bluuebunny/crossref_metadata_2025 huggingface dataset:
In vivo PK/PD assessment of invisibilized IgG cleaving protease for chronic treatment of autoimmune diseases
Authors: Sanmarco Liliana , Pellerin Alex , Green Tobias , Newton Andita , Borah Minasri , Anderson Jordan , Mojta Shanell , Hollfelder Melissa , Higginson-Scott Nathan , Otipoby Kevin , Mascanfroni Ivan
Published: August 2023

Pathogenic autoantibodies are key effectors of inflammation, promoting complement cascade activation, immune cell response, and the consequent tissue damage in autoimmune disease and transplant rejection.

Proteases that cleave IgG antibodies present a novel therapeutic opportunity to treat autoimmune diseases and antibody mediated transplant rejection. Using our IMPACT platform leveraging machine learning, we engineered IgG proteases with reduced immunogenicity for acute and chronic treatment of antibody-mediated diseases. In addition, the invisibilized protease was fused to a Fc to extend its half-life.

To evaluate these IgG proteases in vivo we developed a pharmacokinetics and pharmacodynamics (PK/PD) model. C57BL6 mice were dosed with intravenous immunoglobulin (IVIg) at different time points after protease treatment. Protease and IgG levels were quantified by MSD 15 minutes before and two hours after IVIg injection, respectively. IgG cleavage was detected at early time points which correlated with higher systemic drug levels. Additionally, the Fc-fused protease was detected at later time points with increased systemic enzymatic activity, as compared to the enzyme domain only. Finally, we evaluated the potential of the Fc-protease to cleave antigen bound IgG at the tissue level in a mouse anti–glomerular basement membrane (GBM) induced nephritis model. In summary, our in vivo studies established a PK/PD relationship between exposure and cleaved IgG while showing the benefit of fusing the protease domain to a Fc.
Tissue kallikrein suppresses interferon responses and reduces depressive-like behavior in lupus-prone mice
Authors: Sriram Uma , Nocito Cassandra , Lubinsky Cody , Hand Michelle , Seliga Alecia , Persidsky Yuri
Published: August 2023

Recent studies show that systemic immune responses, including Type I IFNs, affect neuroinflammation and neuropsychiatric responses. Major depression is the most common psychiatric presentation of NPSLE. Reports indicate that neuropsychiatric symptoms, especially depression and anxiety, may be common co-morbidities of childhood-onset of SLE and clinical studies have demonstrated that IFNα can cause depressive symptoms. The management of patients with neuropsychiatric lupus (NPSLE) continues to be a major therapeutic challenge. We have shown that the kallikrein-kinin system (KKS) [comprised of kallikreins (klks), bradykinins (BKs)] and angiotensin converting enzyme inhibitors (ACEi’s), suppressed Type I IFN responses in murine dendritic cells (DCs) from normal and lupus-prone mice and human peripheral blood mononuclear cells (PBMC). Other studies have shown that klk genes are decreased in lupus patients; giving exogenous klk1 ameliorated kidney pathology in mice. We administered (retro-orbitally) adenovirus carrying the kallikrein gene (klk1- a widely expressed tissue klk) in the MRL/lpr lupus-prone mice (at early disease onset) and analyzed immune responses and depressive-like behavior response. Klk1 improved depressive-like behavior, suppressed Interferon Responsive Genes, decreased splenic plasmacytoid DCs, and reduced plasma IFNa levels. Together, these findings suggest that kallikreins and other KKS molecules may be used to control IFNa production/responses and other inflammatory responses in SLE and NPSLE.

This work was supported by NIH Grant R21AR071679 and funds from the Department of Pathology and Laboratory Medicine.
Dynamic single cell lineage recording of endogenous viral specific CD8 T cells reveals multiple potential cell fate pathways
Authors: Abdullah Leena , Emiliani Francesco , Vaidya Chinmay , McKenna Aaron , Huang Yina H
Published: August 2023

In response to viral infection, antigen specific naïve CD8 T cells expand to give rise to a heterogenous pool of effector cells consisting of short-lived effector cells (SLECs) and memory precursor effector cells (MPECs). While these effector populations are phenotypically and functionally well characterized, we still don’t fully understand how they arise from antigen responsive naïve CD8 T cells. We have combined progressive lineage recording with single-cell RNA sequencing and T cell receptor sequencing to delineate the early differentiation of OVA-specific endogenous CD8 T cells in response to acute VSV infection. Transcriptional profiling of CD8 T cells captured at the peak of T cell response confirmed 8 distinct T cell states including a unique interferon responsive cluster. RNA Velocity trajectory analysis supported an asymmetric model of CD8 T cell differentiation where early effector cells gave rise to SLECs while MPECs differentiated into further memory precursors. Moreover, our CRISPR/Cas9-based lineage recorder uncovered T cell clones of various sizes and allowed us to follow up to 5 generations of differentiating CD8 T cells. We observed that expanded clones comprised of memory and effector CD8 T cells while medium size clones preferred memory or effector fate, suggesting that different clones follow different differentiation models. Thus, using RNA-seq based trajectory analyses and a dynamic lineage recorder we uncovered potential differentiation pathways taken by early viral specific CD8 T cells. Our single cell full length TCR-seq will further add to our current models and highlight TCR sequences with better memory potential in response to infection.

