bluuebunny/crossref_metadata_2025 huggingface dataset:
Parainfluenza virus spread into the lower respiratory tract of immunocompromised hosts: Antibodies but not G-CSF reduce viral load and accelerate clearance
Authors: Mostafa Heba , Vogel Peter , Srinivasan Ashok , Russell Charles J
Published: January 2023

In immunocompromised patients, parainfluenza virus (PIV) infections have a higher potential of spread to the lower respiratory tract (LRT), increasing morbidity and mortality. Understanding the immunological defects that facilitate the spread of the infection to the LRT among high-risk populations will help developing better management protocols. In this study, we suppressed the immune system in mice using the widely used anti-leukemic drugs: dexamethasone and/ or cyclophosphamide. We monitored the spread of viral infection into the LRT by non-invasive bioluminescence imaging system using a reporter Sendai virus (murine PIV type 1). Our results show immunosuppression leads to a delay in clearance of infection and higher lung viral loads. A rebound in the peripheral neutrophils counts coincided with initiation of viral clearance. However, treatment of immunosuppressed mice with neutrophil-specific antibodies to delay neutrophil rebound did not delay clearance, and treatment with G-CSF to increase neutrophils counts did not accelerate clearance. On the other hand, treating immunocompromised mice with either Sendai virus-specific polysera or monoclonal antibodies reduced viral progression to the lung and accelerated viral clearance. This study demonstrates the strength of non-invasive bioluminescence in both studying respiratory viral progression and evaluating candidate intervention strategies. The findings challenge the potential benefit of using G-CSF in PIV pneumonia and suggest a therapeutic potential of virus-specific antibodies.
Blood microRNAs correlate with plasma cytokines and clinical severity in patients infected with Influenza B and Rhinovirus/Enterovirus.
Authors: Rosas-Taraco Adrian Geovanni , Arredondo-Loza Erick Roel , Martinez-Castilla Azalia M. , Mata-Tijerina Viviana Leticia , Salinas-Carmona Mario C
Published: January 2023

Acute respiratory infections (ARI) are the leading causes of infectious disease morbidity and mortality in the world. Two of the most common etiological agents are Influenza B virus (IBV) and Human Rhinovirus (HRV). The IBV is responsible for about 25% of laboratory-documented influenza; meanwhile HRV is responsible for 33% to 50% of common cold cases in adults. Little is known about immunological biomarkers in the early stages of the infection caused by this RNA virus and their possible association with symptoms in those patients. The aim of this study was to identify miRNAs and their correlation with cytokines and clinical severity. Adult patients clinically diagnosed with ARI were sequentially recruited from Medical Service and University Hospital of Universidad Autonoma de Nuevo Leon. The study was conducted with approval of the bioethics committee of the School of Medicine of the Universidad Autonoma de Nuevo Leon. Virus was detected by Luminex xTAG RVP kit, levels of nine miRNAs and twenty six plasma cytokines were analyzed by RT-qPCR and multiplex immunoassay, respectively. The severity of symptoms was daily monitored throughout seven days by surveys. The IBV-positive patients showed lower expression levels of miR-146, miR-16 and increased levels of CCL20 when compared with non-IBV. HRV/Enterovirus(EV)-positive patients showed lower expression levels of miR-132, miR-146 and miR-29. Expression levels of miRNAs and plasma cytokines correlated with symptoms severity in both patient groups. In conclusion, our findings suggest that blood miRNAs and plasma cytokines may regulate systemic immune response against IBV and HRV/EV and many of them may be associated with symptoms severity.
Macrophage Bcl6 expression promotes viral dissemination and host inflammation during influenza infection
Authors: Yao Shuyu , Jiang Li , Lu Qingbo , Dent Alexander L. , Sun Jie
Published: January 2023

