bluuebunny/crossref_metadata_2025 huggingface dataset:
Epidemic of COVID-Associated Mucormycosis in India and a road ahead
Authors: Sharma S. , Sharma P. , Chandra U. K. , Javed S. , Nambiar A. , Sharma V. K.
Published: February 2023

COVID-associated Mucormycosis (CAM) was a peak-hour crisis in India. The situation arose from an unprecedented ICU workload of critical COVID-19 cases, insufficient resources and the impaired immune status of the patient. We recommend strong preventive measures, chemoprophylaxis and Amphotericin-B nebulisation for the effective management of CAM.
Systemic lupus erythematosus presenting as acquired factor XIII deficiency and spontaneous subdural haemorrhage
Authors: Kirubaharan A. , Samarasingha S. A. P. N. , Kankanamge H. K. C. A. , Jayanaga W. W. L. A.
Published: February 2023

Factor XIII deficiency is a rare, potentially life-threatening bleeding disorder. While congenital disease is inherited in an autosomal recessive manner, acquired factor deficiency can be associated with autoimmune disease or malignancy. Clinical spectrum may range from mucocutaneous bleeding to intracerebral bleeding.
This case describes a 21-year-old woman who initially presented with low grade fever and cervical lymphadenopathy. In two weeks’ time she developed bilateral spontaneous subdural haemorrhages along with bilateral small and large joint arthritis. Investigations revealed an acquired factor XIII deficiency, positive antinuclear antibodies (ANA) titer and Coomb’s positive autoimmune hemolytic anaemia and was subsequently diagnosed to have systemic lupus erythematosus.
Intracerebral bleeding due to acquired factor XIII deficiency is a rare initial presentation of systemic lupus erythematosus in literature.
Recurrent hypoglycaemia in a patient with thyrotoxicosis due to Insulin autoimmune syndrome
Authors: Sureshkumar S. , Jayanaga W. W. L. A. , Dulanjalie R. B. A. T. , Samarasingha S. A. P. N. , Karunathilaka H.
Published: February 2023

Insulin autoimmune syndrome (IAS) is an immune-mediated hyperinsulinaemic hypoglycemia in individuals previously unexposed to exogenous insulin. A 63-year-old woman developed recurrent hypoglycemic episodes despite being on optimal glycemic support. She was diagnosed with Graves’ disease and was on carbimazole. Her insulin: C peptide ratio was suggestive of exogenous insulin administration or presence of insulin autoantibodies. Her insulin antibody level was elevated. IAS was probably secondary to her carbimazole therapy.
Splenic mycotic aneurysm complicating Streptococcus salivarius mitral valve endocarditis
Authors: Rajapaksha R. W. P. M. R. A. S. , Kalupahana K. L. R. , Eranthaka U.
Published: February 2023

A mycotic aneurysm is a serious, life-threatening complication occurring in 3-10% of patients with infective endocarditis. Splenic artery mycotic aneurysm is an extremely rare complication to occur, and it has not been previously reported with Streptococcus salivarius infective endocarditis. We are reporting a case of a 57-year-old woman with Streptococcus salivarius mitral valve endocarditis, complicated by a leaking splenic mycotic aneurysm which was successfully managed with embolisation. It is important to consider this rare complication in infective endocarditis patients with an acute abdomen.
Thymic microbiota-specific T cells role in intestinal inflammation
Authors: Ruiz Daniel Fernando Zegarra , Kim Dasom , Norwood Kendra , Wu Wan-Jung , Saldana-Morales Fatima , Bettini Matthew , Diehl Gretchen
Published: February 2023

Inflammatory bowel disease (IBD) patients, including Crohn’s disease and ulcerative colitis, exhibit dysregulated immune responses. Although IBD etiology remains uncertain, microbiota-reactive T cells are thought to play a role in pathogenesis. It has been shown that gut microbiota and their metabolites play a role in modulating mucosal immune responses and thus, have the potential to influence the development of IBD. Although host-microbe interactions are required to balance effector and regulatory T cells responses, it remains unknown how microbiota-specific T cells are generated. Here we studied how microbiota-specific T cells develop and how they can drive intestinal inflammation. Colonization of mice at weaning induced the expansion of naive microbiota-specific T cells in the thymus before peripheral distribution. Transfer of naive thymic microbiota-specific T cells into immunodeficient mice induced intestinal inflammation by differentiating into Th1 or Th17 cells. We identified that CX3CR1+ dendritic cells (CX3+DCs) were required for this process, as depleting these cells decreased the expansion of thymic microbiota-specific T cells. Transfer of thymic T cells from mice lacking CX3+ DCs induced less colitis in immunodeficient mice compared to wildtype donors. Moreover, inhibiting CX3+ DCs antigen-presentation suppressed induction of microbiota-specific T cells and transfer of these cells promoted less intestinal inflammation. Together, our data suggests CX3+ DCs drive T cell selection of microbiota-specific T cells in the thymus, which can differentiate into intestinal effector T cells mediating pathology in immunodeficient hosts.
Gut microbiota dysbiosis and altered bile acid catabolism lead to metabolic disorder in psoriasis mice
Authors: HAO YAN , ZHAO QIXIANG , ZOU SONG , ZHOU PEI , HU YAWEN , ZHU YAJUAN , YU JIADONG , ZHOU HONG , WANG ZHEN , ZHENG HUAPING , ZENG FANLIAN , GU LINNA , YUE CHENGCHENG , LI JIONG
Published: February 2023

