bluuebunny/crossref_metadata_2025 huggingface dataset:
Inhibition of JAK/STAT signaling sustain stem-like exhausted CD8+ T lymphocytes with enhanced antitumor effects
Authors: Shourian Mitra , Bourdin Benoite , Decaluwe Helene
Published: February 2023

T-cell exhaustion and reduced memory potential is an obstacle to successful adoptive cell therapy (ACT) of cancer. Ex-vivo expansion protocols require sustained TCR and cytokine stimulation that limit ACT efficacy and long-term persistence. We have shown that IL-2 and IL-15, JAK-STAT dependent-cytokines used in ACT expansion protocols, induce CD8+ T-cell exhaustion both in vitro and in vivo following chronic viral infection. We hypothesized that blocking JAK-STAT signaling during ex-vivo expansion protocols will limit T-cell exhaustion and sustain stemness of adoptively transferred cells. In the current study, we mimicked current ex-vivo expansion protocols using purified murine T-cells stimulated by tumor antigens in the presence of IL-2 or IL-15 and evaluated the impact of adding distinct inhibitors of the JAK-STAT pathway on expanded T cells. We observed that JAK-STAT inhibitors reduced CD8+ T-cell exhaustion and increased the number of Ly108+ TCF1+ stem-like progenitors. The reduction in T-cell exhaustion was observed by decreased expression of exhaustion markers, changes in specific transcription factors, and increased expression of effector cytokines using flow cytometry. Adoptive transfer of ex-vivo expanded T-cells to tumor-bearing mice indicated that CD8+ T-cells expanded in the presence of JAK-STAT inhibitor significantly reduced tumor size and increased mice survival after ACT. RNA-seq analysis identified a reduced exhaustion signature in JAK/STATi treated T-cells. These results suggest that targeting JAK-STAT signaling sustains potent antitumor reactivity of adoptive-transferred cells and may enhance the ex-vivo generation of stem-like progenitor CD8+ T cells for long-term survival in the host.
Lack of stem-like exhausted T cells and increased inhibitory receptor expression distinguishes diffuse large B cell lymphomas from Hodgkin lymphoma.
Authors: Vallée Alexis , Shourian Mitra , Decaluwe Helene
Published: February 2023

Immune checkpoint (IC) blockade has revolutionized the treatment of chemo-refractory solid tumors and has demonstrated promising results for the treatment of resistant blood cancers, including lymphoma. However, clinical response to PD1 blockade depends on the subtype of lymphoma, with durable responses observed in 70 % of Hodgkin lymphoma (HL) versus only 10 to 40% of Diffuse Large B-Cell Lymphoma (DLBCL) and Follicular Lymphoma (FL). The biological aspects that hinder the efficacy of immunotherapy in DLBCL and FL are not well-known. Since DLBCL and FL are more refractory to IC therapy than HL, we hypothesized that TILs from DLBCL and FL present a more advanced state of exhaustion than TILs from HL. We performed an immunophenotyping of TILs from 57 HL, FL or DLBCL lymphoma samples collected at diagnosis and frozen as viable cell suspension. Our data revealed that CD8 TILs from DLBCL express higher levels of ICs (PD1, Tim3 and Lag3) compared to TILs from FL and HL. Boolean analysis demonstrated that DLBCL and FL had a higher frequency of CD8T cells that co-expressed 6–7 ICs, compared to HL, suggesting more advanced exhaustion. In contrast, we observed that HL and FL exhibited significantly more stem-like exhausted cells (PD1+/Tim3−/CD38−/TCF1+) compared to DLBCL. Stem-like exhausted CD8 T cells are driven by the transcription factor TCF-1 and extensively proliferate in response to PD1 blockade. Collectively, our data indicate that TILs from DLBCL and FL express a whole array of ICs. DLBCL further lack protective stem-like CD8 T-cell subsets which correlate with poor clinical outcome. These results strengthen our understanding of the cellular defects precluding potent anti-tumor T cell functions in DLBCL and FL.
Therapeutic cancer vaccines in the Pacific Region
Authors: Sika-Paotonu Dianne
Published: February 2023

The growing burden of cancer globally remains of significant concern, with Pacific communities in Aotearoa/New Zealand and the Pacific Region disproportionately affected by some cancers. In addition, Pacific Peoples remain over-represented in adverse health statistics and inequitable health outcomes relating to cancer. There is potential for immunotherapy based therapeutic approaches to be directed towards helping address inequities with cancer treatment, management and outcomes for vulnerable communities, but will require careful consultation, consideration and contextualisation of the clinical setting for vulnerable communities. The purpose of this work was to ensure appropriate discussion and engagement to explore the potential for therapeutic cancer vaccines within the Pacific setting. Preliminary engagement and contextualisation efforts were undertaken as part of broader discussion and consultation activity, to explore the clinical setting and assess the potential applicability of therapeutic cancer vaccines within the clinical context in Samoa. Appropriate infrastructure and costs associated with therapeutic cancer vaccines would remain important factors given the current resource constrained setting in the Pacific. The urgent need to address late presentations and poor prognostic outcomes for cancer patients was also acknowledged. While therapeutic cancer vaccines hold potential for some cancer patients, the appropriate contextualisation, consultation and consideration will be needed to better understand and guide efforts within the Pacific setting in relation to the potential applicability of therapeutic cancer vaccines for addressing inequities.
Nasal immunization with mast cell activating small molecules protects mice against West Nile Virus infection
Authors: Johnson-Weaver Brandi T , Choi Haewoong , Chan Cliburn , Shterev Ivo , Abraham Soman , Staats Herman F
Published: February 2023

Objective:

Mast cell activators are a novel class of mucosal vaccine adjuvants. The polymeric compound, Compound 48/80 (C48/80), and cationic peptide, Mastoparan 7 (M7) are mast cell activators that provide adjuvant activity when administered by the nasal route. However, mast cell activating small molecules may be an adjuvant alternative that is easily synthesized with high purity compared to M7 or C48/80. The study objective is to identify small molecule mast cell activators that provide adjuvant activity in nasal West Nile virus vaccines.

