bluuebunny/crossref_metadata_2025 huggingface dataset:
Functional evidence for TCR scanning of MHC.
Authors: Kuhns Michael S , Parrish Heather
Published: January 2023

αβ T cells recognize antigenic peptides within their positively selecting self-MHCI or MHCII. While MHC-restriction is well established, the nature of the TCR-MHC interactions that mediate this phenomenon are intensely debated. One possibility is that clonotypic TCRs are positively selected based on their ability to recognize self-peptides within self-MHC (pMHC) in a lock-and-key fashion. Such TCRs would then be limited to scanning for antigenic peptides among those shapes that resemble the selecting pMHC. Alternatively, every TCR may poses germline-encoded specificity for most if not all allelic variants of both MHCI and MHCII. This would allow most TCRs to scan the contents of most MHC for peptides that could mediate positive selection or T cell activation depending on the contributing co-receptor (i.e. CD4 or CD8). Here we asked if increasing the frequency of Lck-associated CD4 molecules in T cell hybridomas would allow for the detection of sub-threshold TCR-MHC interactions that constitute scanning. The reactivity of ten distinct TCRs was assessed in response to selecting and non-selecting MHCII bearing “shaved” peptides with alanine substitutions at TCR contact residues: three of the TCRs were selected on MHCII, two were selected on MHCI, and five were generated in vitro without defined selecting MHC. IL-2 production was observed when each TCR interacted with selecting or non-selecting MHCII presenting shaved peptides. These data provide evidence of TCR-MHC interactions that are independent of the selecting MHC class, allele, or the sequence of the peptide presented by the MHC. We interpret these data as functional evidence for TCR-intrinsic specificity for MHCII.
The MAP kinase, tumor progression locus 2 (Tpl2), activates the Akt/mTOR/S6 ribosomal protein pathway, inhibits Foxp3 expression and limits Treg immunosuppressive functions.
Authors: Li Xin , Acuff Nicole V , Peeks Angela , Kirkland Rebecca , Nagy Tamas , Watford Wendy Tharpe
Published: January 2023

The serine-threonine kinase, tumor progression locus 2 (Tpl2 or Map3k8/Cot), is a potent inflammatory mediator that drives the production of TNF alpha, IL-1 beta, and IFN gamma. We previously demonstrated that Tpl2 modulates TCR signaling and T helper cell differentiation. However, little is known about how Tpl2 alters the development or function of regulatory T cells (Tregs). Tregs are a specialized subset of T cells that express Foxp3 and possess immunosuppressive properties. Because of Tpl2’s role in promoting inflammation, we hypothesized that Tpl2 inhibits Treg development and immunosuppressive functions. Tpl2−/− naïve CD4 T+ cells preferentially differentiated into Foxp3+ inducible Tregs in vitro as well as in vivo in a murine model of OVA-induced systemic tolerance. Treg biasing in Tpl2−/− T cells depended upon the TCR signal strength and correlated with reduced activation of the Akt/mTOR/S6 pathway. Importantly, freshly isolated Tpl2−/− Tregs had increased expression of Foxp3 and immunosupressive cytokines IL-10 and IL-35 and were more suppressive in a T cell transfer model of colitis, as evidenced by reduced accumulation of inflammatory T effectors, systemic inflammatory cytokines TNF, IL-6 and IFN gamma, and colonic inflammation. Therefore, Tpl2 promotes inflammation in part by limiting Foxp3 expression, iTreg differentiation and immunosuppressive functions. These findings suggest that Tpl2 inhibition may be used to preferentially drive Treg induction and limit inflammation in autoimmune diseases.
EphA2 prevents breakdown of the blood-brain barrier in experimental cerebral malaria.
Authors: King Thayer , Mimche Patrice , Bray Christian , Brady Lauren , Meyer Esmeralda , Galinski Mary , Lamb Tracey
Published: January 2023

Cerebral malaria (CM) is a leading cause of death from Plasmodium falciparum infection, yet the mechanisms of disease pathology are poorly understood. While Eph receptors have been linked to several neurological disorders, their roles in malaria remain largely unknown. Experimental cerebral malaria (ECM) is a lethal condition resulting from Plasmodium berghei ANKA (PbANKA) infection of mice on a C57BL/6J background that recapitulates key features of human CM including blood-brain barrier (BBB) breakdown. Here we show that PbANKA-infected EphA2 −/− mice, but not EphA2 +/+ mice, have an intact BBB at ECM onset. CD8+ T cells mediate ECM development, and EphA2 −/− mice exhibit less accumulation of CD8+ T cells in the brain despite normal splenic expansion providing a mechanism by which EphA2 deficiency may preserve BBB integrity. Upregulation of EphA2 mRNA in brains of PbANKA-infected C57BL/6J mice is a feature of ECM absent in non-lethal Plasmodium infections such as PbNK65 where CD8+ T cells are present in the brain but do not disrupt the BBB. Unlike EphA2 +/+ mice, PbANKA-infected EphA2 −/− mice are protected from death even with peripheral and organ-sequestered parasite levels similar to EphA2 +/+ mice suggesting BBB integrity, not parasite burden, is responsible for improved survival in EphA2 −/− mice. Therapeutically targeting EphA2 by blocking EphA2 activation or interaction with membrane-bound ephrin-A ligands on T cells leads to increased survival of PbANKA-infected C57BL/6J mice. We also observe a parasite-dependent upregulation of both ephrin-A mRNA in CD3+ human PBMCs and EphA2 mRNA in human brain endothelial cells. Taken together, our data suggests a novel and crucial role for EphA2 in ECM with potential translation to human CM.
Neutrophils license antifungal monocyte responses via carbonic anhydrase 4 modulation
Authors: Rivera Amariliz , Espinosa Vanessa , Dutta Orchi , Henry Everett K , Siracusa Mark C
Published: January 2023

