bluuebunny/crossref_metadata_2025 huggingface dataset:
Membrane events after cross-linking of the chB6 alloantigen (111.42)
Authors: Funk Phillip , Sanderson Bailey
Published: January 2023

Chickens are one of the classic models of vertebrate immunity. We have been interested in the role of the alloantigen chB6 (formerly called BU1) in the development of B cells within the bursa and have presented evidence that chB6 can trigger apoptosis. Furthermore, the pathway that triggers apoptosis is similar to that triggered by FAS, with caspase 8 as an initiator, caspases 9 and 3 as later caspases, and bcl-xl functioning as an inhibitor of apoptosis. We have been unable to demonstrate a steady state association of chB6 and caspase 8 in DT40, so the mechanism of caspase 8 activation remains unclear. Here we show that chB6 localizes in distinct clusters on the surface of DT40 cells, reminiscent of the SPOTS others have described in Fas signaling. These clusters co-localize with cholera toxin labeled lipid rafts and chB6 appears to be internalized by the B cells. Western blots indicate that chB6 is present in both detergent soluble and insoluble membrane fractions on resting DT 40 cells. However, cross-linking chB6 with a secondary antibody shows that chB6 is internalized selectively from detergent insoluble (lipid raft) fractions of the cell membrane. This suggests an active distribution of chB6 in the membrane and an internal trafficking that may be functionally important. This work offers a new insight into the signaling via chB6. This work supported by a grant from the DePaul University Research Council.
Dissecting the relationship between migration, TCR signaling, and thymic selection in situ (111.44)
Authors: Melichar Heather , Ross Jenny , Herzmark Paul , Robey Ellen
Published: January 2023

T cell receptors (TCRs) on developing thymocytes are screened for appropriate recognition of MHC molecules and self-reactivity before differentiating into mature T cell subsets. However, how thymocyte interactions and TCR signaling relate to developmental outcomes during these selection events in vivo is unclear. We use a thymic slice model, fluorescent calcium reporters and fusion proteins, in conjunction with two-photon imaging techniques, to examine the impact of TCR:peptide affinity and antigen presenting cell type on thymocyte sampling, signaling, and developmental outcome in situ. Low affinity antigen interactions result in multiple, transient interactions, and TCR signals are short and interrupted. In contrast, negative selecting ligands are associated with longer, stable interactions with more sustained signaling. Further, we examined the behavior of male-specific, on-time HY transgenic thymocytes to characterize the behavior and signaling characteristics of cells undergoing positive and negative selection to endogenous antigen in a physiological setting. In sum, these experiments provide insight into the role of cell-cell interactions and TCR signaling in shaping the processes of positive and negative selection in situ.
Differential regulation of V(D)J recombination in multipotent progenitors in bone marrow versus thymus (111.8)
Authors: Borghesi Lisa , Vallejo Abbe , Kardava Lela , Yang Qi , Aites Jennifer
Published: January 2023

V(D)J recombinase activity is expressed in bone marrow (BM) multipotent progenitors (MPPs) that retain the potential to differentiate into B cells in BM and T cells in thymus. However, it remains unclear whether MPPs in each organ exhibit distinct transcriptional requirements for recombinase activity, or whether thymic MPPs imprint the requirements of upstream BM MPPs from which they originate. Here we use V(D)J recombination reporter mice to examine transcriptional requirements in BM versus thymic multipotent subsets. Within BM, the frequency of recombinase+ cells increases from 5% in MPPs to 15% in common lymphoid progenitors (CLPs) and 20% in pre-pro B cells. Within thymus, 10% of early thymic progenitors (ETPs) are recombinase+. While the E47 transcription factor is expressed in both BM and thymus, E47 deficiency selectively ablates V(D)J recombination in the former. BM MPPs, CLPs and pre-pro B cells from E47 knockouts lack detectable recombinase activity. In the thymus, E47 deficiency reduces ETP numbers but V(D)J recombinase activity is maintained. Loss of the related E protein HEB does not diminish recombinase activity in ETPs while functional inhibition of E47:HEB complexes leads to a 40% reduction in TCRβ rearrangements. Thus, V(D)J recombinase activity is regulated differently in MPPs in BM and thymus. E protein transcription factors are essential for recombinase activity in multipotent BM MPPs and CLPs but are dispensable in multipotent thymic ETPs.
Driving HIV-specific vaccine responses in immune suppressed recipients (113.20)
Authors: McEwen Lisa , Bui Cac , Paterson Yvonne , Harn Donald
Published: January 2023

Malaria, TB and HIV-1 remain tremendous disease burdens in much of the world’s population. Vaccines for these diseases are desperately needed. However, vaccine efficacy is likely to be compromised due to the systemic Th2 biasing and immune suppression caused by infection with parasitic helminthes. Our lab and others have shown that helminth infection suppresses Th1-type vaccine-specific responses. Although Sub-Saharan populations will benefit most from these vaccines, a large percentage of these populations live in helminth endemic regions. A goal of our research is to find vaccine vectors that drive significant HIV-specific immune responses in helminth infected recipients, despite the immune status of the individual. In the current study, we demonstrate that administration of an HIV vaccine (Listeria expressing HIV-1 gag protein) drives significant HIV-specific CTL and Th1 responses in mice chronically infected with the helminth parasite Schistosoma mansoni. These results suggest that Listeria vector vaccines likely will drive HIV-specific responses in helminth-infected human populations. Kinetic studies show that the HIV-vaccine-specific responses generated in schistosome infected mice are durable and capable of inducing CD4+ and CD8+ T cell memory. Further, Listeria vectors should be considered in the development of new generation HIV-1, malaria or TB vaccines to be administered to populations in sub-Saharan Africa where helminth infection is endemic.
Vaccine-elicited CD8+ T cells protect against respiratory syncytial virus-induced lung airway obstruction (113.8)
Authors: Lee Sujin , Stokes Kate , Celis Esteban , Moore Martin
Published: January 2023

