bluuebunny/crossref_metadata_2025 huggingface dataset:
A circulating population of CD4+ cutaneous resident memory T cells is diminished in humans with fibrotic disease
Authors: Morawski Peter A , Fahning Mitch , Varkhande Suraj , Beale Emma , Klicznik Maria , Gratz Iris , Campbell Daniel J.
Published: January 2023

Tissue-resident memory T cells (TRM) persist mainly in non-lymphoid tissues where they promote immune recall responses to recurrent insults. We previously defined a population of CD4+CD103+ TRM cells from human skin that become CD69neg and re-enter the circulation, but maintain expression of the skin-associated marker cutaneous lymphocyte antigen (CLA) and the capacity to return to the skin. Comparing blood- and skin-derived CLA+ memory populations, we found that the recirculating fraction of CD103+ TRM are clonally, transcriptionally, and phenotypically most similar to skin-resident CD103+ T cells. We now report that the circulating CD4+CLA+CD103+ T cells are functionally distinct from classically-defined CD4+ T helper lineages, highlighted by the co-production of IL-22 and IL-13. As these cytokines can promote tissue repair macrophage- and epithelial cell-mediated fibrosis and wound healing, we assessed the circulating skin TRM pool during systemic scleroderma (SSc), an autoimmune disorder highlighted by fibrosis of the skin and the underlying organs. Compared to healthy donors, SSc patients had a reduced frequency of circulating CD4+CLA+CD103+ T cells with diminished per-cell IL-13 production, both of which correlated with late-onset disease. Our ongoing work aims to understand how changes in the circulating TRM fraction might be a litmus for the onset or progression of SSc and similar skin diseases. Therefore, circulating CD4+CLA+CD103+ T cells represent a broadly available pool from which the isolation and study of skin TRM cell responses is possible both in health and disease.
Immunological Parameters in Blood (NK, CD4+ numbers, CD4/CD8 ratios, Ig levels, etc.) change with treatment (XRT, Chemotherapy, Immunotherapy etc.): Results in Breast Cancer, Colorectal Cancer and other sites (H&N, Lung, et. al.)
Authors: Williams R Michael , Yunis Edmond J.
Published: January 2023

Our view of cancer and the immune system is that an immune deficiency/failure of immune surveillance results in immune dysregulation including a "hole" in the specificity repertoire that results in active tolerance (Tregs) to cancer antigens and common pathogens (Cell. Immunol. 1978). The genetic basis for cancer survival was described in the P815 mouse model for MHC linked IR-gene control (Williams, Dorf and Benacerraf, Ca. Res. 1974) and the method for application to man was described at AAI2012. At AAI2019 we described the Immunogenetic Theory of Cancer and Aging and defined the disease Acquired Immune Deficiency of Cancer. We described the fact that homozygosity at P2 of the leader peptide of HLA class I and at the NG2A NK cell receptor is associated with older age, longer survival after primary therapy for breast cancer and non-progression if HIV infection occurs. We showed that breast cancer survival is significantly longer among patients with higher numbers of NK cells at initial presentation for breast cancer treatment. Here we present data showing survival in colorectal cancer, just like in breast, is significantly better when NK cell numbers are higher at initial evaluation Presented also are data demonstrating that peripheral blood NK cell numbers decrease during radiation of the breast only to recover to baseline after XRT ends. There is little to no change in numbers of CD4+ cells and other immunological parameters. In colorectal cancer quantitative IgA are related to survival. This is not true in breast cancer. Quantitative IgM and IgG levels are not associated with survival in either breast or colorectal caner. We conclude that simple blood parameters can provide substantial insight about mechanisms of treatment in cancer survival.
Differential Activity of Key Super-Enhancers Drives Disease-Defining Oncogenes in B Cell Cancers
Authors: Payton Jacqueline , Andrews Jared , Pyfrom Sarah , Schmidt Jennifer , Marcy Hannah , Koues Olivia , Cashen Amanda F. , Oltz Eugene M.
Published: January 2023

Normal B cell development depends upon exquisitely timed and calibrated epigenetic and transcriptional changes. Significant perturbations should result in cell death, but rarely, cells escape this quality control system. We compared 3 types of B cell cancer and normal lymphocytes to identify common and distinct epigenetic perturbations that promote oncogenesis. Purified CD19+ B cells from 49 patients (18 Follicular, 12 Diffuse Large B Cell, 19 Chronic Lymphocytic Lymphomas) and 20 donor tonsils were subjected to chromatin immunoprecipitation (ChIP-) and sequencing for H3K4me1, H3K9/14ac, and H3K27ac; FAIRE-seq for open chromatin; RNA-seq for gene expression; and SNP arrays for genome mapping. These integrative studies identified 7,772 differentially bound regulatory regions (DBRs) between lymphoma subtypes and tonsillar B cells. Expression of nearby genes corresponded with ChIP/FAIRE-seq changes at these elements, particularly near DBRs in 1,367 identified super-enhancers. Several of the latter regulate known lymphoma oncogenes, including BCL6, BCL2, and IRF4. We also identified a novel super-enhancer connected to the aberrant expression of FCMR and PIGR. We confirmed enhancer activity using luciferase reporter assays and altered protein expression using immunofluorescence. FCMR regulates the surface expression of BCR; B cells deficient in FCMR have increased tonic BCR signaling and increased differentiation. We suggest that its overexpression may contribute to the chronic active BCR signaling that drives many B cell cancers. In sum, our studies defined distinct enhancer profiles among B cell cancer subtypes and identified a novel regulatory mechanism that may contribute to aberrant BCR signaling in lymphoma.
Epigenetic drivers of Cutaneous T Cell Lymphoma revealed at single cell resolution
Authors: Payton Jacqueline , Andrews Jared , Quinn Chaz , Schmidt Jennifer , Musiek Amy , Mehta-Shah Neha
Published: January 2023

