bluuebunny/crossref_metadata_2025 huggingface dataset:
Neutrophil recruitment to the lymph node in response to S. aureus infection
Authors: Zuba Ania M , Kubes Paul
Published: January 2023

Bacterial skin infections can be cleared and pose no immediate threat to the host. However, if a bacterial infection is not contained and disseminates into the bloodstream, this can lead to system-wide bacteremia and death. Lymph nodes along lymphatic vessels may act as barriers by filtering the lymphatic fluid. This barrier is not merely passive, but is actively modulated in response to inflammation. In tissues, neutrophils are extremely important in controlling bacterial infections and are robustly recruited to the lymph node upon infection with pathogens at a distant site. Intriguingly, how the neutrophils reach the lymph node and their role upon arrival is only beginning to be understood. Neutrophil recruitment to the popliteal lymph node occurs within 2 hours of Staphylococcus aureus infection in the footpad, plateauing at 6 hours. Using intravital multiphoton imaging and the Lysm eGFP reporter mouse we have visualized the behaviour of neutrophils in lymph node blood vessels and in the lymph node tissue. Neutrophils accumulate within wide blood vessels in the lymph node, exhibiting rolling, adhesion, crawling and transmigration from vessel lumen into lymph node tissue. No neutrophils can be seen in afferent lymphatics. Imaging of the subcapsular sinus shows neutrophil accumulation and swarming as well as neutrophils actively phagocytosing S. aureus. This suggests neutrophils are being quickly recruited to the lymph node to prevent dissemination from an initial site of infection via lymphatics. This project aims to define the role of neutrophils as cellular barriers in preventing dissemination of bacteria via lymphatics.
Distinct T cell signatures define subsets of multiple sclerosis patients
Authors: Johnson Mark C , Pierson Emily R , Spieker Andrew J , Nielsen A. Scott , Posso Sylvia , Kita Mariko , Buckner Jane H , Goverman Joan M
Published: January 2023

Studies of the frequencies of myelin-specific T cells in peripheral blood mononuclear cells (PBMCs) from MS patients compared to healthy controls (HCs) have varied in their results, but no previous work has considered the potential influence of lesion localization on these frequencies. While the majority of relapse-remitting MS (RRMS) patients have lesions localized predominantly in the brain, a subset of RRMS patients (~5%) has neuroinflammation localized predominantly in the spinal cord. Importantly, spinal cord localized lesions are associated with a worse prognosis for patients due to their effect on motor function. Animal models of MS suggest that distinct T cell effector functions correlate with brain versus spinal cord lesions. Therefore, we analyzed PBMCs from MS patients with lesions predominantly in the brain or predominantly in the spinal cord and HCs using ELISPOT to assess the frequency of IFN-γ+ and IL-17+ cells responding to two myelin antigens, myelin basic protein (MBP) and myelin oligodendrocyte glycoprotein (MOG). Significant differences in T cell frequencies were determined in some, but not all, comparisons, with MBP-specific IFN-γ+ frequencies showing the greatest separation between our three groups. Strikingly, combining the IFN-γ+ and IL-17+ responses to both MBP and MOG using logistic regression defined distinct T cell signatures that distinguished MS patients with predominantly brain versus predominantly spinal cord lesions. Collectively, this suggests that different myelin-specific T cell responses may influence neuroinflammatory patterns in RRMS patients. Such patterns of peripheral T cell responses may be useful in generating more tailored therapies for patients with MS.
Enhanced IFN-γ signals and STAT5-phosphatase dependent attenuated IL-2 signals in the pathogenesis of rheumatoid arthritis.
Authors: Boland Molly , Vinod Surabhi , Bridges Louis , Raman Chander
Published: January 2023

The importance of type I interferons as a driver of pathogenesis in autoimmunity, including rheumatoid arthritis (RA) is well recognized. However, in this context the contribution of type II interferons (IFN-γ) has been equivocal. This study developed from our recent discovery that the expression of IFN-γ receptor gene Ifngr1 strongly associates with RA disease and expression of Ifngr2 associates with radiographic severity. In this study we show that IFN-γ induced activation is significantly greater in naïve, central memory, Tfh and Treg subsets of CD4+ T cell populations from RA patients compared to healthy controls (HC). Remarkably, such enhanced IFN-γ induced STAT1 activation was not observed in CD8 T cell populations from RA patients. The activation of STAT1 and consequently, IFN-γ signals are regulated by tyrosine phosphatases. Treatment with phenyl-arsine oxide (PAO), a broadly active phosphatase inhibitor, had no effect on IFN-γ induced STAT1 activation in any T cell population from RA or HC. In contrast, such treatment with PAO greatly enhanced IL-2 induced activation of STAT5 in RA CD4 T cell populations (naïve, central memory effector memory, Treg, Tfh) but not from HC or in CD8 T cell populations. This result suggests that the regulatory activity of IL-2 in RA is dampened by a STAT5-specific phosphatase. We further determined that such a STAT5-phosphatase was also expressed and active in regulatory and effector CD4 T cell populations from the spontaneously autoimmune BXD2 mice. Overall our results indicate that CD4 T cell subpopulation dependent enhanced IFN-γ signals and dampened IL2-STAT5 signals contributes to pathogenesis in RA.
Unveiling the specificity of islet-antigen specific regulatory T cells in T1D patient using single cell deep-sequencing
Authors: Xu Daqi , Dong Shen , Martínez-Llordella Marc , Chopra Gaurav , Bluestone Jeffrey
Published: January 2023

