bluuebunny/crossref_metadata_2025 huggingface dataset:
Deficiency of TAM receptor family increases γδT cell population by promoting chemokine-induced gut homing
Authors: Yi Jawoon , Choi Harim , Kim Su-Man , Kang Hyung-Sik
Published: January 2023

Intestinal intraepithelial lymphocytes (IELs) are a various community of lymphoid cells located in between the intestinal epithelial cells (IECs) that configure the intestinal mucosal barrier. The epithelial γδT cells act as a major T cell population in epithelia, which is support in tissue homeostasis and repair. However, we found that the γδT cell population was strikingly increased in IELs in TAM receptor-deficient mice. Based on these data, we tested whether this receptor acts as a crucial regulator, which may generate better homing to the intestinal epithelium. We also tested that adoptive transfer of γδT cells to show γδT cell homing to the intestinal epithelium. Our study shows that a key factor in γδT cell homing into the intestinal epithelium, to maintain homeostasis.
Role for B-cell lymphoma 6 in intestinal innate lymphoid cell homeostasis and inflammatory disease
Authors: Ohayon David E , Seelamneni Harsha , Chaimowitz Natalia S , Cazares Tareian , Waddell Amanda , Huber Alex , Kotey Amos , Rosen Michael J , Miraldi Emily R , Waggoner Stephen N
Published: January 2023

Group 3 innate lymphoid cells (ILC3) are critical for intestinal homeostasis. The functional activity of ILC3 must be tightly regulated as cytokines (e.g. IL-17 and IL-22) produced by these cells are critical for antimicrobial immune defense but can also contribute to intestinal disease when produced in excess. This regulation is achieved via incompletely defined transcriptional networks that include B-cell lymphoma 6 (BCL6), which we recently demonstrated is critical for maintaining ILC1- and ILC3-specific gene expression programs during homeostasis (Pokrovskii et al 2019). We now show that Bcl6-deficient small intestine lamina propria ILC3 exhibit elevated expression of the transcription factors retinoic acid receptor-related orphan nuclear receptor alpha (RORα) and RORγt. These factors are known to support ILC3 cytokine expression, potentially explaining elevated Il17a and Il17f expression by Bcl6-deficient ILC3. Of note, expression of Il22ra2 (IL-22 binding) was also markedly increased in these ILC3, likely limiting bioavailability of IL-22. Accordingly, these mice exhibit marked reduction in IL-22-dependent expression of epithelium-associated anti-microbial peptides in the terminal ileum. Of note, Bcl6ΔILC mice exhibited augmented susceptibility to dextran sodium sulfate-induced inflammatory bowel disease. Thus, BCL6 expression in ILC3-intrinsic BCL6 expression is important for mucosal immune defense and limiting intestinal inflammation.
The importance of distinguishing cell-cell complexes from singlets in flow cytometry and single-cell sorting: a case for an image-based cell sorter
Authors: Burel Julie G , Pomaznoy Mikhail , Arlehamn Cecilia S Lindestam , Seumois Gregory , Vijayanand Pandurangan , Sette Alessandro , Peters Bjoern
Published: January 2023

Our recent work highlights that care needs to be taken when interpreting single-cell data originating from flow cytometry acquisition or cell sorting: We found that doublets involving T cells bound to another immune cell are indeed very often present in the live singlet gate of human samples acquired by flow cytometry, in PBMC as well as in tissues. This hidden ‘contamination’ of cell-cell complexes generates atypical gene signatures of mixed cell lineage in what is assumed to be single cells, which can lead to data misinterpretation, such as the description of novel immune cell types with dual lineage properties. Based on the example of T cell-monocyte complexes, we investigated experimental and data analysis strategies to help distinguishing between singlets and cell-cell complexes in non-imaging flow cytometry and single-cell sorting. We found robust molecular signatures in both T cell-monocyte and T cell-B cell complexes that can distinguish them from singlets at both protein and mRNA level. Imaging flow cytometry with appropriate gating strategy (matching the one used in cell sorting) and direct microscopy imaging after cell sorting were the two preferred methods to validate the presence of cell-cell complexes in suspicious dual-expressing cells. In parallel, we also investigated the molecular profile of T cells and monocytes present in a doublet and found that their association is not random, but enriched for LFA1/ICAM1 interactions and gene expression profiles associated with disease-specific immune responses. Taken together, these data highlight the importance of systematically investigating for the presence of cell-cell complexes in single-cell data derived from non-imaging flow cytometry.
Adaptive optics permits label-free intravital imaging of tissue resident and infiltrating immune cells in the mouse retina
Authors: Chu Colin J , Joseph Aby , Feng Guanping , Dholakia Kosha , Schallek Jesse B
Published: January 2023

The eye is ideally suited for unperturbed longitudinal intravital imaging of immune responses. No confounding inflammatory surgery, externalization or depressurization is required for access as it is uniquely transparent. Directly imaging immune cells in vivo would greatly advance our biological understanding but it is not currently possible in man, and only feasible in mice using exogenous fluorescent labeling or transgenic modification which can result in physiologically unrepresentative results.

We discover both tissue resident and infiltrating immune cells can be visualized in the retina using a custom adaptive optics scanning light ophthalmoscope (AOSLO) employing phase contrast detection of reflected 796nm light. This is analogous to scatter characteristics observed in flow cytometry, with motile cells visible within the laminated structure of the retina.