Methods:

High-throughput screening assessed over 55,000 small molecules for mast cell degranulation activity. Fifteen hit compounds were down-selected to three by in vitro evaluation of immune activation activities, including cytokine production and cytotoxicity, and selection for functional diversity. These three molecules, ST101036, ST048871, and R529877, were then examined for their ability to evoke protection against West Nile virus infections in BALB/c mice when administered nasally with West Nile virus envelope domain III (EDIII) protein.

Results:

All three adjuvants evoked high levels of EDIII-specific antibody and conferred similar levels of protection, ST101036 (67%), ST048871 (73%) and R529877 (75%), which was also comparable to the protection induced by M7 (67%) but markedly higher than the levels seen with mice immunized with EDIII alone (no adjuvant 33%).

Conclusion:

Novel small molecule mast cell activators are as efficacious as previously described mast cell activating peptides and serve as potential replacements in in vivo studies.
Lipid A mimetics BECC438 and BECC470 potentiate durable and balanced immune responses using an ovalbumin murine vaccine model.
Authors: Harberts Erin M , Varisco David J , Fields James K , Izac Jerilyn R , Gardner Francesca M , Sundberg Eric J , Snyder Greg A , Ernst Robert K
Published: February 2023

The need for effective infectious disease vaccines has become an inescapable topic over the past year. Continued development of next-generation vaccines that provide robust protective immunity is imperative. Such vaccines will likely include an adjuvant that avoids excessive adverse reactions and allows for dose and antigen sparing. Bacterial lipid A mimetics BECC438 and BECC470 have recently emerged as lead adjuvant candidates across several experimental models of infectious disease including Yersinia pestis (plague), human papilloma virus (HPV), and influenza-A (flu). To further define BECC438 and BECC470 as immuno-adjuvants, even without antigen from an infectious pathogen, studies presented here use ovalbumin (Ova) as a model antigen in a murine prime-boost vaccine model. Higher magnitude and more balanced production of antibody isotypes IgG1 and IgG2 are observed when BECC adjuvants are compared to classic adjuvants alum and PHAD. This optimal antibody response is durable and maintained for at least 12 weeks post-vaccination. Initial experiments use C57BL6 mice and are expanded to include BALBc and CD-1 (outbred) mice. Observed immune metrics maintained similar trends across male and females, and genetic backgrounds tested. Biacore immunogenicity analysis of C57BL6 serum found an increased half-life of Ova-specific antibodies in BECC438-adjuvanted animals potentially indicative of a higher antigen binding affinity. Toxicity studies conducted in New Zealand White rabbits report that BECC438 was well tolerated with no significant reactogenicity after 50μg and 100μg intra-muscular adjuvant injection. These studies provide continued evidence supporting development of BECC adjuvants in vaccines for human use.
Presence of maternal antibody at initial respiratory syncytial virus (RSV) infection shapes innate and adaptive responses following repeat RSV exposure
Authors: Kosanovich Jessica , Eichinger Katherine , Lipp Madeline , Perkins Timothy , Empey Kerry
Published: February 2023

RSV is the leading cause of childhood hospitalization due to bronchiolitis and a significant risk factor for the development of allergic airway responses. Maternal vaccination has emerged as a promising strategy to protect infants against severe disease. However, the presence of maternal antibody (matAb) at primary RSV infection has been linked to development of wheeze later in life. Therefore, we sought to understand how high levels of matAb during early life RSV infection shapes innate and adaptive immune responses on repeat RSV exposure. Using a murine model of maternal vaccination, we assessed immune responses following adult RSV re-challenge of infants born to unvaccinated and RSV preF-vaccinated dams. Presence of matAb at primary RSV infection resulted in differential activation of resident lung populations at 4 days post-secondary infection (DPSI). Specifically, proliferation of IL-5+ and IL-13+ type 2 innate lymphoid cells were increased in association with elevated numbers of eosinophils in the alveolar space. At 8 DPSI, IL-4-, IL-5-, and IL-13-producing, but not IFNγ-producing, CD4+ T cells were significantly increased, indicating a dominant Th2 response in mice born to vaccinated dams. Importantly, lung inflammation and mucus production were similarly elevated in mice born to immunized and unimmunized dams, suggesting that maternal vaccination did not prevent pathology on repeat infection. Taken together, these data demonstrate that the presence of matAb at primary RSV infection differentially alters innate and adaptive immune responses on secondary RSV exposure. Understanding the mechanisms that drive differential cell activation in the presence of matAb will guide future maternal immunization strategies.
Comparison of young-versus-old cytokine expression profiles in response to TLR agonists in murine and human whole blood.
Authors: Joyner Jason A , Vora Kalpit A. , Monslow Morgan A.
Published: February 2023

