bluuebunny/crossref_metadata_2025 huggingface dataset:
Selective targeting of pathogenic auto-reactive VH4–34 B cells with a rationally developed anti-VH4–34 antibody offers a new therapeutic approach for autoimmune disorders
Authors: Paliwal Shalini , Thakkar Dipti , Chin Wen Jie , Lee Warren , Neo Jacklyn J Y , Tay Lavina , Hanson Brendon , Paszkiewicz Konrad , Ingram Piers , Boyd-Kirkup Jerome
Published: August 2023

B cells expressing B cell receptors (BCRs) specific for self-antigens are normally eliminated, or made anergic, through negative selection during B cell development. VH4–34 is an intrinsically autoreactive antibody (and BCR) heavy chain variable region that includes a germline encoded hydrophobic patch in framework region 1 (FR1). Autoantigens recognized by VH4–34 antibodies include N-acetyl lactosamine chains displayed on RBCs. In healthy individuals, <1% of B cells express VH4–34 encoded BCRs but during the loss of self-tolerance, characteristic of pathogenic disorders, the hydrophobic region of VH4–34 reacts to self-antigen, erroneously driving BCR activation and expansion.

Expansion of VH4–34+ B cells and increased serum concentrations of VH4–34 antibodies has been implicated in systemic lupus erythematosus (50%) and virtually all cases of cold agglutinin disease. To date there have been no therapies against VH4–34 due to challenges of specifically targeting VH4–34 while avoiding other VH regions.

We applied Hummingbird Bioscience’s Rational Antibody Discovery approach to develop anti-VH4–34 antibodies that bind to a computationally predicted and lineage conserved epitope in the hydrophobic FR1 region. The lead antibody, HMBD-011, demonstrates selective and high affinity binding to all tested VH4–34 antibodies, with no off-target binding. The binding epitope of HMBD-011 was confirmed by mutagenesis to be within the FR1 region of VH4–34. Further, HMBD-011 shows selective binding to cells that express VH4–34 BCRs. In vivo, HMBD-011 treatment resulted in rapid elimination of VH4–34+ antibodies and cells.

In conclusion, HMBD-011 represents a promising precision therapy for VH4–34 driven autoimmune disorders.
STAT1 gain of function mutation impairs immune response to viral infections
Authors: Philips Rachael Laura , Liao Yi-Chu , Morrison Tasha , Chaitankar Vijender , Yao Chen , Oikonomou Vasileios , Milner Joshua , Lionakis Michail S , O’Shea John J
Published: August 2023

The coordinated effort of the immune system in host defense is dependent upon the proper release and interpretation of different cytokine signals. Several examples of monogenic diseases targeting the JAK-STAT pathway reveal there is still a critical need to understand basic principles of cytokine signaling and cytokine output. For example, patients with STAT1 gain-of-function (GOF) mutations exhibit a type 1/IFN-gamma bias that antagonizes a type 3/IL-17 immune response important for controlling fungal infection, yet patients also, paradoxically, exhibit chronic and sometimes lethal viral infections. Using a novel conditional knock in STAT1-GOF mouse model, we demonstrate that STAT1-GOF mice respond poorly to viral infections that are easily controlled by WT mice, such as MCMV and LCMV-Armstrong. High-dimensional flow cytometry and scRNA-seq reveal an impaired NK and CD8 T cell effector response in STAT1-GOF mice that is not simply due to an exhaustion phenotype. Instead, STAT1-GOF mice respond poorly to viral infections due to an unexpected defect in IFN-gamma production early during the innate immune response, leading to a cytokine storm, immunopathology, and an impaired adaptive immune response. scATAC/RNA-seq uncovers altered usage of different STAT complexes in STAT1-GOF mice, leading to a misinterpretation of early innate-stimulating cytokines. These results provide insight into why patients with interferonopathies or elevated interferons respond poorly to viral infections and highlight the importance of properly tuning STAT1 during an immune response.

R.L.P. was supported by a Postdoctoral Research Associate (PRAT) fellowship from the National Institute of General Medical Sciences (NIGMS), award number 1Fi2GM137942-01
Single cell transcriptomes from HCMV+ donors reveal a unique NKG2CHigh subset that represents true memory NK cells
Authors: Khalil Mohamed , Malarkannan Subramaniam , Terhune Scott
Published: August 2023

Infection with human cytomegalovirus (HCMV) can result in severe morbidity and mortality to the developing fetus and immunocompromised individuals. Natural killer (NK) cells are cytotoxic lymphocytes that are required to manage and control HCMV infections. The NKG2C receptor expressed by NK cells recognizes and responds to infected cells expressing HLA-E loaded with HCMV UL40-derived peptide. These responding NKG2C+ NK cells have been referred to as ‘adaptive NK’ because they possess features of adaptive immunity and are found at greater percentages in HCMV seropositive individuals. The molecular mechanisms by which adaptive NK cells are generated and their transcriptomic signatures remain unknown. To investigate this, we obtained healthy adult spleens from four HCMV+ and four HCMV− donors and their NK cells were sorted and captured for single-cell RNA-sequencing (scRNA-seq). Donor median age was 59 [IQR 48.5–56.5], 50% (n=4) were female, 50% (n=2) of females were HCMV seropositive and 50% (n=2) of females were HCMV seronegative. Using scRNA-seq, we observed elevated expression and cell numbers of NKG2C in HCMV+ donors when compared to HCMV-donors. We identified a distinct population of NKG2C+ adaptive NK cells displaying elevated levels of CD52, B3GAT1, FCGR3A, CD3E, and IL7R. In addition, our developmental trajectory analysis of adaptive NKG2C+ NK cells revealed a unique branchpoint of true memory NK cells expressing elevated levels of NKG2C and IL7R. Here, we demonstrate that HCMV infection can induce the formation of adaptive NKG2C+ NK cells that display a unique transcriptional and developmental profile. These findings have the potential to influence the future application of adaptive NK cells in cellular immunotherapies.

