bluuebunny/crossref_metadata_2025 huggingface dataset:
IkZF and STAT factor regulatory modules as drivers of TH1 and TFH cell differentiation decisions
Authors: Jones Devin M , Read Kaitlin A , Sreekumar Bharath , Powell Michael Duane , Warren Robert T , Oestreich Kenneth J
Published: January 2023

CD4+ T helper cells are capable of differentiating into subsets that regulate distinct aspects of the immune response. The differentiation of these populations is driven by subset-specific cytokine signals and downstream transcription factor networks that are responsible for promoting the unique gene expression programs of each cell type. It is well-established that cytokine signals are propagated by Signal Transducer and Activator of Transcription (STAT) factors. Recently, we demonstrated that STAT3 and Aiolos, a member of the Ikaros Zinc Finger (IkZF) family, form a novel transcriptional complex that directly induces expression of the T follicular helper (TFH) cell lineage defining factor, Bcl-6. Interestingly, we have also found that a second IkZF factor Eos interacts with STAT5 and forms an opposing complex in T helper 1 (TH1) cells that promotes expression of the Bcl-6 antagonist, Blimp-1. However, whether additional IkZF/STAT complexes function downstream of other cytokine signals in these cell populations is unclear. Here, we report that in vitro-generated TH1 and TFH1-like cells similarly express Ikaros concurrent with increased STAT4 activation downstream of IL-12 signaling. Using co-immunoprecipitation analyses, we were also able to detect interactions between Ikaros and STAT4. Furthermore, as with the other IkZF/STAT complexes, we find that interactions between Ikaros and STAT4 were dependent upon the IkZF C-terminal ZF domain, suggesting that this is a conserved regulatory feature of IkZF/STAT complex formation. Current efforts are now aimed at identifying whether mechanistic interplay between the Ikaros/STAT4 complex and the other IkZF/STAT factors may ultimately dictate TH1 or TFH cell fate and function.
Protective function of DUSP4 in DSS-induced colitis
Authors: Lu Yi , Zhang Yongliang
Published: January 2023

Inflammatory bowel disease (IBD), including Crohn’s disease (CD) and ulcerative colitis (UC), is characterized by chronic inflammation of the gastrointestinal tract. The mitogen-activated protein kinase (MAPK) signalling, an evolutionary conserved cell signalling pathway, is associated with IBD. MAPKs comprises ERK, P38, and JNK, which are activated by upstream MKKs and MKKKs, and are inactivated by dual specificity phosphatases (DUSPs). DUSP4 (MPK2) is a member of DUSP protein family and was found to be involved in various physiological processes. However the role of DUSP4 in IBD is yet to be examined. In this study, we utilized DSS-induced colitis mouse model to investigate the function of DUSP4 in this disease. We found that DUSP4 knockout (KO) mice were more susceptible to DSS-induced colitis, which was demonstrated by more weight loss and higher histological score than wild-type (WT) mice. Consistently, expression of inflammatory cytokines, including IL-6, TNF-α, IL-17A, and CXCL-1, was enhanced in both proximal and distal colon of DUSP4 KO mice compared to WT mice. Moreover, excessive CD8+ and CD4+ cells, especially IL-17A-expressing CD4+ T cells, were identified in the mesenteric lymph node (mLN) of the KO mice. Together, these results demonstrated that deficiency of DUSP4 resulted in increased inflammation in the gut in response to DSS.
Intracellular Signaling triggered by engagement of PD-1 by PD-L1
Authors: Bu Xia , Bu Melissa , Hua Ping , Zhu Baogong , Sharpe Arlene H , Freeman Gordon J
Published: January 2023

Blockade of PD-1/PD-L1 is in effective anti-tumor immunotherapy. To further elucidate the mechanism by which PD-1/PD-L1 engagement causes T cell dysfunction, we investigated intracellular signaling triggered by PD-1/PD-L1 ligation in T cells. We describe a novel antibody that detects PD-1 signaling. This antibody detects phosphorylation of the immunotyrosine switch motif (ITSM) in the intracellular tail of both human and mouse PD-1 (phospho-PD-1). We first examined the expression of PD-1 and phosphorylated PD-1 in T cells from human PBMC. We found that without CD3 and CD28 mAb stimulation, PD-1 and phospho-PD-1 had minimal expression on CD3+CD4+ and CD3+CD8+ cell. Upon stimulation with CD3 and CD28 mAb, we observed increased expression of PD-1 and phospho PD-1 in both CD3+CD4+ and CD3+CD8+ cells. Furthermore, upon CD3 and CD28 mAb stimulation and PD-L1 recombinant protein treatment, the level of phospho PD-1 increased in CD3+CD4+ and CD3+CD8+ cells. Utilizing a co-culture system of Jurkat-hPD-1 and 300-hPD-L1, we mimicked the cell-cell interaction through molecular ligation of PD-1 and PD-L1. Upon stimulation with CD3 and CD28 mAb in this co-culture system, we detected increased phospho PD-1 signal and phospho SHP-2 signal by Western blot. When CD3 and CD28 mAb stimulated Jurkat-hPD-1 cells were treated with recombinant PD-L1 protein, we observed decreased IL-2 secretion which indicates PD-1/PD-L1 ligation suppresses T cell activation and corresponds with phospho PD-1 expression. This study examines the intrinsic intracellular signals upon PD-1/PD-L1 engagement, and the results may promote a better understanding of the mechanism of T cell dysfunction by PD-1/PD-L1 ligation.
Transforming growth factor beta induced PD-L1 expression promotes pro-fibrotic signaling
Authors: Jung Mi-Yeon , Kang Jeong-Han , Leof Edward B.
Published: January 2023

