bluuebunny/crossref_metadata_2025 huggingface dataset:
T cell senescence results in persistent simian varicella virus infection in aged rhesus macaques; a model for herpes zoster (44.4)
Authors: Messaoudi Ilhem , Barron Alexander , Wellish Mary , Li Helen , Wong Scott W , Gilden Donald H , Mahalingam Ravi
Published: January 2023

Herpes zoster (HZ, shingles) is caused by the reactivation of latent varicella zoster virus (VZV) and causes significant morbidity and sometimes mortality in the elderly. Several clinical studies suggest that the waning of T cell immunity with age is the underlying cause of VZV reactivation. However, the exact defects in T cell immunity remain poorly understood due to the fact that VZV infection of laboratory animals, including nonhuman primates, does not result in disease. We have developed a novel animal model wherein rhesus macaques are inoculated with the closely related simian varicella virus (SVV). SVV infection of rhesus macaques reproduces hallmarks of VZV infection in humans: 1) generalized varicella; 2) the development of cellular and humoral immunity; and 3) the establishment of latency with limited transcriptional activity in sensory ganglia. This is the only model where acute infection and latency can be studied in the same animal. Furthermore, whereas adult rhesus macaques can resolve acute SVV infection, aged animals remain persistently viremic. The SVV-specific antibody response generated by elderly animals was comparable to that of adult animals. On the other hand, the SVV-specific T cell response in aged animals was delayed and reduced in magnitude. These data suggest that, similar to clinical observations about VZV, the inability of aged animals to control SVV viremia is due to a reduced T cell response.
The role of the TRAF2/3 binding site in LMP1 and CD40 signaling (44.6)
Authors: Graham John P , Bishop Gail A
Published: January 2023

The Epstein-Barr virus protein, LMP1, is a mimic of the cellular receptor CD40. However, LMP1 signals to B lymphocytes in an amplified and sustained manner compared to CD40. LMP1 contributes to the development of B lymphoma in HIV patients or in immunosuppressed transplant recipients. LMP1 binds the signaling adaptor TRAF2 with lower affinity than CD40, and TRAF2 is needed for inducing CD40-mediated degradation of TRAFs 2 and 3. LMP1 doesn't induce TRAF degradation, and employs TRAF3 as a positive mediator of cell signaling, whereas CD40 signals are inhibited by TRAF3. We thus tested the hypothesis that relative affinity for TRAF2 and/or, distinct sequence differences in the TRAF2/3 binding sites (TBS) of CD40 vs. LMP1, controls the disparate ways in which CD40 and LMP1 use TRAFs 2 and 3, and their distinct signaling characteristics. We examined CD40 and LMP1 mutants in which the TBS sequences were swapped, testing TRAF binding and degradation, and induction of B cell activation. Results revealed that TRAF binding affinity and TBS sequence dictate some, but not all CD40 vs. LMP1 signaling properties. Examination of TRAF binding, degradation, cytokine production, IgM secretion, and the activation of c-Jun kinase and NF-kB revealed that some events are dictated by TBS sequences, others partially regulated, and still others appear independent of the TBS sequence. NIH grant CA099997
STAT1 and STAT2 are required for control of RSV replication in vivo (44.7)
Authors: Cline Troy , Jewell Nancy , Mertz Sara , Kotenko Sergei , Schindler Christian , Durbin Russell , Durbin Joan
Published: January 2023

IFN-α/β are thought to be a critical component of the antiviral immune response, but susceptibility to virus infections is not always impacted by the absence of the IFN-α/β receptor. Using a mouse model of respiratory syncytial virus (RSV) infection we observed that mice lacking both the IFN-α/β and the IFN-γ receptors (IFN-αβ γR-/-) controlled virus replication as well as wild-type mice, whereas STAT1-/- or STAT2-/- mice did not. RSV infection of STAT1-/- or STAT2-/- mice resulted in a 100-fold increase in lung virus titers. We also demonstrated extensive virus spread in the nasal cavity of STAT1-/- and STAT2-/-, but not wild-type or IFN-α/β R-/- mice. The recently discovered type III, orλ, IFNs have also been shown to exert an antiviral effect via activation of the STAT1/STAT2 containing transcription factor ISGF3 which is formed in response to either IFN-α/β or IFN-λ signaling. We found that while RSV infection of wild-type mice induced production of both type I and type III IFNs, we were unable to detect IFN-λ expression or ISGF3 activation following infection of IFN-α/β R-/- mice. These data demonstrate that both STAT1 and STAT2 are necessary for inhibition of RSV replication in vivo, and suggest the existence of a novel pathway leading to their activation in the context of RSV infection.

This research was supported by NIH grants R01 AI047226 and R01 DC00648
Novel roles for T-bet in CD8 differentiation during chronic viral infections (44.8)
Authors: Kao Charlly C. , Intlekofer Andrew I. , Reiner Steven L. , Wherry E. John
Published: January 2023