CD4+CD25hiCD127loFoxp3+ T Regulatory cells (Tregs) are critical in controlling autoreactive T cells. The deficiency of functional Tregs is associated with Type 1 diabetes (T1D) and many other autoimmune diseases. In addition, antigen-specific Tregs suppresses more efficiently compared to polyclonal Tregs in mouse models. Thus, identifying the antigen specificity of Tregs, especially at the site of inflammation, is critical to improve our understanding of Treg deficiencies in T1D and may lead to clinically applicable advances. However, the Treg TCRs recognizing self-antigens in autoimmune diseases are less clarified due to the rare frequency and the anergic status of Treg in vitro. In this study, by the combination of sub-culturing, polyclonal stimulation and antigen stimulation, we can identify islet-antigen specific Tregs from both PBMC and pancreatic draining lymph node of T1D patients using antigen-driven Treg proliferation assay we developed in our lab. The majority of islet antigen-specific Tregs is CD45RO+Tregs compared to the CD45RA+ counterpart. Importantly, we confirmed the enrichment and identified the specificity of the islet antigen-specific Tregs using single cell deep-sequencing. In the future, we will further characterize the uncovered Treg TCR and the phenotype of the identified islet antigen-specific Tregs via comparison between healthy and T1D subjects.
Dysfunction and depletion of human antigen-specific CD4 T cells by HIV: a mechanism for pathogen-specific immune failure in AIDS
Authors: Hu Haitao , Liu Fengliang , Fan Xiuzhen , Ferguson Monique , Sun Jiaren , Soong Lynn
Published: January 2023

HIV infection causes dysfunction and progressive depletion of human CD4 T cells, leading to enhanced host susceptibility to opportunistic infections (OIs). However, loss of immune control over different OIs occurs at different stages of HIV disease in patients; while CMV infection usually causes active disease at late stage with low CD4 counts, mucosal candidiasis caused by fungal pathogen C. albicans can become evident earlier at fairly high CD4 counts. The underlying mechanism is not well defined. We have established an in vitro system to model the interactions between HIV and pathogen-specific CD4 T-cell populations, and have shown that C. albicans-specific CD4 T cells are more permissive to HIV than CMV-specific cells in vitro. We further examined the in vivo impact of HIV on these two groups of pathogen-specific CD4 T cells in HIV-infected patients and the functional parameters involved. We found that compared to CMV-specific CD4 T cells, which manifest a polarized Th1 phenotype, C. albicans-specific CD4 T cells demonstrate mixed Th17-Th1 phenotype, producing high levels of IL-22, IL-17 and IL-2, in addition to IFN-γ and MIP-1β. The Th17 functional subsets (IL-22, IL-17 and IL-2) of C. albicans-specific CD4 T cells are more permissive to HIV in vitro and manifest an earlier impairment in vivo in HIV-infected individuals. Longitudinal analysis of infected subjects with ongoing CD4 depletion showed that C. albicans-specific CD4 response was preferentially and progressively depleted. These data altogether provide compelling evidence for differential susceptibility of pathogen-specific CD4 T cells to HIV infection and dysfunction, suggesting a mechanisms for pathogen-specific immune failure in AIDS.
IL-4 signaling via STAT6 induces human resistin expression
Authors: Batugedara Hashini Maneesha , Jang Jessica C , Patel Jay J , Nair Meera G
Published: January 2023

Human resistin (hRetn) has been implicated as a detrimental protein during helminth infection, where it impairs helminth expulsion. Current literature shows that LPS signaling via NFkB can promote hRetn expression. Promoter analysis of the hRetn gene revealed both NFκB and STAT6 transcription factor binding sites. This suggested that hRetn is not only regulated by TLR ligands but also by Th2 cytokines. Accordingly, we tested the ability of various TLR ligands and IL-4 to stimulate hRetn in bone marrow-derived macrophages (BMDM) from hRetn transgenic (Tg+) mice. We found that LPS and IL-4 stimulation significantly increased hRetn. Further, IL-4-induced hRetn expression was abrogated when BMDM were treated with a STAT6 inhibitor, confirming STAT6 dependent hRetn expression. We tested the effects of IL-4 in vivo by administering IL-4 complexes (IL-4c) intratracheally and intraperitoneally to hRetn Tg+ mice. We observed local and systemic IL-4-induced hRetn mRNA in blood cells, peritoneal exudate cells and the whole lung. To elucidate the relevance of these findings in humans, we stimulated human neutrophils, monocytes and peripheral blood mononuclear cells (PBMC) with IL-4 and discovered that neutrophils produced the most hRetn. Similar to mouse BMDM, the IL-4-induced hRetn in human PBMCs was blocked by STAT6 inhibitors. Collectively, these data suggest that IL-4 signaling via STAT6 induces hRetn and these results are supportive of a novel signaling pathway for hRetn. Future studies will investigate the functional relevance of STAT6 regulation of hRetn, which could provide new insights into controlling the outcome of several Th2 inflammatory diseases such as helminth infection and asthma.
Intracerebral Mycobacterium tuberculosis infection-induced immune response in the central nervous system
Authors: Hernandez Gianna L , Ritter Anna , Mergaert Aisha , Rayasam Aditya , Gerhart Christian , Sandor Matyas , Fabry Zsuzsanna
Published: January 2023