Immune checkpoint proteins such as programmed death-ligand 1 (PD-L1) retain general immune homeostasis and self-tolerance. PD-L1 binds to programmed cell death protein 1 (PD-1) and inhibits T cell activation or protects tumor cells from T cell-mediated killing. PD-L1 has recently been shown to be up-regulated in invasive lung fibroblasts by p53 and focal adhesion kinase (FAK), any potential role of transforming growth factor-beta (TGFβ) in PD-L1 action is unknown in fibrosis. TGFβ is a mysterious protein with various roles in healthy tissue homeostasis/development as well as the development or progression of cancer, fibrotic disorders, and immune modulation, et al. In the current study, we showed that TGFβ induces the expression of the immunoinhibitory molecule PD-L1 in human and murine fibroblasts in a Smad2/3- and YAP/TAZ-dependent manner. Furthermore, PD-L1 knockdown decreased the TGFβ-dependent induction of extracellular matrix proteins, including collagen Ia1 (colIa1) and alpha-smooth muscle actin (α-SMA), and cell migration/wound healing. In addition to an endogenous role for PD-L1 in profibrotic TGFβ signaling, TGFβ stimulated-human lung fibroblast-derived PD-L1 into extracellular vesicles (EVs) capable of inhibiting T-cell proliferation in response to T cell receptor stimulation and mediating fibroblast cell migration. These findings provide new insights and potential targets for a variety of fibrotic and malignant diseases.
IL-7 Receptor Alpha Chain Expression Identifies Human CD8+ Memory T Cell Subsets with Distinct Gene Expression Profiles and TCR Repertoire Compositions
Authors: Chiu Yen-Ling , Chang Shih-Chen , Chia Jean-San
Published: January 2023

Heterogeneity of CD8 T cell play crucial roles in cancer patients and reveals the co-evolution between tumor and immunity. While CD127 expression identifies memory precursor cells during acute infections, the role of CD127 expression in defining memory T cell heterogeneity is not investigated. Here, we analyzed the Vβ-CDR3 usage of human effector memory TEM and TEMRA cells accompanied by functional and gene expression patterns based on surface CD127 expression. We found that, CD127hi subsets are associated with higher level of TCR diversity regardless of their TEM or TEMRA phenotype. CD127lo populations are presented with relatively low TCR repertoire diversity. Moreover, there is a high correlation between CD127hi and CD127lo populations which imply possible differentiation tract between CD127hi and CD127lo T cells, but not between TEM and TEMRA cells. On the other hand, both TEM and TEMRA CD127lo populations showed higher expression of effector genes including perforin, granzyme B, granzyme H, and pan-inhibitory killer immunoglobulin-like receptors, exhibited lower phenotype plasticity upon TCR activation and cytokine-driven proliferation, when compared to their CD127hi counterparts. In other words, depending on the expression of CD127, T cells from either TEM or TEMRA are similar in their gene expression and functionality patterns. These results provide the first evidence that T cell repertoire and gene expression signature can be uncoupled based on distinct CD127-dependent phenotype expression patterns but are delicately balanced in the memory T cell pool.
Organ-specific isoform selection of fatty acid binding proteins in tissue-resident lymphocytes
Authors: Fonseca Raissa , Frizzell Hannah , Christo Susan , Evrard Maximilien , Park Simone L. , Freestone David , Mackay Laura K.
Published: January 2023

Tissue-resident memory T (TRM) cells exist throughout the body where they are poised to mediate local immune responses. Although studies have defined a common mechanism of residency independent of location, there is likely to be a level of specialization that adapts TRM cells to their given tissue of lodgment. It has been shown that TRM cells in the skin rely on the uptake of exogenous fatty acids for their survival and upregulate fatty acid binding proteins (FABP) 4 and FABP 5 as part of their transcriptional program. However, FABPs exist as a larger family of isoforms with different members selected in a tissue-specific fashion that is optimized for local fatty acid availability. Here we show that although TRM cells in a range of tissue widely express FABPs, they are not restricted to FABP 4 and 5. Instead, TRM cells show varying patterns of isoform usage that are determined by tissue-derived factors. These patterns are malleable since TRM cells relocated to different organs modify their FABP expression in line with their new location. As a consequence, these results argue for tissue-specific overlays to the TRM cell residency program, including FABP expression that is tailored to the particular tissue of TRM cell lodgment.
Inflammation-induced lactate leads to the rapid loss of hepatic tissue resident NK cells
Authors: Dodard Garvin , Tata Angela , Erick Timothy , Jaime Diego , Miah Shahjahan , Quatrini Linda , Escalière Bertrand , Ugolini Sophie , Vivier Eric , Brossay Laurent
Published: January 2023

The liver harbors two main innate lymphoid cell (ILC) populations, conventional NK (cNK) cells and tissue-resident NK (trNK) cells also called ILC1s. While the antiviral functions of cNK cells have been well established, trNK cell functions are only beginning to be uncovered. Using the MCMV model of infection, we found that in contrast to liver cNK cells, the trNK cell population initially undergoes a contraction phase prior to a recovery phase to homeostatic levels. The rapid contraction phase is due to apoptosis, while the recovery phase occurs via proliferation in situ. Liver trNK cell apoptosis is not mediated by fratricide, liver lymphocytes, or inflammatory cytokines. Instead, we found that liver trNK cell apoptosis is the consequence of an increased sensitivity to lactic acid. Interestingly, MCMV infection lead to an increase of lactate in the liver, which was supported by changes in lactate dehydrogenase expression. Mechanistic analysis indicates that trNK cell sensitivity to lactate is linked to impaired mitochondrial functions. These findings underscore the unique properties of the liver resident NK cell compartment.
Paneth cells regulate diet-induced obesity and trafficking of inflammatory immune cells into adipose tissues
Authors: Joldrichsen Marisa R , Kim Eunsoo , Cormet-Boyaka Estelle , Boyaka Prosper N
Published: January 2023