Supported by grants from NIH (R01 AI089805, R01 CA254042) and a training fellowship from the Burroughs Wellcome Fund.
Maintenance of the naive T cell population during malnutrition is associated with residency in the bone marrow
Authors: Foster Takesha R , Dadzie Kwesi A , Moore Lindsay , Saravanan Rithanya , Adams Olivia , Washington Aja , Gubbels Bupp Melanie R
Published: August 2023

In mammals, T cell migration is under circadian control, likely to anticipate daily rhythms in infection risk. Glucocorticoids control this process, and malnutrition is associated with increased glucocorticoid levels. Therefore, we evaluated whether malnutrition disrupts the circadian migratory patterns of T cells. Malnutrition did not impact circadian patterns of T cell residency of lymphoid tissues; indicating that fluctuations, rather than specific concentrations, of glucocorticoids are a key circadian signal. Additionally, the total number of CD4+ and CD8+ T cells in the lymph nodes and blood were lower in malnourished as compared to well-nourished mice. However, the percentage and total number of naïve T cells was maintained in the lymph nodes, blood, and spleen of malnourished mice, suggesting preferential preservation of naïve T cells. Interestingly, the percentage and total number of CD4+ and CD8+ T cells in the bone marrow was elevated significantly in mice on a malnourished diet. Additionally, malnourished CD4+ and CD8+ T cells in the bone marrow showed significantly high CCR7 expression and CCL21 expression was increased in malnourished bone marrow compared to control. CCR7 and its chemokine, CCL21, may be responsible for trafficking malnourished T cells to the bone marrow during malnutrition. Overall, these findings suggest that the bone marrow may contribute to naïve T cell preservation during malnutrition.

NSF-MRI [DBI- 1920116] NSF -RUI [IOS-1951881]
Plasticity of clonal memory CD8 T cell differentiation
Authors: Hiam-Gálvez Kamir , Black Mollie , Raposo Colin J , Satpathy Ansuman T
Published: August 2023

Memory CD8 T cells provide highly specific and long-lasting protection against previously experienced pathogens. This potent protection is mediated by hundreds to thousands of unique clones with distinct T cell receptors (TCRs). Higher T cell clonal diversity and recognition of a wider range of pathogen antigens are associated with improved host protection. Therefore, understanding how clonal responses are organized is important for improving T cell function in a range of diseases. To comprehensively test the range of clonal memory T cell differentiation capacity, we deeply profiled polyclonal mouse memory CD8 T cells from acute lymphocytic choriomeningitis virus (LCMV) infection before and after viral rechallenge by paired single cell RNA and TCR sequencing. We found that individual memory T cell clone expansion and differentiation at the peak of the rechallenge response is highly variable. Clones are spread across various patterns of unique phenotypes ranging from highly cytotoxic to stem-like memory states. To quantify clonal differentiation plasticity, we transferred memory T cells into multiple new congenic recipients. Clonal progeny in different hosts acquired highly similar differentiation states suggesting that emergent memory clone behavior is imprinted by events during the primary infection. Indeed, transcriptional state of clones prior to rechallenge was sufficient to predict clone behavior upon rechallenge. This work provides a platform for understanding the mechanisms by which memory T cells make decisions and future studies will aim to perturb clones to elicit specific functions during a memory response.

Supported by grants from the Burroughs Wellcome Fund (Career Award for Medical Scientists [ATS]), Cancer Research Institute (Technology Impact Award, Lloyd J. Old STAR Award [ATS]), American Society of Hematology (ASH Scholar Award [ATS]), Parker Institute for Cancer Immunotherapy (ATS), Stanford School of Medicine (Propel Postdoctoral Scholarship [KHG]), National Institutes of Health (5T32AI007290-38 [CJR])
The age-related changes in lymph node stromal cells underlie defects in peripheral T cell maintenance and immune function decline in old mice
Authors: Sonar Sandip Ashok , Bhat Ruchika , Coplen Christopher P , Nikolich-Zugich Janko
Published: August 2023

Older adults are more vulnerable to infection and cancer, and poorly respond to vaccination. The structural stromal cells in the lymph nodes (LN) are essential regulators of T cell homeostasis and immune response. However, how does aging of LN stromal cells contribute to T cell maintenance defects and functional immunity is incompletely understood. Here we used single cell RNA-Seq of purified stromal cells from adult (3mo) and old (21mo) peripheral LN of C57BL/6 mice to characterize the age-related transcriptional differences in major stromal cell subsets. Our data revealed the enrichment of CCL19hi and CCL19low while diminution of Nr4a1+ and Inmt+ FRC in old LN. The enriched FRC in old LN exhibited a gene signature of cellular and oxidative stress response, mitochondrial defects, and negative regulation of immunity. The ex vivo functional characterization revealed that old LN stromal cells exhibit elevated mitochondrial reactive oxygen species coupled with increased mitochondrial mass and reduced membrane potential, indicative of mitochondrial defects during the senescence. Interestingly, these defects are observed in older but not mid-age LN. Furthermore, in vitro co-culture of LN stromal cells and purified naive T (TN) cells showed that old LN stromal cells poorly support CD8 TN cell survival. Finally, boosting the function of old LN stromal cells using agonistic anti-LTbR or mitigating oxidative stress using N-Acetyl Cysteine reversed this effect, via stabilization of BCL2 in TN cells. Thus, these results suggest that age-related adverse changes in LN stromal cells contribute to defects in maintenance of TN cells, and highlight that these defects could be reversed to restore TN cell homeostasis and functional immunity.