The World Health Organization declared a tuberculosis (TB) global emergency. Central nervous system (CNS) TB is a rare, highly devastating manifestation of extra-pulmonary TB. To date, research has focused on pulmonary TB, with no significant advancements being made in understanding the mechanisms associated with CNS TB. This study uses a small animal model of CNS TB that permits us to investigate the immune responses following CNS Mycobacterium tuberculosis (Mtb) infection. We use intracranial infection of mice with the H37Rv strain of Mtb which has been fluorescently tagged with a TdTomato plasmid, allowing us to track it in vivo, and analyzed the cellular composition of the subsequent immunological response in the brain. At 7 days post infection (PI), we show a robust infiltration of immune cells into the CNS including the infiltration of CD11c+ dendritic cells. We can also see Mtb-containing granuloma-like lesions. We adoptively transferred Mtb-specific P25 CD4+ T and found that these cells also accumulate in the brain and were found activated in the peripheral lymphoid tissue, including the spleen and cervical draining lymph nodes. The P25 T cells present in the brain display extensive proliferation. Moreover, P25 T cells present in the peripheral lymphoid organs of Mtb infected mice show increased proliferation when compared to non-infected controls suggesting a systemic sampling of CNS Mtb antigen. We also show that at 7 days PI there is a significantly higher percentage of activated, INFγ producing, CD4+ T in the brain. Both INFγ and IL-17 producing T cell populations increase in size by day 21. This study will provide knowledge into the pathogenesis of CNS TB, and provide possible treatment strategies to lessen its destructive outcome.
SLAM family receptors 2B4 and CD48 are key drivers of CD8 T cell priming by B cells
Authors: Huang Yu-Hsuan , Tsai Kevin , Kang Sohyeong , Wang Xiaoxia , Ford Mandy L , Harder Kenneth W , Tan Rusung , Priatel John J
Published: January 2023

Mutations in SH2D1A gene that encodes SAP (SLAM-associated protein) result in X-linked lymphoproliferative disease (XLP), a rare primary immunodeficiency defined by exquisite sensitivity to the B-lymphotropic Epstein-Barr virus (EBV) and B cell lymphomas. However, the precise mechanism of how the loss of SAP function contributes to extreme vulnerability to EBV and the development of B cell lymphomas remains unclear. Here, we utilized SAP-deficient (Sh2d1a−/−) mice to investigate the hypothesis that SAP is critical for CD8 T cell immune surveillance of antigen-expressing B cells or B lymphoma cells. Sh2d1a−/− CD8 T cells exhibited greatly diminished proliferation relative to wild type when antigen-presenting-B cells or -B lymphoma cells served as the primary APC. By contrast, Sh2d1a−/− CD8 T cells responded equivalently to wild type CD8 T cells when B cell-depleted splenocytes, melanoma cells or breast carcinoma cells performed antigen presentation. Through application of SLAM family receptor blocking antibodies or SLAM family receptor-deficient CD8 T cells and APCs, we found that CD48 engagement on the B cell surface by 2B4 is crucial for initiating SAP-dependent signaling required for the antigen-driven CD8 T cell proliferation and differentiation. Altogether, a pivotal role for SAP in promoting the expansion and differentiation of B cell-primed viral-specific naïve CD8 T cells may explain the selective immune deficiency of XLP patients to EBV and B cell lymphomas.
ECM remodelling by ADAMTS5 regulates influenza-specific immunity following acute infection
Authors: Stambas John , Ye Siying , Izzard Leonard , Tripp Ralph A , McCulloch Daniel , McMahon Meagan
Published: January 2023

Members of the ADAMTS (A disintegrin-like and metalloproteinase with thrombospondin-1 motifs) metalloproteinase family remodel the extracellular matrix (ECM) to facilitate immune cell migration and cell adhesion. In this current study, we infected Adamts5−/− mice with X31 influenza virus (104pfu/mouse)and measured weight loss, viraemia and T cell immunity following acute infection. Adamts5−/− mice showed greater weight loss and increased viraemia when compared to WT counterparts. Furthermore, we observed reduced NP366 and PA224 influenza specific CD8+ T cells in the spleen and lungs of Adamts5−/− mice with a corresponding accumulation of these T cell specificities in the mediastinal lymph node (MLN) 10 days post infection. Critically, these responses correlated with increased expression of a key ADAMTS5 substrate; versican, in the MLNs of Adamts5−/− mice. We therefore hypothesize that versican accumulation in the MLNs of Adamts5−/− mice results in T cell accumulation and that ADAMTS5 is a key enzyme involved in facilitating T cell migration from the MLN to the periphery following acute influenza virus infection.
The role of bone marrow promiscuous gene expression in peripheral tolerance
Authors: Chen Chih-Yeh , Klein Michelle , Klug Felix , Pinto Sheena , Kyewski Bruno , Beckhove Philipp
Published: January 2023

For the negative selection of autoreactive T cells, peripheral tissue-restricted antigens (TRAs) are ectopically expressed and presented by medullary thymic epithelial cells (mTECs). This physiological property of mTECs, termed promiscuous gene expression (pGE), is predominantly regulated by the transcription factor, autoimmune regulator (AIRE). In the periphery, pGE was also found in secondary lymphoid organs and can induce peripheral immune tolerance. Bone marrow is an important organ known to prime naïve conventional T cells, as well as being a reservoir of regulatory T cells. However, the occurrence of pGE in the bone marrow is hitherto unknown. We hypothesize that a repertoire of auto-antigens is ectopically expressed and presented in the bone marrow, which maintains the peripheral tolerance of T cells. We investigated the presence of AIRE-expressing cells in bone marrow using a transgenic reporter mouse model in which GFP is expressed under the control of the AIRE promoter. AIRE expression was detected at both the RNA and protein levels. The bone marrow-resident AIRE-expressing cells (BMACs) anatomically locate adjacent to clusters of CD4+ T cells and express high levels of CD45, MHC-II and EpCAM. Their tolerogenic characteristic was conferred by the expression of PD-L1 and lack of costimulatory molecules CD80 and CD86. Gene expression analysis showed the AIRE-dependent TRA genes expressed in BMACs differ from those expressed in mTECs. BMACs were also detected at various frequencies in patients carrying different tumor entities. Our result demonstrates that the AIRE-expressing cells which ectopically express TRAs in the bone marrow could comprise the cellular basis for the induction of peripheral T-cell tolerance.
TGF-β1 produced by alternatively activated macrophages synchronizes a tolerogenic iTreg-Th17 cell axis
Authors: Haribhai Dipica , Ziegelbauer Jennifer , Williams Calvin B
Published: January 2023

