bluuebunny/crossref_metadata_2025 huggingface dataset:
T cell expansion is regulated by mutual regulation of ribosome biosynthesis and mTORC1 activity
Authors: Obst Reinhard , Rosenlehner Teresa , Pennavaria Stefanie , Akcabozan Batuhan , Kadam Pallavi , Parlato Rosanna , Straub Tobias , Kranich Jan
Published: August 2023

The clonal expansion of T cells entails a swift and substantial repurposing of cellular physiology towards anabolic pathways to allow for a massive increase of cellular biomass for rapid cell division. Ribosomal biosynthesis is an essential and highly energy demanding process driven by the actvities of all RNA polymerases and ~300 proteins processing and modifying the rRNA and its precursors. How ribosomal biosynthesis is integrated into the process of T cell activation is incompletely understood. We have investigated how the mTOR complexes affect ribosomal biosynthesis, T cell proliferation and TCR signaling. We found that the proliferation of mTORC1-but not mTORC2-ablated T cells is slowed from three divisions per day to one with each consecutive division being delayed, mostly at the G1/S transition. mTORC1 signaling is affected by conventional TCR signaling pathways via PI3K while TCR sensitivity is hampered by mTORC1 inhibition. Upon activation the amount of RNA per cell increases at least 10-fold, which is reduced by half in mTORC1-ablated cells. RNA-seq data showed that this reduction affects all RNA biotypes and that rRNA processing is ineffective in activated T cells. Using 5’ ETS FISH probes of nascent pre-rRNA in imaging cytometry revealed that mTORC1 but not mTORC2 ablation reduces the number and the expansion of nucleoli while translation was not yet affected. Inhibiting RNA Polymerase I by chemical and genetic means blocked not only proliferation but mTORC1 signaling as well. Our data show that TCR signaling, mTORC1 activity and ribosomal biosynthesis are tightly interlinked and regulate each other.

Supported by grants from the German Research Council (CRC1054-B07, OB150/7-1).
Tumor cell-macrophage crosstalk promotes immunotherapy resistance via lipid metabolism
Authors: Liang Zhixian , Cao Jianquan , Tang Wenshu , Wu Haoran , Tu Yalin , Xiong Zhewen , Zhang Lingyun , Zhou Jingying , Chan Stephen L , Cheng Alfred S L
Published: August 2023

Immune-checkpoint blockade (ICB) therapies have transformed the treatment landscapes of hepatocellular carcinoma (HCC). However, the immunosuppressive tumor microenvironment (TME) constructed by tumor cells restricts the responsiveness of ICB therapies to a minority of patients. Triggering receptor expressed on myeloid cells-2 (TREM2) counteracts inflammation and maintains metabolic fitness in myeloid cells. Single-cell RNA-sequencing analysis of our pembrolizumab study (NCT03419481) identified a subset of tumor-associated macrophages over-expressing TREM2 in non-responders. Consistently, our syngeneic ICB-resistant HCC mouse models verified a group of Trem2+ myeloid cells, which were accumulated in the lipid-rich TME of ICB-resistant tumors. Compared to the parental ICB-sensitive tumor cells, the conditional medium (CM) of ICB-resistant tumor cells significantly increased Trem2 expression in macrophages, which was abolished when lipids in the CM were depleted. Notably, lentivirus-mediated Trem2 ablation reduced lipid accumulation in TME and overcame anti-PD-1 resistance with increased cytotoxic CD8+ T cell infiltration. In conclusion, our study highlights TREM2 as an immuno-metabolic target to enhance HCC immunotherapy.

This study is supported by Collaborative Research Fund (C4045-18W) and Li Ka Shing Foundation. Z.L. is supported by Hong Kong PhD Fellowship Scheme.
TGFβ trained immunity in mast cells: do inflammasomes matter?
Authors: Ashbaugh Alexandria M , Lyons David O , Weisen Arianna L , Rasé Viva J , Haughian James M , Pullen Nicholas A
Published: August 2023

Mast cell (MC) contributions to solid cancer microenvironments are controversial and seem tissue dependent. TGFβ is a potent MC chemokine abundantly expressed by high-grade cancers. Previous work reported that TGFβ alone induces biologically relevant expression of IL-6 from MC, which is potentially important for reciprocal paracrine interactions with cancer cells and other leukocytes. Therefore, the objective of the present study was to examine MC in an immunocompetent, high-grade model of breast cancer. Using the 4T1 system in BALB/c mice, we compared mast cell infiltration into primary tumors of wild-type versus 4T1 cell-specific knockouts of IL-6. We also hypothesized that TGFβ institutes a training effect on MC like other metabolically driven mechanisms involving the NLRP3 inflammasome. Using a mixture of flow cytometric and qPCR assays we explored potential MC training in the context of TGFβ.

Results

Contrary to our prediction, MC infiltration into IL-6KO tumors was significantly increased compared to WT. Studies of classic intracellular training mechanisms in response to TGFβ were mixed. Glucose uptake, glycolytic capacity, mitochondrial potential, and ASC speck formation were unaffected by TGFβ, but IL-6 expression increased as previously seen. Additionally, we were unable to detect substantive NLRP3 expression in MC regardless of condition. Despite these results, we were surprised to observe robust increases in expression of IL-1β, IL-18, and caspase-1 with TGFβ stimulus. We conclude that MC are actively recruited to 4T1 tumors dependent on IL-6 (and TGFβ), MC produce select cytokines in response to TGFβ, yet the mechanism explaining this does not involve the NLRP3 inflammasome and remains unresolved.
Reprogramming of tumor-induced emergency granulopoiesis overcomes immune checkpoint therapy resistance
Authors: Tang Hui , Xie Ping , Ahn Ji Hae , Choi Mi Ran , Fan Jie , Orpano Rachel , Zhang Bin
Published: August 2023