The role of Bcl6 in regulating anti-viral immune responses is relatively unclear currently. Further investigation was hindered by the development of lethal inflammation in global Bcl6 deficient mice. To circumvent this and explore the cell type-specific role of Bcl6 in regulating anti-viral immunity, we have generated myeloid cell-specific Bcl6 deficient mice (Bcl6 cKO) by crossing Bcl6 fl/fl mice to LysMcre mice. In contrast to global Bcl6 deficient mice, Bcl6 cKO mice exhibited no signs of autoimmunity and tissue inflammation under homeostatic conditions. Using a murine influenza A/PR8 (IAV) infection model, we found that Bcl6 cKO mice showed drastically increased host resistance to IAV. We observed that although airway virus titers are comparable between control and Bcl6 cKO mice, Bcl6 cKO mice had greatly diminished IAV spreading in the lungs. Correspondingly, Bcl6 cKO mice developed milder pulmonary inflammation compared to control mice, characterized by the decreased levels of airway cytokines/chemokines and diminished inflammatory cell infiltration into the lungs. Interestingly, alveolar macrophage depletion abrogated the phenotype of decreased host morbidity and mortality to IAV infection in Bcl6 cKO mice, suggesting that Bcl6 deficiency in tissue resident alveolar macrophages mediates the effects of enhanced host resistance to IAV infection. Together, our data suggest that Bcl6 expression in lung resident macrophages dampens the efficient development of pulmonary anti-viral immune responses. Current studies are focused on the cellular and molecular mechanisms by which macrophage Bcl6 expression inhibits host innate and adaptive immunity during IAV infection.
Role of T cell receptors in suppressing HIV infection in elite controllers
Authors: Joglekar Alok , Lamothe Pedro A , Ouyang Yong , Liu Zhe , Walker Bruce D , Baltimore David
Published: January 2023

Virtually all HIV-infected patients progress to AIDS if not treated. A group of patients, called Elite Controllers (ECs), can suppress HIV infection without treatment. This ability of ECs is, in part, due to potent CD8+ T cell (CTL) responses against HIV epitopes presented by human leukocyte antigen (HLA) molecules. In this study, we focused on CTL responses restricted to HLA-B27, an allele that is enriched in ECs, but is not sufficient for protective ability. In HLA-B27+ ECs or non-controllers (NCs), immunodominant CTL responses specific to the KK10 epitope in Gag are found. Previous studies have shown that unlike NCs, B27-KK10-specific CTLs from ECs are efficient at eliminating HIV-infected or KK10-peptide pulsed cells. As the potency of CTLs can be modulated by their T cell receptor (TCR), we hypothesized that B27-KK10-specific TCRs play a significant role in suppressing HIV in ECs. Therefore, we cloned B27-KK10-specific TCRs from ECs or NCs, expressed them in primary T cells, and tested their cytotoxicity. We incubated TCR-transduced T cells with either HIV-infected or KK10-peptide pulsed target cells and measured target cell lysis. Unexpectedly, EC-TCRs were not significantly different than NC-TCRs in their cytotoxicity. We then tested the ability of these cells to suppress HIV in humanized mice and found no significant difference between EC- and NC-TCRs. We are currently investigating various aspects of these TCRs such as cross-reactivity to variants of KK10. The result that TCRs from ECs and non-controllers do not recapitulate the phenotype of CTLs is intriguing and warrants further studies to uncover the mechanism of immunologic control of HIV. Our studies also have implications in immunotherapy and vaccine approaches for HIV.
MCMV-specific salivary gland CD8+ TRM cells are formed independently of viral infection of the salivary gland, CD4+ T cell help or CXCR3, but can be transiently boosted by restimulation.
Authors: Dantas Sofia Caldeira , Smith Corinne , Quinn Michael , Snyder Christopher M.
Published: January 2023