Cutaneous T-cell lymphomas (CTCL) are cancers of mature skin-homing T lymphocytes, but the molecular drivers remain poorly understood. We performed whole genome, exome, epigenome, bulk and single cell (sc) transcriptome analyses of serial peripheral blood, skin, and lymph node biopsies from 20 CTCL patients. Initial studies defined markers of therapy resistance and disease progression in pathways that mediate cell migration, cytokine signaling, and epigenetic regulation. Several were novel to CTCL, including BIRC5 (anti-apoptotic); RRM2 (cell cycle); CXCR4 and LAIR2 (migration). On average 7413 cells from 16 serial samples (>100,000 cells total) passed quality thresholds for scRNA- + TCR-seq. We used Seurat, dittoSeq, and SingleR for quality control, dimensionality reduction (TSNE/UMAP), clustering, marker detection, cell type inference, and visualizations, and confirmed the therapy-resistant expression patterns we previously identified, including LAIR2. TCR clonotypes confirmed patient-specific malignant cell populations and defined clonal changes in serial samples. Pseudotime analysis of serial samples with Slingshot identified significant changes in expression related to changes in therapy, particularly in chemokine, TNF, and NFKB signaling. We identified mutations in STAT3&5 in 3/10 and in epigenetic modifiers (KMT2C/D and DNMT3A) in 6/10 patients. Loss of DNMT3A perturbs T cell differentiation and causes leukemia/lymphoma in mouse models. DNMT3A deficiency and additional epigenetic alterations may be previously unrecognized “drivers” of CTCL. These studies define mechanisms of therapy resistance, map clonal evolution, and reveal novel mechanisms of pathogenesis and progression in CTCL.
A Physiologically-Relevant Three-Dimensional In Vitro Model of Human Myeloma Bone Disease
Authors: Cottrell Jessica , Kolaja Kyel , Visconti Richard
Published: January 2023

Multiple myeloma (MM) is a hematologic cancer caused by mature B cell neoplasm that infiltrates the skeleton at several sites. The disease is characterized by uninhibited transformed plasma cell proliferation that disrupts skeletal homeostasis leading to increased bone resorption. Osteolytic lesions (OL) left in the bone, remain long after the treatment of the cancer and indicate disease progression to myeloma bone disease (MBD). Current combinatorial MM therapies inhibit malignant plasma cell proliferation, slow the progression towards MBD, and increase the mean five-year survival rate, but do little to improve osteoblastic function and restore skeletal homeostasis. Several novel MBD treatments have been developed to heal OLs but testing their efficacy against MBD is challenging. Therefore, we created a in vitro three-dimensional microphysiological human MM bone model to facilitate the identification of improved combinatorial treatments that suppress plasma cell proliferation while healing osteolytic lesions. Bone Marrow Stromal Cell derived osteoblasts and bone marrow macrophage derived osteoclasts were capable of bone formation and resorption in a homeostatic coculture as measured by gene expression profiling, alkaline phosphatase deposition, and alizarin red staining. The introduction of human plasmacytoma cell lines induced lesions in the mini-bones decreasing the cumulative hydroxyapatite (HA) content while increasing resorption markers and recapitulating physiological conditions of MBD. In the future, we hope to use this model of human MBD to evaluate the effectiveness of new combinatorial therapies that promise to restore skeletal homeostasis.
Differentiated immunoglobulins and neutrophil-related proteins in bronchoalveolar lavage fluid exosomes of acute respiratory distress syndrome
Authors: Ji Hong-Long , Samten Buka , Hadisurya Marco , Zhao Runzhen
Published: January 2023

Pulmonary immune response is a critical determining factor for acute respiratory distress syndrome (ARDS). Extracellular vesicles released from lung stem and immune cells may serve as inflammatory niches involved in immune response and lung injury repair. The role of exosomal immunoglobulins (Igs) and neutrophil-related proteins in bronchoalveolar lavage (BAL) in lung immunology is unknown. We aimed to examine the content and potential role of exosomal immune proteins of ARDS patients.

The abundance of exosomal proteins was quantified with a proteomic platform. Exosomal Igs and neutrophil-related proteins in BAL were analyzed with a combination of proteomic and bioinformatic software for functional enrichment, cell origin, and network analysis.

There were 4,458 proteins identified and approximately 400 exosomal proteins per stage were significantly altered in patients compared with healthy controls. The number of significantly reduced exosomal Igs was dependent on the severity: 19 for mild, 38 for moderate, and 51 for severe. Of them, 32 Igs were seen in at least two stages, while 19 Igs were occurred only in severe ARDS. Most of these downregulated Igs were either heavy or light chain variable regions, indicating their diverse cell origin. In contrast, no Igs were upregulated in ARDS patients. In addition, four neutrophil-related proteins (MPO, DEFA1, CXCL8, MME) were significantly upregulated in BAL exosomes of ARDS patients. Significant differences in functional enrichment, pathway, cell origin, and network were observed among three stages of ARDS patients.