This work is supported by grants from NIH (P01 AG052359 and R37 AG020719) to Dr. Janko Nikolich-Zugich.
Uncovering the role of STAT5 in CD8+ T cell fate
Authors: Chanda Monica Kasturi , Gu Zehui , Piper Taylor L , Shudde Claire E , Courtney Adam H
Published: August 2023

Long-term protection against infection and disease requires long-lived memory CD8+ T cells. The development of these memory CD8+ T cells requires a differentiation process influenced by cytokines – such as IL-2, IL-7, and IL-15 – that are known to mediate the balance between effector and memory populations. Interestingly, these cytokines act through the common activation of STAT5, however, the mechanisms by which STAT5 dictates CD8+ T cell fate is poorly understood. To elucidate how STAT5 affects CD8+ T cell differentiation, we have developed a strategy to activate endogenous STAT5 in the absence of cytokine. We compared CD8+ T cells cultured in IL-2, a cytokine known to promote effector CD8+ T cells, to those where STAT5 was directly activated. We found that direct activation of STAT5 resulted in decreased IFNγ production in comparison to IL-2 treatment. This observation led us to evaluate the expression of transcription factors and proteins related to effector, memory, and exhausted states. Our results demonstrate that CD8+ T cells that experience direct activation of STAT5 have higher levels of TCF1 and lower levels of granzyme B compared to CD8+ T cells cultured in IL-2. To gain an understanding of phenotypic changes and differentiation state, we performed bulk RNA sequencing to assess global changes in gene expression. Our RNA sequencing results indicate that when compared to CD8+ T cells cultured in IL-2, direct STAT5 activation causes increased expression of genes related to a memory phenotype and a corresponding decrease in those related to an effector or exhausted phenotype. Our findings suggest that STAT5 activation is critical to a memory-like phenotype and could be harnessed for therapeutic benefit in CD8+ T cell-based treatments.

Supported by grants from the V Foundation and the Concern Foundation
Identification of novel genes that regulate B-1 cells
Authors: Rosero Sara , Luo Zheng , Loyd Kent , Baumgarth Nicole
Published: August 2023

IgM is the first antibody produced in ontogeny and in response to an initial antigen exposure. It plays an important role in effective immune protection, and is required for maximal antigen-specific IgG responses. In mice, most circulating serum IgM is generated by fetal-derived B-1 cells in spleen and bone marrow. How B-1 cells preferentially develop into natural IgM secreting cells is incompletely understood. To identify candidate genes critical for B-1 cells development and/or maintenance we generated parts of a larger immunophenotyping database Knock Out Mouse Project (KOMP) providing results of the flow cytometric evaluation of spleen cells from 641 mouse strains, each containing a single-gene deletion as well as 200 individual congenic control mice, identifying 58 distinct leukocyte subsets for each spleen. We generated and curated a complete list of each gene found to be associated with B-1 cell changes. Seven genes were identified that significantly reduced frequencies of CD5+ B-1 cells in males and/or females, of which three were uniquely altering the B-1 cell subset. None of these genes have been associated with B-1 cell regulation previously. Using qRT-PCR we found that 4 showed B-1 cell intrinsic gene expression. Ongoing work is to determine the impact of each gene on serum antibody levels and immune protection to influenza virus challenge.

This work was supported by NIH UM1 OD023221 (KCKL), R0AI148652 (NB), and the Graduate Student Support Program UC Davis (SR).
Aberrant expansion of spontaneous splenic germinal centers induced by hallmark genetic lesions of aggressive lymphoma
Authors: Muppidi Jagan R , Pindzola Grace M , Razzaghi Raud , Tavory Rachel N , Nguyen Hang , Morris Vivian , Darbha Sriranjani
Published: August 2023

Diffuse large B cell lymphoma (DLBCL) is an aggressive heterogeneous clinical entity that is thought to be derived from germinal center (GC) B cells or cells that have recently passed through the GC. Unique molecular vulnerabilities have been identified in the aggressive MCD/C5 genetic subclass of DLBCL. However, the pre-malignant cell-of-origin exhibiting MCD-like dependencies remains elusive. In this study, we examined animals carrying up to four hallmark genetic lesions found in MCD consisting of gain-of-function mutations in Myd88 and Cd79b, loss of Prdm1 and overexpression of BCL2. We discovered that expression of combinations of these alleles in vivo promoted a cell-intrinsic accumulation of B cells in spontaneous splenic GCs. Like MCD, these pre-malignant B cells were enriched for B-cell receptors (BCRs) with evidence of self-reactivity, showed a de novo dependence on Tlr9 and were more sensitive to inhibition of BTK. Mutant spontaneous splenic GC B cells (GCB) showed increased proliferation and IRF4 expression. Mice carrying all four genetic lesions showed a greater than 50-fold expansion of spontaneous splenic GCs exhibiting aberrant histologic features with a dark zone immunophenotype and went on to develop DLBCL in the spleen with age. Thus, by combining multiple hallmark genetic alterations associated with MCD, our study identifies aberrant spontaneous splenic GCB as a likely cell-of-origin for this aggressive genetic subtype of lymphoma.

This research was supported by the Intramural Research Program of the NIH, CCR, NCI.
Borrelia burgdorferi c-di-AMP induces type I IFN response in macrophage through the activation of STING signaling pathway
Authors: Priya Raj , Raghunandanan Sajith , Alanazi Fuad , Yang Frank
Published: August 2023

