Authors: Woods David , Woan Karrune , Wang Dapeng , Yu Yu , Powers John , Sahakian Eva , Cheng Fengdong , Wang Hongwei , Rock-Klotz Jennifer , Villagra Alejandro , Pinilla-Ibarz Javier , Yu Xue-Zhong , Sotomayor Eduardo
Published: January 2023

The roles of individual histone deacetylases in the regulation of T-cell development and function remain largely unknown. Here we provide evidence for HDAC11 as an epigenetic regulator of T-cell inflammatory response as well as CTL central memory formation. To investigate the role of HDAC11 in T-cells, an HDAC11 knockout (HDAC11KO) mouse model was utilized. HDAC11KO mice display no gross phenotypic abnormalities and no alterations in T-cell development or CD4+/CD8+ population distributions. However, HDAC11KO CTLs are hyper-proliferative and secrete significantly higher levels of IL-2, TNF, and IFN-γ upon activation, and HDAC11KO mice accumulate higher percentages of central memory CTLs. In an allogeneic bone marrow transfer model, HDAC11KO T-cells mediate more potent and robust graft vs host disease associated with increased inflammatory cytokines. Mechanistically, we provide evidence that in CTLs HDAC11 interacts with the Eomes gene promoter, a known regulator of IFN-γ production and memory formation. HDAC11KO CTLs, at both basal state and post stimulation, display higher levels of acetylation at the Eomes promoter, indicative of a permissive transcriptional state. Correspondingly, eomes mRNA levels in HDAC11KO cells are elevated. Finally, chromatin immunoprecipitation of HDAC11 reveals interaction between HDAC11 and the Eomes promoter in CTLs. These results support HDAC11 as an epigenetic regulator of CTL function and memory formation through epigenetic regulation of Eomes.
T cell receptor repertoire plasticity in peripheral CD8 T cells (P1446)
Authors: Thomas Paul , Wang George , McClaren Jennifer , Morris Melissa , Doherty Peter , Dash Pradyot
Published: January 2023

Using a technique for the paired amplification of TCR α and β chains from single cells, we have discovered a surprising plasticity in the peripheral TCR repertoire, particularly under conditions of inflammation. This plasticity includes multiple instances of TCR revision, where changes are made in the TCR coding sequence by de novo rearrangement. Here we describe the induction of this process in murine CD8+ T cells derived from influenza-infected lungs and human T cells derived from CMV-infected individuals ex vivo or stimulated in single-cell cultures in vitro. Taking advantage of the co-expression of productive and non-productive Tcra alleles, we demonstrated secondary rearrangement in the TCRβ locus ex vivo, while the in vitro cultures demonstrated a remarkably high rate of revision following strong stimulation. Not surprisingly, revision was found to be RAG-dependent. Utilizing a model of peripheral deletion of RAG2, we showed that mice deficient in revision showed delayed onset and severity of disease in an experimental autoimmune encephalomyelitis (EAE) model, but also produced a less functional T cell response following infectious challenge. Thus, peripheral TCR revision appears to be a robust process that contributes to optimal T cell functionality during infection but may also contribute to the development of autoimmunity.
The intrinsic role of ROG in CD8+ T cell development has an impact on protective immunity against viral infection (P1452)
Authors: Shin Hyun Mu , Kapoor Varun , Welsh Raymond , Berg Leslie
Published: January 2023