Induced regulatory T (iTreg) cells and T helper 17 (Th17) cells develop at mucosal interfaces. While iTreg cells are required for the maintenance of mucosal tolerance, Th17 cells can play divergent roles. Both iTreg and Th17 cells require TGF-β1 for their differentiation. Importantly, the primary source of TGF-β1 in vivo is unknown. Here, we investigate the mechanisms that control iTreg and Th17 cell development. We hypothesize that alternatively activated M2a macrophages support mucosal tolerance through TGF-β1 mediated expansion of both iTreg and Th17 cells.

We pretreat Rag1­­−/− C57BL/6 mice prior to colitis induction or treat mice with established disease with polarized macrophages. In some experiments the macrophages lacked the capacity to produce TGF-β1. Weight loss, colitis disease scores, and FACS analysis of mesenteric lymph nodes and gut infiltrates were used to evaluate the impact of macrophage therapy on the expansion and function of the iTreg-Th17 cell axis.

Pre-conditioning mice with M2a but not M1 macrophages resulted in a 16-fold increase in the number of iTreg cells and a 8-fold increase in the number of Th17 cells when compared to control mice. This expansion required TGF-β1 produced by both the transferred M2a cells and by host myeloid cells. Established colitis was successfully treated by concomitant M2a and nTreg cell transfers, but not by nTreg cell transfers alone.

In conclusion, M2a macrophages that produce TGF-β1 regulate development of a tolerogenic iTreg-Th17 cell axis. The change in iTreg and Th17 cells numbers linked to M2a macrophage polarization is directionally concordant and dependent on T cell produced IL17A, confirming that both iTreg and Th17 cells are required to mediate intestinal homeostasis.
NFIL3 and Ets1 co-regulate a cluster of genes, including Tim-3, that is associated with T cell tolerance
Authors: Zhu Chen , Gu Guangxiang , Zhang Huiyuan , Xiao Sheng , Wang Chao , Xiong Huabao , Garrett-Sinha Lee Ann , Ho I-Cheng , Kuchroo Vijay
Published: January 2023

Inhibitory receptor Tim-3 has critical roles in the suppression of effector T cell function. Here we demonstrate that the expression of Tim-3 is highly associated with T cell tolerance induction. TCR other than CD28 co-stimulatory signal is required for its induction. This process is accompanied with inhibition of IL-2 but upregulation of IL-10. Among the downstream pathways of TCR signaling, the calcium-calmodulin-dependent Kinase II (CaMKII) pathway is essential for Tim-3, IL-10 induction and IL-2 inhibition. CaMKII directly regulates transcription factors Ets1 and NFIL3. While NFIL3 induces Tim-3, IL-10 and suppresses IL-2 expression, Ets1 plays an opposite role for their expression. Accordingly, functional analyses suggest that NFIL3 is required, but Ets1 actually has an opposite role, in maintaining T cell tolerance/anergy. ChIP-Seq results reveal that NFIL3 and Ets1 co-localize in the loci of Tim-3, IL-2 and IL-10, suggesting a functional cooperation between NFIL3 and Ets1 in the regulation of these genes. We further identified a cluster of genes that has direct binding with both NFIL3 and Ets1. RNA-seq analysis reveals that the expression of these genes is associated with ionomycin-mediated T cell anergy induction. Collectively, our study suggests that NFIL3 and Ets1 cooperatively regulate a transcriptional network to control the expression of a cluster of genes, including Tim-3, IL-10, and IL-2, that is functionally important for T cell tolerance/anergy induction.
Separating RORgammat function in Th17 differentiation and T cell development
Authors: Sun Zuoming , Ma Jian , He Zhiheng
Published: January 2023

RORgammat regulates two equally important T cell function: Th17 differentiation and development of thymocytes, and lymph node genesis. By ex vitro structure-function analysis, we identified two amino acid mutations in RORgammat (RORgammatM) that specifically disrupt Th17 differentiation but allow normal T cell development. Mice expressing RORgammatM lack Th17 differentiation and resist to Th17-mediated autoimmune EAE similar to RORgammat−/− mice, however, have almost normal thymic T cell development and lymph node genesis. RORgammatM mice are thus a better model for specifically evaluating RORgammat-regulated Th17 immunity in vivo. Global analysis of RORgammat-DNA binding sites and target genes demonstrated that RORgammatM selectively affected the DNA binding and activating critical RORgammat target genes in Th17 cells. Therefore, our study distinguishes two critical RORgammat functions, which will facilitate the development of RORgammat-based treatments that selectively target Th17-mediated autoimmunity while avoid interfering with RORgammat-regulated development of T cells and lymph nodes.
c-FLIP suppresses p38 MAPK signaling and inhibits dendritic cell activation
Authors: Lai Ming-Zong , Wu Yu-Jung , Wu Yung-Hsuan , Mo Shu-Ting , He You-Wen
Published: January 2023

