bluuebunny/crossref_metadata_2025 huggingface dataset:
Development of syngeneic NOD tumor models to study tumor immunity in autoimmune-prone mice.
Authors: Young Arabella , Nguyen Vinh , Mehdizadeh Sadaf , Sheehan Kathleen C. F. , Serreze David V. , Schreiber Robert D. , Bluestone Jeffrey A.
Published: January 2023

With increasing use of cancer immunotherapies, it is evident that some patients develop immunotoxicity, termed immune-related adverse events (irAEs). Many of these irAEs are a consequence of a peripheral breakdown in self-tolerance but the basis, be it genetic, epitope spreading or environment, remains unclear. This highlights an urgent need to develop preclinical models to profile autoimmunity together with anti-tumor immunity to accurately assess the etiology by which autoimmune pathologies are induced in immunotherapy-treated cancer patients. We generated transplantable syngeneic tumor cell lines in the autoimmune-prone NOD background from spontaneous tumors and methylcholanthrene (MCA)-induced fibrosarcomas. We investigated whether the presence of tumor altered the severity and frequency of autoimmunity in response to cancer immunotherapies. Anti-PD-1, but not anti-CTLA-4, accelerated the onset of autoimmune type 1 diabetes (T1D), similarly observed in a proportion of anti-PD1-treated cancer patients, while also providing improved tumor control and altering the expression of immunomodulatory markers (including CD73, CD39, PD-1) in both pancreatic islets and periphery. In addition, using NOD mice, congenic for the insulin-dependent diabetes (Idd) susceptibility gene Idd9, we determined that the NOR-derived gene locus provided significant protection from anti-PD-1-induced T1D. Tumor studies are underway in these and other Idd congenic NOD mice. Together, these preclinical models provide a platform to assess safety profiles for cancer immunotherapies, identifying cellular mechanisms and therapeutic interventions that inhibit the development of autoimmunity while preserving anti-tumor immunity.
Targeting inflammation to prevent breast cancer metastasis
Authors: Holl Eda K. , Frazier Victoria , Boczkowski David , Sullenger Bruce , Nair Smita K.
Published: January 2023

Biomarkers associated with systemic inflammation correlate with poor prognosis in cancer patients. Circulating cell-free DNA (cfDNA) is released passively and actively by both tumor cells and host cells and cfDNA levels in blood correlate with tumor burden in many tumor types. Notably, studies in breast cancer have shown that inhibition of cfDNA has a significant effect in reducing invasion and migration in vitro. We have previously demonstrated the ability of nucleic acid binding polymers (NABPs) to bind and inhibit the pro-inflammatory effects of cfDNA and RNA in models of systemic lupus erythematosus and influenza. Herein, we demonstrate that in tumor bearing mice, levels of cfDNA increase with tumor burden and decline post NABP in vivo administration. Using an orthotopic breast cancer murine model we demonstrate that NABP treatment, following surgical resection, results in reduced or completely absent lung metastasis. In addition, intravenous delivery of breast cancer cells in the presence of NABPs also results in decreased tumor cell lung seeding and lung metastasis. Taken together, our data suggest that NABPs can be used to target cfDNA and inhibit such DAMPs from activating pathological inflammation, an innovative approach to treat cancer progression and metastasis. In particular, NABPs may be useful in the clinical setting following standard of care surgical tumor resection.
Utilizing the 2017–2018 seasonal influenza vaccine as a treatment for cancer
Authors: Newman Jenna H. , Chesson Charles Brent , Aspromonte Salvatore , Bommareddy Praveen K. , Pepe Russell , Tarabichi Saeed , Li Shengguo , Jhawar Sachin , Herzog Nora L. , Aboelatta Mones , Kaul Eshan , Estupinian Ricardo , Kane Michael P. , Silk Ann , Zloza Andrew
Published: January 2023

In recent years, immunotherapy has yielded increased survival for cancer patients; however, a significant percentage of patient tumors remain refractory to immunotherapy. This is due in part to the absence of an inflamed tumor microenvironment. In our effort to utilize anti-pathogen vaccination to recruit immune cells to un-infiltrated (cold) tumors, we observed that intratumoral injection of influenza A/PR8/H1N1 lysate (but not live virus) reduced B16-F10 melanoma growth (p<0.01). Towards determining whether FDA-approved human vaccines could likewise be utilized, we administered a quadrivalent 2017–2018 human seasonal influenza vaccine via intratumoral injection to mice, significantly slowing tumor growth relative to that of PBS-injected controls (p<0.05). Analysis of RNA from tumor homogenates via the NanoString PanCancer Immune Profiling Panel indicated upregulation of transcripts for chemokines (CCL5, CXCL9), immune checkpoints (PD-L1, LAG3), and HLA molecules – hallmarks of an inflamed (hot) tumor phenotype. In addition to halting tumor growth, intratumoral administration of the human influenza vaccine protected mice from subsequent intranasal influenza challenge. Our findings suggest that intratumoral administration of human vaccines can reduce tumor growth, in part by converting “cold” tumors into “hot” (immune-infiltrated) tumors, while simultaneously providing protection against infection. Future studies will address the efficacy of vaccination in humanized mouse models and the possibility of synergy with checkpoint blockade therapies, in an effort to evaluate the potential of microbial-based therapies as a dual vehicle for cancer treatment and infection prevention.
HIV-1 and HIV-2 induce Interferon-lambda production by plasmacytoid Dendritic Cells
Authors: Hosmalin Anne , Isnard Stéphane , Vimeux Lene , Guillerme Jean-Baptiste , Ramirez Bertha Cecilia , Morel Marina , Cheynier Rémi , Iannetta Marco
Published: January 2023

Type III Interferons (IFN-λ) were barely studied during HIV infection, despite their antiviral properties similar to those of type I IFN and their important role during HCV infection, where IFN-λ3 gene polymorphisms correlate with viral clearance. We tested IFN-λ responses to HIV-1, and also to HIV-2, which is less pathogenic.