Repressor of GATA3 (ROG) is a member of the poxviruses and zinc finger family of transcriptional repressors, and is also known as ZBTB32. Several studies have suggested that ROG may regulate the activation and differentiation CD4+ and CD8+ T cells, and further, may also play a role in plasma cell differentiation. We have investigated the role of ROG in the development of anti-viral memory T cells. Upon acute infection with LCMV Armstrong, Rog-/- mouse accumulated higher proportions and numbers of virus-specific CD8+ T cells, resulting in an increased number of memory cells. Rog-/- mouse exhibited normal viral clearance, but as a consequence of the elevated primary response, the Rog-/- CD8+ T cell population contains enhanced protective memory potential. We also examined the response of Rog-/- mice to infection with a high dose of LCMV-clone 13, which causes a chronic infection leading to exhaustion of the CD8+ T cells. We found that approximately one-half of Rog-/- mice infected with high dose LCMV-clone 13 succumbed to this infection as early as day 9 post-infection and also exhibited severe immune pathology in the lung compared to WT mice. The higher frequency of IFNg-producing virus-specific CD8+ T cells in Rog-/- mice also resulted in enhanced viral control following LCMV-clone 13 infection. These data indicate that ROG plays a unique non-redundant role during acute and chronic LCMV infection, and is an important regulator of CD8+ effector and memory T cell generation.
Functional conditioning of CD8 T cells by thymoproteasome-dependent positive selection (P1408)
Authors: Takada Kensuke , Takahama Yousuke
Published: January 2023

Proteasomes play an important role in the production of peptides presented by MHC class I. The thymoproteasome, which contains β5t catalytic subunit, is expressed specifically in cortical thymic epithelial cells mediating the positive selection of DP thymocytes. In β5t-deficient mice, the cellularity of CD8SP thymocytes and peripheral CD8 T cells is significantly decreased and their TCR repertoire is altered as characterized by defective responses to alloantigens and viral infections. In the present study, we functionally characterized CD8 T cells generated in β5t-deficient mice. We found that CD8 T cells in the secondary lymphoid organs of β5t-deficient mice were enriched with CD44hiCD122hi memory-phenotype cells. This phenotypic alteration was marginal in the thymus but became apparent in the periphery of mice older than 1 week after birth, suggesting that CD8 T cells generated in the thymus of β5t-deficient mice are aberrantly maintained in the periphery. Surprisingly, the enrichment of memory-phenotype CD8 T cells was also seen in monoclonal β5t-/-Rag-/-OT-I mice. In addition, CD44loCD122lo cells obtained from β5t-/-Rag-/-OT-I mice showed enhanced proliferative responses to antigen stimulations. These results suggest that the thymoproteasome-dependent positive selection not only determines TCR recognition specificity but also preconditions T cell function.
Involvement of microglial CD40 in murine retrovirus-induced peripheral neuropathy (P6173)
Authors: Cao Ling , Butler Michael
Published: January 2023

The underlying mechanism of HIV-associated peripheral neuropathy is unclear. C57BL/6 (B6) mice infected with LP-BM5 develop severe immunodeficiency (termed as MAIDS) and peripheral neuropathy. It is known that CD154-CD40 interactions between CD4+ T and B cells are required for the manifestation of MAIDS post-LP-BM5 infection (p.i.) and that HIV encephalitis is associated with increased expression of CD40 on microglia. We aimed to determine whether spinal cord microglial CD40 signaling is involved in LP-BM5-induced peripheral neuropathy. B6-CD40 KO mice were adoptively transferred with either total leukocytes or B cells and examined for mechanical sensitivity, tissue viral loads, and cytokine responses along with the development of MAIDS p.i.. CD40 KO mice transferred either cell population developed MAIDS, the severity of which was correlated with peripheral cytokine responses. CD40 KO mice displayed transient and slightly increased mechanical sensitivity p.i. regardless of cell transfer. In all peripheral tissues examined, increased viral RNA was detected in all infected mice without significant group differences. However, in the lumbar spinal cord, infected wild type B6 mice had the highest viral load and cytokine responses among all groups. Neither type of cell transfer enhanced the LP-BM5-induced increase in viral load and cytokine responses in CD40 KO mice. Together, these data support the involvement of microglial CD40 in LP-BM5-induced peripheral neuropathy.
Adapted HIV-1 epitopes can elicit functional CD8+ T cell responses (P6177)
Authors: Du Victor , Bansal Anju , Sabbaj Steffanie , Heath Sonya , Goepfert Paul
Published: January 2023

