bluuebunny/crossref_metadata_2025 huggingface dataset:
Harnessing Regulatory invariant Natural Killer T cells in Autoimmune Liver Disease Treatment
Authors: Sokke Umeshappa Channakeshava , Hebbandi Nanjundappa Roopa , Babu Kolla Harish
Published: November 2024

Invariant Natural Killer T cells (iNKTs) are pivotal innate T cells that regulate immunity through cytokine secretion. In extra-hepatic autoimmunity, distinct regulatory iNKT cell types (iNKTR) play crucial roles. Recently, we identified a novel liver-specific subset, Maf+LiNKTR1 cells, characterized by its CD4+ TR1-like transcriptional signature. This subset responds to lipid-antigen-presenting nanomedicines, suppressing liver autoimmunity; however, its role under homeostatic conditions, in autoimmunity development, and responsiveness to other iNKT-directed therapies are not known. Using mouse models of Primary Biliary Cholangitis (PBC), here we explored the immunoregulatory role and therapeutic potential of Maf+LiNKTR1 cells. We observed a significant decrease in Maf+LiNKTR1 in PBC, accompanied by increased proinflammatory LiNKT1 subset. Biliary epithelial IL-10 signaling and gut-derived short-chain fatty acids contributed to Maf+LiNKTR1 generation. The reduced Maf+LiNKTR1 frequency correlated with loss of regulatory networks, suggesting a role in maintaining liver tolerance. Importantly, the subset responded to other iNKT-directed drugs, significantly suppressing PBC. Our study extended to the broader regulatory network of Maf+LiNKTR1. Further, it enhances our understanding of Maf+LiNKTR1 in liver health and autoimmunity development, offering potential therapeutic insights for autoimmune and other inflammatory liver conditions.
Investigating the potential of CD8α:MyD88 to restrain terminal exhaustion in tumor-specific T cells
Authors: Albert Gabriella , Cao Phoebe , Davila Eduardo
Published: November 2024

Despite the advancements in immunotherapies for solid tumors, many patients fail to develop durable responses. The immunosuppressive and antigen persistent environment drives T cell exhaustion, interfering with the efficacy of adoptive cell therapies. Terminally exhausted T cells (TTEX) co-express inhibitory receptors, lose effector function and fail to elicit tumor control. Our lab has reported that T cells engineered to express a CD8α:MyD88 fusion protein exhibit enhanced anti-tumor activity and appear less exhausted. We hypothesize that expression of CD8α:MyD88 in T cells restrains exhaustion by transcriptionally repressing inhibitory receptor expression thereby supporting effector function, persistence, and memory. Our RNAseq and RT-qPCR data revealed that expression of inhibitory receptor genes (Havcr2, Entpd1, CD244) was lower in CD8α:MyD88 compared to CD8α T cells. Moreover, collection of CD8α:MyD88 TIL from B16-F10 tumors (10 days post transfer) exhibited reduced frequencies of TTEX (PD1+, TIM3+, CD39+) cells, greater expression of TCF1 and co-stimulatory molecules, and remained functional upon ex vivo restimulation (IFNg). Additionally, a greater frequency of central memory T cells was present in secondary lymphoid tissues. Together, our results suggest that expression of CD8α:MyD88 in T cells may interfere with the expression of inhibitory receptors that dampen the anti-tumor response, thus enhancing the functional longevity and memory potential of T cells.
Novel tumor-specific Vd1/3 gd CAR T cells effectively control solid tumors
Authors: Lopez Bailon Luis , Anadon Carmen , Chaurio Ricardo , Mine Jessica , Plappert Noah , Antonia Scott , Conejo-Garcia Jose
Published: November 2024

Chimeric Antigen Receptor (CAR) T cell therapy has proven to be successful in treating hematological malignancies. However, its effectiveness against solid tumors has been suboptimal so far, due to the strong immunosuppressive conditions and metabolic restrictions present in the tumor microenvironment. Furthermore, the use of autologous T cells for CAR product manufacturing has several disadvantages. Here, we demonstrate the effectiveness of a new CAR T cell platform using cord blood-derived γδ T cell subsets, which can be used allogeneically (“off-the-shelf”) and recapitulate the populations primarily found in solid tumors. As targeting motifs, we used scFv sequences specifically targeting transmembrane molecules expressed in metastatic SCLC and other tumors, but not in vital tissues, which were identified through a comprehensive proteogenomic approach. To minimize PD-1-driven inhibition and enhance metabolic re-programming, we designed a new CD3z-less CAR architecture, which is more effective than conventional second-generation CARs. Our results demonstrate that γδ T cells are natural killers independent of MHC presentation, superior to αβ T cells, allowing the generation of multiples ‘off-the-shelf’ CAR T cell products. Moreover, the new architecture demonstrated better efficiency in killing tumor cells, thus supporting a rationale for future trials aimed at rendering CAR T cells effective against the most frequent and aggressive human solid tumors.
Enrichment using a selectable CD34t marker enhances TCR/CAR transduced T cell functionality and transgene expression in clinically relevant receptors
Authors: Quinn Suzanne , Scurti Gina , Dalheim Annika , Bochnacka Oliwia , Hossain Nasheed , Nishimura Michael
Published: November 2024