Paneth cells regulate many key aspects of gastrointestinal health through the antimicrobial products and cytokines they produce. Loss or defective Paneth cell functions leads to dysbiosis and is one of the causes of Inflammatory Bowel Disease (IBD). The prevalence of obesity is growing in the general population and among recently diagnosed IBD patients, leading to the speculation that obesity increases IBD incidence, but the underlying mechanisms remain to be elucidated. We addressed whether Paneth cells of the small intestine play a role in the development of obesity. For this purpose, control wild-type C57BL/6 and Sox9ΔIEC mice, which lack Paneth cells due to Sox9 gene deletion within the intestinal epithelium (Sox9ΔIEC mice), were fed a high fat diet for 13 weeks. Though the consumption of food was similar between the 2 groups, weekly measurement of body weight showed that Sox9ΔIEC mice gained weight much faster and ultimately became more obese than the wild-type mice. The Sox9ΔIEC mice also developed larger abdominal fats, which included increased numbers of inflammatory macrophages (p<0.05) and neutrophils (p<0.05). Interestingly, B cells were the most increased (p<0.01) immune cells population in white fat tissues of Sox9ΔIEC mice. When compared to control mice, the Sox9ΔIEC mice displayed impaired glucose tolerance (p<0.0001) indicating that these mice also developed a metabolic disorder. Finally, the in vivo FITC-dextran permeability assay showed that Sox9ΔIEC mice on high-fat diet have increased intestinal permeability (p<0.05). These results suggest a new role for Paneth cells as regulators of diet-induced obesity via their impact on gut immune homeostasis and recruitment of immune cells into omental fat tissues.
miR-17-92 regulates MAIT cell development and function
Authors: Liu Tingting , Wang Jie , Zhou Li , Mi Qing-Sheng
Published: January 2023

Mucosal-associated invariant T (MAIT) cells are innate-like T cells that acquire an effector function and differentiate into MAIT1 (T-bet+) and MAIT17 (RORγt+) subsets prior to thymic export. Upon activation, MAIT cells quickly release IFN-γ and IL-17, which modulate a broad spectrum of diseases, including autoimmunity, infection, allergy and cancer. miRNAs are no-coding RNAs regulating immune cell development and function. A recent study indicated defective MAIT cell development in the miRNAs-deficient mice, but, the specific miRNAs that guide their development remain unclear. Previous studies indicated that miR-17-92 regulates different immune cell development and function, however, the role of miR-17-92 in the MAIT cell development remains unclear. Here, we generated T cell specific miR-17-92 knockout (KO) mice to test the role of miR-17-92 in MAIT cell development and function. We found that deletion of miR-17-92 led to a profound defect on the MAIT cells in the number and frequencies in the thymus and peripheral immune organs, including spleen, lymph nodes, and liver, as well as in the lung and peritoneal cavity (P<0.05). The frequency of MAIT17 cells was dramatically reduced in miR-17-92 KO mice, while the frequency of MAIT1 cells was dramatically increased in thymic and peripheral immune organs, compared to wild-type (WT) controls. Furthermore, upon PMA/Ionomycin stimulation, we found a defect of IL-17 production, with significantly increased IFN-γ production by peripheral MAIT cells in miR-17-92 KO mice, compared to WT controls. The detailed miR-17-92 targeting pathway by RNA-Seq and Chip-Seq is under the investigation. Overall, our data indicated that miR-17-92 plays a pivotal role in MAIT cell development and function.
Intestinal intraepithelial lymphocyte repertoires are imprinted clonal structures selected for MHC reactivity
Authors: Schattgen Stefan A , Crawford Jeremy C , Van de Velde Lee-Ann , Chu Hiutung , Mazmanian Sarkis K , Bradley Phil , Thomas Paul G
Published: January 2023

The intestinal mucosa is an important barrier for maintaining homeostasis and immune tolerance. Residing in the gut mucosa are the intestinal intraepithelial lymphocytes (IELs), an unconventional T cell type expressing αβ T cell receptors (TCRs) thought to contribute in immune surveillance and mediate repair of damaged epithelium. IELs are in close contact with a stream of microbial, food, and self-antigens, and while TCR-antigen interaction is presumed the major mechanism governing their functions, little is known about the native TCRαβ-IEL repertoire or its specificity.

Previously, we developed a nearest-neighbor distance measurement (TCRdist) for quantifying convergence within TCR repertoires based on enriched features within their determinants of specificity. Here, we extended TCRdist to develop two novel tools for repertoire analysis: RepDist, for measuring similarity between repertoires, and TStar, for identifying repertoire-enriched features. We applied these to conduct a survey of the ‘normal’ TCR repertoires for three major subsets (CD8αα+, CD8, CD4) of IELs, under varying conditions of microbiome depletion, food antigen deprivation, MHC-deficiency, and combinations thereof. Surprisingly, the only perturbations that disrupted the IEL repertoire were deficiencies in Class I molecules or β2m. Food antigen and microbiome depletion had virtually no effect on the clonotypic composition of all three IEL subsets. MHC-substitution also had little effect, indicating that while Class I is required for CD8αα and CD8 IEL selection, it is haplotype-independent. Taken together, these data demonstrate the native IEL TCR repertoires are selected for self-reactivity, rather than specificity towards exogenous antigens.
BCG vaccination reduces the mortality of Mycobacterium tuberculosis-infected type 2 diabetes mellitus (T2DM) mice through the induction of CXCR3+ T-regulatory cells
Authors: Radhakrishnan Rajesh Kumar , Thandi Ramya Sivangala , Tripathi Deepak , Paidipally Padmaja , McAllister Madeline , Mulik Sachin , Samten Buka , Vankayalapati Ramakrishna
Published: January 2023