c-FLIP is reported to transmit activation signals but the nature of the signaling remains controversial. Here we report a novel function of c-FLIP involving inhibition of myeloid cell activation. Conditional knockout of c-FLIP in dendritic cells (DCs) led to neutrophilia and splenomegaly. Peripheral dendritic cell and macrophage populations were not affected by c-FLIP deficiency in dendritic cells. c-FLIP-knockout conventional DC (cDC), plasmacytoid DC (pDC), and bone marrow-derived DC (BMDC) displayed enhanced production of TNF-α, IL-2, or G-CSF in response to stimulation of TLR2, TLR4, and dectin-1. The enhanced activation of c-FLIP-deficient BMDCs could be partly linked to an elevated activation of p38 MAPK. In contrast, the activation of ERK, JNK, and NF-κB was not affected by c-FLIP-deficiency in BMDCs. In addition, the increased activation in c-FLIP-deficient dendritic cells was independent of caspase-8. Our results reveal a novel inhibitory role of c-FLIP in myeloid cell activation and demonstrate the unexpected anti-inflammatory activity of c-FLIP. In addition, our observations suggest that cancer therapy target c-FLIP downregulation may facilitate DC activation and increase T cell immunity.
Signal regulatory protein α suppresses macrophage inflammatory responses via reducing PKM2 tyrosine phosphorylation
Authors: Zhu Dihan , Pan Chaoyun , Shi Lei , Bian Zhen , Liu Yuan , Ke Zen
Published: January 2023

Signal regulatory protein α (SIRPα) plays an important part to restrain macrophage chemotaxis, secretion and phagocytosis. However, the signals involved in SIRPα-mediated control of macrophage inflammatory responses are poorly defined. Here we report that the inhibitory effect of SIRPα is mainly due to its suppression on macrophage aerobic glycolysis (Warburg effect). Upon ligation by CD47, SIRPα recruits/activates SHP-1 to dephosphorylate pyruvate kinase M2 (PKM2), a rate limiting enzyme of glycolysis. Compared to wild-type mice, SIRPα-deficiency mice (SIRPα−/−) develop a faster and severer macrophage-mediated insulitis induced by multiple low-dose streptozotocin. SIRPα−/− macrophages are M1-preferential and have higher level of phosphorylated PKM2 (p-PKM2) compared to their WT littermates. In addition, activation of macrophages to M1 phenotype, accompanying with reduction of SIRPα but increase of p-PKM2 and aerobic glycolysis level, can be abolished by SIRPα overexpression, as well as glycolysis inhibitors. Overexpression of SIRPα also strongly reduces the levels of macrophage aerobic glycolysis and p-PKM2. Finally, substrate-trapping and mutagenesis study clearly shows the binding of p-PKM2 with SIRPα-recruited SHP-1 complex and identifies PKM2 Tyr105 as a key site mediating SHP-1-PKM2 interaction. In summary, our findings identify SIRPα as a critical molecule that controls both metabolic switch and inflammatory responses in macrophages
Cell culture oxygen and signaling: Supraphysiologic in vitro oxygen signals through the HIF-CXCR4 pathway in human B cells
Authors: Henn Alicia D. , Hilchey Shannon P. , Garigen Jessica , Cipolla Valentina , Perry Sheldon , Farovitch Lorne , Yerden Randy , Zand Martin Stuart
Published: January 2023

Current understanding of B lymphocyte function relies heavily upon in vitro assays. However, standard incubators maintain cells at supraphysiologic oxygen (16–19% oxygen). Physiologic oxygen is far lower (blood 5–10%, secondary lymphoid tissues 1–5%, regions of inflammation, tumor, and bone marrow 0–1%). Previously, we showed that transcripts for hypoxia-induced factor 1 alpha (HIF-1a), an oxygen-sensing transcription factor, are upregulated in peripheral B cells after vaccination1. HIF-1a is also a therapeutic target in multiple myeloma2. Downstream of HIF-1a are genes tied to metabolism, proliferation, and the unfolded protein response. One gene downstream of HIF1a that is critical for B cell migration and function is CXCR4. We set out to determine if room air incubation might affect B cells in vitro, our null hypothesis being that physiologic oxygen would not affect B cell HIF-1a levels or CXCR4 function. We used C-chamber incubator sub-chambers and Pro-Ox 110 gas controllers to control the incubation oxygen and carbon dioxide levels. Setting the reference frame to a range relevant to secondary lymphoid organs (1–5% oxygen), we set the chambers to 5% CO2 and 1% (hypoxic), 4% (physioxic), or 19% (supraphysioxic) oxygen. In primary human B cells and myeloma cell lines, HIF-1a protein levels stabilized at 24 hrs of culture at 1% and 4% oxygen, but not at 19% oxygen. More importantly, B cell migration in response to the CXCR4 ligand, CXCL12, was significantly decreased at low oxygen levels, without affecting cell proliferation or viability, disproving our null hypothesis. This has widespread implications for assessments of B cell function in that incubator oxygen drives HIF-1a degradation and CXCR4 responsiveness in vitro.
Adhesion protein Fibulin-7 (Fbln7) regulate differentiation and polarisation of human monocytes and macrophages.
Authors: Sarangi Pranita P , Chakraborty Papiya , Wahl Larry , Yamada Yoshihiko
Published: January 2023

Fibulin-7 (Fbln7) is a member of the fibulin family of secreted glycoproteins, majorly found in developing teeth, bone and cartilage and shows interaction with other ECM proteins. Recently, it was shown that its C-terminal fragment (Fbln7C) and its peptides show anti-angiogenic effects. Fbln7 is also expressed in immune privileged tissues such as eye and placenta but to date very little is known about its functional significance. The results from this study show that human monocytes adhered to Fbln7 full-length (Fbln7-FL) and Fbln7C via integrin α5β1. Interestingly, actin stress fiber formation of monocytes was promoted by Fbln7-FL and not by Fbln7C. Morphological studies and surface expression analysis of CD14, mannose receptor (CD206), MHC-II and CD11b receptors revealed that both Fbln7-FL and Fbln7C inhibited MCSF induced monocyte differentiation with more significant effect with Fbln7C as compared to Fbln7-FL. Similarly, both protein fragments inhibited IFNγ and IL-4 induced M1 and M2 polarization of human blood monocyte derived macrophages. Collectively, these data suggests that Fbln7 and its fragments could negatively regulate immune cell in the expressed tissues.
High avidity TCR-peptide interactions between CD4 lymphocytes and lymph node dendritic cells promote normal lymphocyte arrest, activation, proliferation, and differentiation independently of dendritic cell ICAM-1 and 2
Authors: Feigelson Sara W , Solomon Adam , Biram Adi , Hatzav Miki , Lichtenstein Maria , Regev Ofer , Curato Caterina , Rashkovan Diana , Lefkowitz Dena , Jung Steffen , Shulman Ziv , Alon Ronen
Published: January 2023