Our proteomic data identified exosomal Igs and neutrophil-related proteins of ARDS that may serve as clinical biomarkers and the regulators of local immune response.
Gamma/delta (γδ) T-cell receptor (TCR)-expressing T cells are clonally expanded in aneurysmal lesions of patients with abdominal aortic aneurism (AAA)
Authors: Nwaneshiudu Ifeyinwa , White John V , Lu Song , Nwaneshiudu Adaobi , Solomides Charalambos , Gaughan John , Monos Dimitri , Oleszak Emilia L , Platsoucas Chris D
Published: January 2023

Several lines of evidence strongly suggest that AAA is a specific antigen-driven T-cell inflammatory disease with a strong genetic component. We tested the hypothesis that γ- and δ-chain TCR transcripts are clonally expanded in AAA lesions. Previously, we showed clonally expanded α-chain (PloS One 2019;14:e0218990) and β-chain (J Immunol. 2014; 192:4897) TCR transcripts in AAA lesions of patients with AAA. We employed two-sided VγI-, VγII-, Vδ1- and Vδ2-specific PCR followed by cloning and sequencing of amplified transcripts to examine whether VγI-, VγII-, Vδ1- and Vδ2-TCR transcripts, respectively, were clonally expanded in aneurysmal lesions of patients with AAA. Clonally expanded VγI-, VγII-, Vδ1- and Vδ2-TCR transcripts were identified in AAA aneurysmal lesions. We identified multiple identical copies of: (i) VγI-TCR transcripts in 16 of 18 patients (range of identical transcripts 30% to 80% in individual patients); (ii) VγII-TCR transcripts in 15 of 15 patients (range of identical transcripts 39% to 80%);.(iii) Vδ1-TCR transcripts in 12 of 12 patients (range of identical transcripts 29% to 94%); and (iv) Vδ2-TCR transcripts in 10 of 10 patients (range of identical transcripts 40% to 91%). These results were statistically significant. PBMC from normal donors (methodological controls) contained almost exclusively unique VγI-, VγII- and Vδ2-TCR transcripts. In contrast, Vδ1 transcripts from PBMC from certain normal donors contained clonally expanded T cells, in agreement with previous reports. In conclusion, clonally expanded γδ TCR-expressing T cells were found in aneurysmal lesions of patients with AAA. These T cells may play an important role in the pathogenesis of AAA. Supported in part by grant RO1 HL64340 from NIH.
Lung ILC2 responses to Alternaria alternata fungal allergen are blunted in “dirty” mice with physiologically transmitted murine microbes
Authors: Block Katharine E , Iijima Koji , Walsh Daniel A , Pierson Mark , Hamilton Sara E , Kita Hirohito , Jameson Stephen C
Published: January 2023

The objective of this study was to test the hypothesis that the increasing global incidence of allergy and atopy are due in part to improved hygiene and decreased microbial exposure. We have adopted a novel mouse model of normalized microbial exposure to test the impact of immune experience on subsequent responses to airway allergens. In this model, specific pathogen free (SPF) B6 mice are cohoused with mice from pet stores and become “dirty” – many commensals and pathogens are transmitted through cohousing and influence the immune cell populations systemically and in the lungs. We treated mice intranasally with a single dose of A. alternata fungal extract (Alt) and assessed production of type 2 cytokines. IL5 and IL13 levels in the lungs and bronchoalveolar lavage fluid were dramatically elevated by Alt in SPF mice but were not significantly increased by Alt in dirty mice. Type 2 innate lymphoid cells (ILC2) are the cells responsible for IL5 and IL13 after acute Alt treatment, and interestingly the number of lung ILC2 was unaltered by cohousing and their activation status appeared similar in both housing conditions. We treated mice with recombinant IL33, the alarmin released by lung epithelial cells in response to allergens, and the results suggest an impaired response to IL33 by dirty lung ILC2. In a repeated Alt exposure model, ILC2 cells expanded in SPF lungs and recruited eosinophils, neutrophils, and T cells. Most dirty mouse lungs contained these cell populations at steady state but were only modestly recruited with repeated Alt exposure. Lung function experiments are in progress. This study suggests that increased microbial exposure leads to more type-2 associated immune cells in the lungs, however responses to airway allergens are dampened.
Investigation of cytokine levels after dupilumb treatment in adults with acute atopic dermatitis
Authors: Chou Ya-Ling , Chou Chun-Jung , Guo Yong-Si , Li Ya-Chi , Lyu Sin-Yi , Chang Deh-Ming , Lu Chun-Chi
Published: January 2023

Objective

Atopic dermatitis (AD) is a chronic, pruritic, inflammatory skin disease that occurs most frequently in children, but also affects many adults. People with AD have high disease load and poor quality of life, without many treatment options. Dupilumab, a monoclonal antibody, approved for treatment inadequately controlled AD, of which the effect on serum cytokines remains unknown. The aim of this study was to compare serum levels of cytokines in adult patients with atopic dermatitis before and after dupilumab treatment and investigate the possible association between such cytokines and dupilumab.

Methods

We compared the profiles of cytokines in 5 adults with AD before and after one course of dupilumab treatment. Assessment of serum cytokine concentrations was performed via commercially-available Milliplex® assays.

Results

Milliplex analysis of cytokines revealed that interleukin 12p70 (IL-12p70), IL-13, IL-21, interferon r (IFN-r) and tumour necrosis factor a(TNF-a) levels were significantly decreased after dupilumab treatment, whereas IL-2, IL-4, IL-6 and IL-10 can not be detected.

Conclusions

These data indicate that serum interleukin 12p70 (IL-12p70), IL-13, IL-21, interferon r (IFN-r) and tumour necrosis factor a(TNF-a) levels were significantly decreased after dupilumab treatment. Further studies are needed to elucidate the role of cytokines and association with dupilumab in adults with AD.
HY209 ameliorates atopic dermatitis through NF-KB and P38 inhibition
Authors: Ghaderpour Aziz , Kim Youn-Hee , Lee Hai-Chon , Seong Seung-Yong
Published: January 2023