The salivary gland is a mucosal site of cytomegalovirus (CMV) replication, persistence, latency and shedding. Using the murine (M)CMV model, we found that MCMV-specific CD8+ T cells localized to the salivary gland within 7 days of infection and adopted a tissue-resident memory (TRM) phenotype by day 14. This timing correlates with viral replication in the salivary gland leading us to hypothesize that viral infection promoted TRM development. However, we found that MCMV-specific TRM developed normally in the salivary gland regardless of the route of infection or the presence of replicating virus. Despite the induction of the chemokines CXCL9 and CXCL10 by salivary gland infection, MCMV-specific TRM developed independently of the chemokine receptor CXCR3 on CD8+ T cells. In fact, salivary gland TRM formed even when activated T cells were transferred into MCMV-naive mice. Moreover, CD8+ TRM were formed independently of CD4+ T cells, which control of MCMV in the salivary gland. These data suggest that T cell activation programs T cells to migrate to the salivary gland, irrespective of local infection or inflammation. Interestingly, the overall numbers of salivary gland TRM were remarkably consistent in these experiments, which could suggest that space was limited in the salivary gland for TRM cells. We found that we could markedly boost the numbers of MCMV-specific TRM by restimulating circulating T cells with a peptide delivered I.V.. However the numbers of salivary gland TRM were only elevated transiently and declined to normal levels over time. Together, these data suggest that salivary gland TRM are formed readily upon antigen stimulation, but that maintenance of TRM in the salivary gland is subject to homeostatic limitations.
Biological Activities of IL-15 superagonist - IL-15 Mutein:IL-15RaFc complex following Intravenous or Subcutaneous Administration
Authors: Liu Bai , Kong Lin , Jones Monica , Noel Terra , Han Kaiping , Rhode Peter R , Jeng Emily K , Cai Weibo , Wong Hing C
Published: January 2023

ALT-803 is a fusion protein complex consisting of IL-15N72D superagonist and a dimeric IL-15 receptor alpha (IL-15Rα) sushi domain IgG1 Fc fusion protein. When administered to mice, ALT-803 is capable of inducing NK and CD8+ T cell proliferation and activation, as well as potent antitumor responses. ALT-803 is currently in clinical studies using an intravenous (iv) route of administration. We were interested in exploring treatment regimens using subcutaneous (sc) administration of ALT-803 as an alternative to iv administration in order to lessen the adverse side effects that were observed. In this study, we compared the pharmacokinetics, immunostimulation, and anti-tumor efficacy of iv and sc administration of ALT-803 in C57BL/6 mice. We found the half-life of ALT-803 to be 7.5 hrs for iv administration vs. 7.7 hrs for sc. The maximal detected serum concentration of ALT-803 was 495 ng/ml at 16 hr time point following sc administration or 3926 ng/ml at 0.5 hr time point following iv administration. Similar bio-distribution of radio-labeled ALT-803 in mice was observed using quantitative PET-scan studies. We also demonstrated that ALT-803 administered iv or sc induced comparable proliferation of CD8+ T cells and NK cells and similarly activated immune cells which resulted in the reduction of tumor burden. A toxicity study of mice receiving multiple injections of ALT-803 for 4 weeks by iv or sc administration revealed that comparable immune system-related changes were observed and ALT-803 was well tolerated. The gradual absorbance into blood stream and lower maximal blood level of ALT-803 in sc injected mice, along with similar antitumor efficacy supports the use of ALT-803 by sc administration in patients with metastatic malignancies.
IL-2 functionalized hydroxyethylstarch nanocapsules for targeting of human T cells with different IL-2 receptor affinities in vitro and in vivo
Authors: Domogalla Matthias Philipp , Wurm Frederik Robert , Mailänder Volker , Landfester Katharina , Steinbrink Kerstin
Published: January 2023

Cell type specific delivery of drugs by use of nanocapsules (NC) is a promising approach for induction of efficient anti-tumor immunotherapies. Here, we report the generation of IL-2 functionalized NC (HES-IL-2) to target CD25+ (IL-2 receptor α chain) T cells. In flow cytometry experiments HES-IL-2 exhibited an enhanced uptake by human CD25+ T cells compared to control capsules. The observed uptake was CD25 specific as CD25high T cells displayed a significantly enhanced NC incorporation compared to CD25− T cells with negligible internalization. In addition blocking of the IL-2 receptor dependent binding by an anti CD25 mAb significantly reduced the HES-IL-2 uptake by human CD25+ T cells. Furthermore, comparing and competitive studies of naïve CD25−, activated CD25+ and regulatory CD25high human T cells revealed a low incorporation of HES-IL-2 in naïve and a moderate and high uptake, respectively in activated or regulatory T cells. Additionally, in order to preferentially address different T cell populations by means of various IL-2 receptor affinities, we generated HES-IL-2 with distinct reduced amounts of IL-2 on their surface. Intriguingly, in contrast to naïve CD25− and activated CD25+ T cells, in which the NC uptake was clearly IL-2 dose-dependent we did not observe any differences when HES-IL-2 with a twofold and tenfold decreased amount of IL-2 were used for studies with regulatory CD25high T cell, indicating a high IL-2 sensitivity of regulatory T cells for a specific targeting. In addition, in vivo studies in human T cell reconstituted RAG2−/−γc−/− mice exhibited a significantly enhanced uptake of HES-D-IL-2 by CD25+ T cells. In summary, we generated IL-2 functionalized NC for targeting of human CD4+CD25+ T cells in vitro and in vivo.
Mkp-1 deficiency strengthens immune defense against fungal infection
Authors: Liu Yusen , Crowell Sara A. , Tang Juan , Kim Victoria Y. , Nelin Leif D. , Zhang Jian
Published: January 2023