Anesthetized mice underwent intraocular injection of LPS to induce acute inflammation before repeated imaging at the same anatomical location across intervals from 6 hours to 2 months. Leukocyte rolling within post-capillary venules and tissue infiltration was visible without exogenous contrast. We confirmed 83% of this population were neutrophils by co-localization following tail vein injection of Alexa Fluor-647 anti-Ly6G antibody. Tracking the same location revealed reduced motility (24 vs 6 hours) despite cellular persistence preceding resolution of inflammation. Microglia could be imaged including their highly motile processes, confirmed using healthy eyes from Cx3cr1GFP/+ reporter mice.

This is the first report of label-free immune cell imaging within the mouse retina and the approach could feasibly translate to existing human AOSLO platforms.
ctDNA for early cancer diagnosis in Pacific Communities
Authors: Sika-Paotonu Dianne , Anitelea Toni , Faatoese Adam , Laing Bridie , Puni Ron , Vaipuna Tevita , Guilford Parry
Published: January 2023

Circulating tumour DNA (ctDNA) refers to small fragments of tumour DNA found in the blood circulation, recognised by the presence of mutations in cancer genes. These DNA fragments can be isolated and quantified to obtain data about the cancer’s size and progression. Since gaining access to and obtaining biopsy samples from solid cancers in people is not always possible, the possibility of utilizing a simple blood sample to allow detection and monitoring of cancer growth is highly desirable. It is anticipated this simple method of detecting and monitoring cancer will enhance the precision of cancer diagnostics and surveillance and also lead to improved cancer health outcomes for Pacific Peoples. The purpose of this work was to support engagement with Pacific communities and share, and discuss and explore current research efforts, that included the potential utilization of ctDNA to improve the diagnosis, care and health outcomes of Pacific Peoples. A Pacific Fono (gathering) was held in the Hutt Valley region to promote appropriate knowledge translation activity, engagement and meaningful dialogue between researchers and the Pacific communities regarding current research works and findings of direct relevance and significance to Pacific communities. The Pacific community response to this research outreach effort was overwhelmingly positive and encouraging. Discussions were respectful and informative with shared information being exchanged between researchers and those in attendance. Feedback included expressed support for regarding the ctDNA research and for other areas of research focus that would be of benefit to Pacific communities. Requests from Pacific community members were also made for future similar events to be held.
ViaKrome Fixable Viability Dyes: A new approach to dead cell discrimination for intracellular multicolor flow cytometry application
Authors: Dupas Nathalie , Dempsey Nancy , Bowers Rita , Burgaud Mégane , Miloud Tewfik
Published: January 2023

The ability to stain live cells with a viability dye and preserve that staining pattern after fixation is critical for intracellular immunophenotyping by flow cytometry. Excluding dead cells from the data allows cleaner separation and identification of cell populations. All commercially available viability dyes are based on NHS ester activated functional groups crosslinking with primary amines on proteins. These functional groups are susceptible to hydrolysis, necessitating the use of non-aqueous solvents, and additional reagent handling steps to protection from water vapor, such as aliquoting the stock solution and reducing freeze-thaw cycles. Here, we propose a new thiol reactive fluorescent dye that covalently binds free thiols present on cellular proteins at a neutral pH. Live cells, impermeable to the FVD, will be weakly stained by the covalent binding of proteins express on the cell surface while dead cells whose membranes have lost structural integrity, will be strongly stained by the covalent binding of intracellular and membrane expressed proteins.

Contrary to current dyes, the ViaKrome dyes are soluble in aqueous solution and do not require DMSO for re-suspension. Hence, we show that the stability of dye ensure robust and reproducible experimental data.

To further increase the ease of use, this new thiol based chemistry is available in a range of dyes excited by the common lasers used in flow cytometry. This choice provides flexibility for multicolor panel design. The compatibility of the ViaKrome dyes, i.e ViaKrome 405, ViaKrome 561, ViaKrome 638 and ViaKrome 808 will be presented in different workflow set-up such as 18-colors immunphenotyping panels and functional assays using PBMCs and whole blood.
Graft vs. host disease leads to elimination of tissue-resident innate lymphoid cells following experimental and clinical allogeneic transplantation
Authors: Vinci Paola , Garcia-Martinez Elena , O’Connor Margaret H. , Egorova Anastasiya , Kuttiyara Jason , Arnhold Viktor , Ponce Doris M. , Hanash Alan M.
Published: January 2023

Innate lymphoid cells (ILCs) are associated with protective mucosal responses after allogeneic bone marrow transplantation (allo-BMT). However, the specific in vivo roles of ILCs after BMT remain unclear. We thus sought to characterize the kinetics and significance of ILC reconstitution after allo-BMT.

In a sex-mismatched acute graft-vs-host-disease (GVHD) model, depletion of recipient ILCs induced significantly worse systemic signs of GVHD, as well as increased thymic injury and intestinal pathology, indicating a contributory role for ILCs in tissue protection post-BMT.

Analysis of ILCs in distinct GVHD target organs after T-cell-depleted BMT demonstrated a high frequency of NK cells and ILC1s in the liver, a higher proportion of ILC2s in lungs, and a higher proportion of ILC3s in the small intestine post-transplant in the absence of GVHD. For all tissues analyzed, the majority of ILCs were host-derived. In contrast, in the presence of GVHD, all ILC subsets were significantly reduced in all tissues analyzed. Furthermore, ILC precursors were significantly reduced in the bone marrow of mice with GVHD, suggesting that alloreactive immunity can impair ILC reconstitution by targeting both mature ILCs and their developmental niche.

To examine the clinical relevance of these findings, human ILCs were analyzed by FACS of lamina propria lymphocytes isolated from patient duodenal biopsy specimens obtained to evaluate symptoms of acute GI GVHD. Consistent with experimental findings, duodenal ILC frequencies were significantly lower in patients with histologic evidence of GVHD.