The two main LFA-1 ligands ICAM-1 and ICAM-2 are constitutively co-expressed on antigen presenting cells (APCs) and on the lymph node stroma. We find abrogated antigen specific CD4 T cell priming in ICAM-1 and ICAM-2 deficient spleens. Nevertheless, the in vivo contribution of these ligands to lymphocyte activation proliferation and differentiation in lymph nodes has been difficult to dissect in total knockout mice due to their multiple roles in lymphocyte entry and priming in these organs. We therefore constructed lethally irradiated chimeric mice reconstituted with ICAM-1 and 2 double knock out (DKO) APCs in which lymphocyte entry into peripheral lymph nodes was reconstituted. Strikingly, these mice elicited normal antigen specific OT-II T cell activation, proliferation and differentiation into Th1 and Tfh effectors. OT-II T cells could also normally arrest on DCs deficient in ICAM-1 and 2. A compensatory role of VCAM-1 in ICAM deficient DCs was ruled out. Nevertheless, polyclonal T cell proliferation in response to endogenous self-antigens required the presence of ICAM-1 and ICAM-2 on endogenous lymph node DCs. Our results are the first indication that high avidity TCR peptide-MHC-II recognition can bypass integrin-mediated lymphocyte adhesions to DCs under specific settings.
CD11c+ T-bet+ IgM memory B cells provide multi-lineage reconstitution of effector and memory B cells following challenge infection
Authors: Kenderes Kevin J , Papillion Amber M , Cabrera-Martinez Berenice , Winslow Gary
Published: January 2023

IgM memory B cells are now recognized as an important component of immunological memory. These cells have been proposed to act as a reservoir of broadly-reactive B cells that differentiate, in germinal centers, into high affinity class-switched effector B cells following antigen encounter. However, we propose IgM memory cells can differentiate into more than just germinal center and class-switched cells. To address this, we monitored the secondary response of CD11c+ T-bet+ IgM memory cells generated by Ehrlichia muris infection, by monitoring eYFP-labeled, flow cytometrically-purified, IgM memory cells following their transfer to naïve recipient mice and subsequent challenge infection. The IgM memory B cells differentiated into IgM-producing plasmablasts early following infection; this resulted in a 4-fold increase in IgM production, relative to control mice that did not receive memory cells. Other donor memory B cells entered germinal centers, down-regulated CD11c, and underwent class switching, generating switched memory B cells. Finally, some donor B cells were maintained as IgM memory cells. Upon secondary transfer and challenge, purified IgM memory cells underwent the same pattern of self-renewal and differentiation. Additionally, when IgM memory cell recipient mice were challenged with a virulent Ehrlichia strain, a reduction in bacterial burden was observed, indicating that IgM memory cells can provide immunity to challenge infection. Our findings also suggest that T-bet+ IgM memory cells act as a self-renewing population capable of repopulating the entire spectrum of effector and memory B cells during secondary infection, and demonstrate an important role for IgM memory cells in maintaining long-term immunity.
IL-6 induces production of VEGF and MCP-1 from human skin mast cells.
Authors: McHale Cody C , Gomez Gregorio
Published: January 2023

Mast cells are the cell type responsible for IgE-mediated allergic reactions, and are involved in innate immunity against bacteria and pathogens. Interestingly, recent research has implicated mast cells in traditionally non-allergic diseases such as cancer. It has been hypothesized that in these non-allergic diseases mast cells may be activated by stimuli other than antigen, such as pro-inflammatory cytokines. Interleukin-6 (IL-6) is a pleiotropic cytokine that has been implicated in several diseases in which mast cells are also believed to contribute, suggesting that IL-6 could be the link that connects mast cells to non-allergic disease. However, the surface expression of IL-6 receptors, and the effect of IL-6 on human tissue-derived mast cells remained to be determined. Here, we show that human skin mast cells express functional membrane-bound IL-6 receptors, and address the role of IL-6 on the release of allergic and inflammatory mediators. Stimulation of skin mast cells with IL-6 resulted in a dose-dependent increase in Signal Transducer and Activator of Transcription 3 (STAT-3) phosphorylation, and increased expression of Suppressor of Cytokine Signaling 3 (SOCS3) mRNA. IL-6 had no effect on degranulation, but enhanced PGD2 biosynthesis and IL-8 production following FcɛRI crosslinking. In addition, treatment with IL-6 alone was sufficient to induce the production of monocyte chemoattractant protein (MCP-1) and vascular endothelial growth factor (VEGF). Overall, these findings clarify the effect of IL-6 on the allergic response from human tissue-derived mast cells, and reveal a potential role for IL-6 in mast cell-mediated angiogenesis.
MRGPRX2 receptor activation as a rapid, high-throughput mechanistic-based approach for detecting peptide-mediated human mast cell degranulation liabilities.
Authors: Lafleur Marc , Lobenhofer Edward K. , Fort Madeline , Werner Jonathan , Fan Fan , Balazs Mercedesz
Published: January 2023

