bluuebunny/crossref_metadata_2025_split huggingface dataset:
CD8 T cell responses to conserved DNAJB1-PRKACA fusion neoantigens in fibrolamellar carcinoma
Authors: Kirk Allison M , Chou Ching-Heng , Awad Walid , Crawford Jeremy Chase , Allen E Kaitlynn , Zhang Xiaoyu , Zamora Anthony E , Strome Scott E , Thomas Paul G 
Published: January 2023

Abstract
Fibrolamellar carcinoma (FLC) is an often-lethal liver malignancy of adolescents and young adults. No systemic therapies are currently approved for FLC, so novel treatments are needed to improve patient outcomes. A promising therapeutic vulnerability in FLC is a highly-conserved gene fusion between DNAJB1 and PRKACA. The resultant fusion protein is identical in more than 90% of patients, making it a potentially ideal neoantigen target for T cell-based immunotherapy. The goal of our study was to test if DNAJB1-PRKACA neoantigens could elicit T cell responses and serve as targets for immunotherapy. Spatial transcriptomic analyses of FLC tumor tissue indicated that CD8 T cells could infiltrate FLC tumors, but expressed exhaustion markers such as TOX. Nevertheless, we expanded T cells from an FLC patient tumor ex vivo and detected T cells that produced IFNγ and TNFα in response to stimulation with a fusion neoantigen. We then used functional response after peptide stimulation or peptide-MHC tetramer staining to identify fusion-specific T cell receptors (TCRs) in both FLC patients and healthy donors. We expressed these TCRs in primary human T cells and found that cells expressing fusion-specific TCRs bound to their cognate tetramer and produced IFNγ, TNFα, and IL-2 in response to stimulation with their cognate peptide. TCR-expressing cells also specifically killed target cells presenting their cognate fusion peptide in vitro. Ongoing experiments will test if cells expressing these TCRs can control growth of fusion-expressing tumors in vivo. Together, these studies have defined the first reported fusion-specific T cell response in an FLC patient, as well as fusion-specific TCRs that hold promise for development in adoptive T cell therapies.
Supported by grants from NIH (R01 AI136514, F31 CA254423) and The Mark Foundation for Cancer Research (Aspire Award)
PIK3IP1/TrIP Immune Regulation on CD8+ T Cells Restricts Anti-Tumor Immunity
Authors: Murter Benjamin M , Banerjee Hridesh , Szymczak-Workman Andrea , Kane Lawrence P 
Published: January 2023

Abstract
The signaling pathways involving phosphoinositide-3-kinases (PI3Ks) are highly conserved and tightly regulated to influence the activation, proliferation, and survival of all cell types. Our lab has recently shown protein TrIP (Transmembrane Inhibitor of PI3K, Pik3ip1) has a distinctly high expression on T cells and is capable of downregulating PI3K signaling in these cells, acting as a negative regulator of T cell immune responses. These studies revealed that CD4+ T cells lacking TrIP expression exhibit a more Th1 inflammatory phenotype compared to WT T cells, both in vivo and in vitro. These data have led us to propose that TrIP restricts the inflammatory activity of T cells more generally, including CD8+ T cells, and that targeting/knockout of this regulator may promote anti-tumor immunity.
Using a conditional TrIP knockout mouse model developed in our lab, we have performed syngeneic tumor challenges in CD8+ T cell-specific TrIP knockout mice (TrIPfl/fl E8icre) which show that TrIP knockout mice are resistant to growth of syngeneic tumors. In addition to this increased resistance, tumors harvested from our knockout mice contain twice as many infiltrating T cells compared to their WT counterparts. Of those, CD8+ T cells appeared to be the main drivers of this increased T cell infiltration as their frequency was double that of the CD4+ population. In addition, these TrIP KO CD8+ T cells have a significantly more inflammatory phenotype detailed through RNAseq analysis.
In conclusion, TrIP plays a significant role in the antitumor immune activity of CD8+ T cells. The TrIP KO mice are resistant to tumor challenge and display an increase in TIL T cell infiltrate. These data describes TrIP as an interesting potential target for immunotherapy.
Supported by grants from the NIH 5R01GM136148-02 and 5T32CA082084-20
Cancer vaccines used in combination with TCR-like antibody blockade of the Qa-1b/Qdm complex, the ligand for the immune checkpoint NKG2A, induce anti-tumor immunity
Authors: Ghaffari Soroush 
Published: January 2023