Fungal infections are a serious threat to people with a compromised immune system. The initiation of an immune response toward fungal pathogens relies on the recognition of fungal molecular patterns through pathways mediated by TLRs and C-type lectin receptors. Activation of these pathways converges on both NF-κB and MAPKs to initiate cytokine and chemokine production, leading to recruitment of inflammatory/phagocytic leukocytes and pathogen elimination. Mkp-1 is a negative regulator of the innate inflammatory response triggered by pathogen recognition, acting to attenuating cytokine production by deactivating p38 and JNK. To understand the role of Mkp-1 in immune defense against fungal pathogens, we assessed the effects of Mkp-1 deficiency on the immune response to C. albicans, the most common fungal pathogen. Contrary to our previous observation with bacterial pathogens such as E. coli or bacterial products such as LPS, Mkp1−/− mice were protected from infection with a lethal dose of C. albicans, which correlates with lower kidney fungal burdens. In response to C. albicans challenge, Mkp1−/− mice produced greater TNF-α. Interestingly, in response to heat-killed C. albicans yeasts and hyphae Mkp-1−/− macrophages also produced significantly greater amounts of TNF-α and had markedly higher p38 and JNK activities than did wildtype macrophages. TNF-α production in both wildtype and Mkp1−/− macrophages in response to heat-killed C. albicans (both yeast and hypha forms) was attenuated by the specific inhibitors of MEK1, JNK, and p38 as well as Syk. Taken together, our results suggest that during fungal infection Mkp-1 acts to restrain the production of cytokines and thus hamper the immune response against fungal infections.
Cytotoxic T lymphocytes in channel catfish, Ictalurus punctatus, can express an inhibitory immunoglobulin-binding receptor
Authors: Spencer David A , Bengtén Eva , Nayak Deepak K , Wilson Melanie
Published: January 2023

Surface immunoglobulin (Ig)-binding receptors, i.e. Fc receptors (FcRs), are well characterized in mammals and play both activating and inhibitory roles in immune cell responses, however how they function in teleosts is less well understood. Here we report an inhibitory IgM-binding receptor on catfish cytotoxic T lymphocytes (CTL). Briefly, flow cytometric analysis revealed a morphologically defined subset of clonal CTL that can express a surface IgM-binding receptor. Chromium release and cell membrane labeling assays reveal that cells within this subset are unable to kill allogeneic target cells. After activation with alloantigen, however, these CTL downregulate the IgM-binding receptor, replicate, and efficiently kill target cells. Importantly, cross-linking the IgMbinding receptor with anti-catfish IgM mAb resulted in cell cycle arrest in the G1/G0 phase with fewer cells transitioning to the cytotoxic effector morphology. Co-immunoprecipitation of surface-biotinylated CTL with anti-catfish IgM identified putative receptor components of 38, 52, and 75 kDa. To identify the protein(s), these gel bands are being analyzed by LC-MS/MS, and Ig-binding of candidate proteins will be examined using transfected insect cells. Overall, this Ig-binding receptor is expressed on >95% of cells in CTL cultures that can no longer be activated to kill target cells. Thus the presence of this receptor may provide a marker for functionally exhausted catfish CTL.
Impaired T cell immunity in the elderly via N-glycosylation
Authors: Hayama Ken L , Khachikyan Khachik , Mkhikian Haik , Li Carey F , Zhou Raymond W , Klaus Suzi , Ly Kim M , Gong Andrew D , Raffatellu Manuela , Demetriou Michael
Published: January 2023