Diabetes is a significant risk factor for the development of active tuberculosis. In this study, we used mouse model of Streptozotocin/Nicotinamide (STZ/NA) induced non-obese type 2 diabetes mellitus (T2DM) to determine the effect of prior BCG vaccination on survival and immune responses to Mycobacterium tuberculosis (Mtb) infection. We found that at 6–7 months post-Mtb infection, 90% of the Mtb-infected T2DM mice died, whereas only 50% of BCG-vaccinated T2DM-Mtb-infected mice died. Moreover, 40% of the PBS-treated uninfected T2DM mice and 30% of the uninfected BCG-vaccinated T2DM mice died, whereas all uninfected and infected nondiabetic mice survived. BCG vaccination was less effective in reducing the lung bacterial burden of Mtb infected T2DM mice compared to Mtb-infected non-diabetic mice, however it reduced immunopathology of lung tissues. Further, we found increased survival of BCG vaccinated Mtb infected T2DM mice is associated with 2-fold expansion of IL-13 producing CXCR3+ T-regulatory cells as measured by flow cytometry, qRT-PCR and confocal microscopy. We also found that prior BCG vaccination restored the immunosuppressive function of T-regulatory cells of Mtb-infected T2DM mice and reduced inflammation. IL-13 producing T-regulatory cells of BCG vaccinated Mtb-infected T2DM mice converted proinflammatory M1 macrophages (iNOS) to anti-inflammatory M2 macrophage (Arg1) phenotype to suppress the inflammation. In contrast, anti-IL-13R antibody inhibited the conversion of macrophages from M1 to the M2 phenotype and enhanced the inflammatory cytokines (IL-6 and TNF-α) production. Our findings suggest a novel role for BCG in preventing excessive inflammation and mortality in T2DM mice infected with Mtb.
Identifying mechanisms that enhance the longevity of tissue-resident memory T cells in the lung
Authors: Lobby Jenna L , Uddback Ida , Cartwright Emily , Hayward Sarah L , Takamura Shiki , Thomsen Allan R , Christensen Jan , Kohlmeier Jacob E
Published: January 2023

Lung tissue-resident memory T cells (TRM) are crucial mediators of cellular immunity against influenza viruses, but the number of these cells in the lung tissue gradually declines in the months following influenza infection. Recently, we showed that intranasal immunization with a replication-deficient Adenovirus that expresses the nucleoprotein from influenza A virus (AdNP) results in long-term maintenance of lung CD8+ TRM for up to 1-year post-immunization. However, the mechanism(s) that promote this enhanced longevity of CD8+ lung TRM remain unknown. Using a combination of mouse infection models, flow cytometry, and RNA-sequencing, we compared CD8+ T cells from the airways, lung, and spleen from AdNP-immunized or influenza x31-infected mice. We found that CD8+ TRM in the lungs of AdNP-immunized mice show increased homeostatic turnover and encounter persistent antigen in the lung. In addition, parabiosis experiments suggest that the influenza-specific CD8+ lung TRM pool is maintained in AdNP-immunized mice by continual recruitment of circulating TEM into the lung TRM pool. RNA-sequencing analysis comparing CD8+ lung TRM from AdNP-immunized and x31-infected mice at 1-month and 1-year post-immunization showed only minor variations that did not fully explain the differences in lung TRM persistence. We are currently investigating how antigen is maintained in the lung following AdNP immunization using microscopy and Cre recombinase-expressing Adenovirus (AdCre) to identify antigen presenting cells harboring antigen long-term in the lung. The results of this study will identify mechanisms that improve the durability of cellular immunity, and will thus inform future design of cell-mediated influenza vaccines.
Dynamic local T cell responses underlie associations in disease severity during infant RSV infection
Authors: Connors Thomas , Dogra Pranay , Mertz Sara , Szabo Peter , Idzikowski Emma , Ramilo Octavio , Mejias Asuncion , Farber Donna L
Published: January 2023

Respiratory syncytial virus (RSV) infections are common throughout infancy, resulting in a wide range of clinical manifestations, with significant morbidity and mortality seen worldwide. The mechanisms leading to severe disease remain unknown. We sought to determine viral and immune mediated factors related to disease severity. Utilizing multi-parameter flow cytometry and single cell RNA sequencing, we correlated local T cell responses with dynamic changes to viral load in samples obtained from infants infected with RSV. Viral burden was highest at time of study enrollment and declined towards clearance in all subjects regardless of disease severity. Peak CD8 T cell response occurred several days after peak viral burden. Disease severity was associated with the accumulation of highly proliferative CD25+/Ki67+ CD8 T effector memory cells in the respiratory tract. Single cell RNA sequencing of T cells revealed distinct transcriptomic profiles of T cell populations. Respiratory tract CD8 T cells upregulated genes for granzyme, perforin, and tissue adhesion (CXCR6, CD49a) while CD4 T cells in the airway were enriched for regulatory T cell genes (FoxP3, CTLA4) compared to blood. Blood samples were defined primarily by gene signatures associated with naïve T cells and no significant upregulation of effector genes, emphasizing the compartmentalized nature of the immune response. Interestingly, clusters of gamma delta T cells upregulating AREG were found in airway samples, supporting a role for immune mediated tissue repair. These results show that disease severity during RSV infection in infancy is associated with localized immune responses and provide insights into how tissue repair may be mediated following severe disease.
Inducible epithelial resistance promotes survival of viral pneumonia by inactivating virus particles and preventing CD8+ T cell dependent immunopathology
Authors: Wali Shradha , Flores Jose , Goldblatt David , Tuvim Michael J , Dickey Burton , Evans Scott E
Published: January 2023