Respiratory syncytial virus (RSV) is the most important pathogen for lower respiratory tract illness in infants. CD8+ T cells clear RSV and may contribute to future RSV vaccines. RSV itself inhibits CD8+ T cell responses. Compared to CD8+ T cells responding to RSV infection, vaccine-elicited anti-RSV CD8+ T cells are less well studied, and mechanisms for achieving optimally functional anti-RSV CD8+ T cells are not defined. We developed a novel RSV vaccination strategy using combined toll-like receptor (TLR) and CD40 stimulation in BALB/c mice as a model for studying antigen-defined RSV-specific CD8+ T cells. Vaccination of mice with TriVax, a mixture of a M2 82-90 peptide representing an immunodominant RSV CD8 epitope, the TLR agonist poly (I:C), and a costimulatory anti-CD40 antibody resulted in robust, functional CD8+ CTL responses. Using a mucus-inducing RSV challenge strain (rA2-line19F), we demonstrate that TriVax vaccination protected against RSV infection and RSV-induced airway mucus expression, mechanical airway obstruction, and cellular lung inflammation. Compared to RSV challenge alone, TriVax vaccination followed by challenge resulted in increased CD8+ T cell cytokine expression and the rapid appearance of RSV-specific CD8+ T cells in the lung. Despite a large number of cytokine expressing and virus-specific CD8+ T cells in the lung, TriVax vaccination did not cause immunopathology. High-quality vaccine-elicited anti-RSV CD8+ T cells were protective in the lung.
Epigenetic effects of transmethylation micronutrients on gene expression in T cells from patients with active lupus. (116.12)
Authors: Ray Donna , Richardson Bruce
Published: January 2023

T cell DNA demethylation contributes to the development of human lupus. However, the mechanisms causing T cell DNA demethylation in lupus patients are incompletely understood. The replication of DNA methylation patterns during mitosis depends on DNA methyltransferase (Dnmt) enzymatic activity and the methyl donor S-adenosylmethionine (SAM), and is inhibited by S-adenosylhomocysteine (SAH). SAM levels are determined by various dietary nutrients including folate and methionine, and previous work demonstrates that patients with active lupus patients have decreased Dnmt activity. We hypothesized that lupus patients will be more susceptible to environmental changes in folate and methionine due to their already low levels of T cell Dnmt activity compared to controls, resulting in aberrant overexpression of genes normally suppressed by DNA methylation. We cultured PHA-stimulated lupus T cells in media containing physiologic high and low concentrations of folate or methionine, and compared effects on methylation sensitive gene expression (CD11a, CD70, KIR and perforin) by flow cytometry. Lupus T cells cultured in lower concentrations of folate and methionine overexpress CD70, KIR and perforin compared to those cultured in standard RPMI 1640 media, relative to control T cells. These results suggest that folate and methionine may contribute to abnormal T cell gene expression in lupus, and that dietary supplementation may have a beneficial effect.
Identification of a tissue resident alveolar macrophage population susceptible to and permissive of Coxiella burnetii infection. (117.14)
Authors: Calverley Matthew , Harmsen Allen
Published: January 2023

Intracellular bacteria, such as the US biological Class B bacterium Coxiella burnetii, are known to infect alveolar macrophages (AM). However, it is not known if a particular subset of AM, within a relatively heterogeneous population of lung macrophages, is particularly susceptible to bacterial infection. Nor is it known whether certain AM are less efficient at resolving infection. We hypothesized that, as a result of tightly regulated immune function, tissue resident AM would prove susceptible to C. burnetii infection and provide a required niche for bacterial persistence. Dye labeling techniques allowed discrimination of AM subsets during the course of infection. Infected cell populations were identified immunohistochemically and through the use of fluorescent protein expressing C. burnetii. We report here that the tissue resident AM are the predominantly infected AM subset, even at low infective doses; these cells remain infected at least nine days after the onset of infection, indicating a permissive niche for bacterial infection. These tissue resident AM undergo a distinguishing phenotypic change during the progression of infection and account for the cell-type predominating in the early uptake of Coxiella. Identification of the AM subset, both susceptible to and permissive of Coxiella burnetii infection, marks an important step in understanding Coxiella pathogenesis providing insight into general pulmonary innate immune function in response to intracellular bacteria.
Heme oxygenase-1 expression were decreased in monocytes from Systemic Lupus Erithematosus patients (119.6)
Authors: Mackern Juan , Llanos Carolina , Herrada Andrés , Leandro Carreno , Gómez Roberto , Anegon Ignacio , Jacobelli Sergio , Kalergis Alexis
Published: January 2023

Systemic Lupus Erithematosus (SLE) is a complex disease characterized by alterations affecting innate and adaptive immune cells. In SLE, monocytes and dendritic cells (DCs) present a stimulatory phenotype that promotes the activation of T cells. One of the molecules that control monocyte function is Heme oxigenase-1 (HO-1) which possesses anti-inflammatory capacity. The aim of this work was to assess HO-1 levels in monocytes of SLE patients (SLEp) and HC. PBMCs were obtained from 43 SLEp and 30 HC. CD14+ monocytes and CD4+ cells were sorted by FACS and HO-1 expression was measured by RT-PCR. HO-1 protein expression was determined by FACS. Parameters of CD4+ T cell activation such as CD69 and CD25 expression and IL2 production after staphylococcal enterotoxin A (SEA) stimulation were used as surrogate to evaluate the stimulatory capacity of monocytes from PBMCs cultures. MHCII and CD86 expression was analyzed by FACS. HO-1 mRNA levels were decreased in monocytes from SLEp compared to HC (P<0.0075, t test). When HO-1 protein expression were assessed by FACS similar results were observed (relative MFI expression to HC; 0.73 ± 0.05; P<0.0001, t test). When CD4+ activation in response to SEA was evaluated, no differences were found. MHCII and CD86 expression in CD14+ cells showed no differences between SLEp and HC. In conclusion, we found a significant decrease in HO-1 expression in monocytes from SLEp, suggesting that an imbalance in HO-1 could play a role in SLE pathogenesis.
The role and mechanisms of type I IFN pathways in suppressing acute colitis (120.15)
Authors: Zhang Lixia , Guo Beichu
Published: January 2023

