bluuebunny/crossref_metadata_2025 huggingface dataset:
Antibody targeting tumor-derived soluble NKG2D ligand sMIC provides dual co-stimulation of CD8 T cells and enables sMIC tumors respond to PD1/PD-L1 blockade therapy
Authors: Dhar Payal , Zhang Jinyu , Larrocha Pablo Saenz-lopez , Zhang Bin , Wainwright Derek A , Wu Jennifer D
Published: January 2023

Insufficient co-stimulation has been demonstrated to account for the suboptimal activation of cytotoxic CD8 T cells (CTLs) and presumably unsatisfactory clinical expectation of PD1/PD-L1 blockade therapy. Tumor-derived soluble NKG2D ligands are associated with poor clinical response to PD1/PD-L1 blockade therapy in cancer patients. One of the most frequently occurring tumor-derived soluble NKG2D ligands, the soluble MHC I chain related molecule (sMIC) can impair co-stimulation to CD8 T cells. We investigated whether co-targeting sMIC can provide optimal co-stimulation to CTLs and enhance the therapeutic effect of PD1/PD-L1 blockades. Single agent therapy of a PD1/PD-L1 blockade antibody or a sMIC-targeting non-blocking antibody or a combination therapy of the two antibodies were implied to well-characterized pre-clinical MIC/sMIC+ tumor models that closely resemble the NKG2D-mediated onco-immune dynamics of MIC+ cancer patients. Therapeutic efficacy and associated effector mechanisms were evaluated. We show that antibody co-targeting sMIC enables or enhances the response of sMIC+ tumors to PD1/PD-L1 blockade therapy. The therapy response of the combination therapy was associated with enhanced antigen-specific CD8 T cell enrichment and function in tumors. Co-targeting sMIC with a nonblocking antibody provides antigen-specific CD8 T cells with NKG2D and CD28 dual co-stimulation, in addition to elimination of inhibitory signals, and thus amplifies antigen-specific CD8 T cell anti-tumor responses. Our findings provide the proof-of-concept rationale and previously undiscovered mechanisms for co-targeting sMIC to enable and enhance the response to PD1/PD-L1 blockade therapy in sMIC+ cancer patients.
Glial TIM-3 shows distinct expression patterns and functions in brain tumor microenvironment
Authors: Kim Hyung-Seok , Kim Ki Sun , Yoon Hee Jung , Park Eun Jung
Published: January 2023

T-cell immunoglobulin and mucin domain containing molecule-3 (TIM-3) is a transmembrane glycoprotein. TIM-3 has diverse immune functions, which differ according to the specific cell type and cell status and appears to play both positive and negative roles in a context-dependent manner. In this study, we questioned whether TIM-3 could be involved in immune surveillance against brain tumor, and thereby affect tumor immunity. To explore whether and how TIM-3 responds to brain tumor and whether it could play a role in specific immune cells of the brain tumor microenvironment, we used an intracranial mouse brain tumor model system. Here, we present that TIM-3 is expressed on both growing tumor cells and their surrounding cells including glial and T cells in an orthotopic mouse glioma model. Comparison of cells from tumor-bearing and contralateral hemispheres of a mouse glioma model shows that TIM-3 level is distinct in tumor-infiltrating CD45midCD11b+ glial cells. We find that TIM-3 affects the expression of several immune-associated molecules in primary glia-exposed conditioned media (CM) from brain tumors. Collectively, these findings suggest that glial TIM-3 actively and distinctively responds to brain tumor, and plays specific immunoregulatory roles in the brain tumor microenvironment.
T cell intrinsic DNA damage and repair response as a novel marker associated with clinical response to PD-1 blockade
Authors: Muroyama Yuki , Huang Alexander C. , Manne Sasikanth , Herati Ramin S. , Mathew Divij , Xu Caiyue , Chilukuri Lakshmi , Zamarin Dmitriy , Friedman Claire F , Wherry E John
Published: January 2023

Despite the high tumor mutational burden (TMB), immune checkpoint blockade (ICB) still fails in some microsatellite instability-high (MSI-H) tumors, suggesting the underlying T cell dysfunction. Immune profiling of PBMC from chemotherapy-resistant MSI-H uterine cancer patients treated with nivolumab identified the proliferative T cell response 2–4 weeks post PD-1 blockade. However, this “immunological response” to ICB was observed regardless of clinical response, suggesting the additional determinants of clinical outcome beyond the proliferative response or high TMB. To test the hypothesis that the gap between immunological and clinical response to ICB was related to T celI-intrinsic response to genotoxic and/or proliferative stress, a novel high dimensional cytometry platform was developed. The platform enables simultaneous analysis of T cell differentiation state and interrogation of multiple DNA damage and repair response (DDR) pathways at single cell resolution. This DDR-Immune platform revealed consistent T cell subset specific patterns of DDR, as well as specific DDR pathways induced by different types of DNA damage. Application of the DDR-Immune platform to this cohort revealed that, in clinical responders, Ki67+ CD8 T cells had rapid increase of pATM, presumably in response to proliferative stress induced by PD-1 blockade. This DDR was not clearly observed in clinical non-responders. In addition, Tregs from clinical non-responders had elevated DDRs compared to responders, suggesting their greater resistance to genotoxic/proliferative stress. Collectively, the new platform reveals previously unrecognized roles for T cell-intrinsic DDR as a novel determinant of immune responsiveness and clinical outcome.
Precision immunology for discovery and development of the Precision Vaccines Program (PVP)-037 small molecule series: imidazopyrimidine adjuvants identified via age-specific human in vitro modeling
Authors: Soni Dheeraj , Borriello Francesco , Scott David A , Ozonoff Al , Brightman Spencer , Smith Jennifer , Shamu Caroline , Ramirez Juan C , Baden Lindsey Robert , Cheng Wing Ki , van Haren Simon Daniel , Pettengill Matthew , Dowling David J , Levy Ofer
Published: January 2023