Mast cells play key roles in allergy, anaphylaxis and pathogen/toxin defense. They contain cytoplasmic granules with a wide spectrum of pleotropic mediators (e.g. histamine, tryptase, serotonin) that are released upon activation. Mast cell degranulation (MCD) occurs upon clustering of the IgE receptor in presence of antigen or via non-IgE mediated mechanisms, one of which is by means of the Mas-related G protein coupled receptor (MRGPRX2). MRGPRX2 is a non-selective, low affinity binding receptor that responds to many basic biogenic amines and peptides. Consequently, MCD is an important potential safety liability for peptide therapeutics. To improve screening of peptides for this liability, we assessed a MRGPRX2-CHO cell-based receptor activation assay using positive (n=30) and negative (n=29) control peptides, which were previously characterized for MCD liability in CD34+ bone marrow-derived human mast cell cultures. MRGPRX2-CHO cells were engineered to selectively activate beta-arrestin upon receptor stimulation. In addition to beta-arrestin, Ca2+ was also measured to determine the potential of ligand bias (selective engagement of G-protein dependent or independent pathways). The MRGPRX2-CHO receptor assay had a sensitivity of 97% (false negative 3%) and specificity of 93% (false positive 7%) against the outcome in human mast cell cultures. No clear evidence of ligand bias was observed. Because MRGPRX2 is overexpressed in CHO cells, the assay may slightly over predict, and does not inform on other mechanisms of MCD not mediated by MRGPRX2. Assessing MRGPRX2 receptor activation provides a rapid, high-throughput and economical mechanism-based approach for detecting peptide-mediated human MCD liabilities.
MALT1 proteolytic activity is necessary for FcɛRI-mediated cytokine production in mast cells
Authors: Alfano Danielle N , Klei Linda R , Lucas Peter C , McAllister-Lucas Linda M
Published: January 2023

IgE mediated mast cell activation triggered by multivalent antigen crosslinking of FcɛRI receptors leads to the release of a wide range of inflammatory mediators. Recent studies revealed that B cell lymphoma 10 (Bcl10) and mucosa-associated lymphoid tissue 1 (MALT1) are required for IgE-induced nuclear factor κB (NF-κB)-induced proinflammatory cytokine production, while dispensible for mast cell degranulation and leukotriene synthesis. MALT1, a cytoplasmic signaling protein, possesses at least two functions: scaffolding activity and proteolytic activity. The contribution of MALT1 proteolytic activity to mast cell function has not yet been evaluated. We hypothesize that MALT1 proteolytic activity is necessary for FcɛRI-dependent proinflammatory cytokine production. In order to test our hypothesis, we compared the activation of bone marrow derived mast cells (BMMC) from wild type mice, MALT1 knockout mice (MALT1−/−), and mice harboring a mutant, proteolytically inactive MALT1 (MALT1PD/PD). We find MALT1 protease is active in wild-type BMMCs as evidenced by RelB cleavage, and is absent, as expected, in MALT1−/− and MALT1PD/PD BMMCs. Cytokine gene expression and production in response to IgE mediated FcɛRI crosslinking is equivalently abrogated in both MALT1−/− and MALT1PD/PD BMMCs as compared to wild type. These findings suggest that the contribution of MALT1 to IgE mediated responses can be largely attributed to its protease activity. The MALT1 protease has been shown to be drugable, and pharmaceutically targeting the MALT1 protease activity may represent a promising approach to abrogating late phase pathologic mast cell activation.
Is MHCII-restricted immunodominance determined by HLA-DM action?
Authors: Osan Jaspreet Kaur , Ferrante Andrea
Published: January 2023

Binding of antigenic peptides to class II MHC molecules (MHCII) and the activity of the “editing” molecule HLA-DM (DM) on the resulting peptide-MHCII complexes are mechanisms critical to immunodominance, yet not fully understood. Our previous results indicate that peptide binding to the human MHCII HLA-DR1 (DR1) is a flexible event in that different thermodynamic components compensate each other to the possible extent to achieve a stable complex, and the energetic mechanism responsible for binding is reflected in the structural lability of the resulting complex. Here we apply our thermodynamic-structural model of peptide binding to sequences derived from HA H1N1, for which a DR1-restricted classification in dominant, sub-dominant, weak and negative has been established. We measured peptide KD and t1/2, and correlated such measurements to the observed level of immunodominance. Our results show that immunodominant peptides have a binding affinity in the micromolar range in the presence and absence of DM, whereas negative and weak showed no binding, with one exception. Interestingly, DM effect on binding affinity is variable for these peptides, altering their affinity values on a sequence-by-sequence basis with no direct correlation to their level of immunodominance. Variations in complex kinetic stability in the absence of DM did not reflect variations in immunodominance, whereas all peptides showed a faster release in presence of DM. Taken together, our results suggest that there is no a unique peptide-binding or kinetic signature, which can be adopted as a proxy for immunodominance. Further investigation in the thermodynamic components of the binding process is required to corroborate or modify these initial conclusions.
Immunomodulatory potential of Weissella cibaria JW15 on dendritic cells..
Authors: Oh Eun young , Lee Sang-Myeong
Published: January 2023