HY209 has been reported to be a ligand for the membrane G protein–coupled receptor, TGR5. HY209 could inhibit inflammatory responses in sepsis by increasing number of immunosuppressive myeloid-derived suppressor cells (MDSC), suggesting the potent role of HY209 in the treatment of atopic dermatitis (AD). Here HY209 was evaluated whether it controls inflammatory immune responses in AD. We employed mouse AD model using a topical treatment of dinitrochlorobenzene (DNCB) on the dorsal skin of Balb/c mice. HY209-treated mice showed significantly reduction in Th2 immune responses, shown as lower IgE serum, and inhibition of T cells mast cells, eosinophilia and neutrophil recruitment accompanied with reduced in epidermal and dermal thickness in skin of HY209 treated mice. Interestingly, regulatory T cells noticeably increased upon to HY209 treatment along with inhibition of type 2 cytokines production. Proteogenomic analyses of mouse skin demonstrated upregulated expression of molecules responsible for tissue regeneration and downregulated expression of proinflammatory molecules. Consistent with these findings, in vitro data indicated that HY209 decreased production of thymic stromal lymphopoietin (TSLP) and CCL17 (TARC) through inhibition of NF-KB and MAPK (P38) pathway. Taken together, HY209 ameliorates AD in mice by normalizing Th1/Th2 imbalance immune responses and inhibiting various pro-inflammatory responses via TGR5-cAMP-PKA axis, Suggesting HY209 could be a promising therapeutic candidate for AD.
COX inhibition enhances allergen-induced IL-33 release and ILC2 responses in mouse lungs
Authors: Zhou Weisong , Zhang Jian , Toki Shinji , Goleniewska Kasia , Norlander Allison E. , Newcomb Dawn C. , Boyd Kelli L. , Kita Hirohito , Peebles R. Stokes
Published: January 2023

The cyclooxygenase (COX) metabolic pathway has regulatory functions in immune responses and inflammation. However, the effect of the COX pathway on innate airway inflammation induced by protease-containing allergens such as Alternaria alternata is not fully defined. Here we show that COX inhibition augmented innate lung type 2 responses after repeated airway exposures to Alternaria extract in mice. We treated wild type BALB/c and IL-33 KO mice with either the COX inhibitor indomethacin or vehicle in drinking water and challenged mice intranasally with Alternaria extract for 4 consecutive days to induce innate lung inflammation. We found that indomethacin significantly increased the numbers of group 2 innate lymphoid cells (ILC2) and IL-5+IL-13+ ILC2 in the lung. Indomethacin also increased type 2 cytokine (IL-5 and IL-13) responses, the percentages of eosinophils, and mucus production in the lung. IL-33 is required for Alt-induced lung ILC2 responses, and indomethacin did not change IL-5 and IL-13 expression in IL-33 KO mice. Consistently, indomethacin increased the release of IL-33 in bronchoalveolar lavage fluid after Alternaria challenge, suggesting that more IL-33 was available for ILC2 activation. Indomethacin also increased reactive oxygen species (ROS) production in the lung, providing a possible mechanism for the increased IL-33 release. Although contrasting effects of PGD2 and PGE2/PGI2 on ILC2 responses have been previously reported, the overall effect of COX pathway on ILC2 function is inhibitory in Alternaria extract-induced airway innate type 2 responses.
Single-cell TCR analysis of mouse lung Vγ4 γδ T cells reveals a link between TCR clonotypes, SLAM family receptor expression, and effector function
Authors: Mistri Somen K , Hampel Kenneth J , Sidoropoulos Nikoletta , Dienz Oliver , Boyson Jonathan E
Published: January 2023

γδ T cells are unusual T cells that are highly enriched in mucosal tissues where they constitute a prominent source of pro-inflammatory cytokines like IL-17 and IFN-γ. The TCR usage of tissue-specific γδ T cells is often associated with specific effector functions. We have recently found that IL-17- and IFN-γ-producing Vγ4 γδ T cells in the mouse lung are marked by the expression of SLAMF1 and SLAMF6 receptors, respectively. The objective of this study was to investigate a possible link between SLAM-associated effector function and γδ TCR usage. We first identified the major Vγ4 γδ TCR clonotypes in the mouse lung (n=13 mice). Vγ4 γδ T cells were single cell-sorted and paired TCR clonotypes were identified using next-gen sequencing of TCR amplicon libraries. The data revealed that TRDV5, TRDV2, and TRDV7 chains accounted for 51.18%, 29.61%, and 14.96% of the productive TCRδ chain rearrangements, respectively. While TRDV5 and TRDV2 CDR3 sequences were limited in diversity, TRDV7 CDR3 sequences were highly diverse. A significant fraction (30.46%) of the TRDV5 sequences were characterized by an invariant germline-encoded Vδ5Dδ2Jδ1 sequence. A comparison between sorted SLAMF1+ and SLAMF6+lung Vγ4 γδ T cells revealed that TRDV5 and TRDV2 chains were predominantly associated with SLAMF1+IL-17+ γδ T cells while the TRDV7 chain was predominantly associated with SLAMF6+IFN-γ+ γδ T cells. Moreover, the invariant germline-encoded TRDV5 sequence was primarily associated with SLAMF1+IL-17+ cells. These data indicate that the lung Vγ4 γδ TCR repertoire is limited in diversity and that specific lung Vγ4 γδ TCR clonotypes segregate with the expression of discrete SLAM family receptors.
STAT4 promotes critical neutrophil functions and is required for antimicrobial immunity in mice
Authors: Keeter W. Coles , Moriarty Alina , Mehrpouya-Baharami Pegah , Melo Paulo , Nadler Jerry , Serezani Carlos H , Kaplan Mark H , Galkina Elena
Published: January 2023