Neutropenia is a significant risk factor for life threatening invasive fungal infections (IFI). Neutrophils are well known as important innate cells that promote the direct eradication of fungal pathogens but whether they mediate antifungal defense beyond their role as effectors is unclear. Here we demonstrate that a pulmonary infection with the clinically relevant fungal pathogen Aspergillus fumigatus induces the diversification of specialized antifungal neutrophils that are required for antifungal CCR2+ monocyte-derived dendritic cell (mo-DC) function. Selective depletion of neutrophils resulted in global transcriptional alterations of the antifungal CCR2+ monocyte response, limited mo-DC differentiation, and diminished conidiacidal activity. Impaired mo-DC antifungal activity in neutropenic mice was accompanied by significant upregulation of carbonic anhydrase 4 (Car4) expression in CCR2+ monocyte precursors. Pharmacological inhibition of Car4 with the FDA-approved drug methazolamide (MZ) rescued the antifungal response of mo-DC and protected neutropenic mice from invasive apergillosis. Thus, beyond their role as effectors, antifungal neutrophils facilitate antifungal mo-DC functions by regulating Car4 activity. Moreover, our data provide proof-of-principle evidence for the importance of carbonic anhydrases in shaping innate cell differentiation as well as for the off-label therapeutic benefit of carbonic anhydrase inhibitors to boost antifungal immunity in susceptible patient populations.
Inflammation and immune cell recruitment in the CNS following disseminated Cryptococcus neoformans infection
Authors: Neal Lori M , Xing Enze , Xu Jintao , Osterholzer John J , Olszewski Michal A
Published: January 2023

Mortality in Cryptococcus neoformans (Cn) infection is chiefly associated with CNS infection but little is known about immune responses against Cn within the CNS. We established an i.v. model of disseminated cryptococcosis that leads to a rapid expansion of Cn in the CNS in order to compare inflammatory readouts in resistant CBA/J with susceptible C57BL/6 mice. Fungal burdens peaked (>107CFU) in the brain by d7 post infection in both models, but in CBA/J mice fungal burden significantly declined between d14 to d21. In contrast, C57BL/6 mice developed persistently high fungal burdens through d21. Consistent with these findings CBA/J mice showed gradual increases in Ly6C+ myeloid, CD4+ and CD8+ T cell infiltration along with production of CCL2, IFNγ and CXCL10, reaching maximum or near maximum levels by day 14. In C57BL6, the development of inflammatory responses was significantly delayed. Production of cytokines and influx of Ly6C+, CD4+ and CD8+ cells was almost absent until d21, but prominently exceeded all readouts from CBA/J at day 21 by several fold. Significant increases in iNOShi CD11bhi MHCIIhi CD11c+ cells and an overabundance of highly activated CD11c+ MHCII+ microglia, hallmarks of rapidly developing CNS pathologies, were also more evident in the brains of C57BL/6 mice at d21. In summary, our findings are consistent with the hypothesis that early recruitment and activation of inflammatory cells in the CNS post-Cn infection, presumably in response to early CCL2-induction, along with subsequent early induction of IFNγ and CXCL10 promotes fungal clearance. In contrast, delayed responses found in C57BL6 mice were ineffective at removing fungal organisms while leading to over exuberant inflammation and detrimental CNS pathology.
Neutrophil recruitment during Leishmania infection: The role of sand fly salivary proteins
Authors: Guimaraes Anderson , Wen Xi , Carvalho Augusto Marcelino , Brzostowski Joseph , Valenzuela Jesus , Oliveira Fabiano
Published: January 2023

Neutrophils are the first cells recruited to the site of an infected sand fly bite and interact with Leishmania parasites. It has been proposed that the recruitment of neutrophils to the site of the bite increases the ability of Leishmania to establish an infection. Here we investigate the effect of sand fly salivary proteins on neutrophil recruitment. We purified bone marrow neutrophils from C57BL/6 mice or from peripheral blood of healthy human donors. Chemotaxis driven by sand fly saliva of vectors of visceral (Lutzomyia longipalpis) or cutaneous (Phlebotomus duboscqi) disease was measured by modified Boyden chamber assay and by imaging with the EZ-TAXIScan assay. Neutrophils migrated towards saliva glands of the vectors P. duboscqi and L. longipalpis, in a dose-dependent manner. Proteinase K treatment of saliva completely abrogated neutrophil recruitment, suggesting a protein as the chemotactic factor. To unearth the identity of the chemotactic factor, we have generated plasmids coding for the 20 most abundant salivary proteins from P. duboscqi and injected them in the ears of C57BL/6 mice. Neutrophil recruitment was analyzed by flow cytometry staining. We have identified 3 different salivary protein-coding plasmids with neutrophil recruitment activity. Finally, we have produced these 3 recombinant proteins in the HEK293 cell line system and we observed that a family of 40kDa salivary proteins acts as chemokines for neutrophils. We plan to validate the role of the salivary chemotactic factor in vivo and test if its neutralization can alter Leishmania infection. We put forward the hypothesis that blockage of this protein will disrupt neutrophil migration early in the Leishmania infection possibly altering the disease outcome.
Exosomes in human semen impair antigen-presenting cell function and decrease antigen-specific T cell responses
Authors: Vojtech Lucia N , Hughes Sean , Levy Claire , Taber Alexandria , Calienes Fernanda , Hladik Florian
Published: January 2023