Patients with psoriasis tend to have significant comorbidities, such as hypertension, diabetes mellitus and obesity, which belong to metabolic disorder. The specific pathways through which psoriasis increases metabolic disorder risk is uncertain. The gut microbiome and gut associated metabolites as important regulators of host energy and substrate metabolism are intricately linked to host metabolic homeostasis. Here we demonstrated that the dysbiotic gut microbiota of aged psoriasis-like model mice(6 months K14-VEGF) exacerbated psoriasis disease and induced the emergence of metabolic disorder when transferred into young mice(2 months K14-VEGF). We used 16S rRNA gene sequencing detected the decrease of Parabacteroides distasonis, an intestinal bacterium, in aged K14-VEGF mice. It has been reported that Parabacteroides distasonis can affect the distribution of bile acid metabolism and benefits the metabolism of host. Metabolomic screening identified an altered distribution of bile acid metabolites, including a decrease in chenodeoxycholic acid(CDCA) in the feces of transferred mice. In addition, supplement with dietary CDCA prevented metabolic disease in K14-VEGF mice. Consequently, we found that aberrant bile acid metabolism may contribute to metabolic disorder in K14-VEGF mice, indicating the possibility to prevent and treat the metabolic disorder by targeting gut microbial metabolites in psoriasis mice.
Eosinophils inhibit breast cancer pulmonary metastatic colonization and directly kill tumor cells via degranulation
Authors: Cederberg Rachel , Bennewith Kevin L
Published: February 2023

Metastatic breast cancer remains incredibly challenging to treat, highlighting the need for an improved understanding of host factors that prevent metastasis, as well as improved therapeutics to treat metastatic cancer. The lungs, which are one of the most common sites of breast cancer metastasis, are host to a variety of immune cell subsets, including eosinophils (Eo), which are innate immune cells that target pathogens via the secretion of cytotoxic granule proteins. Eo have been shown to be anti-tumorigenic when exposed to certain signals from their local microenvironment. To study the role of eosinophils in pulmonary breast cancer metastasis, we utilized transgenic mouse models of eosinophilia (IL5Tg mice) and eosinophil-deficiency (ddGATA mice). EO771 breast cancer cells were injected intravenously (IV) to seed the lungs. We found that IL5Tg mice, which have a systemic expansion of Eo, had significantly lower EO771 lung tumor burden compared to WT mice. We found that Eo-deficient ddGATA mice exhibited accelerated metastatic progression compared to both WT and IL5Tg mice injected IV with EO771 cells. We also found that WT mice had an increased number of lung EO771 tumor cells compared to IL5Tg mice merely 5 days post-IV injection, indicating that Eo may play a role in both initial tumor cell seeding and subsequent metastatic nodule progression. Importantly, we found that Eo co-cultured with EO771 and LLC tumor cells released eosinophil peroxidase, a cytotoxic granule protein, resulting in tumor cell killing. These results highlight a role for Eo in preventing tumor cell colonization to metastatic sites and suggest that developing drugs to trigger Eo degranulation may serve as a viable therapeutic option to treat metastatic disease.
Identification of a macrophage activation threshold associated with malignant transformation and the sensing of candidate “danger signals”
Authors: Jacqueline Camille , Gottschalk Rachel , Minden Jonathan , Finn Olivera J
Published: February 2023

Cancer immunosurveillance is based on the recognition of changes brought by malignant transformation of normal cells, leading to their elimination, immunoediting, or escape. The ability of immune cells to sense changes associated with malignant transformation as early as possible is likely to be important for the successful outcome of cancer immunosurveillance. In this process, the immune system faces a trade-off between elimination of cells harboring premalignant or malignant changes, and autoimmune pathologies. We hypothesized that the immune system has therefore evolved a threshold for distinguishing normal from abnormal cells, perhaps similar to a pathogen recognition threshold at which macrophages distinguish non-dangerous from dangerous microbes. We co-cultured human macrophages with a unique set of genetically related human cell lines that recapitulate breast cancer development: MCF10A (immortalized); MCFneoT (hyperplasia); MCFT1 (atypical hyperplasia); MCFCA1 (invasive cancer). Using cytokines-based assays, we found that the threshold for macrophage activation was between MCFNeoT and MCFT1, with macrophages co-cultured with the atypical hyperplasia and invasive cancer cell lines showing an inflammatory cytokine response. This response was accompanied by an increase in macrophage migration, phagocytosis and capacity to infiltrate MCFT1 spheroids. We are currently validating candidates for “danger signals” that will be shared by MCFT1 and MCFCA1 but absent or expressed at low level in the early state of premalignancy. Using proteomic and transcriptomic approaches, we identified 13 surface or secreted molecules corresponding to this definition, some of which are well-known tumor-associated antigens.
Dendritic cells dysfunction in tumor-draining lymph nodes
Authors: Bandola-Simon Joanna Marta , Roche Paul A
Published: February 2023