We first stimulated PBMC (EFS-Inserm convention) enriched for dendritic cells (DC), the main IFN producers. Purified HIV-1 virions or H9 T cells chronically infected with HIV-1 induced transcription of IFN-λ1 and 2/3 mRNA (RT-qPCR), as well as the secretion of IFN-λ (ELISA). Similar results were obtained with HIV-2-infected cells or virions. Intracellular labeling for IFN-λ1 was detected by flow cytometry in BDCA-4+ plasmacytoid DC (pDC), but not in BDCA-1+ or -3+ conventional DC (cDC). This was confirmed in > 95% purified cell populations. Recognition of HIV-1 or HIV-2 by pDC induced their maturation (surface CD83 and CCR7 overexpression), and bystander maturation of cDC.

Therefore, IFN-λ production by human blood pDC was induced at comparable levels by HIV-1 or HIV-2, either as cell-free viruses or as infected cells, indicating that it is not a clue for the difference in host-pathogen relationship between the two viruses, and that IFN-λ may contribute to the Interferon-Stimulated-Gene response during HIV infection. Knowledge of IFN-λ responses to HIV will help modulating IFN responses toward viral control and against pathogenic immune hyperactivation.
EBV infection modulates the cell viability of gastric cancer cell through modulating miR34a-NOX2-ROS signaling
Authors: Hur Daeyoung , Yun Sun-mi , Choi Dabin , Kim Seon Hyun , Lee Mi Kyung , Kim Yeong Seok
Published: January 2023

Epstein-Barr virus (EBV) nuclear antigen 1 (EBNA1) is a viral protein expressed in all EBV-infected cells that induces malignant transformation. EBNA1 is reported to contribute to tumor progression through an increase in reactive oxygen species via nicotinamide adenine dinucleotide phosphate oxidase. However, the underlying molecular mechanism of EBNA1-induced ROS accumulation in gastric cancer is poorly understood. Here, we demonstrated that miR34a regulation by EBNA1 determined cell fate in EBV-infected gastric cancer cells. ROS content and NOX2 expression were higher in EBNA1-expressing SNU719 cells than in EBNA1-nonexpressing SNU638 cells. Downregulation of NOX2 using siRNA technology in SNU719 cells decreased cell viability and ROS content. Regulation of EBNA1 expression in EBV-associated gastric cancers modulated NOX2 expression, ROS content and cell viability. We also showed that upregulation of NOX2 by EBNA1 was mediated by downregulating miRNA34a. Finally, overexpression of miR34a in EBNA1-expressing SNU719 cells induced typical apoptosis, suggesting that reactivation of miR34a in EBNA1-expressing gastric cancer cells could be a strategy for treatment of EBV-infected gastric cancer cells.
A novel mechanism for IL-23 to up-regulate AICDA and promote pathogenic autoantibodies in lupus
Authors: Hong Huixian , Wu Qi , Yang PingAr , Lou Bao , Li Jun , Li Hao , Cua Daniel J. , Hsu Hui-Chen , Mountz John D.
Published: January 2023

Targeting IL-23 to treat autoimmune disease and chronic inflammation is under development based on its proinflammatory function and pro-Th17 cell effects. IL-23 was previously shown to be important for promoting pathogenic autoantibody production in B6-Faslpr/lpr mice. In contrast, the lack of IL-23 did not influence germinal center (GC) formation and IgG anti-CII autoantibodies in type II collagen immunized DBA/1 mice. We determined if IL-23 regulated development of spontaneous GCs and autoantibody production in lupus-prone BXD2 mice by generating IL-23 deficient BXD2-p19−/− mice. There was significantly decreased total IgG, but increased total IgM and IgM anti-DNA and RF autoantibodies in BXD2-p19−/− mice compared to BXD2 mice. Flow cytometry and confocal imaging analyses showed that both the size and number of GL-7+Fas+ GC B cells were increased, yet there was a decreased development of GC dark zone (DZ) (CD86−CXCR4+) B cells in BXD2-p19−/− mice. Interestingly, the expression of activation-induced cytidine deaminase (AICDA or Aicda) was significantly decreased in GC B cells in BXD2-p19−/− mice. However, the expression of GC program genes (Bach2, Pax5) was increased except Bcl6. In contrast, the expression of key plasma cell program genes Prdm1 in GC B cells was diminished in the absence of IL-23.

Our data suggest a novel pathway for IL-23 to promote IgG autoantibody production by upregulation of AICDA in the GC DZ. Importantly, such pathway is independent of the canonical GC program genes that have been implicated for the induction of AICDA. These results suggest a potential novel action of anti-IL-23 therapies to inhibit adaptive immune responses in addition to its known inhibitory role of innate response in autoimmune diseases.
IL-27 controls allergic airway inflammation via Foxp3+ regulatory T cells
Authors: NGUYEN QUANG TAM , MIN BOOKI
Published: January 2023

