bluuebunny/crossref_metadata_2025 huggingface dataset:
Excessive miR-27 expression impairs regulatory T cell-mediated immunological tolerance
Authors: Cruz Leilani Ong , Hashemifar Somaye Sadat , Cho Sunglim , Wu Cheng-Jang , Lin Ling-Li , Nguyen Duc T. , Khan Aly Azeem , Lu Li-Fan
Published: January 2023

MicroRNAs (miRNAs) are tightly regulated in the immune system as aberrant expression of miRNAs often results in hematopoietic malignancies and autoimmune diseases. Previously, elevated levels of miR-27 in T cells isolated from multiple sclerosis patients has been suggested to facilitate disease progression through inhibiting Th2 immunity and promoting pathogenic Th1 responses. Here we show while mice with T cell-specific overexpression of miR-27 harbor dysregulated Th1 responses and develop autoimmune pathology, these disease phenotypes are not driven by miR-27 in effector T cells in a cell autonomous manner but rather resulted from a perturbed regulatory T (Treg) cell compartment. Excessive miR-27 expression in T cells severely impairs thymic Treg cell development and TGFb-mediated conversion of peripheral Treg cells from conventional T (Tconv) cells. Moreover, Treg cells with exaggerated miR-27-mediated gene regulation exhibit diminished homeostatic potential and suppressor function in vivo. Mechanistically, miR-27 represses a subset of genes that play critical roles in controlling multiple aspects of Treg cell biology. Collectively, our data show miR-27 functions as a key regulator in Treg cell development and function and suggest that proper regulation of miR-27 is pivotal to safeguard Treg cell-mediated immunological tolerance.
Long noncoding RNA MEG3-4 regulates IL-1β during pulmonary bacterial infection by repressing microRNA-138
Authors: Li Rongpeng , Wu Min
Published: January 2023

Long noncoding RNAs (lncRNAs) modulate various biological processes; however, their function in host immunity response against infection is largely unknown. Here, we identify an intergenic lncRNA MEG3 (linc-MEG3) as a tissue specific regulator for pulmonary immunity during Gram-negative bacterial infection. Among the 10 transcripts of linc-MEG3, transcripts 1 and 4 are main regulators as being discovered the most downregulated in mouse lungs after bacterial infection. Overexpression of linc-MEG3-4 in mice led to intensified inflammatory response, severe lung injury, systemic dissemination and ultimately increased mortality. Alveolar epithelial cells and alveolar macrophages (AM) were major cell populations that are targeted by linc-MEG3-4. Due to its 3′ sequences complementary to microRNA-138 (miR-138), linc-MEG3-4 competitively binds miR-138 and thereby freeing its target IL-1β mRNA to intensify inflammatory responses. Our results characterize linc-MEG3 as a novel pulmonary inflammatory regulator of bacterial infection through a miR-138/IL-1β/NF-κB axis.
VprBP, an HIV target protein, is vital for immune responses by controlling T cell growth, proliferation and senescence.
Authors: Wan Yisong , Guo Zengli
Published: January 2023

Naïve mature T cells are maintained in a quiescent state with a low rate of cell size growth (biomass increase due to enhanced metabolism and biosynthesis) and proliferation. Disturbances inflicted by infection and inflammation, often trigger cells to exit quiescence and enter cell cycle, followed by rapid proliferation to restore homeostasis. Cell size growth is imperative for proliferating cells to compensate for the halving of cellular constituents for each division. Thus, proper coordination of cell growth and cell cycle entry of T cells is essential to mount effective immune response. Nonetheless, little is known about how cell cycle entry is controlled and even less about how cell size growth is regulated in T cells.

We studied protein expression during T cell growth and cell cycle entry using quantitative proteomic approach. VprBP, an HIV target protein, was identified to be one of the highly upregulated factors. We found that VprBP was required for the cell growth and cell cycle entry of activated T cells. VprBP deletion abrogated T cell expansion during antiviral and autoimmune responses in vivo. VprBP-deficient cells drastically stabilized p53 protein with elevated expression of p53 target genes. Intriguingly, we found that although p53 deletion failed to correct the T cell growth defect due to VprBP deletion, it largely restored the cell cycle entry of VprBP-deficient T cells. In addition, VprBP deletion led to a premature senescence phenotype in T cells associating with increased cellular aging markers. These results suggest that VprBP controls T cell growth, cell cycle entry and senescence via discrete molecular programs. The recent findings and the underlying mechanisms will be discussed in detail in this presentation.
Molecular mechanisms required for TCR-mediated formation of TCR-CXCR4 heterodimers
Authors: Dinkel Brittney A , Kremer Kimberly , Osborne Douglas Grant , Reed Brendan Kearney , Schrum Adam G , Hedin Karen E
Published: January 2023

