bluuebunny/crossref_metadata_2025 huggingface dataset:
Tissue-resident memory T cells mediate melanoma surveillance and are targets of immunotherapy
Authors: Park Simone L , Buzzai Anthony , Hor Jyh Liang , Effern Maike , Christo Susan N , Mueller Scott N , Holzel Michael , Waithman Jason , Mackay Laura K , Gebhardt Thomas
Published: January 2023

Non-recirculating tissue-resident memory T (TRM) cells reside permanently in epithelial tissues where they are known to protect against infection. TRM cells have now been identified in various human cancers where they correlate with improved patient outcomes, but the mechanisms through which they may mediate protective tumour immunity remain unclear. Using a novel mouse model of transplantable cutaneous melanoma, we show that tumour-specific TRM cells form spontaneously in the epithelial tumour microenvironment where they protect against cancer development independently of circulating memory T cells. A proportion of epicutaneously inoculated mice remained free of macroscopic tumours long after inoculation, and intravital imaging revealed that these mice harboured dormant melanoma cells in their skin that were dynamically surveyed by tumour-specific TRM cells. Depletion of TRM cells from these mice triggered melanoma escape, indicating that TRM cells suppress tumour progression by promoting cancer-immune equilibrium. Further, we found that pre-activated T cells transferred to tumour-bearing mice in a model of adoptive cell therapy gave rise to TRM cells that inhibited the growth of established melanoma. These tumour-specific TRM cells retained their capacity to execute effector functions and protect against tumour growth despite expression of various inhibitory molecules typically associated with T cell dysfunction. Combined, our findings reveal TRM cells to be durable mediators of anti-tumour immunity and suggest that targeting TRM cell responses could serve as a novel immunotherapeutic strategy to drive elimination of solid tumours.
Novel approaches for studying cell-mediated immune responses to influenza vaccination in humans
Authors: Cao Weiping , Mishina Margarita , Ende Zachary , Mboko Wadzanai P , Batra Dhwani , Bohannon Caitlin , Carney Paul , Chang Jessie , Ranjan Priya , Kumar Amrita , Amoah Samuel , Gangappa Shivaprakash , Mittal Suresh , Sheth Mili , Pohl Jan , Stevens James , Sambhara Suryaprakash
Published: January 2023

Vaccination is the most cost-effective public health intervention strategy in the prevention of the spread of infectious diseases. Unfortunately, each year, a substantial proportion of individuals do not respond or respond poorly to vaccination due to age, nutrition, pre-existing medical conditions, vaccine mismatch, or other unknown reasons. To investigate this further, we propose studying the mechanisms and molecular signatures associated with immunogenicity and the efficacy of influenza vaccination in different populations.

The priming environment, induced by the innate immune system is crucial for initiating and fine-tuning antigen-specific adaptive immune responses. Comprehensive antibody panels have been developed to identify the new paradigms in the innate priming environment, focusing on NK, innate lymphoid cells and γδ T cells by flow cytometry in PBMC from vaccine recipients. The specific functions of these cells are then analyzed using single cell sorting and high-throughput transcriptomic and proteomics analysis. To further probe the adaptive response, we have successfully developed receptor binding site mutant HA (H1, H3, B) and HA stem (group 1 and 2) probes that are conjugated to fluorochromes to enumerate and characterize HA-specific B cells. M2e antigen probes are under development. Combining cell sorting, single cell molecular analyses, and the Illumina MiSeq system, paired heavy and light chains of B cell receptors (BCR) from isolated antigen specific B cells are sequenced to dissect antibody diversity.

Through these novel approaches, we aim to identify and characterize critical host factors that can be exploited to develop as novel adjuvants and strategies for more effective influenza vaccines.
Neuraminidase expressed on virus-like particles is superior to inactivated split virus vaccine in conferring cross protection via humoral and cellular immunity
Authors: Kim Ki-Hye , Lee Young-Tae , Park Soojin , Jung Yu-Jin , Kim Yu-Jin , Lee Youri , Kang Sang-Moo
Published: January 2023

Neuraminidase (NA) is the second major surface antigen on influenza virus but often ignored as a vaccine component. In this study, we developed N1 virus-like particles (N1 VLP) containing NA derived from 2009 pandemic H1N1 influenza virus (2009H1N1), and compared with inactivated split virus vaccine in the immune mechanisms of cross protection. Intramuscular immunization of mice with N1 VLP induced IgG antibody responses to 2009H1N1 virus and NA inhibition activity. Mice immunized with N1 VLP induced superior cross protection against lethal infection compared to a current vaccine platform, split virus vaccine (1934H1N1), as determined by body weight changes, lung viral titers, infiltrating innate immune cells, cytokines, and histopathology. Furthermore, N1 VLP immunized mice induced higher levels of IFN-γ effector CD4 T cells, plasma cells, and germinal center B cell (GL7+B220+IgD−CD19+) and plasma cells (CD138+B220+/− IgD-CD19+) than split virus vaccine mice. Also, N1 VLP immune sera provided heterosubtypic cross-protection against H3N2 virus in naïve mice. Therefore, these results suggest that N1 NA-presenting VLP can be developed as an effective cross-protective vaccine candidate standalone or in combination of current influenza vaccination in a supplemented vaccine platform.
Mucosal delivery of nanofiber booster vaccine enhances generation of antigen-specific memory T cells in the lung parenchyma
Authors: Files Megan A , Bharaj Preeti , Saito Tais B , Rudra Jai , Endsley Janice J
Published: January 2023