Aging is met with multifaceted shifts in the makeup and function of immune cell populations, collectively known as “immunosenescence”, which render elderly populations more susceptible to pathogenesis of infectious disease and negatively impact responsiveness to vaccines. Highly efficacious vaccines to protect the elderly remain an unmet medical need. However, it has been demonstrated in recent years that robust vaccine responses can be generated in elderly populations through the use of adjuvants. While the positive impact of adjuvant inclusion in elderly vaccines is evident, a deeper understanding of the adjuvant-related, pathway-specific modulations necessary to overcome immunosenescence is needed. We hypothesize that by evaluating functional and phenotypic data from high-density immunoassays using aged murine and human samples we will identify mechanisms contributing to vaccine non-responsiveness in the elderly. To interrogate deficiencies in aged murine and human cytokine responses to immune pathway modulators, we utilized multiplex cytokine assays to compare young-versus-old in vitro cytokine expression of whole blood stimulated with TLR agonists. The results of these experiments will be discussed and could provide key insights for the rational use of adjuvants in vaccine development that may overcome age-related vaccine non-responsiveness and will also help bridge the gap in translatability of immune dysfunction-related findings between aged preclinical animal species and humans.
Broadly reactive human antibodies prevent and treat pneumococcal infection
Authors: Mousa Jarrod J. , Huang Jiachen , Gingerich Aaron D , Royer Fredejah , Paschall Amy V , Avci Fikri Y
Published: February 2023

Streptococcus pneumoniae remains a leading cause of bacterial pneumonia despite the widespread introduction of vaccines for disease prevention. While vaccines have been effective at reducing the incidence of most vaccine-included serotypes, a rise in infection due to non-vaccine serotypes, and moderate efficacy against some vaccine included serotypes have contributed to high disease incidence, particularly in the elderly. Additionally, numerous isolates of S. pneumoniae are antibiotic resistant or multi-drug resistant. Several highly conserved pneumococcal proteins that are prevalent in the majority of serotypes have been examined and tested as potential vaccines in preclinical and clinical trials. We isolated the first human monoclonal antibodies (mAbs) (PhtD3, PhtD6, PhtD7, PhtD8, PspA16) against the pneumococcal histidine triad protein (PhtD), and the pneumococcal surface protein A (PspA), two conserved and protective antigens. mAbs to PhtD target diverse epitopes spanning the entire PhtD protein, and mAb PspA16 targets the N-terminal segment of PspA. The PhtD-specific mAbs were found to bind to multiple serotypes, while PspA16 serotype breadth was limited. In addition, we examined the prophylactic and therapeutic efficacy of mAb PhtD3 in several mouse models of pneumococcal pneumonia and sepsis. mAb PhtD3 prolonged the survival of infected mice when administered 2 hours before infection or 24 hours after infection. Additionally, mAb PhtD3 was effective against pneumococcal serotypes 4 and 3, the latter of which is a leading cause of invasive pneumococcal disease. Overall, our results provide new therapeutic reagents for disease prevention, and identify regions on PhtD and PspA recognized by human B cells.
Increased disease severity of Ebola virus infection with blockade of CD47
Authors: Rao Deepashri , Carmody Aaron , O’Donnell Kyle L , Hasenkrug Kim , Marzi Andrea
Published: February 2023

Previous work demonstrated that blockade of the anti-phagocytic molecule, CD47 resulted in increased macrophage and dendritic cell activation, enhanced CD8+ T cell responses and significantly improved control of lymphocytic choriomeningitis virus (LCMV) infections of mice. Here, we investigated whether CD47 blockade would be effective in mice infected with Ebola virus (EBOV). Daily treatment with anti-CD47 antibody after infection resulted in increased viral replication, increased body weight loss, and decreased survival. Anti-CD47-treated mice had indications of better immune activation such as higher activation of splenic macrophages and B cells as well as increased CD8+ T cell function. However, CD47 blockade also induced a strong proinflammatory milieu of cytokines and chemokines, a hallmark feature of fatal EBOV infection. Thus, anti-CD47 treatment adversely affected disease severity in EBOV-infected mice.
A Factor H-Fc fusion protein competes with serum FH for binding to Staphylococcus aureus and boosts complement-mediated opsonization
Authors: Sage Megan A.G. , Cranmer Katelyn D. , Wycoff Keith , Sharp Julia A.
Published: February 2023

Staphylococcus aureus is a significant human pathogen that causes a multitude of superficial and invasive infections worldwide. As antibiotic resistance is increasing, novel therapies are in dire need. As a master of immune evasion, S. aureus produces several virulence factors to persist in the host. A primary target of staphylococcal immune evasion is the Complement system, a component of innate immunity central to controlling bacterial infections. We have previously shown that S. aureus binds Complement regulator Factor H (FH) via surface protein SdrE to inhibit Complement. To address the need for novel therapeutics and take advantage of the SdrE:FH interaction, we tested the binding and downstream effect of a fusion protein comprised of the SdrE-interacting domain of FH coupled with IgG1 Fc using a S. aureus clinical isolate known to bind FH.