Inflammatory bowel diseases (IBD), such as Crohn’s disease and ulcerative colitis, is characterized by chronic inflammation of the gastrointestinal tract. The etiology and pathogenesis are poorly understood, dysregulated immune responses in the gut and a loss of tolerance to intestinal microflora are thought to cause IBD in human patients and in animal models. Recent studies show that he IL-23/Th17 axis plays an important role in the development of IBD. Our recent studies revealed a novel immunoregulatory or immunosuppressive function of TLR-mediated type I IFN (IFNβ/IFNα) pathways on the innate and antigen-specific immune response. The goal of this study is to investigate the role and mechanisms of type 1 IFN in the homeostasis of the intestine and development of colitis. Our results show that I IFN receptor (IFNAR) deficient mice were highly susceptible for the development of DSS-induced colitis as indicated by body weight loss. IFNAR KO mice had significantly higher macroscopical and histopathological colitis scores compared to wt mice. We also found increased infiltration of innate immune cells and Th cells including Th17 cells in intestine and colon tissues in IFNAR KO mice. Furthermore, IFNAR KO mice had enhanced expression of proinflammatory cytokines and chemokines in intestinal tissues after DSS-induced colitis. Together, our studies imply the physiological role of endogenous type I IFNs in inhibiting the development of inflammatory bowel disease.
Loss of p27 enhances gastrointestinal eptithelial malignancy in mice with Smad4 deficiency in T cells. (120.17)
Authors: Kim Byung-Gyu , Choi Sung Hee , Robinson Janet , Letterio John
Published: January 2023

We previously reported that selective loss of Smad4 signaling in T cells (Smad4KO) leads to spontaneous gastrointestinal inflammation and cancer. The expression of p27Kip1 is significantly decreased in the colon of Smad4KO mice, thus we investigated the role of p27Kip1 in the maintenance of intestinal mucosal immune and epithelial homeostasis. Smad4KO and p27Kip1 germ line mutation (Smad4/p27DKO) mice develop spontaneous gastrointestinal inflammation and carcinoma at 1-2 months of age and 3 months of age, respectively. Smad4/p27DKO had little in vitro TGF-β-induced Foxp3+CD4+ T cells (iTregs) and decreased natural Foxp3+CD4+ T cells (nTregs) in vivo, whereas Smad4KO had little iTregs and p27KO had decreased nTregs. Furthermore, Smad4/p27DKO have an increased number of mucosal CD4+ T cells and an increased percentage of memory T cells. The production of pro-inflammatory cytokines including IL-6, IFN-γ and TNF-α was greatly increased in the colon mucosa of Smad4/p27DKO mice. The colon epithelium of Smad4/p27DKO mice exhibit an oncogenic signature with a significant elevation in expression of iNOS, p-stat1 and p-stat3 and a repression of the tumor suppressor gene, 15-PGDH when compared with WT, p27KO and Smad4KO mice. Thus, our data indicate that p27Kip1 deficiency promotes gastrointestinal epithelial malignancy through both enhancement of the activation of pathogenic T cells and through promotion of the expression of pro-inflammatory mediators by mucosal epithelial cells.
Bacteroides fragilis decrease intestinal Enterobacteriacease colonization and reverse metronidazole-induced bacterial translocation (120.22)
Authors: Chen Lee-Wei , Wu Ying-Ying , Hsu Ching-Mei
Published: January 2023

Bacteroides, one of the major bacterial groups in the intestine, maintain a complex relationship with the host. In this study, the role of B. fragilis in intestinal permeability and immunity were examined in C57BL/6 mice receiving oral treatment of metronidazole for 6 days. Metronidazole treatment significantly decreased the number of B. fragilis but increased that of Enterobacteriaceae in intestinal mucosa and lumen. Metronidazole treatment increased intestinal permeability and pathogenic Klebsiella pneumoniae translocation. However, metronidazole did not change the bacterial killing activity and expression of TLR4, TLR2, MyD88, RegIIIβ, RegIIIγ, CRP-ductin, RELMβ, as well as defensin proteins in intestinal mucosa. Supplementation of dead B. fragilis in drinking water reversed the effects of metronidazole on intestinal permeability, Enterobacteriaceae colonization, and bacterial translocation (BT), suggesting that B. fragilis maintains intestinal permeability and decreases BT through reducing the colonization of Enterobacteriaceae rather than enhancing the expression of antibacterial proteins or the bacterial killing activity. This is further supported by that B. fragilis monoassociated mice showed a decrease of K. pneumoniae translocation and no change in bacterial killing activity as well as expression of defensin and nondefensin protein expression except of CRP-ductin when compared with germ free mice.
Gut-tropic DC precursors contribute to intestinal immune tolerance (120.4)
Authors: Villablanca Eduardo , Torregrosa-Paredes Patricia , Cassani Barbara , Wagers Amy , Mora Rodrigo
Published: January 2023