Asthma is a chronic inflammatory disease in the lung mediated by allergen reactive Th2 type CD4 T cells producing the Th2 type cytokines. We recently reported that IL-27 plays a key role in Treg functions to control Th17-mediated autoimmune inflammation. However, a role for IL-27 in modulating Treg function during Th2-mediated allergic airway inflammation remains unclear. Here, we report that during cockroach antigen (CA)-induced allergic airway inflammation, IL-27 attenuates inflammatory responses primarily via Foxp3+ Tregs. Sensitizing mice with CA in alum followed by intranasal challenge induces allergic airway inflammation characterized by infiltration of eosinophils and effector CD4 T cells expressing Th2 cytokines in the lung. Recombinant IL-27 administration prior to antigen challenge significantly reduces the inflammation. Accumulation of CD4 T cells expressing inflammatory cytokines is dramatically reduced in the inflamed sites following IL-27 administration. However, IL-27-mediated treatment effects are completely lost when Foxp3+ Tregs are deleted prior to CA challenge. Adoptive transfer of wild type Tregs restores IL-27-mediated inhibition of airway inflammation, suggesting that Tregs may be the direct responders of IL-27. IL-27 stimulation in Tregs increases expression of lymphocyte activation gene 3 (Lag3), a surface molecule implicated in Treg functions. Unlike WT type cells, adoptive transfer of Tregs deficient in Lag3 fails to attenuate airway inflammation even after IL-27 administration. Taken together, these results demonstrate a novel IL-27-dependent Treg function to control allergic airway inflammation
Activation of neonatal mouse lung ILC2s by endogenous IL-33 impacts type 2 responses later in life
Authors: Steer Catherine A. , Shim Brian , Mathae Laura , Takei Fumio
Published: January 2023

Neonatal lung immunity is functionally distinct from adults as responses to environmental antigens are predominantly type 2 biased, and early exposure has been linked to the development of airway allergic diseases. Recently our lab and others have shown that endogenous release of IL-33 due to the stresses of the first few breaths of life leads to expansion and activation of group 2 innate lymphoid cells (ILC2s) from 10 days (D10) to two weeks after birth. However, the amount of IL-33 is lower than in allergen-treated lungs of adult mice, and neonatal lungs do not show signs of type 2 inflammation. Nevertheless, our analyses of IL-33KO and ILC2 deficient pups have suggested that the activation of neonatal lung ILC2s by endogenous IL-33 drives type 2 immunity. As well, neonatal ILC2s labeled with BrdU persist in adulthood. Therefore, we further characterized neonatal lung ILC2s and investigated the effects of neonatal activation on ILC2 functions later in life. Our microarray analysis of neonatal ILC2s showed more similarity to IL-33 activated than naïve adult ILC2s. D10 ILC2s have a high expression of IL-25R, similar to activated adult ILC2s, and can be stimulated by IL-25. We compared IL-33KO and WT ILC2s in adult mice and there was no difference in the phenotype or number of lung ILC2s. However, intranasal (IN) IL-33 injections induced significantly fewer IL-5+IL-13+ ILC2s in IL-33KO mice. We then injected IL-33 into IL-33KO neonates to test the effect on adult ILC2s. Preliminary results show that IN IL-33 into neonatal IL-33KO mice rescue the impaired response of adult IL-33KO ILC2s. These results suggest that the weak activation by the endogenous IL-33 of neonatal lung ILC2s has significant effects on the development of type 2 responses later in life.
Profiling Serum Antibody Specificities in Infants Reveals a Significant Number with Autoreactive Antibodies
Authors: van Oers Nicolai S. , Raj Prithvi , Pichilingue-Reto Patricia , Dozmorov Igor , de la Morena M. Teresa , Wakeland Edward K.
Published: January 2023

The antibody repertoire of an infant develops in response to infections, environmental exposures, and vaccinations. By adulthood, many will produce antibodies that react against self-antigens, often causal to autoimmune diseases such as lupus erythematosus. Limited scientific literature exists as to whether such autoreactive antibodies develop early in infancy. For this reason, the antibody specificities in the serum of healthy infants at 1 and 2 years of age was analyzed. Screening in a cohort of 82 infants revealed that 23 (28%) had moderate to high titered antibodies directed to diverse self-antigens. These numbers are consistent with observed ANA positivity in adults. Ongoing custom targeted DNA sequencing analysis will assess the genetic load of known autoimmune risk alleles in the ANA positive infants. Relationships between the antibody specificities and the genetic risk alleles assembled for each infant will be presented. The comparisons will include clinical information; sex, ethnicity, growth records, vaccination status, infectious history, antibiotic and antiviral treatments, disease status, and family history. Genomic analysis of infants with autoantibodies may facilitate implementation of a new wellness screen to identify antibody positive infants that have significant genetic predisposition and therefore are at risk for developing diverse immune system abnormalities including autoimmune disorders.
Neurokinin 1 receptor-signaling sustains T-cell survival during thymus development and following T-cell activation in secondary lymphoid organs
Authors: Larregina Adriana T. , Sumpter Tina L. , Rojas-Canales Darling M. , Tkacheva Olga A. , Shufesky William J. , Falo Louis D. , Morelli Adrian E.
Published: January 2023

T-cell receptor (TCR)-signaling triggers intracellular Ca2+ increase required for NFAT1/2-mediated IL-2 secretion. This pathway is necessary for thymocyte maturation and survival of activated T cells in secondary lymphoid organs (SLOs). In T cells, cellular Ca2+ levels are regulated by TCR- and G-protein coupled receptor (GPCR)-signaling via the PLCγ and PLCβ subunits, respectively. Nevertheless, the GPCR(s) involved in this phenomenon has not been identified. The neurokinin-1 receptor (NK1R) is a GPCR that induces Ca2+ flux in neurons, and NK1R-signaling by the neuropetides substance P (SP) and hemokinin 1 (HK1) promotes T-cell immunity. We studied the role of NK1R-signaling in T-cell development in the thymus and after T-cell priming in SLOs. By Imagestream, we found that the NK1R and its ligands localize at the site of dendritic cell (DC)-T cell contact. Following CD3-signaling, the NK1R was required for optimal Ca2+flux and NFAT-mediated IL-2 secretion in T cells, effects that were abrogated in NK1RKO or SP/HK1double KO T cells. In the thymus, absence of NK1R resulted in decreased maturation and survival of TCR+ double positive CD4 CD8, single positive CD4, and single positive CD8thymocytes. In SLOs, the NK1R was required for survival of Ag-activated CD4 Th1 and CD8 T cells. In vivo, in a skin model of Th1-DTH induced in NK1RKO T-cell or HK-1/SPdouble KOT-cell bone marrow chimeras, 73±5% of activated CD4 and CD8 T cells died during priming in skin-draining SLOs, and the remaining T cells died in the skin following elicitation. We conclude that the NK1R cooperates with the TCR to increase intracellular Ca2+ necessary for thymocyte maturation and survival of activated T cells in SLOs.