The T-box transcription factor, T-bet, was shown recently to play an important role in memory CD8 T-cell differentiation. During an acute infection, higher levels of T-bet induced the formation of shorter-lived effectors, while lower levels induced long-lived memory CD8s. However, the role of T-bet, or any transcription factor, in regulating CD8 differentiation during chronic infections has not been examined. We infected mice with the chronic Clone-13 strain of lymphocytic choriomeningitis virus (LCMV), whose antigen-specific CD8s become highly dysfunctional (i.e. exhausted). Surprisingly, T-bet levels in these exhausted CD8s exposed to a chronic inflammatory environment are lower than ag-specific cells generated during infection with an acute (Armstrong) strain. Its expression correlated inversely with that of the inhibitory surface receptor, PD-1, which is known to contribute to T cell exhaustion. T-bet deficient mice infected with Clone-13 had higher levels of PD-1; in addition, they exhibited a severe defect in viral control and lost their antigen-specific CD8s after 2-3 weeks. Finally, we show that T-bet binds to the PD-1 promoter in CD8s from LCMV-infected hosts. Thus, during a chronic viral infection, T-bet is critical for the maintenance of antigen-specific CD8 T-cells, and it may regulate T-cell exhaustion, at least in part, by acting directly on the PD-1 locus.
Enhanced T helper 1 (Th1) responses with Fluzone® adjuvanted with a synthetic TLR4 agonist in an emulsion (45.15)
Authors: Baldwin Susan L. , Shaverdian Narek , Goto Yasuyuki , Duthie Malcolm S. , Evers Tara , Mompoint Farah , Vedvick Tom S. , Bertholet Sylvie , Coler Rhea N. , Reed Steven G.
Published: January 2023

Impairments in anti-influenza Th1 responses are associated with the significantly greater risk of influenza related mortality in the elderly population. Developing appropriate adjuvants for seasonal influenza vaccines could therefore improve protection against influenza in the elderly. In this study we compare the activity of three different adjuvants, an oil-in-water emulsion and a synthetic lipid A adjuvant formulated with or without an emulsion, to enhance the immunogenicity of the Fluzone influenza vaccine. Fluzone combined with lipid A plus an emulsion was the most effective vaccine tested in mice, which induced greater vaccine-specific IgG2a and IgG titers, enhanced HI titers and the production of Th1-mediated cytokine responses (IFN-γ and IL-2) to each of the Fluzone components. In contrast, while the Fluzone vaccine plus the emulsion induced good antibody responses and HI titers to all of the vaccine components following a boost, a Th2-mediated cytokine response (IL-5) and IL-10 was induced. We demonstrate that in addition to antibody responses, cytokine responses may also be needed for evaluation and characterization of new adjuvants formulations for influenza vaccines.

This work was supported in part by Grant #42387 from the Bill & Melinda Gates Foundation
Contrasting CTL responses in protective and non-protective models of malaria (45.3)
Authors: Gupta Megha , Takagi Akihide , Richie Thomas L , Kappe Stefan H , Wang Ruobing
Published: January 2023

Protracted sterile protection conferred by a P. yoelii genetically attenuated parasite (PyGAP) vaccine was found to be completely dependent on CD8+ T lymphocytes. Immunization studies with perforin and IFN-γ knock out mice indicated that the protection was largely dependent on perforin as compared to IFN-γ. Both liver and spleen CD8+ T cells from PyGAP immunized mice induced massive apoptosis of liver stage (LS)-infected hepatocytes in vitro without the release of detectable IFN-γ and TNF-a, which directly correlated with GAP vaccine efficacy in vivo. Most importantly, we demonstrated that CD8+ T cells isolated from naïve mice that had survived wild-type P. yoelii sporozoite infection targeted mainly sporozoite-traversed and uninfected hepatocytes, revealing an immune evasion strategy that might have been used by wild-type parasites to subvert host immune responses. Differential processing of the parasite antigens in liver of immunized or naturally infected mice may be responsible for contrasting outcomes of their CTL responses. A controversial role of circumsporozoite protein (CSP) in host immune protection versus immune evasion against malaria is also highlighted by our findings.

This research was supported by SBRI innovation grant.
A study of mycobacterial lipid antigen-specific responses in a novel model of human group 1 CD1 (45.31)
Authors: Felio Kyrie , Nguyen Hanh , Dascher Christopher C , Choi Hak-Jong , Li Sha , Zimmer Michael I , Brenner Michael B , Wang Chyung-Ru
Published: January 2023

Group 1 CD1 presents mycobacterial lipid antigens to various T cell subsets. Although group 1 CD1-restricted responses have been implicated in anti-mycobacterial immunity in humans, little is known about their function, as mice lack group 1 CD1. To study group 1 CD1-restricted immune responses in vivo, we have generated human group 1 CD1-transgenic (hCD1Tg) mice. These mice express group 1 CD1 in a pattern similar to humans and support the development of group 1 CD1-restricted T cells, which can be induced both through infection with Mycobacterium tuberculosis (Mtb) and immunization with Mtb lipid antigens. hCD1Tg mice respond to Mtb epitopes recognized by human group 1 CD1-restricted T cells. The kinetics of these group 1 CD1-restricted responses resembles those of conventional T cells and is more rapid upon secondary exposure to antigen, but is distinct from those of group 2 CD1-restricted NKT cells. Furthermore, we find that the number of group 1 CD1-expressing antigen-presenting cells is altered over the course of Mtb infection, and correlates with the kinetics of group 1 CD1-restricted T cells. Taken together, our data demonstrate the viability of hCD1Tg mice as a model for the study of group 1 CD1-restricted T cells in anti-mycobacterial immunity. Such studies could lead to the development of new vaccines against Mtb. This work is supported by NIH grant NIH-A157460.
A systems biology approach on oral epithelial responses to periodontal pathogens (45.32)
Authors: Perumal Narayanan B , Kollipara Rahul Krishna , Janardhanam Srihari B , Srinivasan Mythily
Published: January 2023