Abstract
Antagonizing antibodies against immune checkpoint inhibitory molecules (ICI) has gained an excellent reputation in immuno-oncology. Anti CTLA-4 and anti-PD-1 are among the first cancer checkpoint immunotherapies that got FDA approval to treat certain malignancies. However, not everyone can benefit from them due to the insufficient expression of the checkpoint marker on the surface of the tumor cells.
CD94/NKG2A receptor is another inhibitory marker known to be expressed by natural killer cells and sub-population of CD8+ T cells. Specific peptides loaded by Qa-1b (mouse) or HLAE (human) on the surface of tumor cells, immune infiltrating lymphocytes, and myeloid cells are NKG2A ligand and are known to have a significant role in modulating anti-tumor effector cell responses.
Our group previously developed a single-domain T-cell receptor-like antibody (EXX-1) with specificity for the Qa-1b/Qdm peptide complex, the ligand for the murine NKG2A/CD94 receptor. We have shown that treating tumor-bearing mice with EXX-1 as a single agent blocking antibody can create a modest delay in mice tumor growth and overall survival time.
The presented study focused on utilizing EXX-1efficacy as a checkpoint blocking antibody in combination with a cancer vaccine. Our findings indicate that cancer vaccine effectiveness can be markedly enhanced when combined with EXX-1 antibody, leading to tumor-free survival in 35% of the mice compared to control groups. Taken together, our results indicate targeting the NKG2A axis through blocking Qa-1b/Qdm peptide complex is an effective means to treat cancer. Also, using HLAE/Qa-1b blocking antibody can improve the efficacy of the cancer vaccines and lead to better clinical outcomes when combined.
T cell receptor revision as a novel mechanism of action of immune checkpoint blockade
Authors: Brice David , Thomas Paul G , McGargill Maureen A 
Published: January 2023

Abstract

Background
Immune checkpoint blockade (ICB) is a promising therapy prescribed to combat tumors. However, acquired resistance to ICB highlights the need for a better understanding of ICB mechanisms. Analysis of T cell receptor (TCR) sequences in individuals before and after ICB showed that a distinct TCR repertoire was observed following ICB, suggesting that T cells with novel specificities contribute to tumor clearance. While the source of these T cells with distinct TCRs is not clear, ICB has been found to lead T cells infiltrating both mouse and human tumors to express components of TCR revision. Therefore, one potential mechanism by which ICB leads to anti-tumor immunity is by inducing TCR revision.

Methods
Wildtype C57BL/6, as well as TCR revision-deficient TCRa+/−.Rag2fl/fl.CD4cre (TCRa+/−.Rag2CD4) mice received subcutaneous injections of either 4.5 × 105 B16F10 or MC38F10 cells. Mice then received either PBS or a combination of 250 μg each of anti-CTLA-4, anti-PD-1, and anti-PDL-1 three times per week starting Day 7 after tumor cell injection. Tumor size and mouse survival were monitored, and baseline splenic and thymic immune cell numbers were measured via flow cytometry.

Results
No differences in T or B cell subsets were detected at baseline between the mouse strains. ICB significantly reduced tumor growth and mortality in the wildtype, but not the TCRa+/−.Rag2CD4, mice for both tumor models tested.

Conclusions
TCR revision capabilities seem to be important in ICB efficacy. Further investigation using these mouse models will help explore the possibility of future targeting of TCR revision pathways to increase ICB efficacy.
Inflammation suppresses elevated TCF1 and FOXO1 expression in vaccine-elicited T cells
Authors: Ivanova Daria Leonidovna , Kilgore Augustus , Klarquist Jared , Thompson Scott , Kedl Ross 
Published: January 2023

Abstract
T cells that express TCF1 display self-renewing capacity and are essential for the long-term T cell response during chronic infections and cancer. Due to their increased responsiveness to checkpoint blockade, TCF1+ T cells are recognized as promising targets for immunotherapies. We found that, in contrast to T cells responding to infections (Tinf), the majority of adjuvanted subunit vaccine-elicited T cells (Tvac) rapidly acquired a memory phenotype (CD127hi) and were TCF1hi. This suggests that Tvacs could be programmed by TCF1 to commit to a memory cell fate. We found that Tvac peak and memory responses were more compromised in the absence of TCF1 than those of Tinf. However, TCF1 KO Tvacs still developed memory, suggesting that additional factors were involved. FOXO1 is important for T cell memory after infections. We found that Tvacs expressed more FOXO1 than Tinfs. In contrast to Tinf, FOXO1 was required for both the peak and memory Tvac responses. Moreover, even though FOXO1 drives TCF1 in Tinfs, Tvacs expressed TCF1 independently of FOXO1. To explore the cause for such high expression of TCF1 and FOXO1 in Tvacs, we next examined the role of inflammatory mediators in their regulation. Tvac TCF1 and FOXO1 inversely correlated with adjuvant-elicited pro-inflammatory signals. Moreover, addition of pro-inflammatory TLR9 ligand CpG reduced Tvac TCF1, FOXO1 and memory. In contrast to prior reports, anti-IL-12 failed to reverse the effect of CpG on Tvacs. Anti-IFNγ, however, restored the Tvac memory phenotype even as CpG drove additional inflammatory mediators. Our data highlight the differences in the transcriptional regulation of vaccine- and infection-elicited T cells and provide insights into the mechanisms regulating TCF1 and FOXO1.
Supported by grants from NIH (T32 AI074491, R01 AI066121)
Cell-specific regulation of antigen-specific germinal center formation and antibody responses by thymic stromal lymphopoietin
Authors: Domeier Phillip P , Ziegler Steven F 
Published: January 2023