Lactic acid bacteria (LAB) are well-known to exert various beneficial health effects. Especially, immunomodulatory activities of LAB have been highlighted by recent studies. Our previous findings demonstrated that Weissella cibaria JW15 possessed immunostimulatory activities through modulation of macrophages, T cells and NK cells. Here, we studied whether this LAB is able to modulate dendritic cells (DC) function since DC are the most effective antigen presenting cells, linking innate immunity with adaptive immunity. Bone marrow derived dendritic cells (BMDCs) from mice were prepared and stimulated with live JW15, heat-killed JW15 and EPS isolated from JW15, followed by evaluation of cytokine production, expression of maturation markers and activation of intracellular signaling pathways. Our results revealed that JW15 as well as EPS induced phenotypic maturation of DC as shown by increased surface expression of CD80, CD86, CD40 and MHC II. In addition, both of JW15 and EPS increased the production of various cytokines in a dose dependent manner including TNF-a and IL-12. We also found that JW15 activated NF-κB and MAPK signal pathways. Collectively, we propose that JW15 and EPS isolated from this bacteria is able to activate dendritic cells and possess a potential to be developed as functional materials to boost immune system. Further studies are needed to provide comprehensive information to support our conclusions and reveal molecular mechanisms by which JW15 modulate immune responses.
Identification of the physiological antigen presenting cell for glycolipid antigens
Authors: Arora Pooja , Porcelli Steven
Published: January 2023

A major subset of T cells that recognize complexes formed by the binding of glycolipids to CD1d are called invariant natural killer T (iNKT) cells. Unlike conventional T cells that need to proliferate and differentiate to become activated effector T cells, iNKT cells exist as an effector population and start responding rapidly after glycolipid recognition. The prototypical iNKT stimulatory antigen is synthetic α-galactosylceramide (αGC), initially designated as KRN7000. Administration of this antigen in vivo results in the production of cytokines associated with both T helper type 1 (Th1) or type 2 (Th2) responses. Several variants of this antigen having different fatty acyl- and phytosphinganine chains have been synthesized and polarize the response towards a predominance of either Th1 or Th2 cytokines, and are therefore described as Th1- or Th2-biasing analogues. It is not clear how the Th1- or Th2-bias in iNKT mediated response is induced, although selective presentation by distinct cell types has been proposed as a possible mechanism. By visualizing glycolipid antigen presentation directly with monoclonal antibodies specific for complexes of αGC bound to CD1d, we showed that the CD8αPosDEC-205Pos dendritic cells were the major APCs in the spleen for a range of αGC analogues, irrespective of their chemical structures and cytokine biasing activities. We found that DCs presenting Th1-biasing agonists upregulated ligands for stimulatory NK receptor molecules, while Th2-agonist presenting cells showed a marked upregulation of ligands for inhibitory receptors. Furthermore, modulation of accessory immune proteins was dependent on CD1d, suggesting direct cell-cell interactions between DC and iNKT cells.
A unique MHCII-binding register correlates with HLA-associated immunogenicity of the major K blood group antigen
Authors: Gunasekera Devi , Zimring James C , Pratt Kathleen P.
Published: January 2023

Kell is a glycoprotein expressed on red blood cells (RBCs). Its K and k isoforms contain either Met (K antigen) or Thr (k antigen) at position 193. Development of anti-K antibodies following K-mismatched blood transfusions can destroy RBCs, while cross-placental transfer of anti-K antibodies can destroy RBC precursors, resulting in Hemolytic Disease of the Newborn. HLA-DR11, DR13 and DR15 are over-represented among K-immunized patient populations. The immunogenicity of overlapping 15-mer Kell peptides with M193 or T193 at every possible position was investigated by Stephen et al (Blood. 2012;119(23):5563-5574). Surprisingly, peptide W179-M193, with the polymorphic M193T site at the peptide’s C-terminus, was the most effective at stimulating CD4 T cells from K-immunized women. The present study utilized MHCII binding prediction algorithms and quantitative peptide-MHCII binding assays to determine the binding registers, anchor residues and affinities of wild-type, truncated, and sequence-modified Kell peptides. Predictions were generated using IEDB and Propred algorithms. Competitive peptide-MHCII binding assays utilized 13 recombinant HLA-DRB1 monomers, Kell peptides and high-affinity reference peptides. Interestingly, the assays identified a unique binding register (W179-S187) restricted to HLA-DR11 and DR15, in addition to the polymorphic site, suggesting stronger MHCII avidity may explain the increased disease susceptibility associated with these alleles.
The role of transmembrane domains in the March-I-mediated downregulation of MHC-II and CD86.
Authors: Walseng Even , Bandola-Simon Joanna , Roche Paul
Published: January 2023

MHC class II molecules on APCs bind to and presents peptides to antigen-specific CD4 T cells. DCs are the most potent subset of APC and can activate naïve T cells. Immature DCs possess relatively low expression of the costimulatory molecule CD86 and have a rapid rate of turnover of MHC-II. This is due in part to ubiquitin-mediated degradation of MHC-II and CD86 by the transmembrane (TM) ubiquitin ligase March-I in immature DCs. Curiously, March-I activity is suppressed by CD83, a TM protein that is upregulated by DC activation. Despite the well-documented role of March-I in specifically regulating MHC-II and CD86 expression, little is known about how these substrates interact with March-I and how CD83 is able to suppress March-I function. In this study we have examined the importance of TM domain interactions in March-I function by replacing TM domain sequences in March-I or CD83 with that of ICAM-1. A March-I TM1 mutant had reduced ability to affect MHC-II expression and had no effect on CD86 expression. Binding studies demonstrated that that March-I TM1 mutant lost all ability to bind to CD86. Whereas wild-type CD83 was able to block March-I downregulation of MHC-II and CD86, replacement of the CD83 TM domain with that of ICAM-1 completely abrogated CD83 suppression of March-I function. Binding studies confirmed that the CD83 TM mutant had lost all ability to bind to March-I. Our data therefore suggest that March-I binds to MHC-II and CD86 by TM domain interactions and that CD83 blocks March-I function by competitively inhibiting the binding of March-I to MHC-II and CD86.