In conclusion, tissue-resident ILCs contribute to tissue protection after allo-BMT, but GVHD leads to ILC elimination and impairs their reconstitution.
Ablation of IRF4 in donor T cells eliminates GVHD while partially preserving GVT in experimental bone marrow transplantation
Authors: Guo Yang , Chen Wenhao
Published: January 2023

Objective

We discovered that the transcription factor IRF4 governs T cell function in heart transplantation (Immunity 47:1114). Here, using a bone marrow (BM) transplantation model, we studied the role of IRF4 in regulating T cell function in graft-vs-host disease (GVHD) and graft-vs-tumor (GVT).

Methods

9 Gy irradiated Balb/c recipients were i.v. injected: 1) with 5 × 106 T cell-depleted CD45.1+ B6 BM cells; 2) with or without 0.1 × 106 Balb/c A20-luciferase lymphoma cells; and 3) with or without 1 × 106 CD45.2+ wild type (WT), Irf4−/−, or Irf4−/−Foxp3sf/Y (Treg-deficient Irf4−/−) B6 donor T cells. GVHD, GVT, survival, and donor T cell states were determined.

Results

In the absence of A20 infusion, all recipients without T cell transfer (n=10), or transferred with Irf4−/− (n=10) or Irf4−/−Foxp3sf/Y (n=5) donor T cells survived long-term (>60 days post BM transplantation). Recipients transferred with WT donor T cells had developed lethal GVHD [mean survival time (MST) = 9.5±2 days; n =8]. Among A20 infused groups, recipients without T cell transfer died due to lymphoma progression (MST=22±2.80 days; n =15). Transfer of Irf4−/− donor T cells led to a moderate GVT effect and significantly prolonged survival (MST=33.4 ± 11.22 days; n =15; p<0.001 vs no T cell). Transferred WT donor T cells exerted potent GVT activity but caused lethal GVHD (12.56 ± 6.02 days; n = 18). Mechanistically, Irf4−/− donor T cells developed into effector T cells, but barely infiltrated into GVHD tissues and were prone to apoptosis.

Conclusions

IRF4 ablation in donor T cells eliminates GVHD in a Treg independent manner while partially preserving GVT. This reveals the possibility to separate GVHD and GVT effects by modulating transcription factors in T cells.
PRMT5 promotes STAT-1 mediated T cell inflammatory response and is a therapeutic target for acute graft-versus-host disease
Authors: Snyder Katiri Jenna , Zitzer Nina C. , Gao Yandi , Choe Hannah K. , Sell Natalie E. , Neidemire-Colley Lotus , Devine Raymond , Behbehani Gregory , Pietrzak Maciej , Vaddi Kris , Baiocchi Robert , Ranganathan Parvathi
Published: January 2023

Introduction

T cell mediated acute graft-versus-host disease (aGVHD) is the main cause of non-relapse mortality in allogeneic hematopoietic cell transplant recipients. Protein arginine methyltransferase 5 (PRMT5) is a post-translational modifier and promotes activation of memory TH cells. We investigate mechanisms by which PRMT5 regulates T cell function and propose PRMT5 inhibition as a therapeutic strategy for aGVHD.

Materials and Methods

PRMT5 expression and function was evaluated in T cells of healthy and aGVHD mice and humans. We assessed T cell proliferation and effector function, using C220, a novel PRMT5 inhibitor. We tested effects of PRMT5 inhibition using in vivo mouse models of aGVHD, where mice received T cell depleted bone marrow + allogeneic splenocytes. In graft-versus-leukemia (GVL) experiments, P815 tumor cells were administered. Mice were treated with C220 or vehicle and monitored for survival and clinical aGVHD scores.

Results

PRMT5 expression and function is upregulated in T cells of mice and humans with aGVHD. Inhibition of PRMT5 reduces T cell proliferation, perturbs cell cycle and downregulates STAT-1 mediated interferon response. Administration of C220 improved survival in aGVHD mouse models and maintained tumor specific CTL response contributing to retention of Graft versus leukemia (GVL) effect.

Conclusions

Inhibition of PRMT5 using C220, down-regulates T cell proliferative and effector response, induces cell-cycle arrest, reduces interferon response and perturbs signaling pathways. C220 shows potent biological activity in vivo by reducing aGVHD clinical severity and prolonging survival in mouse models without compromising GVL. Therefore, PRMT5 is a novel and druggable target for aGVHD.
Recipient vitamin A levels influence the risk of graft-versus-host disease after experimental allogeneic stem cell transplantation
Authors: Pan Pan , Taylor Brian M , Zhu Haojie , Zhou Dian , Chen Xiao
Published: January 2023