T cell immune activation is frequently cross-regulated by CXCR4, which regulates T cell migration through binding its ligand SDF-1 (CXCL12). Here, we define a novel molecular mechanism by which CXCR4 directly impacts T cell immune function even in the absence of SDF-1. Our lab recently reported that, even in the absence of SDF-1, T cell receptor (TCR) stimulation causes CXCR4 to physically associate with the TCR and form a TCR-CXCR4 heterodimer. Moreover, blocking formation of TCR-CXCR4, with the CXCR4 antagonist AMD3100 or by CXCR4 siRNAs, selectively impairs TCR-stimulated signaling, inhibiting T cell production of IL-2, IL-4, IL-10, and TNF but not IFN-λ. Here, we investigate the molecular mechanisms required for TCR-induced TCR-CXCR4 formation in the absence of SDF-1. Using fluorescence resonance energy transfer (FRET) to detect TCR-CXCR4, we discovered important roles for specific TCR-induced kinases and also particular CXCR4 structural motifs, leading us to propose that a kinase-driven receptor trafficking mechanism is responsible for formation of TCR-CXCR4 heterodimers in response to TCR ligation. Furthermore, we have identified CXCR4 structural motifs and an E3 ligase that act as negative regulators of TCR-CXCR4 heterodimer formation. Thus, TCR ligation triggers formation of TCR-CXCR4 complexes that mediate T cell functions. Targeting formation of TCR-CXCR4 complexes may be a novel approach to modulate T cell-mediated immunity.
Environmental cues in Casp8-deficient mice enhance virus-specific T cell expansion and terminal differentiation
Authors: Feng Yanjun , Daley-Bauer Lisa , Roback Linda , Mocarski Edward
Published: January 2023

Cell autonomous death is balanced against survival signaling to establish T cell levels in the course of primary as well as secondary responses to infection. It is known that mitochondrial (intrinsic) cell death contributes to T cell contraction; however, the question remains about the role of extrinsic cell death in the T cell response. Caspase8 (Casp8) initiates apoptosis downstream of death receptor activation while also suppresses RIP3-mediated necroptosis. We have employed wild type (WT), Rip3−/− and Casp8−/− Rip3−/− (DKO) mice to address the contribution of Casp8-dependent apoptosis as well as RIP3-dependent necroptosis in the antiviral T cell response. Although both Casp8 and RIP3 are dispensable for the T cell response to murine cytomegalovirus (MCMV) infection, we found that DKO mice exhibit differences in T cell characteristics compared to WT or RIP3-deficient mice. Interestingly, the T cell populations in naïve DKO mice show increased activation markers (CD44LhiKLRG1hiCD127lo). This pre-activated status results in more abundant virus-specific effector T cells in DKO mice following expansion at 7 dpi. Importantly, DKO T cells respond the same as WT T cells when adoptively transferred into WT environment; however, adoptive transfer of DKO splenocytes into WT recipients results in greater expansion and terminal differentiation of the recipient (WT) T cell population during infection. Altogether, even though Casp8 and RIP3 are dispensable for T cell expansion and contraction when examined directly, Casp8-dependent environmental cues influence T cell expansion during infection and even drive WT T cells to behave like Casp8-deficient T cells when present together during virus infection.
Signaling through PD-1 on CD8 T cells is critical for antigen-independent maintenance of immune memory
Authors: Yuzefpolskiy Yevgeniy , Baumann Florian Martin , Penny Laura Anne , Kalia Vandana , Sarkar Surojit
Published: January 2023

PD-1 is highly expressed on CD8 T cells during activation, both in acute and chronic viral infections. Inhibitory signaling in cytotoxic T cells through the PD-1 axis is well characterized during chronic infections and has led to the development of potent blockade therapies that restore function to otherwise exhausted CD8 T cells. However, the functional significance of transiently increased PD-1 expression on CD8 T cells early after activation and its rapid down-regulation following clearance of antigen during acute infections, remains to be characterized. The enigma is – expression of PD-1 (an immunological brake) during activation temporally coincides with rapid proliferation and production of copious amounts of signature effector cytokines (IFN-γ and TNF-α). We generated PD-1−/− antigen-specific CD8 T cells to study its cell-intrinsic role during acute infection with LCMV. Unexpectedly, PD-1−/− cells did not exhibit increased proliferation early during infection, or enhanced accumulation at the peak of expansion. PD-1−/− CD8 T cells were also unaltered in their polyfunctionality and granzyme B expression. Despite robust effector properties, PD-1−/− CD8 T cells underwent precipitous contraction, leading to near ablation of the memory pool. Mechanistically, in vivo analysis of PD-1−/− memory cells showed a severe defect in antigen-independent homeostatic proliferation due to decreased sensitivity to IL-2, −7, and −15 signals, without evident change in the expression of common gamma-chain cytokine receptors. These studies demonstrate a previously unrecognized role of PD-1 signaling in programming of memory CD8 T cell maintenance and could make it a novel target for manipulating vaccine-induced memory T cell longevity.
Integrating Group-Learning for First-Year Pharmacy Immunology Course to Enhance Students Understanding and Comprehension
Authors: Bowar Benjamin Charles , Royan Ayla , Shellip Alla , Fazal Nadeem , Fazal Nadeem
Published: January 2023

This research hypothesizes that integrating group-learning oriented multiple-choice study guides for first-year pharmacy immunology course will enhance students learning, understanding and comprehension of complicated material. Immunology questions (300 MCQs) were selected based on the criteria of lectures topics, difficulty level and meeting the specific objectives for every lecture that was presented to students and covered in this exam. Study guide was uploaded to Moodle three weeks before the examination and students were given a free access to it with the ability to download and print it. The survey was taken at the end of examination to assess the benefit of study guide to students. Likert-type Ordinary Scale data analysis was used to determine if hypothesis was supported and study endpoints were met. Primary endpoint was to determine the effectiveness of utilizing the study guide in student learning. Secondary endpoint was to determine if students prefer to study in learning groups and what resources were most commonly utilized to find answers to questions. Our studies found that the majority of the students 69% indicated study guide was very beneficial to them and helped to make their study more focused and practicing questions helped them to understand complicated immunology concepts better. Moreover, 65% of the students agreed or strongly agreed that study guide helped them not only to prepare to examination but also to improve the overall exam score. In conclusion, the results of this study suggest that group-based learning approach utilizing a multiple-choice study guide was very beneficial to a first-year pharmacy immunology course and positively supplemented materials being covered in a classroom lecture setting.
Mentoring Undergraduates: Challenges and Choices in a Unique Experience in Entrepreneurship
Authors: Liepkalns Justine S
Published: January 2023