Respiratory viral infections cause significant morbidity and mortality worldwide, necessitating the development of novel strategies to prevent complications of respiratory infections. Our group has discovered the phenomenon of inducible epithelial resistance where mice treated with combination inhalation of Toll-like receptor (TLR) 2/6 and TLR9 agonists (Pam2-ODN) are protected against respiratory pathogens, including viruses. We investigated the mouse survival benefits of paramyxovirus, Sendai (SeV) infection and found that Pam2-ODN treatment one day prior to SeV challenge enhanced survival of mice. This protection was associated with reduced CD8+ T cell inflammation. Depletion of CD8+T cells prior to infection increased baseline virus susceptibility but did not affect protection afforded by Pam2-ODN. Depletion of CD8+T cells after viral burden waned but before peak mortality enhanced SeV pneumonia survival, even in the absence of Pam2-ODN pretreatment, revealing the profound role of CD8+ T cell mediated immunopathology in virus-induced mortality. We further demonstrated that Pam2-ODN treatment controls viral replication by inactivation of SeV prior to epithelial internalization. Epithelial generation of reactive oxygen species induced by Pam2-ODN treatment is necessary for SeV inactivation and preventing CD8+T cell mediated immunopathology. These findings highlight antiviral mechanisms of inducible epithelial resistance and may provide means to protect vulnerable immunocompromised patients against respiratory diseases.
Novel approach for the detection of antigen-specific CD8 T cell responses corresponding to treatment outcome in HPV+ cancer patients
Authors: Sierra Gloria , Ouerkaxi Wushouer , Daftarian Pirouz , Sastry Jagannadha K , George Marybeth , Delcommenne Marc
Published: January 2023

HPV cancer treatment outcome has been positively correlated to the presence of patient immunity against the virus. Six HPV strains account for over 75% of cervical cancer cases worldwide with HPVE6 & E7 protein integration into the host’s DNA being the driving factor in these cancers. However, the sequence of these proteins vary between strains resulting in high diversity in epitope presentation in patients with different HLA haplotypes. Therefore, using one tetramer gives an inaccurate read of a patient’s immunity to the virus. In this study, we set out to develop a method to obtain a more complete read-out of patient immunity to HPV by creating a more comprehensive tetramer staining panel. We first selected 29 in-silico predicted epitopes of both E6 and E7 oncogenic proteins in the 6 most common HPV strains for 3 HLA haplotypes (HLA-A*02:01, HLA-A*11:01 and *24:02). We then utilized a non-UV “peptide exchange MHC tetramer platform” to assess the binding of these peptides to corresponding MHC groove. Since the peptide exchange and affinity of peptides to the cleft of MHC are correlated, we used an algorithm to calculate the measure of the peptide exchange rate. We found that 3 of the 29 peptides showed poor or no binding to the matched MHC, while all others had rates over 81.78%. The MHC tetramers generated by this peptide exchange platform were then used alone or as part of a pool to stain HPV specific CD8+ T cell enriched PBMCs from an HPV positive host. Our results showed no significant difference in the detection of HPV+ CD8 T cells between our generated HPV16 E7 tetramer and the commercially available tetramer. This implies that the use of this peptide exchange tetramer platform can serve as an accurate tool for measuring HPV immunity in cancer patients.
Validation of Immunogenicity Assessments for Non-Clinical Toxicology Evaluation
Authors: Hammonds Tiffany , Newsome Courtni , Freebern Wendy , Thompson Chris
Published: January 2023

To determine the immunogenic potential of biotherapeutics in nonclinical species, assays for anti-drug antibody (ADA) detection must be developed and validated before testing of samples from toxicity studies, to ensure accurate assessment of immunogenicity. ADAs can cause adverse events effecting drug exposure and efficacy complicating the interpretation of the toxicity, pharmacokinetics and pharmacodynamics data. For each compound, a validation is performed prior to study sample analysis. Parameters assessed include Inter analyst, sensitivity, accuracy, drug tolerance, precision, interference and stability of reagents and ADAs. The first step in the validation is to perform precision analysis. Precision is used to determine acceptance criteria for the assay. The inter-analyst experiment is performed by two scientists to show reproducibility. Assay acceptance criteria consists of the acceptable criteria for the pooled negative control (PNC), low quality control (LQC), high quality control (HQC) and LQC/PNC ratio to set the assay cut point. Once this criteria is determined, sensitivity, drug tolerance, interference, and stability are performed to set validation criteria for study samples, reagents, and controls. The drugs to be used on study are labeled with Biotin and Sulfotag and ADAs are assessed utilizing immuno-assay such as a standard ELISA or an electrochemiluminescent immuno-bridging assay. Once study samples are analyzed, it can be determined which subjects have an ADA response elicited by the compound and at what severity. An ADA response helps determine immunogenicity in toxicological evaluations, so it is important to assess for this response when dealing with immunotherapeutic.
Fenton-catalytic nanocomposites for reprograming macrophages towards the improved anti-biofilm activity
Authors: OJAYM ALHUSSAIN A , Yu Bing , Kim Min-Ho
Published: January 2023

Bacterial infections involving the formation of biofilms are associated with diminished bactericidal activity of macrophages, which has been shown to contribute to the persistence in chronic infection. We recently reported that S. aureus biofilm-induced impairment of bactericidal activities of macrophages are associated with attenuation of reactive oxygen species (ROS) generation and NF-κB activation. The objective of this study is to validate the use of biocompatible Fenton-catalytic nanocomposites composed of iron oxide nanoparticles (IONPs), a reducing agent (ascorbic acid), and hydrogen peroxide (H2O2) to take advantage of their ability to stimulate the generation of ROS and activation of NF-kB in macrophages by means of triggering a Fenton reaction. Using an in vitro culture model of NF-kB reporter-RAW 264.7 macrophages, we optimized the range of the concentrations of IONPs, ascorbic acid, and/or H2O2, along with their respective combinations, exhibiting an enhanced NF-kB activity and ROS generation. Based on the findings, we subsequently validated the effect of nanocomposite on the bactericidal activity of macrophages exposed to S. aureus biofilm. Our results revealed that RAW 264.7 cells treated with IONPs resulted in significant reduction in the number of surviving bacteria by ~50% and its efficacy further increased in combination with ascorbic acid and/or H2O2, which were in line with the extent of NF-kB activation and ROS generation. In summary, our study supports that harnessing the characteristic of Fenton-catalytic nanocomposite to tune macrophage polarization to exhibit a bactericidal activity may provide a new strategy for treating infectious diseases.