Abstract
Germinal centers (GCs) are dynamic immune structures that expand upon T-dependent immune challenge to generate high-affinity antibody-producing plasma cells and long-lived memory. GCs require stringent regulation to optimize GC-derived antibody responses. Here, we identified thymic stromal lymphopoietin (TSLP), as a novel regulator of GCs. After immunization with NP-OVA in Alum, TSLP receptor-deficient (TSLPR−/−) mice fail to form antigen-specific GCs and antibodies. TSLPR expression is elevated on T follicular helper cells (Tfh) and GC B cells during inflammation, so we asked how cell-specific loss of TSLPR signaling could disrupt overall GC activity. When TSLPR was deleted in CD4 T cells with a tamoxifen-inducible Cre (CD4CreERT2/+ TSLPRFLOX/FLOX), we observed a similar reduction in T follicular helper cells (Tfh), GC formation, and antibody responses after immunization when compared to CD4CreERT2/+ and TSLPRFLOX/FLOX controls. Conversely, when TSLPR signaling was lost on mature B cells (CD21Cre/+ TSLPRFLOX/FLOX) and GC B cells (AIDCre/+ TSLPRFLOX/FLOX), we observed an increase in antigen-specific GC B cells and antigen-specific memory B cells. TSLPR signaling regulates IRF4 expression in dendritic cells, and we observed a similar reduction in IRF4 expression in Tfh and GC B cells from TSLPR−/− mice compared to TSLPR+/+ controls. Collectively, these findings suggest that TSLPR signaling promotes IRF4 expression in GC B cells and Tfh cells, resulting in distinct roles in the GC. TSLPR and IRF4 signaling in T cells is required for the establishment and maintenance of antigen-specific Tfh cells, whereas TSLPR/IRF4 signaling in B cells inhibits antigen-specific GC B cell retention and the establishment of B cell memory.
Supported by a grant from the NIH (F32 AI154787)
Inhibition of perforin enhances T cell and humoral responses after immunization
Authors: Krishnamurthy Durga , Cevik Hilal , Ali Ayad , Canaday Laura , Mukhopadhyay Eshana , Seibel William , Risma Kimberly , Jordan Michael B , Waggoner Stephen N 
Published: January 2023

Abstract
Cytotoxic lymphocytes such as natural killer (NK) cells release perforin to form pores on plasma membrane of target cells to allow delivery of granzymes that induce apoptosis. We previously showed that NK cells exert a perforin-dependent suppression of follicular helper CD4 T cell and germinal center B cell responses following infection and immunization. Consistent with this activity, genetic ablation of either perforin or NK cells non-additively enhanced the quality and magnitude of humoral immune responses. Therefore, we hypothesize that transient inhibition of perforin represents an innovative means to enhance vaccine efficacy to prevent viral infections such as HIV. Intraperitoneal injection of a selective inhibitor of perforin (35mg/kg) to mice reduced killing activity of NK cells by &gt;50%. A single injection of this inhibitor to mice during alum adjuvanted NP-KLH immunization resulted in a greater than two-fold enhancement of follicular helper CD4 T cell, germinal center B cell, and antigen-specific antibody responses. In summary, transient inhibition of pore-forming protein perforin can block NK-cell immunosuppression to enhance vaccine-elicited T and B cell responses.
National Institute of Heath (NIH) (R01-AI148080)
A Novel Solution to Better Understand the Link Between T Cell Metabolism and Function
Authors: Walls Jessica , Kam Yoonseok , Romero Natalia 
Published: January 2023