Gene-modified TCR transduced T cells and CAR T cells are used clinically and display a range of efficacy across multiple cancers. Many therapies have successfully controlled patient’s tumors; however, some have shown off-target adverse events which are harmful to patients’ overall outcomes. T cell-based therapies have high variation in patient outcome due to multiple factors, such as transduction efficiency and T cell phenotypes. To reduce product variability, our lab developed a selectable marker gene for use in cellular constructs. This selectable marker is a truncated, non-signaling CD34 molecule (CD34t), which can be used to remove the non-transduced T cells that function as bystanders. Our clinical trials have used two TCRs (TIL 1383I and HERV-E) and one CAR (CD19 CAR). Following transduction with these CD34t constructs, we determined transduction efficiency by expression of CD34t. CD34t expression correlates with transgene, allowing us to select high CD34t expressors, and thus high transgene expressors. The selected population produces higher levels of inflammatory cytokines and CD107a expression compared to the nonselected. This improved functionality is likely due to the selected population having higher levels of transgene across the population, whereas the nonselected has a broader range of transgene expressions with fewer expressors total. This selectable marker contributes to a more functional product, which may in turn improve patient response clinically.
Early exposure to exogenous IL-15 subverts ILC precursor priming and the generation of KIR+ NK cells
Authors: Ruesch Michael , Broughton Megan , Eisele Caprice , Kowalski Jessie , Nalin Ansel , Sara Kinda , Elmer Isaac , Anderson Stephen , Oakes Christopher , Mundy-Bosse Bethany , Freud Aharon
Published: November 2024

Killer immunoglobulin-like receptors (KIRs) allow natural killer (NK) cells to recognize and kill tumor cells. The mechanisms that control KIR acquisition during human NK cell development are not known. We previously observed that while human tonsil innate lymphoid cell precursors (ILCPs) could become KIR+ NK cells in immune deficient mice treated with interleukin (IL)-15, ILCPs cultured in vitro with IL-15 and OP9-DL1 stroma generated only rare KIR+ NK cells, suggesting that additional signals are required in vitro. To test this hypothesis, we cultured ILCPs with OP9-DL1 stroma and +/- allogeneic blood CD34+ hematopoietic progenitor cells (HPCs), which on their own can become KIR+ NK cells when cultured as follows: two weeks in Flt3 ligand (FL), c-Kit ligand (KL), and IL-3 followed by two weeks in FL, KL, and IL-15. Remarkably, among the ILCP-derived NK cells, significantly more were KIR+ when derived with HPCs (14.5+/-2.9% w/HPCs vs 1.9+/-0.8% w/out HPCs, p=0.004, n=7). Interestingly, we also observed that when IL-15 was added at day (d)0 instead of at d14, significantly fewer KIR+ NK cells were produced (IL-15 at d0: 3.3+/-0.7% vs IL-15 at d14: 14.5+/-2.9%, p=0.001, n=7). Lastly, we noted epigenetic and transcriptional alterations with IL-15 added at d0 vs d14. Collectively, these data suggest that ILCP differentiation into KIR+ NK cells requires epigenetic priming in a conducive microenvironment and that early exposure to exogenous IL-15 subverts this process.
The SRSF2P95H splicing factor mutation exerts distinct splicing effects across myelopoiesis that converge upon dysregulated G-CSFR signaling
Authors: Rich Abigail , Zinkel Sandra
Published: November 2024

SRSF2 P95H (P95H) splicing factor mutations are initiating genetic lesions in myeloid malignancies. They are associated with myeloid-biased hematopoiesis in mouse models and human disease. Mechanistically, P95H substitutions induce transcriptome-wide alterations in RNA splicing that vary by cell type and genetic background, complicating efforts to understand how P95H drives disease. Efforts to understand P95H mutations focus on splicing in progenitor cell populations, however, to date no studies have investigated this mutation in the mature myeloid cells that comprise >40% of the marrow. We evaluated P95H-associated splicing across myelopoiesis. We subjected promyelocytes, myelocytes, and neutrophils from P95H mice to RNAseq and integrated these data with RNAseq from P95H hematopoietic stem cells and human MDS samples. A cluster-based splicing analysis approach identified cell-type specific splicing with overrepresentation of differentially spliced transcripts involved in myeloid effector function pathways in myelocytes and neutrophils. Missplicing of Csf3r/CSF3R, the granulocyte colony-stimulating factor receptor (GCSFR), was conserved. Notably, P95H mouse bone marrow exhibits impaired activation of GCSFR-dependent pathways (STAT3/5, ERK) that is unique to GCSFR. Overall, these results define P95H-driven alternative splicing as a differentiation-dependent phenomenon and implicate dysregulated GCSFR signaling as conserved feature of P95H-dependent myeloid hematopoiesis.
Ezrin drives adaptation of monocytes to the inflamed lung microenvironment.
Authors: Gudneppanavar Ravindra , Di Pietro Caterina , Oez Hasan , Zhang Ping-Xia , Huang Pamela , Braga Cassia , Tebaldi Toma , Biancon Giulia , Kim Catherine , Gonzalez Anjelica , Halene Stephanie , Krause Diane , Egan Marie , Gupta Neetu , Murray Thomas , Bruscia Emanuela
Published: November 2024

We have previously shown that the actin-binding protein ezrin is reduced and displaced from the plasma membrane in monocytes/macrophages (MΦs) from patients with cystic fibrosis (CF) and CF mice, impacting immune response to bacteria. However, its role in monocyte/MΦ is not fully understood. To investigate how the lack of ezrin affects monocyte/MΦ functions in response to lung infections, we developed a monocyte/MΦ-specific ezrin knock out mouse model (Ez-KOm) . We aerosolized WT and Ez-KOm mice with LPS and assessed the number of lung monocyte/MΦ by flow cytometry and their transcriptional profile via RNA-seq, followed by in vitro functional studies. In WT mice, ezrin is induced by LPS in all lung monocyte/MΦ populations, with the highest induction observed in the interstitial MΦs (IMs) (4-fold higher than monocytes). LPS treated Ez-KOm mice exhibit a significant reduction in IM numbers compared with WT and increased expression of pro-inflammatory markers (Il6, Tnfα, and Cxcl1). RNA-seq revealed differentially expressed genes in Ez-KOm monocytes/IMs related to adhesion, proliferation and cytoskeleton rearrangement. Consistently, Ez-KOm monocytes treated in vitro with LPS on collagen show stunted filopodia, altered F-actin distribution, reduced adhesion and increased cell death due to defective FAK and PI3K/AKT signaling. Our studies suggest that ezrin plays a critical role in the survival and adaptation of monocytes to the lung extracellular matrix during infections.
Non-neutralizing immune-mediated cross-protection against influenza viruses by chimeric M2e-H3 stalk protein or multi-subtype neuraminidase plus M2e virus-like particle vaccine in ferrets
Authors: Kang Sang-Moo , Kim Ki-Hye , Subbiah Jeeva , Bhatnagar Noopur , Liu Rong , Raha Jannatul , Pal Surya , Park Bo Ryoung
Published: November 2024