Exposure to semen is the primary route of transmission for many sexually transmitted infections. Accumulating evidence suggests that components in semen directly impair leukocytes, which could compromise the protective efficacy of vaccine-induced immune responses in the mucosa. Exosomes, small microvesicles with immunomodulatory functions, are present at an average concentration of 2.2 × 1013 particles per ejaculate (n = 18). These seminal exosomes (SE) efficiently and rapidly entered peripheral and vaginal dendritic cells (DCs), whereas T cell uptake was poor. In PBMC cultures, SE impaired memory T cell function, reducing the production of TNF-alpha, IL-2 and/or IFN-gamma in response to virus-derived peptides an average of 60% for CD4+ T cells and 48% for CD8+ T cells, in a dose-responsive manner (n = 6). Reduction of cytokine production by memory CD8+ T cells was reduced to a greater extent for protein antigens as compared to peptide antigens, implying that antigen-presenting cell function was impaired by the presence of SE. Exposing only DCs, as opposed to bulk PBMCs, to SE also blocked subsequent CD8+ memory T cell activation, reducing the proportion of cells making cytokines by 22% (n = 5 donors). Expression of CD107a, a marker of cytotoxic T cell degranulation, was decreased by 42% in PBMC cultures exposed to SE and superantigen (n=4). SE also impaired vaginal T cell cytokine and degranulation responses to superantigen. Thus, SE likely inhibit antigen-specific T cell responses by impairing antigen-presenting cell function. Understanding how programmed immune responses are altered by the presence of semen is important to developing the next generation of vaccine and preventative treatments against sexually transmitted disease
Small intestinal Goblet cells and Goblet cell associated antigen passages assist translocation of pathogenic bacteria
Authors: Kulkarni Devesha Hrishikesh , McDonald Keely G , Knoopr Kathryn , Miller Mark J , Newberry Rodney D
Published: January 2023

The intestinal epithelium forms an essential physicochemical barrier that separates luminal bacteria from underlying immune cells of the intestinal mucosa. Appropriate exposure of the immune cells to luminal antigens is crucial to ensure intestinal homeostasis and to respond to enteric infections. We recently demonstrated a previously unappreciated role of small intestinal (SI) Goblet cells (GCs) in delivering luminal antigens across the epithelium by forming goblet-cell associated antigen passages (GAPs) in the steady state. How pathogenic bacteria affect the formation of SI GAPs and the ability of pathogens to use GAPs as a portal of entry is largely unexplored. Here we report that in conventionally housed mice, luminal exposure to invasive Salmonella typhimurium, but not mutant non-invasive S. typhimurium, results in inhibition of SI GAPs within minutes. Moreover, overriding the ability of GCs to sense the pathogen by deleting MyD88 in GCs or inhibiting EGFR activation downstream of MyD88 during S. typhimurium infection allowed GAP formation, suggesting that GC intrinsic sensing of an invasive pathogen to shut off GAPs is a host defense mechanism. Fluorescent wildtype S. typhimurium preferentially localized around and within GCs forming GAPs. Deletion of GCs prevented the translocation of wildtype S. typhimurium to the SI draining mesenteric lymph node following oral infection, indicating that S. typhimurium uses GCs as a portal of entry. These findings identify a role of GC sensing of an invasive pathogen in inhibiting antigen delivery to the immune system and in limiting the translocation of pathogenic bacteria.
Dysregulation of iCD8α cells promotes susceptibility to intestinal inflammation
Authors: Olivares-Villagómez Danyvid
Published: January 2023

iCD8α cells are a population of innate immune cells present in the intestinal intraepithelial lymphocyte (IEL) compartment, characterized by the expression of CD8αα. We have shown that iCD8α cells play a critical role during immune responses in the intestinal mucosa, however, little is known about the role of iCD8α cells during inflammatory processes of the intestines. The thymus leukemia (TL) antigen is predominantly expressed by intestinal epithelial cells and preferentially interacts with CD8aa. We have shown that CD8αα+ IEL from TL-deficient mice have increased cytokine production and proliferation, indicating that TL controls CD8αα+ IEL effector functions. Rag-deficient mice facilitate the study of innate immune cells, and are an excellent model for the study of iCD8α cells because these cells are the only CD8αα+ IEL present in these mice. Here we present evidence showing that TL regulates the functions of iCD8α cells. Using the anti-CD40 antibody model of colitis, we show that TL−/−Rag-2−/− mice lose more weight, have higher mortality, have more colon damage, and present higher levels of pro-inflammatory cytokines (IFN-γ, IL-6 and OPN) than colons from Rag-2−/− mice. Interestingly, we show that colons and iCD8α cells from TL-deficient mice produce more granzyme than colons or iCD8α cells from TL-competent mice. Moreover, stimulation of iCD8α cells with the pro-inflammatory cytokine IL-12 in vitro induces granzyme secretion. Therefore, we propose that the increased colon inflammation observed in TL-deficient mice is due to: a) dysregulation of iCD8α cells by the lack of TL, and b) increased granzyme secretion by iCD8α cells which may serve as a pro-inflammatory signal.
Balance of Commensal Bacteria-Specific Th17 and RORγt+ Treg Cells in Intestinal Homeostasis and Inflammation
Authors: Xu Mo , Yang Yi , Pokrovskii Maria , Galan Carolina , Littman Dan R
Published: January 2023

A number of Helicobacter species, including Helicobacter hepaticus, have been isolated as the etiological agents of ulcerative inflammatory bowel disease and cancer in their respective hosts. H. hepaticus induces severe Th17-Th1 mediated colitis in a number of immuno-compromised mouse strains, but does not usually cause disease in most wild-type mouse strains. However, how the inflammatory response is carefully contained to be harmless under homeostatic conditions and how perturbations lead to pathogenicity remain elusive questions. To understand this, we developed new research tools, including TCR transgenic mice, as well as MHCII tetramers to interrogate the regulation of H. hepaticus-specific CD4+ T cell function. Interestingly, in contrast to Segmented Filamentous Bacteria (SFB), which induces Th17 cells in the small intestinal lamina propria (SILP) of both IL10+/− and IL-10−/− mice, H. hepaticus mainly induces RORgt+ Treg cells in the large intestinal lamina propria (LILP) of IL-10-sufficient mice, but induces a Th17-Th1 response in the absence of IL-10. We propose that the balance between self/commensal antigen-induced RORgt+ Treg and Th17 cells may be a general strategy to control autoimmune inflammation.
Inducible expression of CXCL1 within the CNS amplifies demyelination in pre-clinical models of multiple sclerosis
Authors: Grist Jonathan Jeffrey , Marro Brett S. , Lane Thomas
Published: January 2023

