bluuebunny/crossref_metadata_2025 huggingface dataset:
IL-33 is required for kidney ischemia-reperfusion-induced injury in mice
Authors: Herbelin Andre , Ferhat Maroua , Giraud Sébastien , Robin Aurélie , Goujon Jean-Michel , Hauet Thierry , Gombert Jean-Marc , Thierry Antoine
Published: January 2023

Introduction

Ischemia-reperfusion (IR) injury during kidney transplantation induces necrosis of renal cells and the release of “alarmins” such as IL-33 that can activate the innate immune system, thereby triggering an inflammatory response and tissue damage leading to renal failure, dysfunction or rejection. The aim of this study was to investigate the contribution of IL-33 in kidney IR injury.

Methods

10–12-week-old wild-type (WT) and IL-33-deficient (IL-33Gt/Gt) male C57/Bl6 mice were subjected to 32 minutes of unilateral kidney ischemia or a Sham operation. After 24 hours, kidneys were harvested and leucocyte infiltration (macrophages, neutrophils, NK and NKT cells) was analyzed by flow cytometry. Renal injury was assessed by measurement of plasma creatinine and histological grading.

Results

Plasma creatinine level and tissue damage significantly increased after renal IR in WT mice, as compared with Sham-operated animals, a difference that disappeared when WT mice were replaced by IL-33Gt/Gt mice. Even though intra-renal neutrophil (CD11b(+)GR-1(+)) were significantly increased 24 hour-post IR in both WT and IL-33Gt/Gt mice, this phenomenon was found to be significantly attenuated in IL-33Gt/Gt mice. Moreover, monocyte/macrophages (CD11b(+)F4/80(+)) and NKT cells, also known for their deleterious effect during renal IR injury, seemed to be less recruited 24 hour-post IR in IL-33Gt/Gt mice compared with WT mice.

Conclusion

IL-33Gt/Gt mice are less sensitive to kidney damage 24 hour-post IR, consistent with a deleterious effect of IL-33 during renal IR injury. This study underlines a new possible role of IL-33 as an innate-immune mediator during kidney IR injury.
NK Cells Play a Critical Role in Mediating Inflammation and Graft Failure During Antibody-Mediated Rejection of Kidney Allografts
Authors: Fairchild Robert L , Kish Danielle , Tanaka Toshiaki , Kohei Naoki , Dvorina Nina , Valujskikh Anna , Baldwin William M. , Fairchild Robert
Published: January 2023

The incidence of antibody-mediated kidney graft rejection has increased, but the key cellular and molecular participants underlying this graft injury remain unclear. We previously reported that kidney allograft rejection in CCR5−/− mice is dependent on donor-specific antibody (DSA). The current study determined if cells expressing cytotoxic function contributed to antibody-mediated kidney allograft rejection in CCR5−/− recipients. Wild type C57BL/6, B6.CCR5−/− and CD8−/−/CCR5−/− mice were transplanted with complete MHC mismatched A/J kidney grafts and intra-graft inflammatory components were followed to rejection. B6.CCR5−/− and CD8−/−/CCR5−/− recipients rejected the allografts by day 35 whereas 65% of allografts in wild type recipients survived past day 80 post-transplant. Allograft rejection in wild type recipients was associated with high titers of DSA, equivalent to those induced in CCR5-deficient recipients. Rejected allografts in C57BL/6, B6.CCR5−/− and CD8−/−/CCR5−/− recipients’ expressed high levels of VCAM-1 and MMP7 mRNA that was associated with high serum titers of DSA. At rejection in wild type and CD8−/−/CCR5−/− recipients, kidney allografts also expressed genes associated with NK cell (Sh2d1B1 and MYBL1) but not with T cell (CXCR6) activity during inflammation. High levels of perforin and granzyme B mRNA expression in kidney allografts peaked on day 6 post-transplant in all recipients. NK cell depletion in CD8−/−/CCR5−/− recipients reduced this expression to background levels and promoted long-term survival of 40% of kidney allografts. These results support a role for NK cells in increasing inflammation during antibody-mediated kidney allograft injury and rejection.
Suppression of Tumorigenicity 2 (ST2) signaling is required for regulatory T cell control of Graft-vs. Host Disease
Authors: Zhang Xiaoli , Matta Benjamin M , Reichenbach Dawn K , Blazar Bruce R , Turnquist Heth R
Published: January 2023

How IL-33-mediated signals impact on ST2-expressing regulatory T cell (Treg) expansion and regulatory functions is poorly understood. To establish the importance of direct IL-33 stimulation of Treg for control of alloimmune responses, we utilized a rodent model of graft-vs. host disease (GVHD) where C57BL/6 mice were irradiated and given a BALB/c bone marrow transplant (BMT) along with BALB/c CD4+ CD25+ Treg from st2+/+ or st2−/− mice at a 1:2 ratio with WT BALB/c CD3+ effector T cells. As expected, WT st2+/+ CD4+ CD25+ cells promoted full protection against GVHD with 90% of recipients receiving st2+/+ CD4+ CD25+ free of GVHD at Day 100 post-BMT. However, only 10% of mice that received st2−/− Treg achieved long-term survival (p=0.0017, st2+/+ v. st2−/−). Associated mechanistic studies revealed that Treg frequency was significantly reduced in recipients receiving st2−/− Treg compared to those receiving st2+/+ Treg. To define the signaling patterns IL-33 generates in Treg, fluorescence activated cell sorting was used to obtain ST2− or ST2+ Treg cells from the splenocytes of Foxp3 reporter mice and IL33 activated signaling downstream of ST2 was quantitated by phosphoflow cytometry. This analysis revealed that IL-33 signaling via ST2 on Treg activates both NF-κB and p38 MAPK. Yet, only IL-33-stimulated p38 MAPK, but not NF-kB, signaling was required for ST2+ Treg expansion. Our studies make two important observations regarding how IL-33 stimulation of Treg supports immune regulation. First, we demonstrate that IL-33 activates p38 MAPK to drive selective ST2+ Treg expansion. Second, we show that IL-33-mediated signals are critical to the capacity of Treg to control the alloimmune responses leading to GVHD.
Development of an Enhanced VETO cells for the generation of Alloantigen-specific Tolerance
Authors: Rajawat Yogendra Singh , McFarland Hugh , Morgan Richard , Zhao Yangbing , Rosenberg Amy S
Published: January 2023