Current influenza vaccine based on hemagglutinin (HA) immunity is not effective in providing cross protection against circulating variants and new pandemic viruses. In prior studies, we developed universal influenza vaccine candidates of multi-subtype neuraminidase and M2 ectodomain virus-like particles (m-cNA-M2e VLP) and chimeric M2e-H3 stalk protein vaccine (M2e-H3stalk), which were effective in inducing cross-protective immunity in mice. Here, we evaluated the immunogenicity and efficacy of these recombinant universal influenza vaccines in ferrets. Our results showed that immunization of ferrets with recombinant universal vaccines induced high levels of IgG antibody responses (M2e, H3stalk, multi-subtype NA), NA inhibition (NAI), antibody-secreting plasma cells in spleens, and IFN-γ secreting blood mononucleate cells. Ferrets with either m-cNA-M2e VLP or M2e-H3stalk vaccine were moderately protected from H1N1 and H3N2 influenza viruses as evidenced by lower viral titers in nasal washes, trachea and lung after challenge. Tests of passive immunity of vaccinated ferret antisera indicate that multi-NA plus M2e vaccine conferred more effective and broader humoral immunity in naïve mice than M2e-H3stalk vaccine. Our findings support that immunity to M2e, HA-stalk, and multi subtypes NA will induce broader cross protection in ferrets, which is likely translational to humans.
Abnormal uterine bleeding after COVID-19 vaccination in South Korea
Authors: Park Dong Choon , Yeo Joon Hyung , Yeo Seung Geun
Published: November 2024

Background: Various adverse reactions have been reported after vaccination against COVID-19.

Objective: Few studies to date, however, have analyzed uterine bleeding after COVID-19 vaccination. The present study therefore evaluated the incidence of abnormal uterine bleeding after COVID-19 vaccination in South Korea.

Methods: Data on suspected adverse reactions to COVID-19 vaccination reported to the Korea Disease Control and Prevention Agency from February 26, 2021, to June 29, 2023 (week 121), were analyzed. Subjects who experienced abnormal uterine bleeding were identified, and the incidence of abnormal uterine bleeding was compared among the types of COVID-19 vaccines administered.

Results: During the study period, 135,894,788 doses of COVID-19 vaccine were administered, with suspected adverse reactions occurring after administration of 483,391 doses (3.56 adverse reactions per 1,000 vaccinations) and abnormal uterine bleeding observed after administration of 6,288 doses (4.9 per 100,000 vaccinations).

Conclusions: Abnormal uterine bleeding was the fifteenth most common adverse reaction after COVID-19 vaccination. Studies are needed to clarify the causal relationships of vaccination and type of vaccine with abnormal uterine bleeding, as well as to develop protocols for the diagnosis and treatment of abnormal uterine bleeding after COVID-19 vaccination.
Abnormal innate immune responses and associated genetic factors in Multisystem Inflammatory Syndrome in Children (MIS-C)
Authors: Khan Rehan , Ji Weizhen , Guzman-Rivera Jeisac , Mishra Abhilasha , Richlin Benjamin , Gaur Sunanda , Singh Aalok , Bukulmez Hulya , Kaelber David , Riordan Mary , Kimura Yukiko , Ganapathi Usha , Michailidis Ioannis , Ukey Rahul , Casseus Myriam , Andrews Tracy , Roy Jason
Published: November 2024

Multisystem inflammatory syndrome in children (MIS-C) is a rare, severe complication of COVID-19 characterized by multi-lineage immune activation and multi-organ system involvement. While baseline immunophenotypes have been characterized, innate immune cell functionality and genetic risk factors remain poorly understood. To address these knowledge gaps, we assessed inflammatory immune responses in children with MIS-C (n = 33) relative to healthy controls (n = 5). We found that Toll like receptor (TLR) signaling is severely dampened in peripheral blood cells in the MIS-C group, with weak cytokine responses to LPS stimulation and increased baseline gene expression of TLR negative regulators, compared to controls. We also found that the cytokine response to LPS was abnormally heterogeneous in the MIS-C group. In particular, cytokine response outliers were enriched for rare and potentially deleterious variants in the lysosomal trafficking regulator (LYST) locus. These LYST variants were associated with markers of lysosomal and mitochondrial dysfunction, altered energy metabolism, and low levels of TLR negative regulator expression. Improved knowledge of immune dysfunctions associated with MIS-C and risk factors may help explain aspects of MIS-C and other manifestations of long COVID and identify targets for therapy of MIS-C and other hyperinflammatory syndromes in children.
THE ONES AND TWOS: PATTERN RECOGNITION RECEPTOR NLRX1 IMPACTS VIRAL REPLICATION OF AND IMMUNE RESPONSE TO SARS-COV-2
Authors: Ivester Hannah , Woolls Mackenzie , Mott Madeline , Gregory Julia , Morrison Holly , Tupik Juselyn , Finkielstein Carla , Duggal Nisha , Weger James , Allen Irving
Published: November 2024