NIH R01 AR068249 and AR071277 to ATL and LDF.
Cell culture oxygen levels affect B cell migration through HIF-1a signaling
Authors: Henn Alicia D. , Garigen Jessica , Cipolla Valentina , Perry Sheldon , Farovitch Lorne , Yerden Randy , Zand Martin Stuart , Hilchey Shannon
Published: January 2023

B lymphocytes in vitro encounter oxygen levels far in excess of what they experience in vivo. Room air incubator O2 is not 21%, the accepted level for outdoor O2[1]. High CO2 and humidity drive incubator O2 down (16–19%), with variability from door openings. However, in vivo O2 is far lower even (tissues 0–7%, blood 5–10%). Hypoxia-induced factor 1 alpha (HIF-1a) is a tightly regulated transcription factor responsive to O2 levels [2]. Downstream of HIF-1a are genes tied to the unfolded protein response, metabolism, proliferation, and migration. A therapeutic target in multiple myeloma [3], HIF-1a protein is stabilized at low O2 levels and rapidly degraded at higher O2 levels. We previously showed in primary human B cells and myeloma cell lines, that HIF-1a protein levels stabilize at physioxic 1% and 4% oxygen, but not at 19% oxygen. B cell migration in response to the CXCR4 ligand, CXCL12, was decreased at low O2 levels. Here we seek to add direct evidence to HIF-1a participation in B cell migration changes by using a lentiviral vector to produce stable transfectants expressing a HIF-1a shRNA to genetically ablate HIF-1a. We used C-chamber sub-chambers and Pro-Ox 110 gas controllers to stably control the gas levels inside a standard incubator. At 1% O2, HIF-1a shRNA expression resulted in undetectable HIF-1a protein levels, and a correlating increase in CXCR4-mediated B cell migration as compared to a lentiviral control vector or non-transfected cells. This adds genetic evidence that directly links incubator O2 levels, HIF-1a activity and B cell migration in vitro.
Methyl-CpG binding domain protein-1 polymorphisms impact immune homeostatic levels of IgG
Authors: Hampton Brea K. , Noll Kelsey E. , Whitmore Alan C. , Plante Kenneth S. , Ferris Martin T. , Heise Mark T.
Published: January 2023

Host immunity plays a key role in protection against infection, however, there is significant individual-to-individual variation in the induction and quality of immune response that is driven in part by genetic factors. A growing body of evidence suggests that variation in immune status prior to specific insult may influence subsequent response to vaccination or pathogen challenge. Therefore, understanding the variation in immune status prior to specific insult is necessary for assessing variability in immune response to vaccination or pathogen challenge. The Collaborative Cross (CC) mouse population is a genetically diverse population that was designed to mimic the genetic and phenotypic diversity that is observed in humans and allows us to investigate mechanistically the contribution of genetics to complex phenotypes. Consequently, we asked whether there was variation in antibody levels in 57 CC strains prior to pathogen challenge and if that variation was associated with specific genetic factors. To assess this, we measured antibody levels for IgM, total IgG, IgG1, IgG2a, IgG2b, and IgG2c by ELISA and mapped quantitative trait loci (QTL) associated with variation in antibody concentrations. We observed substantial variation in all antibody isotypes measured. We also identified a suggestive (p=0.1) QTL on chromosome 18. Allele effects show that the C57Bl/6J allele at the locus of interest is linked to higher levels of total IgG. Subsequent analysis of genes under the QTL identified Methyl-CpG binding domain protein-1 (Mbd1) as the most promising candidate gene associated with total IgG variation. Ongoing studies are evaluating the role of Mbd1 in the regulation of total IgG production.
Development and characterization of a bovine monocyte-derived macrophage cell line
Authors: Chitko-McKown Carol G. , Bartens Marie C. , Gibson Amanda J. , Angela Holder , Vats Ashutosh , Brown Emma , Kolakowski Jeannine , Workman Aspen Mikaela , Heaton Michael P. , Kalbfleisch Theodore S. , Werling Dirk
Published: January 2023

Monocytes are components of the innate arm of the immune response and are one of the first lines of defense again invading pathogens. However, they also serve as host cells for intracellular pathogens such as Mycobacteria, Brucella, and Salmonella. Monocytes represent only a small percentage of circulating leukocytes, thus harvesting sufficient quantities of them for in vitro experiments can be costly, time consuming, and can vary among cattle. It was our objective to develop a monocyte-derived macrophage cell line that could be utilized for studies involving macrophages. Monocytes were obtained from the whole blood of a cross-bred steer by density gradient centrifugation, and monocytes purified by adherence. After extended culture, a population emerged spontaneously that proliferated in culture and could be easily detached from the tissue culture vessels using tryspin-EDTA. These proliferating cells were tested for cell surface determinants indicative of monocyte/macrophage lineage, as well as bactericidal and phagocytic activity. Furthermore, whole genome sequencing on both the cell line and whole blood from the donor steer was performed to determine how they align with the bovine whole genome build, and to identify any genetic mutations. This bovine monocyte-derived macrophage cell line has been passaged over 25 times, resembles macrophages in culture, expresses the CD markers CD14/16, C172a, CD11b, is phagocytic and bactericidal, produces IFN in response to TLR/RLR ligands, and appears to fall into the M2 macrophage category. There is a dearth of bovine macrophage cell lines available to veterinary researchers, and this cell line should be a useful tool for the in vitro study of numerous macrophagetrophic pathogens.
Mechanisms Regulating IL-1β Mediated Inflammation during Secondary Bacterial Pneumonia
Authors: Lupfer Christopher R. , Rodriguez Angeline E. , Freeman Thomas , Rippee-Brooks Meagan D. , Abysalamah Hazzar M. , Smith Amber M. , McCullers Jonathan A. , Kanneganti Thirumala-Devi
Published: January 2023