Tuberculosis (TB) infection caused by Mycobacterium tuberculosis (Mtb) is a serious threat to global health with 10 million new infections each year and 1–2 million deaths. The only licensed vaccine approved for human use, Mycobacterium bovis Bacille Calmette-Guerin (BCG), has been administered to 4 billion people despite variable efficacy. Heterologous vaccine regimens that boost T cell memory generated through BCG priming, however, may be an effective immunization strategy. Our lab previously demonstrated efficacy of a nanofiber (NF) vaccine platform containing co-assembled CD8+ and CD4+ epitopes as evidenced by the generation of polyfunctional CD8+ T cells and protection from aerosol challenge with Mtb. In recent investigations, we observed that boosting BCG-primed mice through a pulmonary route with the NF-based vaccine bearing Ag85B CD4+T cells epitopes generated increased frequency of Mtb antigen-specific T cells in the lung parenchyma as indicated by number of tetramer (I-Ab:Ag85B) positive CD4+T cells. The frequency of Mtb-specific CD4+T cells was further increased by adoptive transfer of dendritic cells pulsed with Ag85B-bearing NF. The Ag85B-specific T cells were further characterized as tissue resident memory (Trm) and effector memory (Tem) T cells based on the surface marker phenotypes (I-Ab:Ag85B+ CD69+/− CCR7− CD44+ CD62Llo). In memory recall experiments, splenocytes from vaccinated mice also expressed polyfunctional cytokines that have been associated with protective immunity. These results suggest that protective immunity following vaccination with a NF subunit vaccine in a prime/boost strategy with BCG is associated with expansion of Trm CD4+T cells in lung parenchyma that recognize the Ag85B epitope.
Natural gene-encoded affinity for cognate antigen facilitates vaccine-amplification of broadly neutralizing antibodies against influenza virus
Authors: Lingwood Daniel
Published: January 2023

B cell receptors display V gene-encoded CDRs and hypervariable CDR3, where the former may harbor innate-like antigen recognition characteristics. To test this in vivo, we deployed transgenic mice containing full human CDRH3 diversity but were constrained to single human VH genes: IGHV1-69*01, a human VH reproducibly used to generate broadly neutralizing antibodies (bnAbs) targeting hemagglutinin stalk of Group 1 influenza A viruses or irrelevant IGHV1-2*02. With this system, we could experimentally evaluate the contribution of V gene encoded CDRs to forming the antibody paratope following immune challenge. Exposure to virus or hemagglutinin vaccine-nanoparticles elicited primary antibody responses targeting the bnAb epitope, but only in IGHV1-69 animals. Repeated exposure amplified off-target responses, however, prime/boost with stalk-only nanoparticle (SS-np) refocused serum antibodies to the target-epitope, expanding the human IGHV1-69 bnAb lineage to elicit prototypic IGHV1-69 Group 1 bnAbs. The bnAb pathway was expanded from a genetically endowed but low frequency population of on target germline B cell precursors (<0.3% of the repertoire). IGHV1-69 therefore endows natural cognate anti-influenza targeting affinity which can be exploited through rational vaccine design to redirect antibodies against an otherwise immunologically silent ‘universal’ flu vaccine target. Remarkably, similar on-target germline B cell levels were seen in human PBMC, suggesting SS-np may elicit bnAbs in humans.
Generation of tissue-restricted CD4 effectors (TFH and ThCTL) requires signals from local antigen & infection during the effector phase
Authors: Devarajan Priyadharshini , Vong Allen M , Castonguay Catherine H , Bautista Bianca L , Swain Susan L
Published: January 2023

Key specialized tissue-restricted CD4 effector subsets such as T helper cytotoxic cells (ThCTL) in the infected tissue and T follicular helper cells (TFH) in secondary lymphoid organs arise after other CD4 effectors (Th1, Th17) peak. In an influenza A virus (IAV) infection model, we find that generation of ThCTL and TFH require that CD4 effectors recognize specific Ag/MHC Class II, 5–7 days after initial infection. We restrict Ag presentation to specific APC subsets and add various APC via different routes, to study which APC and infection conditions drive TFH vs. ThCTL generation at the checkpoint. We find most MHC Class II-expressing APC subsets, including dendritic cells and B cells, are able to drive TFH and ThCTL generation. However, local Ag presentation is crucial. Strikingly, we find that continuing infection, even at this late effector phase, is needed for optimal generation of both subsets. This contrasts with CD4 memory generation, which in our previous studies, was not dependent on infection at this checkpoint. Thus, at the effector checkpoint, Ag presentation in the local tissue drives both optimal specialized effector and most memory generation, whilst signals from infection are required for optimum tissue-restricted effector differentiation. The infection signal can be provided by multiple PRR pathways. We suggest that vaccines will need to be designed to provide additional Ag presentation and the signals associated with infection at the effector checkpoint to induce optimum protective immune responses: to promote strong CD4 memory for broad, heterosubtypic protection and indirectly strong B cell response and memory leading to long-term humoral protection.
CD4+ T cells Promote Humoral Immunity and Viral Control during Zika Virus Infection
Authors: Ngono Annie Elong , Young Matthew P , Bunz Maximilian , Xu Zhigang , Hattakam Sararat , Vizcarra Edward , Regla-Nava Jose Angel , Tang William W , Yamabhai Montarop , Wen Jinsheng , Shresta Sujan
Published: January 2023