Periodontitis, a chronic inflammatory disease, characterized by destruction of soft and hard tissues supporting the teeth affects ~20% in the US. The most widely implicated species in periodontitis are Actinobacillus actinomycetemcomitans (Aa), Porphyromonas gingivalis (Pg), and Fusobacterium nucleatum (Fn). We employ a systems biology approach with bioinformatic, genetic and biochemical methods to study differential responses of oral epithelial cells to specific pathogens. Using public domain microarray data, we compared gene expression profiles of cells infected with 4 different bacteria viz., Aa, Pg, Fn and Streptococcus gordonii (Sg). Various subsets of genes were differentially expressed (two-tailed T test) in these strains and clustered into 3 GO classifications: apoptosis, detection of external stimuli and cytokine activity. We have confirmed gene expression of some TLR genes (real time PCR) and cytokine genes (ELISA). Interestingly, apoptosis related genes were upregulated in cells stimulated with an oral commensal strain (Sg), but downregulated in cells exposed to pathogens (Pg/Aa). Also, many pro-inflammatory cytokines were upregulated in cells stimulated with Pg/Aa as compared to cells stimulated with Sg. In sum, our studies suggest that select periodontal pathogens enhance the survival of infected cells that secrete cytokines and mediate pathology.
PD-L1 expression in the CNS mitigates immune-mediated damage and morbidity at the cost of viral persistence (45.5)
Authors: Phares Timothy W , Atkinson Roscoe , Stohlman Stephen A , Bergmann Cornelia C
Published: January 2023

Interactions of programmed death 1 (PD-1) with its ligand (PD-L1) contribute to negative regulation of T cell function and dampened anti-viral responses. Following neurotropic coronavirus infection, PD-L1 is prominently upregulated on oligodendroglia, but only modestly on microglia and astrocytes. Concomitant expression of PD-1 on CNS infiltrating virus-specific CD8 T cells implicated PD-1:PD-L1 interactions in contributing to viral persistence in oligodendrocytes. This notion was confirmed by infection of PD-L1 deficient mice (B7-H1-/-). Virus is controlled more rapidly with significantly reduced numbers of infected oligodendrocytes in B7-H1-/- mice compared to wild-type. Accelerated control of infectious virus correlated with enhanced IFN-gamma production, critical to mediate anti-viral function in oligodendroglia. However, the lack of PD-L1 significantly increased disease severity and mortality. High Granzyme B levels and expression of TNF-alpha and iNOS were associated with the enhanced morbidity. Moreover, exacerbated disease in B7-H1-/- mice was coupled with greater axonal damage, while myelin loss was similar. The data demonstrate a significant contribution of PD-L1 in hindering viral clearance in glia cells, while protecting CNS tissue from immune-mediated damage. Supported by NIH NS18146.
Hyperactivation of CD8 T cells precedes exhaustion following viral infection (45.9)
Authors: Mackerness Kathryn J , Cox Maureen A , Stefanov Emily , Weaver Casey T , Harrington Laurie E , Zajac Allan J
Published: January 2023

Developing an in-depth understanding of the factors that regulate the induction, quality, and longevity of T cell responses is essential for devising rational strategies to promote anti-pathogen immunity. Cellular immune responses to many persistent viral infections are associated with the development of T cell exhaustion, which is characterized by the inability to produce anti-viral cytokines and proliferate. We have used IFN-γ-Thy1.1 cytokine reporter mice to dissect the initial activation events and functional traits of CD8 T cells as they respond following acute or persistent lymphocytic choriomeningitis virus (LCMV) infections. We show that a positive correlation exists between the expression of the Thy1.1 reporter molecule and IFN-γ production. Significantly, we show that expression of the Thy1.1 IFN-γ reporter molecule is elevated during the early stages of infections associated with the subsequent development of T cell exhaustion but is not as markedly upregulated following infections that become well controlled. This parallels the expression of PD-1, which is known to be upregulated on exhausted cells. Collectively, these data indicate that exhausted T cells, which are ineffective at eradicating persisting antigens, are derived from populations of cells that are initially hyperactivated. Thus, the blockade of this hyperactivation may represent a novel therapeutic approach to enhance cellular immunity against intracellular pathogens.
GATA-3 seals Notch-induced T cell commitment (46.1)
Authors: García-Ojeda Marcos E , Lemaître Fabrice , Richard-Le Goff Odile , Hendriks Rudi W , Cumano Ana , Di Santo James P
Published: January 2023

Transcription factors orchestrate T-lineage differentiation in the thymus. One early checkpoint involves Notch-1 stimulation of thymic immigrants that promotes T-cell commitment at the expense of the B-lineage program. The zinc-finger transcription factor GATA-3 is required for T cell specification, although its mechanism of action is unknown. Here we identify molecular targets of GATA-3 in early thymocytes and show that conditional ablation of GATA-3 expression in committed pro-T-cells unmasks a latent B-cell potential that is undetectable in normal T-cell precursors. Thus, GATA-3 delimits the developmental options in early thymocyte precursors by sealing T-cell fate induced by Notch signals.

M.E. García-Ojeda was funded by a Pasteur Foundation Fellowship. This work was supported by grants from the Institut Pasteur, the Inserm and the Ligue Nationale contre le Cancer. M.E. Garcia-Ojeda and F. Lemaître contributed equally to this work.
Ets1 is required for CD4 shut down during CD8 T cell differentiation in the thymus (46.11)
Authors: Zamisch Monica , Tian Linhua , Grenningloh Roland , Wildt Kathryn F , Ehlers Marc , Ho I-Cheng , Bosselut Remy
Published: January 2023