Gut-associated tolerogenic and inflammatory dendritic cells (DC) develop from at least two different bone marrow (BM)-derived precursors. Tolerogenic, but not inflammatory gut-associated DC, express CD103 and synthesize retinoic acid (RA), which contributes to the induction of Foxp3+ regulatory T cells (TREG). How CD103+ DC and/or precursors are recruited to the intestinal mucosa and then acquire RA synthesizing capacity remains unknown. Here, we show that the BM contains a distinct population of DC precursors expressing the gut-specific α4β7 and CCR9 receptor (gut-tropic pre-DC). Interestingly, vitamin A-depleted mice exhibited a marked decrease in gut-tropic pre-DC in the BM, which correlated with the absence of RA-producing DC in gut-associated tissues. Furthermore, gut-tropic pre-DC lacking β7 integrins exhibited decreased capacity to home to Peyer’s patches and the small bowel lamina propria. Importantly, gut-tropic pre-DC were required to give rise to RA-producing DC in the mesenteric lymph node and to efficiently induce antigen-specific TREG. Consistent with a pro-tolerogenic role of gut-tropic pre-DC, selective depletion of DC derived from β7-expressing cells promoted an inflammatory milieu, as shown by exacerbated inflammation in experimental colitis and by impaired oral immune tolerance. Thus, gut-tropic pre-DC give rise to RA-producing tolerogenic DC in the intestinal mucosa and are required to maintain immune homeostasis during inflammation and steady state
Adaptive immunity impaired by defective lymphocyte motility and activation in mice lacking the actin-bundling protein L-plastin (121.18)
Authors: Morley Sharon Celeste , Todd Elizabeth , Deady Lauren
Published: January 2023

Germinal center development, critical for long-term humoral immunity, requires the migration of T and B lymphocytes to defined locations within and around the B cell follicle. Lymphocyte motility is dependent upon the linkage of extracellular adhesive and chemotactic cues to intracellular changes in the actin cytoskeleton. The molecular mechanisms linking extracellular cues and the actin cytoskeleton are not yet fully defined. We have recently identified the actin-bundling protein L-plastin as a requisite intermediary in both B and T lymphocyte motility. L-plastin is required for establishment of polarity following chemokine stimulation, for the integrin-mediated enhancement of transmigraton, and for the phsophorylation of the integrin-associated kinase Pyk-2. L-plastin is also required for mature synapse formation in T cells. In the absence of L-plastin, cell-intrinsic disruptions of T and B cell motility and activation lead to reduced follicular helper T cell generation, diminished germinal center formation and diminished antibody production to both thymus-dependent and thymus-independent antigens. Probing the specific defects in T and B cell location, activation, and survival in L-plastin-deficient mice will reveal novel insights into critical cell:cell interactions during the germinal center reaction. L-plastin thus serves as a critical bridge between adhesive and chemotactic signals and the morphological changes in actin required for T and B cell function.
Effective detection of phosphorylated Stat5 in FoxP3-positive regulatory T cells (121.24)
Authors: Lee Sun Min , O'Donnell Erika , Haron Jay , Reynolds Sue , Prudencio Joy , Golts Natalie
Published: January 2023

Multicolor flow cytometric analysis is an invaluable tool for analyzing unique cell types within heterogeneous primary cell samples, like peripheral blood mononuclear cells (PBMCs). To identify these specific populations, antibodies against cell surface markers are typically used. However, obtaining greater in-depth knowledge often requires analysis of intracellular events, like phosphorylation of signaling molecules or expression of transcription factors. A challenge to staining cells for both cell surface and intracellular markers is the lack of compatibility of the cell fixation and permeabilization agents with the antibodies required for cell surface and intracellular staining. In this report, we developed an optimized protocol for the simultaneous detection of the cell signaling molecule Stat5 together with transcription factors and cell surface markers. We evaluated human PBMCs for phospho-Stat5 signal induction in Treg cells upon stimulation with IL-2. Using the optimized buffer protocol with antibodies at their optimal concentrations for this intracellular staining condition, we detected the phosphorylation of Stat5 in CD4+/CD25bright/FoxP3+ Treg cells in response to IL-2 stimulation. We also observed differential phosphorylation of Stat5 in various lymphocyte subsets, including memory and naive T cells, when human whole blood was stimulated with IL-2, lysed, fixed, permeabilized, and stained for CD3, CD4, CD45RA, CD45RO, T-bet, and pStat5 expression.
Copper zinc superoxide dismutase 1-deficient mice show increased susceptibility to MOG 35-55-induced EAE (123.12)
Authors: Massilamany Chandirasegaran , Gangaplara Arunakumar , Rathnaiah Govardhan , Steffen David , Lee Jaekwon , Reddy Jay
Published: January 2023

Oxidative stress has been implicated in the pathogenesis of various inflammatory disorders including autoimmune diseases. On the contrary, low levels of reactive oxygen species (ROS) could be beneficial and act as secondary messengers for many physiological processes including cell signaling. The steady state formation of pro-oxidants is normally balanced by their consumption of antioxidants such as superoxide dismutase (SOD), glutathione peroxidase and catalase, thus attaining redox homeostasis. Here we have addressed the role of SOD deficiency in the mediation of central nervous system (CNS) autoimmunity. We demonstrate that copper zinc SOD1-deficient C57BL/6 mice develop more severe EAE induced with myelin oligodendrocyte glycoprotein (MOG) 35-55 as compared to wild type mice. Contrary to the expectations, SOD1-deficient mice showed significant reduction in the expansion of antigen-specific T cells including interferon-γ and interleukin-17A production. Likewise, using major histocompatibility complex class II/IAb MOG 35-55 dextramers, we demonstrate that CNS infiltrates from SOD1-deficient animals contain low frequencies of MOG-specific CD4 T cells. The data suggest that SOD1 deficiency might lead to the elevated levels of ROS and alter EAE severity by modulating T cell responses.
A mouse model of transient diabetes; a new perspective on glucose homeostasis (123.24)
Authors: Girtman Megan , Pavelko Kevin , Pease Larry
Published: January 2023