Lyme disease (LD) which is caused by the spirochete Borrelia burgdorferi (Bb) poses a global health threat. Bb infection induces type I IFN response that is associated with arthritis and other symptoms. However, the ligands, PAMPs, and signaling pathways associated with type I IFN production by Bb are poorly characterized. Stimulator of Interferon Genes (STING) signaling pathway is an intracellular surveillance pathway that detects c-di-nucleotides (CDNs) produced by pathogens through STING and induces type I IFN production. Bb produces CDNs and hence it has the potential to activate the STING signaling pathway. In this study, the role of c-di-AMP in the elicitation of type I IFN response to Bb infection was elucidated. The RAW cell line was infected with Bb and activation of STING signaling was measured by detecting the activation of downstream proteins (pSTING, pTBK1, pIRF3 & pNF-κB), proinflammatory cytokines (IL6, TNF-α) and Type I IFN (IFN-β) response using ELISA, western blotting, immunostaining, and Luciferase Reporter assay. Bb infection to macrophage results in c-di-AMP mediated elicitation of type I IFN response. The induction level of type I IFN response was dependent on the level of c-di-AMP as the Bb mutant strain producing higher c-di-AMP level induced higher cytokine production. Interestingly, c-di-AMP released into the BSK-II medium during the culture of Bb could also stimulate the production of type I IFN response. The induction of type I IFN by c-di-AMP was dependent on STING protein as the RAW STING KO cell line showed a reduction in the level of IFN-β. In conclusion, our results show that Bb-produced c-di-AMP is a major PAMP that triggers type I interferon production in macrophage by activation of the STING signaling pathway.
Removal of the spleen abrogates the long-term hydrosalpinx pathology following Chlamydia genital infection
Authors: Konjufca Vjollca H , Essmyer Zana , Shillova Nita , Hyseni Besmir , Howe Savannah
Published: August 2023

C. trachomatis infections in women may cause pelvic inflammatory disease, chronic pelvic pain, and infertility. In the mouse model, genital Chlamydia disseminates systemically to infect the spleen. Removal of the spleen (splenectomy) abrogates hydrosalpinx pathology in the female reproductive tract (FRT), although it does not affect FRT Chlamydia clearance. Ascension of Chlamydia to the upper FRT (uteri, ovaries, oviducts) is critical for the development of hydrosalpinx. Over the course of genital infection, splenectomized mice exhibit significantly lower Chlamydia burden in the upper FRT and the GI tract compared to controls. Based on the report that Chlamydia-specific IgA secreted in the FRT protects against genital Chlamydia challenge, we hypothesized that splenectomy induces IgA secretion in the FRT. Examination of vaginal washes revealed that unlike controls, splenectomized mice exhibit Chlamydia-specific IgA by 4 weeks post-infection (wpi). However, the presence of IgA at this time did not explain the significant reduction in Chlamydia ascension observed by 2 wpi. Unlike controls, splenectomized mice additionally exhibit Chlamydia-specific IgM antibodies in vaginal secretions as early as 2 wpi. Naïve mice infected with Chlamydia pre-incubated with vaginal washes of splenectomized mice collected at 2 or 6 wpi exhibited reduced Chlamydia burden in the FRT, ILNs, spleen, and the GI tract at 3 and 7 days post-infection compared to controls. Secreted IgM and IgA thus neutralize Chlamydia, diminish its ascension in the upper FRT and thus abrogate the long-term hydrosalpinx pathology. This work reveals a previously unknown role that the spleen plays in the regulation of antibody secretion in the FRT mucosa.

Supported by a grant from NIH (1 R21 AI159743-01)
AIM2-mediated Immunity to Plasmodium Infection in Hepatocytes
Authors: Kurup Samarchith P
Published: August 2023

Malaria, caused by Plasmodium parasites is a severe disease affecting millions of people around the world. Plasmodium undergoes obligatory development and replication in the hepatocytes, before initiating the life-threatening clinical disease of blood-stage malaria. Although the innate immune responses that control Plasmodium infection and development in the liver are known to limit clinical malaria and transmission, the mechanism of such responses remain relatively unknown. Here we demonstrate that the DNA of Plasmodium is sensed by AIM2 (absent in melanoma 2) receptors in the infected hepatocytes, resulting in Caspase-1 activation. However, we observed that Caspase-1 undergoes an unconventional and incomplete proteolytic processing in such hepatocytes, resulting in the activation of the membrane pore-forming protein, Gasdermin D (GSDMD), without the concurrent activation of the inflammasome-associated pro-inflammatory cytokines such as interleukin (IL)-1 and IL-18. Nevertheless, the AIM2-mediated Caspase-1 activation pathway induced the elimination of Plasmodium-infected hepatocytes and a resultant control of malaria in the liver.

Supported by grants from NIH (R01 AI168307)
Induction of protective interferon-β responses in murine osteoblasts following S. aureus infection and the use of nucleic acid nanoparticles to enhance bacterial killing
Authors: Johnson Morgan B , Afonin Kirill , Marriott Ian
Published: August 2023

Staphylococcus aureus is the primary causative agent of osteomyelitis, a severe inflammatory bone infection. Treatment for osteomyelitis includes long-term antibiotic treatment, surgical debridement of necrotic bone, or even amputation. Treatment is challenging due to an inability of antibiotics to penetrate bone tissue and resident bone cells that harbor intracellular bacteria, and the increased incidence of infections associated with antibiotic-resistant S. aureus strains. Following S. aureus infection, resident bone cells including osteoblasts produce immune mediators and bone regulatory factors. While type I interferons (IFNs) are best known for their antiviral effects, it is becoming apparent that they impact susceptibility to many pathogens. Here, we report elevated IFN-β levels in infected femurs in a mouse model of osteomyelitis. RNA Tag-Seq analysis of S. aureus infected osteoblasts showed elevated expression of mRNA encoding IFN-β and ISGs. We confirmed rapid and robust release of IFN-β protein production by osteoblasts following S. aureus challenge. Furthermore, we showed increased protein expression of ISGs by infected osteoblasts and demonstrate that this occurs secondary to the release of IFN-β by these cells. Importantly, we have determined that exposure of S. aureus-infected osteoblasts to IFN-β markedly reduces the viable bacteria harbored by these cells. Finally, we have demonstrated delivery of immunomodulatory nucleic acid nanoparticle (NANPs) scaffolds using lipid-based carriers to murine osteoblasts. These NANPs are promising agents that could induce protective IFNs during S. aureus infection of bone tissue and be further functionalized with antimicrobials to kill intracellular S. aureus.