Abstract
In recent years, metabolism has emerged as a key driver of immune cell function in both healthy and disease settings. Indeed, it has been shown that metabolic reprogramming is a determinant of T cell phenotype and fate. Moreover, reprogramming of the metabolic pathways of T cells can be used as a strategy to improve the antitumor efficacy of adoptive T cell therapies. The Agilent Seahorse XF T cell Metabolic Profiling (XF TCMP) kit is a new solution that, combined with the Seahorse XF analyzer, allows for the simultaneous measurement of basal metabolic demand, metabolic poise and respiratory capacity in T cells. These parameters have been linked with optimal function and improved persistence of T cell therapies. The assay uses an improved uncoupler (BAM15) for more robust measurements of T cell bioenergetic capacity with less optimization than other uncouplers used. The XF TCMP kit was used to evaluate the metabolic phenotype of human T cells expanded in different nutrient conditions. T cells expanded in different cell culture media resulted in populations with different metabolic profiles. These metabolic profiles correlated with different functional markers, supporting the link between metabolism and function. The XF TCMP kit was also used to assess metabolic poise and bioenergetic capacity under nutrient-restriction, allowing us to better understand fuel requirements after expansion. Together, the data show that the XF TCMP kit provides a complete metabolic assessment of T cells in real-time. The assay can be used to develop improved anti-tumor T cell therapies by evaluating strategies that improve the persistence of infused cells or assess how T cells sustain metabolic fitness in nutrient restricted environments to maintain function.
Gene editing at position 71 in DRB1*04:01 blocks collagen sensitization and avoids allorecognition.
Authors: Jha Vibha , Coulombe Marilyne , Rosloniec Edward F , Matsuda Jennifer , Freed Brian M , Roark Christina L 
Published: January 2023

Abstract
DRB1*04:01 is strongly associated with susceptibility to rheumatoid arthritis (RA). Our previous work demonstrated that changing lysine (K) at position 71 in DRB1*04:01 to glutamic acid (E), an amino acid found in resistant alleles, abrogated binding of the immunodominant collagen258–272 peptide. The purpose of this study was to determine if the K71E mutation in DRB1*04:01 would prevent sensitization to collagen and be tolerated by the host immune system in mice. DRB1*04:01 and DRB1*04:01K71E transgenic mice were created on the murine MHC class II knockout background (B6.129S2-H2dlAb1-Ea/J). DRB1*04:01 and DRB1*04:01K71E mice were immunized twice with bovine type II collagen in complete Freund’s adjuvant on days 0 and 21. On day 56, lymph node cells were harvested and stimulated in vitro with collagen258–272 peptide. Introduction of the K71E mutation in DRB1*04:01 resulted in a &gt;90% reduction in proliferation as compared to CD4+ cells from collagen-immunized DRB1*04:01 mice. Next, skin grafts were transplanted from DRB1*04:01K71E mice onto DRB1*04:01 recipients to test for rejection. The skin grafts from DRB1*04:01K71E mice were accepted by the recipient DRB1*04:01 mice with no signs of rejection out to day 70. Our results demonstrate that introducing the K71E mutation in DRB1*04:01 blocks collagen sensitization and avoids allorecognition suggesting that this mutation could safely halt progression of RA by blocking autoimmune T cell responses to collagen.
Supported by IOS-2103367
Novel immunological tools to investigate CD4+ T cell activation in the canine species
Authors: Lang Haeree Park , Dileepan Thamotharampillai , Jenkins Marc K , Friedenberg Steven 
Published: January 2023

Abstract
Opportunities to identify T cell epitopes in the dog have been thwarted by a lack of immunological tools specific for the species. As a result, antigen-specific triggers in diseases that affect dogs like autoimmune disorders and allergies remain unknown. Our objective was to develop immunological tools such as: IFN-γ enzyme-linked immunosorbent spot (ELISpot) and activation induced marker (AIM) assays, and an MHC class II tetramer to help overcome these hurdles. We collected splenocytes from dogs that underwent a routine splenectomy. We used these splenocytes to develop: an ELISpot assay detecting canine IFN-γ production by stimulated T cells, an AIM assay to assess early activation by CD40L expression on CD4+CD5+ canine splenocytes using a cross-reactive anti-human monoclonal antibody, and finally, we produced the first-ever canine MHC II tetramer specific for a common canine DR allele. We confirmed the efficacy of these tools in the presence of positive and negative controls, Staphylococcus aureus enterotoxin B (SEB) and dimethyl sulfoxide (DMSO), respectively. SEB induces a robust canine IFN-γ ELISpot readout quantifiable in canine splenocytes. Similarly, we discovered that canine CD40L expression peaks at 3 hours post-stimulation with SEB. Finally, we confirmed the development of the first canine pMHCII tetramer through size discriminatory SDS-PAGE analysis and peptide exchange assays. Our novel canine-specific reagents will help identify and characterize antigen-specific T cells and their cognate antigens driving canine MHC class II-restricted disease, thus guiding research towards the advancement of antigen-specific immunotherapies.
Supported by a grant from the NIH (K01 OD027058)
Development of swine Fc conjugated Serotype O Foot and mouth disease virus like particles
Authors: Shin Hyun-Jin , Park Jae-Yeon 
Published: January 2023