HIV readily mutates and many of these mutations can be predicted based on the human leukocyte antigen class I (HLA-I) allele of the infected individual. Majority of these HLA-associated HIV polymorphisms may represent escape mutations whereby the epitope adapts to HLA-restricted pressure in every individual. Though these adapted epitopes have escaped recognition by some CD8 T cells, others may readily recognize them. However, to truly answer the question as to whether adapted epitopes induce CD8 T cell responses (primary response), acutely infected patient samples must be tested where we can accurately predict the infecting viral strain. We therefore tested whether CD8 T cells are induced when presented with either adapted or non-adapted epitopes encoded by the infecting viral strain in 8 HIV+ acute patients. Primary responses were more likely induced to non-adapted epitopes compared to the adapted (p=0.041). Furthermore, the few adapted epitopes that were able to elicit responses (primary or not) were predicted to exhibit a significantly higher HLA binding affinity compared to ones that did not elicit a response. CD8 T cells responding to adapted epitopes also shared similar magnitude, functional avidity, and polyfunctionality compared to those elicited to the non-adapted. Our results suggest at least some epitopes with HLA-associated mutations are still potentially immunogenic and may have important implications for T cell-based vaccine development.
Analysis of myelomonocytic populations during HIV infection (P6185)
Authors: Barblu Lucie , Porichis Filippos , Hart Meghan , Kaufmann Daniel
Published: January 2023

We hypothesized that alterations in the monocytic and myeloid-derived suppressor cells (MDSCs) subsets contributed to impaired T cell function in HIV infection. CD14++CD16- classical, CD14+CD16+ intermediate, CD14lowCD16+ non classical monocytes and HLA-DR-CD14+CD33+CD11b+ MDSCs surface markers (PDL-1, CD40, HLA-ABC, HLA-DR, CD86, CCR2) and intracellular molecules (IL-12, TNF-α) were analyzed by FACS. Studies were performed on freshly isolated PBMC of healthy controls (HD) and compared to HIV-infected subjects: ART Treated (ARTC), untreated progressors (CP) and elite controllers (EC). CP subjects had significantly higher MDSCs frequency than HD, ARTC and EC. CP also express higher level of all monocyte subsets than HD and ARTC. Classical monocytes of HIV+ individuals express less HLA Class I and II molecules than HD, with a trend observed for CD86. A marked decrease in IL-12 secretion in CP upon stimulation with recombinant IFN-γ and/or LPS was also present. Also, CCR2 looks like a good marker to evaluate the importance of non classical monocytes and MDSC populations as well. Our data show that compared to HD, myelomonocytic subsets in HIV+ individuals present phenotypic and functional differences that are not fully corrected by spontaneous or therapy-induced control of viral replication. Perturbations of these subsets can contribute to ongoing immune dysfunction in treated and untreated HIV-infected subjects and may represent a target for therapeutic interventions.
Evaluating early differences in regulatory T cell levels in the MAIDS model of AIDS. (P6187)
Authors: Ayala Ramirez Maria , Akalu Yemsratch , Kwaa Abena , Stranford Sharon
Published: January 2023

Murine acquired immunodeficiency syndrome (MAIDS), caused by the murine leukemia virus (MuLV), serves as an animal model to study human AIDS. Infection with the LP-BM5 isolate of MuLV leads to progressive immunodeficiency in MAIDS-susceptible C57BL/6 mice, while resistant BALB/c mice are able to mount a direct and controlled immune response to eradicate the virus. Experiments measuring the protein expression of two natural immunosuppressive molecules, indoleamine 2, 3-dioxygenease (IDO) and interleukin-10 (IL-10), showed significant strain specific differences during the first two weeks after infection, as measured by ELISA. Since IDO and IL-10 expression can be either directly or indirectly upregulated by regulatory T (Tregs) cells, we then compared levels of Treg populations between the two strains in both lymph node and spleen using FACS. Our preliminary findings suggest that BALB/c mice may have a higher percentage of regulatory T cells in both lymph node and spleen as compared to their MAIDS-susceptible counterparts.
The role of Regenerating Islet-derived 1 (Reg1) protein in the MAIDS model (P6199)
Authors: Olokode Ifeoluwa , Stranford Sharon
Published: January 2023