When pandemic virus SARS-CoV-2 (SCV-2) emerged in 2020, research was quickly generated to investigate the virus and host immune responses to it. Much research has focused on the cytokine storm- when overzealous immune responses in severe COVID-19 disease causes rampant production of cytokines following signaling increased by the activation of pattern recognition receptors (PRRs). A unique PRR known as NLRX1 has many diverse roles, including regulation of key antiviral pathways- NF-kB and interferon- autophagy, and understudied roles in cellular metabolism. We show that changes in NLRX1 expression impacts immune responses to and viral growth of SCV-2 in cell culture and in mice. While Nlrx1-/- mice challenged with SCV-2 showed similar overall morbidity to WT counterparts, there were temporary increases in weight loss at multiple time points post infection. SCV-2 infection of Nlrx1-/- mice resulted in more airway inflammation, seemingly contrary to their also having increased viral titer at 2dpi. Data in cell culture report an enhanced replication ability of SCV-2 when NLRX1 expression is lower. Both cell culture and animal studies report differences in immune activation, with differences in cytokine production when NLRX1 expression changes. Together, these experiments: (1) support the enigmatic function of NLRX1 (2) suggest a possible role of NLRX1 in the pathobiology of SCV-2 infection and (3) add to the growing field exploring NLRX1 and its many curious roles.
The role of macrophage phagocytosis on respiratory pneumovirus infection and host defense
Authors: Martinez-Espinoza Ivan , Guerrero Plata Antonieta
Published: November 2024

Lung macrophages play a pivotal role in the defense against viral and bacterial infections. These specialized cells are equipped with multiple mechanisms, such as phagocytosis, to detect the presence of pathogens and clear the infections. Human Metapneumovirus (HMPV) is a leading cause of acute lung infection, affecting particularly young children and the elderly. During and after the HMPV infection, individuals are at an increased risk of secondary bacterial infections due to a decreased immune response capacity, which increases the severity of the disease. However, the cellular contribution of macrophages in the HMPV infection remains understudied. In this work, we investigated the role of macrophage phagocytosis in HMPV infection and the effect of HMPV on macrophage phagocytic activity. For that, we used THP-1- and PBMC-derived macrophages as experimental models. We found that macrophages were susceptible to HMPV infection and that phagocytosis is one of the mechanisms used by the virus to infect the cells. However, macrophages do not replicate the virus efficiently. Furthermore, viral infection decreases the capacity of macrophages to phagocyte bacteria, as demonstrated by the activity of intracellular pH indicator dye. These data highlight the importance of phagocytosis as a mechanism to control respiratory viral infections and the subversion of the host immunity by HMPV that may contribute to subsequent opportunistic bacterial infections.
The role of CMKLR1 in lymphocyte activation and localization within the CNS during West Nile Virus Encephalitis
Authors: Cortes Alejandro , Durrant Douglas
Published: November 2024

Continued West Nile virus (WNV) outbreaks causing neuroinvasive disease in the US highlight the urgent need to decipher CNS viral control mechanisms. DCs are pivotal in recruiting and reactivating CNS-infiltrating lymphocytes for viral clearance. However, the molecular mechanisms that regulate DC recruitment and activation remain unknown. In CNS-related autoimmune disorders, CMKLR1, a chemokine receptor expressed by a DC subset, aids their recruitment and activation via its ligand, chemerin. To investigate the role of CMKLR1 during WNND, we infected WT and CMKLR1-/- mice with WNV. CMKLR1-/- mice exhibited a slight increase in mortality but elevated clinical scores accompanied by increased CNS viral loads compared to WT. We hypothesize that CMKLR1 restricts WNV neuropathogenesis by regulating the activation and localization of antiviral T cells within the CNS during WNV encephalitis. Determining the role of CMKLR1 in effector T cell activation and migration may provide insights into local molecular mechanisms controlling lymphocyte function and fate within the CNS during WNV.
Improving immunity against chronic virus infection using an autoimmunity associated Ptpn22 allele.
Authors: Shaikh Anam , Bevis Alec , Barnes Jenna , Schwarting Nancy , Cockerham Tammy , Orozco Robin
Published: November 2024

Dysfunctional dendritic cells (DC) lead to inadequate T cell activation causing chronic virus infection. Enhancing both DC and T cell function can prevent chronic virus infection. An approach to enhance immune function is to harness the immune-activating phenotype driven by the autoimmunity-associated allele of PTPN22, which is expressed in all immune cells. We previously found that mice expressing the Ptpn22 autoimmunity-associated allele (Ptpn22-R619W) successfully clear Lymphocytic choriomeningitis virus clone 13 (LCMV-cl13) unlike wildtype (WT) mice. This correlates with a more immunostimulatory DC phenotype and enhanced T cell function. However, the mechanism Ptpn22-R619W enhances DC and T cell function remains unclear. Using our CRISPR/cas9 generated Ptpn22-R619W mice, we addressed the hypothesis that Ptpn22-R619W increases DC immunostimulatory proteins which increases anti-viral T cell activation during virus infection. Post infection, Ptpn22-R619W DCs have reduced infection, increased CD86, and decreased PD-L1 expression both in vivo and ex vivo. Additionally, Ptpn22-R619W DCs have a differential gene expression profile. Also, Ptpn22-R619W mice have enhanced CD8 T cell function against the LCMV-cl13 minor epitope, GP276, but not the major epitope. Lastly, co-culture experiments demonstrate that Ptpn22-R619W in both DC and T cells is necessary to enhance T cell function. These results show how Ptpn22-R619W prevents DC and T cell dysfunction during virus infection.
NEK7 phosphorylation amplifies NLRP3 inflammasome activation downstream of gasdermin D and potassium efflux
Authors: Xu Jie , Zhang Lingzhi , Duan Yanhui , Sun Fangyuan , Odeh Nouha , He Yuan , Núñez Gabriel
Published: November 2024