This work is supported by the National Center for Advancing Translational Sciences, National Institutes of Health, through grant number TL1TR001437
Oral tablet vaccination against SARS-CoV-2 spike boosts cross-reactive serum and mucosal neutralizing antibodies following mRNA vaccination: a phase 2 clinical trial
Authors: Johnson Susan , Jegede Clara B , Gutierrez Samanta , Greco Susan , Cummings James , Gottlieb Keith A , Tucker Sean N
Published: August 2023

Current SARS-CoV-2 intramuscular vaccines do not induce the breadth of immunity needed to halt the development of new variants. New strategies that elicit immunity at the site of infection could induce cross-reactive mucosal responses that decrease transmission.

We developed a room-temperature stable oral tablet vaccine composed of recombinant adenovirus type 5 expressing the spike gene of SARS-CoV-2. In an open label phase 2 clinical trial (NCT05067933), 66 subjects, either naïve or previously mRNA vaccinated, received 2 doses of a low or a high dose 28 days apart. The vaccine was well tolerated with a benign safety profile.

Among previously vaccinated subjects, oral tablet vaccination was able to boost serum IgG and IgA at 29 and 57 days post-vaccination. The serum neutralizing antibodies increased 1.6-fold, from 481 AU/ml to 778 AU/ml at Day 57. The subjects with lower starting titers had greater increases after oral boosting. Most subjects in the vaccinated cohorts had an increase in SARS-CoV-2 IgA in the nose or saliva. This mucosal IgA was not only cross-reactive against SARS-CoV-2 variants but also SARS-CoV-1 and MERS. These samples demonstrated surrogate neutralizing activity against both Wuhan and omicron BA4/5, despite the vaccine expressing Wuhan antigens, highlighting the ability of mucosal vaccination to elicit cross-reactive responses.

Overall, this vaccine was safe and well tolerated. It induced cross-reactive immunity in both the serum and more importantly, in the mucosa, the site of infection. This offers an ideal platform for boosting immune responses that could decrease transmission rates and extend protection to emerging variants. It also offers unique advantages in distribution to curb future pandemics.
An in vivo overexpression system to study the functions of complement activated products in a mouse model of lupus
Authors: Wang Fang , Wu Yee ling
Published: August 2023

Complement cleavage products generated during activation serve a wide range of functions from immune defense against microbes to homeostasis. However, the functions of complement activated products such as C4a, albeit present at high levels in lupus, remain unknown. Studying the functions of complement activated products is challenging. Studies using purified complement proteins can be confounded by minute contaminants with potent effects. Transgenic animals often provide more unequivocal insights. However, the generation of transgenic animals is highly involved and time-consuming, especially when backcrossing to genetically susceptible backgrounds is required for the development of diseases. Here, as an illustration for studying the functions of a complement activated product in vivo, we employed hydrodynamic injections to deliver expression plasmids encoding C4a to mouse liver. This technique allows hepatocytes to synthesize and directly secrete C4a into the circulation without disrupting intact C4. Using this system, we were able to detect exogenous C4a protein >2 months post-injection in the lupus prone MRL/lpr mice. Preliminary phenotyping on MRL/lpr mice overexpressing C4a revealed two immunologically relevant features: 1) the frequency of T cells was reduced and 2) the levels of autoantibodies against double-strand DNA were elevated comparing with vector controls. These data suggest that C4a can potentially modulate lupus manifestations. While detailed analyses are ongoing to determine the functions of C4a in lupus, our study illustrates that in vivo overexpression using hydrodynamic injections represents a relevant approach for studying the functions of complement activated products in disease models.

supported by the Lupus Research Alliance
Spatial immunoprofiling of tissue sections with ChipCytometry&[trade]
Authors: Sindelar Tim , Stanslowsky Nancy , Northcutt Adam
Published: August 2023

Highly multiplexed spatial biomarker analysis has demonstrated the potential to advance our current understanding of the immune system and its role in cancer, from tumor initiation to metastatic progression. Previously, a trade-off between plex and spatial context meant that our understanding of immune cell involvement in cancer was limited to providing deep information on either cell phenotypes or their spatial context, but not both. ChipCytometry is a novel, highly multiplexed technology that preserves both plex and spatial context to deeply profile immune cell diversity at single-cell resolution. ChipCytometry uses commercially available antibodies and combines iterative immuno-fluorescent staining with high-dynamic range imaging to profile dozens of protein biomarkers in a single tissue specimen. Cellular phenotypes are identified with via flow cytometry-like hierarchical gating from standard multichannel OME-TIFF images, compatible with a variety of computational tools being developed for multiplexed analysis and visualization. Here, we use ChipCytometry to identify and quantify key immune cell subtypes in FFPE tissues. The results show precise expression levels for each biomarker in the assay in each individual cell in the sample, while maintaining spatial positioning of each cell. Spatial analysis reveals quantifiable heterogeneity of immune cell infiltration within the tumor samples, demonstrating the utility of the ChipCytometry platform for the in-depth immune profiling in FFPE samples.
High-Throughput Antibody Discovery Using Barcode Enabled Antigen Mapping (BEAM
Authors: Shahi Payam , Adams Bruce , Reyes Daniel , Maheshwari Shamoni , Mousavi Nima , Krishnan Sreenath , Ramen Nandhini , Tsai FuNien , Kumar Poornasree , Finnegan Peter , Fernandes Sunjay , Bali Gunmeet Kaur , Terry Jessica
Published: August 2023

Multi-analyte single cell technologies have potential to greatly accelerate antibody discovery by identifying antigen-specific B cells. Barcode Enabled Antigen Mapping (BEAM) provides a high-throughput, multimodal analysis of antigen-bound B cells (BEAM-Ab) using 10x Genomics Chromium Single Cell Immune Profiling v2 Solution and novel computational approaches to discover B-cell receptor (BCR) sequences for further functional characterization.