The functional role of the ELR+ chemokine CXCL1 in host defense and disease in two pre-clinical mouse models of the human demyelinating disease multiple sclerosis (MS) was assessed. Mice in which expression of CXCL1 is under the control of a tetracycline-inducible promoter active within GFAP-positive cells were generated and infected intracranially with the neurotropic JHM strain of mouse hepatitis virus (JHMV) or immunized with myelin oligodendrocyte glycoprotein (MOG)35-55 to induce experimental autoimmune encephalomyelitis (EAE). In both JHMV-induced neurologic disease and EAE, overexpression of CXCL1 by astrocytes resulted in increased clinical disease severity. Immunophenotyping cells infiltrating into the central nervous system (CNS) revealed a selective increase in Ly6G+CD11b+ neutrophils (but not Ly6G−Ly6C+CD11b+ monocytes) present within the CNS. In JHMV infected mice and MOG35-55-immunized mice, the T cell response to antigen with regard to proliferation and cytokine production was not altered, nor was trafficking into the CNS affected. In both models, increased CXCL1 expression within the CNS resulted in increased morbidity that correlated with selectively elevated neutrophil infiltration, diminished numbers of mature oligodendrocytes, and an increase in the severity of demyelination. Neutrophil ablation in CXCL1-transgenic mice reduced the severity of demyelination in mice arguing a role for these cells in white matter damage. Collectively, these findings illustrate that sustained CXCL1 expression amplifies the severity of white matter damage and neutrophils can contribute to this process in two different mouse models of MS.
Vector mediated in vivo induction of antigen-specific regulatory T cells abrogates clinical and histological signs of EAE
Authors: Hoffman Brad E , Keeler Geoffrey D
Published: January 2023

Induction of peripheral immune tolerance requires regulatory T cells (Tregs). Protocols designed to manipulate antigen (Ag) specific-Treg populations in vivo, represent a powerful approach for treating autoimmune disease such as Multiple Sclerosis (MS). It has been shown that Ag-specific Tregs can play a critical role in the protection and recovery of MS, and its animal model, experimental autoimmune encephalomyelitis (EAE). Unfortunately, methods to induce endogenous CNS-targeting Ag-specific Tregs that achieve therapeutic efficacy are lacking.

To address this, we developed an immune tolerance induction therapy using an AAV vector expressing the full coding sequence for the neuro-protein MOG under the control of a strong liver specific promoter. Our data demonstrates that peripheral injection of this vector induces functionally suppressive transgene-specific Tregs, and mice pre-treated with this vector are protected from developing EAE. More notably, when vector is administered during mild to moderate disease, there is a nearly complete reversal of EAE, both clinically and histopathologically. When treatment is withheld until mice exhibit moderately severe EAE, our therapy still significantly reduced the neurological symptoms and clinical score, although to a lesser degree. Thus, to increase efficacy of the vector induced Tregs, a short course of rapamycin treatment was given concomitantly which had a synergistic affect, resulting in a long-term significant reversal of disease. Using our vector platform to deliver full-length proteins offers a superior MHC/HLA independent approach for in vivo induction of Ag-specific Tregs compared to other ex vivo or epitope restricted Treg mediated therapies being investigated.
Towards the repair of marginal liver grafts: Ex vivo Mesenchymal stromal cell perfusion
Authors: Bartczak Agata , Ma Xue-Zhong , Manuel Justin , Zhang Xing-Hua , Cifuentes Juan Andres Echeverri , Linares Ivan , Selzner Markus , Keating Armand , McGilvray Ian
Published: January 2023

More than 25% of persons listed for a liver transplant in Canada die before obtaining a suitable liver graft. This occurs despite the recent inclusion of livers from deceased after cardiac death (DCD) organ donation. Over half of possible DCD livers undergo long periods of ischemia that sensitize the liver to reperfusion injury (HIRI) and render the liver grafts unsuitable (marginal) for transplantation. Mesenchymal Stromal cells (MSC) protect against HIRI by decreasing the production of inflammatory mediators secreted by liver resident macrophages. We hypothesized that MSC-treatment of marginal DCD grafts on an ex-vivo perfusion circuit (NEVLP) would repair HIRI damage. Kupffer Cells (KCs) are central to HIRI: they are activated after reperfusion and release various inflammatory cytokines, including TNFα, which promote hepatocyte death. We have developed a DCD liver graft model to evaluate the potential of MSC treatment. In this model, liver grafts undergo 45-minute warm ischemia followed by 2-hours of cold ischemia before being placed on a normothermic ex-vivo perfusion unit for 6 hours. We found that the infusion of MSCs into the ex-vivo perfusion circuit decreases the levels of TNFα in the perfusate. This data was confirmed in in vitro studies where the co-culture of LPS-activated KC with MSCs decreased the amount of TNFα in culture supernatants compared to KC alone cultures. The levels of TNFα in the supernatant were further decreased in cultures treated with LPS-primed MSCs. We propose that the combination of MSC treatment and NEVLP perfusion of DCD livers will repair HIRI-injured and can be rapidly translated to the clinic to expand the donor organ pool.
Response to extracorporeal photopheresis in chronic graft versus host disease correlates with levels of IL-10 producing regulatory B cells
Authors: Chandra Daniel J , Hosoba Sakura , Giver Cynthia , Waller Edmund K
Published: January 2023