Infection is a significant cause of mortality and immunization is the most promising biomedical intervention to reduce this burden. However, multiple booster doses are often required to protect those with distinct immunity such as the young, elders, and immunocompromised individuals. Adjuvants are molecules that potentiate vaccine responses. There is a need for a larger toolbox of adjuvants, since Alum is the only adjuvant currently incorporated into licensed vaccines in the pediatric setting. We utilized a unique in vitro approach to discover new adjuvants, screening a ~10k small molecule library via a no-wash 384-well quantitative TNF immunoassay in peripheral blood mononuclear cells (PBMCs) from 3 adult healthy human donors. Preliminary hits were based on induced TNF production in at least two of the three donors screened. We identified a novel imidazopyrimidine (IMP) scaffold named PVP-037 which demonstrated efficacy, potency, and adjuvanticity. PVP-037 demonstrated broad age-independent immune-stimulatory ability in human PBMCs and dendritic cells, including key Th-polarizing cytokines such as IL-12, and significant enhancement murine humoral responses to recombinant hemagglutinin (rHA) proteins in the quadrivalent influenza. PVP-037 further demonstrated limited activity in several common cell lines (THP-1) and murine/porcine primary leukocytes. Structure activity relationship (SAR) studies identified the analog PVP-144-3 with greater in vitro efficacy across species (porcine, non-human primates) and murine in vivo adjuvanticity. Overall, we discovered an IMP adjuvant via high throughput screening of human mononuclear cells in vitro which correlated with enhanced influenza immunization in mice.
Corn based nanoparticle delivered inactivated influenza virus vaccine intranasally augments mucosal immune response in pigs
Authors: Gourapura Renukaradhya J , Feliciano-Ruiza Ninoshkaly , Renu Sankar , Lu Fangjia , Han Yi , Schrock Jennifer , Dhakal Santosh , Patil Veerupaxagouda , HogenEsch Harm
Published: January 2023

Swine influenza A virus (SwIV) causes respiratory tract infection in pigs. Available SwIV vaccines fail to provide cross-protective immunity in pigs. Nano-11 is an amphiphilic nanoparticle (70–80nm) obtained from sweet corn-derived phytoglycogen. Nano-11 carries high surface positive charge and thus facilitates easy preparation of nanoparticle based vaccine by electrostatic interaction with killed SwIAV antigen (KAg) or peptides (negative charge). Earlier we showed that Nano-11 bound killed SwIV H1N2 Ag (Nano-11+KAg) delivered intranasally in pigs induced mucosal antibody response, but the challenge heterologous H1N1 SwIV load was not substantially reduced in the airways. In this study, KAg or conserved ten IAV peptides co-adsorbed with adjuvant Poly(I:C) (negative charge) on Nano-11 [Nano-11+KAg/peptides+Poly(I:C)] was vaccinated to influenza-free pigs intranasally, twice, and challenged with a heterologous SwIV. We observed increased SIgA and IgG responses in the airways and enhanced proliferation of IFN-g+ gd T cells in PBMCs in Nano-11+KAg+Poly(I:C) vaccinates compared to control. In Nano-11+peptides+Poly(I:C) vaccinates noticed an increased proliferation of IFN-g+ gd T cells and IFN-g+ cytotoxic T cells in PBMCs compared to control. Commercial vaccine group induced higher IgG response in serum and proliferation of IFN-g+ T-helper/memory cells in PBMCs compared to control. However, reduction in challenge virus load in any of the vaccinated groups was not statistically significant. In conclusion, inclusion of Poly(I:C) in Nano-11 flu vaccine improved the T cell response, but further improvements in the vaccine formulation is required to take advantage of this easy to prepare particle based mucosal flu vaccine.
Trispecific antibody against HIV-1 demonstrate anti-viral activity in vivo and have potent Fc-dependent effector functions
Authors: Almasri Cassandra , Xu Ling , Dias Joana , Promsote Wanwisa , Wei Ronnie , Lord Dana M , Rao Ercole , Beninga Jochen , Yang Zhi-yong , Pegu Amarendra , Mascola John R. , Nabel Gary J.
Published: January 2023

Broadly neutralizing antibodies (bNAbs) against HIV-1 have been suggested as a complementary immunotherapy to current combination small molecule anti-retroviral therapies (cART) for treatment of HIV-1 infection. Monoclonal bNAbs when used as a monotherapy demonstrate high susceptibility for escape in HIV-1 infected individuals, thus a trispecific bNAb targeting three independent HIV-1 envelope determinants was developed to achieve more robust suppression. Upon treatment of viremic simian-human immunodeficiency virus (SHIV)-infected macaques with the trispecific bNAb, plasma viral suppression of up to 1000-fold was observed. To elucidate a mechanism for the observed plasma viremia control, in vitro assays using the trispecific bNAb were conducted to determine Fc-mediated effector functions. First, we determined that the trispecific bNAb bound to HIV-1 infected cell lines with an equal or higher affinity than its parental bNAbs alone. We then conducted antibody-dependent cell cytotoxicity (ADCC) and antibody-dependent cell phagocytosis (ADCP) assays and found that the trispecific bNAb had comparable or better activity compared to its parental bNAbs alone. Similarly, when we measured direct virolysis of HIV-1 virus using an antibody-dependent complement-mediated virus lysis (ADCML) assay, the trispecific bNAb had comparable or better activity compared to its parental bNAbs alone. These studies demonstrate that the trispecific bNAb shows anti-viral activity in vivo and has potent Fc-dependent effector functions that target both free virus and virally infected cells compared to monoclonal bNAbs alone. Thus, the trispecific bNAb is a promising single immunotherapeutic protein for treatment of HIV-1 infection.
Role of antibody in vaccine-mediated protection against pneumonic tularemia in the rabbit model
Authors: Reed Douglas S , Olsen Emily , Bowling Jennifer , Willett Katherine , Hazlett Karsten , Barry Eileen
Published: January 2023