To demonstrate the antigen sensitivity of BEAM-Ab, we spiked in 5% Hen Egg Lysozyme (HEL) and 5% gp120 transgenic B cells in 90% wild type non-transgenic splenocytes. The cells were screened using a panel of barcoded antigens and then CD19+PE+ (antigen+) B cells were isolated using flow cytometry. Single cell gene expression, BCR, and antigen barcode analysis indicated clonotype specificity to the respective antigens in the sorted cells. Furthermore, we demonstrated BEAM-Ab specificity by analyzing a convalescent COVID patient sample against an antigen panel containing five different COVID antigens and a negative control. Analysis of this COVID sample suggests the clonal expansion of B cells recognizing the ectodomain of the wild type SARS-CoV2 spike protein, receptor binding domain of spike protein, and the ectodomain of the D614G mutant spike protein. We did not observe any clonal expansion to Omicron-specific antigen or the non-specific negative control in our dataset.

Our data demonstrates that BEAM-Ab is an effective antibody discovery tool. BEAM-Ab unlocks the ability to rapidly screen large numbers of samples with an accurate single cell resolution and antigen specificity, with the entire workflow generating the candidate sequences just one week after sample processing.
Multiplex assays to determine correlation of binding and neutralizing antibodies following SARS-CoV-2 infection and vaccination
Authors: Venkateswaran Neeraja , Sarwar Jawad , Nelson William Max , Venkateswaran Kodumudi S
Published: August 2023

The COVID-19 pandemic has led to the rapid development of many different therapeutics and vaccines to reduce the burden of the disease. Most vaccines target the highly immunogenic spike (S) protein for generating functional immune responses to block the entry of the virus into host cells. However, the mutations in the S-protein have led to the emergence of many variants that escape the immune response. We developed two multiplex assays using Luminex® xMAP® technology to assess antibody profiles after infection and vaccination; 1) to quantify the binding antibodies against SARS-CoV2 receptor binding protein (RBD), nucleocapsid protein (NP), S1-protein (S1), and Trimer having S1, S2, and extracellular domain protein; 2) a multiplex surrogate virus neutralization antibody assay targeting CoV2 wild type and various mutant viruses. The quantitative binding assay uses the WHO standard for determining the binding antibody unit (BAU)/mL. Surrogate virus neutralization assay measures the inhibition of ACE2 protein binding to wild-type and S protein variants coupled to beads in the presence of immune or convalescent sera. We determined the negative threshold using 81 pre-pandemic serum samples collected during 2017–18. We used 116 samples collected from 45 vaccinated and 15 infected subjects for antibody quantification. Binding antibodies to RBD, S1, and trimer were higher in vaccinated individuals with minimal reactivity to NP, whereas the NP antibody response was higher in the infected individuals. We observed a direct correlation between spike protein binding and neutralizing antibodies using the two assays. We saw the most elevated neutralization antibodies against variants from individuals with breakthrough infections.
Heterologous versus homologous boosting regimens elicit qualitatively distinct, BA.5 cross reactive T cells in transplant recipients
Authors: Thompson Elizabeth A , Ngecu Wabathi , Stoddart Laila , Johnson T Scott , Chang Amy , Cascino Katherine , Alejo Jennifer , Abedon Aura T , Tobian Aaron A R , Segev Dorry , Werbel William A , Karaba Andrew H , Blankson Joel N , Cox Andrea L
Published: August 2023

The Omicron BA.5 subvariant of SARS-CoV-2 markedly escapes neutralizing antibodies induced by vaccination due to mutations in the Spike (S) protein. Solid organ transplant recipients (SOTRs) suffer high COVID-19 morbidity, yet show poor ancestral strain recognition after COVID-19 vaccination. CD8+ T cell responses may provide a crucial second line of defense. Therefore, it is critical to understand which vaccine regimens induce robust, conserved T cell responses. We evaluated anti-S IgG titers, subvariant pseudo-neutralization, and S-specific CD4 and CD8 T cell responses in SOTRs following homologous mRNA (mRNA-1273 or BNT16b2) or heterologous Ad26.COV2.S (JNJ-78436735) boosting. Homologous boosting with three mRNA doses induced the highest anti-S IgG titers. However, antibodies induced by both vaccine regimens demonstrated significantly lower pseudo-neutralization against BA.5 compared to the ancestral strain. In contrast, S-specific T cells maintained cross-reactivity against BA.5 compared to ancestral recognition. Homologous boosting induced higher polyfunctional CD4 T cells with phenotypes associated with metabolic and immunologic activation. Comparing two mRNA platforms, mRNA-1273 induced significantly higher IL-21+ peripheral T follicular helper cells and increased antibody titers compared to BNT16b2. Heterologous boosting with Ad26.COV2.S did not increase CD8 responses compared to homologous boosting, as CD8 responses were low across all individuals. These data demonstrate that boosting with the ancestral strain can induce cross-reactive T cell responses against emerging variants of concern in SOTRs, but currently available boosting strategies do not induce robust CD8 T cell responses.