The NLRP3 inflammasome plays a critical role in innate immunity and inflammatory disease. The interaction of NEK7, a member of the NIMA-related kinase family that is essential for inflammasome activation, with NLRP3 is enhanced by potassium efflux. However, the mechanism by which potassium efflux promotes the interaction between NEK7 and NLRP3 remains unknown. Here, we show that following NLRP3 activation NEK7 is rapidly phosphorylated at a specific threonine residue by JNK1 downstream of gasdermin D-mediated potassium efflux. NEK7 phosphorylation enhances the binding between NEK7 and NLRP3, which further promotes inflammasome assembly and activation. Mutant mice and macrophages in which the threonine residue of NEK7 was replaced by valine were impaired in NEK7 phosphorylation, NLRP3 inflammasome activation and IL-1β secretion. Thus, NEK7 phosphorylation is an important event that acts downstream of gasdermin D and potassium efflux to further enhance NLRP3 inflammasome activation.
Regulatory T cells tonically inhibit spontaneous activation of naturally arising memory-phenotype CD4+ T lymphocytes
Authors: Li Jing , Yang Ziying , Kawajiri Akihisa , Sato Kosuke , Tayama Shunichi , Ishii Naoto , Kawabe Takeshi
Published: November 2024

Foxp3- CD4+ T lymphocytes consist of naïve (CD44lo), foreign antigen-specific memory, and self antigen-driven memory-phenotype (MP; both CD44hi) cells at homeostasis. We recently showed that self-reactive MP cells spontaneously proliferate and differentiate into the T-bet+ subset. How excess proliferation and Th1 differentiation of MP cells are inhibited in steady state remains unclear. Regulatory T cells (Tregs) can inhibit several immune responses, posing a possibility that the same lymphocytes may suppress spontaneous MP cell activation. Here we show using Foxp3-diphtheria toxin receptor-transgenic mice that both preexisting MP and naïve CD4+ T cells spontaneously proliferate and differentiate into Th1 cells upon Treg depletion. MP as compared to naïve CD4+ T cells more rapidly proliferate and differentiate three to five days post Treg depletion whereas these responses are mainly attributed to by the latter T cells at a later time point. Moreover, we demonstrate that these early MP proliferation and differentiation are driven in a manner dependent on CD28 and IL-2 signals, respectively. Furthermore, our data suggest that such activation of MP and naïve CD4+ T cells contribute to development of multi-organ inflammation at early and later time points, respectively. Together our results argue that Tregs tonically inhibit early, spontaneous proliferation and Th1 differentiation of MP CD4+ T lymphocytes, thereby contributing to maintenance of T cell homeostasis.
You're (Cyto)Toxic: HDAC5 controls the activation of cytotoxic CD8+ intraepithelial lymphocytes
Authors: Borregard Megan , Jabri Bana , Ciszewski Cezary
Published: November 2024

In Celiac Disease, upregulation of IL-15 by distressed intestinal epithelial cells results in the pathogenic reprogramming of cytotoxic CD8+ intraepithelial lymphocytes (IE-CTLs), which then promote tissue destruction. We found that IL-15 induced major transcriptional changes associated with effector function in IE-CTLs, but did so without impacting chromatin accessibility. Here, we explore how the intestinal microenvironment controls IL-15-mediated activation of IE-CTLs without restructuring their epigenetic landscape. We found that butyrate, a known immunomodulatory molecule, reduced expression of Granzyme B and NKG2D in ex vivo human IE-CTLs. Further, butyrate broadly impacted the expression of genes linked to survival and activation in a histone deacetylase (HDAC)-dependent manner. Using small interfering RNA, we determined that HDAC5 controlled the IL-15-mediated activation of IE-CTLs without impacting their survival. HDAC5 has little to no deacetylase activity against histone tails, and our data suggest that it remains localized in the cytoplasm following stimulation. We believe that HDAC5 may deacetylate key signaling molecules to promote their activation, and thus regulate the IL-15-mediated licensing of IE-CTLs. Understanding how HDAC5 controls activation of IE-CTLs could shed light on novel therapeutic options that specifically target IE-CTL activation responses in inflammatory T cell disorders, like Celiac Disease, without impacting their survival in the tissue.
CARD11-dependent signaling in T cells is critical for the activation of multiple AP-1 transcription factor complexes.
Authors: Bauman Bradly , Stinson Jeffrey , Frank Andrew , Sukumar Gauthaman , Dalgard Clifton , Snow Andrew
Published: November 2024

The CARD11 scaffold enables TCR-induced NF-κB, mTORC1 and JNK signaling. While the requirement of CARD11 for NF-κB stimulation is well known, it remains unclear if/how CARD11 influences other transcription factors (TFs) essential for T cell activation: AP-1 and NFAT. We sought to delineate the importance of CARD11 in activating AP-1- or NFATc1-dependent transcription in human T cells, employing mitogen-activated wild-type (WT) or CARD11 KO Jurkat T cells. RNA-seq analysis revealed significant downregulation of AP-1-mediated transcriptional programs in CARD11 KO cells, with a concomitant increase in NFATc1 expression. Although JNK inhibition in WT cells had a surprisingly minimal impact on AP-1 activation, loss of CARD11 substantially reduced mitogen-induced AP-1 component expression and activity, encompassing all canonical JUN and FOS family members. We further demonstrated that deleterious CARD11 variants derived from CARD11-associated Atopy with Dominant Interference of NF-κB Signaling (CADINS) disease patients also impaired AP-1 function. Conversely, JNK inhibition in WT T cells enhanced NFATc1 activity with concomitant upregulation of the NFAT-dependent TH2 master regulator GATA3, congruent with enhanced GATA3 induction in CADINS patient T cells. Our novel findings indicate that CARD11 governs the activation of AP-1 and NFAT TF complexes originally thought to be induced via CARD11-independent pathways, with implications for human T cell differentiation and function.
Resolving haplotype variation and complex genetic architecture in the human immunoglobulin kappa and lambda chain loci in individuals of diverse ancestry
Authors: Engelbrecht Eric , Rodriguez Oscar , Shields Kaitlyn , Schultze Steven , Tieri David , Jana Uddalok , Lees William , Smith Melissa , Watson Corey
Published: November 2024