We have induced acute diabetes following immune attack on the pancreas with islet specific CD8+ T cells, but surprisingly many animals subsequently recovered blood glucose homeostasis. The recovered animals exhibited nearly normal glucose tolerance and produced normal levels of blood insulin following glucose challenge. Remarkably very few islets were present in these animals and those remaining were virtually insulin free. In this model B6 RIP-OVAhi mice receive 100 to 600 naïve OT-1 CD8+ T cells prior to activation by an infection with Theilers murine encephalomyelitis virus encoding the SIINFEKL ovalbumin antigen. When greater numbers of T cells are transferred, this protocol causes acute and fatal diabetes. However, when limiting numbers of T cells are used, animals can recover from the acute phase of the disease. Under these limiting conditions, we find that the mice develop acute pancreatitis by day 5 following infection and stop producing insulin by day 8. By day 11 to 14 some animals begin to recover. Early in the recovery process, insulin positive beta cells are evident in islets. However, late after recovery (days >56) animals that regulate blood sugar normally are found devoid of insulin producing beta cells, though insulin levels are near normal. This raises important questions about how these animals recover glucose homeostasis following cellular immune attack on their islets. Our hypothesis is that insulin is produced outside of the pancreas in these animals.
GILT modulates CD4+ T cell tolerance to cutaneous autoantigen TRP1 (123.32)
Authors: Rausch Matthew , Hastings Karen
Published: January 2023

Maintaining tolerance to skin-restricted antigens is critical for avoiding cutaneous autoimmunity. We have shown that gamma-interferon-inducible lysosomal thiol reductase (GILT) is essential for efficient MHC class II-restricted processing of the melanocyte differentiation antigen, tyrosinase-related protein 1 (TRP1). In GILT-/- TRP1-specific TCR transgenic (Tg) mice, CD4+ TRP1-specific T cells escape thymic deletion, but do not induce autoimmune destruction of melanocytes (vitiligo). GILT-/- Tg mice have fewer peripheral CD4+ TRP1-specific T cells compared to TRP1-deficient mice. CD4+ T cells in GILT-/- Tg mice have marked reduced IL-2 and IFN-γ production and a portion are antigen-experienced. Since tolerance is only partially mediated by an increased percentage of TRP1-specific regulatory T cells in GILT-/- mice, we sought to characterize other mechanisms that constrain the activity of TRP1-specific T cells, such as anergy, exhaustion and deletion. Here, we demonstrate that fewer TRP1-specific T cells were recovered from skin-draining lymph nodes following adoptive transfer of CD4+ T cells from GILT-/- Tg compared to TRP1-deficient Tg mice. Furthermore, TRP1-specific T cells from GILT-/- Tg mice underwent diminished antigen-specific proliferation in vitro compared to T cells from TRP1-deficient Tg mice. These findings suggest multiple peripheral tolerance mechanisms inhibit CD4+ T cell mediated autoimmunity to an endogenously expressed cutaneous autoantigen.
Transaldolase Deficiency Enhances the Development of Experimental Autoimmune Encephalomyelitis (123.46)
Authors: Perl Andras , Bartos Adam , Niland Brian , Fernandez David
Published: January 2023

Transaldolase (TAL) regulates mitochondrial hyperpolarization (MHP), a reversible step of T-cell activation. Here, we investigated the role of TAL in T-cell mediated experimental autoimmune encephalomyelitis (EAE), a mouse model multiple sclerosis. Direct immunization with myelin oligodendrocyte glycoprotein peptide 35-55 (MOG35-55) elicited EAE in TAL-deficient (TAL-/-) mice at an accelerated rate relative to wild-type littermates (TAL+/+; 2-way ANOVA, p=0.0005). Adoptive transfer of MOG35-55 peptide-immunized TAL-/- lymphoblasts triggered EAE in naïve Rag2-/- recipients at a 2.10±0.42 greater higher degree relative to TAL+/+ lymphoblast (p < 0.0001). The increased potency of TAL-/- lymphoblasts was associated with an expansion of CD4+ T cells (62.74±4.48) as compared to TAL+/+ controls (57.34±4.48, p=0.007). In contrast, the frequency of CD4+/CD25+/Foxp3+ regulatory T cells was reduced in TAL-/- mice (2.91 ± 1.02%) relative to TAL+/+ littermates (3.64 ± 1.02%),; p=0.002) In vivo administration of N-acetylcysteine (NAC) reversed the skewing of CD4+ effector and regulatory T cells and ameliorated the EAE disease score in NAC-treated TAL-/- mice (p<0.05). These data identify the metabolic control of MHP by TAL, as a novel checkpoint of T cells lineage specification that affects the expansion of effector and regulatory CD4+ T cells and thus modulates the onset and severity of EAE. This checkpoint is redox-controlled and represents a target for therapeutic intervention with NAC.
Improved identification of leukocyte sub-populations using new bright fluorochrome reagents in multi-color flow cytometric analysis. (124.11)
Authors: Stall Alan , Luan Feng-Jun , Edinger Mark , Jaimes Maria , Elia Jeanne , Chen Yu
Published: January 2023