Development of specific immunotherapies to prevent graft rejection and graft-versus-host disease offers the promise of selectively deleting only allospecific immune responses. Veto activity is the ability of a cell to specifically suppress/delete only T cells directed against antigens of the veto cells themselves, but not against third-party antigens. In the present study, we reasoned that providing TCR-like signaling to allogeneic major histocompatibility complex (MHC) molecules on the surface of CD8+CTLs might result in enhanced veto function. To generate the donor antigen specific veto cells, we have utilized the Chimeric Antigen Receptor (CAR) T cell approach, in which CD8+CTLs express an MHC alloantigen fused to signaling molecule apparatuses. We fused mouse MHC class I alloantigen (H-2Dd) to the hinge and trans-membrane domains of the mouse CD8 chain plus intracellular signaling sequences derived from mouse 4-1BB, and CD3-ζ chain creating various CAR constructs. These CARs were cloned into retroviral vectors. Ex-vivo transduced CD8+T cells (FVB-Dd) were adoptively transferred in the recipient parental mice strain (FVB-H-2q). 4–6 weeks post adoptive transfer, we detected low levels of Dd+ CD8+T cells in lymph node and spleen, ranging from 2–3% of total CD8+T cells. Dd expressing skin grafts from 3604 transgenic mice (H-2Dd transgenic mice on FVB-H-2q background) were transplanted on recipient mice and preliminary data suggests that skin graft survival was extended for 4 months (usual rejection in naïve mice is by 15–18 days).Veto activity of these Dd+CD8+T cells will be examined by in vitro Dd specific CTL assays, proliferation assays.
Protein Kinase C-θ and vimentin modulate multiple facets of Regulatory T-cell function.
Authors: McDonald-Hyman Cameron , Thangavelu Govindarajan , Saha Asim , Muller James , Zhang Guoan , Kumari Sudha , Koehn Brent H. , Mitchell Jason S. , Fife Brian T , Serody Jonathan S , Osborn Mark J , Hippen Keli L , Kelekar Ameeta , Munn David H , Altman Amnon , Neubert Thomas , Dustin Michael L , Blazar Bruce R
Published: January 2023

Regulatory T-cells (Tregs) prevent autoimmune and alloimmune reactions. Augmenting Treg function may enhance Treg therapies for these diseases. Treg-specific inhibition of protein kinase C-theta (PKC-θ) enhances Treg function. However, it is unclear whether PKC-θ inhibition can boost Treg function in systemic inflammatory conditions.

In a mouse model of acute GVHD, we found that Tregs treated with the PKC-θ inhibitor AEB071 reduced GVHD mortality and severity significantly better than DMSO treated Tregs. Compared to DMSO, AEB071 treated Tregs significantly reduced conventional T-cells (Tcon) proliferation on D4 after transplant. Multi-photon microscopy showed that AEB071 treated Tregs significantly increased Tcon velocity and displacement compared to DMSO.

Mechanistically, AEB071 augments expression of Neuropilin-1 (Nrp1) and Lymphocyte activation gene 3 (Lag3). Antibody blockade of Nrp1 and Lag3 in transwell suppression assays reduced the effect of AEB071 on Treg function. PKC-θ inhibition also reduces phosphorylation of mTORC2 targets FoxO3a and Akt phospho-site S473, but not mTORC1 targets S6, 4E-BP1 or Akt phospho-site T308. Compared to DMSO, AEB071 treatment significantly increased fatty acid uptake and oxygen consumption rate (OCR).

Phosphoproteomic analysis identified a significant alteration in the interaction between PKC-θ and the intermediate filament vimentin after AEB071 treatment, which was confirmed by confocal. Vimentin siRNA treatment also significantly reduces PKC-θ/vimentin interaction, increases Treg function, Nrp1 expression and OCR.

In summary, PKC-θ and vimentin modulate multiple aspects of Treg function, and altering these molecules may enhance the efficacy of Treg therapeutics.
Targeting PI3K signaling to ameliorate chronic graft versus host disease.
Authors: Paz Katelyn G , Flynn Ryan , Du Jing , Dong Shuai , Okkenhaug Klaus , Stark Anne-Katrien , Vanhaesebroeck Bart , Johnson Amy , Tannheimer Stacey , Turka Lawrence A , Byrd John , Queva Christophe , Blazar Bruce R
Published: January 2023

The phosphoinositide 3-kinase (PI3K) pathway is a key signaling pathway necessary for T cell activation, differentiation and metabolism. T effector cells rely on increased PI3K signaling to fuel glycolysis for their metabolic needs, while T regulatory cells downregulate PI3K and favor lipid oxidation. The metabolic processes of lymphocytes modulating chronic graft versus host disease (cGVHD) have remained largely unexamined and represent a novel therapeutic strategy for this disease. Here, we investigated the role of PI3K signaling in a murine model of cGVHD that is etiologically linked to up regulated germinal centers (GCs) and characterized by multisystem organ disease; including fibrosis of the lung, which results in pulmonary dysfunction. We hypothesized that inhibition of PI3K signaling would alter the activation and/or function of GC-facilitating T follicular helper (TFH) cells resulting in lessened disease. The findings in this study are that mice treated with a PI3Kd inhibitor had decreased pulmonary dysfunction similar to that of the control, non-cGVHD mice. The frequencies of splenic TFH cells as well as GC B cells were decreased by a PI3K delta inhibitor compared to non-treated cGVHD controls. In a similar manner, mice that received PI3K kinase delta dead Tregs also had decreased TFH frequency as well as reduced pulmonary dysfunction. Our results indicate the differential requirement for signaling through PI3K delta and suggest that targeting this pathway may be a potential new therapy for treatment of cGVHD. Additional studies are required to validate the potential therapeutic use.
Comprehensive pipeline for bioinformatics prediction of immunogenic minor histocompatibility antigens
Authors: Efimov Grigory , Bykova Nadezda , Vdovin Alexander , Filkin Serguei , Postovskaya Anna , Malko Dmitry
Published: January 2023