Chronic graft versus host disease (cGVHD) continues to be a major source of morbidity and mortality in allogeneic hematopoietic stem cell transplant (allo-HSCT) patients, and current therapies are limited by severe toxicity and immunosuppression. Numerous studies have confirmed the efficacy of extracorporeal photopheresis (ECP) in the treatment of steroid refractory cGVHD without inducing immunosuppression. Although studies have posited a role for immunoregulatory cells in the mechanism of ECP, the immunological effects of ECP are not completely understood. The purpose of this study was to evaluate the role of IL-10 producing regulatory B cells (Bregs) in ECP, and we hypothesized that patients who responded to ECP would have a higher frequency of Bregs over the course of their treatment. We evaluated 10 patients with various stages of cGVHD who received ECP therapy at Emory University from 2013 to 2015. ECP responders were defined as patients who were able to reduce their steroid dose by 50% within 4 months of starting treatment with stable or improved clinical GVHD scores. By measuring CD19+ IL-10+ cells as a proportion of total lymphocytes, we found that ECP responders had a significantly higher percentage of Bregs at 2 months (mean 3.7 vs. 0.08; p = 0.02) and 4 months (mean 2.2 vs. 0.15; p = 0.02) after initiating ECP compared with non-responders. When analyzing for overall B cell frequency, we found a significantly higher percentage of B cells in ECP responders at 2 months (mean 27.6 vs. 0.47; p = 0.02) and 4 months (mean 10.1 vs. 0.77; p = 0.02) post-ECP. Our results are consistent with studies reporting a Breg defect in cGVHD and suggest that there is a correlation between clinical response to ECP therapy and Breg frequency.
The effect of lymphodepletion on specificity and function of cytomegalovirus-responsive CD8 T cells in transplant recipients
Authors: Higdon Lauren E , Margulies Kenneth B , Maltzman Jonathan S
Published: January 2023

Polyfunctional CD8 T cells produce multiple cytokines and effector molecules in order to control chronic viral infections, such as cytomegalovirus (CMV), a cause of allograft damage in transplant patients. Healthy individuals resolve primary CMV infection rapidly and control viral reactivation with memory T cells. IFNγ production by CD8 T cells protects from CMV, but the role of polyfunctional T cells in immunosuppressed transplant patients is not fully understood. We measured responses to CMV polypeptides IE-1 and pp65 to characterize the effects of T cell depletion on T cell specificity and polyfunctionality. In this study we identified polyfunctionality as co-expression of IFNγ, TNFα, and/or the degranulation marker CD107a. We analyzed T cell responses in pre- to a year post-transplant PBMC samples from cardiac and renal transplant patients. Treatment included anti-viral prophylaxis and 3 drug immunosuppression, with T cell depleting induction therapy in some patients. The 3 normal donors and 12 pre-transplant samples analyzed fall into 3 groups on the basis of IE-1 and pp65 specificity: equal response to both or dominant for either IE-1 or pp65. Almost all cells with exactly two functions were TNFα+. In some patients, lymphodepletion led to an early loss of polyfunctional T cells. However, polyfunctional CD8 T cells returned to pre-transplant levels by day 180. In addition, 3 patients displayed shifts from their pre-transplant IE-1 vs pp65 specificity post-transplant, while specificity was consistent over a year post-transplant in the other 8. In conclusion, pre-transplant TNFα expression is predictive of polyfunctionality, and further study is needed to determine the effect of T cell depletion on fine antigen specificity.
Islet encapsulation with tannic acid-containing nanothin coatings modulates immune responses and restores euglycemia upon islet transplantation
Authors: Tse Hubert Ming , Padgett Lindsey E. , Pham-Hua Dana , Xue Bing , Anderson Brian , Zeiger Michael , Kozlovskaya Veronika , Kharlampieva Eugenia
Published: January 2023

Type 1 diabetes (T1D) is an autoimmune disease resulting in pancreatic b-cell lysis via reactive oxygen species (ROS), pro-inflammatory cytokines, and islet-infiltrating leukocytes. One promising treatment for T1D is islet transplantation, but its clinical application is constrained due to islet availability, adverse effects of immunosuppressants on islet function, and declining graft survival. Islet encapsulation may provide an immunoprotective barrier to aid in preserving function and curbing immune-mediated rejection post transplantation. We previously demonstrated that a cytoprotective nanothin multilayer coating consisting of tannic acid (TA), an immunomodulatory antioxidant, and poly (N-vinylpyrrolidone) (PVPON), curtailed immune responses involved in transplant rejection. We hypothesized that (PVPON/TA) coating may hinder the synthesis of redox-sensitive chemokines that recruit immune cells to sites of islet engraftment and blunt ROS-dependent cues necessary for M1 macrophage differentiation. (PVPON/TA) coatings elicited a 7-fold decrease in M1 macrophage-derived pro-inflammatory chemokines CCL5 and CXCL10, blunted M1 macrophage activation markers, and curbed diabetogenic CD4 T cell migration approximately 2-fold. Importantly, murine islet encapsulation with (PVPON/TA) material restored euglycemia in immunodeficient-diabetic mice post transplantation. Our studies demonstrate that (PVPON/TA) multilayer coatings represent a promising immunomodulatory biomaterial for islet transplantation. Future studies will evaluate the efficiency of PVPON/TA coated islets in curbing hyperglycemia by allotransplantation into diabetic rodent models.
Obese mice exhibit accelerated immune and myeloid reconstitution after congenic hematopoietic stem cell transplantation
Authors: Khuat Lam Tan , Mirsoian Annie , Pai Chien-Chun Steven , Abedi Mehrdad , Murphy William J
Published: January 2023