Graft-versus-host disease (GVHD) remains a major complication after allogeneic hematopoietic stem cell transplantation (HSCT). Retinoic acid (RA), the active metabolite of vitamin A, has been implicated in GVHD pathogenesis. However, whether recipient vitamin A levels modulate GVHD risk remains controversial based upon recent clinical data. To gain insights into this clinically relevant question, Balb/c mice were kept on diets containing normal (VAN) or excessive (VAH) amounts of vitamin A. Lethally irradiated VAN and VAH mice were then transplanted with bone marrow and splenic T cells from MHC-mismatched C57BL/6 mice to induce GVHD. There was no significant difference in the expansion of donor CD4 and CD8 T cells in the spleen and CD4 T cells in the mesenteric lymph nodes between the two groups early after HSCT. There was also no significant difference in the expression of gut-homing molecule α4β7 on donor T cells in VAN versus VAH mice. However, there was a significant reduction in serum LPS levels in VAH mice compared with those of VAN mice in later stage of GVHD development, indicating a better maintained intestinal barrier integrity. Gene expression of proinflammatory cytokines including IFN-γ and TNF-α was also significantly decreased in the colon tissues of VAH mice compared with that of VAN mice. Despite reduced intestinal inflammation, VAH mice unexpectedly died significantly faster than VAN mice after transplantation. Collectively, our studies indicated that higher vitamin A levels are associated with accelerated GVHD-associated mortality after experimental HSCT. Therefore, some caution should be exercised when considering high dose vitamin A supplementation for GVHD prevention as proposed in recent clinical trials.
Intestinal dysbiosis predicts lower gastrointestinal tract acute graft-versus-host (aGVHD) disease development
Authors: da Silva Marina Burgos , Ponce Doris M. , Gomes Antonio L.C. , Moore Gillian , Slingerland John B. , Pamer Eric , Taur Ying , Peled Jonathan U. , Van Den Brink Marcel R.M.
Published: January 2023

Introduction

aGVHD is a major complication after hematopoietic cell transplantation (HCT) and is driven by alloimmune targeting of intestine, liver, and skin. As aGVHD has been linked to intestinal dysbiosis and the majority of the intestinal microbiota biomass resides within the colon, we hypothesized that certain microbiota features confer protection to the lower GI tract.

Methods

We evaluated 16S stool profiles of 849 samples from 215 recipients of unmodified allo-HCT. Patients were classified in four groups according to aGVHD organ involvement: upper GI only (UGI, n =53), lower GI with/without upper GI (LGI, n=56), no GI involvement (non-GI, n=30) and no GVHD (n=76). Samples were grouped into pre-onset (day −20 to 0) and post-onset (day 1 to 20) aGVHD.

Results

Pre-onset, LGI patients showed a decreased strict anaerobes to facultative anaerobes (A/F) ratio vs. non-GI (p=0.036) and a low A/F ratio was associated with reduced overall survival (p=0.004) from aGVHD onset. After aGVHD onset, LGI patients had reduced microbial alpha diversity, abundance of predicted butyrate-producing (BP) bacteria and A/F ratio vs. non-GI patients (p=0.017, p=0.003 and p=0.020, respectively). LGI patients also had less BP bacteria, members of genus Blautia, and lower A/F ratio (p=0.0.017, p=0.008 and p=0.048 respectively) vs. UGI patients.

Conclusions

LGI patients had distinct microbial features consistent with dysbiosis, when compared to UGI and non-GI patients. Importantly, we observed microbiota disruption in the 20 days preceding aGVHD onset, suggesting a predictive marker of LGI aGVHD and an association with overall survival, which have potential practical implications for GVHD risk stratification and therapeutic interventions.
Tc17 cells cause gut graft-versus-host disease through induction of dysbiosis and depletion of CX3CR1hi phagocytes
Authors: Song Qingxiao , Wang Xiaoning , Wu Xiwei , Qin Hanjun , Zhao Dongchang , Jenq Robert R , Van Den Brink Marcel RM , Riggs Arthur D. , Martin Paul J , Chen Yuanzhong , Zeng Defu
Published: January 2023

Th/Tc17 but not Th/Tc1 cells are resistant to steroid-induced apoptosis or inhibition of cytokine production, but the role of Th/Tc17 in pathogenesis of steroid-resistant intestinal graft versus host disease (SR-Gut-GVHD) remains controversial. Here, we show a reduction of IFN-g-producing Th/Tc1 cells and expansions of IL-22-producing Th17 and especially Tc17 cells in the gut tissues of mice with SR-Gut-GVHD. IL-22 from Tc17-dervied from IFN-g−/− donor CD8+ T cells augments host-tissue production of RegIIIg, leading to dysbiosis. IL-22 also augments host tissue production of IL-18 that sustains Tc17 expansion. In the absence of donor-derived IFN-g, host-tissue PD-L1 depletes donor-derived CX3CR1hi intestinal mononuclear phagocytes (MNP), leading to increased bacterial translocation and loss of IL-18 binding protein that neutralizes IL-18. Depletion of CX3CR1hi MNP in the colon is also observed in mice with SR-Gut-GVHD. Taken together, our results indicate that depletion of donor-derived CX3CR1hi MNP in the colon plays an important role in Tc17-mediated gut GVHD pathogenesis. These results call for caution in clinical testing of IL-22 agonists or IFN-g antagonist in patients with SR-Gut-GVHD, and they offer a new avenue toward understanding mechanisms that account for SR-Gut-GVHD.
Long term time-course monitoring of NK cell-mediated ADCC using the Celigo Image Cytometer
Authors: Chan Leo , Wucherpfennig Kai W , De Andrade Lucas
Published: January 2023