Hematopoietic stem cell transplantation (HSCT) is widely used for treatment of haematological malignancies. Both raft versus tumor (GVT) effect and potentially lethal graft versus host disease (GVHD) may develop after transplantation. Current approach to limit GVHD, is based on donor selection and immunosuppression. But as alloreactivity is not restricted to the HLA locus, up to 40% of fully HLA-matched transplantations are complicated by severe forms of GVHD. Non-synonymous single nucleotide polymorphisms (SNPs) in the genome coding regions can give rise to polymorphic peptides – minor histocompatibility antigens (MiHA) – recognized by the immune system as foreign.

Limiting alloreactivity to SNPs in hematopoietic genes could allow to discriminate between GVHD and GVT. Thus information of which polymorphisms are immunogenic is of great importance.

So far only a limited number of MiHA have been discovered. Methods for verification of SNP immunogenicity are slow and laborious so there is a need for algorithms predicting potentially immunogenic polymorphisms.

We have developed TIE predict (TransImmuno Epitope predictor) which is a comprehensive pipeline for in silico prediction of MiHAs based on exome data of donors and patients. This pipeline utilizes several algorithms for peptide to MHC binding prediction, which increases the overall accuracy of prediction. TIE predict is linked to TransImmuno Epitope database holding the data on common genetic poymophisms genomic sequences and annotations. We have tested this approach on publically available genetic data and currently starting to use it on patients and donors undergoing HSCT in combination with immunogenicity assays.
Group 3 NCR+ Innate Lymphoid Cells accumulate in human lung cancer
Authors: Ferlazzo Guido , Mingari Mariacristina , Carrega Paolo
Published: January 2023

Tertiary lymphoid structures (TLS) are a common finding in non-small-cell lung (NSCL) cancers and are predictors of favorable clinical outcome. Here, we show that NCR+ Innate-Lymphoid-Cells (ILC)3s are present in the lymphoid infiltrate of human NSCL carcinomas and are mainly localized at the edge of tumor-associated TLS. This intra-tumoral lymphocyte subset is endowed with lymphoid tissue-inducing properties and, upon activation, produces IL-22, TNF-α, IL-8, IL-2 and activates endothelial cells. Tumor-NCR+ ILC3 may interact with both lung tumor cells and tumor-associated fibroblasts, resulting in the release of cytokines primarily upon engagement of the NKp44 activating receptor. In patients, NCR+ ILC3s are present in significantly higher amounts in stage I/II NSCL cancers than in more advanced tumor stages. Our results indicate that NCR+ ILC3s are associated with human NSCL cancer and may contribute to the formation of protective tumor-associated TLS.
IP3 3-Kinase B Suppresses B Cell Lymphoma by Antagonizing PI3K/mTOR in B cells
Authors: Sauer Karsten , Conche Claire , Deng Yisong , Jin Hyun-Yong , Bethel Kelly , Rigaud Stephanie M , Westernberg Luise , Siegemund Sabine , Xiao Changchun
Published: January 2023

Accounting for ~4% of all cancers in the US, Non-Hodgkin Lymphoma (NHL) is the most prevalent blood cancer. Diffuse large B-cell Lymphoma (DLBCL) is the most common and aggressive type of NHL. Here, we identify inositol-trisphosphate (IP3) 3-kinase B (Itpkb) as a novel tumor suppressor whose deficiency triggers DLBCL in mice. We found that aging Itpkb−/− mice die prematurely with anemia and splenomegaly. 15% of these mice showed multiorgan-infiltration with neoplastic germinal-center phenotype B cells reminiscent of DLBCL. Itpkb−/−B cell lymphomas are mono-or oligoclonal, transplantable, and constitutively hyperactivate the oncogenic phosphoinositide 3-kinase (PI3K) signaling pathway. Growth of Itpkb−/−B cell lymphoma xenografts in immunodeficient mice is sensitive to treatment with PI3K or mTOR inhibitors. In humans, subsets of DLBCL patients carry Itpkb missense mutations, deletions or copy number reductions. Additionally, Itpkb expression is altered in several other hematopoietic and non-hematopoietic human cancers. We propose that Itpkb suppresses tumors by producing inositol-tetrakisphosphate (IP4), a soluble analog of the PI3K product phosphatidylinositol-trisphosphate (PIP3). We and others have previously shown that IP4 antagonism with PIP3 for Akt effector kinase binding prevents excessive Akt/mTORC1 signaling in hematopoietic cells. Our new results suggest that this mechanism prevents oncogenic PI3K signaling in the B cell lineage and possibly in other cell types. Thus, Itpkb might be a novel biomarker for tumor aggressiveness or prognosis, and Itpkb activating drugs or IP4 might have therapeutic potential.
IFNγ has variable effects on expression of MHC Class I and ICAM-1 in pediatric cancers which alter sensitivity to NK cell mediated lysis
Authors: Aquino-Lopez Arianexys , Senyukov Vladimir , Romain Gabrielle , Varadarajan Navin , Lee Dean A.
Published: January 2023