U01AI138897 U54CA260491
Pre-existing immunity modulates responses to mRNA boosters
Authors: Dangi Tanushree , Sanchez Sarah , Lew Min Han , Awakoaiye Bakare , Visvabharathy Lavanya , Richner Justin , Koralnik Igor Jerome , Penaloza-MacMaster Pablo
Published: August 2023

mRNA vaccines have shown high efficacy in preventing severe COVID-19, but breakthrough infections, emerging variants and waning antibody levels have warranted the use of boosters. Although mRNA boosters have been widely implemented, the extent to which pre-existing immunity influences the efficacy of boosters remains unclear. In a cohort of individuals primed with the mRNA-1273 or BNT162b2 vaccines, we observed that lower spike-specific antibody levels before boost were associated with higher fold-increase in spike-specific antibody levels after boost, suggesting that pre-existing antibodies to SARS-CoV-2 downmodulate the immunogenicity of mRNA-based vaccines. Similar effects were observed in mice that received mRNA-based SARS-CoV-2 vaccines and HIV-1 vaccines. Our subsequent studies in mice show that pre-existing antibodies against the antigen encoded by the mRNA vaccine itself accelerate the clearance of vaccine antigen following a booster vaccination, via Fc-dependent mechanisms, limiting the amount of antigen available to prime B cell responses. These data suggest that transient downmodulation of antibody effector functions may improve the efficacy of mRNA boosters against SARS-CoV-2 and other diseases.

Start-up chart string, Northwestern University National Institute on Drug Abuse (NIDA, DP2DA051912)
Interferon-lambda modulates dendritic cell IL-10 production and CD8 T cell immunity during influenza A virus infection
Authors: Disla Lisbeth , Gong Qiaoke , Hemann Emily A
Published: August 2023

Influenza A virus (IAV) infection remains a major global health burden, requiring development of novel strategies to limit infection and drive durable protective immunity. Type III interferon (IFN), IFN-lambda, plays an important role in regulating IAV infection and immunity, but it’s contribution to regulating adaptive immunity remains less understood. Our previous research found mice lacking the IFN-lambda receptor (Ifnlr1−/−) are more susceptible to heterosubtypic rechallenge with influenza A virus. Ifnlr1−/− mice also have reduced effector and memory IAV-specific CD8 T cells compared to WT, highlighting a unique role for IFN-lambda in regulating protective CD8 T cell immunity against IAV. However, this defect in the CD8 T cell response was not T cell-intrinsic, but rather due to aberrant dendritic cell (DC) function in the absence of IFN-lambda signaling. RNA-Seq analysis of DC from the draining lymph nodes of mice following IAV infection revealed enhancement of an immunosuppressive network containing IL-10 in Ifnlr1−/− not present in WT. We hypothesize that IFN-lambda suppresses IL-10, playing a crucial role in regulation of protective CD8 T cell responses against IAV. Our results demonstrate IL-10 production is increased in bone marrow-derived DC (BMDC) in vitro and CD103+ DC in vivo following IAV infection in Ifnlr1−/− compared to WT. Ongoing experiments will unravel the contribution of IFN-lambda regulated IL-10 in regulation of CD8 T cell responses during IAV infection in vivo. Overall, understanding the mechanisms by which IFN-lambda signaling regulates DC function and protective CD8 T cell immunity will inform therapeutic potential of this cytokine during respiratory virus infection.

Supported by NIH grant K22 AI146141
Dissecting the role of H-2Db class I molecule in the development of brain atrophy during Theiler’s murine encephalomyelitis virus (TMEV) infection
Authors: Wininger Katheryn M , Goddery Emma , Ayasoufi Katayoun , Tritz Zachariah , Wolf Delaney , Jin Fang , Hansen Michael , Johnson Aaron J
Published: August 2023

Brain atrophy is a common feature of many neurological diseases as diverse as Alzheimer’s disease, cerebral palsy, Huntington’s disease, multiple sclerosis, epilepsy, encephalitis, neurosyphilis, neuroAIDS, and Covid-19 infection. We have developed a murine model of brain atrophy using the Theiler’s murine encephalomyelitis virus (TMEV) infection mouse model of multiple sclerosis. In this study, we investigated the contribution of the MHC class I molecule, H-2Db, in generating an immune response associated with the onset of brain atrophy using mice with a C57BL/6 background. The H-2DbClass I molecule not only contributed to significantly more ventricular enlargement in a T2-weighted MRI at 45 d.p.i., but this ventricular enlargement correlated to CD8 T cell infiltration. To further define the cellular and molecular mechanisms of MHC class I molecules in TMEV induced brain atrophy we investigated the cell specific contribution of brain resident antigen presenting cells (APCs) in ventricular atrophy. We developed novel bi-transgenic mouse lines with tamoxifen induced conditional ablation of the H-2Db class I molecule in Cx3CR1+ brain resident myeloid cells. Cx3CR1cre+/Db Lox P mice presented with significantly less ventricular atrophy at 45 d.p.i., compared to cre- littermates along with a decrease in CD8 T cell infiltration. These results strongly imply that antigen presentation by H-2Db on myeloid cells, at least in part is responsible for the development of brain atrophy and provided insight into the molecular mechanism of this poorly understood neuropathology.