Aging is associated with a decline in immune function, increasing the frequency and severity of infection in the elderly. We have previously shown that Asparagine (N) – linked protein glycosylation is a critical negative regulator of T cell immunity in both mice and humans. Here we report N-glycan branching in T cells increases with age, leading to T cell hypo-activity and increased susceptibility to infection. Reducing N-glycan branching rejuvenates T cell activation, proliferation and pro-inflammatory TH17 over anti-inflammatory Treg differentiation in aged T cells. Susceptibility of aged mice to Salmonellae typhimurium invasion/dissemination was reduced by lowering N-glycan branching. A critical metabolic precursor of N-glycosylation and branching is N-acetylglucosamine (GlcNAc), a common amino sugar that is part of the regular human diet. GlcNAc is endogenous to human serum, increases with age and correlates with N-glycan branching in T cells. T cell specific deletion of N-acetylglucosamine kinase, thereby blocking salvage of GlcNAc into N-glycans, reduces branching in aged T cells and rejuvenates T cell activation. Collectively, our results suggest that age-dependent increases in N-glycan branching, via increased supply of GlcNAc, impairs T cell immunity in the elderly. Therapeutic inhibition of N-glycan branching may provide a novel mechanism to rejuvenate T cell responses and reduce infection risk in the elderly.
A Prime-Boost Dengue Vaccination Strategy Induces Durable Multivalent Memory B Cell Responses
Authors: Hatch Kristin , Friberg Heather , Koren Michael , Keiser Paul , Lin Leyi , Jarman Richard G , Currier Jeffrey R
Published: January 2023

Denguevirus is a mosquito-borne flavivirus that is comprised of four antigenically related but distinct serotypes. A higher risk for the most severe clinical manifestations of dengue infection, dengue fever and dengue hemorrhagic fever, is associated with sequential infections with heterologous serotypes. While there currently is no precisely defined correlate of protection, durable tetravalent immunity is the desired outcome of dengue vaccination. A Phase 1 clinical trial was conducted at the Walter Reed ArmyInstitute of Research (WRAIR), WRAIR #2136, using live attenuated virus (LAV)and purified inactivated virus (PIV) vaccine candidates containing all fourserotypes. The two products were tested in a prime-boost regimen (PIV followed by LAV and vice versa) in different schedules to determine if dosing schedule or order of administration affected neutralizing antibody levels and/or cell mediated responses. Utilizing a three color Fluorospot assay to measure DENV-specific IgG, IgA, and IgM secreting cells, we tested 6-months post second dose memory B cell cultures to determine if the response was tetravalent and if a change in vaccination strategy affected the valency. We also investigated how memory B cell responses correlated to neutralizing antibody data. Results show that a majority of subjects have a robust multivalent DENV-specific IgG memory B cell response at 6 months post-vaccination. A better understanding of memory B cell responses and how they relate to neutralizing antibody will help find a suitable DENV vaccine that provides durable tetravalent immunity.
CHARACTERIZATION OF INTERFERON-γ PRODUCING CELLS IN THE PERIPHERAL BLOOD OF PATIENTS WITH TUBERCULOSIS
Authors: Ruiz-Sánchez Bibiana Patricia , Cruz-Zárate David , Hernández-Solis Alejandro , García-Paredes Víctor Gabriel , Cicero-Sabido Raúl , Serafín-López Jeanet , Estrada-García Iris Citlali Elvira , Wong-Baeza Isabel
Published: January 2023

Tuberculosis (TB) caused 1.4 million deaths in 2015. The infection starts when Mycobacterium tuberculosis is inhaled and interacts with immune cells in the respiratory tract. Alveolar macrophages, neutrophils and dendritic cells (DCs) engulf the mycobacteria; these DCs migrate to the draining lymph node and induce the activation of mycobacteria-specific Th cells. These Th cells migrate back to the lung and produce IFNγ and TNFα, two cytokines essential for mycobacterial control. In addition to Th cells, other cells, such as Tγδ and NK cells, produce IFNγ in response to mycobacteria; innate lymphoid cells (ILCs) are also able to produce IFNγ, but it still unknown if ILCs, which have been detected in human lungs during cancer, produce this cytokine in response to mycobacteria.