Antibody-dependent cell-mediated cytotoxicity (ADCC) assay has been widely performed for immuno-oncological research. Traditionally, ADCC assays are conducted by measuring the amount of released Chromium, calcein AM, or Lactate dehydrogenase (LDH) molecules after the target cancer cells are killed by effector/antibody pair. These methods can be inaccurate due to indirect measurement of supernatant at the end point of 4 hour, however, there is a need to characterize the effects of antibody-dependent cytotoxicity for longer than 72 hours. In this work, we demonstrated the time-course monitoring of NK cell-mediated ADCC of A375 cells in the presence or absence of IL2 and antibodies for 76 hours. First, A375-ZsGreen cells were seeded into a 96-well plate with and without target antibodies. Next, NK cells with and without IL2 were added to the wells at 10:1, 5:1, 2:1, and 1:1 E:T ratios. The co-culture was allowed to incubate for 76 hours and the image cytometer was used to scan and analyze at times from 0 to 76 hours. The software was able to count individual cells as well as confluence % at each time point by segmenting highly fluorescent objects in the images. We were able to show time-dependent ADCC killing with decrease in ZsGreen positive cells as the read-out. In addition, the uniformity of cell killing can be observed in the Celigo obtained whole well images. One of the most important findings was that there was regrowth of target cells after the initial 30 hours of cell killing, indicating the activated NK cells without antibodies did not eradicate the cancer cells. Therefore, the ability to perform long term time-course monitoring of ADCC is highly important to better characterize the effects of target antibodies on cell killing function.
Immune pressure against ovarian cancer depends on antigen-specific CD69+CD103+TRM T cells
Authors: Galindo Carmen Maria Anadon , Yu Xiaoqing , Gonzalez Ricardo A Chaurio , Payne Kyle K , Biswas Subir , Costich Tara Lee , Perez-Sanz Jairo , Mandal Gunjan , Martin Alexandra L , Harro Carly , Powell Chase , Mine Jessica A , Wang Zhitao , Conejo-Garcia Jose
Published: January 2023

The relationship between stem-like, tissue resident memory (TRM) and exhausted T cells at tumor beds is incompletely understood. We found that more than 50% of the CD8+ T cells in human ovarian carcinomas are TRM cells. RNA seq of TRM and their re-circulating counterparts infiltrating multiple human ovarian carcinomas showed a very distinctive phenotype, characterized by co-upregulation of effector and exhaustion markers, along with increased clonality and marked proliferative and lipid metabolism signatures. Interestingly, both populations showed very little overlap in TCR repertoire. Single cell analysis of TRM population showed that this is a heterogeneous population composed of different clusters defined by gene expression and TCR repertoire. A distinctive cluster shows higher expression of cytotoxic mediators and exhaustion markers, along with higher clonality and higher proliferation rate, with a TCR repertoire shared by CD103+TCF7+ stem-like cells. Single cell ATACseq showed that TRM and their counterpart have different chromatin structure and, within both populations also appear variables epigenetic landscapes. In addition, the presence of TRM is correlated with better prognosis, while tumor antigen-specific TRM T cells transferred into syngeneic tumor-bearing mice were more effective at delaying tumor growth than their tumor antigen-specific re-circulating counterparts. Together, our data indicate that productive immune pressure against malignant progression depends on a subset of CD8+ stem-like T cells differentiating into a narrow cluster of TRM T cells of ~300 TCR clones, which represent true tumor antigen-specific effector lymphocytes.
Immunogenicity and the CXCL12-coat control T cell accumulation in murine pancreatic cancer
Authors: Yan Ran , Wang Zhikai , Gao Ya , Janowitz Tobias , Fearon Douglas T
Published: January 2023

Improving the efficacy of immunotherapies in pancreatic cancer can increase patient survival rate. Microsatellite-stable pancreatic adenocarcinoma (PDA) exclude T cells and resist immunotherapy. This may occur by cancer cells coating themselves with CXCL12, a chemokine produced by cancer associated fibroblasts (CAFs). However, PDA with microsatellite instability (MSI-H) is infiltrated with T cells and responsive to PD-1 checkpoint inhibition. We found that human MSI-H pancreatic cancer cells display the CXCL12-coat, suggesting additional factors control pancreatic cancer immune accessibility. We hypothesized that expression of neoantigens by MSI-H PDA is an independent variable that overcomes T cell exclusion mediated by the CXCL12-coat. To test this, we established a mouse hepatic PDA metastases model in which PDA cells express doxycycline-induced ovalbumin (OVA), and adoptively transferred OVA-specific OT-I CD8+ T cells report intra-tumoral accumulation of T cells. Inducing OVA expression in established metastases increases intra-tumoral accumulation and activation of OT-I cells, despite the presence of the CXCL12-coat. We are currently investigating whether high- and low-affinity antigen-expressing cancers are subject to T cell mediated elimination in the presence and absence of the CXCL12-coat, respectively. The CXCL12-coat on PDA cells and the expression of neoantigens can independently control immune response. As the formation of the CXCL12-coat is facilitated by the Transglutaminase-2 (TGM2) and can be inhibited by TGM2 inhibitors (Z Wang et al., bioRxiv, 2019), these findings reveal potential combination therapies to overcome pancreatic cancer immunotherapy resistance.
Improving Specific Targeting of Tumors Through Bispecific SNIPER Antibodies
Authors: Erbe Amy K , Hernandez Reinier , Gerhardt Daniel , Hammer Bonnie , Felder Mildred , Bercher Mark , Dennin Jennifer , Massey Christopher , VandenHeuvel Sabrina , Feils Arika S , Heck Mackenzie , Hank Jacquelyn A , Glasser Bryan , Green Roland , Sondel Paul M
Published: January 2023