Interferon-γ (IFNγ), a cytokine secreted by effector lymphocytes upon activation, is known to cause upregulation of MHC class I in tumor cells, which has been associated with natural killer (NK) cell inhibition. NK cells are very heterogeneous with respect to receptor expression and their function depends on an interplay between these inhibitory and activating signals. In order to optimize NK cell therapy, it is essential to identify the NK cells expressing the optimal combination of receptors for a given target. In this study we evaluated tumor cell lines from the Pediatric Preclinical Testing Program to determine the impact of IFNγ on their expression of NK cell activating and inhibitory ligands, death receptors, and adhesion molecules using mass cytometry, and their correlation with tumor lysis. We found that IFNγ treatment can significantly increase ICAM-1 and/or HLA-ABC levels together or independently. When HLA-ABC increases without concomitant ICAM-1 upregulation, this is associated with decreased lysis. Conversely, when ICAM-1 upregulation exceeds HLA-ABC upregulation this is associated with increased conjugate formation and better NK cell cytotoxic activity. This increase in cytotoxicity and conjugate formation is lost after blocking ICAM-1. Time-lapse imaging of the NK cell interaction with tumor cells shows that NK cells require less time to form stable contacts with IFNγ treated tumor cells when compared to untreated tumor cells. This suggests that IFNγ induced upregulation of ICAM-1 enhances NK cell cytotoxic activity by reducing the time required for NK cells to form stable contacts with their target.
NKG2D receptor activation makes CD8+ T cells resistant to the suppressive effects of TGF-β
Authors: Prajapati Kushal , Perez Cynthia , Burke Brianna , Plaza-Rojas Lourdes , Guevara-Patino Jose Alejandro
Published: January 2023

Natural killer group 2, member D (NKG2D) is a stimulatory receptor majorly present on the surface of natural killer and CD8+ T cells. We and others have shown that NKG2D receptor enhances the function, persistence and proliferation of CD8+ T cells. On the other hand, a major hurdle in the field of cancer vaccine and adoptive T cell transfer therapy (ACT) is diminished effectiveness and persistence of CD8+ T cells due to immuno-suppressive factors. TGF-β is a highly prevalent immuno-suppressive factor that negatively regulates the function and persistence of CD8+ T cells. We hypothesized that NKG2D activation opposes the suppressive effects of TGF-β on CD8+ T cells, and enhances their anti-tumor activity in tumor microenvironment. Here, we show that activating the NKG2D receptor makes human CD8+ T cells resistant to TGF-β’s suppressive effects on the activation, proliferation and granzyme B expression. Further, we found that NKG2D knock-out mouse CD8+ T cells are more susceptible to these TGF-β mediated suppressive effects in vitro. Interestingly, under influence of TGF-β, NKG2D KO CD8+ T cells’ ability to secrete multiple cytokines in vivo was significantly less than their WT counterparts. Our next objective is to determine the molecular mechanisms by which NKG2D makes CD8+ T cells resistant to TGF-β. For the proximal mechanism, we will determine NKG2D’s immediate signaling pathway, namely PI3K or Grb2-SOS-Ras, mediating resistance to TGF-β. Moreover, our preliminary data shows that NKG2D activation increases the levels of regulator of G protein signaling 3 (RGS3), inhibitor of TGF-β signaling, in CD8+ T cells. We will investigate if up-regulation of RGS3 is a distal mechanism underlying NKG2D mediated resistance to TGF-β in CD8+ T cells.
Killed Klebsiella pneumoniae treatment reduces tumor burden in murine melanoma
Authors: Costa Amanda M , Perekslis Thomas , Alexander Matthew P , Sanders Kiah , Gunn Hal , Mullins David W
Published: January 2023

Acute infection in a cancer-bearing organ has been associated with improved prognosis and enhanced survival, although the anti-cancer immune mechanisms remained undefined. In collaboration with Qu Biologics, we hypothesized that stimulating pathogen-specific immunity, using bacteria that have previously infected specific organs, may induce protective anti-cancer immune responses in those target organs. We evaluated Klebsiella-mediated anti-cancer efficacy in metastatic-like B16 melanoma using heat-killed Klebsiella pneumoniae (Qu Biologics’ drug product QBKPN Site Specific Immunomodulator [SSI]). Subcutaneous injection of QBKPN SSI significantly reduced tumor burden. Furthermore, subcutaneous treatment with heat-killed E. coli, (QBECO SSI) reduced lung tumor burden but not as substantially as with QBKPN. This suggests that subcutaneous immune induction using a lung specific-pathogen activates a lung-specific antitumor response.

We next asked whether pre-exposure to K. pneumoniae would improve anti-tumor efficacy. We exposed mice to live K. pneumoniae via intratracheal infection prior to subcutaneous injections with QBKPN SSI. Pre-exposure to K. pneumoniae significantly enhanced QBKPN SSI-induced anti-tumor immunity and control of metastatic-like B16 melanoma in the lungs. The anti-tumor efficacy in exposed mice correlated with an influx of monocytes and neutrophils, but did not correlate with an influx of T cells into the lungs. Collectively, these data suggest pre-exposure to K. pneumoniae may induce tissue resident memory cells that mediate tumor cytolysis, and thus explain the lack of T cell infiltration into the lungs.
Critical roles of Noxa and Mcl-1 in the signaling mechanisms of bortezomib in TRAF3-deficient mouse B lymphoma and human multiple myeloma cells
Authors: Luo Chang , Han Yeming , Liu Yingying , Edwards Shanique K E , Xie Ping
Published: January 2023