An effective anti-tumor response requires activation of both CD4 helper T cells (through MHC-II) and CD8 cytotoxic T cells (through MHC-I). Activation of tumor-specific T cells depends on tumor antigen presentation by DCs. However, it is known that the tumor microenvironment largely suppresses immune responses. The focus of our study is to characterize the functional status of DCs in tumor-draining lymph nodes and to identify the mechanisms that lead to defective presentation of tumor antigens. We transplanted tumors into the flank of mice and analyzed DCs from both tumor-draining lymph node (TDLN) and non-draining lymph nodes (NDLN) as our model system. Cross-presentation of OVA to CD8 T cells was not suppressed in TDLN in comparison to the NDLN either in vivo or ex vivo. However, both in vivo and ex vivo CD4 T cell activation by DCs from TDLNs was defective. Subset analysis revealed that both migratory and resident DCs in TDLN show a less mature phenotype as compared to their counterparts isolated from NDLN. Functional assays revealed defects in antigen uptake and processing in DCs from TDLN. We also identified differences in the proportion of MHC-II molecules containing the Invariant chain CLIP peptide in DCs from TDLN in comparison to NDLN. These features of DC function, supported by RNA-Seq analysis, contribute to defective antigen presentation to CD4 T cells and may explain the dysfunction of DCs in tumor-bearing mice. Further analysis should enable moving towards “correcting” DC defects that would allow CD4 T cell priming to provide help to anti-tumor CD8 T cells.
Gemcitabine treatment modulates myelopoiesis progenitors and monocytes to promote lung metastasis
Authors: Ding Chuanlin , Yan Jun
Published: February 2023

Metastasis-associated macrophages (MAMs) play important roles in tumor metastasis through the formation of a pre-metastatic niche. In mouse models of lung metastasis, interstitial macrophages (IMs) markedly accumulate in the lungs and differentiate into MAMs. Regarding the origin of IMs/MAMs, tissue-resident macrophages and bone marrow (BM)-derived classical monocytes (BM-Mo) contribute to the pool of MAMs. Chemotherapy offers long-term clinical benefits to many cancer patients. However, several pre-clinical studies have demonstrated that certain cytotoxic drugs enhance metastasis by multiple mechanisms. Our recent studies suggest that Gemcitabine (GEM) promotes the immunosuppressive function of monocytic MDSC (M-MDSC) in the tumor microenvironment via the tumor-derived GM-CSF and efferocytosis signaling. In this study, we hypothesize that host responses to GEM treatment enhance the accumulation of BM-derived monocytes in lung, which promotes lung metastasis. Multi-dose of GEM treatment induces the increase of BM LSK cells and multipotent progenitors (MPPs) in tumor-free mice, which is associated with the increase of treatment induce mitochondrial ROS (mtROS). Thus, the percentage of BM Ly6C+ monocytes is significantly increased because of increased progenitor monocyte differentiation potential and monocyte proliferation. Further, GEM treatment increases the percentages of lung CCR2+Ly6C+ monocytes and IMs. Consequently, GEM pre-treated mice have a significantly higher number of pulmonary metastases compared to untreated mice in the experimental metastasis model. These results suggest a novel mechanism for chemotherapy induced metastasis via the modulation of myelopoiesis progenitors.
Blockade of diacylglycerol transferase 1 reprograms lipid accumulation in myeloid-derived suppressor cells and suppressed tumor progression
Authors: Adeshakin Adeleye Oluwatosin , Yan Dehong , Adeshakin Funmilayo O. , Zhang Mengqi , Afolabi Lukman O. , Wan Xiaochun
Published: February 2023

Immunotherapy, a way of restoring immune response and anti-cancer immunity, has revolutionized cancer therapy. However, immunosuppressive cells such as myeloid-derived suppressor cells (MDSC) still represent a significant impediment to immunotherapy, contributing to therapy failure and poor clinical responses. We previously reported that uptake of polyunsaturated fatty acid (PUFA) by MDSCs led to lipid accumulation via upregulation of fatty acid transport protein 2 (FATP2) to induce ROS-mediated immunosuppressive function thereby impairing T-cells activation to demonstrate their anti-tumor effect. Here lipidomics analysis revealed the exact lipid species accumulated in MDSCs from tumor-bearing tissues to be phospholipids and triglycerides (TAG). Interestingly, we observed significantly increased expression of a key enzyme involved in TAG synthesis - diacylglycerol transferase 1 (DGAT1), in MDSCs of Lewis lung carcinoma (LLC) and melanoma (B16F10)-bearing mice. Inhibiting DGAT1 reduced neutral lipid accumulation in MDSCs. Also, DGAT1 blockade downregulated MDSCs immunosuppressive genes and decreased MDSCs immunosuppressive function. Remarkably, blockade of DGAT1 expression reduced MDSCs and macrophages, thus abrogated tumor growth. Altogether, our study identified an unreported lipid metabolic target in MDSCs that promises a novel therapeutic option in clinical settings to enhance cancer immunotherapy.

This work was supported by the National Key R&D Program of China (2019YFA0906100), National Natural Science Foundation of China (Grants 82071772, 81501356 and 81373112), Key-Area R&D Program of Guangdong Province (2019B020201014).
Negatively charged nanoparticles inhibit tumor growth by altering tumor-associated macrophages and cancer-associated fibroblasts
Authors: Donthireddy Laxminarasimha Reddy , Vonteddu Prashanthi , Elhofy Adam , Gabrilovich Dmitry I
Published: February 2023

In this study, we show that a negatively charged biodegradable nanoparticles, CNP-301, have strong anti-tumor activity in several tumor models. This effect appears to be driven by shifting polarization of tumor associated macrophages (TAMs) to an anti-tumor M1 type and by downregulation of pro-tumorigenic cancer associated fibroblasts. Moreover, we show that CNP-301 treatment induces apoptosis of cancer associated fibroblasts (CAFs) in the tumor supporting a mechanism where perturbations of the TME and the associated stroma results in anti-tumor effects.
Development of surface engineered antigenic exosomes as vaccines for respiratory syncytial virus
Authors: Hong Suyeon , Ruan Shaobo , Greenberg Zachary , He Mei , McGill Jodi L
Published: February 2023