Immunoglobulins (IGs), critical components of the human immune system, are composed of heavy and light protein chains encoded at three genomic loci. The IG Kappa (IGK) chain locus consists of two large, inverted segmental duplications. The complexity of IG loci has hindered effective use of standard high-throughput methods for characterizing genetic variation within these regions. To overcome these limitations, we leverage long-read sequencing to create haplotype-resolved IGK assemblies in an ancestrally diverse cohort (n=36), representing the first comprehensive description of IGK haplotype variation. We identify extensive locus polymorphism, including novel single nucleotide variants (SNVs) and a common novel ∼24.7 Kbp structural variant harboring a functional IGKV gene. Among 47 functional IGKV genes, we identify 141 alleles, 64 (45.4%) of which were not previously curated. We report inter-population differences in allele frequencies for 14 IGKV genes, including alleles unique to specific populations within this dataset. In addition, we identify haplotypes carrying signatures of gene conversion that associate with enrichment of SNVs in the IGK distal region. Finally, we identify extensive polymorphism in the IG Lambda (IGL) locus, including copy number variation for genes encoding IGLC and IGLJ genes. These data lay a foundation for advancing our understanding of how genetic variation in IG loci impacts expressed antibody repertoires in human populations.
Diversity and affinity from plasma vs. B cells for monoclonal antibody development
Authors: Hawksworth David , Moore Jeff , Larkin Isaac
Published: November 2024

Diagnostic assay development requires a diverse panel of high affinity antibodies. Historically, our lab has used splenic CD19+ B-cells to generate mouse hybridoma cell lines. However, literature suggests that, as a group, terminally differentiated, CD138+ plasma cells secrete the highest affinity antibodies, making these cells an attractive target for hybridoma development. To compare the use of these two lymphocyte populations for generating mouse hybridomas, three fusion experiments were completed whereby cells were separately isolated and fused. Hybridomas were plated in semi-solid media, expanded, and selected based on size and antibody secretion. An average of 246 (+ 73) plasma cell hybridoma colonies were available for selection, per 1x106 specific lymphocytes used for fusion, compared to 19 (+ 13) B cell hybridoma colonies. Cells from selected colonies were transferred to 96-well plates, grown for 7 days and the antibodies screened for antigen binding by enzyme immunoassay. Across the three fusion experiments, 40 (+19)% of the wells with growth from the plasma cell hybridomas were antigen reactive, vs. 6 (+ 0.5)% from the B cell hybridomas. Our data suggests a higher fusion efficiency from the plasma cell population, which allows for a greater sampling of the mouse immune repertoire, leading to identification of more antigen specific antibodies with a wider range of affinities and greater sequence diversity.
Immune dynamics in response to risk group 4 pathogens in nonhuman primate models
Authors: Rivera Deja , Barr Bobbi , Marketon Anthony , Bernbaum-Culter Rebecca , Pahar Bapi , Anthony Scott
Published: November 2024

Despite the critical importance of emerging infectious disease research, the underlying immune responses to risk group 4 pathogens remain largely understudied relative to many other pathogens. Spectral flow cytometry has dramatically increased the capacity of fluorescence-based cell analyses, allowing for the development of comprehensive panels across a variety of cell types. We have developed a 33-color phenotypic panel that incorporates a plethora of markers to simultaneously identify numerous distinct cellular populations within circulation and in tissue with a deliberate and extensive characterization of monocytes, dendritic cells, and tissue-resident T cells. This panel utilizes multiple approaches to accurately identify appropriate cell markers that can be easily distinguished with minimal to no compensation, despite containing fluorochromes with similar excitation and emission spectra. Importantly, this high-complexity panel was implemented on both fresh and frozen samples of whole blood, peripheral blood mononuclear cells (PBMCs), and cells isolated from multiple peripheral tissues across several nonhuman primate (NHP) models and on frozen human PBMCs, making this a broad cross-reactive panel enabling direct comparisons among NHP species and with humans. This new phenotypic flow panel allows for broad insight into underlying immune responses to infections across species while remaining amenable to additional potential markers of interest.
StarBright Dyes — Expanding the Availability and Choice of Fluorophores to Improve Immunophenotyping Data in Veterinary Studies
Authors: Sanderson Sharon , Rosenwasser Oliver , Blundell Michael , Long Tracey
Published: November 2024

Flow cytometry is an important application for veterinary studies that is commonly used for diagnostics, drug screening, and for the fundamental understanding of veterinary diseases. However, the range of fluorophores that flow cytometry veterinary antibodies are conjugated to is limited. This restricts the choice available for multiplexing panels and therefore panel size, which is especially important for rare samples as the information that can be determined from a single sample will be reduced. In addition, less choice means that best practice for panel design cannot always be followed, often resulting in poor cell resolution and data quality.

To address this problem, Bio-Rad is expanding the range of antibodies that are conjugated to StarBrightTM Dyes, to include canine, bovine, and porcine targets. Antibodies conjugated to StarBright Dyes are bright, easy to use, and will work with most protocols and cytometers. This means you have the choice and flexibility to follow best practice and make bigger panels, getting better results, saving time and resources.