With many cell surface markers expressed at very low levels the ability to identify (and sort) biologically relevant sub-populations by flow cytometry is often limited by the availability of fluorescent reagents sufficiently bright to resolve these markers. In the past year multiple new fluorochromes have been developed which have significantly improved brightness compared to previously available reagents. In particular, BD Horizon™ PE-CF594 and BD Brilliant Violet™ 421 reagents can be 2-5 times brighter than equivalent PE reagents which have typically been the brightest available. These brighter reagents, when used in multicolor analyses, provide multiple benefits including full resolution of small, dim sub-populations resulting in more accurate enumeration and enrichment via sorting. Data will be presented demonstrating this improved resolution using model systems including human T memory and Treg cell sub-populations and murine CD34+ bone marrow cells.
A proof-of-concept screen for identification of novel natural product modulators involved in mouse Treg/Th17 differentiation by using high-throughput Flow Cytometry (124.2)
Authors: Wang Zichun , Cheng Hongsheng , Shen Yongchun , Hansen Hans , Suh Ted
Published: January 2023

The recent discovery of two novel subsets of CD4+ T lymphocytes (Th17 and Treg) has led to a paradigm shift in the understanding of how autoimmune responses are mediated and regulated. Interestingly, both Th17 and Treg cells can develop from the same naïve CD4+ T cell precursors and in some circumstances they can convert to each other. Several recent publications highlight the role of the aryl hydrocarbon receptor (Ahr) in regulating Treg and Th17 cell differentiation and possible application to human diseases. Depending on the ligands, Ahr activation can control naïve CD4+ T cell differentiation towards either a Treg (TCDD) or a Th17 (FICZ) phenotype. Identifying novel agents that promote or inhibit differentiation of naïve CD4+ T cells to Treg or Th17 could be beneficial in the area of Oncology and autoimmune diseases. Here we describe an assay developed for identification of new modulators based on T cell differentiation by using high-throughput flow cytometry. With TGF-β or TGF-β/IL-6 stimulation isolated mouse naïve CD4 cells can be differentiated efficiently into Treg or Th17 lineages. Treg or Th17-specific expression was detected by intracellular staining of FoxP3 or IL-17, respectively. A proof-of-concept screening against 1600 pure natural products (NPs) was performed and seventeen Th17 specific hits were identified
Plasma ST2 predicts therapy-resistant graft-versus-host disease and survival (126.13)
Authors: Vander Lugt Mark , Braun Thomas , Ferrara James , Hanash Samir , Levine John , Harris Andrew , Couriel Daniel , Paczesny Sophie
Published: January 2023

Graft-versus-host disease (GVHD) is the primary limitation of allogeneic hematopoietic cell transplantation (HCT). Although we have validated a panel of 6 GVHD plasma biomarkers with diagnostic and prognostic value, current blood tests do not predict a patient's response to therapy. We compared plasma from responders (R) and non-responders (NR) using proteomics as a discovery engine, and high-throughput ELISA as validation. Five proteins significantly elevated in NR and 6 validated GVHD markers were measured at therapy initiation in 381 patients with acute GVHD and treated with steroids. In multivariate analysis including the clinical GVHD grade (the strongest clinical predictor), suppressor of tumorigenicity 2 (ST2), the IL33 receptor, significantly predicted response to GVHD therapy. Then, we compared ST2 and grade to predict mortality 180 days post-therapy, we found that patients with high grade and low ST2 had a good prognosis while patients with low grade and high ST2 had a poorer prognosis, suggesting that ST2 provides important prognostic information above the clinical grade. We then analyzed the ability of ST2 measured 14 days post-transplant to predict the occurrence of GVHD and mortality. Patients with high ST2 were 1.4 times more likely to develop GVHD by day 100 post-transplant and 3.2 times more likely to die 180 days post-transplant. Therefore, measurement of ST2 significantly enhances the accuracy of outcome prediction in allogeneic HCT recipients.
Memory T cells are resistant to depletion due to their intrinsic characteristics and anatomical location (126.22)
Authors: Ayasoufi Katayoun , Valujskikh Anna
Published: January 2023

Memory T cells are known to be less sensitive to depletion therapies than naïve T cells. We tested whether anatomical location of memory T cells determines their resistance to depletion. We injected naïve B6 mice with rabbit anti-mouse lymphocyte serum, ALS. Two days after the last injection, more than 95% of CD8 and 60-80% of CD4 T cells were depleted from lung, liver, bone marrow, peripheral blood and spleen, but not from the thymus, with the residual cells being predominantly CD44hi. The in vivo binding of rabbit IgG to T cells in ALS-treated mice did not necessarily correlate with the depletion efficiency. Residual CD4 T cells were isolated from various organs and intravenously injected into groups of ALS-treated or control IgG-treated B6 mice. The distribution of injected cells in the adopted hosts was similar regardless of their origin. The CD4 T cells from lung, liver and thymus were further depleted when relocated into circulation, suggesting that their location is critical in the depletion resistance. The greatest depletion was observed within spleen and liver of the ALS-treated adopted hosts implicating these organs as possible sites of depletion. However, CD4 T cells obtained from BM and spleen were not further affected by relocation suggesting that they are intrinsically resistant to depletion. Understanding the phenotype and functions of depletion-resistant memory T cells from different organs may guide the use of lymphodepletion in transplant patients.
Tracking natural antitumor T cell responses at primary and metastatic tumor sites (127.6)
Authors: Dong Haidong , Liu Xin , Gibbons Rachel
Published: January 2023