Human respiratory syncytial virus (HRSV) is one of the main drivers of lower respiratory tract illness in infants and young children worldwide. There are no licensed vaccines for HRSV available. Exosomes are cell-derived extracellular nanovesicles containing various biomolecules for intracellular communication. Exosomes secreted by antigen presenting cells such as dendritic cells can elicit immune responses by carrying MHC-I with antigenic peptide complex and other co-stimulating factors. Therefore, exosomes have emerged as potential vaccines to prevent viral infections or to treat cancers. Our prior work has demonstrated that a PDMS microfluidic culture chip device can be used to generate dendritic cell-derived tumor antigenic exosomes, with the capacity to activate tumor-specific CD8+ T cells. Herein, we further extended the work to surface engineer dendritic cell-derived exosomes with antigenic peptides from RSV (M187–195 and NS161–75). A mouse model of RSV infection was used to define the immunogenicity of surface engineered exosomes for activating virus-specific immune responses in vitro and in vivo. In vitro assays demonstrated that surface engineered exosomes successfully carried the engineered peptides of interest and had the capacity to elicit IFNγ production by activated, virus-specific CD8+ T cells. However, vaccination with engineered exosomes did not prime an in vivo activation of antigen-specific CD8+ T cell response, although surface engineered exosomes exhibit the safe administration. Additional experiments are necessary to optimize the potency of surface engineered exosomes as a vaccine platform for the prevention of viral infections such as RSV.
Reduce inflammatory cytokine profile in subspecialized neonatal APCs responding to viral pathogen-associated molecular patterns has implications for vaccine design
Authors: Lu Zhongyan , Guggenbiller Mathew , Tornbom Tora , Malloy Allison Marie Wahl
Published: February 2023

Young infants experience a higher burden of morbidity and mortality due to respiratory viruses and incomplete understanding of distinct age-dependent immune responses limits precision vaccine design in this age group. Subspecialized DCs and monocytes have evolved to detect viral pathogen-associated molecular patterns (PAMPs) to defend against infection and drive the adaptive immune response. We hypothesized that viral PAMPs and IFN I differentially activate neonatal DC and monocyte subsets resulting in age-dependent cytokine responses. To test this hypothesis, we isolated mononuclear cells from cord blood (CBMC) and peripheral blood (PBMC) and analyzed their responses through novel multiparameter spectral flow cytometric assays to PAMPs mimicking viral RNA. We discovered that conventional (c) DC2 in early life respond to Resiquimod (R848, TLR7/8 agonist) with similar upregulation of activation markers (CD80/86, CD70, CD38) and expression of TNFa, but more IL-6 compared to their adult counterparts. Unexpectedly, R848 and polyinosinic: polycytidylic acid (PIC, TLR3 agonist) resulted in significant CD25 upregulation by adult cDC2 and monocytes, in contrast to neonatal cells, suggesting decreased sensitivity to IL-2. In addition, in response to the antiviral type I IFN, cDC2 and monocytes from adults produced higher levels of CXCL10. Together our data suggest that neonatal DCs and monocytes have distinct functional responses to viral PAMPs, R848 and PIC, exhibiting reduced proinflammatory cytokine profiles. This may limit the potential for cytokine storm, but also delay the responsiveness to viral infections and vaccines that implement viral RNA in their design.
Transcriptional repressor BACH2 is a key regulator of stem-like CD8 T cells during chronic viral infection
Authors: Wu Tuoqi
Published: February 2023

During chronic viral infection and cancer, a CD8 T cell subset with stem-like characteristics maintains long-term T cell immunity and mediates the response induced by immune checkpoint blockade. The transcriptional and epigenetic programs of stem-like CD8 T cells diverged from those of other CD8 subsets early in the immune response. Using mice with chronic lymphocytic choriomeningitis virus (LCMV) infection, we found that the gene encoding transcriptional repressor BACH2 was highly expressed in stem-like CD8 T cells but was epigenetically silenced in terminally exhausted CD8 T cells early after infection. Ectopic expression of BACH2 enforced the transcriptional and epigenetic programs of stem-like CD8 T cells and restrained antiviral CD8 T cells in the lymphoid tissues. BACH2 deficiency impaired the differentiation of stem-like CD8 T cells and dysregulated their molecular program. We further demonstrated that BACH2 promoted stem-like CD8 T cell differentiation by suppressing BLIMP1 and RUNX3 pathways. In summary, our results showed that transcriptional repressor BACH2 drove stem-like CD8 T cell differentiation by guarding them against alternative cell fate.
CD25-targeted IL-2 promotes improved outcomes of influenza infection and boosts memory CD4 T cell formation
Authors: Strutt Tara M , Prokop Emily , Flores-Malavet Valeria
Published: February 2023