Several Zika virus (ZIKV) vaccines designed to elicit protective antibody (Ab) responses are currently under rapid development, but the underlying mechanisms that control the magnitude and quality of the Ab response remain unclear. Here, we investigated the CD4+ T cell response to primary intravenous and intravaginal infection with ZIKV. Using the LysMCre+Ifnar1fl/fl (myeloid type I IFN receptor-deficient) C57BL/6 mouse models, we identified six I-Ab-restricted ZIKV epitopes that stimulated CD4+ T cells with a predominantly cytotoxic Th1 phenotype in mice primed with ZIKV. Intravenous and intravaginal infection with ZIKV effectively induced follicular helper and regulatory CD4+ T cells. Treatment of mice with a CD4+ T cell-depleting Ab reduced the plasma cell, germinal center B cell, and IgG responses to ZIKV without affecting the CD8+ T cell response. CD4+ T cells were required to protect mice from a lethal dose of ZIKV after infection intravaginally, but not intravenously. However, adoptive transfer and peptide immunization experiments showed a role for memory CD4+ T cells in ZIKV clearance in mice challenged intravenously. These results demonstrate that CD4+ T cells are required mainly for the generation of a ZIKV-specific humoral response but not for an efficient CD8+ T cell response. Thus, CD4+ T cells could be important mediators of protection against ZIKV, depending on the infection or vaccination context.
Evaluation of HIV-Specific T-Cell Responses in HIV-Infected Aging Patients with Controlled Viremia on Long-term Antiretroviral Therapy
Authors: Bradley Nicole Elizabeth , Wertheimer Anne , Klotz Stephen A , Ahmad Nafees
Published: January 2023

While long-term antiretroviral therapy (ART) has helped many human immunodeficiency virus type 1 (HIV-1) infected aging patients to achieve controlled viremia and restored CD4 T-cell counts, the role of HIV-specific T-cell responses in these patients is not clearly understood. We have investigated HIV-specific CD4 and CD8 T-cell responses in 100 HIV-infected patients (HIV+) who have aged on long-term antiretroviral therapy (ART) with controlled viremia and restored CD4 T-cell counts. We show the median circulating HIV-specific CD4+ and CD8+IFN-γ T-cells in HIV+ were higher than uninfected controls (HIV−), including increased levels of HIV-specific CD4+IFN-γ T-cells and decreased levels of CD8+IFN-γ T-cells with increasing CD4T-cell counts in HIV+. In addition, HIV-specific CD8+TNF-α T-cells were higher in HIV+ than HIV−, including decreasing levels were seen with increasing CD4T-cell counts of HIV+. Furthermore, the CD8+ T-cell mediators, Granzyme B and CD107a, were higher in HIV+ than HIV−, however these mediators decreased with increasing CD4 T-cell counts in HIV+. More importantly, HIV-specific CD4+ and CD8+ T-cells produced higher levels of IFN-γ and CD8+ T-cells TNF-α, Granzyme B and CD107a with increasing age of HIV+ probably to counter residual HIV and reduced immunity in older individuals. In conclusion, while HIV-infected patients with controlled viremia and improved CD4 T-cell counts showed reduced CD8 T-cell responses, the CD8 T-cell responses were higher in HIV+ with lower CD4 T-cell counts as well as in HIV+ patients with increasing age.
PSGL-1 is a negative regulator of effector and memory T cells
Authors: Tinoco Roberto , Bradley Linda M
Published: January 2023

Effective T cell responses depend on adhesion mechanisms that guide T cells to sites of infection and inflammation. P-selectin glycoprotein ligand-1 (PSGL-1) has been shown to impact T cell migration; however, its role in the differentiation of responding T cells has not been investigated. Since PSGL-1 is highly expressed on naïve, effector, and memory T cells with varying degrees of glycosylation that impact ligand binding, we hypothesized that PSGL-1 may regulate T cell fates. Using mice deficient in PSGL-1 and we found that PSGL-1 limited the magnitude of the ensuing T cell response. PSGL-1-deficient mice had an increased accumulation of anti-viral CD4+ and CD8+ T cells after acute LCMV infection. Virus-specific PSGL-1-deficient T cells had enhanced survival and were not hyper proliferative. Furthermore, PSGL-1-deficient T cells had superior effector function, downregulation of PD-1 and other inhibitory receptors and changes in their transcriptional signature. After viral clearance, more CD4+ and CD8+ PSGL-1-deficient T cells survived to form memory cells. Interestingly, memory T cells from PSGL-1-deficient mice sustained PD-1 downregulation implying that PSGL-1 may regulate inhibitory receptors in memory T cells. Our findings highlight a previously unrecognized role for PSGL-1 in negatively regulating T cell differentiation during acute viral infection.
The pleiotropic effects of PTPN22 and its pro-autoimmune alternative allele on the antiviral immune response and T cell exhaustion
Authors: Orozco Robin C , Marquardt Kristi , Mowen Kerri A , Pratumchai Isaraphorn , Teijaro John R , Sherman Linda A
Published: January 2023

A major alternative allele of the tyrosine phosphatase, PTPN22 1858T, is present in 5–10% of the North American population and is strongly associated with numerous autoimmune diseases. Extensive work understanding this allele in the context of autoimmune disorders has defined a role for PTPN22 in TCR and BCR signaling, toll-like receptor activation, and cytokine production. However, despite the importance of these inflammatory factors in controlling viral infection and promoting T cell exhaustion, very little work has been done to understand how PTPN22 affects these processes. Using mice deficient in expression of PTPN22, our lab previously identified a role for this gene in promoting T cell exhaustion during chronic viral infection. Yet, the mechanism by which it is acting and how the alternative allele effects T cell exhaustion remains unknown. To examine these questions, we used CRISPR/Cas9 to generate mice expressing the human-relevant PTPN22 alternative allele (PTPN22 R619W). Using the well-established LCMV-cl13 infection model in our mice, we tested the hypothesis that pleiotropic effects of PTPN22 contributes to T cell exhaustion and chronic viral infection. We address this through examining R619W expression in various immune cells contributing to viral clearance. Importantly, we determine T cells expressing R619W have increased antigen specific activation during viral infection. Additionally, we show that expression of R619W in CD8α-conventional dendritic cells reduces PD-L1 expression during viral infection and elucidate the underlying mechanism. Results of this study provide a new mechanism through which PTPN22 tempers the immune response that may be applicable to other diseases beyond chronic viral infection.
Small GTPase Rab8 plays a critical role in B cell antigen presentation
Authors: Mattila Pieta K , Vainio Marika , Perez Sara Hernandez , Sustar Vid , Rajala Johanna
Published: January 2023