Viral bacterial coinfections are known to cause severe pneumonia due to enhanced pathogen growth and excessive or altered immune responses. Despite knowing the overall causes for disease severity, current treatments fail to improve disease in many patients. Thus, an improved understanding of the specific immune pathways involved is needed to improve therapeutic treatment design. Interlukine-1β (IL-1β) is an important immune signaling cytokine responsible for inflammation. It exists as an inactive precursor that can be activated by caspase-1 containing inflammasomes. We found that coinfection of Influenza A virus and Streptococcus pneumoniae results in inflammasome activation mainly through the NOD-like receptor protein NLRP3. IL-1β levels are synergistically enhanced during coinfection, but not overall inflammasome activation. Instead, enhanced activation of the transcription factor NF-κB results in increased production of pro-IL-1β, which is subsequently processed into its active form by the inflammasome. In vivo, we discovered that IL-1β regulates lung pathology, and mice treated with a combination of antibiotics and IL-1β neutralizing antibodies have improved weight loss and mortality compared to either treatment individually.
T cell exhaustion during murine cutaneous and visceral leishmaniasis.
Authors: Valadares Diogo Garcia , Davis Richard E. , Kiser Ellen , Chen Yani , Borbon Tiffany , Wilson Mary E.
Published: January 2023

Leishmaniasis is a chronic progressive disease usually controlled by TH1-type IFN-γ producing cells. Human or murine leishmaniasis can progress despite the presence of T cells capable of producing IFN-γ. It is reported that CD8 cells in humans with visceral leishmaniasis (VL) express markers of T cell exhaustion. We hypothesized that myeloid cells play a role in exhausting T cells at the local sites of Leishmania species infection inhibiting proper adaptive responses. We addressed this hypothesis in BALB/c models of cutaneous leishmaniasis (CL) or VL caused by L. major or L. donovani infection, respectively. We examined inhibitory receptors PD1, LAG3, CTLA4 and TIM3 on lymphoid cells, and counter-receptors PDL1, MHCII, and CD80 in myeloid cell by flow cytometry throughout 4 weeks of infection. Both models showed significant increases in PD-1 in circulating dendritic cells after 1 week of infection. However, after 3–4 weeks when infection was chronic, PDL-1 was expressed on neutrophils in the blood and at the local sites of infection (infected ear tissue and draining lymph nodes of L. major infected mice; spleen cells of L. donovani-infected mice). Furthermore, PD1 was expressed on both CD4 and CD8 T cells in blood and lymph nodes of mice infected with L. major at the same time. In contrast, only CD4 T cells, but not CD8 T cells) displayed higher PD-1 in blood and spleens of mice infected with L. donovani. In conclusion, both disease models elicited the exhaustion counter-receptor PDL1 on neutrophils in the blood and the local site of parasite growth. The T cell exhaustion marker PD1 was expressed on T cells at the same sites, although both CD4 and CD8 T cells displayed PD1 in the CL model, whereas PD1 expression was more profound on CD4 T cells in the VL model.
Exogenous induction of type I IFN protects mice from Cryptococcus neoformans and Cryptococcus gattii infections by cellular immunity and iron restriction.
Authors: Davis Michael J. , Moyer Shannon , Sionov Edward , Mayer-Barber Katrin D. , Barber Daniel L. , Cai Hongyi , Walter Peter J. , Jenkins Lisa , Chang Yun C. , Kwon-Chung Kyung J.
Published: January 2023

Rational

Cryptococcus neoformans (Cn) causes deadly mycosis in AIDS patients whereas Cryptococcus gattii (Cg) infects mostly non-HIV patients. As HIV infection induces type I IFN (t1I) we hypothesized that exogenously induced t1I would differentially affect infections by Cn and Cg.

Results

Cn or Cg infected mice were dosed with poly(I:C)LC (PIC) to induce t1I. PIC treated mice were protected from Cn and Cg infection mortality with reduced fungal burdens. In Cn infected mice, PIC protection was mediated by classical Th1 immunity as CD4 T cells and IFNγ were required and Cn mediated Th2 polarization and eosinophilia were reversed by PIC. In contrast, PIC protection from Cg did not require any of the immune factors previously associated with Cn immunity; T cells, B cells, NK cells, IFNγ and monocyte derived myeloid cells were all dispensable for protection. Interestingly, PIC protection depended on β-2-microglobulin (B2m). In addition to its role as a co-factor for several immunological proteins, B2m is involved in iron trafficking suggesting that PIC protection may function by limiting Cg’s access to iron. In support of this, iron supplementation reversed PIC protection and iron chelation mediated similar protection as PIC. PIC also induced expression of several proteins involved in iron trafficking.