GD2 is expressed on neuroblastomas, as well as melanomas, small cell lung cancers and sarcomas. Anti-GD2 mAb can be used to treat these cancers and is part of the standard care for neuroblastoma. Expressed minimally on normal tissues, GD2 is expressed on some nerve cells. Thus, anti-GD2 treatment can cause neuropathic pain. To increase tumor specificity, we developed a bispecific SNIPER antibody, INV721, to simultaneously target 2 tumor antigens. One arm of INV721 is specific to GD2 and the other arm is specific to B7H3. B7H3 is overexpressed on multiple tumor types, including those listed above, with minimal expression on most normal cells (some expression on liver cells), and absent on nerve cells. The Fab arms targeting GD2 and B7H3 are each low-moderate affinity, such that INV721 will only bind with high affinity when both arms of INV721 bind to their antigens on the same cell. This high-affinity binding requires expression of both GD2 and B7H3, resulting in high-specificity of the SNIPER to tumor cells. To test the in vivo binding affinity of our bispecific antibody, INV721 was radiolabeled with 89Zr. Mice bearing GD2/B7H3-expressing tumors were intravenously injected with 89Zr-labeled INV721 and its longitudinal in vivo biodistribution was monitored via positron emission tomography imaging. 89Zr-INV721- showed elevated and persistent accumulation in the tumor with minimal uptake in normal tissues. 89Zr-radiolabeled isotype control antibody displayed significantly lower tumor uptake demonstrating the specificity of INV721. The goal of this SNIPER-antibody is to enhance the tumor-specific delivery of therapeutic mAbs, which may decrease toxicity and improve efficacy for cancers expressing both GD2 and B7H3.
Synthetic long peptide vaccines possessing a universal helper epitope can unmask the therapeutic effects of MHC I-restricted neoepitopes
Authors: Swartz Adam , Riccione Katherine , Congdon Kendra , Sanchez-Perez Luis A , Nair Smita K , Sampson John H
Published: January 2023

Synthetic long peptide (SLP) vaccines targeting neoantigens arising from patient-specific missense mutations may offer a means to address the antigenic heterogeneity of glioblastoma (GBM) tumors by simultaneously targeting a multitude of tumor-specific antigens expressed throughout these heterogenous tumors. This approach is challenged by uncertainties pertaining to vaccine design, including peptide composition required for immunogenicity and efficacy. To this end, we investigated the mechanism of an efficacious SLP vaccine targeting the neoantigen Odc1, expressed within the mouse astrocytoma SMA560. This led to the identification of three generalizable principles governing the effectiveness of neoantigen-targeting SLPs: (1) SLPs containing an MHC I-restricted neoepitope may activate neoantigen-reactive CD8+ T cells, which drive direct antitumor effects; (2) to induce robust neoantigen-reactive CD8+ T-cell responses, CD40L-mediated T cell "help" is required; and, (3) CD40L interactions are conferred by an SLP only when a “helper” epitope is physically conjoined to an MHC I-restricted neoepitope. To leverage these findings for clinical translation, we developed a rationally-designed vaccine comprised of an MHC I-restricted neoepitope linked to a universal “helper” epitope. This design was capable of maintaining the effects of the Odc1 vaccine and, remarkably, unmasked the therapeutic effects of, otherwise, poorly immunogenic MHC I-restricted neoepitopes. Together, these findings are significant because they elucidate mechanisms required for efficacious SLP vaccines and demonstrate a clinically-tractable approach with the potential to expand the therapeutic breadth of neoantigen vaccines.
An in vivo humanized mouse model for determining donor-dependent cytokine release syndrome in response to checkpoint and bispecific antibody therapy
Authors: Draheim Kyle M , Yang Jiwon , Jiao Jing , Cheng Mingshan , Keck James G
Published: January 2023

Although immuno-oncology therapies such as checkpoint inhibitors, bispecific antibodies and CAR-T cells have been successfully used as cancer treatments, they can have severe adverse effects such as cytokine release syndrome (CRS). The animal models and in vitro human PBMC assays presently in use do not reliably predict CRS in patients. Currently, the only widely accepted predictors of CRS are cancer burden and therapeutic dose. Despite this, most pre-clinical assays used to evaluate CRS do not incorporate cancer cells. A predictive assay that can screen for patient/cancer/therapy combinations at risk for developing CRS would improve the safety of immuno-oncology drug development.

We have developed a novel, rapid, sensitive and reproducible in vivo PBMC humanized mouse model that is able to differentiate human PBMC donors based on individual cytokine release response. Using this model, we evaluated cytokine release in response to immune checkpoint inhibitors and bispecific T-cell engaging antibodies. Additionally, this mouse model uses luciferase labelled cell lines to allow the measurement of tumor burden at the time of treatment as well as assess short term tumor response. We have ongoing efforts to further develop this assay in order to evaluate efficacy and CRS of at the same time, and progress will be discussed. This mouse model has immediate utility in current and future drug development.
Vaccine efficacy against primary and metastatic cancer with in vitro-generated CD103+ conventional dendritic cells
Authors: Zhou Yifan , Slone Natalie , Chrisikos Taylor T , Kyrysyuk Oleksandr , Babcock Rachel Lauren , Medik Yusra B. , Li Haiyan S , Kleinerman Eugenie S , Watowich Stephanie S
Published: January 2023