Upon recognition of specific antigens, B cells commit to receptor-mediated endocytosis and subsequent processing of antigen into peptides for presentation on MHC class II (MHCII) molecules. This highly specific and efficient antigen presentation is a prerequisite for B cells to be able to receive T cell help and initiate high-affinity antibody responses. The intracellular vesicle trafficking and the identity of the compartments vital for antigen processing and peptide-MHCII presentation remain poorly known. We set to characterize the vesicular pathway of internalized antigen in B cells via extensive co-localization analysis using various different markers. Our data reveal involvement of both classical endosomal markers and more specialized vesicular regulators. It appears that B cells process antigen in atypical endosomes that share both early (EEA1+, Rab5+) and late (Rab7+, Rab9+, LAMP1+) endosomal markers together with Cathepsin-S, required for proteolytic processing. Interestingly, a small GTPase Rab8, linked to specialized protein secretion pathways in the literature, strongly colocalizes with antigen throughout different stages of vesicle transport. To investigate the functional role of Rab8 on B cell antigen processing, we went on to generate Rab8-deficient B cell lines by CRISPR/Cas9. Notably, we detected a dramatic loss of antigen presentation in the cells deficient in Rab8. Our preliminary data points towards a role for Rab8 both in the transport of antigen as well as MHCII.
Peptide editing and MHC binding mechanisms of Tapasin and TAP binding protein related, TAPBPR
Authors: Kim Ellen J , Natarajan Kannan , Boyd Lisa F , Jiang Jiansheng , Mage Michael G , Margulies David H
Published: January 2023

Cell surface major histocompatibility complex class I (MHC-I) molecules play a crucial role in immunity to microbes and tumors by presenting intracellular peptides for recognition by CD8+ T cells. Since the repertoire of bound peptides is crucial to MHC-I stability as well as to T cell and NK cell recognition, the molecular mechanism by which peptides are selectively loaded onto MHC-I molecules is of considerable interest. A key role is played by the well-characterized chaperone tapasin as indicated by reduced cell surface MHC-I levels and altered peptide repertoires in tapasin deficient cell lines and mice. Despite extensive studies of the functional role of tapasin in shaping peptide repertoires, direct structural information on its interaction with MHC-I is lacking. Following the recent biochemical and crystallographic studies of the interaction of the tapasin homolog, TAPBPR, with MHC-I, we have engineered soluble versions of tapasin for comparison with TAPBPR. Using surface plasmon resonance assays of purified, untethered proteins, we observe that tapasin binds directly to an MHC-I molecule loaded with truncated peptides but with lower apparent affinity than TAPBPR. Similarly, using fluorescence polarization, we find that tapasin facilitates peptide exchange onto MHC-I with a lower efficiency than TAPBPR. These experiments permit direct comparisons of tapasin/MHC-I and TAPBPR/MHC-I interactions and help elucidate the functional relationship of these two distinct chaperones in shaping the MHC-I peptide repertoire.
Self-adjuvanting properties of peptide nanofiber vaccines are dependent on autophagy
Authors: Rudra Jai S , Khan Arshad , Endsley Janice J , Zloza Andrew , Jagannath Chinnaswamy
Published: January 2023

A crucial challenge in subunit vaccine development is to minimize adjuvant-associated toxicity and balance immunogenicity with safety. Due to the chemical heterogeneity and toxicity associated with most plant- or pathogen-derived adjuvants, those clinically approved in the U.S. are limited to alum and AS04 (alum combined with MPLA). Therefore, there is a compelling clinical need for new adjuvant strategies that potentiate strong immune responses and are chemically defined and minimally toxic. We have previously demonstrated that supramolecular peptide nanofibers are potent adjuvants that elicit protective antibody and cellular immune responses in animal models of infectious diseases, cancer, and addiction. Critically, the adjuvant potential was non-inflammatory and the exact mechanism of action was unknown. Here, we report that self-assembling peptide nanofibers bearing CD4+ or CD8+T cell epitopes are processed through mechanisms of autophagy in antigen presenting cells (APCs). Using in vitro antigen presentation assays, we confirmed loss and gain of adjuvant function using pharmacological modulators of autophagy and APCs deficient in multiple autophagy proteins. Incorporation of microtubule associated protein 1A/1B-light chain 3 (LC3-II) into the autophagosomal membrane, a key biological marker for autophagy, was confirmed using microscopy. Our findings indicate that autophagy plays an essential role in the mechanism of adjuvant action of supramolecular peptide nanofibers.
MHC class II conformers drive differential B cell activation
Authors: Drake James R
Published: January 2023

MHC class II molecules present exogenous antigen-derived peptides to CD4 T cells to elicit T cell-based help/regulation. While these peptides represent a robust source of heterogeneity, the class II molecules themselves are also heterogeneous. MHC class II molecules exist in two distinct conformational states, based on alternative pairing and differential cholesterol binding of the molecule’s α and β chain’s transmembrane domains. What have been termed “M2 paired” class II (M2 class II) represents the majority of cell surface class II, whereas M1 paired class II (M1 class II) is a smaller fraction of surface molecules. However, M1 class II is enriched in “lipid raft” membrane domains, is highly stimulatory for CD4 T cells and is selectively linked to B cell activation signaling pathways. Moreover, in B lymphocytes M1 class II selectively associates with intracellular antigen-B cell receptor (BCR) complexes and is selectively loaded with peptide derived from the processing of BCR-bound cognate antigen. In contrast, peptides from non-cognate antigen internalized by fluid-phase endocytosis (as would happen for self-antigens) is loaded onto both M1 and M2 class II. Here, we show that while selective engagement of M1 class II drives B cell calcium signaling (an activation event), co-engagement of M1 and M2 class II allows an M2 class II-associated inhibitory activity to block M1 class II signaling. These findings provide new insight into the molecular mechanisms of MHC class II signaling by revealing the distinct signaling properties of M1 and M2 class II. The results also suggest a B cell-intrinsic mechanism by which T cell recognition of M1 vs. M2 peptide-class II complexes could regulate B cell activation via differential class II signaling.
Molecular mechanisms underlying the glycation induced-impairment of antigen processing and presentation in diabetic (T2DM) patients and ob/ob mice
Authors: Santambrogio Laura , Clement Cristina , Negroni Maria Pia , Nanaware Padma , Morozova Kateryna , Stern Lawrence
Published: January 2023