In established cancer in mice and humans, “emergency” myelopoiesis usually occurs to facilitate malignancy progression. Neutrophils and immature myeloid cells accumulated in the bone marrow, periphery and tumor are potent mediators of immunosuppression and tumor promotion. Here, we demonstrated that Cysteinyl leukotriene receptor 1 (CysLTR1), a key player for asthma, and other inflammatory conditons, sustained tumor-induced emergency granulopoiesis. Genetic ablation and pharmacological inhibition of CysLTR1 resulted in diminished tumor growth with enhanced antitumor immunity. The anti-tumor effect of CysLTR1 inhibition was linked with transcriptomic rewiring of granulopoiesis and neutrophil reprogramming toward an anti-tumor immune phenotype; this process required the distinct transcriptional factors to specifically dictate neutrophil progenitor commitment and differentiation in association with controlled degranulation. Furthermore, single-cell RNA sequencing analysis provided a comprehensive transcriptional landscape of neutrophil maturation, function and fate decision in tumor bearers. CysLTR1-mediated changes in the differentiation and degranulation also occurred in human neutrophils and correlated with the survival and ICT responsiveness in cancer patients. Consistently, targeting CysLTR1 with clinically available antagonists overcame resistance to immune checkpoint PD-1 inhibitors in several mouse tumor models. These findings thus have important implications for understanding the abnormal myelopoiesis during cancer progression and suggest new therapeutic approaches to improve ICT.

Supported by grants from NCI (R01CA250101) and DoD (W81XWH-20-1-0024).
NCG-M humanized mice - an excellent model for human immune reconstitution of myeloid lineages
Authors: Chen Santi Suryani , Wang Huiyi , Fan Jialu , Guo Shiying , Zhang Mingkun , Li Shuai , Li Zhiying , Moore Mark , Zhao Jing , Xiang Gao , Ju Cunxiang
Published: August 2023

Severely immunodeficient mice engrafted with human hematopoietic stem cells (HSC) have been extensively used in immuno-oncology studies to evaluate the efficacy of cancer therapies. However, because of species differences in the immune system between human and mouse, murine cytokines provide limited support to human immune cells, thus human immune system mice have limited human immune cell engraftment. Increasing evidence has shown that myeloid cells, especially macrophages and dendritic cells, are critical for the induction of anti-tumor immunity. We developed a mouse model, NCG-M, that can support human T, B, NK, and various myeloid and granulocyte cells such that in vivo evaluation of agents that require the interplay between these immune cells can be examined. This model was genetically engineered on the severely immunodeficient strain NCG and can produce human granulocyte/macrophage colony-stimulating factor 2 (GM-CSF, also known as CSF2), interleukin-3 (IL-3) and stem cell factor (SCF, also known as KITLG). Upon human CD34+ HSC cell engraftment, a significant increase in myeloid lineage cells, such as granulocytes, monocytes, neutrophils, macrophages, dendritic cells, and mast cells, was observed in the NCG-M cohort compared to NCG mice. The NCG-M mouse also supports the development of human T cells, and preliminary data showed increased B cells and NK cells. Importantly, the increased efficiency of immune cell reconstitution did not affect the morbitity and mortality of this mouse. The NCG-M is an appropriate mouse model for studying the efficacy of therapeutic agents that require human T cells and myeloid cells.
Cancer promotes immune escape via transdifferentiating B cells into macrophage-like cells
Authors: Biragyn Arya , Chen Chen , Park Bongsoo , Bodogai Monica , Lee Jung-Min , Beerman Isabel
Published: August 2023

Previously we reported that breast cancer uses the generation of regulatory TGFb+ B cells to facilitate its metastasis via inducing FoxP3+Tregs and educating MDSCs 1,2. To do this, the cancer secretes TSLP to cause premature emigration of B-cell precursors from the bone marrow (BM) and accumulation in the spleen as a source of tBregs 3. However, it remains unclear why other cancers similarly cause premature emigration and accumulation of B-cell precursors, although they do not generate tBregs. Here, we report that this is to use these B-cell precursors to generate macrophage-like cells (termed B-MF) 4. We found that cancer transdifferentiate a small but bona fide Csf1R+Pax5Low subsets of BM B cells(pre-B and immature IgM+ B cells) into B-MF in the tumor. This alternative pathway of the generation of tumor-associated macrophages (TAM) is phenotypically and functionally distinguishable from that of BM monocyte-derived TAMs, as B-MFs more efficiently phagocytize apoptotic cells, suppress proliferation of T cells, and induce FoxP3+regulatory T cells. Our modeling studies in mice with cancer reveal that B-MFs support tumor progression and metastasis presumably by retarding tumor-infiltrating IFNγ+CD4+ T cells. Since B-MF-like cells and their transcriptional signature can be detected in patients with breast and ovarian cancers, we think that this transdifferentiation pathway is clinically relevant and hence could serve as an immunotherapeutic target.