Interleukin-2 is a pleotropic cytokine with potent pro- and anti-inflammatory effects. These divergent impacts can be directed in vivo by forming complexes of recombinant IL-2 and anti-IL-2 monoclonal antibodies (IL-2C) to target IL-2 to distinct subsets of cells based on their expression of subunits of the IL-2 receptor. Herein we show that treatment of mice with a prototypical anti-inflammatory IL-2C, JES6-1-IL-2C, best known to induce CD25+ regulatory CD4 T cell expansion, surprisingly causes robust induction of a suite of inflammatory factors in the lung. However, treating mice infected with influenza A virus (IAV) with this IL-2C reduces lung immunopathology and is associated with marked expansion of regulatory CD4 T cells and innate lymphoid cells. We compare the spectrum of inflammatory proteins upregulated by pro- and anti-inflammatory IL-2C treatment and uncover a pattern of expression that reveals potential beneficial versus detrimental aspects of the influenza-associated cytokine-storm. Moreover, we show that JES6-1-IL-2C can deliver survival signals to CD4 T cells responding to IAV that improve their memory fitness, indicating a novel application of IL-2 to boost pathogen-specific T cell memory while simultaneously reducing immunopathology.
Role of heterotypic T cell memory following influenza re-challenge and secondary bacterial infection.
Authors: Cipolla Ellyse , Alcorn John F
Published: February 2023

Following recovery from influenza infection, an immunological T cell memory compartment is established. Memory T cells are able to respond and react to conserved pieces of the virus providing heterotypic strain protection. The role that cellular memory plays in influenza re-challenge is a topic of ongoing interest. The potential to harness cellular immune memory, in particular tissue resident memory, could provide researchers with the means to develop a universal influenza vaccine able to elicit a powerful immune response that is poised and localized in the lung environment.

Secondary bacterial infections in conjunction with influenza infection lead to increased instances of morbidity and mortality when compared to influenza infections alone. The role of heterotypic T cell memory and susceptibility to secondary bacterial pneumonias has yet to be elucidated. We hypothesize that prior exposure to influenza alters the memory response to a heterotypic super-infection re-challenge event. Mice were challenged with A/X-31(H3N2) and re-challenged 54 days later with strain A/PR/8/34 (H1N1), then challenged with MRSA on day 60. Sample analysis methods included histology, bacterial plating, flow cytometry, and RTPCR. Results suggest that mice with an established memory compartment are able to control secondary bacterial infections against MRSA as compared to their acute infected counterparts. Analysis of lung damage via histology also suggests that memory experienced mice are more protected from damage caused by secondary bacterial infection. This research suggests that the T cell memory compartment can limit the effects of bacterial super-infection in the context of heterotypic influenza strain challenge.
CD4 Effectors must re-encounter Antigen Locally in the Presence of Signals from Infection to Fully Differentiate into Specialized Tissue-restricted TFH and ThCTL effectors
Authors: Devarajan Priyadharshini , Vong Allen M , Castonguay Catherine H , Bautista Bianca L , Jones Michael C , Kugler-Umana Olivia , Swain Susan L
Published: February 2023

Functionally specialized, tissue-restricted CD4 effector subsets such as T helper cytotoxic cells (ThCTL) in the infected tissue and T follicular helper cells (TFH) in secondary lymphoid organs develop after Th1 and other effectors. Following influenza A virus infection, we find that CD4 effectors must recognize cognate Ag/MHC-II to become ThCTL and TFH but that this does not require a specific APC subset. While intrasplenic and intravenous Ag/APC delivery support spleen TFH, they do not support lung ThCTL or DLN TFH. Conversely, intranasal Ag/APC delivery supports lung ThCTL and DLN TFH indicating that Ag presentation in the site of residence is crucial. Strikingly, we find that continuing Ag-independent signals from infection are also needed for optimal generation of both subsets, even at this late effector phase. This contrasts with CD4 memory generation from effectors which is dependent only on Ag recognition and not on infection. Thus, this implies a pivotal checkpoint at which the fate of CD4 effectors is determined. In the absence of Ag most contract, while Ag alone can generate memory, and with continuing infection-mediated signals, distinct specialized effectors. This guards against unnecessary responses when pathogen is not present or has been cleared, but ensures the development of potent effectors in tissues if it persists. We propose that for vaccines to induce stronger immunity, they will need to be designed to provide additional presentation of CD4 T cell Ag and signals associated with infection at the effector checkpoint. This will provide the TFH needed for strong B cell Ab response and strong tissue-restricted CD4 T cell memory to ensure frontline response and broad, heterosubtypic protection.
STAT1 protects CD4 T cells from deletion by NK cells during influenza infection and promotes Th1 identity that can be enhanced through STAT4 to improve their anti-viral impact
Authors: Finn Caroline M , Dhume Kunal , McKinstry K Kai
Published: February 2023

Most CD4 T cells responding to influenza A virus (IAV) fit prototypical Th1 criteria including high expression of the transcription factor T-bet, the Th1 ‘master regulator’, and strong IFNg production. Optimal Th1 development in vitro also requires cytokine-dependent activation of the transcription factors STAT4 and STAT1, but how these pathways impact anti-viral responses is not clear. Here, to focus on the roles of CD4 T cell-intrinsic STAT4 and STAT1, we transferred STAT-deficient (STAT−/−) or wildtype CD4 T cells recognizing virus to congenic wildtype hosts, challenged with IAV, and analyzed donor effector responses in the infected lungs. STAT4−/− and wildtype cells reached similar numbers with no differences seen in their Th1 identity. In contrast, STAT1−/− donor cells were barely detected in host mice but reached similar numbers to wildtype donors when NK cells were depleted. Analysis of STAT1−/− cells in NK cell-depleted hosts revealed severely compromised Th1 identity that mirrored T-bet−/− donor cell responses. While these findings suggest that full Th1-polarization during IAV infection does not require STAT4, treatment of mice with IL-12 dramatically increases Th1 attributes in wildtype but not STAT4−/−CD4 T cells. This indicates that the wildtype IAV-primed CD4 T cell response is not strongly Th1-polarized. Finally, priming CD4 T cells in vitro with STAT4- and STAT1-activating cytokines promotes effector cells better able to combat IAV when transferred to unprimed mice than when only STAT1 activating signals are present. Enhancing Th1 identity through harnessing CD4 T cell-intrinsic STAT4 activation can thus improve their anti-viral impact during infection, with important implications for vaccine design.
Validating cell surface markers as accurate predictors of tissue residency
Authors: Scott Milcah C , Stolley J. Michael , Pierson Mark J , Wijeyesinghe Sathi , Gavil Noah Veis , Masopust David
Published: February 2023