NS103212, NS122174
Adrenergic receptors regulate T cell differentiation in viral infection and cancer
Authors: Globig Anna-Maria , Zhao Steven , Roginsky Jessica , Avina-Ochoa Natalia , Heeg Maximilian , Chaudhary Omkar , Hoffmann Filipe Araujo , Chen Dan , O’Connor Carolyn , Emu Brinda , Kaech Susan
Published: August 2023

CD8+ T cells are critical components of the immune reaction against viral infections and cancer and have the potential to eliminate infected or malignant cells. However, when the antigen persists, CD8+ T cells enter an exhausted state. Chronic disease can lead to increased systemic noradrenaline (NA) levels, but it is currently still unclear how NA impacts the differentiation and function of exhausted CD8+ T cells.

We here set out to characterize the effects of β adrenergic signaling on CD8+ T cells in chronic viral infection with LCMV-clone 13 and in murine cancer models. We observed that chronically infected mice had elevated systemic NA levels and CD8+ T cells expressed higher levels of the β-1 adrenergic NA receptor, Adrb1. NA impaired T cell receptor signaling of ADRB1-expressing CD8+ T cells, and reduced T cell proliferation and function. Conversely, genetic ablation of ADRB1 prevented terminal CD8+ T cell differentiation in chronic viral infection and ADRB1-blockade enhanced T cell functionality in combination with immune checkpoint blockade (ICB) in an ICB-sensitive melanoma model. Expanding these observations to an ICB-resistant model of pancreatic cancer, we found that pharmacological blockade of adrenergic receptors synergized with ICB to genetically reprogram CD8+ T cells towards a tissue resident memory T cell-like state and improved T cell functionality, resulting in decreased tumor size.

In summary, our data suggest that β adrenergic receptors represent a novel immune checkpoint that modulates CD8+ T cell differentiation and function in the context of chronic antigen exposure and that targeting adrenergic receptors may synergize with ICB in cancer patients.

AMG was supported by the German Research Foundation (Deutsche Forschungsgemeinschaft, GL 991/1-1). SZ was supported by K00CA222741. This work was supported by grants from the NIH 5 R01 CA240909 (SMK) and 5 R01 CA216101 (SMK).
Indirect IgG ELISA and serotype-specific neutralizing antibody titers are associated with protection against dengue in children in Cebu, Philippines
Authors: Odio Camila D , Daag Jedas , Crisostomo Maria Vinna , Adams Cameron , White Laura J , Deen Jacqueline , de Silva Aravinda M , Katzelnick Leah C , Ylade Michelle
Published: August 2023

Standard measures of dengue virus (DENV) antibodies have not been established as protective against dengue. We assess whether an indirect dengue IgG ELISA (PanBio; Brisbane, QLD, Australia) and serotype-specific neutralizing antibody titers measured by a two-dilution focus reduction neutralization test (FRNT) are associated with protection against dengue. Healthy children (n = 1,214) aged 9–14 years in Cebu, Philippines were enrolled in a prospective population-based cohort study in May – June 2017 and were tested for dengue when they reported a febrile illness through March 2020. Serum samples were collected at enrollment, and all were tested by ELISA. FRNTs were performed using the WHO DENV1–4 reference strains on a random subset to quantify serotype-specific neutralizing antibodies (nAbs). Inverse probability weighting was used to adjust for subset size. The odds of dengue as predicted by antibody measures were assessed by logistic regressions and adjusted for age, gender, and residential site. Dengue naïve children were used as the reference group in all models. The probability of dengue was 9.2% (95% CI: 4.5 – 17.9) for the naïve group, 9.5% (4.8 – 17.8) for those with ELISA < 2, but 4.5% (2.8 – 7.2) for those with ELISA >= 2 (p=0.04). The probability was even lower at 1.4% (0.6 – 3.1) for those with the highest DENV1–4 geometric mean titers (GMT, p = 0.0001). Pre-vaccine nAbs to DENV2 and DENV3 were protective against a homotypic dengue case (DENV2: OR 0.28, 95% CI 0.12 to 0.65; DENV3: OR 0.27, 95% CI 0.13 to 0.57), but not heterotypic dengue. We show a commercial ELISA and serotype-specific nAbs are associated with the probability of dengue, providing useful assays for use in population and vaccine trials.

This work was supported by the Philippine Department of Health, the Intramural Research Program of the National Institute of Allergy and Infectious Diseases (NIAID), and NIH Extramural Grant: P01AI106695.
Identity of virus-specific and cross-reactive T cell memory to mpox
Authors: Adamo Sarah , Gao Yu , Cai Curtis , Müller Thomas R , Niessl Julia , Akhirunnesa Mily , Ljunggren Hans-Gustaf , Sällberg Matti , Bergman Peter , Ekström Anna-Mia , Grifoni Alba , Sette Alessandro , Price David A , Buggert Marcus
Published: August 2023

The unprecedented spread of mpox (previously known as monkeypox) in several countries worldwide has led the WHO to declare the mpox outbreak a global health emergency in 2022. T cell responses are likely to play a central role in the resolution of the mpox infection. However, the prevalence and phenotype of mpox-specific T cells have not been investigated in detail, and the extent of cross-reactive immunological memory following previous smallpox vaccination is not known. Here, we assessed T cell responses to mpox infection in n=22 convalescent patients and n=53 healthy blood donors of different age groups, using CD4- and CD8-targeted peptide pools and optimal peptides through activation-induced marker (AIM), functional, and tetramer assays. We observed considerable CD4 and CD8 T cell activation among mpox patients in response to the peptide pools. Surprisingly, we did not detect differences between age groups of non-infected donors, despite previous exposure of older individuals to smallpox vaccination. Investigation of n=3 mpox-cross-reactive immunodominant vaccinia epitopes revealed that memory cells, although present, were maintained at comparable frequencies to naïve T cell precursors in older individuals. CD8+ T cells specific for the cross-reactive epitopes were significantly expanded and acquired an effector memory phenotype of elevated T-bet and Granzyme-B levels after mpox infection. Our data demonstrate low magnitudes of cross-reactive T cells against mpox across different age groups, whereas mpox infection leads to robust induction of effector memory T cells with strong recall potential.