Murine acquired immune deficiency syndrome (MAIDS) caused by the LP-BM5 isolate of Murine Leukemia Virus (MuLV), is an animal model used to study HIV-induced AIDS. After infection with the virus, the MAIDS susceptible C57BL/6 strain becomes immunocompromised and develops symptoms similar to that of humans infected with HIV-1. Conversely, MAIDS resistant BALB/c animals overcome their MuLV infection and gain adaptive immunity against subsequent challenge. Although it has been established that T cells and the genes found in the major histocompatibility complex (MHC) play a vital role in the outcome of MuLV infection, recent studies have identified genes outside the MHC that also play a role in MAIDS susceptibility. One resistance-associated gene, regenerating islet-derived 1 (Reg1), was found to be highly expressed in the lymph node and spleen of recently infected BALB/c mice, with much lower expression seen in the susceptible BL/6 strain. The aim of this study was to design an enzyme-linked immunosorbent (ELISA) assay that could be used to identify the murine Reg1 protein and quantify its concentration in mouse tissue samples. Preliminary results with this assay show up to two-fold more Reg1 protein in the spleens of BALB/c versus BL/6 animals at three days post MuLV infection. Evaluation of lymph node samples and the possible role of this protein in susceptibility to MAIDS are currently under investigation.
Early HIV-1 infection differentially alters responses of KIR-expressing Natural Killer cells in individuals carrying cognate HLA-C haplotypes (P6203)
Authors: Korner Christian , Granoff Mitchell , Amero Molly , Jost Stephanie , Altfeld Marcus
Published: January 2023

“NK cell licensing” describes the acquisition of functional competence of KIR-expressing NK cells in the presence of cognate HLA ligands. However, whether licensed NK cells contribute to the control of viral infections is not clear. Therefore, we investigated the response of KIR+ NK cells in association with cognate HLA-C haplotypes in HIV-1 infected individuals. 18 HLA-Bw4(-), KIR2DL1/2/3(+) patients with early HIV-1 infection have been enrolled. NK cell responses and KIR expression were measured by flow cytometry. NK cell responses were defined by levels of degranulation and cytokine production after co-incubation with 721.221 target cells. While the frequency of KIR2DL1+ NK cells was increased in the presence of the cognate C2C2 haplotype, the proportion of KIR2DL2/3+ NK cells was reduced as compared to individuals carrying the C1C1 or C1C2 haplotype (p<.05 for each). Of note, levels of degranulation and cytokine production of bulk NK cells did not differ among the three groups. However, within KIR2DL1+ NK cell subsets the C2C2 haplotype was associated with increased NK cell activity (p<.05), while the C1C1 haplotype had no effect on the respective KIR2DL2/3+ NK cell subset. Our results indicate a C2C2 haplotype-dependent expansion and increased responsiveness of KIR2DL1+ NK cells during early HIV-1 infection. A potentially more efficient licensing of KIR2DL1+ NK cells in C2C2 carriers might explain the observed increased responses in the setting of early HIV-1 infection.
Impact of NK cells on control of HIV infection in humanized BLT mice (P6207)
Authors: Jost Stephanie , Chang Judy , Rands Keith , Tomezsko Phillip , Doyle Erin , Daillere Romain , Dudek Timothy , Vrbanac Vladimir , Allen Todd , Tager Andrew , Altfeld Marcus
Published: January 2023