Funding support: Supported by NIH R01 NR 015674
Critical roles of TIGIT+ human regulatory B cells in the suppression of antibody-mediated allograft rejection
Authors: Hasan Md Mahmudul , Wang Yuanyuan , Blaji Uthra , Gu Jinghua , Nattamai Durga , Nyarige Verah , Thompson-Snipes LuAnn , Klintmalm Goran B. , O’Leary Jacqueline G. , Wang Junwen , Joo HyeMee , Oh SangKon
Published: January 2023

TIGIT (T cell immunoreceptor with Ig and ITIM domains) is a co-inhibitory receptor, highly expressed by Tregs, and contributes to the suppressive function of Tregs by limiting pro-inflammatory Th1 and Th17 cell responses. Herein, we report that subsets of human regulatory B cells (Bregs) express surface TIGIT and can thus play a critical role in immune regulation. Using data from human B cell surface arrays and RNA-seq, we identified two major TIGIT+ human Bregs subsets, exhibiting CD24hiCD27+CD39hiIgD+IgMhiCD1chi marginal zone (MZ)-like and CD24hiCD27+CD39+IgD−IgM+CD1c+ memory Bregs. We further demonstrated that TIGIT+ Bregs were also capable of suppressing immune responses by their ability to express not only IL-10 and PD-L1, but also granzyme B and TGFb1. In addition, TIGIT+ Bregs can also suppress immune responses by altering dendritic cell functions. In line with these observations, liver allograft recipients with donor-specific alloantibody (DSA) had significantly lower numbers of TIGIT+ Bregs than those without DSA. Moreover, the frequency of TIGIT+ Bregs correlated with that of CD4+CD25+CD127lo Tregs, while it inversely correlated with the frequency of follicular helper T cells. We thus concluded that TIGIT+ Bregs play critical roles in immune regulation in liver allograft recipients.
Antibody-drug conjugates targeting CD45 plus Janus kinase inhibitors permit allogeneic hematopoietic stem cell transplantation with minimal treatment-related toxicity
Authors: Persaud Stephen P , Cooper Matthew L. , Ritchey Julie K. , Rettig Michael P. , DiPersio John F.
Published: January 2023

Allogeneic hematopoietic stem cell transplantation (alloHSCT) is a potentially curative therapy for hematologic diseases. However, toxicities from chemotherapy- or irradiation-based transplant conditioning, as well as graft-versus-host disease (GvHD), limit the use of alloHSCT to the most severe diseases. Novel conditioning regimens able to achieve donor engraftment with minimal adverse effects for the patient could greatly expand the safe applicability of alloHSCT to a wider variety of diseases. Antibody-drug conjugates targeting CD45 (CD45-ADC) have been shown previously to permit engraftment in murine syngeneic HSCT models. We set out to extend this approach to the allogeneic setting, developing CD45-ADC-based regimens able to both make marrow space for donor stem cells and overcome immune barriers to their engraftment. CD45-ADC combined with CD4+ and CD8+ T cell depletion was sufficient for engraftment in MHC-matched (BALB to DBA/2) and MHC-mismatched (CB6F1 to B6) murine alloHSCT models. The selective Janus kinase inhibitor baricitinib, shown by our lab to prevent GvHD, also inhibited host-versus-graft responses and allowed allogeneic engraftment when combined with CD45-ADC. In our alloHSCT models, up to 90% donor chimerism was achieved in over 70% of mice treated with CD45-ADC plus baricitinib. Finally, unlike mice conditioned with irradiation, mice treated with CD45-ADC did not develop GvHD when infused with allogeneic splenocytes. In conclusion, CD45-ADC plus baricitinib provides a safe, effective approach to alloHSCT conditioning in mice. These studies demonstrate the promise of immunotherapeutic strategies to achieve transplant tolerance while minimizing treatment-related morbidity and mortality.
Galactin-1 favors engraftment in sickle cell disease patients after haploidentical hematopoietic stem cell transplantation and in murine MHC-mismatched allotransplantation
Authors: Shaikh Ahmad , Gangaplara Arunakumar , Olkhanud Purevdorj , Kone Abdoul , Almengo Katherine , Patel Sajni , Saxena Ankit , Lopez-Ocasio Maria , Gucek Marjan , McCoy Philip , Fitzhugh Courtney D
Published: January 2023