Abstract
Foot-and mouth disease virus (FMDV) cause a highly contagious infection in cloven-hoofed animals. The current conventional FMD vaccines are produced by chemical inactivated virulent FMDV virions, but conventional FMD vaccines risky and limitation such as respect to accidental release of FMDV and high-level biosafety facilities were required to handle FDMV. FMDV virus like particle (VLP) could be alternative choice for future vaccine against FMDV because of efficacy and safety. Receptor for Fc portion of antibody play and important role in the activation of immune response for infection of virus and bacteria. Therefore, immunization with a complex of virus antigen and Fc has the potential to be an improved. Here we report our vaccine studies with Fc conjugated serotype O FMDV VLP for alternative FMDV vaccine.
Identifying B cells in hatchling and adult Trachemys scripta, red-eared slider turtles
Authors: Paton Hanna D , Mool Allison , Fairow Christen , Hurst Whitney , Bowden Rachel , Vogel Laura 
Published: January 2023

Abstract
Evidence suggests there are important differences between reptile and mammalian immune strategies, yet little is known about humoral immune responses in reptilian vertebrates. Our laboratory is interested in reptile B cell development and function and our undergraduate research project involved B cell detection using flow cytometry in spleens of hatchling and adult turtles. We made use of a previously developed monoclonal antibody (HL673) that recognizes turtle light chain protein. The murine HL673 mAb was purified using a protein A affinity column and reactivity was tested by ELISA. Some of the purified antibody was biotinylated and incubated with blood and spleen samples from adult and hatchlings. Bound antibodies were detected and B cell populations identified using flow cytometry. We were able to identify B cell containing populations based on scatter and specific staining. Our results showed detection of turtle B cells using the labeled mAb in both adults and 6 week old hatchlings. Future studies will use this reagent to further investigate the distribution and function of B cells.
This work was supported by NSF 1725199 and NIH 1R15AI140118 – 01.
Flt3 and its ligand as an ancient regulators of dendritic cells: evidence in the amphibian Xenopus laevis
Authors: Paiola Matthieu , Roy Sobhan , Ma Siyuan , Patrick Charkira , Pavelka Marin , Adams Erin J , Robert Jacques 
Published: January 2023

Abstract
The evolution of hematopoietic-derived dendritic cells that are so critical for T cell activation is still poorely understood. In mammals, fms related tyrosine kinase 3 (Flt3) and its ligand (Flt3lg) are important regulator of dendritic cell homeostasis. Flt3 and Flt3lg co-evolution was investigated in all jawed vertebrates with special focus in an ancient allotetraploid tetrapod Xenopus laevis. flt3 synteny and predicted amino-acid structure are conserved in all jawed vertebrate including in the duplicated S and L homeologous chromosomes of X. laevis. Flt3lg gene is conserved among X. laevis homeologous chromosomes and most jaw vertebrates with the exception of ray-finned fish. Amino acid comparison, 3D structure modelling and tissue distribution suggest a subfunctionalization of Flt3 and Flt3lg homeologs. Tagged recombinant Flt3lg.S and Flt3lg.L were produced to investigate cell surface expression of Flt3s. Both Flt3lg.S and L specifically stain a splenic population of leukocyte MHC class IIhigh+ and CD8+ phenotypically similar to the recently described X. laevis dual follicular/dendritic-like XL cells. Our results indicate that Flt3lg.S and L are both involved in XL cell homeostasis and that XL cells have hematopoietic origin. In X. laevis, the non-polymorphic MHC class I-like XNC4 restricting innate-like T cells is critical for tadpoles’ resistance against the non-TB mycobacteria M.marinum. Preliminary experiments indicate that XL cells are XNC4+ and are being recruited to the infection site together with XNC4-restricted T cells. This suggests that XL cells are involved in T cell activation, which is consistent with the an ancestral dual follicular/dendritic cell function of XL cells.
Supported by NIH (R24-AI059830 – R21-AI139718), NSF (1754274) and 2021 Rochester Vaccine Fellowship