Bortezomib, a clinical drug for multiple myeloma (MM) and mantle cell lymphoma, exhibits complex signaling mechanisms, which vary depending on the cancer cell type and the critical genetic alterations of each cancer. In the present study, we investigated the signaling mechanisms of bortezomib in mouse B lymphoma and human MM cells deficient in a new tumor suppressor gene, TRAF3. We found that bortezomib robustly induced up-regulation of the pro-apoptotic protein Noxa, cleavage of the anti-apoptotic protein Mcl-1, and accumulation of the oncoprotein c-Myc. Interestingly, bortezomib exhibited contrasting roles on canonical versus non-canonical NF-kB pathways. Overexpression of Noxa was sufficient to induce apoptosis and inhibit proliferation in human MM cells by inducing cleavage of Mcl-1 and caspase 3. Furthermore, we demonstrated that oridonin (an inhibitor of NF-kB1 and NF-kB2) or AD 198 (a drug targeting c-Myc) drastically potentiated the anti-cancer effects of bortezomib in TRAF3-deficient malignant B cells. Taken together, our findings provide new insights into the signaling mechanisms of bortezomib, which would aid the design of novel bortezomib-based combination therapies. Our results also provide a rationale for clinical evaluation of the combinations of bortezomib and oridonin (or other inhibitors of NF-kB1/2) or AD 198 (or other drugs targeting c-Myc) in the treatment of lymphoma and MM, especially in patients containing TRAF3 deletions or relevant mutations.
Altered host immune responses following infection with miR-150 expressing influenza viruses
Authors: Xia Yingju , Izzard Leonard , Ye Siying , Stambas John
Published: January 2023

Current influenza vaccines offer considerable protection for the majority of the population. However, vaccine efficacy amongst high risk populations, such as the elderly, is variable. Novel approaches are therefore required to improve vaccine efficiency in these susceptible populations. MicroRNAs (miRNAs) play important roles in the regulation of immune responses by modulating gene expression. Our work describes the generation and delivery of engineered influenza viruses with the ability to express defined immunomodulatory miRNAs. MicroRNA-150 (miR-150) has been shown to be involved in the development of T cell and B cell. Furthermore, evidence suggests that circulating miR-150 can be actively transported to target cells where it can influence gene expression. In this study, we first investigated the ability of recombinant influenza viruses to deliver immunomodulatory miR-150, and alter cytokine production in vitro. miR-150 was inserted into an artificial intron between the NS1 and NEP genes of the PR8 H1N1(PR8-150) and X31 H3N2 (X31-150) influenza A viruses and was rescued using reverse genetics. Mature miR-150 expression was confirmed using qPCR and by knockdown of its target gene, c-Myb, using a dual-luciferase assay. Moreover, cytokine mRNA expression levels (IL-6, IFN-β, CXCL10 and CCL5) were enhanced following infection with PR8-150 in A549 cells. Mice infected intranasally with recombinant X31-150 virus showed significantly higher numbers of neutrophils, macrophages and influenza specific CD8+ T cells post infection compared to X31 wild type controls. These results demonstrate that recombinant influenza viruses can be used to deliver functional miRNAs with the ability to modulate immunity both in vitro and in vivo.
Seasonal vaccination with the inactivated influenza vaccine generates antibody profiles that are distinct from those generated by the live-attenuated influenza vaccine
Authors: Shagal Ayelet , Monto Arnold , Hertz Tomer
Published: January 2023

Vaccination is an effective strategy for preventing influenza infection and is now recommended annually by the CDC for all individuals age 6 months and above. There are currently two licensed seasonal influenza vaccine formulations in the US: the inactivated influenza vaccine (TIV) and the live-attenuated influenza vaccine (LAIV). A previous clinical trial (FluVacs) conducted in 2007–2008 comparing the efficacy of these two vaccines in adults aged 18–65 reported that both vaccines were efficacious, but the TIV had an overall higher efficacy than the LAIV. We hypothesized that the two vaccines will also generate distinct antibody signatures partially due to the difference in the reported efficacy. We therefore compared the antibody profiles generated by the two vaccines. Profiles were generated using antigen microarrays (AMs) which are a high-throughput binding assay. We generated peptide AMs spotted with overlapping 20mer peptides from a seasonal vaccine strain (2012) and used serum samples of 150 participants from the FluVacs study taken at day 21 post vaccination and at the end of the season. We also used baseline samples to account for previous antibody memory responses. We found that both vaccines generated relatively weak responses when compared to baseline. However, in-line with our hypothesis, the antibody titers generated by the TIV vaccine were higher than those of the LAIV. We then used unsupervised clustering to cluster the post-vaccine responses of all trial participants. We found distinct clusters, each corresponding to a different response pattern. In conclusion we used a novel assay to profile influenza vaccine induced antibody responses and found that the TIV and LAIV vaccines generate different antibody profiles
Immunization with a VCG-based vaccine protects against Chlamydia-induced infertility in the mouse genital infection model
Authors: Eko Francis , Pais Roshan , Omosun Yusuf , He Qing , Blas-Machado Uriel , Igietseme Joseph U. , Fujihashi Kohtaro
Published: January 2023

Chlamydia trachomatis genital infections constitute a major public health challenge due to the significant morbidity that includes pelvic inflammatory disease, ectopic pregnancy and tubal factor infertility. An effective vaccine is required to protect against infection and prevent development of upper genital tract pathology. Previous studies showed intramuscular immunization with a Vibrio cholerae ghost (VCG)-based chlamydial vaccine induced cross protective immunity in mice but its ability to protect against upper genital tract pathology was not evaluated. We sought to assess the ability of rectal immunization with this vaccine to protect against upper genital tract pathology following infection with a heterologous chlamydial serovar in the mouse model. Female mice were rectally immunized with VCG expressing select C. trachomatis serovar D outer membrane proteins and challenged intravaginally with live serovar E chlamydiae 2 weeks postimmunization (PI) to evaluate cross protection. To evaluate upper genital tract pathology, mice received a repeat challenge infection 8 weeks after initial challenge. Rectal immunization elicited high levels of antigen-specific Th1 cell-mediated and humoral immune responses in mucosal and systemic tissues. Furthermore, immunization induced cross-protective immunity that reduced the length and intensity of genital chlamydial shedding and protected against Chlamydia-induced infertility and tubal pathologies. The ability of the VCG vaccine candidate to elicit heterotypic genital tract immunity and protect against Chlamydia-induced upper genital tract pathology is remarkable and highlights its potential for development as a universal chlamydial vaccine candidate.
Secreted heat shock protein gp96-Ig vaccine for malaria prophylaxis
Authors: Strbo Natasa , Romero Laura , Podack Eckhard R , Villasante Eileen F , Edgel Kimberly A , Strbo Natasa
Published: January 2023