Undergraduate research has the benefit of helping students make a transition from passive learning to active learning; however, students new to research are presented with a catch-22 when trying to identify a research laboratory to work in. I present a case study of a unique mentoring experience and what I have been learning about mentoring writ large. Among issues faced included fostering independent thinking and enthusiasm, attribution of credit, calibrating mentor involvement, establishing “protected time” and regular meetings, and building a relationship while still maintaining authority. This case includes an added layer of complexity: entrepreneurship. Two freshmen and I began an online public fundraising campaign for a small laboratory project involving a screening method for Ebola. This allowed me, as a post doctoral fellow, to start a lab project within a different discipline with lab space provided by a supportive biology department. The resulting screening method was filed for a preliminary patent at the United States Patent and Trademark Office. The students had experience in giving an oral presentation at a conference and participating in local outreach during a local science fair. We were awarded a booth at the Atlanta Science Festival. One of the students has received additional funding and gave an invited TEDx talk; the other wrote an essay published in a peer-reviewed journal. Starting this project through unconventional means and maintaining a trusting mentoring relationship was important in shaping a platform from which to propel these students’ careers forward.
Immunology education within a Postgraduate Nursing Science program in New Zealand
Authors: Sika-Paotonu Dianne
Published: January 2023

Immunology is key to understanding the cellular basis for certain diseases. Postgraduate Nursing Science students with a limited undergraduate background in Biomedical Science may find Immunological course components challenging. The goal was to successfully incorporate and establish a substantial Immunological component within the postgraduate Pathophysiology course for Nursing Science and Health students at Victoria University of Wellington in New Zealand. In-class teaching sessions were conducted that covered the fundamental concepts of Immunology and included Innate, Adaptive Immunity, Inflammation and Immunopathophysiology lessons. A variety of teaching and learning approaches and strategies were employed to facilitate in-class learning for students and support understanding, and improve participation and student engagement with in-class activities. These included the use of Youtube video clips, animations depicting key cellular and mechanistic interactions, one-on-one peer discussions and traditional lecture sessions interspersed with brief question and answer interactions designed to gauge understanding and also consolidate learning. Cumulative data showed favourable student outcomes were achieved for Immunological assessment components. Student satisfaction and teaching scores based on formal student evaluation processes also showed positive responses regarding the Immunology course elements. This work highlights the successful incorporation of essential Immunological components into a teaching curriculum for Postgraduate Nursing Science education in New Zealand.
The role of Type I and Type II Interferon in the adaptive response to blood stage malaria infection.
Authors: Hahn William O , Gale Michael J , Stetson Dan , Liles WC , Pepper Marion
Published: January 2023

Background

The adaptive immune response is critical during the host response to blood stage malaria infection but the cytokine networks important in the development of an appropriate adaptive response are unclear. Type I and Type II gene networks are upregulated in response to blood stage malaria infection in both humans and mice.

Methods

In a murine model of blood stage malaria infection (Plasmodium yoelli), we utilized a tetramer-based approach to examine the effects of Type I and Type II interferon on the antigen specific adaptive immunologic CD4+ T-cell and B-cell response. Disruptions in both Type I and Type II interferon had substantial effects on the cellular immune response and also affected the course of infection. Mice with disruption in Type I Interferon signals had worsened parasitemia early whereas mice with disruption in Type II interferon signals had problems with delayed clearance of the parasite.

Conclusion

We conclude that Type I and Type II Interferon responses have distinct roles in the host response to blood stage malaria infection. We conclude that early Type I Interferon signals are important for appropriate adaptive response whereas early Type II interferon signals are disruptive for late clearance of the parasite.
Lytic granule convergence is essential for NK cells to promote targeted killing while preventing collateral damage
Authors: HSU HSIANG-TING , Viswanath Dixita , Mace Emily , Christakou Athanasia , Wiklund Martin , Önfelt Björn , Orange Jordan
Published: January 2023

NK cell activation triggers serial events leading to lysis of diseased cells: 1) granule convergence, preformed lytic granules rapidly cluster to the microtubule-organizing center (MTOC), 2) granule/MTOC polarization to the immunological synapse, and 3) degranulation, exocytosis of lytic contents onto the target cell. Granule convergence requires LFA-1 signaling and dynein motor function. Yet, it remains elusive how lytic granule convergence contributes to NK cell cytotoxicity. Using Drosophila S2 cells, we regulate NK cell signal inputs to precisely control granule convergence and degranulation. In the absence of LFA-1 signaling, the average distance of lytic granules to the MTOC increased >1.3 fold, indicating diffuse granule localization. Using imaging flow cytometery, we determined that without LFA-1, the synaptic accumulation and degranulation of lytic granules decreased by 3.3 and 2.5 fold, respectively suggesting undirected granule release. Using an ultrasound-guided-acoustic-trap-microscopy system to enforce live single NK cell with multiple target cell contacts, we determined killing efficiency with real-time granule tracking. NK cells activated by CD16 alone without co-engaging LFA-1 showed ~40% lower targeted killing and ~30% higher non-specific killing of neighboring unlabeled “bystander” S2 cells. Finally, we used ciliobrevin D, a dynein inhibitor, to physically block granule convergence in NK cells conjugated with physiologically relevant target cells. Dynein-inhibited NK cells caused ~36% increase in bystander killing. Thus, we demonstrated that NK cells converge lytic granules to improve the efficiency of targeted lytic granule secretion and prevent collateral damage to neighboring tissue.
A context-dependent role for IL-21 in modulating the differentiation, distribution, and abundance of effector and memory CD8 T cell subsets
Authors: Tian Yuan , Cox Maureen A , Kahan Shannon M , Ingram Jennifer T , Bakshi Rakesh K , Zajac Allan J
Published: January 2023