This work was supported by grant 1R01AI170012-01 to IM and MBJ from the National Institute for Arthritis and Musculoskeletal and Skin Diseases.
Immune recognition of latent neuronal Toxoplasma gondii infection
Authors: Eberhard Julia N , Bunkofske Molly , Shallberg Lindsey , Hunter Christopher A
Published: August 2023

Toxoplasma gondii is a neurotropic parasite with high mortality rates in immunocompromised individuals. Successful therapies have been developed against the acute tachyzoite stage, but no treatment options exist to target the latent form of the parasite – bradyzoites that form cysts within neurons. It is thought that residence of cysts in neurons shelters T. gondii from immune recognition and clearance. However, a decline in cyst numbers over time suggests the presence of anti-cyst immunity. Additionally, neuronal deletion of STAT1 results in increased cyst size and number, but no change in tachyzoite replication, providing evidence for IFN-γ-mediated cyst control. T cells are a major source of IFN-γ and are crucial for parasite control in the CNS, but it remains unclear if T cells directly target cyst-infected neurons. The use of transgenic parasites with bradyzoite-restricted OVA expression (BagOVA) reveals that cyst-derived antigen can induce OT-I CD8+ T cell responses in the brains of chronically infected mice. These cyst-specific OT-Is have distinct effector phenotypes and cytokine production when compared to T cells responding to antigen expressed by both tachyzoites and bradyzoites (ptubOVA). To determine whether neurons directly present cyst antigen to induce CD8+ T cell responses in BagOVA infection, mice with neuronal-specific loss of MHCI were generated. Infection of these mice showed no significant change in the magnitude of T cell responses or parasite burden in the CNS. Together these findings reveal that T cell responses can be mounted against cyst antigens, but another cell type likely mediates cross presentation of cyst derived antigens.

Supported by grants from NIH (R01 580-5803-4-580697-XXXX-2000-3000)
Pulmonary Immunopathogenesis of Granulibacter bethesdensis
Authors: Sharma Atul , Chauhan Arun , Ramesh Ashwin , Mishra Bibhuti B , Sharma Jyotika
Published: August 2023

Granulibacter bethesdensis (Gb) is a Gram negative bacterial pathogen reported to cause disease exclusively in patients with Chronic Granulomatous Disease harboring mutations in one of the five subunits of NADPH oxidase complex. The pathogen has been shown to cause chronic as well as recurrent infections in CGD patients that can be fatal. Although systemic and septic infection have been reported, the pathogenesis of Gb in respiratory microenvironment is completely unknown. Here we compared the disease progression and infection outcome of wild-type (WT) and murine CGD model Gp91−/− mice following intranasal infection with a clinical isolate of Gb. We found that compared to the WT mice, Gp91−/− CGD mice are highly susceptible to respiratory infection with Gb. While the WT mice displayed transient increase in inflammatory cytokines and moderate leukocyte infiltration characteristic of a mild, resolving infection, CGD mice displayed hypercytokinemia but attenuated antimicrobial function of phagocytic cells. A significant upregulation of Type 2 inflammatory cytokine IFN-γ in lungs Gb infected gp91−/− prompted investigation the its role in GB pathogenesis. Neutralization of this cytokine mitigated the inflammatory response and reduced bacterial burden in infected Gp91−/− CGD mice resulting in significantly improved overall survival. Mechanistically, IFN-γ neutralization improved antimicrobial function of neutrophils. Taken together, our findings suggest that in the absence of a functional superoxide production machinery, G. bethesdensis is capable of causing a chronic respiratory infection with fulminant sepsis and IFN-γ produced during the infection has a detrimental effect on the bacterial clearance.
DUSP8 induces TGF-β-stimulated IL-9 transcription and Th9-mediated immune responses by inactivating a transcriptional repressor
Authors: Chuang Huai-Chia , Tan Tse-Hua
Published: August 2023

Dual-specificity phosphatase 8 (DUSP8) is a MAPK phosphatase that dephosphorylates and inactivates the kinase JNK. DUSP8 is highly expressed in T cells; however, the in vivo role of DUSP8 in T cells remains unclear. Using T-cell-specific DUSP8 conditional knockout (T-DUSP8 cKO, DUSP8 f/f; CD4-Cre) mice, mass spectrometry, and chromatin-immunoprecipitation sequencing, we found that DUSP8 stimulated interleukin-9 (IL-9) gene expression and Th9 differentiation. TGF-β stimulated DUSP8 phosphatase activity in T cells. Mechanistically, DUSP8 dephosphorylated a transcriptional repressor upon TGF-β signaling, leading to the nuclear export of this transcriptional repressor and subsequent IL-9 transcriptional activation. Reduction of IL-9 mRNA levels in T cells of T-DUSP8 cKO mice was reversed by knocking out the transcriptional repressor in double knockout mice. Furthermore, IL-9 mRNA levels were induced in T cells lacking the transcriptional repressor. In addition, T-DUSP8 cKO mice displayed a reduction of IL-9 and Th9-mediated immune responses of allergy, autoimmune responses, and anti-tumor immunity in animal models. In contrast, the reduced allergy phenotype in T-DUSP8 cKO mice was reversed by knocking out the transcriptional repressor in double knockout mice. Collectively, DUSP8 induces TGF-β-stimulated IL-9 transcription and Th9-mediated immune responses by promoting the nuclear export of a transcriptional repressor.
Identification of a novel interleukin-9 receptor complex in non-hematopoietic cells
Authors: Cheung Cheuk Lam , Kaplan Mark H
Published: August 2023