Type 1 conventional dendritic cells (cDC1s) possess efficient antigen presentation and cross-presentation activity, yet the efficacy of immunotherapy utilizingthis population is understudied. We used in vitro-generated CD103+cDC1s in vaccination strategies to test their ability to control melanoma and osteosarcoma tumors. In vitro-generated CD103+cDC1s produced cDC1-associated factors such as IL-12p70 and CXCL10, and demonstrated antigen cross-presentation activity upon stimulation with the Toll-like receptor 3 (TLR3) agonist polyinosinic:polycytidylic acid (poly I:C). In vitro-generated CD103+cDC1s also migrated to tumor-draining lymph nodes (TdLNs) following poly I:C treatment and intratumoral (i.t.) delivery. Vaccination with poly I:C-activated and tumor antigen-loaded CD103+cDC1s enhanced tumor infiltration of tumor antigen-specific and IFN-g+CD8+ T cells, and suppressed melanoma and osteosarcoma growth. CD103+cDC1s showed superior anti-tumor efficacy compared to vaccination with monocyte-derived DCs (MoDCs), and led to complete regression of 100% of osteosarcoma tumors in combination with CTLA-4 antibody-mediated checkpoint blockade. In addition, CD103+cDC1s effectively protected mice from pulmonary melanoma and osteosarcoma metastases. Our data indicate an in vitro-generated CD103+cDC1 vaccine elicits systemic and long-lasting tumor-specific T cell-mediated cytotoxicity, which restrains primary and metastatic tumor growth. The CD103+cDC1 vaccine was superior to MoDCs and enhanced response to immune checkpoint blockade. These results indicate the potential for new immunotherapies based on use of cDC1s alone or in combination with checkpoint blockade.
Oncolytic peptide LTX-315 enhances anti-melanoma immunity by inducing MyD88-dependent maturation of dendritic cells
Authors: Yang De , Li Xiao-Qing , Alam Md Masud , Rekdal Øystein , Oppenheim Joost J
Published: January 2023

LTX-315 is a synthetic cationic oncolytic peptide with potent anticancer cytotoxicity. LTX-315 induces both immunogenic tumor cell death and the generation of tumor-specific immune response. Given the central role of dendritic cell (DC) maturation in the induction of antigen-specific immune responses, we investigated the effect of LTX-315 treatment on the maturation of tumor-infiltrating DCs (TiDCs) and the generation of anti-melanoma immunity. The results reveal that LTX-315 induces the maturation of DCs through three distinct mechanisms: 1) release of DAMPs/alarmins; 2) complexes of LTX-315 with nucleic acids that act immunostimulatory; and 3) direct induction of the maturation of both conventional and plasmacytoid DCs by triggering TLR7. Importantly, LTX-315-induced maturation of DCs in vitro and in vivo was dependent on the presence of the signal transducer MyD88 as well as distinct TLRs. Our data also substantiate the notion that reagents like LTX-315 with both tumor cell cytotoxic and immunostimulaing capacities are more effective immunotherapeutics.
Reprogramming leukemia cells into antigen presenting cells as a novel cancer vaccination immunotherapy
Authors: Linde Miles H , Dove Christopher G , Gurev Sarah F , Phan Paul , Zhao Feifei , Gars Eric J , Marshall Payton L , Miller Lindsay P , Majeti Ravindra
Published: January 2023

B-cell acute lymphoblastic leukemia (B-ALL) is an aggressive hematopoietic neoplasm characterized by recurrent genetic lesions resulting in malignant transformation. Despite an arrest in B cell maturation, human B-ALL blasts retain the capacity for reprogramming to the myeloid lineage. To test the therapeutic implications of reprogramming, we generated murine models of B-ALL capable of myeloid lineage reprogramming via ectopic expression of CEBPα and PU.1. Once reprogrammed, B-ALL cells acquired an APC phenotype and stimulated antigen-specific T cells. In vivo B-ALL reprogramming in immunodeficient mice led to a modest survival benefit, however, reprogramming in immunocompetent mice led to tumor eradication and protection from subsequent re-challenge, demonstrating successful generation of durable, systemic, and curative immunity. This therapeutic benefit is dependent on the presence of both CD4+ and CD8+ T cells, and characterized by an increased frequency of bone marrow-infiltrating T cells and oligoclonal T cell expansion. Moreover, local myeloid reprogramming of a primary tumor drove systemic immune activation and eradication of distant, non-reprogrammed lesions. Our data suggests that B-ALL cells reprogrammed to the myeloid lineage are potent APCs capable of presenting tumor-associated antigens, and in vivo reprogramming elicits robust, durable, tumor-eradicating, and systemic T cell-mediated immunity. Recent efforts have further identified sarcoma and carcinoma models amenable to myeloid-lineage reprogramming. Thus, reprogramming of malignant cells into APCs represents a novel immunotherapeutic strategy with potential clinical utility in the treatment of a broad array of human malignancies.
Evaluation of a live attenuated influenza A vaccine expressing an IgA-inducing peptide in swine
Authors: Brand Meghan J Wymore , Gauger Phillip , Ferreri Lucas , Cáceres C. Joaquín , Perez Daniel , Vincent Amy , Rajão Daniela
Published: January 2023

Influenza A (IAV) is a significant disease for both human and swine health, as well as a zoonotic concern. The pig is an ideal model for human disease and evaluation of novel IAV vaccines. The objective of this study was to evaluate a bivalent (H1N1 and H3N2) live attenuated influenza A vaccine (LAIV) containing the IgA-inducing peptide gene (IGIP) in swine. Pigs were vaccinated with IGIP-LAIV or LAIV, challenged with heterologous H1N1 or H3N2, and necropsied at 5 or 14 days post infection (DPI). Peripheral blood mononuclear cells (PBMCs) and cells isolated from bronchoalveolar lavage fluid (BALF) were evaluated by flow cytometry and assessed for IgA-secreting cells (IgA-SCs) by ELISPOT. Pigs vaccinated with IGIP-LAIV and LAIV had decreased lesion scores compared to non-vaccinated pigs after challenge with either virus, and there was a trend for decreased lesion scores in the IGIP-LAIV compared to LAIV group. At 0 DPI, PBMC CD21+B cells were increased in IGIP-LAIV pigs compared to LAIV and naïve pigs. There was no difference in circulating total IgA-secreting cells between any groups post-challenge, though at 14 days post boost both the IGIP-LAIV and LAIV groups had increased virus-specific IgA-SCs to both vaccine viruses. In BALF at 5 DPI, the IGIP-LAIV and LAIV had increased total IgA-SCs, as well as increased virus-specific IgA-secreting cells to both vaccine viruses regardless of challenge virus. In conclusion, IGIP-LAIV and LAIV protected from heterologous challenge and induced virus-specific IgA-SCs in blood and BALF. IGIP-LAIV increased circulating B cells and had a trend for improved protection compared to LAIV, though no difference was observed in IgA-SCs between vaccines. Thus, the IGIP-LAIV represents an alternative LAIV vaccine.
The transcriptional landscape of porcine peripheral blood immune cells
Authors: Herrera-Uribe Juber , Byrne Kristen A , Liu Haibo , Becker Sage , Loving Crystal L , Tuggle Christopher K
Published: January 2023