Conclusions

These data show that t1I induction protects mice from Cn and Cg but protection was mediated by distinct immune mechanisms. PIC mediated protection from Cn requires CD4 T cells and IFNγ. However, this classical immune pathway to cryptococcosis is not involved in PIC-mediated protection from Cg. Instead, PIC protection from Cg seems to involve iron limitation suggesting a novel role for t1I in regulating host iron in the lungs.
A2B Adenosine Receptor Signaling on Epithelial Cells Promotes Type 2 Immunity Against Helminths
Authors: El-Naccache Darine W. , Chen Fei , Palma Mark , Wu Wenhui , Csoka Balazs , Hasko George , Gause William C.
Published: January 2023

Intestinal helminth infections trigger potent type 2 immune responses, although the mechanisms that initiate these host protective responses against these multicellular parasites remain uncertain. We have previously reported that inoculation with the murine intestinal nematode parasite Heligmosomoides polygyrus (Hp) results in rapid increases in intestinal epithelial surface expression of the ectonucleotidases, CD39 and CD73, which in turn degrade extracellular ATP into adenosine. Adenosine binds to adenosine G-protein-coupled cell surface receptors (A1, A2A, A2B and A3 –AR). Mice deficient in A2BAR have an impaired type 2 immune response and delayed worm expulsion during helminth infection. We now report that mice lacking A2BAR specifically on intestinal epithelial cells (Villin-Cre-A2BARfl/fl), but not on myeloid cells (LysMCre-A2BARfl/fl), have an impaired type 2 memory immune response with delayed worm expulsion and decreased egg production after Hp secondary inoculation. pSTAT6 levels in CD4+ T cells and B cell MHC-II surface expression (an indicator of IL-4 activity) from mesenteric lymph nodes of Villin-Cre-A2BARfl/fl mice were significantly decreased after primary Hp inoculation. Taken together, our data demonstrate a key role for epithelial cell specific A2BAR signaling in the initiation of the protective type 2 immune response to helminth infection. These studies indicate that extracellular adenosine binding to epithelial cell A2BAR acts as an endogenous danger signal that is released following helminth infection, which triggers host protective type 2 immune responses.
Human gut commensal Prevotella histicola ameliorates disease as efficiently as Copaxone and Interferon-β in a preclinical animal model of multiple sclerosis
Authors: Shahi Shailesh K. , Freedman Samantha N. , Gibson-Corley Katherine , Karandikar Nitin J. , Murray Joseph A. , Mangalam Ashutosh K.
Published: January 2023

Multiple sclerosis (MS) is a demyelinating disease of the central nervous system. We and others have shown that gut microbiota play an important role in disease pathogenesis of MS as there is an enrichment or depletion of specific gut bacteria compared to healthy controls (HC). Thus the specific human gut bacteria found depleted or showing lower abundance in MS patients can be used as potential drug to treat MS. Further, we and others have shown that patients with MS have either lower abundance of Prevotella compared to HC or an increased level of Prevotella in patients receiving disease modifying therapies such as Copaxone and/or Interferon beta (IFN-β). We have previously identified a specific strain Prevotella histicola within Prevotella genus which can suppress disease in experimental autoimmune encephalomyelitis (EAE) animal model of MS. In this present study we compared disease suppressing ability of P. histicola to Copaxone or IFN-β in HLA-DR3.DQ8 transgenic mouse model of MS. We observed that P. histicola suppresses EAE as efficiently as Copaxone and IFN-β. P. histicola treated mice showed higher level of CD4+FoxP3+ regulatory T cell frequencies and decreased levels of pro-inflammatory cytokines IFNγ and IL17 suggesting that P. histicola suppresses disease through immunomodulation. Fecal microbiota analysis of different groups showed that group receiving Prevotella histicola had a distinct gut microbiota than the control group suggesting that P. histicola as might suppresses disease through modulation of gut microbiota. In conclusion, our study suggests that human gut commensal Prevotella histicola can provide an alternate treatment option for MS patients.
SA-FasL-engineered PEG microgels as a novel means of modulating immune response to allogeneic islet grafts
Authors: Shirwan Haval , Woodward Kyle B. , Headen Devon M. , Zhao Hong , Shrestha Pradeep , Tan Min , Bowen William S. , Coronel María M. , Hunckler Michael D. , Weaver Jessica D. , Yolcu Esma S. , García Andrés J.
Published: January 2023

Allogenic islet transplantation is an effective therapy for type 1 diabetes in the clinic. Sustained graft survival requires chronic immunosuppression that has adverse effects. Islet graft rejection is initiated and perpetuated by T effector cells (Teffs). Teffs upregulate Fas death receptor on their surface and undergo apoptosis following engagement with FasL. Therefore, FasL has the potential as an immunomodulator to establish immune tolerance to islet grafts by eliminate alloreactive Teffs. This study used PEG microgels engineered with a novel form of FasL, SA-FasL, for the prevention of allogeneic islet graft rejection. Poly(ethylene glycol) microgels were produced with biotin on their surface. Biotinylated microgels were engineered with SA-FasL (1 μg/103 microgels) by taking advantage of the high affinity interaction between biotin and the streptavidin (SA) domain of the molecule. SA-FasL-PEGs induced apoptosis in Fas expressing A20 lymphoid cells in vitro. Co-transplantation of SA-FasL-PEGs with naïve islets led to prolonged survival of grafts as compared with the control group (islets + unmodified PEGs), with 20% surviving for a 200-day observation period. A short (15 days) treatment with low dose rapamycin further improved the immunomodulatory efficacy of SA-FasL-PEGs, with > 90% of grafts surviving for a 200-day observation period. CD4+CD25+FoxP3+ Tregs were required for graft survival, as depletion of this cell population on day 50 post-transplantation resulted in prompt rejection. These findings provide strong proof-of-efficacy and feasibility for the use of SA-FasL-engineered microgels as an off-the-shelf product for the modulation of alloreactive responses with significant therapeutic potential.
CD45 ligation reduces Tregs’ motility and enhances recognition of activating signals in vitro
Authors: Lee Jounghyun Helen , Lee Joanne Haeun , Rothstein David , Kam Lance C.
Published: January 2023