The activation of naïve CD8 T cells typically results in the formation of effector cells (TE) as well as phenotypically distinct memory cells that are retained over time. Memory CD8 T cells can be further subdivided into central memory (TCM), effector memory (TEM) and tissue-resident memory (TRM) subsets, which cooperate to confer immunological protection. Using mixed bone marrow chimeras and adoptive transfer studies in which CD8 T cells either do or do not express the IL-21 receptor (IL-21R), we discovered that under homeostatic or lymphopenic conditions IL-21 acts directly on CD8 T cells to favor the accumulation of TE/TEM populations. The inability to perceive IL-21 signals under competitive conditions also resulted in lower levels of TRM phenotype cells and reduced expression of granzyme B in the small intestine. Furthermore, IL-21 differentially promoted the expression of the chemokine receptor CX3CR1 and the integrin α4β7 on circulating CD8 T cells in the mixed bone marrow chimeras and on CD8 T cells primed in vitro. Thus, IL-21 may influence CD8 T cell migration by modulating the expression of CX3CR1 and α4β7. The requirement for IL-21 to establish CD8 TE/TEM and TRM subsets was overcome by acute lymphocytic choriomeningitis virus infection. Nevertheless, memory virus-specific CD8 T cells remained dependent on IL-21 for optimal accumulation in tissues under lymphopenic conditions. Overall, this study reveals a context-dependent role for IL-21 in sustaining effector-phenotype CD8 T cells and influencing their migratory properties, accumulation, and functions.
Liver sinusoidal endothelial cells induce the tolerogenic and regulatory feature of autoreactive CD4+ recent thymic emigrants
Authors: Ge Qing , Xu Xi , Zhang Shusong , Wang Ke
Published: January 2023

Mechanisms of peripheral tolerance play a critical role in preventing T cells that escape from negative selection in the thymus from initiating autoimmune reactions. We found that a fraction of CD4+ recent thymic emigrants (RTEs) were retained in the liver independent of the secondary lymphoid organs. Following transfer into RAG−/− recipients, CD4+ liver RTEs induced more severe organ-specific inflammation and cell infiltration than lymph node RTEs, indicating that more autoreactive RTEs were retained in the liver. Compared to RTEs from the lymph nodes, more CD4+ RTEs from the liver acquired activated phenotype (CD44hiCD62LloCXCR3hiPD-1loFoxp3−) with high expression of LAG3, FasL, IL-10, and IFN-γ. These liver RTEs also acquired regulatory functions as they could suppress the proliferation of naïve T cells in vitro. We further found that liver sinusoidal endothelial cells could induce the tolerogenic and regulatory phenotype of liver RTEs via cellular interaction. The expression of MHC, Aire, and Notch all contributed to RTEs’ acquisition of liver tolerance. These results suggest that the liver is the first checkpoint in the periphery to filter, retain, and enforce tolerance to autoreactive CD4+ thymic emigrants that escape from negative selection.
Irreversible effector CD8+ T cell differentiation is linked to asymmetric, intra-divisional TCF1 silencing
Authors: Lin Wen-Hsuan Wendy , Yen Bonnie , Nish Simone A , Kratchmarov Radomir , Bhandoola Avinash , Xue Hai-Hui (Howard) , Reiner Steven L
Published: January 2023

During specific immune responses, naive T cells give rise to terminally differentiated effector T cells and pluripotent, self-renewing central memory T cells. We recently provided evidence that, after approximately 3 cell divisions, TCF1, a key transcription factor of memory CD8+ T cells, is unequally expressed by sibling T cells undergoing cytokinesis. We now show that repression of TCF1 by one sibling is cell cycle-dependent and subsequently irreversible. Using pharmacological inhibitors, we found that cells which fail to progress through cell cycle are incapable of silencing TCF1. Using a transcriptional reporter of TCF1, we found that cells that extinguish TCF1 expression do not revert to TCF1-expressing cells. Although inhibition of PI3K/mTOR signaling blocks initiation of TCF1 repression, the same blockade fails to alleviate established silencing of TCF1. T cell progeny that maintain TCF1 expression undergo self-renewal and appear to remain pluripotent, while progeny that lose TCF1 expression appear destined to become functional, but short-lived, effector cells. TCF1-expressing cells generated in initial cell divisions, prior to irreversible TCF1 loss, appear to become quiescent, memory-like cells, whereas those cells that retain TCF1 expression when their sibling cells undergo irreversible TCF1 loss have more effector-like characteristics. The unidirectional quality of TCF1 silencing is compatible with a model wherein central memory cells represent cellular progeny that have yet to repress TCF1, rather than descendants of effector cells that lost TCF1-expression. Asymmetric, intra-divisional differentiation may explain the regenerative quality of lymphocyte clones during immunity.
Plasmablasts induced by experimental Plasmodium infection suppress protective humoral immunity through PD-L1
Authors: Guthmiller Jenna , Graham Amy C , Pope Rosemary L , Zander Ryan A , Butler Noah S
Published: January 2023