During the latest steps of thymocyte differentiation, T cell precursors that have rearranged TCRbeta and TCRalpha genes and express both CD4 and CD8 (double positive, DP) are selected into MHC II-restricted CD4 T cells, or MHC I-restricted CD8 T cells. Several transcription factors regulate this developmental transition: Gata3 and Zbtb7b promote the generation of CD4 cells, whereas Runx3 is important for CD8 T cell development by promoting the cessation of CD4 expression. The transcription factor Ets1 was reported to contribute to CD8 cell development, but its function in this process remains unclear. The present study has tested the hypothesis that Ets1 participates in CD8 cell differentiation by enabling Runx3 function. Expression of the P14 MHC I-restricted transgenic TCR in Ets1-deficient mice resulted in the generation of a population of CD8 lineage cells that failed to downregulate CD4, suggesting that Ets1 is required for the down-regulation of CD4. Transient transfection analyses failed to reveal any Ets1-Runx3 cooperation on CD4 expression. Rather, we found that Ets1 is necessary for the proper expression of Runx3 during CD8 thymocyte differentiation, and enforced expression of Runx3 restores CD4 shut-down in Ets1-/- MHC I-restricted thymocytes. These findings identify Ets1 as a key player during CD8-lineage differentiation and show that it acts at least in part by promoting Runx3 expression.
Impaired development of human Th1 cells in patients with deficient expression of STAT4 (46.14)
Authors: Chang Hua-Chen , Han Ling , Goswami Ritobrata , Nguyen Evelyn Thi , Pelloso David , Robertson Michael J. , Kaplan Mark H.
Published: January 2023

IL-12 activates STAT4, which is a critical regulator of inflammation and Th1 lineage development in murine systems. The requirement for STAT4 in the generation of human Th1 cells has not been examined thoroughly. Expression of the Th1 signature markers IFNγ, IL-12Rβ2, TNFα, and CXCR3 is greatly reduced in Th1 cultures of CD4 T cells isolated from Non-Hodgkin's Lymphoma patients following autologous stem cell transplant, which have acquired STAT4 deficiency, compared to control Th1 cultures. T-bet expression is not affected whereas IL-4 and IL-5 production is increased in patient Th1 cultures. STAT4 deficiency does not result in defects in the development of Th2 cells. Reconstitution of STAT4 in patient T cells allowed recovery of Th1 signature markers IFNγ, IL-12Rβ2, and CXCR3. In contrast, ectopic expression of IL-12Rβ2 in patient Th1 cells, which was also decreased with acquired STAT4-deficiency, did not rescue STAT4 expression, though it did increase IFNγ production to levels intermediate between control and patient samples. These results demonstrate that as in murine systems, STAT4 is required for optimal human Th1 lineage development.
Bcl6 is required for the development of T follicular helper cells marked by downregulation of P-selectin glycoprotein ligand-1 (46.7)
Authors: Poholek Amanda C , Hernandez Sairy , Chandele Anmol , Dong Xuemei , Odegard Jared , Kaech Susan M , Dent Alexander L , Craft Joe
Published: January 2023

Follicular helper T cells (TFH), defined by expression of the surface markers CXCR5, PD-1 and ICOS, are located in germinal centers where they provide help for B cell maturation through the secretion of IL-21. The transcriptional repressor Bcl6 is upregulated in TFH cells; however, the role that it plays in their development is unknown. We describe downregulation of P-selectin glycoprotein ligand-1 (PSGL1) as a new surface marker for TFH cells, and show that Bcl6 is required for the development of TFH cells that are PSGL1lo. Activated antigen-specific CD4 PSGL1lo T cells are enriched for expression of CXCR5 and PD-1, and highly upregulate IL-21 and Bcl6. Over-expression of Bcl6 leads to an increase in PSGL1lo TFH cells; however, it cannot rescue TFH cell development in the absence of ICOS. Consistent with others, we find B cells are required for full development of TFH cells, but not for downregulation of PSGL1, suggesting there are multiple steps in development of the subset. This data demonstrates that Bcl6, independent of ICOS signaling, is critical for the development of this lineage.
Fas induced apoptosis and Restimulation Induced Cell Death (RICD) is restricted to human effector memory CD4+ T subsets (46.8)
Authors: Ramaswamy Madhu , Cruz Anthony C , Siegel Richard M
Published: January 2023

FasL, a member of the TNF super family and its receptor Fas, are critical for peripheral tolerance of T cells via a mechanism of TCR-induced apoptosis of activated cells, termed RICD. Studies of RICD have shown that human CD4+T cells require TCR crosslinking to maximally undergo apoptosis when stimulated with Fas. However, the relative sensitivity of different CD4+ T cells subsets to Fas stimulation have not been addressed.

We found that essentially all cells sensitive to Fas stimulation derive from human memory CD4+ T cells and within the memory T cell pool, 'effector' cells lacking CCR7 and CD27 were consistently the most sensitive to Fas-induced apoptosis. Fas resistant 'central memory' as well as Fas-sensitive 'effector' cells had similar levels of Fas, induced FasL as well as pro-survival Bcl2/BCLxL levels. Biochemical studies indicate a highly efficient formation of the death-inducing signaling complex (DISC) in memory and effector T cells, which correlates with their apoptotic response. Finally, in ALPS patients with Fas mutations, resistance to Fas apoptosis was found predominantly in the effector memory subsets.

These results suggest for the first time that within a heterogeneous population of CD4+ T cells, effector memory T cells are specifically programmed for sensitivity to Fas-induced apoptosis compared with activated naïve T cells.
Differential sensitivity of Th1, Th2 and Th17 cells to Fas-mediated apoptosis (47.15)
Authors: FANG Yujiang , YU Shiguang , MULLEN Helen
Published: January 2023