Regulatory T cells (Tregs) play a pivotal role in modulating immune response hence supporting immune tolerance. Recently, CD45 ligation with anti-CD45RB mAb MB23G2 (anti-CD45RB) was reported to enhance graft-survival; evidence shows it was by homeostatic Treg proliferation in vivo. While many in vivo data support the notion that anti-CD45RB supports Tregs expansion, it is still unclear if the ligation ex vivo is sufficient to enhance Tregs’ avidity for activation and further promote their expansion and function. Here, we were to see if anti-CD45RB ligation can enhance Treg’s sensitivity to perceive activating signals in varying strength in vitro. To test this hypothesis, mouse CD4+ T cells (Foxp3-GFP linked B6 mouse, both Foxp3+ and Foxp3−, or Tregs and Tconvs, respectively) were seeded onto surfaces micropatterned with an antibody targeting the CD3 on T cell (anti-CD3) with and without the presence of anti-CD45RB. These surfaces were created by first microcontact printing of 2-μm diameter features of anti-CD3 at various concentrations (i.e., 1, 5, and 20 μg/mL) on glass coverslips and back-filled with ICAM-1. The effect of anti-CD45RB on Treg and Tconv activation was compared by the ratios of phosphorylation of Lck at Tyr 394 to 505 sites, cell registration on anti-CD3 dot array, and motility of each cell type on varying concentration of activation signals. Overall results show that anti-CD45RB specifically slow-down Treg motility and, therefore, enhance the probability to contact to activating signals (cell registration) and improve T cell activation indicated by ratios of phospho-Lck at active over inactive sites. This study assures the importance of further investigation on the role of anti-CD45RB in Treg activation mechanism.
TIPE2, an inflammation transfer switch, regulates the on-and-off immunosuppressive function of MDSCs in tumors
Authors: Yan Dehong , Wang Jinghui , Adeshakin Adeleye O. , Xu Meichen , Chen Youhai H. , Wan Xiaochun
Published: January 2023

Inhibition of the tumor-promoting inflammation of myeloid-derived suppressor cells (MDSCs) is an important determinant of successful clinical immunotherapies. However, the critical checkpoints governing MDSC function in cancer remain poorly characterized. We report here that the critical role of the essential negative regulator of inflammation tumor necrosis factor-α–induced protein 8-like 2 (TIPE2) in the accumulation and immune inhibitory function of MDSCs. TIPE2-deficient MDSCs decreased immune-regulatory activity and enabled the activation of T cell function and the induction antitumor responses. TIPE2 expression in MDSCs was induced by tumor-derived reactive oxygen species (ROS). Importantly, TIPE2 promoted CCAAT/enhancer-binding protein-β (C/EBPβ) by negative feedback inhibiting phospho-AKT/phospho-GSK-3β/phospho-C/EBPβ signaling axis, leading to decreased interleukin-6 (IL-6) and IL-10 production and STAT3 phosphorylation. IL-6 overexpression restored immune-suppressive activity of TIPE2-deficient MDSCs. These findings suggest a critical role of TIPE2 in tumor-induced tolerance and a novel therapeutic strategy of targeting TIPE2 in MDSCs to enhance the potential of cancer immunotherapy.
Profiling the effects of carbonic anhydrase 9/12 inhibition on tumor associated macrophage/microglia polarization in glioma
Authors: Boyd Nathaniel , McFarland Braden , McDonald Paul , Dedhar Shoukat , Hjelmeland Anita
Published: January 2023

Brain tumor microenvironments, including hypoxia and acidic stress, contain a subset of stem-like neoplastic cells termed brain tumor initiating cells (BTICs) that are resistant to chemo- and radiotherapy, providing a reservoir for tumor recurrence and a desirable target for glioma treatments. In prior studies, we have shown that inhibition of carbonic anhydrases 9 and 12 (CA9/12) in combination with chemotherapeutic temozolomide effectively delayed in vivo growth in a BTIC xenograft model. CA9/12 are hypoxia-responsive genes shown to be elevated in tumors that modulate the intra- and extracellular pH in tumor microenvironments. Importantly, downstream effects of hypoxia have shown CSF-1R induction, which can polarize tumor associated macrophages/microglia (TAM) toward an immunosuppressive phenotype and further exacerbate tumor progression. We have profiled CA9/12 inhibitor-treated GBM tumors using integrative omics (kinomics/transcriptomics) and discovered a shift in kinomic activity and gene expression that suggests an immunosuppressive transition in TAMs. Validating this observation, we characterized surface expression of CD11b+ TAMs post-treatment and discovered decreased expression of MHCII (inflammatory) and increased expression of CD206 (immunosuppressive). Together, our data suggest that by understanding the immunophenotypic shift that happens in response to CA9/12 inhibition in glioma, we may be able design a treatment strategy to improve upon the effectiveness of inhibitor therapy.
Mutation of the CD28 costimulatory domain confers increased CAR T cell persistence and decreased exhaustion
Authors: Boucher Justin C. , Li Gongbo , Shrestha Bishwas , Zhang Yongliang , Vishwasrao Paresh , Cabral Maria L. , Guan Lawrence , Davila Marco L.
Published: January 2023

The therapeutic promise of chimeric antigen receptor (CAR) T cells was realized when complete remission rates of 90% were reported after treating B cell acute lymphoblastic leukemia (B-ALL) with CD19-targeted CAR T cells. However, with increasing numbers of patients treated, challenges have become evident, especially regarding poor CAR T cell responses, which might be caused by exhaustion. Detailed understanding of the mechanism of CD28-dependent exhaustion in CAR T cells will allow the design of a CAR less prone to exhaustion and reduce relapse rates. We hypothesized that by mutating the CD28 subdomains YMNM and PRRP and leaving only the PYAP CD28 subdomain active we would optimize CAR T cell signaling and reduce exhaustion. We found mutated CAR T cells with only a functional PYAP subdomain secrete 50% less IFNγ, IL6, and TNFα after 24hr stimulation as well as having significantly less TCR signaling as measured by Nur77 compared to non-mutated CD28 CAR T cells. Additionally, mutated CAR T cells injected into mice had decreased expression of PD1 (82% to 62%) in the BM after 2 months. Studies in Rag−/− mice also showed mutated CAR T cells were increased 35% in the BM and 92% in the spleen compared to non-mutated CD28 CAR T cells. After challenge with tumor, exhausted mutant CAR T cells had increased IFNγ (42%), TNFα (62%) and IL2 (73%) secretion compared to exhausted non-mutated CD28 CAR T cells. This suggests that CAR T cells with only the PYAP subdomain are able to persist and be effective longer because they have optimized CD28 signaling. This work allows for development of an enhanced 2nd generation CAR T cell therapy for B cell malignancies by optimizing CAR T cell activation and persistence which may reduce relapse rates and severe toxicities.
Failure to upregulate calmodulin underlies the suppressed KCa3.1 function and enhanced sensitivity to adenosine in CD8+ T cells of head and neck cancer patients
Authors: Chimote Ameet A. , Gawali Vaibhavkumar S. , Madis Erick , Wise-Draper Trisha , Conforti Laura
Published: January 2023