We evaluated, by flow cytometry, the frequency and IFNγ production of Th, Tγδ, NK and ILCs (ILC1, ILC2 and ILC3) in the peripheral blood of healthy individuals (negative and positive for the Quantiferon [QTF] test, which detects latent TB), and patients with active pulmonary TB. The numbers of Th, Tγδ, NK cells and ILCs (ILC1, ILC2 and ILC3) were decreased in TB patients. Peripheral blood was stimulated with M. tuberculosis soluble extract for 5 h in the presence of brefeldin A. In healthy, QTF-negative individuals, IFNγ production was increased in Tγδ and ILC2 after stimulation; in healthy, QTF-positive individuals, IFNγ production was increased in Th, Tγδ and ILCs, and in TB patients, IFNγ production was increased in NK cells and ILC2. These results suggest ILCs produce IFNγ during TB infection.

This project was funded by SIP-IPN-México (20170616).
Long Term Impact of Acute Infection on Lymphatic Function
Authors: Zaretsky Arielle Glatman , Han Seong-Ji , da Fonseca Denise Morais , Hand Timothy , Belkaid Yasmine
Published: January 2023

The lymphatic system facilitates critical processes for immunity, including immune cell trafficking, lipid transport, and the maintenance of tissue homeostasis. In the mesentery, these lymphatic vessels (LVs) allow drainage and cell trafficking from the gastrointestinal (GI) tract through the mesenteric adipose tissue (mAT) to the mesenteric lymph nodes (mLNs). During GI infection, this vasculature is altered by inflammation, which can induce lymphatic leakage. Repair of LVs after infection is resolved is necessary to restore lymphatic function. Although preliminary studies have found acute lymphatic leakage is conserved between GI pathogens, in an acute Yersinia pseudotuberculosis infection, mice control bacterial growth, but develop a chronic mesenteric lymphadenopathy and the increased permeability of LVs does not resolve after the infection is cleared. Consequently, the migratory CD103+CD11b+dendritic cells (DP DCs) cannot reach the mLN and instead pass through the dysregulated LVs into the mAT. The absence of DP DCs in the mLN is associated with impaired development of Th17 adaptive immunity to oral vaccination. Little is known about the mechanisms that regulate lymphatic integrity, however these studies reveal a remodeling of the adipose tissue after infection, and are ongoing to understand the long-term increased permeability of LVs and the potential to restore function. Our results support the idea that acute GI infections are associated with permanent changes in DC traffic, which interfere with the activation of immune responses and can have severe implications for GI tract integrity and predisposition to inflammatory diseases and suggest that dysregulation of the mAT may contribute to the prolonged disruption.
Activation-induced chromatin decondensation in the lymphoid lineage
Authors: Rawlings Jason S , Knaub Alynna , McDonald Sierra
Published: January 2023

It has previously been demonstrated that chromatin decondensation is required for αβ T cell proliferation. B cells, γδ T cells, and Natural Killer cells share the same lineage as αβ T cells, originating from hematopoietic stem cells and a common lymphoid progenitor. In this study, we investigated the hypothesis that chromatin decondensation is required for proliferation of these other cells of the lymphoid lineage. We assessed chromatin decondensation by using Western Blot to test for Histone H3 solubility and Flow Cytometry to test Histone H3 accessibility. We show that Lipopolysaccharide, a known activator of B cells, induces the decondensation of chromatin in B cells. To determine if both T and B cells can decondense chromatin at the same rate, both were stimulated with PMA and Ionomycin. B cells and ab T cells decondense chromatin in a similar manner, although there appears to be differences in kinetics between the two. Preliminary data collected on NK cells and γδ T cells suggest that their rate of decondensation is similar to αβ T cells in response to PMA and Ionomycin stimulation. Recently, our lab demonstrated that calcium is required and sufficient to initiate chromatin decondensation in αβ T cells, future studies will investigate if calcium can also regulate the decondensation of B cells, γδ T cells, and NK cells, establishing a conserved mechanism to control proliferation of the lymphoid lineage.
The expression profiles and regulation of PD-L1 in tumor-induced myeloid-derived suppressor cells
Authors: Lu Chunwan , Savage Natasha , Singh Nagendra , Liu Kebin
Published: January 2023