Francisella tularensis, a gram negative coccobacillus, is the causative agent of tularemia, a severe zoonotic infection. When inhaled, as few as 15 cfu is sufficient to cause disease. There is concern that tularemia could be used as a biological weapon so vaccines are urgently needed. We have previously shown that the New Zealand White (NZW) rabbit is a good model of human disease; after exposure to small particle aerosols containing virulent F. tularensis at doses as low as 66 cfu, naive NZW rabbits develop fever within 3 days and succumb within 4–7 days. Disease pathology in the NZW rabbit strongly resembles what has been reported in humans. Recombinant derivatives of a virulent strain of F. tularensis are highly attenuated in NZW rabbits and protect against aerosol challenge with virulent F. tularensis. The level of protection is dependent on the strain used, the route of vaccination, and the number of vaccinations. Using a prime-boost small particle aerosol vaccination regimen, 73% percent of rabbits survived a robust (100 LD50) challenge when vaccinated with a strain that is deficient in the aromatic amino acid synthase pathway. Pre-challenge, post-vaccination plasma IgG and IgM titers against heat-killed F. tularensis correlate with protection. A single inoculation of plasma from hyper-immune rabbits extended the survival of naïve rabbits by one day. We will report our efforts to evaluate the role of antibody in protection of NZW rabbits against tularemia, including passive immunization of mice using purified immunoglobulin from hyper-immune rabbits and in vitro assays of antibody function.
In silico design of a universal carrier protein: A potential solution to the variable immunogenicity observed in past vaccines against drugs of abuse
Authors: Saylor Kyle Lucas , Donnan Ben , Zhang Chenming
Published: January 2023

Vaccines against drugs of abuse (VADAs) show promise as a possible therapeutic for the alleviation of addiction due to their ability to modulate intake behavior via the altering of drug pharmacokinetics. In the past, however, these vaccines have failed to show clinical efficacy due to extensive variability in the magnitude of antibody response between individual vaccinees. Correspondingly, the future success of VADAs hinges directly on whether or not researchers can find ways to engineer more broadly effective vaccines. Differences in response must, to some extent, be the result of variable capacities for each individual’s immune system to process and/or present antigens. As such, we speculate that future VADAs will need to target the three key stages of vaccine processing, recognition, processing, and presentation, in order to achieve clinical effectiveness. Here, we describe a project aiming to engineer a broadly effective carrier protein based on this concept. Using haplotype frequency data provided by Be the Match, HLA-DQ and -DR haplotypes covering 99% of the population were selected for analysis. Next, the most common carrier proteins used in conjugate vaccine formulations were analyzed for MHC II epitope content using these haplotypes and NetMHCIIPan 3.2 prediction software. Finally, a modified version of the prediction output was used to excise the most likely MHC II epitopes. The highest-ranked epitopes were then stitched together using linkers designed with chemical conjugation sites and cathepsin cleavage sequences and subsequently tethered to cholera toxin subunit B. These results present the successful design of an in silico derived, broadly effective carrier protein to be used in future VADA formulations.
Cell-based cancer immunotherapy using chimeric antigen receptor (CAR)-engineered natural killer (NK) cells surface-modified with drug-loaded nanoparticles targeting adenosine receptors
Authors: Cinay Gunce Ezgi
Published: January 2023

The immunosuppressive tumor microenvironment (TME) plays a key role in failed eradication of solid tumors and tumor recurrence upon cancer treatment, as resident immune cells lose their cancer surveillance abilities. One of the underlying mechanisms responsible for the loss of adoptively transferred immune cell function in the TME is the A2a adenosine receptor (A2aR) inhibitory pathway. Encapsulating SCH-58261 (SCH), a potent antagonists of A2aR, in crosslinked multilamellar liposomal vesicles (cMLVs) was shown to efficiently deliver the drug to adoptively transferred cells in tumors. We genetically modified human NK92 cells to express anti-mesothelin CAR (ameso.CAR.NK) to be used in this study.

We hypothesized that the efficacy of NK cell adoptive transfer therapies could be enhanced if CAR.NK cells were used as active carriers to direct SCH-loaded cMLVs to the immunosuppressive TME to block the A2aR pathway. We first covalently attached cMLVs to NK cell surface. We next verified that cMLV conjugation did not have adverse effects on NK cell viability and migration. In addition, surface conjugation of cMLVs did not reduce IFN-γ secretion and cancer cell killing ability of NK cells, suggesting similar levels of cognate antigen-specific activation of ameso.CAR.NK cells by mesothelin-overexpressing ovarian cancer cells.