Beyond cell-cell contact, communication at a distance requires the secretion of cytokines that bind to specific receptors on responsive cells. Interleukin (IL)-9 is a cytokine with pleiotropic effects not only on immune cells that contribute to the progression of tumor and autoimmunity, but also on structural cells including airway and intestinal epithelial cells involved in the development of inflammatory and allergic diseases. Typically, IL-9 receptor (IL-9R) functions as a complex of two subunits – the α-chain (IL-9Rα) and a common γ (γc) chain, that is shared with other cytokine receptors. Although structural cells express IL-9Rα, they lack expression of the γc chain. Thus, it is still unclear how IL-9 transmits a signal through a single IL-9Rα chain in non-hematopoietic cells. This study aimed to explore the possibility that like the type II IL-4Rα, the IL-9Rα chain can pair with the IL-13Rα1 chain, forming a type II IL-9R and can mediate IL-9 responses in epithelial cells. We demonstrated that the IL-9Rα/IL-13Rα1 complex was detected in situ in mouse tracheal epithelial cells and human small airway epithelial cells (SAEC) using proximity ligation assay. STAT3 was activated after exposure to IL-9 in vitro in SAEC when co-cultured with neutralizing antibody targeting γc chain but STAT3 activity returned to its basal level in the presence of anti-IL-13Rα1 antibody, providing further evidence of a type II IL-9R on epithelial cells. Strikingly, asthma-associated genes are upregulated in SAEC upon IL-9 stimulation in RNA-seq analysis. Collectively, our results identify a putative receptor complex - a type II IL-9R composed of IL-9Rα and IL-13Rα1, providing a mechanism for responses of structural cells to IL-9 and a potential role in asthma.
IL-2 rescues impaired T cell function upon loss of Itk via metabolic reprogramming
Authors: Kaul Zenia , Gomez-Rodriguez Julio , Golec Dominic P , Reilley Julie , Schwartzberg Pamela L
Published: August 2023

IL-2 inducible T cell kinase (Itk) is a critical component of T cell receptor (TCR) signaling required for activation of PLCg and downstream transcription factors, that regulate expression of effector molecules. Loss of Itk impairs T-cell activation by modulating the strength of TCR signal. Itk−/− mice have defects in T cell development with decreased proliferation and impaired differentiation to multiple CD4 T helper lineages. Most severe defects are seen in differentiation of Th9 cells, which produce the cytokine, IL-9. Under Th9 differentiation conditions, Itk−/− cells initially appear similar to WT cells but at 72hrs fail to sustain long-term expression of effector molecules. Th9 differentiation could be rescued by inclusion of IL-2. Itk−/− CD4 T cells show decreased mTORC1 signaling and do not maintain expression of IRF4, c-Myc, pRb and multiple nutrient transporters, including the Glucose transporter 1 (GLUT1), transferrin receptor (CD71), and amino acid co-transporter (CD98), all of which were rescued by IL-2. IL-2 is not required initially but can be added as late as 48hrs into culture. Using SCENITH—a flow cytometry-based technique that profiles metabolism—we found that WT differentiated Th9 cells were primarily glycolytic. In contrast, Itk−/− Th9 cells were less synthetic and more dependent on mitochondrial respiration. Inclusion of IL-2 in Itk−/− cultures rescued metabolic profiles, comparable to WT cells. Our results suggest that metabolic flexibility is central to dictating the functional/effector fate of CD4 T cells and that the Tec kinase Itk and IL-2-mediated pathways are required for maintenance of metabolic integrity, providing novel insight into mechanisms by which IL-2 can rescue T cell functional defects.

This work was supported by the Intramural Research Program of NIAID, NIH.
Quantitation of low abundant cytokines in human blood samples using SMC® high sensitivity immunoassays
Authors: Hwang Joseph B , Walden Zara , Banerjee Munmun , Gilliam Brooke , Xiao Qiang
Published: August 2023

Quantitation of biomarkers in blood is crucial for the understanding of inflammatory diseases, which can lead to exploration into new therapeutic applications. Although the mechanism of disease pathogenesis may be unknown, pro-inflammatory cytokines (IL-1, IL-6, IL-17, IL-18, IL-23, IFN-γ, and TNFα) and anti-inflammatory cytokines (IL-4, IL-10, and IL-13) have been implicated in the pathogenesis of a variety of diseases. In basic research, standard immunoassays, such as an enzyme-linked immunosorbent assay (ELISA), have provided valuable understanding of both normal and pathological processes for many decades. However, in more recent decades, new immunoassay technologies have uncovered numerous biomarkers, such as cytokines, in blood that were once undetectable using traditional ELISAs. In this study, we have developed high-sensitivity immunoassays, using Single Molecule Counting (SMC®) technology, to meet this new challenge of quantifying low abundant proteins in blood. The SMC® IL-4, IL-17A and IL-23 assays consistently demonstrate LLoQ of 0.04 pg/mL, 0.03 pg/mL, and 0.1 pg/mL, respectively. More importantly, these assays were able to accurately quantify cytokines in healthy and patient samples, which were once not quantifiable using traditional ELISAs. Most notably, the dynamic range of these ultrasensitive SMC® kits allowed quantitation of both healthy and disease blood samples in a single assay. In summary, SMC® high sensitivity cytokine immunoassay kits can provide a powerful non-invasive biomarker tool in studying inflammatory diseases.
Human CCR6+ Th cells show both an extended stable gradient of Th17 activity and imprinted plasticity
Authors: Singh Satya , Parween Farhat , Edara Nithin , Zhang Hongwei H , Chen Jinguo , Otaizo-Carrasquero Francisco , Cheng Debby , Oppenheim Nicole A , Ransier Amy , Zhu Wenjun , Shamsaddini Amirhossein , Gardina Paul J , Darko Samuel W , Singh Tej Pratap , Douek Daniel C , Myers Timothy G , Farber Joshua M
Published: August 2023