Supported by SNF Post-doc mobility grant (P500PB_211069)
Age upon RSV infection and epitope-specificity influence the memory differentiation of antiviral T cells
Authors: Lu Zhongyan , Turfkruyer-Husson Mathilde , DaMata Jarina , Nhan Paula , Malloy Allison M W
Published: August 2023

Background

Despite the high rate of respiratory syncytial virus (RSV) infection and severe symptoms in infants, subsequent infections occur, indicating impaired mucosal immune memory at early life. CD8 T cells are associated with reduced disease in human adult challenge trials and murine models, demonstrating their protective roles. However, since primary RSV infection occurs in infancy, a better understanding of the memory differentiation of RSV-specific respiratory CD8 T cells in neonates is required.

Methods

CB6F1/J mice were infected at 7, 14, 25 days of life or adulthood to characterize the RSV-specific memory T-cell development. Cells from mediastinal lymph nodes (MLN) and lungs were analyzed by flow cytometry at 7, 11, 14, and 40 days post RSV infection (dpi) after staining with fluorescent antibodies and tetramers specific for M and M2 epitopes in RSV.

Results

RSV-specific CD8 T cell responses were overall lower in neonates from peak to memory phase, but differ by epitope. At 40dpi, M-specific CD8 T cells preferentially differentiated to central-memory (CM) and resided in the MLN in both adult and neonatal mice, whereas M2-specific CD8 T cells preferentially differentiated to effector-memory (EM) and resided in the lungs in adult but not neonatal mice. Importantly, lung tissue resident memory cell differentiation was limited in early life and increased with age upon infection.

Conclusion

Our results indicate that the magnitude and memory development of RSV epitope-specific CD8 T cells differ by tissue and age at infection, suggesting that age-associated mucosal immune factors contribute to the development of RSV-specific memory T cells.

This work was supported by funding through the NIAID R01AI154619 (A.M.W.M.). Supplemental support provided by USUHS Department of Pediatrics grant PED-86-3658 (A.M.W.M.).
B cell responses to membrane-presented antigens require the function of the mechanosensitive calcium channel Piezo1
Authors: George Rachel , Sohn Haewon , Kwak Kihyuck , Torgbor Charles , Manzella-Lapeira Javier , Brzostowski Joseph , Pierce Susan K
Published: August 2023

It has become increasingly evident that even though B cells respond to both soluble and membrane-presented antigens in vitro, the primary mode of antigen recognition in vivo appears to be of antigens on the surfaces of antigen-presenting cells. To determine how B cells sense that an antigen is membrane associated, we investigated the role of the Piezo1 mechanosensing cation channel in human B-cell activation. We discovered that human B cells flux calcium when simply poked by a glass probe or placed on a ridged glass surface, responses that were blocked by Piezo inhibitors. Piezo1 opens in response to changes in membrane tension and we showed that B cells placed on glass but not on soft planar lipid bilayers showed increased plasma membrane tension as measured by fluorescence lifetime imaging with a membrane tension reporter. B cell calcium responses to glass surfaces and to the Piezo1 antagonist Yoda1 were independent of BCR engagement. However, critically, B cell responses to membrane-presented antigens but not to soluble anti-Ig were inhibited both by Piezo1 inhibitors and by siRNA knockdown. In the absence of Piezo-1 function B cells engaging membrane-associated anti-Ig failed to flux calcium, spread on the membrane, form an immune synapse, increase plasma membrane tension, promote actin polymerization, increase expression of CD69 or proliferate. We conclude that Piezo1 plays a pivotal role in initiation of human B cell activation to membrane antigens.

Postbaccalaureate Intramural Research Training Award - NIH OITE
Discovery of a toxin for skin immune defense in African lungfish
Authors: Salinas Irene , Posavi Marijan , Benedicenti Ottavia
Published: August 2023

African lungfish estivate every dry season to survive unfavorable environmental conditions. The estivation process is associated with a drastic remodeling of the skin characterized by severe inflammation, granulocyte infiltration, epithelial damage and stem cell depletion which ultimately lead to formation of a cocoon with potent antimicrobial functions. We recently identified a novel toxin molecule that can be detected in the lungfish mucus and cocoon using proteomics. There are two genes in the African lungfish genome encoding for this toxin, one with six exons and the segmented duplicated gene with five exons. The toxin is also found in the Australian lungfish genome as a single exon molecule but appears to be lost in South American lungfish. Our studies show that dermal stem cells express this toxin at the steady state. Upon estivation, toxin mRNA levels are upregulated up to 200-fold and immunofluorescence microscopy shows it co-localizes with extracellular trap markers. 3D structure modeling predicts a pore-forming delta-endotoxin with potential insecticide functions. Current experiments are trying to elucidate the biological roles of this new toxin in the lungfish skin during freshwater and estivating stages.
Elucidating the role of TCR expression in Mycosis Fungoides: A Preliminary Analysis of Skin of Color biospecimens from Howard University Department of Dermatology
Authors: James Nia , Nwade Nina , Desse Sachi , Nagarkar Akanksha , Yildiz-Altay Ummugulsum , Okoye Ginette A , Richmond Jillian M , Byrd Angel S
Published: August 2023