Plasmodium infections cause malaria, which remains a global public health crisis. Germinal center derived anti-Plasmodium antibody responses are essential for control of blood-stage parasite replication and clearance. However, antibody responses are dominated by extrafollicular, short-lived plasmablasts that secrete low-affinity antibodies. The factors that regulate plasmablast expansion and the contribution of plasmablasts to protective anti-Plasmodium humoral immunity are not well understood. Utilizing experimental murine Plasmodium yoelii infection, we identified that early, splenic plasmablast responses (CD138hi B cells) are dominated by innate B1b (CD43+CD5−) B cells and are comprised of both parasite- and host-specific cells. We also found that CD138hi B cell responses are largely governed by IFN-γ inflammation, not parasite burden. Strikingly, adoptive transfer of CD138hi B cells reduced germinal center B cell class-switching and parasite-specific IgG secretion in Plasmodium infection-matched recipients. Moreover, T follicular helper cells recovered from CD138hi B cell recipient mice were inefficient at promoting germinal center B cell differentiation. In vitro, Plasmodium infection-induced CD138hiB cells suppressed CD4 T cell proliferation. Mechanistically, we linked the in vivo and in vitro suppressive activity of CD138hi B cells to their expression of the inhibitory ligand PD-L1. Together, our data identify that Plasmodium infection-induced plasmablasts suppress the induction of protective humoral immunity during experimental malaria. This knowledge can be used to develop immune-based therapies that limit or prevent innate plasmablast responses.
Human resistin promotes helminth infection-induced tissue inflammation through binding to Toll-like Receptor 4
Authors: Jang Jessica , Wang Spencer , Le Gros Graham S , Lazar Mitchell , Nair Meera G
Published: January 2023

While murine resistin-like molecules (RELM) can modulate the immune response following helminth infections, the relevance of human RELM proteins in helminth infection is unknown. In helminth-infected patients, we found that circulating human resistin is increased compared to non-infected individuals and positively correlated with parasite burdens and proinflammatory cytokines. To examine the function of human resistin, we utilize transgenic mice in which the human resistin gene was inserted. Following infection with the helminth Nippostrongylus brasiliensis (Nb), resistin expression was upregulated in infected tissue, contributing to exacerbated Nb-induced inflammation characterized by monocyte infiltration and production of proinflammatory cytokines. This increased inflammation resulted in elevated Nb egg burdens and delayed worm expulsion. Genome-wide transcriptional profiling of infected lung tissue revealed that resistin promotes expression of proinflammatory cytokines downstream of toll-like receptor signaling, leading us to investigate resistin signaling through TLRs. Using cell binding assays with lung cells, we show that resistin does not bind as efficiently to TLR4−/− monocytes compared to WT monocytes. Furthermore, resistin directly induced TNFα production in bone marrow derived macrophages, but this activation was abrogated in TLR4−/− macrophages. Together, these studies identify resistin as a detrimental factor induced by helminth infections, where it promotes proinflammatory cytokines and impedes parasite clearance through TLR4. Targeting the resistin/proinflammatory cytokine axis may provide new diagnostic or treatment strategies for helminth infection and associated pathology.
Activation of human keratinocytes by Leishmania spp.: divergent effects of Leishmania infantum versus Leishmania major.
Authors: Scorza Breanna , Wacker Mark , Messingham Kelly , Fairley Janet , Wilson Mary E
Published: January 2023

All Leishmania infections are initiated in skin, but clinical manifestations vary greatly with infecting species. Leishmania major (Lm) causes localized cutaneous lesions, whereas L. infantum (Li) causes potentially fatal disseminated disease without skin pathology. Early responses at the skin infection site influence adaptive responses to Leishmania, yet little is known of the role of keratinocytes. We hypothesized that Leishmania induce keratinocytes to produce factors influencing the immune response. Incubation of Li with primary or immortalized human keratinocytes caused a significant increase in pro-inflammatory cytokine transcripts il6, il8, tnfa, and il1b measured by RT-qPCR. However, keratinocytes exposed to five distinct Lm isolates did not induce these transcripts, highlighting a species-specific difference in inflammatory response. Similar to live parasites, Li-derived exosomes induced more il8 mRNA compared to control (p<0.01) or Lm-derived exosomes (p<0.05). Western blotting confirmed NFkB-p65 activation in keratinocytes exposed to Li but not Lm. To examine whether soluble keratinocyte factors influence nearby immune cells, Li-infected human monocytes were cocultured with keratinocytes across a transwell membrane. Soluble products of Li-exposed keratinocytes improved monocyte control of parasite replication compared with unexposed controls (p<0.01). However, culture with Lm-exposed keratinocytes across the transwell did not affect monocyte Lm infection. These data suggest that (1) activated keratinocytes may increase monocyte leishmanicidal activity and (2) keratinocytes support an early inflammatory environment, uniquely tailored to each Leishmania species, at the infection site.
Cellular and humoral immune response in human Chagas’ disease after treatment with Benzonidazol
Authors: Rodrigues Virmondes , Llaguno Mauricio Llaguno , Correia Dalmo , Silva Marcos Vinicius
Published: January 2023