Tissue resident memory T cells (TRM) represent the dominant antigen-experienced T cell subset that mediate local immunosurveillance in non-lymphoid tissues (NLT). Most accurately defined using migration assays, including parabiosis surgery and tissue grafting, TRM remain situated within NLT without recirculating through the blood. Such migration assays are challenging and often impossible to perform given animal-use approval or experimental constraints. These limitations have necessitated the use of cell surface markers such as CD69 and CD103 as surrogates for residency in the absence of more stringent approaches. Yet reliance on these cell surface markers alone often fails to accurately predict residency in tissues. As such, we performed a systematic interrogation of the correlation between flow cytometric staining profiles and the property of durable residence as assessed using parabiosis. In multiple infection models, we found that no consistent marker combination accurately identifies and captures the abundance and heterogeneity of TRM in diverse NLT. By evaluating the usefulness of cell surface markers as diagnostic of residency on a tissue- and infection-specific basis, we identified tissue specific phenotypes that reproducibly predicted residency in three infection models. These findings are important because refined analyses of TRM populations is key to addressing deficiencies in our understanding of how TRM mediate immunosurveillance. Ongoing work will further validate these tissue-specific phenotypes in additional infection models and ‘dirty mice’ that contain CD69+ T cells within the equilibrating T cell populations.
STAT1 gain of function mutation impairs immune response to viral infections
Authors: Philips Rachael Laura , Yao Chen , Milner Joshua D , Lionakis Michail S , O’Shea John J
Published: February 2023

Regulation of T cells by interferons and other cytokines, which act via STAT family transcription factors, is critical for host defense. Patients with STAT1 gain-of-function (GOF) mutations exhibit heightened STAT1 activation after cytokine stimulation and develop lifelong ailments, including chronic infections, autoimmunity, and cancer. A characteristic feature of this disorder is chronic mucocutaneous candidiasis (CMC), related to exagerated type 1/IFNγ bias that antagonizes a type 3/Th17 response required for fungal clearance. However, this type 1/IFNγ bias does not explain why almost 40% of reported patients exhibit chronic and sometimes lethal viral infections. This observation is unexpected, as cells with STAT1-GOF mutations exhibit increased expression of interferon stimulated genes following IFNα/β and IFNγ stimulation and patients show more IFNγ-producing CD4 T cells. This paradox emphasizes the critical need to understand basic principles of cytokine signaling, specifically how a STAT1-GOF mutation alters cytokine output. Using a novel conditional knock-in mouse expressing a common GOF STAT1 mutation, T385M, we found that this model recapitulates observations in patients (i.e. elevated STAT1 activation, increased total STAT1 protein, susceptibility to CMC), as well as a strong type 1/interferon bias at steady state. Despite this type 1 bias, STAT1-GOF exhibit impaired NK and CD8 T cell response to systemic viral infections. These results reveal that STAT1 activity must be tuned for proper immune response to viral infections.
Loss of the Class II Accessory Molecule HLA-DO leads to altered TCR avidity
Authors: Welsh Robin A , Song Nianbin , Foss Catherine , Sadegh-Nasseri Scheherazade
Published: February 2023

HLA-DO (DO in human; H2-O in mice) is a highly conserved non-classical MHC class II accessory molecule. Unlike the main Class II peptide editor HLA-DM, expression of DO is restricted to the thymic medulla, B cells and certain dendritic cell subsets. Although DO forms a stable complex with HLA-DM, its biological functions remain poorly understood. Previously, our lab provided biochemical evidence in support of a model where DO enhances the binding of DM-resistant peptides, and reduces the binding of DM-sensitive peptides to the HLA-DR1 (DR1) molecules in vitro. Changes in the quantities of either DM-resistant of DM-sensitive peptides could therefore lead to alterations in both thymic deletion, and peripheral activation of CD4 T cells. Here, using both in vivo antigen presentation as well as a novel avidity measurement tool, z-Movi™ (LUMICKS), we found that: (1) brain recovered APCs from diseased DO-KO mice present less of the MOG(35–55) epitope, (2) naïve DO-KO CD4 T cells have a more avid interaction with their own APCs, and (3) that hemagglutinin specific, DR1 restricted CD4 T cells had a less avid interaction with B cells from flu vaccinated DR1+DO-KO mice as compared to B cells from vaccinated DR1+DO-WT mice. These avidity changes could explain two things: (1) why we find that despite DR1+DO-KO mice have similar precursor levels of collagen specific CD4 T cells as DR1+DO-WT mice, they are more susceptible to the development of Collagen Induced Arthritis, even though we previously showed that the immunodominant collagen epitope is DM-sensitive. And, (2) why we find increased levels of regulatory T cells in the absence of DO.
Unconventional Endo-Lysosomal Assembly of Human Leukocyte Antigen B (HLA-B)
Authors: Olson Eli C , Raghavan Malini
Published: February 2023