S. aureus bound significantly more FH-Fc than Fc-control proteins indicating a greater affinity for SdrE compared to S. aureus Fc-binding proteins Protein A and Sbi. In the presence of normal human serum, FH-Fc competitively bound to S. aureus and reduced S. aureus recruitment of serum FH compared to Fc-control. When examining the effect of FH-Fc on complement-mediated opsonization of S. aureus, treatment with FH-Fc resulted in a slight increase in C3-fragment deposition and a significant increase in C5a generation compared to control. This may be attributed to the cumulative effect of serum FH displacement plus a boost in classical pathway activation via additional Fc presence afforded by SdrE:FH-Fc binding. Taken together, these data support FH-Fc as a potential anti-staphylococcal therapeutic. Future studies will explore how FH-Fc affects S. aureus survival when challenged with phagocytes.
Reading the writing of immunizations in mice – the quantitative assessment of secreted antibodies to evaluate the quality of immunizations
Authors: Eyer Klaus
Published: February 2023

Vaccination needs to prepare the immune system to rapidly secrete specific antibodies upon a future encounter with the pathogen. Assessments of this feature are often challenging to perform, as measurements using antibody titers represent only a poorly resolved average of the rapidly evolving repertoire. We recently published and described a novel, droplet-based microfluidic technology that allows the quantitative characterization of humoral immune responses with single-cell and -antibody resolution. The employed snapshot analysis in this system enables studying even rapidly evolving repertoires. Additionally, the method extracts for each secreted antibody its production rate, specificity, and affinity, offering a high-resolution view of immunization. Consequently, this system enables the investigation of the immunization-generated secreted antibody repertoire with high temporal, analytical, and spatial resolution.

This study in mice used the above method to characterize the quality of the immunization-induced immunoglobulin-G repertoire, and we correlated these measures with the recall-accessible repertoire. Here, we showed that introducing variation in the immunization schemes led to significant differences in the secreted repertoire. Additional analysis after recall allowed quantifying the accessible antibody repertoire, and due to the single-cell resolution, our data allowed us to compare these two repertoires on a phenotypical level. We showed that the relationships between the two repertoires are not trivial and strongly depend on immunization itself. In summary, the developed approach introduced a novel, quantitative, and functionally resolved alternative to study the quality of immunizations.
Disease-associated microglia are implicated in neuropsychiatric manifestations of systemic lupus erythematosus
Authors: Makinde Hadijat M , Chen Shang Yang , Mike Elise V , Putterman Chaim , Winter Deborah R , Cuda Carla M
Published: February 2023

Systemic lupus erythematosus (SLE) is an autoimmune disease affecting multiple organs, including the brain. Though 50% of patients may experience neuropsychiatric symptoms (NPSLE), disease mechanisms remain largely unknown. Microglia, the resident innate immune cell of the brain, have recently been implicated in NPSLE. Microglia are heterogenous and may be associated with a homeostatic microglia (CD11clo) or a disease-associated microglia (DAM; CD11chi) state common to neurodegenerative disease and aging. Yet, few studies have examined microglia subsets in NPSLE. We showed that expression of a shared NPSLE transcriptional signature and DAM-associated genes correlates with the severity of behavioral deficits in microglia isolated from two NPSLE models prior to overt systemic disease. Further, our single-cell RNA-seq data identify homeostatic microglia and DAM states in control and NPSLE-prone mice. DAM in NPSLE are enriched for genes associated with antigen presentation but depleted for genes associated with phagocytosis, which is in contrast to DAM in neurodegenerative disease that are critical for phagocytic functions. Moreover, NPSLE CD11clo microglia upregulate genes linked to synapse pruning and phagocytosis, consistent with exacerbated synaptic pruning by microglia in models of NPSLE. We also find that restricted expression of the DAM transcriptional program in NPSLE DAM corresponds to improved behavioral outcomes in NPSLE-prone mice following treatment with fingolimod, a sphingosine-1-phosphate receptor modulator that reduces microglia activation and improves blood brain barrier integrity. These discoveries mark the first to implicate DAM as a potentially pathogenic microglia subset in NPSLE.
Quaternary Ammonium Compound Disinfectants Reduce Lupus-Associated Splenomegaly by Targeting Neutrophil Migration and T-Cell Fate
Authors: Abdelhamid Leila , Cabana-Puig Xavier , Mu Qinghui , Moarefian Maryam , Swartwout Brianna , Eden Kristin , Das Prerna , Seguin Ryan P. , Xu Libin , Lowen Sarah , Lavani Mital , Hrubec Terry C. , Jones Caroline N , Luo Xin M.
Published: February 2023

Hypersensitivity reactions and immune dysregulation have been reported with the use of quaternary ammonium compound disinfectants (QACs). We hypothesized that QAC exposure would exacerbate autoimmunity associated with systemic lupus erythematosus (lupus). Surprisingly, however, we found that compared to QAC-free mice, ambient exposure of lupus-prone mice to QACs led to smaller spleens with no change in circulating autoantibodies or the severity of glomerulonephritis. This suggests that QACs may have immunosuppressive effects on lupus. Using a microfluidic device, we showed that ambient exposure to QACs reduced directional migration of bone marrow-derived neutrophils toward an inflammatory chemoattractant ex vivo. Consistent with this, we found decreased infiltration of neutrophils into the spleen. While bone marrow-derived neutrophils appeared to exhibit a pro-inflammatory profile, upregulated expression of PD-L1 was observed on neutrophils that infiltrated the spleen, which in turn interacted with PD-1 on T cells and modulated their fate. Specifically, QAC exposure hindered activation of splenic T cells and increased apoptosis of effector T-cell populations. Collectively, these results suggest that ambient QAC exposure decreases lupus-associated splenomegaly likely through neutrophil-mediated toning of T-cell activation and/or apoptosis. However, our findings also indicate that even ambient exposure could alter immune cell phenotypes, functions, and their fate. Further investigations on how QACs affect immunity under steady-state conditions are warranted.
Interferon alpha promotes extrinsic apoptosis of keratinocytes following exposure to ultraviolet B radiation.
Authors: Estadt Shannon N , Moore Tyson M. , Kahlenberg J Michelle
Published: February 2023