This work was supported by the Intramural Research Program, NIA/NIH.
Inhibition of TCR-MHC-II interactions abrogates Treg-mediated immune suppression and augments anti-tumor immunity
Authors: Panda Abir Kumar , Shevach Ethan M
Published: August 2023

CD4+Foxp3+ Tregs play a non-redundant role in controlling autoimmunity. Continuous TCR signaling and the availability of exogenous IL-2 from CD4+ effectors regulate the size of the Treg pool and their suppressive function in the steady state. Homeostatic proliferation of Tregs and memory phenotype (MP) CD4+Foxp3−T cells depends on CD80/CD86-CD28 signaling. Acute blockade of TCR-MHC-II interactions in vivo by anti-MHC-II mAb significantly enhances memory phenotype (MP) Treg, CD4+, and CD8+ T cell expansion by a CD28-dependent pathway. IL-15 trans presentation by APC also contributes to Treg, MP CD4+ and MP CD8+ proliferation following blockade of TCR-MHC-II interaction by a CD122-dependent pathway. Combined CD28 activation and IL-15 stimulation together with IL-2 deprivation secondary to MHCII-TCR blockade result in decreased expression of CD25, but enhancement of Foxp3 expression by Treg. In addition, Treg exhibit decreased pAKT/NFAT signaling, but enhanced pSTAT5/mTOR signaling. Mice transplanted with several different transplantable tumors manifest enhanced tumor immunity in the presence of MHCII-TCR inhibition. Our results demonstrate that in the absence of TCR-MHC-II interactions, Treg proliferate, but lose their identity and manifest disabled suppressor function resulting in enhanced innate and adaptive immunity against cancer progression.

This work was supported by the Intramural Research Program of NIAID, NIH.

This work was supported by the Intramural Research Program of NIAID, NIH.
IL-27 promotes pathogenic Tfh-like CD4 effectors and exhausted-like CD8 T cells in a mouse model of Sjögren’s
Authors: Debreceni Ivy L , Lieberman Scott
Published: August 2023

Sjögren’s is an autoimmune disease that targets the exocrine glands leading to profound dryness, extra glandular manifestations, and increased risk of developing lymphoma with no therapy to treat the underlying immune dysregulation. Nonobese diabetic (NOD) mice spontaneously develop T cell mediated inflammation of the lacrimal gland, similar to human disease. IL-27 is elevated in the salvia and serum of people with Sjögren’s and is required for lacrimal gland autoimmunity in NOD mice. Utilizing IL-27Rα KO NOD mice, I have previously found both CD4 and CD8 effector T cells require IL-27 to transfer lacrimal gland autoimmunity to NOD-SCID mice. Here, we utilized an adoptive co-transfer system that promotes infiltration of lacrimal glands by both WT and IL-27Ra KO T cells to define the T cell intrinsic effects of IL-27 signaling in lacrimal gland infiltrating T cells through 28 color spectral flow cytometry and performed unsupervised dimensionality reduction analyses to identify subsets of T cells reliant on intrinsic IL-27 signaling. Among the total concatenated T cell population including both WT and KO T cells, 12 total subsets were identified. Strikingly, when visualizing WT and KO separately, we identified populations of CD4 effector T cells and CD8 T cells that were either heavily skewed or only present in WT. Through phenotyping these populations, I found IL-27 signaling induced Tfh-like PD-1+ICOS+ CD4 effector T cells and PD-1+ICOS+CD39+ exhausted-like CD8 T cells in the glands of recipient mice. Taken together, these data demonstrate a role for IL-27 in promoting pathogenic T cells in the immune dysregulation of Sjögren’s.

Supported by grants from NIH(T32 AI007485, R01 EY027731)
Cutaneous T cells promote distinct outcomes in human skin structural cells associated with inflammatory disease
Authors: Morawski Peter A , DeBerg Hannah , Pribitzer Stephan , Fahning Mitch , Stefani Caroline , Lacy-Hulbert Adam , Gratz Iris , Campbell Daniel J
Published: August 2023

The skin is an immunologically active barrier tissue specialized to deal with a litany of external stresses. Immune cells coordinate with structural cells in the skin to promote appropriate inflammatory responses and subsequent restoration of barrier integrity following insult. Recent single cell analyses of human skin have defined previously unappreciated transcriptional heterogeneity in structural cells, including disease-associated changes in keratinocytes and fibroblasts. Cutaneous T cell activity is implicated in the development of inflammatory skin disease, but the mechanisms by which T cells promote disease-associated changes in the skin remain unclear. We now show that distinct subsets of circulating and resident CD4+ cutaneous T cells promote a diverse array of cytokine-dependent transcriptional outcomes in human keratinocytes and fibroblasts. We used these in vitro generated transcriptional signatures to identify T cell-dependent gene modules associated with inflammatory skin disease in vivo. For example, we find that Thelper17 cells regulate the expression of epigenetic modifiers in keratinocytes, genes that are enriched in the skin of patients with psoriasis and normalized in response to anti-IL-17 therapy. Thus, we have identified T cell-dependent cytokine-driven transcriptional changes associated with inflammatory skin disease that can be used to dissect immune mechanisms of anti-cytokine therapeutic activity.
Targeting Pim2 improves effector function and longevity of T cells in cancer immunotherapy
Authors: Wu Yongxia , Tian Linlu , Hitchingham Brianna , Choi Heejin , Yang Kaipo , Yu Xue-Zhong
Published: August 2023

The Provirus Integration sites for Moloney murine leukemia virus (Pim) kinase family has been studied extensively in tumorigenesis. We previously reported a key role of Pim2 in negatively regulating T-cell responses to alloantigen. We further investigated how Pim2 regulates T-cell mediated anti-tumor responses using murine models of adoptive cell therapy (ACT) and autologous hematopoietic cell transplantation (HCT). We found that Pim2−/− Pmel CD8 T cells showed enhanced ability for controlling B16 melanoma growth following ACT. Pim2−/− T cells were more potent to control C1498 leukemia growth after syngeneic HCT. Mechanistically, Pim2−/− CD8 T cells exhibited increased effector cytokine production, metabolic activities, and unexpectedly CD62L expression than WT controls following activation. Activated Pim2−/− CD8 T cells had less apoptosis and exhaustion during expansion in IL-2. In vivo, Pim2−/− CD8 T cells differentiated more into effector, but less into exhausted or suppressive subsets in tumors. On the other hand, Pim2−/− CD8 T cells exhibited increased longevity in spleen and tumor draining lymph nodes. Our data indicates that Pim2 negatively regulates CD8 T-cell anti-tumor activity by controlling their persistence in secondary lymphoid organs and effector function in tumors. Blocking Pim2 with a Pim2-specific inhibitor improved graft-versus-leukemia activity after autologous HCT and enhanced anti-melanoma effects after ACT. Pim2 inhibition also increased anti-tumor activity of human melanoma-specific T cells and CD19 CAR-T cells. Taken together, targeting Pim2 may serve as a novel strategy for improving cancer immunotherapy through enhancing differentiation and persistence of effector T cells.