Signal transducer and activator of transcription 4 (STAT4) is a transcription factor mainly associated with Th1 development, yet its role in neutrophil biology is unknown. We are the first to demonstrate that in vitro murine neutrophils stimulated with IL-12 directly activate STAT4 in a JAK2-dependent manner resulting in distinct transcriptional changes in antimicrobial response genes. STAT4-deficiency also impairs several neutrophil functions in vitro. Interestingly, IL-12 directly induces STAT4-dependent ROS formation at 14 hours, which is not due to cell death. Neutrophil extracellular trap (NET) formation in response to either LPS or heat-killed Pseudomonas aeruginosa results in decreased nuclear translocation of myeloperoxidase, decreased neutrophil elastase release, and decreased DNA release in STAT4-deficient neutrophils. In vivo neutrophil migration to the peritoneal cavity is blunted in Stat4−/− mice following either thioglycollate, CXCL1, or GM-CSF injections. Finally, we have generated Stat4floxLysMCre and Stat4floxS100A8Cre mice which are deficient in STAT4 in either myeloid cells or specifically neutrophils, respectively. Following in vivo infection with methicillin-resistant S. aureus, bacterial burden is increased in the blood and peritoneal cavity of Stat4floxLysMCre and Stat4floxS100A8Cre mice. Gene expression analysis of peritoneal exudate cells from infected mice show decreased expression of Ly6G/CD11b, further highlighting defective homing of Stat4−/− neutrophils in vivo. All together these data reveal an undiscovered role of STAT4 in neutrophil functions and a subsequent role of STAT4 in innate immunity and antimicrobial defense mechanisms.
β-1,3-glucan induces natural killer cell memory-like immunity
Authors: Li ShuShun , Mody Christopher H
Published: January 2023

Natural killer (NK) cells are considered innate lymphocytes that are able to kill pathogenic microorganisms, including bacteria, parasites and fungi as well as virus-infected cells and tumor cells without prior stimulation. Recent studies suggest that they possess a memory-like property to previously encountered pathogens (e.g., cytomegalovirus). We showed that NK cells directly kill Cryptococcus and Candida, which are fatal opportunistic fungi often infecting immunocompromised individuals. We determined that NKp30 is the activating pattern recognition receptor that mediates NK cell killing of these fungi, and that β-1,3-glucan is the ligand for NKp30. Interestingly, we found that soluble β-1,3-glucan enhanced NK cell killing of C. neoformans and C. albicans. Since humans and animals are exposed to β-1,3-glucan through inhalation of components of the cell wall of microbes or consumption of dietary grains, we asked whether isolated β-1,3-glucan plays a role in shaping NK cell immunity. To determine whether β-1,3-glucan induces a training or memory effect, YT cells, an NK cell line, were treated for 24 hours with laminarin or Saccharomyces β-1,3-glucan and cultured for various times. After an initial period of enhanced C. neoformans killing, killing was reduced to the untreated level on the second week after the initial treatment. However, when cryptococcal killing was assessed on the fourth week, a much greater killing was observed compared to the enhanced killing. This suggests that a recall immune response was induced. We conclude that β-1,3-glucan may induce NK cell memory- like immunity.
Evidence for serial antibody-dependent cell-mediated cytotoxicity (ADCC) by primary unstimulated human natural killer (NK) lymphocytes
Authors: Sung Alexander P , Navarrete-Galvan Lydia T , Guglielmo Michael , Smith-Gagen Julie , Redelman Douglas D , Merica Rebecca , Hudig Dorothy
Published: January 2023

Serial killers are more desirable than single-hit attackers when it comes to treating cancer. Antibody-dependent cell-mediated cytotoxicity (ADCC) mediated by NK lymphocytes is a significant component of many monoclonal antibody anti-cancer immunotherapies. However, there is a significant problem associated with ADCC: the NK cells lose their CD16A Fc-receptors and can no longer recognize target cells coated with antibodies, due to the action of NK cell metalloproteases. In this report, we searched for evidence for serial ADCC before the effector cells lose their receptors. Human ADCC measured by 51Cr-release at multiple NK effector to tumor cell ratios (E:Ts) was largely finished after 4 hours when the targets were Daudi B leukemia cells coated with non-fucosylated obinutuzumab anti-CD20. We reasoned that serial killing is evident when the number of dead targets exceeds the number of initial CD16A NK effectors. We assessed the dead Daudi per NK effector (D/E) at an E:T of 1:4 to detect serial killers given an excess of targets. For 28 healthy human subjects, the total ratio of dead targets per CD16Apos NK effector cell ranged from 0.6 to 2.2, with a median of 1.5 and average of 1.5 +/− 0.4. Four donors (14%) had D/Es >2. The D/E values were similar for CD16A AA158 F/F donors compared to F/V & V/V donors, even though the V-positive donors had greater initial NK cellular levels of CD16A. We conclude that this gross measurement (D/E) can detect serial ADCC even without the information provided by time-lapse cinematography to indicate the actual fraction of effectors that are serial killers. This simple measurement may be useful to screen for promotion of serial killing by reagents that reduce proteolytic loss of NK cell CD16A.
A rho GDP dissociation inhibitor produced by a subset of T-regulatory cells enhances mitophagy in Mycobacterium tuberculosis infected human macrophages
Authors: Paidipally Padmaja , Thandi Ramya Sivangala , Radhakrishnan Rajesh Kumar , McAllister Madeline K , Samten Buka , Vankayalapati Ramakrishna , Tripathi Deepak
Published: January 2023

Previously, we found that a Rho GDP dissociation inhibitor (D4GDI), produced by apoptotic T regulatory cells inhibit Mycobacterium tuberculosis (Mtb) growth in human macrophages through reactive oxygen species (ROS) mediated IL-1β production. Mitochondria is an important source of ROS and critical for antibacterial properties. In the current study, we evaluated the effects of D4GDI on mitochondrial homeostasis and anti-microbial scheme during Mtb infection. Recombinant D4GDI (rD4GDI) treatment of Mtb infected human macrophages enhanced the accumulation of LC3B protein on the mitochondria and triggered mitophagy. rD4GDI treatment significantly enhanced the expression of a mitophagy receptor BCL2 and adenovirus E1B 19-kDa-interacting protein 3-like (BNIP3L). BNIP3L siRNA inhibited rD4GDI dependent accumulation of LC3B protein on the mitochondria and enhanced Mtb growth in human macrophages. Further we found that rD4GDI treatment of Mtb infected macrophages induces the expression of antimicrobial peptide phospholipase family protein phospholipase A2 group VII (PLA2G7). Further studies are underway to determine the interactions between BNIP3L, PLA2G7 and mitophagy in rD4GDI treated macrophages to inhibit Mtb growth in human macrophages.
Activation of TREM1 Signaling in Kupffer Cells Following HIV and HCV Stimulation Augments Hepatic Inflammatory Responses
Authors: Thomas Emmanuel , Hyun Jinhee
Published: January 2023