The limited ability of the cytotoxic CD8+ T cells to infiltrate solid tumors presents a major roadblock to developing effective immunotherapy. Adenosine (Ado) accumulates in high concentrations in solid tumors where it contributes to the suppression of T cell function. We have previously shown that Ado reduces the chemotactic ability of peripheral blood CD8+ T cells (PBTs) from head and neck squamous cell carcinoma (HNSCC) patients by suppressing KCa3.1 channel function. Herein, we conducted experiments to elucidate the mechanism of KCa3.1 dysregulation in HNSCC PBTs. KCa3.1 channels are calcium-activated and require binding of calmodulin (CAM). PBTs were isolated from HNSCC patients and healthy donors (HD), and activated with CD3/CD28 antibodies. CAM levels decreased post-activation in HNSCC PBTs (by ~24%, n=7) while they increased in HD PBTs (by ~37%, n=6, p=0.001). To study whether CAM downregulation contributes to KCa3.1 dysfunction, we transfected HD PBTs with siRNA against CAM (siCam). siCam transfection decreased CAM expression and KCa3.1 currents, but not KCa3.1 expression. We then studied whether CAM downregulation affects the T cell chemotactic response to Ado in HD PBTs. Control (scrRNA-transfected) HD PBTs migrated towards CXCL12 in the presence of Ado, but downregulation of CAM abrogated their chemotactic ability in the presence of Ado (~71% inhibition, n=8, p<0.012). Activation of KCa3.1 channels with 1-EBIO restored the ability of siCam-transfected PBTs to migrate towards CXCL12 in the presence of Ado. Our data suggest that downregulation of CAM decreases KCa3.1 activity and suppresses chemotaxis in HNSCC PBTs which may contribute to their limited ability to effectively penetrate Ado-rich tumors.
Expansion and exhaustion of alloantigen-specific cytotoxic T lymphocytes (CTL) in channel catfish, Ictalurus punctatus.
Authors: Spencer David A. , Bengtèn Eva , Wilson Melanie
Published: January 2023

CTL are well studied in mammals, however less is known about the dynamics of memory CTL expansion and exhaustion in teleost fish. Here we provide insights into teleost CTL dynamics using our model alloantigen-specific catfish clonal CTL cell line called TS32.15. Briefly, TS32.15 was cloned from peripheral blood leukocytes isolated from fish immunized with allogenic 3B11 B cells. This clonal line is not synchronized and consists of small resting T cells and larger granular effectors. TS32.15 cells exhibit strict target specificity for 3B11s and they require stimulation with irradiated 3B11 B cells every 7–10 days for their continuous growth. When exposed to irradiated targets, the small T cells initially do not lyse targets, but replicate and differentiate to morphologically distinct cytotoxic effectors. These effectors do not replicate, but efficiently lyse 51Cr-labeled 3B11s. After lysing targets, or with a prolonged absence of targets, effector cells transition to a non-cytolytic exhausted stage and become apoptotic. Late stage apoptotic CTL bind IgM on their surface and are preferentially cleared by myelocyte-enriched head kidney leukocytes. These results indicate teleost CTL likely undergo clonal expansion and differentiation in response to antigen similar to their mammalian counterparts, and establish binding of exogenous IgM is a marker for monitoring exhausted CTL in catfish. Additionally, our studies show that repeated stimulation of a TS32.15 culture mimics a chronic infection and results in CTL exhaustion.
Nlrp12 mediates adverse neutrophil recruitment during influenza virus infection
Authors: Hornick Emma , Banoth Balaji , Miller Ann , Zacharias Zeb Ralph , Jain Nidhi , Wilson Mary E. , Gibson-Corley Katherine , Legge Kevin L. , Bishop Gail A. , Sutterwala Fayyaz S. , Cassel Suzanne
Published: January 2023

Exaggerated inflammatory responses during influenza A virus (IAV) infection are typically associated with severe disease. Neutrophils are among the immune cells that can drive this excessive and detrimental inflammation. In moderation, however, neutrophils are necessary for optimal viral control. In this study, we explore the role of the nucleotide-binding domain leucinerich repeat containing receptor (NLR) family member Nlrp12 in modulating neutrophilic responses during lethal IAV infection. Nlrp12−/− mice are protected from lethality during IAV infection and show decreased vascular permeability, fewer pulmonary neutrophils, and a reduction in levels of neutrophil chemoattractant CXCL1 in their lungs compared to wild-type (WT) mice. Nlrp12−/− neutrophils and dendritic cells (DCs) within the IAV-infected lungs produce less CXCL1 than their WT counterparts. Decreased CXCL1 production by Nlrp12−/− cells was not due to a difference in CXCL1 protein stability, but instead to a decrease in Cxcl1 mRNA stability. Together, these data demonstrate a previously unappreciated role for Nlrp12 in exacerbating the pathogenesis of IAV infection through the regulation of CXCL1 mediated neutrophilic responses.