Chagas’ disease (caused by Trypanosoma cruzi) remains a leading neglected tropical disease in the Americas, associated with 546,000 disability adjusted life years and 10,300 annual deaths. Even today there is a lack of studies proving the effectiveness and effects of treatment with benznidazole in chronic patients, as well as its effects on the cellular immune response. Therefore, we evaluate the clinical and immunological changes in chronic chagasic patients treated with benzonidazol after 48 months of therapy. We assessed the variation of anti T. cruzi IgG antibodies and their isotypes, before and after treatment and the production of cytokines and immunophenotypic profile of T lymphocytes after treatment with benzonidazol. We observed that the treatment did not alter the clinical outcome. In patients with cardiac form or with clinical deterioration was observed a decrease of IgG3 specific antibody and an increase in T. cruzi specific IgG1 antibodies. In patients with clinical deterioration, there was a decrease of Th2, Th17 and Treg cells in cultures stimulated with mitogens. We conclude that, in the chronic phase of Chagas’ disease, after 4 years of treatment, a higher number of Th17 and Treg cells are associated with no disease progression.
Follicular dendritic cells modulated by formyl peptide receptor ligands are capable of enhancing the germinal center reaction in mucosal immune system
Authors: Jang Yong-Suk , Kim Sae-Hae , Kim Ju , Kim Yu Na , Park Jisang
Published: January 2023

In various vaccination protocols, induction of germinal center (GC) is one of the critical factors involved in successful immune induction because GC formation results in the production of high affinity antibodies via B cell clonal expansion, somatic hypermutation, and affinity-based B cell selection. Although GC formation in Peyer’s patch (PP) is slowly and spontaneously induced by intestinal microbiota, it is unknown whether follicular dendritic cells (FDCs) in PP directly contribute to the antigen-specific immune response induction. In this study, we found the expression of formyl peptide receptor (FPR-2), a receptor for cathelicidin LL-37 which could play a role as an adjuvant in mucosal vaccine, on FDCs. The interaction between LL-37 and FPR-2 on FDCs promotes the expression of CXCL13 which is closely associated with the GC formation. Based on these findings, we conclude that the adjuvant role of LL-37 in mucosal immune induction is closely associated with the enhancement of GC reaction in PPs by FDC stimulation.
Germinal responses in murine Peyer’s Patches are regulated by a subpopulation of microbiota-induced IL-21high Tfh cells
Authors: Jones Leigh A , Ho Wen Qi , Ying Sze , Ramakrishna Lakshmi , Srinivasan Kandhadayar G , Yurieva Marina , Ng Wan Pei , Subramaniam Sharrada , Hamadee Nur H , Joseph Sabrina , Dolpady Jayashree , Atarashi Koji , Honda Kenya , Zolezzi Francesca , Poidinger Michael , Lafaille Juan J , de Lafaille Maria A Curotto
Published: January 2023

IL-21 is the signature cytokine produced by T follicular helper (Tfh) cells and is vital in driving both the germinal centre (GC) reaction and formation of high affinity antibodies. However, the degree of Tfh cell heterogeneity and function is not fully understood. By utilising a novel IL-21eGFP reporter mouse carrying a diphtheria toxin receptor (DTR)-enhanced green fluorescent protein (eGFP) fusion gene, we identified a subpopulation of highly differentiated Tfh cells within Peyer’s Patches (PPs). This subpopulation demonstrated highest expression of the key Tfh molecules - Bcl6, ICOS and IL-21, and was found within germinal centres and surrounding B cell areas. TCRb repertoire analysis of GFP+Tfh cells revealed that these were polyclonal and closely related to GFP− Tfh cells, indicating selection by common antigens. In vitro stimulation of IL-21eGFP CD4+ cells in the presence of IL-6, TGFb and retinoic acid led to the induction of GFP+ cells that did not co-express IL-17 or Foxp3. Treatment of IL-21eGFP mice with antibiotics led to an ablation of PP GFP+CD4+ cells meaning that the presence of these cells relies on an intact bacterial microbiota. Furthermore, both polyclonal CD4+ T cell and B cell activation is necessary to support the differentiation of GFP+Tfh cells. Finally, GFP+ cell depletion resulted in a reduced frequency of PP GC B cells and IgG1+ cells and small but significant shifts in gut microbiome composition. Therefore, using a new IL-21-reporter mouse we have identified a subpopulation of Tfh cells induced by the gut microbiota and necessary for optimal PP GC and IgG1 responses.
Nanoimaging granule dynamics and subcellular structures in activated mast cells using soft X-ray tomography
Authors: Chen Huan-Yuan , Chiang Meng-Lin , Lin Zi-Jing , Guttermann Peter , Schneider Gerd , Lai Lee-Jene , Liu Fu-Tong
Published: January 2023