In the future, we will test the in vitro function of irradiated CAR.NK cells, given the requirement of irradiating NK92 cell lines prior to clinical use. Next, we will test our platform in an ovarian cancer xenograft mouse model and examine the effects of our therapy on tumor growth. Finally, we will investigate the potential of this therapy to reverse immune suppression on the TME-resident tumor-infiltrating NK cells.
The length of the intracellular domain conditions the antitumor efficacy of CAR-T cells
Authors: Ramello M Cecilia , Roselli Emiliano , Abate-Daga Daniel
Published: January 2023

Among the variables affecting CAR-T cell function, evidence shows that CAR architecture is a common factor influencing clinical outcomes. Previously, we demonstrated that a 2nd generation (gen.) CAR targeting the prostate stem cell antigen (PSCA) was more efficient than a 3rd gen. counterpart, in a preclinical model of pancreatic adenocarcinoma. This superior anti-tumor capacity was associated with greater tonic signaling. To further characterize this phenomenon, we compared the antitumor activity and phenotype of two 2nd gen. CAR-T cells (CAR-Ts) that differed in the length of the intracellular domain (ICD) by only 24 amino acids: PSCA-8t28Z vs. PSCA-8t(+24)28Z. Upon adoptive transfer into tumor-bearing mice, PSCA-8t28Z CAR-Ts displayed significantly greater antitumor efficacy. This was not due to differences in CAR surface expression, or in trafficking or persistence of T cells, since CAR-T numbers in spleen and tumor were comparable among groups. However, it was associated with significantly higher phosphorylation of CD3ζ in PSCA-8t28Z CAR-Ts in spleen, suggesting spontaneous CAR-signaling activity. Unsupervised analysis revealed that proliferation was similar between CAR-T and GFP T cells in spleen. In contrast, tumor-infiltrating (TI) PSCA-8t28Z CAR-Ts exhibited a cluster of Ki-67hi cells that was absent in the other groups. Moreover, TI PSCA-8t28Z CAR-Ts showed the highest expression of inhibitory receptors, suggesting antigen-induced CAR signaling within the tumor. Together, our results show that shorter ICDs are associated with higher tonic signaling and higher T cell activation after antigen recognition. These data highlights the importance of CAR-ICD length for optimal CAR-T function in solid tumors.
A commensal Clostridium species restricts systemic alphavirus dissemination through a type I interferon signaling axis
Authors: Winkler Emma S , Thackray Larissa B , Hykes Barry L , Handley Scott A , Droit Lindsay , Andhey Prabhakar , Mack Matthias , Shrihari Swathi , Swain Amanda , Cella Marina , Colonna Marco , Artyomov Maxim , Stappenbeck Thaddeus S , Diamond Michael S
Published: January 2023

The role of the gut microbiota in shaping antiviral immune responses at non-intestinal sites has been described in multiple models of viral pathogenesis. Although chikungunya virus (CHIKV), an emerging alphavirus, is a substantial global health threat, the impact of the microbiota in modulating immune responses to alphavirus infection is entirely unexplored. Here, we show that CHIKV infection of oral antibiotic-treated or germ-free mice resulted in increased viremia within one day of infection, and this enhanced viral dissemination. Microbiota depletion resulted in greater CHIKV infection of circulating monocytes, which was linked to dampened systemic type I interferon (IFN) responses. Differences in CHIKV tropism and replication following microbiota depletion depended on MyD88 signaling in plasmacytoid dendritic cells, which contributed to systemic type I IFN production within hours of infection. Colonization of antibiotic-treated mice with a single bacterial microbiota-derived Clostridium species resulted in MyD88-dependent type I IFN responses that restricted CHIKV infection in blood. We propose that select bacterial symbionts impact antiviral immunity to alphaviruses within hours of infection through a type I IFN signaling axis and by virtue of effects on viremia may determine disease severity and affect vector transmission and epidemic spread.
HLA-B recycling and trafficking pathways in primary human monocytes
Authors: Olson Eli C , Raghavan Malini
Published: January 2023

Human leukocyte antigen class I (HLA-I) molecules are a heterotrimeric protein complex responsible for presenting peptide antigens to CD8+ T cells. Typically, peptides are loaded onto the HLA-I complex in the ER and trafficked to the cell surface through the secretory pathway. Efficient peptide loading in the ER involves specific chaperone interactions to stabilize open conformers. While peptide loading is a tightly regulated process, HLA-B allotypes vary in their assembly and expression characteristics. We have recently found that some HLA-B allotypes such as B*35:01 are expressed at high levels in monocytes when compared with other allotypes such as B*08:01. Monocytes contained high levels of intracellular HLA-I, which is accumulated in a non-ER AP-1+ compartment. We hypothesized that monocytes possess a secondary mode of assembly involving recycling through the endo-lysosomal pathway. We find that HLA-B accumulates in various endo-lysosomal compartments. Additionally, endosomal recycling appears to be important for maintaining surface expression. Certain allotypes exhibit greater cell-surface rescue when lysosomal acidification is inhibited, consistent with a model of differential dependence on ER assembly factors. Taken together, these results indicate that endosomal pathways play a role in maintaining constitutive HLA-B surface expression, and that the inhibition of recycling pathways results in a loss of surface HLA-B. Some allotypes are more susceptible to degradative trafficking due to their chaperone-dependent assembly mechanisms, leaving them more vulnerable to peptide dissociation in the endosomes and trafficking to lysosomes.
Isoform repertoire of Blood mononuclear cells from SLE patients
Authors: Carmen Silvina del , Singh Prashant , Nath Pulak , Zhao Yuqi , Huynh Richie , Chung Cheng-Han , Rossi Robert , Pascual Virginia , Banchereau Jacques
Published: January 2023

Systemic Lupus Erythematosus (SLE) is characterized by numerous immunological alterations, including increased expression of immune activators, such as IFNa, along with alterations in cell composition, such as expansion of plasma cells that produce autoantibodies.