Systemic lupus erythematosus (SLE) is an autoimmune disease characterized by sensitivity to ultraviolet radiation (UVR). UVR triggers cutaneous and systemic disease flares, yet mechanisms driving this are not well characterized. Type I interferons (IFNs) are overexpressed in non lesional SLE skin and promote death of SLE keratinocytes (KCs) after UVR, but the manner in which they do so is not known. This study explores activation and regulation of cell death pathways in KCs exposed to type I IFNs and UVR. We treated immortalized human KCs (N/TERTs) overnight with IFNα prior to UVB exposure. Four hours post UVB, IFNα priming significantly increased the percentage of cleaved-caspase-3+ (CC3+) and Annexin V+ propidium iodide− (AV+PI−) cells compared to treatment with UVB alone. Further, IFNα promoted apoptosis at a lower dose of UVB. Inhibiting RIP1, RIP3, or caspase-1 had no effect on death suggesting no involvement of necroptosis or pyroptosis. Use of a pan-caspase inhibitor or a caspase-8 inhibitor significantly reduced AV+PI− cells following IFNα and UVB treatment while caspase-9 inhibition did not, suggesting enhanced activation of extrinsic apoptosis. Treatment with IFNα increased expression of the death ligand TRAIL, an inducer of extrinsic apoptosis. Further studies will determine if IFN-induced upregulation of TRAIL is responsible for UVB-enhanced apoptosis in an autocrine fashion. Together, these data suggest that photosensitive responses exhibited by SLE patients may be due to type I IFN priming of KCs that sensitizes cells to undergo increased extrinsic apoptosis after minimal exposures to UVB. Continued investigation into mechanisms by which this occurs will provide important prophylactic options to prevent SLE flares.
NOX2-deficient neutrophils facilitate joint inflammation in immune-mediated arthritis
Authors: Liao Yi-Chu , Chen Peng-Chieh , Shieh Chi-Chang K
Published: February 2023

Immune-mediated inflammatory arthritis, including rheumatoid arthritis (RA), is characterized chronic inflammatory in the joints. NADPH oxidases 2 (NOX2) complex in phagocytes mediates the generation of high concentrations of reactive oxygen species (ROS) to kill the engulfed microbes. Inherited mutations in the subunits of NOX2 result in chronic granulomatous disease (CGD). CGD patients experience recurrent infections and diverse autoimmune conditions including arthritis. In our previous studies, we found that NOX2-deficient mice tended to have more severe inflammatory arthritis and neutrophils accumulation. Based on the hypothesis that NOX2-deficient neutrophils play a role in facilitating immune-mediated arthritis, we investigated the activity of neutrophil from inflamed joints. We found that while neutrophils from both NOX2-deficient and WT mice weakened the anti-CD3 and anti-CD28 induced T lymphocyte responses, NOX2-deficient neutrophils were less suppressive. Down-regulated PD-L1 expression on NOX2-deficient neutrophils was observed, which could be a cause for their impaired suppressive function. Moreover, pro-inflammatory genes, including IL-1b, NOS2, and Cxcl3 were up-regulated in NOX2-deficient neutrophils according to our RNA-seq results. In humans, neutrophils from RA joints were found to suppress the T cell proliferation. Consistent with our mice model, carbonylated protein and total GSH level, the oxidative markers, were lower in synovial fluid of RA patients. Our results implicate that neutrophil-mediated immune regulation may be an important factor in controlling inflammation in the joints, and this regulation is affected by the NOX2-produced ROS.
Loss of the TAM receptor Mer contributes to Sjögren’s syndrome-like disease in mice
Authors: Witas Richard M , Nguyen Cuong Q
Published: February 2023

Sjögren’s syndrome (SjS) is a chronic autoimmune disease primarily involving the exocrine glands where the involvement of the innate immune system is largely uncharacterized. Mer signaling has been found to be protective in several autoimmune diseases but remains unstudied in SjS. Here we report that soluble Mer (sMer) levels were elevated in SjS patient sera and positively correlated with focus score, ocular staining scores, rheumatoid factor, and Ro60 autoantibody levels. Increased sMer was also detected in C57BL/6.NOD-Aec1Aec2 (SjSS) mouse sera, coinciding with higher ADAM17 activity, the enzyme responsible for cleavage of Mer, and inactivation of such. Mer signaling outcomes were observed to be diminished in SjSS mice as evidenced by impaired efferocytosis in SjSS mouse macrophages and decreased Socs1 and Socs3 expression in SjSS salivary gland (SG). SjS diagnostic criteria were examined in MerKO mice to evaluate the protective role of Mer in SjS disease pathogenesis. MerKO mice developed SG infiltrates of B and T lymphocytes, SG apoptotic cells, antinuclear antibodies, and reduced saliva flow. Together, our data suggest a sequence of events where enhanced ADAM17 activity increases Mer inactivation, depresses Mer signaling, and removes a protection against the loss of self-tolerance and onset of autoimmune disease in SjSS mice.
Inflammation targets CDK11 in autoimmune diabetes as a protective mechanism against beta cell apoptosis.
Authors: Mora Conchi , Sala Ester , Vived Celia , Luna Júlia , Saavedra-Ávila Noemí-Alejandra , Sengupta Upasana , Haba Laura , Pizarro Javier , Verdaguer Joan , Stratmann Thomas , Vázquez-Carrera Manuel , Lahti Jill M
Published: February 2023