Th17 and related (type 17) cells have been investigated in mice for their contributions to autoimmune diseases and have demonstrated roles in immune-mediated disorders and host defense in humans. However, the pathways of differentiation of type 17 cells and the structure and heterogeneity of the type 17 memory population in humans are not well understood; such understanding is critical for manipulating these cells in vivo. By exploiting differences in levels of surface CCR6 and analyzing cells in bulk and as single cells, we found that human type 17 memory cells, including individual T cell clonotypes, form an elongated continuum of type 17 character along which cells can be driven by increasing the signature regulator, RORγt. On this continuum, cells are marked by extensive but progressively diminishing co-expression of genes of alternative lineages. Single-cell analysis of cytokine gene expression also revealed that Th cells initially undergo pan-activation of effector pathways rather than choose one lineage, and that these pan-activated cells persist in the memory population with multi-lineage character - reflecting both an efficient mechanism for jump starting an initial immune response and the availability of multi-potential cells in case of a repeat challenge. Along the type 17 continuum, the cells’ phenotypes and epigenomes are stable across cell divisions under homeostatic-like conditions. Nonetheless, strong activation in non-polarizing and polarizing environments can induce additional functionalities by both previously imprinted and environment-induced mechanisms that contribute differentially across the continuum to yield the unusual plasticity ascribed to even highly differentiated type 17 cells.

This study was supported by the Intramural Research Program of NIAID, NIH.
Epicutaneous Staphylococcus aureus exposure triggers lung inflammation via epithelia-intrinsic IL-36R signaling
Authors: Nolan Sabrina J , Saito Yoshine , Dikeman Dustin A , Pontaza Cristina , Ahmadi Michael , Youn Christine , Zhang Jing , Archer Nathan K
Published: August 2023

Allergic diseases have been increasing recently and affect nearly 20% of the human population, including 200 million people with atopic dermatitis (AD), 250 million people with food allergies, and 600 million people with allergic rhinitis or asthma. The atopic march is the concept whereby having early-in-life AD and a dysfunctional skin barrier during initial allergen sensitization increases the predisposition of allergic diseases at distant epithelial sites (e.g., asthma). We have previously shown that IL-36R signaling was required to exacerbate the progression from AD-like skin inflammation to subsequent allergic lung inflammation in mice. However, whether IL-36R acts directly on the skin and/or lung epithelia to trigger the progression towards lung inflammation is not entirely clear. To address this gap in knowledge, we performed our allergic lung inflammation model in WT, IL-36R−/−, keratinocyte-specific IL-36R deficient (K14-cre×IL-36Rfl/fl), and lung epithelial-specific IL-36R deficient (Nkx2-1-cre×IL-36Rfl/fl) mice, whereby first epicutaneous Staphylococcus aureus and cockroach antigen (CrA) exposure initiates AD-like skin inflammation, which is followed by intratracheal CrA exposure to trigger subsequent allergic lung inflammation. Evaluation of skin and lung inflammation by image, histologic and flow cytometric analyses revealed that both keratinocyte- and lung epithelial-intrinsic IL-36R signaling were required for the development of lung inflammation, weight loss, and neutrophil lung infiltration despite no differences in circulating neutrophils. Collectively, our findings suggest that IL-36R signaling in skin and lung epithelia is a critical mechanism for the progression of the atopic march.

NIAMS: R01AR073665 and LEO Foundation: LF-OC-22-000953; SJN is funded by NIAMS 1T32AR074920
Dictionary of immune responses to 86 cytokines in vivo at single-cell resolution
Authors: Cui Ang , Huang Teddy , Li Shuqiang , Ma Aileen , Perez Jorge , Sander Chris , Keskin Derin , Wu Catherine , Fraenkel Ernest , Hacohen Nir
Published: August 2023

Cytokines mediate cell-cell communication in the immune system and represent important therapeutic targets. A myriad of studies have underscored their central role in immune function, yet we lack a global view of the cellular responses of each immune cell type to each cytokine. To address this gap we created the Immune Dictionary – a compendium of single-cell transcriptomic profiles of over 20 cell types in response to each of 86 cytokines in murine lymph nodes in vivo, representing over 1,500 cytokine-cell type combinations. A cytokine-centric view of the dictionary revealed that most cytokines induced highly cell type-specific responses. For example, the inflammatory cytokine IL-1β induced distinct gene programs in almost every cell type. A cell type-centric view of the dictionary identified multiple cytokine-driven cellular polarization states in each immune cell type, including previously uncharacterized states such as an IL-18-induced polyfunctional NK cell state. Based on the dictionary, we developed companion software, Immune Response Enrichment Analysis (IREA), for assessing cytokine activities and immune cell polarization from gene expression data, and applied it to reveal cytokine networks in tumors following immune checkpoint blockade therapy. Our dictionary generates new hypotheses for cytokine functions, illuminates pleiotropic effects of cytokines, expands our knowledge of activation states of each immune cell type, and provides a framework to deduce the roles of specific cytokines and cell-cell communication networks in any immune response.