Haploidentical hematopoietic stem cell transplantation (haplo-HSCT) is associated with increased risk of allograft rejection. Understanding the mechanisms of graft rejection and tolerance induction are critical for improving HSCT outcome. Recently, we performed a proteomic analysis using plasma samples from sickle cell disease (SCD) patients who underwent haplo-HSCT and found that galectin-1 (Gal-1) levels were significantly higher in engrafted patients. Here, we studied major histocompatibility complex (MHC)-mismatched allo-HSCT murine model to better understand the role of Gal-1 in immune tolerance. Transplanted mice were classified into either rejected (cyclophosphamide/sirolimus alone) or engrafted (cyclophosphamide and sirolimus) based on our previous findings. In rejected mice we noted significantly higher frequencies of CD4+T, CD8+T, NK cells, IFN-γ TNF-α producing CD4+T cells, and also IFN-γ producing DCs and macrophages. Importantly, in engrafted mice we found significantly increased frequencies of Tregs and IL-10 producing CD4+T and Tr1 cells, and the proportion of these cells expressing Gal-1 was significantly higher. Further, we detected a significant increase in Gal-1 levels in plasma of engrafted mice, and this corroborated with our data from engrafted patients. Here, we have shown for the first time that not only Gal-1 favors engraftment in SCD patients who underwent haplo-HSCT, but Gal-1 also contributes to HSC engraftment in our MHC-mismatched murine model. Taken together, our results demonstrate that Gal-1 expressing Tregs and Tr1 cells may play an essential role in inducing immune tolerance and stable mixed chimerism after HSCT.
The Role of PPARd in Alloreactive CD8 T cell Metabolism
Authors: Brown Rebecca Audrey , Dobbs Andrea , Denby Elisabeth , Waltz Gail , Rajeevan Arathi , Byersdorfer Craig A
Published: January 2023

Graft-versus-host disease (GVHD) is the most common cause of non-relapse mortality following allogeneic hematopoietic stem cell transplantation. Using a pre-clinical model of GVHD, previous work has found that CD8 T cells recovered on day 7 post-transplant upregulate the transcription factor, peroxisome proliferator activated receptor delta (PPARd). While PPARd has been shown to be a key regulator of fatty acid oxidation (FAO) in other tissues, it’s role in T cells is not well studied. We hypothesize that PPARd drives FAO in alloreactive CD8 T cells and that CD8 T cells lacking PPARd will be unable to oxidize fat and thus unable to cause GVHD. To investigate this hypothesis, ex vivo FAO was measured in day 7 WT versus PPARd KO CD8 T cells by quantitating conversion of 3H-palmitate to 3H2O. Production of 3H2O decreased by 50% in KO CD8 T cells, suggesting an inability to fully oxidize fat. To understand the mechanism underlying this decrease, we performed RNA sequencing and identified thirty genes that were differentially expressed in WT versus KO CD8s including 8 genes related to FAO. Strikingly, despite a decreased ability to oxidize fat, PPARd KO CD8 T cells were recovered in equal numbers on day 7 post transplantation, suggesting that CD8 T cells lacking PPARd adopt alternative metabolic pathways to generate sufficient energy for in vivo proliferation and activation. Ultimately, our analyses indicate that PPARd is required for FAO in alloreactive CD8 T cells, but this process is dispensable for short term proliferation and/or survival of CD8 T cells during GVHD. Future directions will identify compensatory metabolic pathways utilized by PPARd KO T cells through a combination of in vitro nutrient drop out experiments and metabolomic flux analyses.
PD-1 blockade later but not at post-transplant attenuates chronic graft-versus-host disease
Authors: Rhee Stephanie , Aguilar Ethan George , Paz Katelyn , Flynn Ryan , Kuchroo Juhi , Sage Peter , Sharpe Arlene H , Freeman Gordon J , Ahmed Rafi , Blazar Bruce R
Published: January 2023

PD-1 and its ligand PD-L1 are widely appreciated as targets of immune checkpoint blockade. We sought to investigate the role of the PD-1 pathway in the context of chronic graft-versus-host disease (cGVHD). To induce cGVHD, mice are conditioned with Cytoxan and radiation prior to MHC disparate bone marrow (BM) + T cells (70k) and develop multi-organ symptoms with bronchiolitis obliterans lung manifestations. CGHVD mice have high survival rates past 50 days, with <15% weight loss over this period. Anti-PD-1 mAb given to mice with established cGVHD resulted in disease attenuation assessed by significantly improved pulmonary function tests compared to irrelevant mAb controls. PD-1 blockade significantly reduced germinal center (GC)-promoting GC B and T follicular helper (Tfh) cell and increased GC-suppressing T-follicular regulatory (Tfr) cell frequencies. However, in studies with mice given five percent higher T cell dose, resulting in T cell rather than GC-mediated disease, anti-PD-1 mAb dramatically worsened cGVHD severity. To assess GC cell-specific effects, recipients were given PD-1-deficient cells in graft compartments. Mice receiving PD-1 KO T cells predictably exhibited marked escalation of cGVHD severity and lethality, while mice receiving PD-1 KO B cells showed diminished disease, with significant improvement in pulmonary function. Co-culture of isolated B, Tfh and Tfr cells from NP-OVA immunized mice to determine effects of individual GC cell populations on Ig class switching demonstrated significant increase in Tfr suppressive capacity with addition of anti-PD-1 mAb. Together, these data provide evidence for PD-1 support of the GC reaction and identifies the PD-1/PD-L1 axis as a potential target for cGVHD therapy.
Conventional type 2 dendritic cells and natural killer cells mediate control of early metastatic seeding
Authors: Weizman Orr-El , Krykbaeva Irina , Bosenburg Marcus , Iwasaki Akiko
Published: January 2023

The multistep progression of transformed primary tumors to metastatic dissemination and seeding of distant site is a lethal determinant of disease. Although the role of immune cells and immune sensors in promoting or restricting metastatic growth is well described, whether immune cells are capable of detecting and controlling metastatic cells during peripheral tissue seeding is unknown. Here we examine if local immune cells can confer early anti-metastatic control in the lung using intracardiac injection to mimic metastatic spread. Using a syngeneic mouse model of YUMMER1.7 melanoma expressing GFP and luciferase (YMR1.7-G/L), we demonstrate that lung resident conventional type 2 dendritic cells (cDC2) and extravascular NK cells confer host anti-tumor immunity during the first three days following intracardiac injection. Rapid production of IFN-γ by NK cells serves an essential role to limit metastatic burden. Using mice deficient in innate sensors and signaling pathways, we demonstrate that the transcription factors IRF3 and IRF7 are required for early metastatic control in a type I IFN-independent manner. In contrast, ablation of adaptive immune cells had no effect to initial metastatic burden. Collectively our results highlight a novel cDC2 - NK cell axis that orchestrates a non-redundant innate immune response program to limit initial metastatic burden in the lung.
Response to anti-PD-1 and anti-LAG-3 immune checkpoint blockade (ICB) is associated with induction of pro-inflammatory Tregs
Authors: Sturgill Elizabeth Rowland , Mick Courtney , Kaufmann Johanna K , Redmond William L
Published: January 2023