We present data showing multiplexing panels on canine, bovine, and porcine peripheral blood, acquired on the ZE5 Cell Analyzer from Bio-Rad. These immunophenotyping panels include antibodies which are conjugated to traditional fluorophores as well as new StarBright Dyes. We demonstrate how multiple cell populations and subpopulations can easily be detected, such as B cells, T cells, and natural killer cells.
Enhanced Annotation of CD45RA to Distinguish T cell Subsets in Single Cell RNA-seq via Machine Learning
Authors: Ran Ran , Brubaker Douglas
Published: November 2024

T cell heterogeneity presents a challenge for accurate cell identification, understanding their inherent plasticity, and characterizing their critical role in adaptive immunity. With the advent of single-cell RNA sequencing (scRNA-seq), researchers can now investigate the gene expression profiles of these surface proteins at the single-cell level, having a deeper understanding of cell identity. However, CD45RA, the isoform of CD45 which distinguish between naive/central memory T cells and effector memory/effector memory cells re-expressing CD45RA T cells, cannot be well profiled by scRNA-seq due to the difficulty in mapping short reads to genes. In order to facilitate cell type annotation in T cell scRNA-seq analysis, we employed machine learning and trained a CD45RA+/- classifier on single-cell mRNA count data annotated with known CD45RA antibody levels provided by cellular indexing of transcriptomes and epitopes sequencing (CITE-seq) data. Among all algorithms we tested, the trained support vector machine (SVM) with a radial basis function (RBF) kernel with optimized hyperparameters achieved a 99.96% accuracy on an unseen dataset. The multilayer Perceptron (MLP) classifier, the second most predictive method overall, also achieved a decent accuracy of 99.74%. Our simple yet robust machine learning approach provides a valid inference on the CD45RA level, assisting the cell identity annotation and further exploring the heterogeneity within human T cells.
Unleashing the Power of γδ-T Cells with REAlease Technology
Authors: Brühl Vanessa , Donner Christin , Dhana Ermanila , Räty Lotta , Hildalgo Jose , Höher-Peters Susanne , Winkels Gregor
Published: November 2024

γδ-T cells represent a unique subset of immune cells that share characteristics of the innate and adaptive immune system. They are not MHC-restricted, making them promising candidates for allogeneic CAR T cell therapies. Enrichment of γδ T cells allows to simplify research, improve understanding of their biology and facilitate developments for therapeutic applications. The cutting-edge REAlease technology offers a direct means of enriching unlabeled cells from diverse blood materials. The option of full automation contributes to the standardization of routine separations, concurrently minimizing hands-on time. Evaluation of separation performance and functionality was conducted through flow cytometric analyses and in-vitro assays. Demonstrating the utility of the newly developed REAlease γδ T-cell MicroBead Kits, we achieved high-purity enrichment from PBMC, BC, WB and leukopak. The ratio of γδ T subtypes in enriched fractions was analyzed,, as well as the activation status. Isolated cells showed no expression of activation marker CD69 and remained inactive in culture. Stimulation effectively activated the cells showcasing their full functionality. Subsequent analysis examined cytokine secretion in non-stimulated and stimulated cells. The isolation strategy presented minimizes cell manipulation and reduces interference with downstream processes. It can thus make an important contribution to the progress of disease research and the exploration of new therapeutic possibilities.
An Alternative Immune Cell Labeling System Based on the New Two-Dimensional Nanomaterials MXenes
Authors: Giro Linda , Fusco Laura , Gazzi Arianna , Benjamin Chacon , Shuck Christopher , Ley Klaus , Orecchioni Marco , Gogotsi Yury , Delogu Lucia
Published: November 2024

Deepening our understanding of immune cell behavior is vital for creating safe, effective treatments, particularly as immune cell therapy, also known as cellular immunotherapy or cell-based immunotherapy, represents a groundbreaking approach in the treatment of various diseases, including cancer. Transition metal carbides and nitrides (MXenes), a novel family of 2D nanomaterials, show promise as advanced trackers for immune cells—key for precise diagnostics and therapies. Traditional cell labeling methods have stagnated due to limited chemical options, impeding progress in applied medicine. Moreover, these methods are incompatible with single-cell mass cytometry by time-of-flight (CyTOF), a globally adopted technology that improves classical flow cytometry. We propose an innovative solution utilizing MXenes to overcome these challenges. Our method, Label-free sINgle-cell tracKing of 2D matErials by mass cytometry (LINKED), leverages a novel, biocompatible, multiplexed, label-free detection approach via CyTOF and Mass Ion Beam Imaging by Time-of-Flight (MIBI-TOF). This technique overcomes chemical limitations and integrates seamlessly with CyTOF, allowing for nanomaterial detection and simultaneous measurement of diverse immune cell and tissue features. Our work promises to advance immunological research significantly, offering refined cell labeling and tracking techniques crucial for the advancement of translational medicine.
Single cell analysis of melanoma reveals a prognostic myeloid cell signature associated with survival and response to immunotherapy in advanced melanoma
Authors: Ahmad M Usman , Heo Lyong , Nair Ramesh , Sharma Saurabh , Kirane Amanda
Published: November 2024

INTRODUCTION: We sought to elucidate myeloid sub-type differences to characterize biology and support therapeutic strategies in melanoma patients with diverse biogeophaphies.

METHODS: scRNA-seq of melanoma were combined from 6 datasets with 126 patients. After integration, cells were clustered with scCCESS, using an artificial neural network. We conducted Recursive Partitioning Analysis (RPA), differential expressed gene (DEG) analysis, and pseudobulk comparison. We validated the signature using MuSiC deconvolution on bulk RNA data.