NIH R01 CA258440 and R21 CA263140 to Xue-Zhong Yu, MCW Center for Immunology Pilot Award to Yongxia Wu
BRD4 and BET proteins regulate CD8 T cell exhaustion in mouse models of cancer
Authors: Pratt Brandon M , Green William D , Mullins Genevieve , Green Jarred , Milner Justin
Published: August 2023

Harnessing the cytotoxic capacity of CD8 T cells is central to the efficacy of immunotherapy and has revolutionized the fight against cancer. However, despite encouraging success for select indications, immunotherapies still fail to achieve durable and effective outcomes for certain cancer types and in many patients. Failure of immunotherapies often results from CD8 T cells differentiating into an epigenetically controlled and reinforced state known as T cell exhaustion. Therefore, resolving the molecular programs governing T cell differentiation within the tumor microenvironment (TME) is critical to enhancing treatment. Using mouse models of breast cancer and pancreatic cancer, we investigated the role of the bromodomain and extraterminal (BET) family of proteins in mediating T cell differentiation and exhaustion. Depletion of BRD2, BRD3, and BRD4 in CD8 T cells differentially affected CD8 T cell accumulation and the exhausted phenotype within the tumor microenvironment. Further, small molecule inhibition of the BET family similarly impacted CD8 T cell differentiation and exhaustion in vivo. Our findings may inform new strategies to circumvent CD8 T cell exhaustion during infection.

supported by a training grant from the UNC Pharmacology Department (5T32GM135095-02)
Novel mechanisms of androgen receptor-centered transcriptional regulatory network in regulating CD8+ T cell exhaustion and sex bias in cancer
Authors: Xiao Tong , Schafer Johanna , Song No-joon , Wang Cankun , Weltge Payton , Li Xue , Ma Qin , Li Zihai
Published: August 2023

Sex bias in cancers arising from nonreproductive organs is known but poorly understood. We recently reported on a T cell-intrinsic role of androgen receptor (AR) in driving CD8+ T cell exhaustion which underlies the poorer tumor control in males (Kwon et al., Sci Immunol, 2022). This study aims to understand how AR regulates CD8+ tumor-infiltrating lymphocytes (TILs) by identifying its genome-wide targets. We created a CD8+ T cell-specific AR knockout (KO) mouse model by crossing E8I-cre mice with Ar-floxed mice, which we challenged with syngeneic bladder tumor MB49. We then monitored tumor growth and performed spectral flow cytometry using a T cell exhaustion panel. Further, we used Cleavage Under Targets and Tagmentation – sequencing (CUT&Tag-seq) to map the entire AR targets in CD8+ TILs. Loss of AR in CD8+ T cells significantly slowed the growth of MB49 in male but not female mice. CD8+ TILs from male CD8 AR KO mice showed reduced TOX+ TCF1− terminally exhausted subset (69%; P≤ 0.05) and TOX expression (72%; P = 0.0583) compared to controls. Similarly, TOX expression decreased by 43% in AR-deleted CD8+ T cells following chronic TCR stimulation in vitro (P ≤ 0.0001). Finally, using CUT&Tag-seq, we found that AR binds directly to promoters of multiple key transcriptional regulators of T cell exhaustion, including Tcf7 and Tox.

Through identifying the genome-wide targets of AR in CD8+ TILs, we characterized the cell-intrinsic mechanism by which AR regulates CD8+ T cell exhaustion. Our work suggests AR is a master transcriptional regulator that negatively impacts CD8+ T cell immunity, which may contribute to poorer survival in males and supports combining androgen deprivation therapy with immune checkpoint blockage for cancer treatment.

Special Institutional Funding
A new slow-releasing formulation of butyrate prolongs allograft survival
Authors: Sepulveda Martin N , Chen Luqiu , Cao Shijie , Wang Ruyi , Hubbell Jeffrey , Nagler Cathryn , Alegre Maria-Luisa
Published: August 2023

The microbiota consists of diverse microbes producing metabolites that can influence the immune system locally as well as systemically. These bioactive metabolites (postbiotics) have therapeutic potential and may prove useful to facilitate graft acceptance. Short-chain fatty acids (SCFA) promote intestinal homeostasis and have been linked to health and disease. The Hubbell-Nagler laboratories at the University of Chicago have developed a micelle-based platform that can deliver the SCFA butyrate systemically, with long-term retention and induction of tolerogenic responses. We hypothesized that the administration of this butyrate formulation will improve graft survival by suppressing the alloimmune response. Our preliminary data showed that only oral delivery of the butyrate formulation prolonged survival of a C57Bl/6 male to female minor-mismatched skin graft significantly, whereas sub-cutaneous and intra-peritoneal administration were ineffective. Improved graft survival was associated with diminished production of TNFα by splenic and skin-draining lymph node myeloid cells upon LPS stimulation, suggesting that butyrate modulates the myeloid compartment to reduce levels of pro-inflammatory cytokines. The postbiotic also enhanced survival of a BALB/c to C57Bl/6 major-mismatched skin graft, without the use of immunosuppressive drugs, and resulted in lower levels of donor-specific alloantibodies. Prolonging the administration of butyrate post-transplantation ameliorated graft survival even further, confirming the micelle-based platform has therapeutic potential. Current efforts focus on determining if the butyrate formulation drives myeloid cell polarization toward an anti-inflammatory phenotype.