Evidence suggests natural killer (NK) cells can mediate antiviral activity in HIV-infected humans. Here, we sought to investigate whether the level of NK cell activation in primary HIV infection not only influences the NK cell mediated elimination of HIV infected cells, but also affects the longevity and effectiveness of HIV-specific T cell responses. To address this hypothesis, we monitored HIV viral loads and HIV-specific adaptive immune responses in a humanized BLT mouse model of HIV infection, comparing NK cell-depleted and control mice. Depletion was achieved through the use of an anti-NKp46 antibody prior to HIV infection. Proportions of immune cell subsets and viremia were assessed at baseline and then once a week for at least 5 weeks post-infection using flow cytometry and qRT-PCR. We observed that the impact of NK cells on plasma viral loads varied between batches of mice reconstituted with different tissues. One set of NK cell depleted mice displayed a 4-fold increase in viremia as well as decreased levels of T cell immune activation at 3 weeks post-infection. In a second group of mice reconstituted with independent human tissue, NK cell depletion had no significant effect on viral loads. These results suggest that further investigation is warranted to identify factors accounting for an increased control of HIV replication associated with NK cells in some individuals and not others, such as expression of a protective combination of KIR and HLA alleles.
CD8+ DC trans-presentation of IL-15 to naïve CD8+ T cells produces antigen inexperienced T cells in the periphery with memory phenotype and function (P1315)
Authors: Kedl Ross , Sosinowski Tomasz , White Jason , Cross Eric , Marrack Pippa , Gapin Laurent
Published: January 2023

Various populations of memory phenotype CD8+ T cells have been described over the last 15-20 years, all of which possess elevated effector functions relative to naïve phenotype cells. Using a technique for isolating antigen specific cells from unprimed hosts, we recently identified a new subset of cells, specific for nominal antigen, but phenotypically and functionally similar to memory cells arising as a result of homeostatic proliferation (HP). We show here that these “Virtual Memory” cells are independent of previously identified “innate memory” cells, arising as a result of their response to IL-15 trans-presentation by lymphoid tissue-resident CD8α+ DCs in the periphery. The absence of IL-15, CD8+ T cell expression of either CD122 or Eomes, or of CD8a+ DCs all lead to the loss of Virtual Memory cells in the host. Our results show that CD8+ T cell homeostatic expansion is an active process within the non-lymphopenic environment, is mediated by IL-15, and produces antigen inexperienced memory cells which retain the capacity to respond to nominal antigen with memory-like function. Preferential engagement of these “Virtual Memory” T cells into a vaccine response could dramatically enhance the rate by which immune protection develops.
Effects of senescence on immune parameters in baboons shows differential susceptibilities (P1321)
Authors: Bonnel Caroline , Arteaga-Cortes Lourdes , Rice Karen , Leland M. , Dube Peter , Kraig Ellen
Published: January 2023

Vaccine efficacy declines significantly with age, yet old baboons immunized with Yersinia pestis LcrV antigen generated vigorous humoral responses [JI 181:109 (2008)] while aged rhesus macaques did not. To assess whether these non-human primate (NHP) species are differentially impacted by immunosenescence, animals were immunized with Y. pestis F1, a plasmid-encoded protein which is known to elicit naïve B and T cell responses. Interestingly, age-associated decreases in antibody titers to F1 were observed in both species. However, defects in T cell proliferative responses were seen only in the macaques; T cell reactivity to both mitogen and antigenic stimulation was similar in the old and young baboons. We therefore asked whether other aspects of immune regulation are affected by age. The loss of naive, relative to memory, T cells was seen, as expected. Moreover, there was a decrease, albeit relatively small, in the production of naive T cells (TRECs). However, several parameters known to change with aging were not affected. For example, AID induction was maintained in most, but not all, old baboons. Moreover, the T cell fine specificity was similar in old and young animals. Lastly, TCR-Vβ spectratype analysis revealed that the age-associated repertoire skewing reported in old rhesus and humans is not seen in baboons, even in the CD8+ T cell subset. Thus, baboons do appear to be less affected by aging and this NHP species may provide novel insights into healthy immune aging.
Bioartificial collagen matrix limits IL-7 and IL-15-induced T-cell homeostatic proliferation without affecting T-cell receptor signaling (P1327)
Authors: Mailloux Adam , Epling-Burnette Pearlie
Published: January 2023