Mycosis fungoides (MF) is the most prevalent Primary Cutaneous T Cell Lymphoma, comprising up to 60% of known cases. It is primarily a disease of resident memory T-lymphocytes that undergo phenotypic changes as a result of several gene alterations including ones coding for T-cell receptor (TCR) expression. The upregulation and downregulation of TCRs may give insight into the complex etiology of MF. There is a clear discrepancy in the morbidity and mortality of MF in the skin of color (SOC) population due to a myriad of factors including delayed diagnoses, biological differences, and limited research. Differences in the physiology of skin of color can cause atypical presentations, often mimicking other pigmentary and inflammatory dermatoses, causing delays in diagnosis and higher mortality. Therefore, further investigation into the molecular physiology of MF is warranted to better understand the disease presentation, progression, and appropriate therapies for SOC patients. Previous studies have identified the most common TCRs present in predominantly White cohorts including TCRVb3, TCRVb5, TCRVb6, TCRVb7, TCRVb20 and TCRVb21. Using MF biopsies from SOC patients at Howard University Dermatology, we analyzed lesional TCR gene expression and compared it to healthy SOC patients with the goal of considering novel therapeutic targets and analyzing how these molecular findings impact the diagnosis and treatment. Preliminary data shows that TRBC1 and TRBC2 are enriched in MF SOC patients whereas TRBV7–8 was enriched in the healthy margins. Because TCRs can be used as a biomarker to identify MF, this knowledge also has the potential to aid in the early diagnosis of MF, thus improving the disparity in outcomes seen in patients of color.

Sachi Desse - recipient of the Dermatology Foundation DRSA Jillian M. Richmond - recipient of the Skin of Color Society Research Grant Angel S. Byrd - recipient of the American Skin Association Milstein Research Scholar Award for Melanoma/Non-Melanoma Skin Cancer
Multiomic and spatial immunophenotyping reveals a prominent Runx3+ resident T cell population in the healthy human breast
Authors: Longworth Aaron J , Mallya Sharmila , Lev Tatyana , Pein Maren , Adam Isam , Lincy Antony , Naef Pascal , McMullen Timothy P , Halas Paige , Kessenbrock Kai , Lawson Devon A
Published: August 2023

The role of Runt-domain related transcription factor 3 (Runx3) in the early development of T cells is well established, however recent work has implicated Runx3 as a driver of residency in barrier tissues such as the lung and intestinal epithelium, as well as in the tumor infiltrating lymphocytes of murine and human melanomas. We have identified a subpopulation of resident T cells in the healthy human breast exhibiting constitutive expression of Runx3. Employing single-cell RNA sequencing paired with concurrent sequencing of T cell receptors (TCRseq, 10× Genomics) and oligo-tagged antibodies (Totalseq, BioLegend) specific to canonical markers of memory and effector T cells, we compared the clonality and transcriptome of T lymphocytes isolated from reduction mammoplasty or tumor-patient-derived contralateral mastectomies to those obtained from matched patient peripheral blood. We subsequently utilized highly-multiplexed spatial immunophenotyping through Co-detection by indexing (CODEX, Akoya Biosciences) to infer the role of Runx3+ resident T cells in the healthy human breast based on informatic analysis of the associated cellular neighborhoods. Our analysis finds an abundant population of highly clonal CD8+ Runx3+ cytotoxic T lymphocytes consistently located in the milk-producing lobular and secretory ductal regions of the healthy adult breast, postulating roles in barrier immunity and tissue reconstruction. Furthermore, marked presence of these cells in the contralateral mastectomies of tumor-bearing patients suggests a protective antitumoral role in nonaffected or cleared tissue.

Supported by NIH grant R01 (5R01CACA237376-03), T32 (T32-NS121727-01)
RGC-32 facilitates reactive astrocytosis by modulating the expression of axonal guidance molecules
Authors: Rus Horea G , Tatomir Alexandru , Cuevas Jacob , Nguyen Vinh , Cudrici Cornelia , Badea Tudor , Rus Violeta
Published: August 2023

Response Gene to Complement (RGC)-32 modulates TGF-β-induced extracellular matrix secretion and the ability of astrocytes to undergo reactive changes in vivo during experimental autoimmune encephalomyelitis (EAE). However, the molecular pathways underlying these effects are still not well understood. In this study, we investigated how lack of RGC-32 affects the transcriptomic profile and the expression of axonal guidance molecules (AGM) in astrocytes during EAE. We performed next-generation RNA sequencing on brain neonatal astrocytes isolated from wild type (WT) and RGC-32 knock-out (KO) mice, either unstimulated or stimulated with TGF-β. Results were then validated by using Real-Time PCR. Spinal cords from WT and RGC-32 KO mice with EAE (at days 0 and 14) were stained by immunohistochemistry for the astrocyte marker GFAP, AGM, and for nuclear factor IA (NFIA), a gliogenic factor and transcriptional regulator of AGM. Lack of RGC-32 had a significant impact on the transcriptomic programs normally associated with brain development whose re-expression is usually seen in reactive astrocytes. Connectivity analysis revealed that genes coding for AGM were particularly affected. We found lower transcript levels of ephrin receptor A type 7 (Epha7), plexin A1 and Slit guidance ligand 2 in RGC-32 KO astrocytes. Moreover, our results showed that NFIA and EPHA7 are expressed by astrocytes during EAE. We found a lower number of astrocytes expressing EPHA7 and NFIA in RGC-32 KO mice with acute EAE when compared with WT mice. These results suggest that RGC-32 might facilitate reactive astrogliosis during acute EAE through regulating the expression of AGM and NFIA.