Spontaneous antitumor immunity remains challenging to detect and monitor largely due to the absence of established biomarkers. We selected the integrin CD11a as a biomarker to define antitumor T cell responses. CD11a is up-regulated on effector and memory T cells and is involved in the killing of tumor cells by cytotoxic T lymphocytes. In primary (subcutaneously inoculated) and metastatic (intravenously injected) tumors, CD11a high CD8 T cells were detected in the primary (skin) or metastatic (lung) tumor sites, but not in secondary lymphoid organs. Once tumors were established, CD11a high CD8 T cells were identified in tumor draining lymph nodes and peripheral blood. Phenotype analysis revealed that CD11a high CD8 T cells were proliferating (Ki67+) activated T cells (CD62L low, CD69 high, PD-1 high). CD11a high CD8 T cells expressed the transcription factor T-bet but not Foxp3. Live, but not dead, tumor cells, induced expansion of CD11a high CD8 T cells in the lung, suggesting that the access of tumor cells to target tissues is required to induce CD11a high CD8 T cell responses. Our results suggest that CD11a may provide a new T cell biomarker to measure ongoing natural tumor-reactive T cell responses and to predicate the efficacy of tumor immunotherapy.
Antigen reactivity of B cells from patients at risk to develop Multiple Sclerosis demonstrates a specific somatic mutation pattern. (159.28)
Authors: Ligocki Ann , Rounds William , Li Min , Courtney Ardith , Frohman Elliot , Greenberg Benjamin , Stowe Ann , Ward E. , Monson Nancy
Published: January 2023

Multiple Sclerosis (MS) is the leading disease of the central nervous system and a significant, costly cause of disability in young adults. Patients at risk for MS often present with a single demyelinating event, typically called a “clinically isolated syndrome” (CIS). A CIS can occur in the brain, spinal cord (Acute Partial Transverse Myelitis, APTM), or optic nerve (Optic Neuritis, ON). Our laboratory discovered a unique pattern of antibody gene mutation accumulation (i.e. antibody gene signature, or “AGS”) that is exclusive to B cells from the cerebrospinal fluid (CSF) and brain lesions of MS patients and CIS patients that develop MS in the future. Our goal for this study was to determine whether CIS and MS patient B cells that carry this pattern of somatic hypermutation accumulation in their antibody genes react to brain tissue. Thirty-four antibody genes of B cells isolated from the CSF of MS and CIS patients carrying this AGS pattern were cloned into antibody expression vectors to generate recombinant full-length human antibodies (rhAbs). Preliminary DAB staining using a subgroup of these rhAbs on cryo-sectioned mouse brain demonstrates binding to CNS tissue. Cortical staining of varied intensities was found among the rhAbs tested. Patterns of staining and details of antigen reactivity will be presented. This result provides promise in continuing to identify the auto-antigens that are recognized by the AGS B cells shared among patients at risk to convert to MS.
Interferon beta-1a modulation of B cells (161.12)
Authors: Huang Hui , Ito Kouichi , Dangond Fernando , Dhib-Jalbut Suhayl
Published: January 2023

Clinical studies have shown that IFNβ-1a reduces relapses and slows disease progression in relapsing multiple sclerosis (MS). Although the effect of IFNβ-1a on T cells has been extensively investigated, its effects on B cells are poorly understood. We examined the effect of IFNβ-1a on IL-10 production in B cells. B cells isolated from healthy donors were cultured with IFNβ-1a, and production of IL-10 was measured. IFNβ-1a enhanced IL-10 production in B cells in a dose dependent fashion. We also found that IFNβ-1a treatment increased the percentage of B-cells expressing B7.2 (CD86). To examine how IFNβ-1a mediated increase of B7.2 affects the proliferation of CD4+ T cells, CD4+ T cells were cultured with IFNβ-1a-treated or non-treated B cells, with or without anti-CD3. Anti-CD3 mAb-mediated T-cell proliferation was partially suppressed when B cells were treated with IFNβ-1a. Addition of anti-CTLA-4 mAb partially reversed the IFNβ-1a-mediated suppression of T-cell proliferation. These data suggest that IFNβ-1a-induced increase of B7.2 in B-cells may reduce the proliferation of CD4+ T cells through increased interaction of B7.2 with CTLA-4. In conclusion, in in vitro experiments, IFNβ-1a increases IL-10 production in B cells and can indirectly inhibit T-cell proliferation through up-regulation of B7.2 on B cells. These findings shed light on the mechanism of action of IFNβ-1a in MS.
Increase in natural killer T (NKT) Cell activation in hypoxic environments via the induction of HIF1alpha (162.16)
Authors: East James , Travis Candice , Webb Tonya
Published: January 2023

Tumors often experience hypoxic microenvironments due to poor vascular access in the early stage of neoplastic growth and continue to experience oxygen deficits in late stage progression despite the increase in neovascularization to the primary tumor or metastasis. This persistent hypoxic state selects for tumors cells that are able to compensate for low oxygen levels by increasing use of the glycolytic cycle under aerobic conditions (The Warburg Effect), suppressing apoptosis and inducing decreased genomic stability. These effects have been shown to be mediated, at least in part, by the transcription factor hypoxia inducible factor (HIF)-1alpha. Natural Killer T (NKT) cells are a subset of T cells that recognize a wide range of lipid antigens presented by the MHC Class I-like CD1d molecule. In this study, we sought to investigate the effects of hypoxia induction on CD1d-mediated NKT cell responses. Pharmacological agents were used to induce HIF-1a in LCD1d, and then we assessed their ability to present endogenous antigen to NKT cells. It was found that the induction of HIF-1a resulted in a significant increase in CD1d-mediated NKT cell activation, as assessed by IL-2, GM-CSF, and IL-13 production. The increase in NKT function was not due to with an increase in cell surface expression of CD1d. These results suggest that hypoxia in tumor cells may alter the repertoire of endogenous lipids presented in the context of CD1d which permits their recognition by NKT cells.
Ym1 is a stem cell secreted factor that augments NK cell killing of tumor cells (162.31)
Authors: Manzar Gohar , Kim Eun-Mi , Tabayoyong WIlliam , Zavazava Nicholas
Published: January 2023