Mast cells play an important role in allergic responses. During activation, these cells undergo degranulation, a process by which numerous mediators stored in the granules are released. Granule homeostasis in mast cells has mainly been studied by electron microscopy (EM), where the fine structures of subcellular organelles are partially destroyed during sample preparation. Migration and fusion of granules have not been studied in detail in three dimensions (3D) in unmodified samples. Here, we utilized soft X-ray tomography (SXT) coupled with fluorescence microscopy to study the detailed structures of organelles during mast cell activation. We observed granule fission, granule fusion to plasma membranes, and small vesicles budding from granules. We also detected lipid droplets, which become larger and more numerous as mast cells become activated. We observed dramatic morphological changes of mitochondria in activated mast cells and 3D-reconstruction revealed the highly folded cristae inner membrane, features of functionally active mitochondria. We also observed giant vesicles containing granules, mitochondria, and lipid droplets, which we designated as granule-containing vesicles (GCVs), and we verified their presence by EM in samples prepared by cryo-substitution, albeit with a less clear morphology. Thus, our studies using SXT provide significant insights into mast cell activation at the organelle level.
An update on IgBLAST, a tool for Ig and TCR sequence analysis
Authors: Ye Jian
Published: January 2023

The variable domain of B and T cell receptor is encoded by multiple genes, including the variable (V) gene, the diversity (D) gene and the joining (J) gene. Previously we developed an analysis tool, IgBLAST, for immunoglobulin sequences (http://www.ncbi.nlm.nih.gov/igblast/). Here we describe our recent update on this tool. The new functionalities include capability to analyze T cell receptor sequences (TR), annotating the CDR3 region, extending alignment to 5′ end of the V region, more flexibility in search criteria and providing summarized information to aid large-scale sequence analysis. We use examples to demonstrate these new functionalities and their implications in Ig and TCR sequence analysis.
Antibody immunotherapy targeting a novel B-cell lymphoma secreted biomarker
Authors: Sutkowski Natalie A , Bouchard Mollie , Bryant John Matthew , Sun Wei , Haque Azizul
Published: January 2023

We have used a patented (US 8,745,713) in vitro antibody platform technology to isolate fully human antibodies against a novel B cell lymphoma secreted biomarker, which is an isoform of the Fc Receptor-Like (FCRL) protein family. The FCRL isoform appears to inhibit CD4+ T cell recognition, and consequently would obstruct T cell immunosurveillance of B cell lymphoma. Targeted antibody immunotherapy is now a first-line treatment of B cell lymphoma due to the success of rituximab, a chimeric antibody that binds to and eliminates cells expressing the pan-B cell marker CD20. We envision that antibody targeting the secreted FCRL isoform would have a completely different mechanism of action from antibodies that bind to and eliminate cells expressing tumor antigens. Instead, anti-FCRL would be more analogous to antibodies that target immunoinhibitory molecules like CTLA4, or PD1 and its ligand PD-L1. Antibodies blocking these inhibitory interactions restore T cell function, ostensibly by promoting T cell priming in the case of CTLA4 blockade, and by preventing T cell exhaustion during PD1/PD-L1 blockade. Antibody targeting the FCRL lymphoma biomarker is instead expected to boost CD4+ T cell antigen presentation by DC and other professional APC, allowing for more robust T-cell anti-tumor responses. Furthermore, targeting the secreted FCRL isoform is not expected to have serious side effects like tumor lysis syndrome, nor affect the patients’ normal B cells. Moreover, since the FCRL biomarker was identified in serum of lymphoma patients, a clinical diagnostic screening assay could readily be developed to monitor for tumor progression and treatment response, as well as, identify candidate patients for FCRL targeted antibody immunotherapy.
True Sensitivity of Immunoassays: Are Concentrations of Low Abundance Analytes Real or Artifacts?
Authors: Oberoi Pankaj , Suschak Jessica , Stenglin Martin , Aghvanyan Anahit , Nikolenko Galina , Roy Daisy , Gardner Louis , Lockney Dustin , Shukla Animesh , Porter Erica , Miller Thomas , Biesso Adrianna , Sardesai Laukik , Sivakamasundar Ramou , Mathew Anu , Glezer Eli N , Wohlstadter Jacob N
Published: January 2023

We developed immunoassays with fg/mL detection limits, 100 to 1,000X more sensitive than current ELISA technology. We measured concentrations of low-abundance cytokines and other biomarkers in the serum of healthy donors, and found that these analytes are present at lower concentrations than had been reported in previous studies. This raised the question of how to demonstrate the reliability of immunoassay measurements for analytes present at sub-pg/mL concentrations. To establish whether measured concentrations are due to the true analyte or an artifact, we depleted the analyte from clinical samples by using high quality antibodies conjugated to Dynabeads. We hypothesized that high abundance serum or plasma components that bind non-specifically to immunoassay components would not be depleted, and that the true analyte would be depleted by the antibodies depending on affinity.

IL-2, IL-4, IL-6, IL-10, IL-17A, IL-21, GM-CSF, TSLP, TNF-a, Cardiac Troponin I, and Tau were used as model analytes. The limits of detection achieved for these S-PLEX™ assays ranged from ~0.2 to ~20 fg/mL. Spike recovery and dilution linearity were between 80% – 120%. Depletion experiments were performed with up to six antibodies per assay. All samples could be depleted by high quality antibodies, including antibodies that were not part of the assays. This indicates that the assays measured the true analyte.