In response to T cell receptor crosslinking, naïve CD4+ T cells can differentiate into three major subsets, Th1, Th2 and Th17 cells. Th17 cells play an important role in the pathogenesis of autoimmune diseases, but little is known about the regulation of apoptosis in Th17 cells. This study was undertaken to directly determine and compare the sensitivity of in vitro polarized Th1, Th2 and Th17 cells to Fas-mediated apoptosis and to determine if the anti-apoptotic molecule FLIP could inhibit apoptosis of each of the three subsets. Using several different methods, the order of sensitivity of T cell subsets to Fas-mediated apoptosis is: Th1>Th17>Th2. The greater sensitivity of Th17 cells compared to Th2 cells correlated with their higher expression of FasL. The decreased sensitivity of Th17 compared to Th1 cells correlated with higher expression of FLIP by Th17 cells. Transgenic overexpression of FLIP in T cells protected all three subsets from Fas-mediated apoptosis. Using splenocytes from IFNγ-/- mice, Fas-mediated apoptosis of T cells activated under Th1 polarization-inducing conditions were shown to be dependent on IFN-γ, while Fas-mediated apoptosis of Th17 and Th2 cells was IFN-γ independent. These findings provide new knowledge for understanding how survival of different subsets of T cells is regulated. This information might be useful for the design of new strategies to treat autoimmune diseases (NIH DK35527).
A European inter-laboratory pre-validation of in vitro assays to evaluate immunotoxicity (47.18)
Authors: Vandebriel Rob , Kleensang Andre , Koeper Lydia-Mareen , Biola-Vidamment Armelle , Fiechter Danielle , Carfi Maria , Gribaldo Laura , Corsini Emanuela , Pallardy Marc , Pieters Raymond , Vohr Hans-Werner , Van Loveren Henk
Published: January 2023

The new European Union REACH (Registration, Evaluation and Authorisation of new and existing CHemicals) policy will increase the use of laboratory animals if alternative methods are not available. The immune system is a target for many chemicals and drugs, with potential adverse effects on human health. We performed an inter-laboratory pre-validation of a set of in vitro assays to detect immunosuppressive capacity.

Human PBMC and murine and rat splenocytes were used. Six immunosuppressive compounds (benzo(a)pyrene, cyclophosphamide, methotrexate, rapamycin, dexamethasone, and urethane) and one non-immunosuppressive compound (D-mannitol) were tested. Basal cytotoxicity was assessed by measuring LDH-release. Human PBMC were stimulated with anti-CD3/anti-CD28, murine splenocytes with anti-CD3/anti-CD28, Con A, LPS, and anti-CD40/IL-4, and rat splenocytes with Con A and LPS. Cell proliferation and IFN-γ and TNF-α production were measured.

Five out of 6 immunosuppressive compounds could be identified as such, and the non-immunotoxic one came out as negative. This suggests that in vitro assays are able to detect immunosuppressive capacity. The sensitivity of the various species/stimulant/endpoint combinations ranged between 38 and 61%, while the specificity ranged between 67 and 100%. This suggests that from these results a choice for specific species/stimulant/endpoint combinations cannot be made.
Simultaneous detection of human IL-17, FoxP3 and other Th cytokines and surface markers by multi-color flow cytometry (47.4)
Authors: Li Li , Wu Xiaowei , Boyce Christopher , Wei Aili , Rohrer Jurg
Published: January 2023

Naïve CD4+ T helper cells can be induced to differentiate towards at least four types of committed helper T cells, namely T helper 1 (Th1), Th2, Th17 and regulatory T (Treg) cells according to the local cytokine milieu. The committed cells are characterized by the expression of specific transcription factors and by the cytokines released. Th17 cells are acknowledged to be instrumental in the response against microbial infection, but are also very efficient inducers of tissue inflammation and crucial initiators of organ specific autoimmunity. FoxP3+ Treg cells are necessary and sufficient to prevent autoimmunity throughout the life span of an individual. Immune and autoimmune responses are regulated by a fine balance between effector and regulatory T cells. There is a reciprocal relationship between pathogenic Th17 cells and Treg cells. Therefore the need to simultaneously detect multiple transcription factors and cytokines to define these populations is becoming increasingly important. In this report, we describe the simultaneous detection of IL-17 and FoxP3 as well as other Th cytokines (IFN-gamma and IL-4) and cell surface markers (CD3, CD4, CD25, CCR4, CCR6, CD45RA) using optimized buffer systems and protocols with multi-color flow cytometry.
Regulation of effector CD4+ T cell functions by Tbet (48.13)
Authors: Yeh Wen-I , Harrington Laurie E.
Published: January 2023

Many chronic inflammatory diseases, including inflammatory bowel disease and multiple sclerosis, are reported as Th1 mediated disorders; however, recent data from animal models suggests that Th17 cells are responsible for disease. Interestingly, IFNγ does not appear necessary for disease induction, but the key Th1-associated transcription factor Tbet is required. Therefore, we investigated the relationship between Tbet expression and effector CD4 T cell functions. Colitis induced by adoptive transfer of IFNγ KO CD4 T cells resulted in rapid disease and surprisingly over 60% of CD4 T cells expressed Tbet. We next examined the kinetics and functions of Tbet in vitro during Th1 and Th17 polarization. Tbet was upregulated within 24 hrs of stimulation under Th1 conditions, but remained low in Th17 cells. Moreover, Th17 differentiation was unaffected by Tbet deficiency. In order to define molecules regulated by Tbet, we examined gene expression in WT and Tbet KO CD4 T cells by microarray analysis. Consistent with our earlier results, Th1 associated cytokines were reduced but Th17 and Th2 associated genes were elevated in Tbet KO CD4 T cells. Notably, both the mRNA and protein levels of IL-10, an inhibitory cytokine, are increased in Tbet KO CD4 T cells suggesting a potential role for IL-10 in vivo. In conclusion, we propose that Tbet is critical in maintaining pathogenic Th1 cells and suppressing IL-10 expression.
Cellular Damage Determination in Newborn Balb/c Mice Skin Through Ro60, Hsp27, 60, 70 and 90 expression. (48.32)
Authors: Aguilera Galaviz Luis Alejandro , Olarte Saucedo Maricela , García Cabral Fernando , Lopez Luna Maria Argelia , Sanchez Rodriguez Sergio Hugo
Published: January 2023