Patients with obesity, metabolic syndrome, or type-2 diabetes (T2D) often exhibit hyperglycemia and hyperlipidemia. A consequence of the dysmetabolism is non-enzymatic coupling of reactive sugars and lipids with proteins and DNA inducing glycation and glycoxidation. Biochemically, these modifications disrupt the cellular proteome by changing the protein chemical structure and inducing protein unfolding, cross-linking and aggregation. We investigated protein post-translational modifications (PTM) induced by glycation and glycoxidation in PBMC from non-diabetic, pre-diabetic and diabetic individuals as well as CD11c+ dendritic cells purified from mice fed a high-fat diet, obese (Ob/Ob) mice and relative controls. By employing label-free bottom-up proteomic and bioinformatics analysis we mapped several cellular pathways affected by the oxidative PTMs, including the antigen processing and presentation machinery. Functional assays of endosomal processing, using transthyretin and insulin as model antigens, determined how the oxidative PTMs interfered with antigen processing. Glycation and glycoxidation moieties on MHC II proteins were mapped at sites known to interact with peptides, with the peptide exchange factor DM, and with the TCR. In vitro glycation of human HLA-DR1 resulted in impaired interaction with the peptide exchange factor HLA-DM. In vivo and in vitro impaired immune response to a model antigen (I-Ea protein ) was also observed in Ob/Ob mice. Our results shed light on the biological implications of the role played by glycation and glycoxidation, as observed in T2D and metabolic syndrome, on the antigen processing machinery as well as its functional consequences for MHC II restricted immune responses.
Optimizing steroid administration in murine models of Citrobacter rodentium-induced acute phase colitis
Authors: Clarke Sandra T , Shelton Tara , Boras Valerie , Brumer Harry , Wang Chang Qing , Abbott D. Wade , Xing Xiaohui , Uwiera Richard R.E. , Inglis G. Douglas
Published: January 2023

This study used Citrobacter rodentium-infected C57BL/6 mice as a model of acute colitis to assess treatment with steroids prednisolone (pred) and dexamethasone (dex). Both germ-free (GF) and normal flora (NF) adult mice were used to determine an optimal steroid dosing strategy to mitigate intestinal inflammation caused by C. rodentium attachment/effacement lesions. Male and female mice (6-weeks of age) were orally inoculated with C. rodentium (1.0 × 108 CFU/100 μL) for two consecutive days. Five days post-infection, mice were gavaged with pred (1 mg/kg/d or 3 mg/kg/d), dex (0.6 mg/kg/d or 1.5 mg/kg/d) or controls for five days (GF n = 3 and NF n = 4 for each treatment). GF mice had longer colons (base of cecum to anus) than NF mice (7.1 ± 0.1 cm vs. 6.2 ± 0.2 cm; P < 0.001) and lighter total liver weight than NF mice (1.0 ± 0 g vs. 1.1 ± 0.2 g; P = 0.03). Colonic cytokines were quantified by ELISA. Steroid treatment decreased concentrations of colonic KC (CXCL1; P < 0.001) in NF mice; and high-dose dex decreased KC more effectively than low-dose dex (3207.5 ± 583.5 pg/g vs. 9217.6 ± 1375.4 pg/g; P = 0.019). Although C. rodentium increased colonic concentrations of KC (P < 0.001), MIP-2 (CXCL2; P = 0.19), TNF-α (P = 0.031), IL-22 (P = 0.018) and myeloperoxidase (P < 0.001) in NF mice, no differences were attributable to steroids or sex in GF and NF mice. Colonic MIP-2 (P < 0.001), IL-10 (P = 0.032), TNF-α (P < 0.001), IL-17 (P < 0.001) and IL-22 (P = 0.005) were higher in NF than GF mice, likely due to underdeveloped gut-associated lymphoid tissue in GF mice. In summary, high-dose dex (1.5 mg/kg/d) but not high-dose pred (3 mg/kg/d) mitigated production of the potent neutrophil chemoattractant KC in a C. rodentium induced model of acute-phase colitis.
Enhancement of gut barrier function by microbial metabolite, urolithin A via AhR-Nrf2 dependent pathways in IBD
Authors: Jala Venkatakrishna R , Singh Rajbir , Chandrashekarappa Sandeep , Bodduluri Sobha Rani , Becca Baby V , Hegde Bindu , Kotla Niranjan , Hiwale Ankita A , Saiyed Taslimarif , Patel Paresh , Vijay-Kumar Matam , Langille Morgan , Douglas Gavin M , Dryden Gerald , Cheng Xi , Rouchka Eric , Waigel Sabine J , Alatassi Houda , Zhang Huang-Ge , Haribabu Bodduluri , Vemula Praveen K
Published: January 2023