Proposed vaccine principle relies on secreted gp96-Ig chaperoning Plasmodium falciparum (Pf) AMA1 and PfCSP sporozoite proteins that are efficiently taken up and cross presented by activated DC via MHC I to CD8 CTL, thereby stimulating an avid, antigen specific, cytotoxic T cell response. The generation of a powerful, cytotoxic anti-sporozoite CD8 CTL response by the vaccine is expected to provide prophylactic immunity against malaria by killing infected liver cells, thereby preventing blood stage infection.

We performed mouse immunogenicity experiments using 293-gp96-IgPfAMA1-PfCSP vaccine cells that produce gp96-Ig and express PfAMA1 and PfCSP. One million 293-gp96-IgPfAMA1-PfCSP cells were injected in B6 mice by intraperitoneal (IP), subcutaneous (SC), intramuscular (IM) and intradermal (ID) routes. Control mice received 293-gp96-Ig. Five days later, mice were sacrificed. Total spleen cells were cultured overnight in medium only or with pool of overlapping PfAMA1 and PfCSP peptides. Frequency of PfAMA1- and PfCSP-specific CD8 T cell responses in the spleen was measured by intracellular cytokine staining assay and analyzed by flow cytometry.

We found that vaccine cells delivered by IP and SC routes induced the strongest PfAMA1- and PfCSP-specific immune response systemically (1.8±0.4% and 1.3±0.3% PfAMA1- and PfCSP-specific spleen CD8 T cells, respectively). In addition, after 293-gp96-IgPfAMA1-PfCSP vaccination frequency of liver-infiltrating CD8+ T cell was significantly increased (70% of all liver CD3+ cells were CD8+CD44+CD62L− T cells).

Our findings are strongly supportive of the novel gp96-Ig malaria vaccine as unique systemic and liver-homing, sporozoite specific CD8 CTL vaccine strategy.
Fc-dependent effector functions of neutrophils induced by hemagglutinin stalk-specific antibodies
Authors: Mullarkey Caitlin E , Bailey Mark J , Golubeva Diana , Azarm Kristopher , Nachbagauer Raffael , Miller Matthew S , Palese Peter
Published: January 2023

Broadly neutralizing antibodies that recognize the conserved hemagglutinin (HA) stalk have emerged as exciting new biotherapeutic tools to combat both seasonal and pandemic influenza viruses. Our general understanding of the mechanisms by which stalk-specific antibodies achieve protection is rapidly evolving. It has recently been demonstrated that broadly neutralizing HA stalk-specific IgG antibodies require Fc-FcR interactions for optimal protection in vivo. Here we explore the relationship between such stalk-specific antibodies and neutrophils. Neutrophils represent a critical innate effector cell population and play an important role in orchestrating downstream adaptive responses to influenza virus infection. Yet, at present the interplay of HA stalk-specific IgG, Fc-FcR engagement, and neutrophils has remained largely uncharacterized. Using an in vitro assay to detect the production of reactive oxygen-species (ROS), we show that both human and mouse monoclonal HA stalk-specific IgG antibodies are able to induce the production of ROS in neutrophils, while antibodies specific to the HA head domain do not. Furthermore, our results indicate that the production of ROS is dependent on Fc receptor engagement and phagocytosis. We went on to assess the ability of monoclonal HA stalk-specific IgA antibodies to induce ROS. We found that monoclonal IgA lead to a greater induction of ROS as compared to monoclonal IgG. Our data demonstrate this activity is dependent on the engagement of FcαR1. Taken together our findings describe a novel FcR-dependent effector function induced by HA stalk-specific IgG and IgA antibodies.
Responses to fibroblast-adapted human cytomegalovirus (HCMV) vaccines conform to CD8 T cell recognition paradigms
Authors: Murray Susan E , Nesterenko Pavlo , Munks Michael , Smart Savannah , Vanarsdall Adam , Lee Ronzo , Cox Josephine , McVoy Michael , Adler Stuart P , Hill Ann B
Published: January 2023

Recently, a rhesus CMV-based SIV vaccine that clears SIV from half of vaccinated monkeys was shown to elicit unconventional CD8 T cell responses to both SIV and RhCMV (IE1) antigens in all immunized monkeys. These responses were characterized by i) recognition of an unusually large number of peptide epitopes within each antigen, and ii) MHCII restriction by ~2/3 of responding CD8 T cells (Hansen et al, Science, 2013). Unconventional responses were only elicited by fibroblast-adapted rhCMV strain 68-1 that lacks ability to infect cells other than fibroblasts due to loss of pentameric viral glycoprotein expression.

To determine whether fibroblast-adapted human CMV (HCMV) vaccines also elicit unconventional CD8 T cell responses, we exploited an ongoing phase I clinical trial of live HCMV vaccines. These vaccines have similar fibroblast-restricted tropism to rhCMV68-1, although the precise genetic lesions differ. We analyzed CD8 T cell responses in 12 seroconverters by stimulating PBMC with overlapping peptide pools covering 20 HCMV antigens. Seven subjects in this low dose trial (100 or 1000 pfu) made consistent, measurable CD8 T cell responses. All 7 responded to IE1; 3 also responded to 1–2 additional antigens. We have mapped responses to individual 15mers in 3 subjects, and all revealed highly focused responses to 1–3 epitopes within IE1. In one HLA-B08 positive subject, the two responses mapped to known immunodominant B08-restricted IE1 epitopes. We have mapped 4 other epitopes to date; of these, 3 are 9mers and are likely conventional, but one may be a 13mer. Thus far, in contrast to RhCMV in monkeys, the CD8 T cell response to fibroblast-adapted HCMV vaccines in humans appears to be predominantly conventional.