We previously observed that embryonic stem cell-derived hematopoietic progenitor cells (HPCs) were resistant to NK cells in vitro despite their poor expression of MHC antigens. In contrast, HPCs were readily deleted by NK cells in vivo. This discrepancy led us to study protein expression by HPCs pre and post-transplantation using proteomics. The rationale was that in response to serum cytokines in vivo, HPCs likely upregulated certain proteins that enhanced NK cell cytotoxicity. Upon sequencing, most of these proteins were determined to be housekeeping proteins except for one protein, which we identified as Ym1, also known as chitinase 3-like protein 3. Subsequently, recombinant Ym1 was generated in E. coli. Ym1 strongly enhanced NK cell cytotoxicity to HPCs in vitro by activating NK cells into the highly cytotoxic Mac1hi subsets. This process was APC-dependent, suggesting cross-presentation of IL-15 to NK cells by APCs. In vivo, Ym1 induced the emergence of mature CD27loMac1hi peripheral blood NK cells, which have been found to be most effective at lysing tumor cells. Further, Ym1 caused a significant increase in the production of IFN-γ, perforin and granzyme B. Indeed, Ym1 remarkably enhanced target cell killing by 3-4 fold, which is a novel finding that has promising implications for the application of Ym1 in cancer treatment.
Enhancement of NK cell activity and Th1 immunity in healthy subjects by orally administered fucoidan mix. (162.35)
Authors: Miyazaki Yoshiyuki , Nakamizo Masaji , Tsuji Hiromitsu , Kirino Tomomi , Saito Yuzu , Kawahara Kazuhiro , Otsuka Ken , Tachibana Hirofumi , Yamada Koji , Fujii Makoto , Tachikawa Daisuke
Published: January 2023

Fucoidan is a sulfated polysaccharide derived from brown seaweeds which mainly consist of L-fucose. In Asian countries such as Japan, Korea and China, peoples eat seaweeds willingly as nourishing health foods. We previously showed that the tumor formation by transplanted Sarcoma180 cells was suppressed in mice fed 2% fucoidan mix containing diets, and NK cell activity was enhanced in the fucoidan-fed mice. The results suggest that the activated NK cells participated in elimination of the tumor. However, in human, immune enhancing activities of fucoidan have hardly been proved. Therefore, we performed an open-label trial in this study. Five healthy volunteers (47 ± 6 years old, 2 male and 3 female) took 2.5 g fucoidan mix per day for 30 days. There were no abnormal observations in the values of WBC, RBC, platelet count and blood profile. On the other hand, NK cell activity was augmented by 1.42 times in the average at end of the trial than the initial value. The ratio of IFN-gamma-producing (Th1) cells also increased to 114% for the initial value by the day 30 by the fucoidan mix intake, whereas IL-4-producing (Th2) cells decreased to 77%. As a result, the rise of Th1/Th2 ratio by 1.46 times in the average was observed with 4 subjects among 5 examinees. Furthermore, CD8+ cells tended to expand with the all subjects. These results suggest that intake of fucoidan is effective to augment anti-tumor immunity in human.
Hematopoietic progenitor kinase 1 (HPK1) influences regulatory T cell functions (163.26)
Authors: Sawasdikosol Sansana , Alzabin Saba , Cary Chris , Zha Ren Yuan , Burakoff Steven
Published: January 2023

HPK1 is a negative regulator of TCR-initiated signal transduction. HPK1-/- mice are more susceptible to experimental autoimmune encephalomyelitis, suggesting that the ability to enforce peripheral tolerance may have been compromised. Paradoxically, we observed that more natural regulatory T cells were found in the periphery of HPK1-/- mice than in wild type C57BL/6. We determined whether HPK1-/- Foxp3+ Tregs retained their ability to suppress TCR-induced effector T cell proliferation and found that they lack the ability to suppress. Because the ability to suppress T cell proliferation is a function conferred to Tregs by Foxp3-dependent suppression of IL-2 transcription, we assessed whether HPK1-/- Foxp3+ Tregs retain the ability to repress TCR-induced IL-2 production. We found that HPK1-/- Tregs produce a significant amount of IL-2 in response to TCR engagement, suggesting that Foxp3 is unable to carry out transcriptional repression. This aberrant phenotype implicates HPK1 as an important regulator of Treg functions.
IL-15 restores defective NK anti-fungal activity in a primary Hemophagocytic lymphohistiocytosis Patient (164.1)
Authors: Li Shushun , Kyei Stephen , Wright Nicola , Mody Christopher
Published: January 2023

Hemophagocytic lymphohistiocytosis (HLH) is a rare yet potentially fatal clinical syndrome caused by various congenital defects that result in defective perforin-dependent cytotoxicity of NK cells. This leads to increased T cell activation and proliferation and excessive cytokine production including IL-1, IL-6, IFN-γ, TNF-α and GM-CSF that causes sustained activation of macrophages that is manifest as extensive inflammation and tissue damage. One study showed that 50% of deaths from familial HLH were due to invasive fungal infections. We sought to investigate whether NK cells from a patient with HLH have defective anti-fungal activity and seek a potential therapeutic approach. NK cells (CD56+/CD3-) were isolated from a two-month-old boy with c1090-1091del of the perforin gene resulting in p.Leu364>Stop consistent with the FHL-2 form of HLH. Defective NK killing was observed for both Cryptococcus neoformans and Candida albicans. Remarkably, the defective anti-Candida activity of NK cells could be restored within 24 hours by IL-15 treatment that was similar to the enhanced anti-Candida activity of NK cells from healthy donors. We speculate that IL-15 enhances antifungal activity by stimulating NK cells to employ an alternate microbicidal effector mechanism. This result is the first to show that IL-15 can restore defective cytotoxic activity of NK cells from an HLH patient, and further studies might be considered to explore the mechanism and potential form of immunotherapy.