Inflammatory bowel diseases (IBD) consisting of Crohn’s and ulcerative colitis are resultant of dysregulation of the immune system leading to intestinal inflammation and microbial dysbiosis. Numerous studies in recent years highlighted the pivotal role of gut microbiota and their metabolites in host physiological processes including in IBD. Urolithin A (UroA) is a microbial metabolite derived from polyphenolics (e.g., ellagitannins/ellagic acid) of pomegranate and berries. We also synthesized a potent structural analogue of UroA (UAS03) and tested their efficacies in preventing and treating colitis in pre-clinical models. Our studies showed that UroA/UAS03 significantly enhance gut barrier function in addition to blocking unwarranted inflammation. We demonstrate that UroA and UAS03 exert their barrier functions through activation of aryl hydrocarbon receptor (AhR)- nuclear factor erythroid 2–related factor 2 (Nrf2)-dependent pathways to upregulate epithelial tight junction proteins. In addition, treatment with these compounds attenuated colitis in pre-clinical models by remedying barrier dysfunction and blocking increased inflammatory mediators such as IL-6, TNF-α and IL-1β. UroA/UAS03 failed to induce tight junction proteins and protect against 2,4,6-Trinitrobenzenesulfonic acid (TNBS)-induced colitis in AhR−/− and Nrf2−/− mice suggesting an obligatory requirement of AhR and Nrf2 pathways for UroA/UAS03 mediated beneficial activities. Overall, the results highlight how microbial metabolites provide two-pronged beneficial activities at gut epithelium by enhancing barrier functions and reducing systemic and local inflammation to protect from colonic diseases.
Neuroantigen-specific regulatory CD8+ T cell responses as a therapy for relapsing autoimmune demyelinating disease
Authors: Brate Ashley A , Itani Farah R. , Pewe Lecia L. , Harty John T , Karandikar Nitin J.
Published: January 2023

Multiple Sclerosis (MS) is an autoimmune demyelinating disease of the central nervous system (CNS). We have shown that CNS-specific CD8+ T cells possess a disease suppressive function in MS and its animal model, experimental autoimmune encephalomyelitis (EAE). In humans, this suppressive function of CD8+ T cells is deficient during MS relapses. We thus evaluated the role of CD8+ T cells in the context of the relapsing-remitting model (RR-EAE), using SJL mice. We demonstrate here that PLP178–191- and MBP84–104-specific CD8+ T cells ameliorated disease severity in an antigen-specific manner. Moreover, PLP178–191 CD8+ T cells reduced the number of relapses in PLP178–191-induced disease, even when transferred during ongoing disease. We were further able to ascertain that the suppressor population of CD8+ T cells express CD25, and show that a very low number of CD25+ CD8+ T cells are sufficient to suppress RR-EAE. We also probed the role of endogenously generated CNS-specific CD8+ T cell responses by using Listeria monocytogenes (LM) encoding CNS antigens to preferentially prime suppressive CD8+ T cells in vivo. Using this system, we show infection with LM expressing PLP175–194 endogenously induced disease suppressive CD8+ T cells that protected and treated PLP178–191 disease. Importantly, a combination of CD8+ T cell transfer boosted by LM infection also successfully treated ongoing disease induced by a non-cognate peptide (PLP139–151), indicating that this approach could be effective even in the context of epitope spreading. These studies support a potential immunotherapeutic strategy using LM vaccination to prime disease regulatory CD8+ T cells.
The Aryl Hydrocarbon Receptor as a regulator of CNS autoimmunity
Authors: Kenison-White Jessica , Jhaveri Aditi , Tezza Sara , Nowakowska Dominika , Sherr David , Quintana Francisco Javier
Published: January 2023

Therapeutic approaches aimed at controlling the balance between effector T cells and regulatory T cells in an antigen-specific manner are of high clinical significance in the field of autoimmunity. The aryl hydrocarbon receptor (AhR) has been identified as a key regulator of the adaptive immune system and thus represents a strong targetable candidate for the induction of immune tolerance. Here, we report the generation of a novel liposomal platform to co-administer a tolerogenic AhR ligand, ITE, and an autoimmune disease-specific T cell antigen. Liposomes containing ITE and a T-cell epitope from myelin oligodendrocyte glycoprotein (MOG)35–55 are taken up by dendritic cells and induce a tolerogenic transcriptional program which promotes the differentiation and expansion of long-lasting antigen-specific regulatory T cells. Treatment with these liposomes almost completely prevented the development of experimental autoimmune encephalomyelitis (EAE), an experimental model of relapsing-remitting multiple sclerosis (MS), and reversed symptoms in mice with ongoing EAE. This disease suppression was associated with the expansion of antigen-specific FoxP3+ regulatory T cells (Treg cells) and type 1 regulatory T cells (Tr1 cells) as well as the suppression of CNS-infiltrating effector T cells. Furthermore, treatment with liposomes containing ITE and MOG reversed symptoms of disease in chronic progressive EAE in nonobese diabetic mice, an experimental model resembling several aspects of secondary progressive MS. Taken together, these results suggest that our liposomes represent a novel therapeutic platform to induce antigen-specific tolerance for the treatment of autoimmune diseases.
Myelin basic protein specific TCR-engineered regulatory T cells from multiple sclerosis patients are suppressive to MBP-specific T effector cells
Authors: de Paula Alves Sousa Alessandra , Kim Yong Chan , Jacobson Steven , Scott David W
Published: January 2023