Autoimmune diabetes (T1D) is caused by the immune-mediated destruction of insulin-producing beta cells in the pancreas leading to hyperglycemia. Apoptosis is the main mechanism responsible for beta cell demise, including Fas-receptor engagement and Perforin release as essential death executers. However, the chronic inflammatory milieu pervading pancreatic islets prior to diabetes onset, alters beta cells function and primes beta cells for apoptosis long before the debut of the disease. To identify those genes that are targeted by inflammation and are causally related to beta cell fitness and viability we have used the microarray technology. We have identified several candidate genes being downregulated by inflammation, one of this genes is Cdk11, a cyclin-dependent kinase which is involved in transcription (CDK11p130), mitosis and apoptosis (CDK11p58). L-type cyclins are the partners for Cdk11p130, and Cyclin D3 is the partner of CDK11p58. The Cdk11 gene, when is transcribed, generates a single mRNA form, that can be translated either into CDK11p130 or CDK11p58 by a differential IRES (Internal Ribosome Entry Site) usage, being CDK11p58 only expressed during mitosis, while CDK11p130is expressed during all cell cycle phases. We have addressed the role of CDK11 in autoimmune diabetes using the T1D-prone NOD mouse model hemideficient in CDK11, since the CDK11 null mutation is embryonically lethal. First of all, we have confirmed the effect of inflammation on Cdk11 mRNA expression in islet cells by submitting NODSCID mice to adoptive transfer of increasing amounts of diabetogenic NOD splenocytes, and, observed that Cdk11 expression in islet endocrine cells is downregulated by inflammation in a dose-response dependent manner.
Vitamin A Deficiency Deteriorates Lupus Nephritis.
Authors: Abdelhamid Leila , Cabana-Puig Xavier , Swartwout Brianna , Zhu Jing , Li Yaqi , Ross A. Catharine , Cecere Thomas E. , Ruiz Phillip , Reilly Christopher M. , Luo Xin M
Published: February 2023

Vitamin A deficiency (VAD) is observed in both human and mice with lupus nephritis (LN). However, whether VAD is a driving factor for LN progression is unclear. Here, we investigated the effect of VAD on the progression of LN in a lupus-prone mouse model, MRL/lpr. In these mice, while LN is not apparent before 8 weeks of age, the inflammation of lymphoid tissues suggesting initiation of lupus is evident at weaning. Thus, we initiated VAD either before or after weaning to reveal a potential time-dependent effect. At around 15 weeks of age with both types of VAD, we found exacerbated LN that was characterized by deteriorated kidney inflammation, impaired renal epithelial integrity, and dramatic squamous metaplasia of the renal pelvic urothelium. Both types of VAD provoked severe neutrophilic tubulointerstitial nephritis and accelerated renal failure. This was concomitant with significantly higher mortality in all VAD mice. Looking at an earlier time point of 7 weeks of age and before the onset of clinical LN, both types of VAD increased splenomegaly, lymphadenopathy, and circulating autoantibodies; three additional hallmarks of lupus, as well as renal infiltration of conventional and plasmacytoid dendritic cells. These results suggest VAD-driven deterioration of LN regardless of the time of VAD initiation. Our findings raise significant public health concerns that concurrent lupus and VAD may lead to more severe SLE. Future studies will elucidate the underlying mechanisms of how VAD modulates myeloid cells to accelerate the initiation of LN.
Specific traits of the B cell response to self-antigens revealed by single cell transcriptomics
Authors: Castrillon Carlos Eduardo , Simoni Lea , van den Broek Theo , van der Poel Cees , Akama-Garren Elliot H , Carroll Michael C
Published: February 2023

In an autoimmune environment, B cells spontaneously participate and are selected in germinal centers (GC). In previous work, we investigated epitope spreading in a mixed bone marrow chimera model that combines B cells from lupus-like mice with those from wild type (WT) B6 mice (Degn et al Cell 2017). In the current study, we identify novel biomarkers of autoimmune B cells at the population and single cell level.

Cohorts of autoimmune and WT immunized chimeric mice were prepared. To track WT-derived B cells, we used the tamoxifen inducible AID-CreERT2-EYFP mice. WT chimeras were immunized and boosted. Splenic B cells were sorted based EYFP+ signal and processed for droplet-based gene expression and BCR repertoire single cell RNAseq. To detect specific traits of autoreactive B cells relative to immunized, unsupervised cluster analysis, differential gene expression and pseudotime analysis were used.

Results show higher antibody repertoire diversity in the autoimmune relative to immune B cells, but striking similarity for transcriptomic usage. However, distinct gene expression was identified for the autoreactive B cells such as CXCL10 usage by GC B cells and SLPI expression by autoreactive plasma cells. Importantly, we were able to identify DN2- and DN4-like memory B cells in both conditions. DN2 cells, presumed to derive directly from naïve B cells, have been recently pointed as a source of pathogenic extrafollicular antibody secreting cells in humans.