The obesity rate is increasing in the United States with more than a third of U.S. adults being obese. Obesity is a form of sterile chronic inflammation, termed “meta-inflammation”, which is local, chronic and triggered by metabolical pathways. Therefore, there is a need to uncover the potential role of obesity in the development of immune cell subsets post-HSCT. In this study, we wanted to assess the impact of obesity on immune reconstitution following congenic HSCT in lean and DIO (diet-induced obese) recipients. C57BL/6 mice received lethal radiation dose at 1100cGy and 5 million T cell-depleted bone marrow cells from H2-matched B6.SJL mice. Our data demonstrated that DIO recipients displayed a more rapid and robust reconstitution affecting both myeloid and lymphoid compartments. DIO recipients had increased neutrophils, platelets and total lymphocytes post-HSCT compared to control mice. Colony forming unit-granulocyte macrophage (CFU-GM) assays showed significantly higher numbers in DIO recipients at day 14 post-HSCT. Organ cellularity from thymus, spleen and bone marrow were increased in DIO recipients compared to lean mice at day 14 and 21 post-HSCT. Higher numbers of CD4+CD8+ thymocytes, as well as higher number of NK cells and B cells in the spleen and lymph nodes were also observed. Splenocytes from DIO recipients had greater proliferative ability either after stimulation with concanavalin A or in mixed lymphocyte reaction compared to lean controls. Taken together; these results indicated that obesity results in acceleration of engraftment and donor immune reconstitution.
Human cytokine-induced memory-like NK cells expand in patients with AML and display enhanced anti-leukemia responses.
Authors: Berrien-Elliott Melissa M , Romee Rizwan , Wagner Julia , Rosario Maximillian , Jewell Brea , Schappe Timothy , Leong Jeffery , Abdel-Latif Sara , Willey Sarah , Yu Liyang , Oh Stephen , mulder Arend , Class Frans , Fehniger Todd A.
Published: January 2023

NK cells are innate lymphoid cells that mediate anti-tumor responses. Relapsed/refractory AML patients have a poor prognosis and new therapy approaches are needed. Recently, memory-like properties of NK cells have been discovered, including cytokine-induced memory-like NK cells. Human NK cells briefly pre-activated with IL-12, IL-15, and IL-18 and then allowed to differentiate in vitro have greater re-stimulation responses compared to control cells. We hypothesized that such human memory-like NK cells possess enhanced anti-leukemia functionality. Indeed, memory-like NK cells produced increased IFN-γ in response to primary AML blasts in vitro, compared to controls. Multi-dimensional mass cytometry analyses revealed minimal alterations of memory-like NK cell inhibitory KIR repertoire, and both KIR-ligand matched and mismatched memory-like NK cells responded to primary AML blasts. In the context of a first-in-human clinical trial, allogeneic memory-like NK cells expanded to become the majority of the cells in the blood and bone marrow from AML patients 7–14 days after infusion. Furthermore, donor memory-like NK cells displayed enhanced leukemia-triggered IFN-γ production compared to recipient NK cells in the same samples, and clinical responses have been observed in 3 of 6 evaluable patients. In one patient, mass cytometry revealed expansion of CD3− CD56+ CD8+ NK cells with restricted KIR expression and multiple activating receptors. Deep NK cell immunophenotyping of in vivo expanded memory-like NK cells in additional patients is ongoing. Since memory-like NK cells expand in vivo and display enhanced anti-leukemia functionality, they represent a promising immunotherapy approach for patients with AML.
Generation of catalytic antibodies is an intrinsic property of an individual’s immune system: a study on a large cohort of renal transplant patients
Authors: Mahendra Ankit , Peyron Ivan , Thaunat Olivier , Dollinger Cécile , Gilardin Laurent , Sharma Meenu , Wootla Bharath , Rao Desirazu N. , Padiolleau-Lefevre Séverine , Boquet Didier , More Abhijit , Varadarajan Navin , Kaveri Srini , Legendre Christophe , Lacroix-Desmazes Sébastien
Published: January 2023

The origin and physiopathological relevance of catalytic antibodies is not well understood owing to the fact that catalytic antibodies have been studied in relatively small cohorts of patients with rare diseases and/or without systematic follow-up. In the present study, we have followed the evolution of the levels of catalytic IgG in a large cohort of 100 renal transplant patients over a 2-year period. Prior to transplant, hydrolysis of the generic substrate PFR-MCA was greater for patients’ IgG than for a therapeutic preparation of pooled IgG from healthy donors (6.6±0.9 vs 0.65±0.03 fmol/min per pmol). Renal transplant was marked by a drastic decrease in levels of catalytic IgG over 3 months (6.6±0.9 vs 2.4±0.2 fmol/min/pmol; P<0.0001) followed by a steady increase at 12 months (3.2±0.3 fmol/min/pmol; P=0.015) and further at 24 months (5.1±0.6 fmol/min/pmol; P=0.004). When divided into quartiles based on the rates of IgG-mediated PFR-MCA hydrolysis measured in pre-transplant samples, the IgG catalytic activity in the upper quartile of patients was significantly high both pre-transplant (12.03±1.6 vs 2.7±0.2 fmol/min/pmol, P<0.0001) and 24 months post-transplant (6.8±1.2 vs 4.6±0.7, fmol/min/pmol, P=0.0004). Interestingly, IgG-mediated hydrolysis of a model protein substrate, pro-coagulant factor VIII, did not correlate with that of PFR-MCA prior transplantation, while it did 12 months post-transplant (P<0.0001, R2=0.4). Taken together, our results suggest that the level of circulating catalytic IgG under pathological conditions is an intrinsic property of each individual’s immune system, and that recovery of pre-transplant levels of catalytic IgG is accompanied by changes in the repertoire of target antigens.
Levels of anti-Gal IgG2 negatively correlate with the survival of porcine islet graft in non-human primates under immunosuppression of anti-CD154
Authors: Kang Hee Jung , Lee Haneulnari , Park Eun Mi , Kim Jong Min , Park Chung-Gyu
Published: January 2023

Background

Anti-CD154, the most successful immunosupppression (IS) regimen in porcine islet transplantation (PITx) is prohibited for clinical use. Anti-CD40, an alternative to anti-CD154, is not as effective as anti-CD154. To understand the reason, the relationship between anti-Gal responses and the graft survival was investigated in rhesus monkeys of PITx.