This project was supported by grant NIH/NIAID 2R01AI115716
Prohibitin is Upregulated on Donor T cells in Murine Acute Graft-versus-Host Disease and Promotes Th17 Response
Authors: Snyder Katiri Jenna , Gao Yandi , Neidemire-Colley Lotus M , Sell Natalie , Ranganathan Parvathi
Published: August 2023

Introduction

Acute graft-versus-host disease (GVHD) is a T cell-mediated disorder and the main cause of non-relapse mortality in allogeneic hematopoietic cell transplantation recipients. Here, we investigate PHB in Th17-mediated inflammation and as a therapeutic target for acute GVHD.

Methods

Irradiated mice received T cell depleted bone marrow cells and allogeneic or syngeneic splenocytes. At day 25 post-transplant, blood, spleen, liver, and colon were harvested and analyzed for surface PHB, IFNγ, and IL-17 on donor T cells by flow cytometry. Splenocytes were cultured in the presence of PHB pharmacological inhibitor rocaglamide (RocA) and analyzed for PHB and IL-17 expression in donor T cells. Naïve murine CD4 T cells were cultured in Th17 polarizing conditions with RocA. CRISPR/Cas9-mediated knockout of PHB was performed followed by evaluation of Il17a, Il17f, and Il22 gene expression by quantitative PCR.

Results

Surface expression of PHB is increased in murine allogeneic donor T cells in liver, spleen, colon, and peripheral blood in acute GVHD. PHB-expressing splenic T cells secrete IL-17, which is reduced with treatment with a PHB inhibitor in ex vivo experiments. PHB inhibition of murine CD4 T cells in vitro impairs differentiation into the Th17 subtype resulting in reduced IL-17 secretion and downregulation of Il17a, Il17f, and Il22 genes. Knockout of PHB reduces transcription of Il17 and Il22 genes.

Conclusions

These findings show relevancy of PHB in Th17-mediated inflammation and suggest a potential role for PHB in acute GVHD. To our knowledge, this study is the first to investigate PHB in acute GVHD, and our findings will offer greater support for clinical use of PHB inhibition to treat this fatal disease.

Supported by grants from AAI (KS, PR), ACS (PR; RSG-22-053-01-IBCD), and NIH NCI (PR; R01 CA252469).
Gene Expression Profiling and Signaling Pathway Analysis of Tolerant T cells Circulating in Adult Patients after PTCy Haplotransplantation
Authors: Chen Xiaohua , Nee Taylor , Rajasundaram Dhivyaa , Farah Rafic , Szabolcs Paul
Published: August 2023

The mechanisms for the signaling pathways of T cell-dependent tolerance after haploidentical hematopoietic stem cell transplantation (haplo-HSCT), are incompletely understood. We tested 8 patients (Pt) with leukemia or myelodysplastic syndromes (median age:51-yrs old) following IRB approved consenting. All received Flu+TBI (+/− Cy) pre-conditioning and haplo-HSCT with post-transplant Cytoxan (PTCy) and FK506 as GvHD prophylaxis.

The circulating T cells were hyporeactive in mixed lymphocyte reaction (MLR) assay to recipient DC in Pts at 1yr (or 2yrs in 1 Pt) post-haplo-HSCT (~6m post-ISD withdrawal), while these T cells responded vigorously to 3rd party APC. Neither depletion of Tregs, nor blockade of IL10R or PD1 could reverse T-cell hyporeactivity. Low-dose IL2 triggered mild anti-host responses. Cytokine profiling further confirmed the MLR responses. ImmunoSEQ™ revealed the disappearance of alloreactive T-cell clones from peripheral pool with time, even though a few alloreactive T clones remained. Low dose IL2 had minimal impact to reverse the diminishment.

Unlike graft T-cell responses against host DC, tolerant T cells in circulation against hDC showed relative downregulation of allograft rejection, T-helper response, co-stimulation, and cell proliferation pathway, that was paired with upregulation of co-inhibition, PD1-PDL1, Ferroptosis and CREB pathways.

In summary, hyporeactivity of donor-derived circulating T cells to recipient DC were evident. We found Treg, Tr1 activity to be insignificant in maintaining long-term tolerance. T-cell clonal deletion appears to be the dominant mechanism, along with systemically altered signaling pathways in tolerant T-cells.
Characterization of ZG16b protein exocrine function and biomarker potential for salivary gland damage in chronic graft vs. host disease
Authors: Mody Drashty Paresh , da Silva Ana Costa , Sharma Rubina , Mays Jacqueline
Published: August 2023

The oral cavity is a frequent target of chronic Graft versus Host Disease (cGVHD) following allogenic hematopoietic stem cell transplant (allo-HSCT). Detection of early cGVHD-induced damage to exocrine tissues, such as salivary glands, is challenging to detect prior to irreversible tissue fibrosis. Recent work in our laboratory used shotgun proteomics to identify putative salivary biomarkers at salivary gland cGVHD onset. Saliva samples from patients affected with oral cGVHD post allo-HSCT had a drop in protein content of zymogen granule 16b (ZG16b), recapitulated in immunohistochemical staining of human minor salivary gland (MSG) biopsy sections. ZG16b loss correlated with CD45+ cells infiltration. This protein is poorly characterized, and immunofluorescence tissue staining suggested ZG16b protein localization to acinar cells. ZG16b cellular expression localized to serous and serous mucous acinar cell populations in single cell RNAseq (scRNA seq) data from healthy human labial MSG. Little is known about the function of this ZG16b in other exocrine tissues such as pancreas. scRNA seq analysis of repository data suggests ZG16b colonization in the pancreatic ductal cells. Similar to MSG, the pancreatic Zg16b gene was expressed in mucus secretion pathways and additional interactions with cell migration and homeostasis markers. Ongoing work is being done to further probe for binding partners for ZG16b (endogenous proteins, components of the oral microbiome and immune cell receptors) to distinguish the protein from its homologs. In parallel, the utility of salivary ZG16b as a cGVHD and general salivary gland damage biomarker is being tested. This work will bring clarity Zg16b’s role in exocrine tissues and alloimmune disease.