Alternative Splicing (AS) is a biological process that regulates post-transcriptional alterations of the RNA. Pathogenic AS events have been associated to several diseases. Furthermore, the description of SLE autoantibodies that target components of the spliceosome such as anti-Sm/RNP, anti-Ro/La, and anti-dsDNA has led us to hypothesize that SLE is associated to abnormal AS events.

RNA-seq uses standard Short-Read technologies which involve RNA or cDNA fragmentation for the generation of 150 bp sequencing libraries. Since most mammalian mRNAs are 1–2 kb long, using RNA-seq to identify novel alternatively spliced isoforms is extremely challenging. However, emerging Long-Read sequencing technologies allow full-length mRNA sequencing, de novo transcriptome assembly, and isoform profiling. Our lab has built a computational pipeline that combines Long-Read sequencing with RNA-seq data to identify and quantify novel transcripts in peripheral blood mononuclear cells (PBMCs) from SLE patients compared to healthy controls. From our computational analysis, we have observed that SLE patients display differential exon-inclusion and exclusion events in transcripts coding for immune activating and regulatory proteins, respectively. We conclude that SLE might be associated with pathogenic AS events that might place the immune balance under double jeopardy, through activation of immune activating receptors and inhibition of immune inhibiting receptors.
Characterization of new circular RNA products from the Pax-5 gene in B-cells
Authors: Robichaud Gilles A. , Hannay Brandon , Martin Danick , Veilleux Vanessa , Finn Nicholas
Published: January 2023

Circular RNAs (circRNAs) represent a new class of RNAs which are highly expressed and conserved in mammalian cells. CircRNAs regulate many biological processes and their aberrant expression is often associated with disease progression. We have identified and characterized circRNAs generated from the Pax-5 gene, an essential transcription factor for B-cell lineage commitment and also, a potent oncogene. Given the pivotal role of Pax-5 gene products in B-cell development and function, we set out to profile Pax-5 circular RNA (circPax5) expression and elucidate their role in B-cell biology. We identify and map five major circPax5 transcript isoforms which are found in B-cell lines and primary B-cells isolated from peripheral blood mononuclear cells (PBMCs) of healthy donors. These circPax5 variants consists of circular single-stranded RNA sequences corresponding to: i) exons 2–3; ii) exons 2–4; iii) exons 2–5; iv) exons 2–6; and, v) exons 2–8 of the human Pax-5 gene. Using TaqMan assays designed to target each unique circPax5 junction region, we found that circPax5 expression profiles vary with B-cell developmental stages. Furthermore, circPax5 exons 2–5 and 2–6 are overexpressed in patients suffering from chronic lymphocytic leukemia (CLL). Functionally, we observe that circPax5 products modulate B-cell processes and interact by sponging of specific micro-RNAs. Altogether, we identify new genetic products (i.e. circRNAs) generated from a prominent immune-regulating gene (Pax-5). The functional elucidation of circPax5 products will potentially bring insight into the function and signaling of B-cell processes and disease.
Missense mutation in non-recombinase domain of RAG1 results in B-cell developmental defect
Authors: Bodansky Aaron L , Burn Thomas , Behrens Edward M
Published: January 2023

Objective

To examine how a single amino acid change to a region of RAG1 that is not related to VDJ recombination affects B-cell development.

Methods

We studied B-cells within a Rag1 P326G missense mutant mouse (Rag1PG) utilizing flow cytometric analysis to phenotype B-cell development and peripheral populations, as well as biochemical studies examining the B-cell Receptor (BCR) molecular complex and signaling function. We also created a mixed bone marrow chimera to determine which alterations were cell-intrinsic.

Results

We find that overall there is an ~50% decrease in total B-cells in the Rag1PG mouse compared to wildtype (WT) due to a Pro-B to Pre-B-cell developmental block. Peripheral B cell cytopenias are in the Follicular Zone B-cell compartment, with sparing of the Marginal Zone B-cells. There is decreased Syk expression in Rag1PG—however no differences in BCR signaling strength were appreciated in Rag1PG. In the mixed bone marrow chimera, we find a competitive advantage in the WT developing B-cells, but peripheral B cell populations survive equally well.

Conclusion

Despite the Rag1PG protein being a single amino acid change to a region of RAG1 not involved in recombination, we nevertheless find a developmental defect consistent with leaky SCID. Given that there is no evidence of altered BCR signaling, and the developmental block occurs at the stage in B-cell development in which VDJ recombination is first required, these data suggest a recombination defect. The mutation is in a ubiquitin ligase domain, and it is possible this alters currently unappreciated roles of RAG1, such as histone degradation to allow RAG1 access to DNA. Our findings illustrate how mutations in the non-recombinase domain of RAG1 may result in leaky SCID.
Landscape of bone marrow T cells in acquired aplastic anaemia revealed by single-cell sequencing
Authors: LIU YAN , Wang Shuwen , Zuo Xinyi , Peng Jun
Published: January 2023

Objective

Acquired aplastic anaemia (AA) is a T-cell-mediated autoimmune disease characterized by hematopoietic stem cell destruction in the bone marrow (BM). The aim of our study is to explore the cellular heterogeneity, lineage and functional states of T cells involved in the pathogenesis of AA.