In this study, we sought to examine inflammatory responses following HIV or HCV stimulation of macrophages or Kupffer cells (KCs), that may contribute to virus mediated inflammation and subsequent liver disease. KCs are liver-resident macrophages and reports have provided evidence that HIV can stimulate and infect them. In order to characterize HIV-intrinsic innate immune responses that may occur in the liver, we performed microarray analyses on KCs following HIV stimulation. Our data demonstrate that KCs upregulate several innate immune signaling pathways involved in inflammation, myeloid cell maturation, stellate cell activation, and Triggering Receptor Expressed on Myeloid cells 1 (TREM1) signaling. TREM1 is a member of the immunoglobulin superfamily of receptors and it is reported to be involved in systemic inflammatory responses due to its ability to amplify activation of host defense signaling pathways. Our data demonstrate that stimulation of KCs with HIV or HCV induces the upregulation of TREM1. Additionally, HIV viral proteins can upregulate expression of TREM1 mRNA through NF-κB signaling. Furthermore, activation of the TREM1 signaling pathway, with a targeted agonist, increased HIV or HCV-mediated inflammatory responses in macrophages due to enhanced activation of the ERK1/2 signaling cascade. Silencing TREM1 dampened inflammatory immune responses elicited by HIV or HCV stimulation. Finally, HIV and HCV infected patients exhibit higher expression and frequency of TREM1 and CD68 positive cells. Taken together, TREM1 induction by HIV contributes to chronic inflammation in the liver and targeting TREM1 signaling may be a therapeutic option to minimize HIV induced chronic inflammation.
Nerve associated lung resident interstitial macrophage subset exhibits distinct localization and polarization
Authors: Yeung Stephen T , Ural Basak Burcu , Damani-Yokota Payal , Devlin Joseph C. , De Vries Maren , Samji Tasleem , Jang Geunhyo , Loke P’ng , Dittmann Meike , Reizis Boris , Khanna Kamal M
Published: January 2023

Tissue resident macrophages are a diverse population of cells that perform specialized functions: sustaining tissue homeostasis and tissue surveillance. Here we report an interstitial subset of CD169+ lung resident macrophages that are transcriptionally and developmentally distinct from alveolar macrophages (AM). They are primarily localized around the airways and in close contact with the innervating pulmonary nerves. These nerve and airway associated macrophages (NAMs) are tissue resident, yolk sac derived, self-renewing, and do not require CCR2+ monocytes for development or maintenance. We show that unlike AMs, CSF-1, but not CSF-2 is required for their development. Bulk population and single cell transcriptome analysis indicates that NAMs are distinct from other lung resident macrophage subsets and highly expressed immune regulatory genes under steady state and inflammatory conditions. NAMs proliferated robustly following TLR activation and influenza infection, and in their absence, the inflammatory response was remarkably augmented resulting in excessive production of inflammatory cytokines and innate immune cell infiltration. We show that AMs contribute to viral clearance while NAMs, contribute to regulating virus-induced inflammation in the lung. Overall, our study relates structure to function at a fundamental level, and provides essential insights into a distinct subset of resident macrophages in the lung that function to maintain immune and tissue homeostasis.
IL-4R signaling mediated polarization of neutrophils promoted monocytes derived alveolar macrophages function
Authors: Chen Fei , El-Naccache Darine W. , Wu Wenhui , Inclan-Rico Juan M. , Ponessa John J. , Espinosa Vanessa , Lothstein Katherine E , rivera-Medina Ameriliz , Siracusa Mark C , Gause William
Published: January 2023

We have reported that alternatively activated neutrophils (N2) enhance lung anti-helminth trained macrophage effector function in response to infection with N. brasiliensis(Fei Chen, et al., NI, 2014). However, it is not clear how these neutrophils develop and what lung macrophage subsets actually mediate resistance. We report here that IL-4R signaling is required for the polarization of neutrophils within 2 days after N. brasiliensisinfection and that this characteristic N2 phenotype can develop in Rag−/− but not Rag2cg−/− mice, suggesting a role for innate lymphoid cells in initiating N2 cell differentiation. N2 neutrophils further showed significant proliferative capacity and oxidative phosphorylation at day 2 after N. brasiliensisinfection. Using CD45 congenic transfers and fate mapping Cx3cr1creER-YFP/+R26tdTomato/+ mice, we further show that monocyte-derived alveolar macrophages are the major effector macrophage subset mediating parasite killing through Arginase 1 dependent mechanisms. In vivo depletion of neutrophils with anti-Ly6G Ab blocked the development of this anti-helminth pulmonary macrophage subset and RNAseq analyses show marked differences from tissue resident alveolar macrophages. These studies demonstrate heterogeneity in lung macrophage and neutrophil subsets during helminth infection resulting in distinct activation pathways required for helminth resistance.
Simplified size-based immune cell monitoring using a hand-held particle counting device
Authors: Smith Janet L , Cappione Amedeo
Published: January 2023