NIH, NIDCR Intramural program
Elevated exhausted effector tissue resident CD8+ T-cells in Chronic Graft versus Host Disease patient Oral mucosa unveiled by spatial transcriptomics and scRNA seq analysis
Authors: Sharma Rubina , da Silva Ana Costa , Martin Daniel , Pavletic Steve , Mays Jacqueline
Published: August 2023

Chronic graft-versus-host disease (cGVHD), an autoimmune-like disease following allogeneic hematopoietic stem cell transplantation where T cells can attack host tissue. Oral mucosa (OM) is a most common cGVHD effector site in the oral cavity. We studied T cell populations and tissue-specific signals that regulate their differentiation and functionality. Patient OM biopsies from ongoing clinical trials (NCT03602599, NCT00331968) were taken at 6 months post-transplant. We found enrichment of immune cells in the OM mainly in the CD8 T-cell cluster and identified two unique populations in the OM of cGVHD patients: one characterized by expression of proliferative markers and the other more prominent, characterized by exhaustion makers (TIM3, LAG3, TIGIT). The latter had low expression of CD69 but expressed markers of tissue residency; CD103, BLIMP1, PD1, FABP5. Interestingly, this population showed high expression of BATF, IRF4, and RUNX3, transcription factors that induce sustained effector CD8 T-cell programming. These exhausted cells displayed higher expression of effector molecules IFNG, GZMB, PRF1 inducing sustained CD8 T-cell effector function. Ligand-receptor analysis revealed strong interactions between CXCL9-CXCR3 in CD8 T-cells and myeloid cells. Spatial transcriptomic analysis confirmed the CD8 T-cell infiltration in the submucosal region of OM in cGVHD colocalizing with the elevated markers of exhaustion, surrounded by myeloid cells, thus confirming the striking interaction of CXCL9 and CXCR3. These exhausted CD8 T-cells may play an important role in cGVHD pathogenesis causing by driving sustained tissue damage and could serve as a might be used as a novel target for cGVHD therapy.

This work was supported by the intramural programs of the NIDCR and NCI.
Amelioration of graft versus host disease in humanized mice by exopolysaccharide from a commensal bacterium
Authors: Kalinina Olga , Knight Katherine L
Published: August 2023

Graft versus host disease (GVHD) is the major and often lethal complication of allogenic bone marrow transplantation. Prophylactic regimens for GVHD are given as standard pre-transplantation therapy; however, up to 60% of these patients develop GVHD, and require additional immunosuppressive intervention. We identified a probiotic exopolysaccharide (EPS) that induces inhibitory macrophages and tolerogenic dendritic cells (DCs) that inhibit T cell proliferation. We tested if EPS-treated human DCs, derived in vitro from CD34+ cord blood cells, would inhibit allogeneic T cell proliferation in a mixed lymphocyte reaction (MLR) and found that they did, demonstrating that EPS can induce tolerogenic DCs in vitro. We translated these studies in vivo using a humanized mouse model of aGvHD and demonstrated that EPS-treated human DCs administered at the time of transfer of human peripheral blood mononuclear cells (hPBMCs) significantly increased survival of these mice compared to mice given PBS-treated DCs, or no DCs. Several subsets of DCs have been described, e.g., cDCs, moDCs and plasmacytoid DCs, each with different phenotypes and functions. Using labeled EPS, we showed that the tolerogenic CD34-derived DCs generated by EPS treatment are conventional DCs (cDCs). We found that EPS binds to two cDC populations, conventional DC1 (cDC1) and conventional DC2 (cDC2), but only cDC1 were inhibitory in our MLR cultures. Our data indicate that EPS-DCs, potentially cDC1, can be used as a cell-based therapy to mitigate aGvHD in humans.

NIH 1R41AI155281-01
Deleting Runx3 in CD4+ T Cells Disrupts Effector Functions and Facilitates Tolerance Induction in Solid Organ Transplantation
Authors: Xie Stephen Zhiyuan , Allocco Jennifer B , Cassano Alexandra , Chen Luqiu , McKeague Michelle L , Chong Anita S , Alegre Maria-Luisa
Published: August 2023

Long-term transplantation tolerance depends on the control of graft-reactive T cells. In particular, the alloreactivity of CD4+ T cells needs to be suppressed as these cells are both necessary and sufficient for cardiac allograft rejection. Although experimental protocols such as anti-CD154-mediated co-stimulation blockade can stably induce donor-specific tolerance in MHC-mismatched cardiac allografts, other more immunogenic organs such as skin are less susceptible to tolerogenic treatments, in part due to additional reactivity of the host against donor commensals that accompany the graft. The transcriptional mechanisms that enable CD4+ T cells to drive rejection and whose targeting can promote tolerance are not well understood. RNAseq comparison of the transcriptome in graft-reactive T cells from rejecting versus tolerant heart allograft recipients revealed several transcription factors differentially expressed between rejected and tolerant mice in both spleen and graft. Here, we report that the transcription factor Runx3 in alloreactive CD4+ T cells promotes Th1 and cytotoxic response against the allograft and resistance to tolerance induction. Conversely, ablating Runx3 in alloreactive CD4+ T cells augments the efficacy of anti-CD154 blockade treatment enabling skin graft acceptance.
Tear cytokine profiling for ocular graft versus host disease utilizing a new multiplex assay
Authors: Li Andrew J , Martinez-Guasch Fernando , Sajjan Seema , Cao Xuefang , Atanackovic Djordje , Sunshine Sarah
Published: August 2023