Intermittent homeostatic growth governed by STAT3/5-associated common γ chain (γc) cytokines IL-7 and IL-15 is the principal source of new T-cells after thymic involution. Lymphoproliferation, as seen in large granular lymphocyte leukemia and autoimmune lymphoproliferative syndrome (ALPS), results from failed anti-apoptotic and hyper IL-15 response. Contextual constraints in the microenvironment may also regulate homeostasis. Using primary human naïve and memory CD4+ and CD8+ T-cells, we found that bioartifical collagen matrix limits proliferation by IL-7 and IL-15, but not by anti-CD3/CD28. Flow cytometry was used to analyze collagen receptor expression on dividing T-cells. The expression of known activating collagen receptors, discoidin domain receptor (DDR)1, DDR2, and gpVI was not observed. However, the inhibitory collagen receptor, leukocyte associated IgG-like receptor-1 (LAIR-1), was increased with each cellular division, reaching 2-3 fold over non-dividing cells in response to IL-7 and IL-15. Such changes were not observed with anti-CD3/CD28 stimulation. Western blot analysis revealed no difference in cyclins, cyclin-dependant kinase (CDK)4, p21, p27, or STAT3/5 phosphorylation in collagen matrix, but did reveal reduced expression of the cell cycle regulator CDK6. These data provides rational for the future study of LAIR-1 in T-cell homeostasis and indicates that collagen adhesion may provide spatial repression from the microenvironment during homeostatic expansion.
NLRP12 protein function in the regulation of MHC-I expression in monocytes of patients transplanted with HPCs and their relationship to the reconstitution of CD8 T lymphocytes. (P1333)
Authors: Hernandez Marcela , Ortiz-Navarrete Vianney
Published: January 2023

The reconstitution of the T CD8 lymphocyte population in patients subject to hematopoietic cell transplantation (HCT) depends largely of homeostatic peripheral expansion (HPE) of the T CD8 lymphocytes that were not eliminated during the pre-transplant conditioning regimen or the ones that are contained in the transplanted cells. In order to have an efficient HPE, MHC-I molecules, IL-7 and IL-15 are required. Evidences obtained in our lab indicate that the low and/or heterogeneous MHC-I levels of expression correlate with a inefficient reconstitution of T CD8 lymphocytes in patients that received allogeneic HCT. The deficiency in MHC-I expression could be due to a decrease in the expression of the NLRP12 protein, which has been proposed as a positive regulator of classical and non-classical MHC-I molecules expression. Therefore the objective of the following project is to determine the involvement of NALP12 in the MHC-I expression in monocytes from patients subject to HCT. We studied a cohort of 11 patients, 6 of which received autologous transplant and 5 with allogeneic transplant. Most of the patients present normal levels of T CD8 lymphocytes and a homogeneous expression of MHC-I. The patients that received autologous transplantation show a correlation of r=0.56. Our results suggest that NALP12 could act as an enhancer for the expression of the MHC-I molecules and that this expression could be affected in patients subject to allogeneic HPC transplantation.
Distinct apoptotic machinery and selective gene regulation by glucocorticoids in Th17 cells (P1294)
Authors: Banuelos Jesus , Lu Nick
Published: January 2023