The immune system surveys the body for foreign or mutated structures with the help of the human leukocyte antigen class I (HLA-I) protein complex, which displays protein fragments called peptides on the cell surface. The genes encoding the HLA-I heavy chain in humans are highly variable, which results in predisposition to or protection from diseases based on individual HLA-I variants. We have observed that sub-optimal assembly conditions for certain HLA-B variants produce peptide-deficient molecules on the cell surface. The monocyte immune cell type overcomes this deficiency in assembly, reversing the previously observed trends. Intracellular HLA-B accumulates in monocytes and monocyte-derived dendritic cells (moDCs) in endo-lysosomal compartments, a predicted location for supplemental peptide assembly. Current findings suggest that certain HLA-B variants are better able to efficiently assemble with intracellular and extracellular antigens in the endolysosomal compartments, conferring a selective advantage in infection conditions where traditional antigen presentation is blocked.
Roles of CX3CR1 and probiotic lactobacilli on the development of autoimmunity in MRL/lpr mice
Authors: Puig Xavier Cabana , Abdelhamid Leila , Swartwout Brianna , Zhu Jing , Liao Xiaofeng , Reilly Christopher M , Luo Xin M
Published: February 2023

Systemic lupus erythematosus (SLE) is a multi-system autoimmune disease with no known cure. Gut microbiota and the immune system have a close relationship that controls inflammation. Alterations in the gut microbiota, known as dysbiosis, can trigger SLE in both human and mouse. We previously published differences between the gut microbiota in lupus-prone MRL/Mp-Faslpr (lpr) mice compared to healthy controls, with lower Lactobacillaceae abundance in lpr mice. Restoring the gut microbiota with a mix of 5 different strains of this family significantly attenuated lupus-liked disease in lpr mice. In this study, we sought to understand the potential benefit of this mix in lpr mice with Cx3cr1 deficiency (KO). KO mice on C57BL/6 background display increased translocation of commensal bacteria into the mesenteric lymph node (MLN) and increased susceptibility to intestinal inflammation. However, whether CX3CR1+ macrophages play a role in controlling systemic inflammation was unclear. After backcrossing the KO allele to lpr mice, we found that KO lpr mice exhibited exacerbated splenomegaly and lymphadenopathy, as well as increased proteinuria. Interestingly, treating the KO lpr mice with the 5 Lactobacillus strains attenuated the enlargement of MLN and abrogated the increase of proteinuria. Furthermore, the effects of the probiotic bacteria were accompanied by downregulation of serum endotoxin, suggesting the reversal of a leaky gut that may explain their benefits on KO lpr mice. Together, our results show an important role of CX3CR1+ macrophages in controlling systemic inflammation in lupus, and that probiotic Lactobacillus spp. may be an effective treatment against SLE through preventing bacterial translocation.
Ageing promotes non-remitting experimental autoimmune encephalomyelitis with persistent meningeal inflammation and subpial demyelination in A/T SJL/J mice.
Authors: Le Zuo Michelle Xiao , Ramaglia Valeria , Ma Iris , Gommerman Jennifer L
Published: February 2023

Current therapies for multiple sclerosis (MS) reduce the frequency of relapses but fail to prevent transition from relapse-remitting into progressive MS. Subpial demyelination, meningeal inflammation and neurodegeneration are hallmarks of progressive MS, however there is no single animal model that mimics all these features. Adoptive transfer of proteolipid protein (PLP)-primed Th17 cells into young (~12 weeks old) SJL/J recipient mice induces experimental autoimmune encephalomyelitis (EAE) characterized by subpial demyelination associated with meningeal tertiary lymphoid tissues (TLTs), however young mice develop remitting, non-progressive disease. Since age is a risk factor for transitioning to progressive MS, we hypothesized that adoptive transfer of PLP-primed Th17 cells into old (~12 months old) SJL/J recipient mice would promote a progressive phenotype. Indeed, transfer of cells into aged recipient mice resulted in a non-remitting EAE compared to young recipients despite a similar ability to produce T cell derived cytokines in the CNS at peak disease. However, old mice showed persistent meningeal inflammation as evidenced by more B/T cell rich TLTs in the meninges compared to young mice, and developed increased CNS pathology (subpial demyelination, microglia/macrophage activation, glial limitans disruption, axonal injury, loss of synapses), whereas young EAE mice had recovered most of the pathology seen at peak of disease by day 25 post-transfer. In summary, our model provides an opportunity to assess how subpial demyelination associated with meningeal inflammation impacts the transition from relapse-remitting to progressive MS in a single animal model.
TRAF3 enhances type I interferon receptor (IFNAR) signaling in T cells through modulation of PTPN22
Authors: Hornick Emma , Wallis Alicia Michelle , Houtman Jon , Bishop Gail A
Published: February 2023

T cell responses are critical for successful resolution of many infectious and non-infectious challenges. Robust T cell activation and acquisition of effector functions relies on receipt of at least three signals: 1) antigen recognition by the T cell receptor, 2) co-stimulation, and 3) cytokines. TNF receptor-associated factor 3 (TRAF3) is an adapter protein with diverse cell type- and receptor-specific functions, that regulates signals one and two of conventional T cell activation. In this study, we examine the role of TRAF3 in signaling downstream of the potent signal three cytokine, type I interferon (IFN I). Work from our laboratory has shown that in other contexts, TRAF3 regulates cytokine receptor signaling by facilitating association of phosphatases with JAKs at the cytokine receptor. We therefore hypothesize that TRAF3 regulates IFNAR signaling in T cells through affecting recruitment of the phosphatase PTPN22 to the IFNAR complex.