Multiple sclerosis (MS) is an autoimmune disease of the central nervous system (CNS), characterized by inflammation, demyelination and axonal injury. Expanded T regulatory cells have been used in clinical trials for autoimmune diseases, but CNS-specific cells are rare in polyclonal populations and have the potential to be non-specifically immunosuppressive. Previously, we developed MS-specific Tregs by transducing a MBP-specific T-cell receptor (Ob2F3 TCR) into expanded human Tregs. Ob2F3 TCR-transduced Tregs successfully suppressed MBP-specific Ob2F3 T effector cells in vitro as well as MOG-induced EAE in vivo (Kim et al., J. Autoimm. 92: 77–86, 2018). However, Tregs in MS patients have been reported to be impaired in their suppressive function. Herein, we transduced Tregs from MS patients and healthy controls and tested them for suppression of effector T-cells to determine whether this activity is significantly different with patient Tregs. Treg (CD4+CD25hiCD127low) and naïve T-cells (CD4+CD25lowCD127hi) were isolated from peripheral mononuclear cells and retrovirally transduced with specific the Ob2F3 MBP-specific TCR, expanded and tested for suppression in vitro. We found that MBP-specific Tregs isolated from MS patients maintain Treg markers and are able to inhibit autologous MBP-specific T effector cells as well as MBP-specific Tregs from healthy controls. Our data suggest that Tregs obtained from MS patients can retain their suppressive function upon expansion and transduction with the MBP-specific TCR, and could be used therapeutically to restore a tolerance in patients. (Supported by NMSS RG1612-26484 and NIH Intramural funds)
Photobiomodulation therapy (PBMT) regulates the production of IL-10 and IFN-Ɣ by peripheral blood mononuclear cells (PBMC) and CD4+ T cells isolated from subjects with Multiple Sclerosis (MS)
Authors: Tolentino Miguel , Cho Chi C. , Lyons Jeri-Anne
Published: January 2023

Multiple sclerosis (MS) is a CD4+ Th1/Th17 mediated autoimmune disease characterized by myelin destruction, neurodegeneration and mitochondrial dysfunction. Current therapies suppress inflammation during early stages but are not effective in chronic stages characterized by oxidative damage. New therapies to slow progression, offer neuroprotection, and ameliorate the symptoms at any stage are urgently needed. Photobiomodulation therapy (PBMT) with light at 600–1100 nm is demonstrated to improve inflammatory and neurodegenerative disorders by down-regulating pro-inflammatory mediators and up-regulating anti-inflammatory mediators.

Our lab showed that PBMT with 670nm light reduced clinical severity in the C57BL/6 experimental autoimmune encephalomyelitis (EAE) model of MS by down-regulation of oxidative stress, apoptosis and pro-inflammatory cytokines, and up-regulation of anti-inflammatory cytokines. Data presented here evaluate the effect of PBMT on cytokine production by immune cells isolated from MS patients and healthy donors. PBMC and CD4+ T cells activated with Phytohaemagglutinin (PHA) or CD3/CD28 activator, respectively, received PBMT in vitro and cytokines were measured in cell culture supernatants by ELISA. Light at 670nm and 830nm increased IL-10 and reduced IFN-Ɣ produced by PBMC from MS patients. Light at 670nm, 735nm and 830nm increased IL-10, whereas 670nm and 830nm, but not 735nm, light reduced IFN-Ɣ produced by CD4+ T cells. Light at 670nm and 830nm reduced IFN-Ɣ produced by PBMC, while 830nm light increased IL-10 production by CD4+ T cells isolated from healthy donors. Our results demonstrate that PBMT differentially regulates the immune response in persons with MS and healthy donors.
Alcohol consumption leads to sex-specific amelioration of disability in a mouse model of multiple sclerosis
Authors: Caslin Blaine , Karmakar Aditi , Maguire Cole , Helmsdoerfer Kristen , Mohler Kailey , Kirwin Stefanie , Wylie Dennis , Melamed Esther
Published: January 2023

Multiple sclerosis (MS) is a neuroinflammatory autoimmune disorder and the leading cause of neurological disability in young people. Alcohol consumption is known to have both neuroinflammatory and immunosuppressive effects, yet despite high rates of alcohol abuse among MS patients, its effects on MS disease progression have not been well studied. The few studies on alcohol consumption during MS have produced conflicting results and suggest opposing clinical outcomes at different alcohol doses. In this study, we hypothesized that low-dose daily alcohol consumption could ameliorate the clinical course of experimental autoimmune encephalomyelitis (EAE), an animal model of MS. Male and female C57BL/6 mice were fed a 2.6% ABV Lieber-deCarli diet or isocaloric control prior to MOG35–55-mediated EAE induction and monitored for 40 days. Male alcohol consumers (AC) experienced significantly greater remission compared to controls or AC females. Histological analysis revealed significantly fewer Iba1+ cells in the dorsal horns and lateral columns of AC male cervical and thoracic spinal cords compared to the other groups. 16S rRNA gut microbiome sequencing revealed distinct flora patterns during EAE onset and remission, and showed increased abundance of immunoregulatory species A. muciniphila and A. equolifaciens in AC males. These results demonstrate that moderate alcohol consumption can ameliorate EAE by modulating male microglial activation while simultaneously promoting an anti-inflammatory gut microbiome.
Increased activated platelet binding to T cells in lung cancer patients is correlated with history of thrombosis
Authors: Meikle Claire K , Meisler Adam , Garg Priyanka , Kelly Clare A. , Jeffries Joseph A. , Gao Tess , Bird Cara M. , Willey James C. , Worth Randall G.
Published: January 2023

This study aimed to characterize the role of platelet-leukocyte aggregation in lung cancer patients in context of history of thrombosis. Whole blood from lung cancer patients and healthy volunteers was labeled with fluorescently-conjugated antibodies, then fixed prior to flow cytometry. Platelet-leukocyte aggregates were quantified by detecting the number of platelets within all leukocyte-positive events.