Methods

CD3+CD4+ and CD3+CD8+ mature T cells, isolated from BM of five newly diagnosed severe AA patients and two healthy controls by FACS, were subjected to single-cell RNA sequencing (ScRNA-seq) and T cell receptor (TCR) sequences using 10X Genomics Chromium Single Cell 5′ and Single-Cell V(D)J protocols. Libraries were sequenced on Illumina NovaSeq platforms.

Results

We obtained transcriptome data for 47,508 cells from severe AA patients and 20,758 cells from controls. Bioinformatic analysis of the transcriptional profiles and TCR sequences revealed their molecular and functional properties and developmental trajectory. All clusters are not library specific, but consisted of cells from 7 different donors. We identified all major cell types and signature genes for each subset. We discovered a marked decrease of three heat shock protein family A (Hsp70) members (HSPA6, HSPA1A and HSPA1B) as a signature of AA patients. Interestingly, this phenomenon occurred for all T cell subpopulations. GO annotations related to these genes included response to temperature stimulus, unfolded protein binding and protein folding. We suspected these Hsp70 members were closely related to the pathogenesis of AA.

Conclusions

Our results establish a reference map of human BM T cells in health and AA patients. and demonstrate that T cells have unique characteristics in AA patients, which may represent a pathogenesis of this autoimmune disease.
IFNγ Contributes to Splenic B and T cell Activation and Cytopenia in STING Gain-of-Function Autoinflammation
Authors: Gao Kevin MingJie , Motwani Mona , Marshak-Rothstein Ann , Fitzgerald Kate A.
Published: January 2023

cGAS-STING is a cytosolic dsDNA sensing pathway whose regulation is vital to immune homeostasis. Constitutively active STING mutations cause an autoinflammatory syndrome known as STING Associated Vasculopathy with onset in Infancy (SAVI). Pediatric SAVI patients develop treatment resistant lung fibrosis associated with immune abnormalities, including lymphopenia with hyperactivity of remaining lymphocytes and an interferon-stimulated gene signature. Unlike other cGAS-STING driven autoinflammatory syndromes in which Type 1 interferons (T1IFNs) mediate pathology, loss of the T1IFN receptor fails to prevent disease in mice expressing the STINGV154M SAVI (VM) mutation. Instead, lymphocytes play a critical role in SAVI, as VM Rag1−/− mice fail to develop lung disease. Thus, we sought to test whether T cell production of IFNγ (Type 2 IFN) could be a causal mechanism of disease in SAVI, and predicted that IFNγR1 and TCRβ deficiency ought to rescue SAVI disease in a similar fashion. In alignment with our expectations, we found that B cell lymphopenia and hyperactivity were equally rescued in VM IFNγR1−/− and VM TCRβ−/− mice. Moreover, IFNγR1 deficiency also led to improvements in thymic development, T lymphopenia, and hyperactivity. However, despite resolution of splenic lymphocyte defects in VM IFNγR1−/− and VM TCRβ−/− mice, these mice continued to develop fibrotic lung disease. This finding indicates a T cell and IFNγ independent process in SAVI lung disease pathogenesis. In concordance with this, we have recently found that B cells accumulate in the VM lung with a highly activated phenotype independent of αβ T cells, suggesting a potential role for B cells in the etiology of SAVI fibrotic lung disease which we are actively investigating.
Immunodietica: towards a data-driven approach to dissect interactions between diet and autoimmune disease
Authors: Ferreira Leonardo , Burov Andrey A. , Miao Brenda Y. , Morais Vasco H. , Gershteyn Iosif M.
Published: January 2023

50 Million people in the United States are estimated to suffer from an autoimmune disease, and this number is predicted to continue growing. Up to 70% of cases could be due to environmental factors. Therefore, an understanding of how diet, likely a major component of the environmental factors described, impacts the immune system merits exploration. We sought to develop a system of comparison between human epitopes implicated in seventy autoimmune diseases and the overlap between them and epitopes found in various commonly consumed animals and plants. The aim of the comparison was to construct a comprehensive database of overlap to provide an index, the Gershteyn-Ferreira index, of potential auto-immunogenicity of various diet-derived proteins. The construction of the database utilized the automation of querying and recording the epitopes with the IEDB (Immune Database and Analysis Resource) system. We created a comprehensive index for twenty-four organisms. Of note, our system personalizes the epitope overlap of the organisms per individual by integrating the human leukocyte antigen (HLA) alleles of the subject and re-running the overlapping epitopes to filter for in silico HLA binding. In addition, we mapped the tissue expression pattern of each epitope to gauge the risk of being exposed to it in the diet. With this new system, Immunodietica, we can determine exact diet-derived epitopes that could be responsible for the pathogenesis or symptom severity of autoimmunity, as well as generate a personalized ‘heat-map’ of potential triggers, the Gershteyn-Ferreira sensitivity passport, for use by autoimmune disease patients.
An amino acid change in IRF7 increases SLE risk through transcriptional regulation of type I interferons
Authors: Javier Ellen F , Dunn Katelyn , Donmez Omer , Forney Carmy , Roe Margaret , Rowden Hope , Miller Daniel , Javier Jose , McDaniel Margaret M , Pasare Chandrashekhar , Waggoner Stephen N , Weirauch Matthew T , Kottyan Leah C
Published: January 2023