This work was supported by the NIH Grant RM1HG006193 and an Adelson Medical Research Foundation Grant to N.H. This work was also supported by a Natural Sciences and Engineering Research Council of Canada (NSERC) Doctoral Fellowship, Whitaker Health Sciences Fund Fellowship, and Wellington and Irene Loh Fund Fellowship to A.C., and NCI Research Specialist Award (R50CA251956) to S.L.
GRK3 Promotes Ligand-mediated Endocytosis and Limits Signaling of C-C Chemokine Receptor 7 (CCR7)
Authors: Sanchez Anahi , Bassuk Daniel W , Bill Colin A , Vines Charlotte M
Published: August 2023

C-C chemokine receptor 7 (CCR7) binds to two different ligands, which are differentially internalized, and mediate distinct signaling pathways. G protein coupled receptor (GPCR) kinases (GRKs) can phosphorylate ligand activated GPCRs. Therefore, we sought to determine if GRKs functioned differentially following binding of CCR7 ligands, C-C Chemokine Ligand 19 (CCL19) or CCL21 to promote distinct signaling events. Both the CRISPRCas9-modified human T-cell line, (CEM-GRK3−/−), in which both loci were disrupted, and primary murine T-cells homozygously deleted for GRK3 (T-GRK3−/−), were unable to fully internalize ligand bound CCR7. To determine if homozygous loss of GRK3, affected migration we examined chemotaxis to CCL19 and CCL21. Primary T-GRK3−/− were unable to migrate in response to CCL19 or CCL21. To examine signaling we measured mobilization of Ca2+ in primary T-GRK3−/−. In contrast to Ca2+ mobilization in CEM parental cells, which revealed sustained release of approximately 10% Ca2+ for both ligands over a 5-minute span, we found that both ligands induced a three-fold increase in Ca2+ signaling, suggesting that GRK3 plays a role in limiting the Ca2+ mobilization in T-cells to allow for migration. Overall, these observations reveal a novel role for GRKs in CCR7 signaling pathways.

Supported by grant from NIH SC1-GM111172
The Ikaros zinc finger transcription factor Aiolos as a novel regulator of T cell migration
Authors: Leonard Melissa , Read Kaitlin A , Jones Devin M , Pokhrel Srijana , Warren Robert T , Oestreich Kenneth J
Published: August 2023

Chemokine signaling is an integral component of lymphocyte migration, activation, and survival. The chemokine receptor CXCR3 is highly expressed on CD4+ T helper 1 (TH1) cells, which play a critical role in the adaptive immune response against intracellular pathogens. While CXCR3 helps direct TH1 cells to sites of inflammation or infection, it has also been implicated in a multitude of autoimmune diseases and cancers. Thus, understanding the molecular mechanisms that regulate CD4+ T cell migration is imperative for preventing immunopathology. Here, we identify the Ikaros zinc finger (IkZF) transcription factor Aiolos (IkZF3) as a positive regulator of CXCR3 expression in CD4+ T cells in a murine influenza model. Initially, we show decreased numbers of Aiolos-deficient antigen-specific CD4+ T cells in the lung, which correlates with decreased CXCR3 expression. RNA-seq and ATAC-seq analyses of in vitro-generated WT and Ikzf3−/− TH1 cells revealed decreased Cxcr3 transcript and reduced chromatin accessibility at regulatory regions of Cxcr3 in the absence of Aiolos, respectively. Collectively, these data imply a novel role for Aiolos in positively regulating CXCR3 expression in TH1 cells. Given these findings, we seek to define the functional effects of Aiolos deficiency on T cell migration and the molecular mechanism(s) by which Aiolos directly and/or indirectly regulates migration. Future work is aimed at determining whether Aiolos affects CXCR3 expression through direct association or indirectly via impacts on other factors.

K. J. O. is supported by a grant from The National Institutes of Health (NIH) AI134972, as well as from The Ohio State University College of Medicine and The Ohio State University Comprehensive Cancer Center.
Single-cell RNA sequencing identifies distinct transcriptomic signatures between PMA/Ionomycin and aCD3/aCD28 agonistic antibodies stimulation on primary human T cells
Authors: Lee Jung-Ho , Lee Brian H , Nam Hyo Jeong , Jeong Soyoung , Kim Hyun Je , Park Chung-Gyu
Published: August 2023

Immunologists have activated T cells in-vitro using various stimulation methods including PMA/Ionomycin and anti-CD3/CD28 agonistic antibodies. PMA stimulates protein kinase C, which is followed by activation of NF-κB, and Ionomycin translocates to the plasma membrane and increases intracellular calcium levels, resulting in activation of NFAT. On the other hand, anti-CD3/CD28 agonistic antibodies can activate T cells through ZAP-70, which phosphorylate LAT and SLP-76 to activate T cells. Despite the two different in vitro T cell activation methods that have been utilized for decades, the differential effects on primary human T cells following chemical-based or antibody-based activation have not been comprehensively described. As single-cell RNA sequencing (scRNA-seq) technologies enable the dissection of gene expression at single-cell resolution unbiasedly, we decided to compare the transcriptome profiles of the non-physiological and physiologic activation methods on four independent human PBMC-derived T cells. Remarkable differences in expression of cytokine and their receptor’s transcriptomes were identified. Also we identify activated CD4 T cell subsets (CD55+) enriched specifically by PMA/Ionomycin activation. We found that expression of CD40L gene was elevated on PMA/Ionomycin-treated CD4 T cells while expression of OX40 gene was elevated on aCD3/aCD28 agonistic antibody-treated CD4 T cells. We believe this human T cell transcriptome atlas derived from two different activation methods will enhance our understanding of the in vitro T cell activation assays.