Blood consists of different immune cells with essential functions given by distinct molecular signatures including transcriptomic profiles. The porcine immune system shares many similarities with that in human; however, the porcine immune cell transcriptome has not been as comprehensively studied. To identify distinct transcriptomic profiles of major porcine peripheral blood immune cells, we employed magnetic separation and fluorescence activated cell sorting methods to isolate 9 different cell types from blood PBMCs from two healthy pigs, representing monocytes, neutrophils, NK cells and specific populations of T and B cells. We obtained deep individual cell type transcriptomes using RNA-seq, detecting up to 10,974 genes. Principal component analysis revealed appropriate clustering of replicate samples and the separation of T, B, NK and myeloid cell groups. Pairwise comparisons revealed significant expression of a large number of genes specific for monocytes, neutrophils and NK cells only, while enrichment analysis identified highly differentially expressed genes for all cell types. Gene Ontology term analysis showed high enrichment of biological processes related to the nature of each cell type (i.e., B cell antigen presentation enriched in B cell transcriptome). The gene enrichment and specific genes of porcine immune cells is being further validated in silico to human datasets using gene set enrichment analysis (GSEA), and in vitro using qRT-PCR analysis. Taken together, the gene expression profiles reported here is the first comprehensive transcriptomic study of circulating porcine immune cell types and provides a valuable resource to elucidate molecular markers for porcine immune cell identity and function.
Mast cell histamine mediates intestinal immune response to early life stress in piglets via histamine 1 receptor (H1R)
Authors: Thelen Kyan , Wilson Neco , Berrios-Vazquez Glorian , Moeser Adam J
Published: January 2023

Mast cells are important immune modulators of stress-related gastrointestinal (GI) disorders. Our previous studies investigating early life stress in piglets showed that histamine, a major mast cell granule mediator is released within minutes of early weaning stress and is followed by increased intestinal mucosal expression of histamine receptor subtypes. The precise contribution of histamine to stress-induced GI immune responses is unknown. The objective of this study was to test the hypothesis that stress-induced intestinal immune response is mediated through the histamine 1 receptor (H1R). Fifteen-day-old female Yorkshire piglets were administered either saline vehicle or the H1R antagonist, desloratadine (2 mg/kg; intramuscular), 30 minutes prior to early weaning stress. At weaning, piglets were weaned from their dams and housed in nursery pens with ad libitum access to water. At 24 hours post-weaning, mid-jejunum and mesenteric lymph nodes (MLN) were collected for subsequent qPCR gene expression as well as IHC localization and protein expression analyses. Unweaned control piglets remained with the sow and were collected immediately at weaning. Gene expression for H1R was not influenced by weaning; however, immunohistochemical analysis of H1R revealed increased expression and localization in lamina propria cells and myofibroblasts in jejunal villi and expression in the longitudinal and circular muscle layers in early weaned piglets. In the MLN, weaning increased the expression of TNFα, TGFβ, and β-Integrin. This response was reduced in the group treated with the H1R antagonist. Together, these data demonstrate that histamine via H1R plays an important role in early weaning stress-induced intestinal immune responses.
The Immune response of Susceptible or Resistant Bat Species to White Nose Fungus
Authors: Lupfer Christopher R , Anderson Briana N. , Naffa Keslie S. , Tomasi Tomas E.
Published: January 2023

North American bats are being decimated by White Nose Syndrome (WNS), a disease caused by the psychrophilic fungus Pseudogymnoascus destructans (P.d.). Bats are an important part of the ecosystem and have global economic impact through consuming insect pests or pollination of crops. Thus, we need to understand why some bat species are susceptible and some resistant to WNS to develop strategies to protect bats from this disease. However, WNS has decreased populations of some temperate bat species to levels where conducting research on live bats must be curtailed. We have generated transcriptomic profiles derived from primary wing fibroblast cell cultures of resistant and susceptible bat species that were exposed to P.d. Our data indicate that fibroblasts from susceptible species of bats have an enhanced immune response whereas the immune response in resistant species is muted. Importantly, these results were confirmed in samples collected from live bats prior to population declines. We have now developed qRT-PCR assays to test the activity of several immune genes that were upregulated in the cell-cultures (transcriptomics analysis). Generally, we found that wing tissue from tricolored bats (WNS-susceptible) late in hibernation showed increases in gene expression compared to pre-inoculation with Pd, but wing tissue from big brown bats (WNS-resistant) did not change (or decreased). These results, though preliminary, suggest that wing fibroblast cell-cultures may indeed be a good surrogate for using living bats in some kinds of WNS research. Our data also suggest that the cause for high mortality in WNS susceptible bat species is immune activation instead of immune tolerance.