Methods

Intraportal PITx was performed in 18 diabetic monkeys: 3 received various IS without CD40 pathway blockade (Tx-control); 12, IS with anti-CD154 (Tx-anti-CD154); and 3, IS with anti-CD40 (Tx-anti-CD40). Blood samples were weekly obtained from the monkeys after PITx until the graft ceased to function, and additionally from 3 monkeys without transplantation (non-Tx-control) for 6 months. Levels of anti-Gal IgG, IgG1, IgG2 and IgM in the samples were analyzed by in-house ELISA, and levels of D-dimer and high-sensitive CRP by turbidimetry.

Results

Intraportal PITx led to strong induction of anti-Gal IgG1 response, not IgG2, in Tx-control, which was suppressed in both Tx-anti-CD154 and Tx-anti-CD40. The level of anti-Gal IgG2 revealed negative association with the levels of CRP and D-dimer in Tx-anti-CD40 and non-Tx-control, not in Tx-anti-CD154. The time-weighted average levels of anti-Gal IgG2 in Tx-anti-CD154 were significantly lower than in non-Tx-control. Additionally, the levels of anti-Gal IgG2 and D-dimer negatively correlated with the graft survival in Tx-anti-CD154.

Conclusions

The strong correlation between the suppression of anti-Gal IgG2 response and the graft survival in Tx-anti-CD154 suggests that IgG2 response is implicated with the effector mechanism mediating rejection, possibly NK cell activation which is known to promote IgG2 response.
Biological role of T Memory Stem Cells as a Cellular Reservoir for Graft-versus-Host Disease
Authors: Tkachev Victor , Furlan Scott N , Watkins Benjamin K , Panoskaltsis-Mortari Angela , Blazar Bruce R , Kean Leslie S
Published: January 2023

T memory stem cells (Tscm) are a subset of antigen-experienced, long-lived, self-renewing and multipotent T cells, which reconstitute immunity following hematopoietic cell transplantation (HCT). Here we investigated the biological role of Tscm in acute Graft-versus-Host Disease (GVHD) using a translational non-human primate model.

Tscm cells were tracked longitudinally following autologous or allogeneic HCT under different immunoprophylaxis regimens, resulting either in fulminant Th1-dependent ‘Primary GVHD’, late-onset Th17-driven ‘Breakthrough GVHD’, or GVHD-free immune tolerance.

Following HCT, Tscm acquired an activated phenotype (Ki67+, CD69+, PD-1+ and CCR5+), but produced low amounts of IFNγ, IL-2, IL-17A, TNFα and Granzyme B. Tscm were a minor population in donor grafts but expanded in peripheral blood (with a reciprocal decline of naïve T cells) early after HCT and then contracted (with a concomitant increase of effector T cells) when recipients developed clinical GVHD. The kinetics of Tscm expansion/contraction correlated closely with clinical disease: Tscm expanded slower and contracted later in animals progressing to Breakthrough compared to Primary GVHD, and Tscm numbers remained stable under tolerizing conditions. At necropsy, animals with GVHD had increased numbers of Tscm in both lymphoid organs (blood, lymph nodes, spleen) and non-lymphoid GVHD-target organs (colon, lungs, liver) compared to GVHD-free animals.

Our data demonstrates that Tscm dynamics correlate with GVHD-free survival and suggest that pathogenic T cells infiltrating both lymphoid and non-lymphoid organs transition from the naïve into the effector state via Tscm. Thus Tscm may play a key role as a cellular reservoir for GVHD.
EOMESODERMIN-EXPRESSING INNATE-LIKE CD8+ T CELLS IN HUMANS ARE IMPAIRED IN CHRONIC MYELOID LEUKEMIA PATIENTS AT DIAGNOSIS
Authors: Herbelin Andre , Jacomet Florence , Cayssals Emilie , Desmier Deborah , Barbarin Alice , Basbous Sara , Levescot Anaïs , Robin Aurélie , Giraud Christine , Barra Anne , Guilhot François , Roy Lydia , Gombert Jean-Marc
Published: January 2023

Starting with our recent identification in healthy individuals of a new distinct CD8+ T cell subset harboring Eomes and exhibiting a marked “innate” (panKIR/NKG2A+) phenotype, we present here the first report on this unique T cell population in a physiopathological context, namely chronic myelogenous leukemia (CML), a clonal myeloproliferative disorder. The current first-line therapies for this hematopoietic malignancy are tyrosine kinase inhibitors such as Imatinib (IM). Knowing that the generation of innate Eomes-expressing CD8+ T cells in mice depends on PLZF-expressing NKT cells and that iNKT cells are functionally deficient in CML, we surmised that panKIR/NKG2A+Eomes+ CD8+ T cells could be altered during CML. Accordingly, in CML patients at diagnosis, the pool of blood KIR/NKG2A(+)Eomes(+) CD8(+) T cells was found to be severely reduced. Moreover, like for iNKT and NK cells, the functions of these innate-like CD8(+) T cells were impaired, as attested by their lost of potent antigen-independent cytotoxic activity and IFN-γ production in response to innate-like stimulation by IL-12+IL-18. Remarkably, both innate-like CD8(+) T cells and iNKT cells returned to normal after complete CML remission upon IM therapy, supporting evidence for an iNKT cell-dependent generation of circulating KIR/NKG2A+Eomes+ CD8+ T cells in CML patients, as in mice. Our study reveals a possible contribution of iNKT/innate-like CD8(+) T cells as a new innate immune axis to tumor control in CML.
The Hepatic Recruitment of Innate Natural Killer T cells and Immune-Microenvironment Modulation after Colorectal Cancer Liver Metastasis
Authors: Feng Hao , Lu Aiguo , Thasler Wolfgang
Published: January 2023

Aims

To investigate the modulation of the immune microenvironment by human NKT cells and the crosstalk between NKT cells and Kupffer cells (KC), dendritic cells (DC) in colorectal cancer liver metastasis.