Biological samples are often heterogeneous cell mixtures that differ by type and functional or responsive capacity. Beyond differences in protein expression, many cell types and physiological states are also distinguishable by size (cell diameter). The ability to distinguish population subsets is critical to many aspects of research. Such variation in cellular attributes is most commonly determined by multicolor fluorescent antibody detection of cell type-specific surface markers using flow cytometry. While flow cytometers are complex, trained operator-dependent platforms of core facilities, the availability of simplified methods for identifying activation status and measuring differences in the relative cell frequencies of more than one cell type in samples or co-cultures is somewhat limited. Moreover, despite the need for speed and accuracy, hemocytometry, the most widely used technique for cell counting, is quite tedious and fraught with user-driven variability. To address these needs, we present application of a hand-held cell counter that operates on the basic Coulter Principle of measuring changes in electrical impedance produced by particles (cells or beads) as they pass through a defined aperture. Specifically, instrumentation has been collapsed to the size of a pipette; the precision-made, microfluidic sensor contains the cell-sensing zone that enables size discrimination at the submicron with high counting accuracy. To demonstrate performance, the platform was used to assess cell distribution in human PBMC isolates and determine immune cell activation in response to CD3/CD28 co-stimulation or PHA exposure. All experiments were conducted in comparison to flow cytometry and a standard Coulter device.
Functional responses of salivary gland resident T cells are suppressed by extracellular adenosine
Authors: Springer Lauren E , Smith Corinne , Snyder Christopher M
Published: January 2023

Tissue-resident memory T cells (TRM) survive and persist within non-lymphoid tissues throughout the body, and are therefore subjected to an array of tissue-specific environments. Previous work showed that T cells extracted from the salivary gland were markedly dysfunctional for cytokine secretion upon ex vivo stimulation. This finding is important since the salivary gland is a site of shedding for several viruses. Cytomegalovirus (CMV) is a herpesvirus that persists in the salivary gland and is shed in saliva. Using the natural mouse pathogen murine (M)CMV, we previously showed that most MCMV-specific T cells in the salivary gland readily become TRM. Interestingly, CD8+ TRM in the salivary gland, but not T cells in the spleen, expressed both CD39 and CD73 ectoenzymes that break down extracellular adenosine triphosphate (ATP) to the immunosuppressive molecule adenosine. Expression of CD73 was dependent on TGF-β and neither CD39 nor CD73 expression was dependent on MCMV infection. Importantly, blocking the adenosine receptor A2AR during stimulation restored the function of CD8+TRM recovered from the salivary gland, resulting in IFN-γ and TNF-α production by salivary gland TRM that was comparable to T cells from the spleen. These data suggest that extracellular adenosine inhibits the function of salivary gland CD8+ TRM and that any ATP released during infection may be converted to adenosine by the TRM cells themselves, an avenue we are currently pursuing by use of mass spectrometry with and without MCMV infection.
CD6-ALCAM signaling regulates multiple effector/memory T cell functions
Authors: Ampudia Jeanette , Young-Greenwald William W. , Badrani Jana , Gatto Sole , Pavlicek Adam , Doherty Taylor , Connelly Stephen , Ng Cherie T
Published: January 2023

CD6 is a T-cell costimulatory receptor that has been implicated in the pathogenesis of multiple autoimmune and inflammatory diseases. Primarily expressed on CD4 T cells, CD6 promotes immune synapse formation, T-cell activation and T-cell migration via interaction with its ligand activated leukocyte cell adhesion molecule (ALCAM). While the contribution of CD6 to T cell activation has been well described, less is known regarding the role of CD6 on effector and memory T cells (Teff). Thus, to better characterize this, we examined phosphorylation signaling patterns during CD6 co-stimulation together with the impact of CD6 on differentiated Teff functions. Profiling of ~100 phosphorylation targets associated with T-cell receptor signaling revealed that CD6 co-stimulation on T cells activates factors in pathways involved in actin polymerization, motility, integrin activation and T-cell activation. Comparison of Tnaive cells vs. Teff demonstrated differing levels of phosphorylation in response to CD6 stimulation. Furthermore, CD6 signaling on Teff sustained phosphorylation of these pathways at later timepoints compared to CD28 stimulation. Blockade of the CD6 pathway, using the clinically tested anti-CD6 mAb itolizumab during re-stimulation of CD4 Teff cells in the presence of ALCAM, inhibited multiple effector functions including proliferation and changes in blast size. This effect was observed exclusively in the presence of ALCAM, indicating that the effect was specific to blockade of the CD6-ALCAM pathway. These findings demonstrate that the CD6-ALCAM pathway is a key regulator of effector T-cell functions and further support targeting this pathway to directly inhibit both naïve and effector T cell populations.
Antigen presenting group 3 innate lymphoid cells control neuroinflammation
Authors: Grigg John B , Shanmugavadivu Arthi , Regen Tommy , Parkhurst Christopher , Eberl Gerard , Waisman Ari , Sonnenberg Gregory F
Published: January 2023

Pro-inflammatory T cells in the central nervous system (CNS) are causally associated with multiple demyelinating and neurodegenerative diseases, but the pathways controlling these responses remain unclear. Here we define a population of group 3 innate lymphoid cells (ILC3s) that are uniquely enriched in the CNS during a mouse model of multiple sclerosis. CNS-associated ILC3s exhibit distinct transcriptional profiles, localize in proximity to infiltrating T cells, and express major histocompatibility complex class II (MHCII) and co-stimulatory molecules. Critically, antigen presentation by ILC3s was required to selectively promote pro-inflammatory T cell responses in the CNS and the development of multiple sclerosis-like disease. In contrast, antigen presenting ILC3s in the periphery lack co-stimulatory molecules and did not appear to contribute to disease induction, but rather could induce tolerance in autoimmune T cells and prevent multiple sclerosis-like disease when experimentally targeted to present myelin antigen. Collectively, our data demonstrate an essential role for CNS-associated ILC3s in promoting T cell-dependent neuroinflammation and reveal the potential to harness peripheral ILC3s for the prevention of autoimmune disease.
Laquinimod arrests development of experimental autoimmune uveitis (EAU) and inhibits related immune processes, in the context of altered gut microbiota
Authors: Xu Biying , Jia Xiuzhi , Tang Jihong , Caspi Rachel R , Gery Igal
Published: January 2023

EAU is an animal model for autoimmune uveitis in humans and is used to study mechanisms and potential therapeutics for this blinding disease. Here, we tested the immunosuppressive efficacy of Laquinimod (LAQ), a potent AhR (aryl hydrocarbon receptor) ligand on development of EAU.