In conclusion, using depletion experiments, we demonstrated that our assays are highly specific and measure the correct analytes. This test of “True Sensitivity” can be applied to all assays claiming to measure low abundance biomarkers.
Using transgenic expression of a MHC Class II/peptide restricted T cell receptor in mature CD8+ T cells to target auto-antigen decorated antigen presenting cells for destruction
Authors: Lees Jason R , Nurmukhambetova Saule , Kim Yong Chan , Zhang Ai-Hong , Scott David W
Published: January 2023

CD4+ T cells are thought to be necessary for MS neuroinflammation, coordinating the destructive immune response following recognition of central nervous system (CNS) antigens in the context of MHC class II molecules on professional antigen presenting cells (APC). Recognition of CNS antigens within the CNS is required to drive CD4 re-activation and subsequent production of cytokines and chemokines crucial to the recruitment of secondary immune effector cells. This CD4+ T cell driven recruitment in turn results in induction of immune pathology while also priming APC to enhance future T cell-APC interactions. Recent studies in cancer immunotherapy have defined protocols for inducing expression of chimeric antigen-specific T cell receptors (CAR) in mature CD8 T cells for the purpose of inducing destruction of antigen bearing cells, apparently without modulating the underlying effector profile of the transduced T cell (i.e., CD8 cells are still cytotoxic through perforin and granzymes). Here we detail the use of defined MHC class II/peptide restricted T cell receptor expression in mature CD8 T cells in an effort to target for destruction MHC class II expressing professional antigen presenting cells containing CNS antigen while sparing MHC II negative neurons and oligodendrocytes. Crucially, an effective antigen elimination strategy would not only modulate the cognate antigen recognized by the transgenic TCR but, given the propensity of APC to present multiple antigens derived from the same phagocytized material, should also impact uncharacterized CNS antigen epitopes.

This study examines a novel potential therapeutic modality for its efficacy in neuroinflammation. Supported by NMSS pilot grant PP3361.
Selective inhibition of matrix metalloproteinase-9 reduces clinical severity in a murine model of Multiple Sclerosis via a reduced immune response
Authors: Onwuha-Ekpete Lillian C. , Tokmina-Roszyk Dorota , Fields Gregg B.
Published: January 2023

Matrix metalloproteinases (MMPs) are a family of proteolytic enzymes that mediate the degradation of extracellular matrix components. Their functions are essential for normal physiological processes such as wound healing, but their dysregulation is associated with various pathologies including Multiple Sclerosis (MS). During MS, CD4+ T-cells activated in the periphery penetrate the blood-brain barrier (BBB), recruit immune cells, initiate destruction of the myelin sheath, and cause axonal loss. Experimental Autoimmune Encephalomyelitis (EAE) is a well-established murine model of MS. In EAE, MMP-9 is required for penetration of the BBB and generation of auto-antigens. Recent studies have demonstrated that MMP-9 contributes to normal intracellular function of various cell types including antigen activated T-cells; however, the intracellular role of MMP-9 in immune cell activation during EAE pathogenesis is not known. In our in vitro studies of immune cells we assessed their response to mitogen following treatment with an MMP-9 inhibitor (MMP-9I). We demonstrated that the MMP-9 intracellular activity is important for homeostatic maintenance as well as a robust immune response. Our in vivo studies using MMP9I resulted in reduced disease severity and a reduction in key EAE hallmarks, weight loss and clinical score. Furthermore, proliferation of immune cells in the periphery following disease induction was reduced and the cellular infiltrates across the BBB. These results further emphasize the importance of MMP-9 as a therapeutic target in autoimmune diseases in addition to its well-established extracellular roles. Thus, it presents a two pronged approach to the therapeutic treatment of autoimmunity.
BBB-permeable peptide conjugated cytoplasmic domain of CTLA-4 inhibits Th1 and Th17 responses and pathogenesis of multiple sclerosis
Authors: Lim Sangho , Kim Won-Ju , Kim Yeon-Ho , Lee Sohee , Koo Ja-Hyun , Lee Jung-Ah , Kim Hye-Mi , Park Hong-Jai , Kim Do-Hyun , Lee Hong-Gyun , Yoon Heeseok , Kim Ji Yun , Shin Jae Hun , Kim Lark Kyun , Doh Junsang , Kim Hongtae , Bothwell Alfred L , Lee Sang-Kyou , Suh Minah , Choi Je-Min
Published: January 2023

Multiple sclerosis (MS) is one of the most severe autoimmune disease, which cause severe inflammation in central nervous system (CNS). In the MS, CNS-infiltrating effector T cells are regarded that play critical roles. However, current drugs for MS could not targeting infiltrated T cells due to the limitations of drug delivery, which cannot penetrate blood-brain barrier (BBB) and deliver into the CNS. Here, we identified a novel BBB-permeable peptide, dNP2, which effectively delivered proteins into the CNS in vivo. It transduced cargo proteins into the cells with higher efficiency than other cargo delivery peptides. Moreover, it localized in resident neuron, astrocytes and microglia of the mouse brain through blood vessels by penetrating the BBB. Also, we found that dNP2 can deliver its cargo protein into the infiltrated T cells in the CNS of MOG35-55 immunized MS model mice. When we treated the dNP2-conjugated cytoplasmic domain of cytotoxic T lymphocyte antigen 4 (dNP2-ctCTLA-4), it efficiently downregulated cytokine production in activated T cells, and showed inhibitory impact on experimental autoimmune encephalomyelitis (EAE) in both preventive and therapeutic schemes. This was accompanied with reductions of demyelination and infiltration of T helper 1 (Th1) and T helper 17 (Th17) cells in the CNS. This study suggests that dNP2 is a novel BBB-permeable peptide and that dNP2-ctCTLA-4 could be an effective agent for treating CNS inflammatory diseases such as MS.