Methods

The numbers of iNKT cells and other cells from tumor margin, tumor or adjacent normal tissues were analysed by flow cytometry and AMNIS system. Ex vivo co-culture, 3D culture, Elisa array were established for this study.

Results

In primary colorectal cancers, percentage of iNKT cells was higher in early stage patients (T0~2). In the metastatic CRC, CXCR6+ cells are recruited and enriched at the invasive margin, elevated CXCR6 density was negatively related with Rees’ score, which suggests better prognosis. iNKT cells were the major population (30~40%) of CXCR6+cells, and the proportion and counts of iNKT cells were higher at the invasive margin (<2cm from liver- tumor interphase). CXCL16+ cells were found to be enriched at the invasive margin using immunofluorescence. 85±14% of CXCL16+ (the ligand of CXCR6) cells were CD68+ macrophage, of which 89% cells were Clec4f+Kupffer cells. And the Ex vivo chemotaxis experiments showed that rhCXCL16 and Clec4f+Kupffer cell conditional medium promote iNKT cells recruitment. The ex vivo and in vitro experiments showed that iNKT cells could lead KC to M2 differentiation, however, antigen presented by KC restricted the CRC elimination capability of iNKT cells. In contrast, antigen presenting by DC enhanced clearance of CRC but at meanwhile also induce hepatocytes cell death. Furthermore, we found by in situ staining and FACS that the majority of the KC was M1 KC at intra-tumor area and M2 KC in the adjacent normal tissues. The iNKT cells as well as KC showed Th2 polarization at the invasive margin.
VGX-3100 Drives Regression of HPV16/18 CIN2/3 and Robust Cellular Immune Responses in Blood and Cervical Tissue in a Blinded, Randomized, Placebo-controlled Phase 2b Study
Authors: Morrow Matthew P , Trimble Connie , Shen Xuefei , Dallas Michael , Weiner David , Boyer Jean , Yan Jian , Kraynyak Kimberly A , Sylvester Albert J , Giffear Mary , Marcozzi-Pierce Kathleen , Shah Divya , Broderick Kate , Khan Amir S , Lee Jessica , Humeau Laurent , Sardesai Niranjan , Bagarazzi Mark
Published: January 2023

Objectives

Assessment of the safety and efficacy and immunogenicity of VGX-3100 in women with biopsy-proven CIN2/3 with concurrent HPV16 and/or HPV18 infection.

Methods

The randomized, placebo-controlled, double-blind study, which was stratified by age and severity of CIN, evaluated cervical tissue changes after three 6 mg intramuscular doses of VGX-3100 followed by electroporation with Inovio’s CELLECTRA2000 device at weeks 0, 4, and 12.

Results

Among 167 vaccinated women, the study met its primary efficacy endpoint; the percentage of patients who had regression of CIN2/3 to CIN1 or no disease at 6 months post third dose was significantly higher in the VGX-3100 group compared to placebo (p=0.034). In addition, the trial demonstrated the ability of VGX-3100 to clear HPV infection concurrent with regression of CIN2/3 (p=0.003). Post-hoc immune analysis also revealed significantly elevated immune responses in treated patients who had CIN2/3 regression concurrent with HPV clearance when compared to those who did not. This included the presence of CD8+ T cells in the blood exhibiting CD137 expression concurrent with perforin (p=0.032) as well as perforin in addition to granzyme A (p=0.036) as well as an influx of CD8+ T cells into cervical tissue (p=0.008).

Conclusion

The successful phase 2b results represent a significant milestone in the development of active immunotherapies to treat HPV-related dysplasia and cancer. The data generated from the trial reveal statistically significant correlations between antigen specific T cell activity and clinical benefits afforded by VGX-3100. Thus VGX-3100 has the potential to provide an important alternative or adjunct to surgery in treating CIN 2/3 based on HPV specific T cell activity.
Salmonella promote T cell-mediated anti-tumor immuno-therapy
Authors: Xiao Yichuan , Wang Yan , Liu Chenli
Published: January 2023

B16 melanoma is widely used as an animal model to investigate the efficacy, mechanism and immune response of anti-tumor immuno-therapy. In this study, the anti-tumor efficacy was examined by using a Salmonella strain 7207, which cannot survive in the circulation system and aerobic tissues, but can thrived in the anaerobic environment of solid tumors. We found that Salmonella injection could significantly inhibited tumor growth in B16 tumor-bearing mice. Interestingly, injection of heat-killed Salmonella also showed comparable anti-tumor efficacy as alive Salmonella, with no bacteria detectable in the tumor and circulation system. In addition, both alive and heat-killed Salmonella could promote robust pro-inflammatory gene induction in bone marrow-derived macrophages and dendritic cells, and increased tumor-infiltrating IFN-g-producing cytotoxic T cells (CTL). Moreover, deletion of T cells abolished the anti-tumor effect by injecting of alive or heat-killed Salmonella in Rag1-deficient tumor-bearing mice, suggesting Salmonella -mediated anti-tumor effect is due to the cytotoxic function of CTL. Taken together, our study provides a new insight of the development of anti-tumor immuno-therapy, and a potential therapeutic strategy for B16 melanoma.