C57BL/6J mice were challenged for EAU with 300ug of the uveitogenic antigen, IRBP 651–670 and were treated from day 0 or day 7, relative to immunization, with 25mg/Kg LAQ, or PBS, by oral gavage. EAU progression was monitored by fundus examination and confirmed by histology on day 14 post immunization (p.i.). Treatment with LAQ from day 0 p.i. completely prevented EAU development and inhibited proliferation and production of pro-inflammatory cytokines to peptide 651–670. Treatment with LAQ from day 7 p.i. also significantly inhibited the development of EAU and cellular immune responses, but to a lesser degree than in day 0 mice. In contrast to its inhibitory effect on pro-inflammatory processes, LAQ treatment increased the proportions of T-regulatory FoxP3+CD4+ T cells in treated mice, as compared to the control group.

Metagenomic analyses revealed that immunized mice treated with LAQ had altered composition of gut microbiota when compared with PBS controls, with an increased population of Bifidobacteriaceae, and decreased Clostridaceae 1, Deflubiitaleaceae and Anaeroplasmataceae bacteria.

Our results demonstrate that LAQ, a water-soluble AhR ligand, is a potent inhibitor of EAU development and exerts its effect primarily during the inductive phase of the disease. The connection of the observed changes in gut microbiota to its inhibitory effects on disease is under investigation.
T cell-intrinsic C5L2 activation protects against uncontrolled inflammatory Th responses and autoimmunity
Authors: West Erin E , Freeley Simon , Kolev Martin , Niyonzima Nathalie , Mogensen Trine Hyrup , Woodruff Trent M , Monk Peter , Lappegard Knut , Espevik Terje , Mollnes Tom Eirik , Christiansen Mette , Kemper Claudia
Published: January 2023

Intracellular/autocrine complement proteins have emerged as critical regulators of human Th1 induction and contraction. T cells contain both intracellular C3 and C5 activation systems, with intracellular C3aR1 and C5aR1 stimulation driving T cell homeostatic survival and normal Th1 IFN-gamma production, respectively. Here we demonstrate how the intracellular/autocrine C5 system is regulated by using T cells from the first described patient with C5aR2 deficiency. This patient suffers from an autoinflammatory syndrome, with enhanced inflammatory Th responses and a profound loss of naïve CD4 T cells (95% of CD4 T cells in the blood have a memory T cell phenotype). Thus, in contrast to intracellular C5aR1 stimulation, cell surface expressed C5aR2 is an important negative regulator of inflammatory Th induction. While both C5aR1 and C5aR2 can bind C5a, we found that the carboxypeptidase-processed form of C5a, C5a-desArg, was twice as potent as C5a in reducing Th1 induction. In addition, carboxypeptidase M (CPM) expression was highly induced upon T cell activation indicating that CPM may be mediating T cell-derived C5a-desArg generation and thus, C5aR2 stimulation. In this vein, activation of T cells in the presence of a CPM inhibitor induced Th1 hyper-induction, which was rescued by addition of a C5aR2 agonist and reduced by adding C5a-desArg, but not C5a. The in vivo importance of T cell-expressed CPM and autocrine C5aR2 activation is demonstrated by the fact that CPM−/− mice have enhanced inflammatory Th responses and CPM−/− T cells cause increased pathology in an in vivo T cell transfer colitis model. These data highlight the complex auto-regulatory functions of complement in T cell responses.
Toll like receptor 4 ligation promotes B7 family costimulatory molecules expression in a time and cytokine dependent fashion
Authors: Soto Henry Velazquez , Real Fernanda , Salazar Alberto , Jiménez Martínez Maria C
Published: January 2023

Introduction

The interplay between costimulatory and coinhibitory molecules allows adequate integration of instructions for T cell fate. In this study, we explored how antigen concentration and time exposition to a pathogen associated molecule pattern may modulate the expression of B7 family costimulatory molecules CD86, ICOSL, PDL1, PDL2 and the Interferon Regulatory Factor 4 in human monocyte-derived dendritic cells (MoDCs).

Methods

MoDCs were obtained from healthy donors. MoDCs were stimulated with different concentrations of E. coli lipopolysaccharide and stimulated for 12, 24 or 48 h. Supernatant from stimulated MoDCs were collected for soluble cytokines determination using cytometric bead arrays. Cells were collected and membrane stained with fluorochrome-conjugated monoclonal antibodies to evaluate PDL1, PDL2, CD86 and ICOSL trough flow cytometry. Intracellular staining was performed for IRF4 after cell permeabilization.

Results

TNF α and IL-6 were significantly increased at 12 h, while IFN-γ and IL-2 were increased at 48 h. Costimulatory molecules showed a differential expression pattern. ICOSL was preferentially incremented at 48 h post stimuli independently of concentration used. PDL1 was induced at low concentrations and short stimulation periods, maintaining their expression all along with the stimulation kinetic. PDL2 increased its expression at short time of stimulation. CD86 and IRF4 did not show statistically significant differences.

Conclusion

Our results suggest that costimulatory molecules have time-dependent expression profile, possibly influenced by the cytokines contained in the microenvironment and a lesser by the antigen concentration.