Veterans Administration Merit Award I01BX001458 (to HR)
Interferon-alpha plays an important role in glial-mediated symptoms of neuropsychiatric systemic lupus erythematosus
Authors: Lin Stacie L , Aw Ernest , Zhang Yingying , Carroll Michael C
Published: August 2023

Systemic lupus erythematosus (SLE) is an autoimmune disease that leads to clinical pathology across organs, including the central nervous system (CNS). In up to 80% of patients with SLE, CNS manifestations can range from anxiety and fatigue to overt psychosis. While studies have demonstrated a hallmark increase in circulating interferon alpha (IFNα) in the peripheral blood and cerebrospinal fluid, the cellular mechanisms leading to neuropsychiatric lupus (NPSLE) remain unclear (Crow et. al., 2014). Using a mouse model of lupus which expresses an elevated IFNα signature and exhibits behaviors of NPSLE, we found a spatial pattern of interferon-stimulated genes (ISGs) in the brain parenchyma utilizing the multiplexed spatial transcriptomics platform MERFISH (Moffitt et. al., 2016). Guided by unbiased single nucleus sequencing highlighting astrocytes and oligodendrocytes as major glial cells enriched in ISG expression, we hypothesized that this spatial distribution implicates IFNα-driven glial activity against neuron function, thus disrupting regions of the CNS that manifests as NPSLE. We also investigated the role of IFNα on microglia activity and behavior changes using a cell-specific Cre mouse model. Further functional assays reveal increased synapse engulfment by microglia upon IFNα activation. Despite waning ISGs levels in the periphery, levels in the brain persist along with behavior severity, pointing to a CNS source of IFNα. This body of work demonstrates how IFNα released locally by glial cells can mediate NPSLE development via disruption of neuron function. Deeper understanding of targetable pathways in IFNα signaling will expand the therapeutic potential of type 1 interferon-driven neuropsychiatric diseases.
Hepatic regulatory T-cells promote colorectal liver metastasis in NASH
Authors: Hu Jiannan , Wang Han , Zhang Hongji , Ren Jinghua , Cheng Xiang , Yao Yonghong , Tsung Allan , Huang Hai
Published: August 2023

Background

Non-alcoholic steatohepatitis (NASH), a progressive inflammatory form of NAFLD, underlies an extremely versatile and dynamic inflammatory microenvironment along with aberrant metabolism and ongoing liver regeneration. Regulatory T cells (Tregs), as essential immune regulatory cells, are critical for the inhibition of tumor-promoting inflammation and cancer cell escape. Our recent study found that Tregs were selectively increased in both mouse models and patients with NAFLD, which promotes us to study the role of Tregs in NASH-HCC. However, the role of Tregs in the development of hepatic metastasis in NASH liver remains unknown.

Methods

A Western diet mouse model and the STAM mouse model were utilized for NASH development. MC38 murine colorectal tumor cells were injected via portal vein at 12 weeks of feeding or orthotopically injected into cecum subserosa. Tumor development will be determined 3 or 4 weeks after injection. Depletion of Tregs by FoxP3-DTR mice or CD25 antibody treatment. Transcriptomic profiling of leucocytes in hepatic immune microenvironment was determined by scRNA-seq.

Results

There is a positive correlation between increased Tregs and hepatic tumor growth in NASH liver. Depleting Tregs inhibits the development of hepatic metastasis in NASH liver compared with control liver. NASH development led to a significant transcriptomic shift in hepatic immune cells, such as Kupffer cells, Tregs and MDSCs. Blocking the interaction of Tregs and PMN-MDSCs in the TME prevents tumor growth in the NASH liver.

Conclusions

Tregs can suppress immunosurveillance in the premalignant stages of NASH. Therapies targeting Tregs could offer a potential strategy for preventing hepatic metastasis in patients with NASH.

RO1GM137203
T Follicular Helper Cells Restrain Obesity-Related Metabolic Disease by Damping Intestinal Inflammation
Authors: Wang Haiguang , Barrow Fanta , Florczak Kira , Abujamra Maya , Parthiban Preethy , Fredrickson Gavin , Revelo Xavier S
Published: August 2023

The intestinal immune system is a central modulator of obesity-associated metabolic disease. Particularly, high-affinity intestinal IgA maintains gut homeostasis after its generation in a germinal center (GC) response that requires functional T follicular helper (Tfh) cells in Peyer’s patches (PP). The objective of this study was to determine how obesity affects Tfh function leading to metabolic disease. We investigated the phenotype of Tfh cells in diet-induced obese mice. Obese mice showed enlarged PP featured by an expansion of CXCR5high ICOShigh Tfh cells. Surprisingly, intestinal Tfh cells in obese mice showed evidence of dysfunction as they expressed substantially lower PD1 and BCL6 but higher CCR7. As a result, the GC response and the generation of IgA+ B cells are also defective in obese mice. To determine if Tfh directly regulate intestinal inflammation and metabolic disease during obesity, we fed Tfh-deficient (Cd4Cre Bcl6flox) mice a western diet and investigated their microbiota composition, barrier integrity, and inflammatory status. In the gut, Tfh-deficient obese mice showed a disturbed microbiota, reduced regulatory T cells (Tregs), compromised intestinal barrier, and a loss of the microbiota-derived short-chain fatty acid butyrate. Moreover, we observed exacerbated liver inflammation in Tfh-deficient mice, suggesting a fundamental role for Tfh in dampening inflammation in the gut-liver axis. Mechanistically, Tfh cells express lower levels of insulin receptors and increased Foxo1, suggesting that reduced insulin signaling underlies the dysfunction of Tfh cells during obesity. Overall, our findings highlight an unexpected role for Tfh cells as essential regulators of obesity-related metabolic disease.