Supported by NIH R21 AI116107
Optimizing antiviral humoral responses the natural (killer) way
Authors: Rydyznski Carolyn E. , Waggoner Stephen N
Published: January 2023

Many vaccine efforts to prevent deadly viral diseases aim to generate high-affinity, class-switched virus-specific antibodies, which arise mainly from germinal center (GC) reactions. However, vaccine-induced GCs are sometimes inadequate to prevent infections with major human pathogens, including HIV. An improved understanding of the mechanisms regulating the GC reaction will improve future vaccines. We discovered that natural killer (NK) cells suppress the generation of GC B cells and follicular helper T (Tfh) cells in a perforin-dependent manner following acute viral infection. Remarkably, a fraction of activated NK cells up-regulate the B-cell follicle homing receptor, CXCR5, which potentially facilitates interactions between cytotoxic NK cells and GC-inducing cells in or near B-cell follicles. We performed RNAseq analysis of this unique subset of NK cells to uncover potential mechanisms of humoral immune suppression. We also interrogated qualitative changes in antigen-specific B cells induced by NK cells following immunization with the hapten-carrier immunogen, NP-KLH. NK cells restricted the NP-KLH-induced GC response and number of NP-specific B cells. In the absence of NK cells, there was an increase in NP-specific plasma cells in the bone marrow at early time points and a higher ratio of high- to low-affinity NP-specific IgG in sera at late time points, suggesting that NK cells impair the quality of the humoral response that develops following immunization. To date, our data provide evidence that NK-cell suppression of GCs represents a previously underappreciated regulatory axis, potentially mediated by a unique subset of NK cells, which could be manipulated to enhance future vaccine efforts.
A unique subset of CD11c+ B cells upregulate FasL expression in response to IL-12p40 during lethal dose influenza virus (IAV) infections.
Authors: Zacharias Zeb Raplh , Legge Kevin L
Published: January 2023

Our recent work has detailed a novel regulation of the IAV-specific CD8 T cell response during lethal dose IAV infections. During such infections, FasL+ plasmacytoid dendritic cells (pDC) within the regional lymph nodes (LN) target and eliminate the developing Fas+ effector CD8 T cell response limiting immunity and driving severe disease. While our studies have shown that pDC are sufficient to drive this mechanism (i.e. transfer of wild type pDC into gld mice results in elimination of activated CD8 T cells), antibody targeted elimination of pDC in wild type mice does not completely reverse CD8 T cell apoptosis. This suggested that an additional cell type might contribute to the overall loss of CD8 T cells during lethal/high pathogenic IAV infections. Here we describe a CD11c+B cell population that shares some common markers with both pDC and several defined B cell subsets, but can be distinguished from both based on unique marker expression. Our data suggests that these CD11c+ B cells are recruited to the LN in greater numbers compared to pDCs and appear to express FasL upon exposure to IL-12p40 homodimer in lethal dose IAV infections. Further our results suggest that FasL+ expression with BST2+ expression within CD11c+ B cells. Our current studies are focused on further determining the factors regulating this CD11c+B cell population and its relative contribution to the overall FasL-dependent control of the magnitude of the CD8 T cell response during lethal dose IAV infections.
TCF1 regulates antiviral T follicular helper (Tfh) cell responses through a negative feedback loop with IL-2 and Blimp1
Authors: Wu Tuoqi , Shin HyunMu , Moseman E. Ashley , Ji Yun , Huang Bonnie , Harly Christelle , Sen Jyoti M , Berg Leslie J , Gattinoni Luca , McGavern Dorian B , Schwartzberg Pamela L
Published: January 2023

Th1 cells and T follicular helper cells (Tfh cells), are generated early after viral infection. However, the molecular pathways that drive the differentiation of these two distinct subsets remain unclear. Here, we demonstrate that a molecular circuit involving TCF1, Blimp1, and IL-2 regulates the differentiation of viral-specific Tfh and Th1 cells. Tcf7 gene (encodes TCF1) is actively transcribed in naïve and Tfh cells but strongly repressed in Th1 cells. TCF1 expression was negatively correlated with CD25 and Blimp1 expression, and was suppressed by IL2 signaling and Blimp1. Notably, loss of Tcf7 in T cells led to defective Tfh-cell and GC responses after viral infection. Tcf7-deficient Tfh cells had reduced Tfh transcription signatures and increased Th1 transcription signatures. Moreover, Tcf7-deficient Tfh cells had lower mitochondrial mass and membrane potential, and showed reduced expression of genes related to oxidative metabolism. Il2ra and Prdm1 (encodes Blimp1) were ectopically expressed in Tcf7-deficient Tfh cells and were bound by TCF1, suggesting the existence of a negative feedback loop linking TCF1 to IL-2 and Blimp1. Over-expression of Bcl6 rescued Tfh defects caused by Tcf7 deficiency, demonstrating that TCF1 is upstream of the Bcl6-Blimp1 axis. Finally, we found that Tcf7-deficiency did not cause evident defect in Tfh responses in Th2 immunization models. Therefore, TCF1 specifically regulates antiviral Tfh responses.
Regulatory T-cells are essential to promote proper CD4 T-cell priming upon mucosal infection
Authors: Lund Jennifer M , Soerens Andrew , Costa Andreia
Published: January 2023