Platelet-CD4 T cell and platelet-CD8 T cell aggregates were both significantly increased in lung cancer patients (both p < 0.0001). Lung cancer patients had significantly more CD4 and CD8 T cells aggregated with activated platelets compared to healthy volunteers (p < 0.01 and p < 0.001, respectively). Lung cancer patients were then separated into groups based on history of thrombosis: No previous thrombosis, arterial thrombotic event (ATE), and venous thromboembolism (VTE). ATE patients had significantly more CD8 T cells aggregated with platelets than patients with no history of thrombosis (p < 0.05). VTE patients had significantly higher expression of platelet activation markers in CD4 and CD8 T cell aggregates than patients with no history of thrombosis (p < 0.01 and p < 0.001, respectively) and ATE patients (p < 0.05 and p < 0.001, respectively).

Platelet-T cell aggregates were significantly increased in lung cancer patients compared to healthy volunteers, and platelet activation within aggregates was significantly correlated with history of VTE in patients with lung cancer. These findings indicate that interactions between platelets and T cells in cancer patients may contribute to a procoagulant phenotype.
Preclinical development of natural compound emodin as an agent to halt breast cancer metastasis
Authors: Fan Daping , Liu Qing , Hodge Johnie
Published: January 2023

Our previous studies demonstrated that a natural compound, emodin, suppresses breast cancer growth and metastasis by modulating tumor-associated macrophages (TAMs), we aim in this study to further develop emodin as an agent to diminish breast cancer post-surgery metastatic recurrence. Using various mouse and human breast cancer cell lines, we demonstrated that emodin not only directly reversed TGF-β1-induced epithelial to mesenchymal transition (EMT), migration and invasion, but also indirectly attenuated EMT by suppressing TAM polarization and TGF-β1 production. Emodin also suppressed the stemness and progenitor properties of tumor-initiating cells (TICs). The EMT process and TIC populations in primary breast tumors were diminished by emodin in vivo, and pretreating tumor cells with emodin suppressed breast cancer lung metastasis in a stem cell-like circulating tumor cell-seeding model. In orthotopic metastasis mouse models, administration of emodin before surgery suppressed the metastatic recurrence of breast cancer and improved the overall survival. Mechanistic study revealed that emodin inhibited both canonical and non-canonical pathways of TGF-β1 signaling in breast cancer cells. Taken together, our study demonstrated that emodin can be developed as a therapeutic agent to prevent post-surgery metastatic recurrence of breast cancer by suppressing TGF-β1-mediated crosstalk between breast cancer cells and TAMs.
RELT stains prominently in B cell lymphomas and binds proteins associated with leukemia
Authors: Cusick John K , Alhomsy Yasmeen , Talbott George , Sumida Cara , Aghaians Dino , Hejazi Nazila , Jacobs Aaron
Published: January 2023

Receptor Expressed in Lymphoid Tissues (RELT) is a human TNFR that is expressed prominently in the hematopoietic system and negatively regulates T-cell activation in mice. RELT has two identified homologous binding partners, RELL1 and RELL2. This study sought to further elucidate the function of RELT by identifying novel protein interactions with RELT family members and to study the localization of RELT in both normal and diseased tissues. A yeast two-hybrid screen identifed Phospholipid Scramblase 1 (PLSCR1) and MyoD family inhibitor domain-containing protein (MDFIC) as potential RELT-family member binding proteins that were confirmed by in vitro co-immunoprecipitations. PLSCR1 has been demonstrated to possess anti-leukemic properties, and RELT expression results in an altered cellular localization of PLSCR1 as determined by immunofluorescence (IF). The MDFIC gene encodes for a transcription factor and is located proximally to regions of chromosome 7 (7q31.1) frequently lost in AML patients. MDFIC was observed to co-localize with RELL1 at the plasma membrane and co-localize with RELT in intracellular compartments as determined by IF. Since RELT, PLSCR1 and MDFIC are prominently expressed in the hematopoietic system, we sought to characterize the expression of RELT in both normal lymph nodes and B cell lymphomas. Immunohistochemistry revealed a higher intensity of RELT staining in germinal centers in comparison to surrounding lymph node regions. Interestingly, the level of RELT staining increased progressively in low and high-grade B cell lymphomas versus normal lymph nodes. Collectively, these results further our understanding of proteins that interact with RELT and identify an association of RELT with B cell lymphomas.
Epigenetic mediated immune exhaustion persists after successful Tuberculosis therapy
Authors: DiNardo Andrew R , Rajapakshe Kimal , Nishiguchi Tomoki , Mtetwa Godwin , Maphalala Gugu , Makedonas George , Coarfa Cristian , Mandalakas Anna M
Published: January 2023

BACKGROUND

Chronic antigen stimulation from HIV, HPV, LCMV and schistosomiasis induce epigenetic-mediated immune exhaustion. Individuals with successful anti-TB therapy (ATT) have a thirteen-fold increased risk of developing recurrent TB.

METHODS

From a cohort of HIV infected and uninfected individuals with TB and their asymptomatic household contacts (n=32), genome-wide DNA methylation (DNA MethylEPIC) and epigenetic deconvolution (EDEC) identified cell-specific DNA methylation. Cell-specific results were validated with MSRE-PCR, gene expression and flow cytometry-based Mtb-specific multi-dimensional immune profiling.

RESULTS

Participants with TB had DNA hypermethylation in the IL-2, TCR and IFN-γ signalling pathways. The IFN-γ signalling pathway was affected in both innate (NK cells and monocytes) as well as adaptive (CD4 and CD8 T cells) lineages. Select DNA hyper-methylated differences were validated using MSRE-PCR. Pioneer and Transcription factors critical for cell-mediated immunity were also significantly differentially methylated with enrichment scores compatible with previous epigenetic-mediated immune exhaustion. After overnight stimulation with IFN-γ, TB patients have decreased IFN-γ-inducible gene expression. Six months after the completion of successful ATT, participants with TB have persistent DNA hypermethylation.

DISCUSSION

Persistent DNA hypermethylation may be a potential mechanism by which individuals with TB are at increased risk of recurrent disease. This may also assist in identifying potential new targets for host-directed therapy.