Many patients who have undergone allogeneic hematopoietic stem cell transplantation (aHSCT) as part of cancer therapy achieve remission. However, nearly 50% of these patients develop Graft versus Host Disease (GVHD), a chronic autoimmune disease that can damage eyes. Quantifying specific cytokine changes in tears may reveal biomarkers and future treatment targets for ocular GVHD (oGVHD). Compared to other assays, the IsoLight Codeplex Secretome multiplex assay (Isoplexis) has advantages of high-throughput analyses and small sample requirements, but has yet to be validated for tears. This study evaluates the tears of a patient with oGVHD utilizing Isoplexis and correlates the cytokine profile with clinical disease severity.

As a pilot study, we collected normal and oGVHD patient tears which we analyzed on two backgrounds– standard Bovine Serum Albumin (BSA) or artificial tears (ATs). The controls were ATs (negative) and a concentrated cytokine solution (positive).

The cytokine levels of artificial tears alone were below the limit of detection (LOD) as expected. Out of a 22 cytokine panel, oGVHD patient tears had elevated TNF-α, TNF-β, perforin, MIP-1α, MIP-1β, MCP-1, IL-2, IL-4, IL-5, IL-7A, IL-9, IL-13, IL-15, IFN-γ, granzyme B, and GM-CSF with ATS background, but no cytokines above the LOD in the BSA background plate. The control tears had elevated IP-10. Elevated cytokines for the oGVHD patient corresponded to clinical symptom findings.

The results suggest that using ATs as the background improves sensitivity to detect tear cytokines on Isoplexis. Elevated IL-7A and GM-CSF in oGVHD tears parallel literature findings. Further sample evaluation will continue to validate Isoplexis for tear cytokine analyses.

Supported by Program for Research Initiated by Students and Mentors (PRISM), University of Maryland School of Medicine Office of Student Research and Eversight, Cigarette Restitution Fund, University of Maryland Greenebaum Comprehensive Cancer Center
Therapeutic improvement of Parkinson’s disease transplantation therapy through modulating the immune system
Authors: Park Tae Yoon , Kim Kwang-Soo
Published: August 2023

We report clinically the implantation of patient-derived midbrain dopaminergic progenitor cells (mDAPs), differentiated in vitro from autologous iPSCs, in a patient with idiopathic Parkinson’s disease (PD). The patient-specific mDAPs were produced under Good Manufacturing Practice conditions and characterized as having the phenotypic properties of substantia nigra pars compacta neurons; testing in a humanized mouse model indicated an absence of immunogenicity to these cells. The cells were implanted into the putamen (left hemisphere followed by right hemisphere, 6 months apart) of a patient with PD, without the need for immunosuppression. Clinical measures of symptoms of PD after surgery stabilized or improved at 18 to 24 months after implantation. Although our previous data (Schweitzer*, Park* et al., NEJM, 382(20) p1926–1932, 2020) showed that using autologous mDAPs can avoid graft rejection, cell survival and functional recovery in the first patient as assessed by 18F-DOPA PET scan analysis and clinical motor assessments was modest, suggesting that survival of mDA neurons may have been suboptimal even in autologous implantation. We have developed a safer and more effective treatment by not only increasing mDA neuron survival by simultaneously controlling the causative immune response, but also by modulating the problematic proliferative property, which is a characteristic of progenitor cells after implantation. In conclusion, our new study suggests that initial, specific mDA neuron survival in these grafts is strongly affected by the host innate immune response, while long-term graft survival is subject to the adaptive immune response, elucidating a two-phase involvement of the immune system for graft survival.

This work was supported by NIH grants (NS070577 and OD024622), the Parkinson’s Cell Therapy Research Fund at McLean Hospital and Massachusetts General Hospital, and the Masson Family Endowed Scholar in Neurosurgery, and the George A. Lopez, MD Endowed Chair in Neurosurgery.
Identification of Novel Immunosuppressive Drug Targets for Liver Transplantation Using Genome-Scale Metabolic Modeling
Authors: Aghamiri Sara Sadat , Puniya Bhanwar Lal , Amin Rada , Murry Daryl J , Helikar Tomáš
Published: August 2023

The global database on donation and transplantation reported 129,681 worldwide organ transplants in 2020, with kidneys and livers being the most commonly transplanted organs. Rejection of the transplanted organ by the host’s immune system is a common complication and a major reason for graft failure and patient mortality. To address these limitations, we developed genome-scale metabolic models of liver and CD4+ T cells using the pipeline for MetAbolic Drug Repurposing IDentification-MADRID and integrating our data analysis results from RNA-seq, proteomics, and microarray human sample datasets (step 1). Drug targets obtained from the ConnectivityMap database are mapped to metabolic genes in the model where systematic knockouts are performed on each gene which leads to identifying differential fluxes (step 2). Our human-cell-specific models identified novel immunosuppressive drug targets by integrating differentially expressed genes in post-liver transplanted non-tolerant versus healthy control individuals and differential fluxes (step 3). We identified 193, 217, and 196 potential metabolic gene targets for T-helper 1, 2, and 17, respectively, against patients with a non-tolerant phenotype after liver transplantation. This genome-scale metabolic modeling approach for liver transplantation can assist in personalized treatment by using any individual’s sample data at step 1 and 2. It can also save time and resources in preclinical drug development studies by predicting more precise drug targets and drug repurposing based on patients’ omics data. By using this approach, we aim to improve the success rates of liver transplantations and reduce the complications associated with organ rejection.