Systemic Lupus Erythematosus is an autoimmune disease, it could be multi-systemic and characterized by auto-antibodies, especially antinuclear (anti-Ro, anti-La, anti-DNA), damage is mediated by immune complexes. The injuries in the skin of patients with SLE are aggravated by sun exposition (UV light and heat), altering the cellular expression and distribution of Ro60 antigen. In order to understand the damage mechanism involved in skin of patients with LES by environmental stressors, we developed a murine model focused in Ro60 and the heat shock proteins family (Hsp) expression. Objective: Determinate the cellular damage of the skin of newborn Balb/c mice, which was measured through the expression of the Ro60, Hsp27, 60, 70 and 90. Methods: Balb/c mice were exposed to different physical and chemical stressors like UV irradiation, heat (40ºC), H2O2, ethanol, HgCl2, camptotecine and cicloheximide drugs, in presence or absence of caspase 3 inhibitor. Skin extracts were obtained and the proteins Hsp27, 60, 70 and 90 were characterized and immunodetected by PAGE-SDS and Western blot using ECL, densytometric analysis was performed. Results: The Ro60 expression is modified with physical and chemical stressors, also it is increased mainly with UV light and heat, and decreased with caspase 3 ihnibitor. Hsp27, Hsp70, Hsp90 was similar to Ro60. The over expression of those proteins during the stress have the same profile. However using caspase 3 inhibitors before the stress, a down regulation are observed.
LILRA3 protein is constitutively expressed in normal serum and increased in patients with active rheumatoid arthritis (49.11)
Authors: Tedla Nicodemus , Chandra Vasudha , An Hongyan , Bryant Katherine
Published: January 2023

Leukocyte immunoglobulin-like receptor A3 (LILRA3) belongs to a family of cell surface receptors that have activating or inhibiting functions in leukocytes through their intracytoplasmic immunoreceptor tyrosine-based activating motifs (ITAMs) and inhibitory motifs (ITIMs). LILRA3 lacks transmembrane and cytoplasmic domains, suggesting that it may be secreted. Although LILRA3 function is unknown, its high structural homology to activating LILRA1 and A2 indicate that it may act as antagonist or agonist to these receptors. Clinically, LILRA3 may have a role in a number of immune-related diseases. A recent finding showed that individuals who lack the LILRA3 gene have a higher incidence of multiple sclerosis, suggesting that LILRA3 may regulate inflammatory responses. However to date, expression studies of LILRA3 have been limited to mRNA. Here we developed an ELISA to detect LILRA3 protein in serum. To investigate whether LILRA3 plays a role in chronic inflammation, we examined its expression in serum and synovial fluid of patients with rheumatoid arthritis (RA). For the first time, we demonstrate LILRA3 protein expression in serum with increased levels in serum and synovial fluid of patients with RA compared to osteoarthritis or normal subjects. The level of LILRA3 in RA correlated with disease activity score. These findings indicate that LILRA3 may play a role in the pathogenesis of RA.
Autoimmune Transcriptional Profiles in Early Rheumatoid Arthritis Peripheral Blood Cells (49.12)
Authors: Olsen Nancy J , Ligon John , Davis Laurie S
Published: January 2023

Blood samples from early rheumatoid arthritis (RA) patients were examined for unique transcriptional profiles. Whole blood (WB) was collected in PAXgene Blood RNA tubes (Qiagen) from seropositive early RA patients with mean disease duration of 28 weeks, established RA patients (n=2) and healthy controls (HC; n=5). Total RNA purified on Qiagen columns was assessed by Human 6-BeadChip Illumina arrays for expression of 26,098 annotated genes, >21,000 ESTs and 1550 control transcripts. Differentially expressed transcripts were identified using GeneSpring 7.3.1 (Agilent). Of 33,318 transcripts passing filtering, 678 were differentially regulated at least 2 fold in RA vs. HC and 600 were statistically significant by Welch ANOVA analysis (p<0.05). Most (592) were significant by Benjamini and Hochberg false discovery rate multiple testing (p<0.05) and 28 were significant after more stringent Bonferroni family-wise error analysis. Transcripts strongly expressed in early RA included RNA helicases (DDX3), synthases (PGS1), adaptor proteins (PIK3AP1), membrane proteins (CD107b), and signal transduction molecules (DUSP1, FRAT2). A majority of transcripts (18/29) previously identified as RA class predictors were upregulated in early RA. Real-time PCR carried out to confirm gene expression in a larger early RA cohort suggests unique patterns in early RA and lupus. Prospective studies will be of interest to determine gene expression patterns correlating with phenotypes.
QTL associated with collagen-induced arthritis in CXB mice. (49.13)
Authors: Marshall Dana Robin
Published: January 2023

B6 mice are of intermediate susceptibility to collagen-induced arthritis (CIA) while Balb/c mice are resistant but are, in turn, highly susceptible to proteoglycan-induced arthritis. Antigen presentation is H-2 directed but the disease that results is thought to be driven by regions outside of the MHC therefore, CXB mouse strains afford the opportunity to look at the influence of these regions on CIA. H2-b CXB strains are predicted to show variation in disease parameters relative to B6, depending on which Balb/c chromosome regions are present. 9/13 CXB strains are H-2b while one has a recombined H-2 region (CXB9). The four H-2d strains were crossed with C57Bl/6ByJ to generate heterozygous F1 mice that could present collagen via the B6-contributed H-2b locus. A range of incidence and severity of disease was seen across the strains, and mice were assigned to susceptibility groups based on collected parameters, including incidence. QTL analysis using WebQTL, has identified regions on chromosomes 13, 15 and 19, that correlate with susceptibility to CIA. Genes in these regions include some associated with inflammation via TGFá and IL-8, and a T-cell gene linked to rheumatoid arthritis in humans. Additionally, 3/14 identified genes are relatively specific to chondrocytes, with cartilage being the target tissue for autoimmune arthritis. These data are encouraging in that autoimmune disease involves some type of immune deregulation that in certain individuals, acts on a specific target. This model may, therefore, lend clues as to why cartilage becomes a specific target for immunological attack.
DR1001 presents ‘altered-self’ peptides by accepting citrulline in its binding pockets (49.20)
Authors: James Eddie A , Moustakas Antonis K , Bui John , Papadopoulos George K , Kwok William W
Published: January 2023