Overall, results reveal the variety of characteristics that B cells, in their many states, adopt in the response to self and should open the way to new approaches to control their pathogenicity in autoimmune disease.
Role of Extracellular Vesicles in human uveitis
Authors: Agashe Vrushali Vinay , Lee Jung Wha , Maclean Mary , Nath Pulak Ranjan , Sen H. Nida
Published: February 2023

Non-infectious uveitis (NIU) is a significant cause of blindness. It is an immune mediated disease however the exact pathogenesis remains elusive. Considerable evidence exists on the immunomodulatory effects of Extracellular Vesicles (EVs) in various diseases; but little is known on the role of EVs in uveitis patients. We have previously shown that cytokine stimulated ARPE19 cells, release EVs; these EVs are capable of inhibiting T cell proliferation, inducing proinflammatory cytokine production from monocytes and monocyte cell death, in PBMCs from uveitis patients. The purpose of this study was to isolate EVs from uveitis patients and to determine their immune-modulatory potential in-vitro. EVs were isolated from the serum of NIU patients using Size Exclusion Chromatography (SEC) and set up in culture with PBMCs or ARPE19 cells; after stimulation cells were stained to determine activation and cytokine production. A higher number of EVs were isolated from patients, as compared to healthy controls; but EVs from both, were of the appropriate size, had a low protein content and were positive for the exosome associated markers CD9, CD63 and CD81. EVs from patients expressed increased levels of CD8, CD29, CD44, and HLA-DR/DP/DQ, indicating their potential to activate cells in-vivo as compared to controls. An upregulation of CD69 and CD25 on T cells and increased IFNg production was observed when the cells were cultured with EVs from patients. Incubating ARPE19 cells with the EVs from patients, increased the expression of MCP1 and IL8 in the cells. Preliminary data indicates that EVs isolated from uveitis patients can induce a robust activation of cells in-vitro. Current experiments are underway to further substantiate these results.
Dissecting the complexity of heart infiltrates in post-infectious myocarditis induced with CVB3 infection by single-cell RNA sequencing analysis
Authors: Lasrado Ninaad , Borcherding Nicholas , Arumugam Rajkumar , Starr Timothy K. , Reddy Jay
Published: February 2023

Viral myocarditis is a predominant cause of heart failure in children and young adolescents that can lead to dilated cardiomyopathy (DCM). Experimentally, Coxsackievirus B3 (CVB) is commonly employed to study the pathogenesis of myocarditis. Post-infectiously, affected mice can develop DCM, and viral RNA may be present with or without inflammation raising a question as to the events culminating in the development of DCM. To dissect this complexity, we sought to characterize heart infiltrates from CVB infected mice by single-cell RNA sequencing and compared the profiles with healthy mice. These analyses revealed 26 subtypes of cells with fibroblasts, T cells and macrophages as the major constituents. Other cell types include endothelial cells (EC), smooth muscle cells, granulocytes, B cells, and innate lymphoid cells. By analyzing the transcriptomes, we noted differential expression of transcription factors (TFs) in various cell types. One such cell type is EC, in which, activating transcription factor 4, interferon regulatory factor 1 (IRF1) and signal transducer and activator of transcription 6 (STAT6) that have a role in apoptosis and inflammation were upregulated. Correspondingly, expression of Myc and nuclear factor erythroid-derived 2-like 2 implicated in EC survival and angiogenesis was downregulated. The data suggests the ECs may play a role in the pathogenesis of CVB infection through a proinflammatory transcriptional program.
Zfp36 family members redundantly protect against T cell-mediated autoinflammation and premature mortality
Authors: Cook Melissa Erin , Bradstreet Tara R , Webber Ashlee , Kim Jongshin , Santeford Andrea , Schwarzkopf Elizabeth A , Stumpo Deborah J , Apte Rajendra S , Blackshear Perry J , Edelson Brian T
Published: February 2023

Cytokine production must be tightly regulated in order to prevent auto-inflammatory diseases. The zinc finger 36 (Zfp36) family of RNA-binding proteins, including Zfp36, Zfp36l1, and Zfp36l2, are known to negatively regulate mRNA stability or translation of many transcripts, including cytokines. Polymorphisms in ZFP36L1 and ZFP36L2 have been identified in GWAS studies of a variety of human autoimmune diseases, necessitating understanding their functions. While there are reports of all three family members regulating T cell cytokine production, delineating the exact functions of these genes is challenging due to spontaneous phenotypes upon global deletion of single genes and potential redundancy in their functions. To overcome this, we generated Cd4-Cre+ Zfp36fl/fl Zfp36l1fl/fl Zfp36l2fl/fl mice. Only upon triple deletion, but not individual or paired deletions, do mice spontaneously develop an inflammatory disease characterized by early mortality and immune cell infiltration into various organs, including the central nervous system, kidneys, and liver. These mice have drastically elevated levels of many cytokines in their sera. RNA-sequencing reveals that deleted T cells are enriched in gene pathways involving inflammation, proliferation, and apoptosis. Our findings demonstrate a novel redundancy of the Zfp36 family members in regulating T cell homeostasis and suppressing cytokine-driven inflammation. We are investigating the mechanisms and mRNA targets that control this phenotype. Understanding the individual and redundant functions of the Zfp36 family members may lead to opportunities to target them for suppression of T cell-driven autoimmunity or for activating anti-tumor and anti-pathogen T cell responses.
An Exon 2-deficient isoform of the Foxp3 gene fails to protect against IgE-mediated cutaneous autoimmunity after ultraviolet light exposure.
Authors: Domeier Phillip P , Chang Sarah , Utz Paul J , Zhou Baohua , Ziegler Steven F
Published: February 2023