Regulatory T-cells (Tregs) limit autoimmunity and immunopathology using a variety of suppressive mechanisms, but their roles during pathogen-directed immune responses remain unclear. Following Herpes Simplex virus-2 (HSV-2) infection, mice lacking Tregs fail to control viral replication, pointing to a role for Tregs in facilitating productive immune responses. Using adoptive transfer of TCR transgenic CD4 T-cells into Treg-sufficient or Treg-depleted mice prior to HSV-2 infection, we found that Tregs are required for timely accumulation of HSV-2-specific CD4 T-cells within the infected tissues. Further, Tregs are critical for appropriate trafficking of dendritic cells (DCs) from the vaginal mucosa to the dLN, which results in fully effective CD4 T-cell priming, activation, and ultimately migration to the infected tissues. Using CTLA-4 conditional knockout mice, we demonstrate that Tregs impact DC migration through a CTLA-4-mediated mechanism. Together, our data highlight the critical role of Tregs in proper potentiation of adaptive immune responses to microbial infection.
Immunosequencing reveals diagnostic signatures of pathogen infection and HLA type in the T-cell Receptor Repertoire
Authors: DeWitt Will , Emerson Ryan O. , Vignali Marissa , House Steven , Desmarais Cindy , Rieder Mark J. , Carlson Christopher S. , Gravley Jenna , Hansen John A , Robins Harlan S.
Published: January 2023

We analyzed the T-cell response to CMV by sequencing rearranged TCRs in 528 subjects (240 CMV+ and 288 CMV−). We assessed concordance between ~80 million unique TCRs and CMV serostatus, looking for significant associations. We identified 104 CMV-associated TCRs at FDR ≈ 0.20. Training a binary classifier on these features, we predict CMV serostatus in a cross-validation procedure with a diagnostic odds ratio of 17 on the same cohort. A second independent cohort of 120 subjects with known CMV serostatus yielded a diagnostic odds ratio of 31.

Next, we studied the HLA-restriction of each CMV-associated TCR by assessing enrichment of particular HLA types among subjects that carry each CMV-associated TCR. Of 104 CMV-associated TCRs, 41 were HLA-restricted at p ≤ 10−3, and none of these were significantly associated with multiple alleles in any locus.

We find concordance between our data and previously-reported CMV-specific TCRs: most previously-reported CMV-specific TCRs are present in our data, however only 5 CMV-associated TCRs have been previously reported. Of these, four were significantly HLA-restricted in these data, and agreed with previous findings.

We investigated the association of TCRs with each HLA allele present in the cohort, with significant results for several higher frequency alleles. Cross validation with binary classifier training resulted in high accuracy prediction for these alleles, indicating that HLA type can be inferred from immunosequencing data.

We demonstrate the validity of association studies using immunosequencing for detection and HLA-association of public T-cell responses to infection, and report that assessing the presence of associated T-cell responses can serve as a powerful diagnostic classifier.
Superior isolation of antigen-specific brain infiltrating T cells using manual homogenization technique
Authors: Garcia Luz Milbeth Cumba , Huseby April M , Johnson Aaron J
Published: January 2023

Effective recovery of activated brain infiltrating lymphocytes is critical for research investigations involving murine neuroimmunological disease models. To optimize lymphocyte recovery, we compared isolation methods using brains harvested from 7 day Theiler’s virus infected mice. Brains were processed using either a mechanical dounce based approach or were enzymatically digested using type IV collagenase. The resulting cell suspensions from these two techniques were transferred to a Percoll gradient, centrifuged, and lymphocytes were recovered. Flow cytometric analysis of CD45hi cells showed greater percentage of CD44hiCD62loactivated lymphocytes using dounce method. Also, a 3-fold greater recovery of activated virus specific CD8 T cells for the immunodominant virus epitopes Db: VP2121-130 and Kb: OVA257-264 was achieved through mechanical dounce homogenization approach as compared to collagenase digest. Greater percentage of viable cells was also achieved with the dounce homogenization. We therefore conclude that manual homogenization is a superior approach to isolate activated T cells from the mouse brain.
The role of the RIG-I like receptors in mediating CD8+ T cell immunity during virus infection
Authors: Aguilar-Valenzuela Renan A , Ma Daphne Y , Suthar Mehul S
Published: January 2023

West Nile virus (WNV) is a mosquito-borne Flavivirus that is a major cause of virus-induced encephalitis in the US. Upon virus infection, pattern recognition receptors (PRRs) recognize viral PAMPs to trigger potent antiviral defense programs. In particular, the RIG-I-like receptors (RLRs) are a family of cytosolic PRRs that comprise of RIG-I, MDA5 and LGP2 and are expressed in nearly every host cell, including antigen-presenting cells. We have previously demonstrated that RLRs as well as their central adaptor protein mitochondria antiviral signaling (MAVS) are critical for mediating immune protection against WNV infection. Recently, we discovered that the RLRs function in a non-canonical manner within CD8+ T cells to promote T cell expansion, survival and effector function during virus infection. Specifically, we found that the RLR LGP2 plays an essential role in promoting virus-specific CD8+ T cell survival and effector functions. To better understand this novel RLR pathway in T cells, we evaluated the cell-intrinsic roles of RIG-I and MAVS, two known interacting partners of LGP2. Following in vitro CD8+T cell stimulation, we found that RIG-I−/−, but not MAVS−/−, CD8+ T cells displayed higher proliferation rates than wild type (WT) cells. Adoptive transfer studies into RAG−/− mice revealed that WNV-specific CD8+ T cells from RIG-I−/− or MAVS−/− mice were present in lower percentages than WT CD8+ T cells during virus infection. To define the non-canonical RLR signaling, we are undertaking a genetics approach to screen proteins associated with the RLR interactome in CD8+ T cells. Altogether, our data indicate that RLRs function in a noncanonical manner within CD8+ T cells to regulate antigen-specific responses during virus infection.