We investigate the role of TRAF3 in IFNAR signaling using a TRAF3-deficient human T cell lymphoma line and T cells from a T cell specific TRAF3 knockout mouse (T-Traf3−/−). TRAF3−/− T cells display a marked defect in STAT1 activation, leading to defects in IFN-stimulated gene expression and CXCL10 secretion. We show TRAF3 and PTPN22 association with JAK1 and IFNAR1 upon IFN I stimulation in WT T cells, and increased association of PTPN22 with JAK1 and IFNAR1 in TRAF3−/− T cells. Inhibition of PTPN22 restores IFNAR signaling in TRAF3-deficient cells to near WT levels. Together, these data indicate that TRAF3 modulates IFNAR signaling by modulating PTPN22 association with the IFNAR signaling complex, broadening our understanding of the crucial signals that drive T cell activation and polarization.
Impact of aging on monocyte subset production by bone marrow progenitors
Authors: Goodridge Helen Sara , Barman Pijus K , Shin Juliana , Wu Di , Kang Seokjo
Published: February 2023

Monocytes can patrol the circulation or be recruited into tissues to become macrophages or monocyte-derived dendritic cells (moDCs). We previously showed that monocytes arise via two independent pathways in mouse bone marrow: Granulocyte-Monocyte Progenitors (GMPs) produce neutrophils and monocytes, and Monocyte-DC Progenitors (MDPs) yield monocytes and conventional and plasmacytoid DCs (cDCs and pDCs). Notably, both pathways produce monocytes that can differentiate into macrophages, but moDCs arise exclusively via the MDP pathway. We also showed that the microbial stimuli LPS and CpG promote monocyte production by GMPs and MDPs, respectively. We are now investigating how aging impacts the balance of monocyte production by GMPs and MDPs. scRNAseq analysis revealed elevated Cd74 and H2 gene expression by steady-state classical monocytes from old mice compared to young mice, with a larger proportion of monocytes expressing these genes. Consistent with this, we observed proportionally more monocytes expressing CD74 and MHCII in the bone marrow and circulation of old mice by flow cytometry, and a higher yield of CD11c+ MHCII+ moDCs in GM-CSF cultures of MDPs and monocytes from old bone marrow. These findings are indicative of elevated moDC production during aging, perhaps reflecting the more inflammatory microenvironment. In ongoing studies, we are investigating whether imbalanced monocyte production by the GMP and MDP pathways underlies increased moDC production during aging.
Developing a model of thymic regulatory T cell development with single-cell RNA sequencing
Authors: Owen David L. , LaRue Rebecca , Farrar Michael A.
Published: February 2023

CD4SP thymocytes that receive moderate to strong TCR signals upregulate either FOXP3 or CD25, generating distinct FOXP3lo or CD25+ Treg progenitor (TRP) populations, respectively. Stimulation of these TRP populations with IL-2 induces co-expression of CD25 and FOXP3, completing thymic Treg differentiation. The alterations in gene expression that drive these respective Treg differentiation programs, as well as the identification of agonist selected cells within conventional CD4SP thymocytes remain unclear. To address these questions, we performed single-cell RNA-Seq with paired TCR sequencing of conventional and Treg lineage thymocytes, including recirculating Tregs. Interestingly, we found that IL-2 was exclusively produced within the most immature CD25+ TRP undergoing agonist selection. TCR analysis demonstrated that some TCRs were enriched in CD4SP receiving agonist stimulation that gave rise to Tregs, or were subjected to negative selection, while others were excluded from this pathway. Finally, we found several distinct subsets of recirculating Tregs, likely pointing to a variety of functions these cells play in thymic homeostasis and function. We are currently carrying out spatial transcriptome studies to identify the specific localization of distinct Treg intermediates within the thymus. We are using these combined single cell data sets to generate a comprehensive model of thymic Treg development.
CCN3 aggravates intestinal inflammation in inflammatory colitis through regulating NLRP3 inflammasome in macrophages
Authors: Wei Yingying , Duan Lihua , Zhong Jixin
Published: February 2023

CCN3 is one of member in CCN family that regulates several cellular activities including cell adhesion, proliferation and migration. However, the role of CCN3 in the pathogenesis of inflammatory bowel disease (IBD), an idiopathic intestinal disease characterized by chronic inflammation of the gastrointestinal tract, remains unclear. Herein, we established a DSS induced colitis model that mimic human inflammatory bowel disease by using the CCN3 conditional knock-out mice (LysMCre-ccn3f/f). We found that the mortality and colitis disease score were significantly reduced in LysMCre-ccn3f/f mice, compared to the control group (LysMCre). To further evaluate the contribution of macrophages and neutrophils to the protective effect of CCN3 deletion on colitis, we separately depleted macrophages and neutrophils in these mice before colitis induction. The difference in disease severity between these two groups was abolished after the depletion of macrophages, but not neutrophils, suggesting that CCN3 expression in macrophages plays a critical role in intestinal inflammation. In addition, we found that the production of IL-1b and expression of NLRP3 in LysMCre-ccn3f/f bone marrow-derived macrophages (BMDM) were significantly lower than those in LysMCre, indicating that NLRP3 inflammasome activation may be involved. In conclusion, our data provide a direct evidence that CCN3 may play a role in colitis by regulating NLRP3 inflammasome activation in macrophages.