HLA-DRB1*1001 (DR1001) is associated with rheumatoid arthritis, containing the shared epitope (residues 70-74) within its third hypervariable region. It has been suggested that shared epitope alleles bias T cell selection and amplification, perhaps favoring the recognition of common self-peptides. However, the binding motif of DR1001 and its epitopes remain relatively uncharacterized. One recent paper inferred a binding motif for DR1001 by aligning the sequences of eluted peptides, but only two epitopes were available to validate these findings. The objective of this study was to assess the capacity of DR1001 to present RA associated self-peptides. This was accomplished by defining high affinity non-self epitopes, characterizing amino acids (including citrulline) that can bind at DR1001 anchor positions and using this information to predict putative citrullinated epitopes within RA associated proteins. We identified 15 novel DR1001 restricted epitopes using class II tetramers. Using two of these as a template, we defined the amino acid preferences for each peptide binding pocket. Citrulline was accepted at positions 4, 7 and 9. Based on the observed binding preferences we identified several arginine containing sequences from within RA associated proteins that would be expected to bind after citrullination. For 12 of these, we synthesized unmodified and citrullinated versions and measured their binding to DR1001. Five sequences bound to DR1001, implicating these as potential 'altered-self' epitopes.
Analysis of SLAM family receptors 2B4, CS1 and NTB-A in patients with Systemic Lupus Erythematosus (SLE) (49.22)
Authors: Mathew Stephen O , Kim Jong R , Pertusi Raymond M , Patel Rahul K , Mathew Porunelloor A
Published: January 2023

Systemic lupus erythematosus (SLE) is a chronic autoimmune inflammatory disease, characterized by the improper regulation of B lymphocytes that leads to the production of auto-antibodies. Several studies have identified a genetic susceptibility region called Sle 1 in mice and some human subjects. Recently, genomic characterization of the Sle1b locus, identified a highly polymorphic cluster of genes coding for the signaling lymphocyte activation molecule (SLAM)-related receptors (SRRs). 2B4 (CD244), CS1 (CD319, CRACC) and NTB-A are members of SRRs and are expressed on NK, T, B cells and monocytes. The aim of this study was to investigate expression of members of the SRRs in peripheral blood mononuclear cells (PBMC) from patients with SLE. We analyzed PBMC from forty-six patients with SLE and thirty-one healthy individuals. The surface expressions of 2B4, CS1, and NTB-A on T cells, B cells, NK cells, and monocytes were analyzed by flow cytometry. 2B4 expression was upregulated on T cells but down-regulated on NK cells and monocytes in patients with SLE. B cells from patients with SLE significantly overexpressed CS1 compared to B cells from healthy controls. The surface expression of NTB-A was upregulated on T cells and monocytes, but down-regulated on B cells in patients with SLE. Our results suggest that altered expression of SRRs may be related to the abnormal B cell hyperactivity in patients with SLE. (Research supported by grants from Texas Higher Education Board and UNTHSC).
The Epigenome of SLE (49.26)
Authors: Sullivan Kathleen E , Zhang Zhe , Song Li , Petri Michelle A
Published: January 2023

Systemic lupus erythematosus (SLE) is a polygenic disorder affecting 1:1000 adults. Recent data have implicated interferons in the pathogenesis. We examined H4 acetylation and gene expression in monocytes from patients with SLE to define alterations to the epigenome. Monocytes from 14 controls and 24 patients were used for analysis by chromatin immunoprecipitation for H4 acetylation and gene expression arrays. H4 acetylation is significantly altered in monocytes from patients with SLE. Although there was a relationship between increased H4 acetylation and gene expression the two gene sets were not concordant. The most significant relationship for both genes with increased H4 acetylation and increased gene expression was the potential for transcriptional regulation by IRF1. Alpha-interferon was a significant contributor to gene expression patterns in SLE, but the greatest concordance was seen in the enrichment of certain transcription factor binding sites upstream of genes with increased H4 acetylation in SLE and genes with increased H4 acetylation after alpha-interferon treatment. We conclude that SLE is associated with a globally altered epigenome. Multiple lines of evidence implicate the interferon pathway. Altered epigenetic patterns could contribute to the perpetuation of disease by molding the gene set competent for expression.
SR16951, a new small molecule antagonist of MCP-1 for the treatment of autoimmune diseases (50.13)
Authors: Lam Jennifer S , Alt Carsten , Bernard Claude , Laborde Edgardo , D'Andrea Annalisa
Published: January 2023