FOXP3 is required to maintain immune tolerance, but the functional contributions of FOXP3 isoforms to the maintenance of tolerance is poorly understood. Mice express a single isoform of the Foxp3 gene, but humans encode an additional exon 2-deficient splice variant isoform of FOXP3 with no known function. To determine how exon 2 of FOXP3 contributes to Treg activity, we generated mice that only express the exon 2-deficient FoxP3 variant (called B6.FoxP3ΔExon2). In the steady-state, B6.FoxP3ΔExon2 mice develop mild autoimmunity (splenomegaly, autoantibodies and kidney nephritis) with enlarged germinal centers and elevated titers of skin-reactive IgE. We also observed skin-reactive IgE antibodies in SLE patients with active photosensivity. Therefore, We challenged B6 and B6.FoxP3ΔExon2 mouse skin with ultraviolet B (UVB) light to determine if UVB damage can increase the production of self-reactive IgE. When compared to B6 controls, B6.FoxP3ΔExon2 mice developed greater skin inflammation with increased infiltration of IgE+ plasma cells (B220lowCD138+Blimp1 +) at the site of UVB damage. B6.FoxP3ΔExon2 mice also had higher numbers of IL4-producing T effector cells (IL4+CD4+CD44+Foxp3−) and IgE-producing B cells in draining lymph nodes of UVB-challenged skin as compared to untreated control lymph nodes. Finally, UVB-treated B6.FoxP3ΔExon2 mice also had worsened kidney nephritis and proteinurea, phenocopying multi-systemic flares that occur in SLE patients with photosensitivity. Collectively, we found that exon 2 of the Foxp3 gene is required to control cutaneous inflammation though the regulation of skin-reactive IgE, and IgE-producing B cells could be targeted to limit the severity of SLE-associated photosensitivity.
Lupus-like extrafollicular B cell responses and autoreactivity are hallmarks of severe COVID-19
Authors: Woodruff Matthew C , Ramonell Richard P , Lee F. Eun-Hyung , Sanz Ignacio
Published: February 2023

In late 2019, the emergence of the coronavirus SARS-CoV-2 resulted in a global pandemic, costing over 2 million lives. A hallmark of the disease resulting from SARS-CoV-2 infection, COVID-19, has been it’s heterogeneity in presentation. To understand the immunological underpinnings of this variability in symptoms, we performed a deep characterization of the B cell responses generated in patients within the NIH-defined mild/moderate or severe/critical categories. Somewhat surprisingly, patients with severe disease had higher neutralizing antibody titers than their mild/moderate counterparts, but showed strong evidence of extrafollicular B cell activation by flow cytometry – previously described in humans primarily in autoimmune disease. Further analysis of the B cell repertoire through single cell VDJ sequencing revealed low (or fully absent) levels of somatic hypermutation in the antibody secreting cell compartment, phenocopying the low selective pressures previously associated with extrafollicular responses. Together, these findings suggested the possible relaxation of peripheral tolerance in severe patients. This was confirmed through clinical autoreactivity testing, revealing more than 70% of the most severe COVID-19 patients, despite no history of autoimmunity, test positive for anti-nuclear antibodies or rheumatoid factor. Altogether, these findings provide the immunological context for an emerging consensus in the literature whereby severe COVID-19 is characterized by robust extrafollicular B cell activation, resulting not only in neutralizing antibody production, but also autoreactive B cell targeting with the potential for direct involvement in severe disease pathology.
DOCK8 mutations in COVID-19 and MIS-C Cytokine Storm Syndrome
Authors: Cron Randy Q , Zhang Mingce , Cron Remy R , Absher Devin , Bridges John M , Schnell Amanda , Bhatraju Pavan K , Vagrecha Anshul , Lozinsky Shannon , Acharya Suchitra , Levy Carolyn Fein , Chatham W. Winn
Published: February 2023

Background

We recently identified DOCK8 as a novel gene associated with cytokine storm syndrome (CSS). Heterozygous missense mutations in DOCK8 diminish NK cell lytic function and contribute to increased pro-inflammatory cytokine production (CSS). CSS is a potential complication of COVID-19 with severe consequences. Children are at risk of a SARS-CoV-2 post-infectious CSS, multisystem inflammatory syndrome in children (MIS-C). Host genetic factors associated with COVID-19 CSS and MIS-C CSS are unknown.

Methods

To date, 16 adult patients enrolled in a COVID-19 CSS clinical trial at UAB had whole genome sequencing. Four (25%) had rare heterozygous DOCK8 mutations (3 missense, 1 intronic). A COVID-19 CSS adult patient in Seattle also had a DOCK8 missense mutation. In addition, DOCK8 missense mutations were identified in 5 children (UAB & Northwell) hospitalized with MIS-C. DOCK8 mutations, or wild-type (WT) sequence controls, were introduced into human NK-92 cells by FOAMY virus transduction. WT and mutant DOCK8-expressing NK-92 cells were incubated with K562 target cells and compared for cytolysis and degranulation (CD107a).

Results

One COVID-19 patient DOCK8 mutation (Gly523Arg) reduced NK cell degranulation by 30% and cytolysis by 23% (n=3). Similar studies of 3 MIS-C patients with DOCK8 missense mutations revealed up to 31% reduced NK cell degranulation and 48% reduction in cytolysis by 3 mutations (n=3). Two-way ANOVA analysis revealed statistically significant (p<0.05) differences in NK cell degranulation and lysis for 4 unique DOCK8 mutations.

Conclusion

Heterozygous DOCK8 missense mutations may contribute to severe COVID-19 and MIS-C CSS by partial dominant-negative effects yielding decreased NK cell cytolysis.