Systemic Lupus Erythematosus (SLE) is an incurable, debilitating autoimmune disease characterized by widespread inflammation and rampant production of autoantibodies. The most prominent and highly replicated set of genes up-regulated in the immune cells of patients with SLE are the type I interferons (IFN-I) and IFN-responsive genes. IFN-I are predominantly made by plasmacytoid dendritic cells (pDCs), and their expression is directly regulated by the transcription factor interferon regulatory factor 7 (IRF7). While IRF7 is an established SLE risk locus, the variants responsible for disease pathology remain unknown. We hypothesize that an amino-acid changing SLE risk variant in IRF7 (rs1131665) alters expression of disease-relevant IFN-I in clinically-relevant cells to increase SLE risk. The functional genomic consequences of the SLE-associated variant were assessed in human cell lines and in genome-edited mice with an introduced SLE-risk variant at Irf7. Our data demonstrate greater than 2-fold genotype-dependence in IFN-stimulated response element-driven luciferase activity and inflammatory cytokine secretion detected in supernatant after toll-like receptor-7 stimulation. Gene expression differences in cells with IRF7/Irf7 risk variants are consistent with those dysregulated in SLE patients. In the present study, we demonstrate the functional consequences of an amino acid substitution in a critical type I interferon regulator. Understanding these mechanisms will enhance development of more effective clinical practices for autoimmune patients expressing the risk variant for IRF7.
Understanding human Crohn’s disease: T cell immunoprofiling and in vitro primary intestinal epithelial monolayer models
Authors: Kemp Roslyn , Angus Hamish , Fan Lisa , Butt Grant , Schultz Michael
Published: January 2023

Crohn’s disease (CD) is a type of inflammatory bowel disease caused by uncontrolled inflammation and is associated with loss of tolerance to commensal bacteria. There is no current consensus on how CD forms; however, four susceptibility criteria are required for disease: a microbial presence, an immune response, an environmental trigger, and genetic susceptibility.

We investigated the peripheral and intestinal immune response of people with CD compared to healthy people. CD peripheral blood (PBMC) contained higher baseline frequencies of pro-inflammatory Th17 cells compared to those from healthy controls, and regulatory T cells from CD patients increased in frequency in response to the commensal Faecalibacterium prausnitzii, whereas those from healthy controls did not. CD patient PBMCs secreted higher concentrations of the pro-inflammatory cytokines: IL-6, TNF, and IL-17A, compared to healthy controls.

We developed a human 2-dimensional large intestine organoid monolayer transwell model that replicates the in vivo intestinal epithelial barrier as the cells of the epithelial monolayer are arranged into a selectively semi-permeable polarised membrane, separating the apical and basal regions of the system, emulating the luminal and lamina propria intestinal regions. Co-culture of CD patient-derived monolayers with patient PBMCs and heat-killed F. prausnitzii caused disruption of monolayer integrity, whereas control patient monolayers were unaffected. IL-6 was present in control monolayer supernatants but not those from CD patients. We hypothesise that CD patient PBMCs have inherent abnormal responses to commensal bacteria, and in the context of this model, IL-6 may be required for maintenance of monolayer integrity.
Role of eosinophils and MMP-9 in autoimmune blistering disease bullous pemphigoid
Authors: Liu Zhi , Burette Susan , Diaz Luis , Li Ning
Published: January 2023

Bullous pemphigoid (BP) is an autoimmune inflammatory subepidermal blistering disease associated with autoantibodies against two hemidesmosomal components, the BP230 and BP180 and an inflammatory infiltrate including eosinophils and neutrophils. BP180-specific IgG autoantibodies from BP patients induce BP-like skin lesions in BP180 humanized mice (hBP180 mice). Subepidermal blistering induced by anti-BP180 IgG depends on complement, mast cells, and neutrophils. Since mouse eosinophils lack IgE receptors for human IgE, we generated a humanized IgE receptor FceRI mouse strain (hFceRI) and crossed hFceRI mice onto hBP180 mice to generate hFceRI/hBP180 mice. Anti-BP180 IgE, when injected i.d. into hFceRI/hBP180 mice, trigger infiltration of dominant eosinophils and minor neutrophils and subsequent subepidermal blistering 48 h post IgE injection. In this study, we tested if anti-BP180 IgE-induced BP depends on eosinophils, neutrophils or both. We found that anti-BP180 IgE induce BP in hFceRI/hBP180 mice and neutrophil-deficient but not eosinophil-deficient hFceRI/hBP180 mice. Eosinophil-deficient hFceRI/hBP180 mice reconstituted with eosinophils but not neutrophils from hFceRI/hBP180 mice became susceptible to anti-BP180 IgE-induced BP. Increased MMP-9 was present in diseased mouse blister fluid. MMP-9-deficient and eosinophil-deficient hFceRI/hBP180 mice reconstituted with MMP-9-sufficient but not MMP-9-deficient eosinophils when injected with anti-BP180 IgE developed BP. These data suggest that eosinophils and MMP-9 are critical for BP and could be therapeutic targets for BP treatment.
Telomere length predicts mortality in the Black Women’s Experiences Living with Lupus (BeWELL) Study
Authors: Chae David H. , Chung Kara , Martz Connor D. , Drenkard Cristina M. , Lim S. Sam , Lin Jue , Epel Elissa S.
Published: January 2023