The work was supported by University of Nebraska collaboration initiatives.
Serum proteomic analysis of peripartum cardiomyopathy reveals a unique disease with distinctive dysregulation of inflammatory and cholesterol metabolism pathways
Authors: Lovell Jana , Bermea Kevin , Rousseau Sylvie , Cohen Charles D , Bhalodia Aashik , Zita Marcelle Dina , Yu Jinsheng , Head Richard D , Blumenthal Roger S , McNamara Dennis M , Sharma Garima , Adamo Luigi
Published: August 2023

Background

The pathophysiology of peripartum cardiomyopathy (PPCM) and its defining features when compared to other forms of cardiomyopathy remain unclear, thus limiting the development of targeted therapies.

Methods

To address this knowledge gap, we utilized a systems biology approach and performed high-throughput aptamer-based proteomic analysis (SOMAscan 7K) of serum samples from women with PPCM (n = 67) or non-peripartum cardiomyopathy (NPCM, n = 31), and age-matched peripartum and non-peripartum healthy women (n = 10 each). Samples were obtained from the Investigation in Pregnancy-Associated Cardiomyopathy and the Intervention in Myocarditis and Acute Cardiomyopathy studies. Comparative analyses were performed based on the presence of cardiomyopathy and peripartum status.

Results

Principal component analysis revealed distinct proteomic signatures of PPCM, NPCM, and each healthy control group (p<0.00001 for all pairwise comparisons). Comparative analyses of PPCM and NPCM identified hundreds of differentially expressed serum proteins before and after normalization to healthy controls. Pathway enrichment analyses of proteins differentially expressed in PPCM highlighted dysregulation of several biological pathways related to inflammation and cholesterol metabolism, including upregulation of the anti-inflammatory liver-X receptor/retinoid X receptor (LXR/RXR) pathway.

Conclusions

Serum proteomic profiling of PPCM indicates that PPCM is a unique disease entity characterized by the dysregulation of pro-inflammatory and cholesterol metabolism-related anti-inflammatory pathways. These pathways may represent potential novel therapeutic targets in PPCM.
Complement activation as a biomarker and a pathogenic factor in rhabdomyolysis-induced acute kidney injury
Authors: Roumenina Lubka T , Boudhabhay Idris , Grunenwald Anne
Published: August 2023

Rhabdomyolysis is a life-threatening condition caused by skeletal muscle damage with acute kidney injury (AKI) being the main complication dramatically worsening the prognosis. Specific treatment for rhabdomyolysis-induced acute kidney injury (RIAKI) is lacking and the mechanisms of the injury are unclear.

We discovered intra-kidney complement activation in tubules and vessels of patients and mice with rhabdomyolysis-induced acute kidney injury. Moreover, elevation of complement proteins and activation fragments in the urine of RIAKI patients compared to rhabdomyolysis without RIAKI and healthy donors by complement multiplex assays supported a pathogenic role of the complement system. Urinary C5a distinguished AKI, while Ba was elevated even for the patients without AKI, suggesting infra-clinical manifestations. Complement deficient mice (C3−/−, FB−/−) were protected from RIAKI. The transcriptomic signature of RIAKI mice included a strong inflammatory and apoptotic component, which were C3/complement-dependent, as they were normalized in C3 knockout mice. The intra-kidney macrophage population expressed a complement-sensitive phenotype, overexpressing CD11b and C5aR1. A kinetic mouse model of RIAKI revealed that heme toxicity is the first pathogenic mechanism to injure the tubules, evidenced by kidney transcriptomic signature (QuantiGene RNA assay), followed by neutrophils and inflammatory monocytes, upregulating C5aR1 (flow cytometry). Thus, our results demonstrate a direct pathological role of heme and complement in rhabdomyolysis-induced acute kidney injury.

CSL Behring research grant
Circulating Antibodies Specific for Dietary Fiber Glycans
Authors: Patnode Michael , Vega Giovanni , Hernandez-Kaempf Nicolette , Deshpande Nikita
Published: August 2023

The mammalian gut lumen contains an extremely diverse collection of microbial species and dietary constituents. Protein and carbohydrate antigens on commensal bacterial surfaces stimulate the production of specific antibodies, which can be secreted and bind their epitopes in the lumen. Despite the high abundance of plant polysaccharides (dietary fiber) in the intestine, it remains unclear whether these foreign carbohydrates stimulate the production of secreted anti-fiber antibodies. Here, we characterize antibody specificity in mice using a panel of artificial food particles composed of microscopic magnetic beads, each simultaneously tagged with a fluorescent barcode and coated with one of 64 different dietary or host glycan preparations. Using this approach, we detected secreted antibodies in the cecum and circulating antibodies in serum that selectively bind subsets of dietary glycans. Alteration of the host diet to include supplemental dietary fiber led to a significant increase in anti-fiber antibodies in serum. In contrast, cecal fiber-specific antibody reactivity was not increased after fiber feeding. Analysis of the gut microbiota revealed that relative abundances of Akkermansia muciniphila were significantly higher in mice fed supplemental fiber relative to mice fed a control diet. This work raises the possibility that taxa such as A. muciniphila are involved in the production of antibodies specific for dietary components.

Supported by a grant from NIH (K01 DK124445)