Glucocorticoids (GCs) are extensively used in the treatment of inflammatory disorders and surprisingly, are ineffective in suppressing Th17 responses. We sought to determine the mechanisms underlying the insensitivity of Th17 cells to GCs. We found that in vitro polarized murine Th1 and Th2 cells, but not Th17 cells, were sensitive to GC-induced apoptosis as determined by various apoptotic markers such as annexin-V staining. At the mRNA level, GCs suppressed IL-4 and IFN-γ, and unexpectedly IL-22 but not IL-17 (A and F). Despite the distinct GC responses, Th1, Th2, and Th17 cells showed similar levels of GC receptor (GR) isoforms. Using PCR array to survey 84 genes involved in apoptosis, we found significant differences in the apoptotic machinery between Th1 and Th17 cells. Th17 cells showed increased expression of anti-apoptotic survivin and Bfl-1 whereas Th1 cells showed increased expression of pro-apoptotic Bim, Fas ligand, caspase 1, and death receptor 5. GCs induced Bim in both Th1 and Th17 cells while the same treatment decreased the expression of Nod1 and Bcl-xL in Th1 but not Th17 cells. Altered apoptotic machinery and differences in genes regulated after GC treatment may underlie the inability of GCs to induce apoptosis in Th17 cells. These studies on the mechanisms underlying the selective insensitivity of Th17 cells to GCs may provide a basis for improved treatment regimens for severe asthma and other Th17-mediated disorders.
A synthetic compound, PPY-345, ameliorates ovalbumin-induced asthma (P6021)
Authors: Chung Hwan-Suck , Jeong Jin-Hyun , Shin Minkyu , Hong Moochang , Park Soojin , Kim Hankyum , Yoon Moonsik , Kim Hyunseong , Bae Hyunsu
Published: January 2023

Eosinophilia is one of the characteristic signs of allergic inflammation. Previously, we discovered a new compound, PPY, derived from the fruit of Vitex rotundifolia L. and evaluated its anti-inflammatory and anti-asthmatic properties. In this study, we synthesized a new modified compound, PPY-345, which was based on the PPY skeleton, and we evaluated its anti-asthmatic effects. To evaluate the anti-asthmatic effect of PPY-345 in vitro, A549 lung epithelial cells were stimulated with TNF-alpha, IL-4 and IL-1-beta to induce the expression of CCL11, a chemokine involved in eosinophil chemotaxis. PPY-345 treatments significantly reduced CCL11 secretion. PPY-345 treatment did not inhibit the translocation of NF-κB into the nucleus but suppressed the phosphorylation of signal transducers and activators of transcription 6. To characterize the anti-asthmatic properties of PPY-345 in vivo, we examined the influence of PPY-345 in an ovalbumin (OVA)-induced asthma model. PPY-345 treatment significantly reduced airway hyperreactivity as measured by whole-body plethysmography. PPY-345 further reduced total cells, including eosinophil, macrophage and lymphocytes, in the BAL fluid, goblet cell hyperplasia and myosin light chain 2 positive smooth muscle cell area in the lung tissue. Additionally, PPY-345 significantly suppressed the levels of OVA-IgE present in the serum. These results suggested that PPY-345 could improve asthma symptoms in OVA-sensitized mice.
CD4+TH2, not TH17 cells mount antigen specific recall response and drive allergic inflammation (P6029)
Authors: Liu Bo , Chen Chun-Yu , Lee Jeeboong , Burwinskel Karen , Wang Yui-Hsi
Published: January 2023

Allergic asthma is a heterogeneous inflammatory disease of the airway. In addition to CD4+ TH2 and TH17 cells of the adaptive immunity, recently described innate lymphoid cells (ILC2) also contribute to the pathogenesis of allergic asthma. However, it remains unclear that which cell type is responsible for mounting antigen-driven immune response at the recall phase of allergic inflammation. Repeated intranasal OVA re-challenges induced a significant expansion of TH2, not TH17 or ILC2 cells in rested mice that were previously sensitized with OVA plus papain. Consequently, the amount of IL-5 and IL-13, not IL-17 or IFN-γ production in the lung of mice re-exposed to OVA antigen were significantly elevated than those of mice sensitized only. Mice ablated of CD4+ T cells failed to respond to repeated OVA antigen re-challenges and exhibited ameliorated allergic airway inflammation, while their lung ILC2 remained intact. Furthermore, STAT6-deficient mice that lacked TH2 cells also failed to mount allergic recall response to OVA antigen, despite of their competency in eliciting TH17 immune response. Collectively, our results suggest that lung resident antigen specific CD4+TH2 cells, not TH17 or ILC2 cells, induced after sensitization are the primary cell type responsible for mounting allergen recall response that exacerbates allergic inflammation.