Monocyte Chemoattractant Protein-1 (MCP-1) is a chemokine that plays a role in the inflammatory response by attracting monocytes, memory T cells, dendritic cells, natural killer cells, and endothelial cells to the site of injury. MCP-1 and its receptor, CCR2, have been implicated as potent mediators in a variety of inflammatory and autoimmune diseases, such as multiple sclerosis (MS), rheumatoid arthritis, and atherosclerosis. Thus, the ability to block the actions of MCP-1 can be a powerful therapeutic approach for the treatment of these diseases. Here we report on SR16951, a small molecule antagonist of MCP-1. SR16951 inhibits MCP-1-induced migration of monocytic cells in vitro. Moreover, SR16951 showed a significant effect at preventing and therapeutically ameliorating the clinical symptoms that manifest during Experimental Autoimmune Encephalomyelitis (EAE), a model for MS, and Collagen-Induced Arthritis. In order to gain a better understanding of the mechanism of SR16951, we focused on differences in intracellular signaling events following MCP-1 stimulation in the presence or absence of SR16951. We found that SR16951 inhibits MCP-1-induced phosphorylation of Akt, a downstream target of the PI3K/Akt signaling pathway. These preliminary studies would indicate that SR16951 inhibits MCP-1-induced chemotaxis by interfering with early signaling events that take place after binding of MCP-1 to CCR2.
Immune modulation by chondroitin sulfate and its degraded disaccharide in the development of experimental autoimmune encephalomyelitis (EAE) (50.2)
Authors: Zhou Juhua , Nagarkatti Prakash , Zhong Yin , Nagarkatti Mitzi
Published: January 2023

We attempted to understand the immune modulation mediated by chondroitin sulfate A (CS-A) and its disaccharide degradation product (CSPG-DS) in the development of experimental autoimmune encephalomyelitis (EAE), an animal model of multiple sclerosis. Clinical symptoms in myelin oligodendrocyte glycoprotein peptide (MOG35-55)-induced EAE mice significantly enhanced following CS-A injection, whereas CSPG-DS injection completely inhibited EAE development. When a clinically more severe form of EAE was induced, CS-A accelerated the clinical disease, whereas CSPG-DS significantly delayed EAE. Further studies demonstrated that CS-A up-regulated STAT4 expression, and thus induced interferon-gamma production and Th1 CD4 T cell differentiation. CS-A also up-regulated STAT3 expression and increased IL-17 producing T cell populations. CSPG-DS treatment both in vivo and in vitro decreased tumor necrosis factor - alpha production from splenocytes. In vitro and in vivo assay indicated that CSPG-DS treatment significantly blocked T cell migration, whereas CS-A treatment increased lymphocyte infiltration in the brain when compared to MOG-treated control mice. Our results suggest that while CS-A exacerbates the development of EAE, CSPG-DS administration may be useful in its treatment. (Supported in part by NIH grants R01AI053703, R01ES09098, R01 AI058300, R01DA016545, R01HL058641 and P01AT003961).
The natural antioxidant lipoic acid (LA) alters immune cell function: implications for multiple sclerosis (50.3)
Authors: Salinthone Sonemany , Schillace Robynn V. , Yadav Vijayshree , Marracci Gail , Bourdette Dennis , Carr Daniel W.
Published: January 2023

Multiple Sclerosis (MS) is an often disabling disease of the central nervous system. Current FDA approved therapies are only partially effective, associated with side-effects and costly. Thus, there is a need for development of novel treatment alternatives for MS. The antioxidant lipoic acid (LA) reduces disease severity and T lymphocyte migration into the spinal cord in a model of MS, experimental autoimmune encephalomyelitis. A pilot clinical trial demonstrated that oral LA is well tolerated in MS patients. Because the mechanism of action of LA is unknown, we sought to determine the effects of LA on immune cell function and identify a signaling pathway that may mediate this response. We discovered that LA stimulates production of cAMP in immune cells obtained from MS patients after oral ingestion of LA. Studies using radiolabeled prostaglandin E2 (PGE2) indicate that LA competes for binding of the PGE2 receptors. LA suppresses the production of interleukin (IL)-2, IL-6 and IL-17 in these cells, and inhibits T cell proliferation. Furthermore, studies blocking activation of the cAMP signaling pathway illustrates that cAMP mediates LA suppression of interferon gamma (IFNγ) production. These novel findings suggest that LA may inhibit immune cell function via the cAMP signaling pathway and has implications for MS therapy.

This work is supported by the Dept. of Veterans Affairs Biomedical Laboratory Research & Development Service.
Granulocyte-Macrophage Colony-Stimulating Factor (GM-CSF) induces tolerogenic dendritic cells that delay onset of type 1 diabetes by enhancing CD4+CD25+Foxp3+ regulatory T cells (50.33)
Authors: Ganesh Balaji B , Cheatem Donald , Gopisetty Anupama , Bhattacharya Palash , Vasu Chenthamarakshan , Prabhakar Bellur S
Published: January 2023

Impairment in dendritic cell (DC) functions is implicated in defective immune regulation that leads to type-1 diabetes (T1D) in both humans and the established non-obese diabetic (NOD) mouse model. In this study, we examined the roles of DC modulators such as GM-CSF and Flt3-L to affect DC function and prevent T1D development in NOD mice. Both GM-CSF and FLT3-L could suppress the development of T1D if treatment was initiated earlier in life prior to the development of clinical diabetes. However, unlike Flt3-L, GM-CSF was far more effective in suppressing T1D even when administered at later stages of insulitis. Ongoing periodic treatment of NOD mice with GM-CSF prevented the development of T1D and the animals remained healthy until 3 months after cessation of the treatment. Adoptive transfer of CD4+CD25+ T cells from GM-CSF-treated mice could suppress effector T cell response and T1D in NOD-SCID mice, and this suppression was associated with enhanced IL-10 and TGF-β1 production. Moreover, adoptive transfer of GM-CSF-exposed DCs to naive NOD mice resulted in an expansion of Foxp3+ T cells and a significant delay in T1D onset. These results indicate that primarily the GM-CSF renders DCs tolerogenic resulting in subsequent Treg induction and delayed onset of T1D in NOD mice. NIH 1RO1AI058190