Systemic lupus erythematosus (SLE) is a chronic autoimmune disease marked by inflammation that disproportionately impacts Black women. Telomeres are repetitive sequences of DNA capping the ends of chromosomes that shorten more rapidly in response to inflammation. Studies suggest that the telomere maintenance system may be disrupted in those with SLE, who have been shown to have shorter telomeres compared to healthy controls. Among those with SLE, Blacks have been found to experience more accelerated telomere shortening compared to those of other race groups. We examined the relationship between telomere length (TL) and mortality using data from the Black Women’s Experiences Living with Lupus (BeWELL) Study. Participants were 438 Black women with a validated diagnosis of SLE living in the Atlanta, Georgia metropolitan area, recruited between April 2015 and May 2017 predominantly from a large population-based registry. TL was assayed from dried blood spots collected at baseline and measured as the relative telomere to single copy gene (T/S) ratio. Mortality was assessed prospectively through April 2019. A total of 24 participants died during this time period. There was a negative association between TL and mortality, with shorter TL being associated with greater mortality risk (hazard ratio = 0.01, p=0.02). This relationship remained statistically significant after controlling for age and years since diagnosis, and further adjustment for a range of socio-demographic and health-related factors, including SLE damage and activity. Findings suggest that TL may forecast mortality among Black women with SLE. The association between TL and mortality among people with SLE should be further explored.
Similar cardiovascular disease histopathology and macrophages in heart tissues of younger adult SIV-infected and uninfected aged rhesus macaques
Authors: Kuroda Marcelo J , Petkov Daniel I , Liu David , Allers Carolina , Didier Peter J , Didier Elizabeth S
Published: January 2023

Cardiovascular disease is a major cause of morbidity in aging and in HIV-infected individuals despite efficacy of cART, and furthermore, is associated with macrophage activation and inflammation. Nonhuman primates simulate human disease, and we previously reported that distinct macrophage populations play different roles in the pathogenesis of disease during SIV infection in rhesus macaques. For example, in the lung, short-lived macrophages were more readily destroyed by SIV and contributed to AIDS pathogenesis. Long-lived alveolar macrophages on the other hand were not easily destroyed by virus and thus may contribute to a long-term virus reservoir that also promotes chronic inflammation. The present studies extend this work to characterize macrophages in heart and skeletal muscles affecting cardiomyopathy observed during SIV/HIV infection as well as in heart tissues of uninfected younger and older macaques. The results demonstrated that macrophages are distributed uniformly throughout the heart tissues in animals of all age groups. Increased percentages of macrophages, however, were observed in hearts of aged macaques with similar increased percentages of macrophages in SIV-infected younger adult macaques. Interestingly, the increased macrophages of both aged uninfected and younger SIV-infected macaques was not due to increases in the absolute numbers of macrophages but rather to decreased heart muscle cellularity. This suggested that cardiovascular disease observed in aged as well as HIV-infected individuals may be due to similar mechanisms of decreased number of heart muscle cells and dysfunctional inflammatory long-lived macrophages of the heart.
Single cell transcriptomics of the T cell response to Mycobacterium Tuberculosis reveals phenotypic diversity within and between infected individuals
Authors: Kirschman Junghwa Choi , Ogongo Paul , Ernst Joel , Altin John
Published: January 2023

Tuberculosis (TB) is an airborne infectious disease and caused by Mycobacterium Tuberculosis (MTB) and the leading infectious cause of adult mortality in the world. Despite its high transmission rate and high fatality in untreated individuals, currently there is only one widely-used vaccine and it does not protect adults from TB. The development of a more efficacious vaccine has been hampered by limitations in our understanding of the type of immunity required to protect against TB. Using single cell transcriptomics, we have developed a method of identifying Mtb-reponsive T cell clones and performing deep phenotyping on them. Our study of circulating cells in latently-infected individuals reveal robust differences in the transcriptional phenotypes of Mtb-responsive CD4 T cells, both within and between individuals. We expect that this transcriptome-wide view of Mtb-specific cells, and a simultaneous view of the inciting antigens at peptide resolution, will enable a next-generation studies of the response to Mtb in natural history and vaccine trial cohorts, with the potential to reveal correlations of protection and therefore new vaccine targets and mechanisms.
Prognostic value of autophagy markers in sepsis- a clinical case report
Authors: Mallarpu Chandra Shekar , Ponnana Meenakshi , Prasad Sudhir , Singarapu Maneendra , Chelluri Lakshmi Kiran , Madiraju Charitha
Published: January 2023

Sepsis is characterized by a dysregulated response to infection that is initiated by recognition of pathogen-associated molecular patterns from invasive microorganisms by pathogen recognition receptors to trigger an immune response. The goal is to establish regulatory mechanisms governing sepsis-induced cell death and correlate cell death mechanisms with clinical biochemistry pre- and post- antibiotic treatment. Towards this end, we present a case report of a 48-year-old male patient with a clinical history of adenocarcinoma, colostomy, chronic kidney disease and suspected sepsis admitted to emergency department. Patient was on an antibiotic treatment regimen (Monocef 1 g single dose and Flucloxacillin 1 g single dose for three days). Among several promising biomarkers for sepsis, the role of cell death marker p62/SQSTM1 was evaluated for its prognostic role in this particular case of sepsis. Flow cytometry and ELISA analysis of septic patient’s peripheral blood mononuclear cells (PBMCs) and serum samples were performed and correlated with serum electrolyte and serum biochemistry values pre- and post- antibiotic dose. Flow cytometry data analysis of PBMC samples from septic patient showed significantly lower p62 post- antibiotic when compared to pre- antibiotic dose [p < 0.05]. Notably, comparison of pre- and post- antibiotic treatment regimen showed significant modulation of serum ELISA levels of autophagy marker, p62/SQSTM1, that correlated with alteration in serum potassium levels [p < 0.05]. Our current study demonstrates the impact of serum potassium levels on autophagy in a patient’s case of sepsis.