T cells upregulate the immune checkpoint, LAG-3, which is associated with reduced T cell effector function following repeated antigen exposure. Further, high LAG-3 expression on Tregs has been reported in patients with varying cancer types; thus, we hypothesized that the combination of aLAG-3 with another common ICB, aPD-1, would synergize to potently reinvigorate T cells to promote tumor regression and increase survival.

Combined aPD-1/aLAG-3 immunotherapy significantly improved the survival of CT26 (BALB/c; colon carcinoma)and MCA-205 (C57BL/6; sarcoma) tumor-bearing mice compared to monotherapy. In-depth analysis of tumor-infiltrating lymphocytes (TIL) revealed that aPD-1/aLAG-3 therapy significantly increased the overall percentage, cytotoxicity (granzyme A), and cytokine production (TNF-a and IFN-g) of CD8+ TIL, and increased CD4+ Teff/Treg ratios compared to aPD-1 or aLAG-3 alone. These effects were enriched in responders to aPD-1/aLAG-3 therapy, which were designated as those exhibiting decreased tumor size on the day of harvest compared to maximum tumor growth post-implant. Surprisingly, Tregs isolated from tumors responding to aPD-1/aLAG-3 displayed increased proliferation, secretion of pro-inflammatory cytokines (TNF-a and IFN-g), and increased LAG-3 expression. In summary, these data suggest that aPD-1/aLAG-3 immunotherapy increased CD8+ TIL exhibiting enhanced effector function, increased CD4+ Teff/Treg ratios, and induced Treg pro-inflammatory responses. Together, these positive immunological changes led to a more immune stimulatory tumor microenvironment capable of supporting tumor regression and significantly improved tumor-free survival.
Modulation of piglet’s immune system development with fecal microbial transplantation
Authors: Blais Mylene , Verso Luca Lo , Thibodeau Alexandre , Harel Josée , Fravalo Philippe , Lessard Martin
Published: January 2023

The aim of the study was to investigate fecal microbial transplantation (FMT) as a novel strategy to modulate the postnatal development of porcine immune system and the establishment of the intestinal microbiota in newborn piglets. Ten litters were used for the experiment. At birth, four piglets were identified in each litter and randomly assigned to the control (CTRL) and the FMT groups. At 3,4,8,9, and 10 days of age, piglets in the FMT group were orally administered with FMT material suspended in 10% bovine colostrum solution. The FMT was prepared with an equal mixture of fecal material harvested from healthy suckling and fully weaned piglets. Control group was inoculated with the vehicle solution. Animals were then euthanized at d 22 to identify the leukocyte subsets in mesenteric lymph nodes (MLN) and blood by flow cytometry and to characterize the microbiota from ileum, caecum and colon by 16S rRNA amplicon sequencing. Results showed that FMT reduced the overall number of T lymphocytes (CD3+) both in blood and MLN (P<0.05). Blood percentages of Th lymphocytes (CD3+CD4+CD8α−) were also reduced in piglets receiving FMT (P<0.05), as well as γδ T lymphocyte (CD3+γδ+) and monocyte/macrophage (SWC3+CD14+) concentrations in the MLN (P<0.05). On the other hand, FMT increased the blood percentage of NK cells (CD3−CD4−CD8α+CD16+) (P<0.01). Results obtained from the microflora analysis confirmed that FMT administration affected piglet’s colon microbiota. In conclusion, although it had a mild impact on hindgut microflora, we observed that FMT has the potential to modulate the immune system development in newborn piglets. Further studies are necessary to fully understand the long-lasting effects of FMT on animal health.
Identifying autoantibody biomarkers for Addison’s disease in dogs
Authors: Treeful Amy , Friedenberg Steven
Published: January 2023

Addison’s disease is a life-threatening endocrinopathy caused by the immune-mediated destruction of the adrenal cortex. Only dogs and humans develop the disease naturally. In humans, autoantibodies against the adrenocortical steroidogenic enzyme 21-hydroxylase can predict patients at high risk of developing the disease. No comparable biomarker exists for canine Addison’s disease. We hypothesized that, similar to humans, newly diagnosed dogs with Addison’s disease will have consistently detectable autoantibodies against adrenocortical steroidogenic enzymes. Serum was collected from dogs in four study cohorts (diagnosed within 30 days and healthy age-matched controls; long-term cases and healthy age-matched controls) in three dog breeds with a high prevalence of Addison’s disease. These samples were evaluated for anti-adrenocortical autoantibodies using both immunofluorescence assays and Western blotting. Initial results from immunofluorescence assays on bovine adrenal sections probed with serum IgGs from the recently diagnosed cohort showed positive staining in the adrenal cortex and negative staining for the unaffected cohorts. Additionally, preliminary data from Western blots on canine adrenal homogenates probed with serum IgGs identified a band at ~42 kD unique to the recently diagnosed group from each breed. These findings have been replicated using five canine adrenal homogenates, as well as bovine and porcine homogenates. Our results suggest that dogs with a new diagnosis of Addison’s disease may have autoantibodies targeting a ~42 kD adrenal protein. Ongoing work includes testing reproducibility and identifying the specific antigenic target using 2D gels with spot cutting followed by LC-MS/MS.