RESULTS: 102,971 single cells from 126 patients generated myeloid cells of 6 cell types. RPA analysis of 42 patients with melanoma brain metastases revealed a single myeloid population associated with median survival months (13 vs 32 vs 52, p=0.029). Partial Response (p=0.010) and adjuvant targeted therapy (p=0.036) differed. Myeloid cell type 1 was enriched with CLEC10A, AXL, and MHC Class II genes. 42 patients on nivolumab were used to validate signature with on treatment biopsy with improved median survival months (13.25 vs 30, p=0.046) and Partial Response (p<0.001). 19 patients shifted high infiltration groups during treatment with 11% dropping, 44% increasing, and 47% remaining high.

CONCLUSIONS: Increased infiltration of myeloid cells showed improved survival and treatment response in melanoma brain metastases. Validation of the signature showed an association with nivolumab response and survival.
Efficacy of IFN-g, CD40L, and poly(I:C) treated bone marrow-derived macrophages in murine mammary carcinoma
Authors: Kurt Robert , Roberts Meghan , Finn Joshua , Lass Melissa , Oviedo-Bermudez Ernesto
Published: November 2024

Here we explored methods to generate anti-tumor bone marrow-derived macrophages (BMDM) and how delivery of the BMDM at early tumor sites would impact disease progression. BMDM treated with IFN-g, CD40L, poly(I:C), and a combination of the three were assessed. Treatment with CD40L alone had no significant impact on cytokine production, phagocytic activity, or MHC expression by the BMDM. Treating BMDM with IFN-g impacted IL-1b expression as well as MHC Class II and CD80 expression. While poly(I:C) alone had a greater impact on the BMDM than IFN-g alone, treatment with poly(I:C), IFN-g, and CD40L had the greatest impact on the BMDM. Treatment with all three agonists resulted in increased IL-1b, TNF-a, and IL-12 expression, decreased expression of arginase and mrc, increased phagocytic activity, increased nitrite production, increased MHC Class II and CD80 expression, and these BMDM significantly impacted growth of two murine mammary carcinoma models; EMT6 and 168. However, the BMDM had no impact on progression of the 4T1 mammary carcinoma. Interestingly, BMDM treated with poly (I:C) alone significantly decreased growth of the EMT6 tumor, had no impact on growth of the 168 tumor, and enhanced growth and metastasis of the 4T1 tumor. Collectively, these data show that treating the BMDM with poly(I:C) alone exhibited varying results depending upon the tumor type, and that treating BMDM with poly(I:C), IFN-g, and CD40L generates BMDM with more consistent anti-tumor activity.
Target specificity in the ADAR editing landscape explains the unique immunoregulatory function of the interferon-inducible isoform ADAR1-p150
Authors: Pfaller Christian , Herrmann Maike , Krebs Yvonne
Published: November 2024

The adenosine deaminase acting on RNA (ADAR) family includes three enzymes, ADAR1-p150, ADAR1-p110, and ADAR2 that bind endogenous and pathogen-derived double-stranded RNA (dsRNA) and convert adenosine (A) to inosine (I). This process called A-to-I editing disrupts dsRNA structures and counteracts activation of innate immune sensors MDA-5 and PKR, preventing autoimmune disorders like Aicardi-Goutieres-Syndrome. We and others have shown that the interferon-inducible ADAR1-p150, but not the other ADARs, protects cells and organisms from aberrant innate immune activation by self-derived dsRNA, even though A-to-I editing is performed by all three ADARs. We hypothesized that ADAR1-p150 edits specific transcripts that cannot be edited by the other enzymes. Here we present a novel approach to quantify RNA editing in transcriptomic data. Our tool TSniffer detected more than 40,000 regions of A-to-I editing distributed over the entire human transcriptome, affecting 30% of protein-coding transcripts and 10% of long non-coding RNAs. Differential editing frequencies in cell lines with knock-outs for the different ADARs revealed transcripts exclusively edited by ADAR1-p150. In contrast, editing by ADAR1-p110 and ADAR2 was cooperative and more redundant. Our analyses fill gaps of knowledge about the A-to-I editing landscape, highlight the dominant immunoregulatory function of ADAR1-p150, and provide new insights in the role of aberrant A-to-I editing in disease development.
The FLRT3-UNC5B Pathway is a Novel Regulator of T Immunosurveillance
Authors: Flies Dallas , Yan Chuan , Yang Qiqi , Arbitman Steven , Fitzgerald Daniel , Sharee Sasan , Shaik Jahangheer , Bosiacki Jason , Myers Kayla , Paucarmayta Ana , Johnson Dorothy , O'Neill Tom , Cusumano Zachary , Langermann Solomon , Langenau David , Patel Shashank
Published: November 2024

Our understanding of molecular cell-surface interactions that control T cell responses in cancer remains limited despite many advances in the field. We hypothesized that tumor cells take advantage of developmental protein-protein interactions such as axon guidance interactions that could inhibit or repel tumor-immune interactions in the TME to evade T cell anti-tumor immunity. We used gain-of-function screening to evaluate a gene-set of axon guidance proteins for inhibitory function in human T cells and identified and validated Fibronectin and Leucine-Rich Transmembrane protein-3 (FLRT3) as an inhibitor of T cell activity. FLRT3 inhibited human T cell function and promoted tumor growth in humanized tumor cell-derived xenograft (CDX) models. FLRT3 inhibited T cells through interaction with Unc-5 Netrin Receptor B (UNC5B) expressed on activated T cells. A FLRT3 monoclonal antibody designed to specifically block FLRT3 interactions with UNC5 family proteins reduced tumor growth in CDX models that was dependent on T cells. FLRT3 blockade also enhanced CAR-T and BiTE + T cell activity and infiltration in humanized zebrafish tumor models. These studies support the notion that aberrant axon guidance protein expression in the TME act as T cell checkpoints, and the FLRT3:UNC5B pathway can be targeted therapeutically to improve T cell-based immunotherapy activity and infiltration.