Programmed death-ligand 1 (PD-L1) is an inhibitory ligand that binds to PD-1 to suppress T cell activation. PD-L1 is constitutively expressed and inducible in tumor cells, but the expression profiles and regulatory mechanism of PD-L1 in myeloid-derived suppressor cells (MDSCs) are largely unknown. We report that PD-L1 is abundantly expressed in tumor-infiltrating immune cells in human colon carcinoma, and 25–67% CD11b+CD33+ myeloid-derived suppressor cells (MDSCs) from peripheral blood of human colon cancer patients are PD-L1+. Using a spontaneous colon cancer mouse model, we observed that PD-L1+MDSCs increased significantly in tumor-bearing mice as compared to MDSCs from tumor-free mice. Interestingly, the highest PD-L1+ MDSCs were observed in the tumor microenvironment in which 56–71% tumor-infiltrating MDSCs are PD-L1+ in vivo. In contrast, PD-L1+ MDSCs are significantly less in secondary lymphoid organs and peripheral blood as compared to the tumor tissues, whereas, bone marrow MDSCs are essentially PD-L1− in tumor-bearing mice. IFNg is highly expressed in cells of the tumor tissues and IFNg neutralization significantly decreased PD-L1+ MDSCs in the tumor microenvironment in vivo. However, IFNg-activated pSTAT1 does not bind to the cd274 promoter in CD11b+Gr1+PD-L1+ myeloid cells. Instead, pSTAT1 activates expression of IRF1, IRF5, IRF7 and IRF8 in MDSCs, and only pSTAT1-activated IRF1 binds to an unique IRF-binding sequence element in vitro and chromatin in vivo in the cd274 promoter to activate PD-L1 transcription. Our data determine that PD-L1 is highly expressed in tumor-infiltrating MDSCs and in a less degree in lymphoid organs, and the pSTAT1-IRF1 axis regulates PD-L1 expression in MDSCs.
Evidence that Th1-polarized Tfh cells drive the differentiation of T-bethi atypical memory B cell expansion in human malaria
Authors: Obeng-Adjei Nyamekye , Crompton Peter D
Published: January 2023

Many chronic infections, including malaria and HIV, are associated with a large expansion of CD21−CD27−CXCR3+CD11c+ ‘atypical’ memory B cells (MBCs) that exhibit reduced effector functions. Little is known about the conditions or transcriptional regulators driving atypical MBC differentiation. Here we show that atypical MBCs in malaria-exposed individuals highly express the transcription factor T-bet. Moreover, a longitudinal study of malaria-exposed children demonstrated a positive correlation between the incidence of febrile malaria and the expansion of T-bethi B cells. The Th1-cytokine containing supernatants of malaria-stimulated PBMCs and B cell receptor (BCR) cross- linking induced T-bet expression in peripheral and tonsilar B cells that were blocked by neutralizing IFN-γ. Accordingly, recombinant IFN-γ plus BCR cross-linking drove T-bet expression in peripheral and tonsilar B cells. Consistent with this, Th1-polarized Tfh (Tfh-1) cells more efficiently induced T-bet expression in naïve B cells. These data illuminate the mechanisms underlying atypical MBC differentiation.
Circulating memory CD4 T follicular helper cell subsets correlate with clinical immunity against human malaria
Authors: Furtado Raquel M , Lauvau Gregoire , Daily Johanna P
Published: January 2023

Malaria remains a global burden with over 200 million infections and half a million deaths annually. While studies in human and surrogate mouse models reveal that B cells and CD4 T cells are essential, it is not known if and how sterilizing immunity against malaria may be achieved. Residents of endemic areas develop clinical immunity to Plasmodium falciparum over time yet protection is partial and requires repeated exposures. Herein we conducted a 2 year longitudinal study on patients with mild malaria in a hyperendemic region in Malawi. We categorized patients with a high number and short interval between reinfections (‘susceptible’) and with a low number and longer interval between reinfections (‘protected’). We hypothesized that clinical immunity correlates with the presence of specific immune cell subsets in protected versus susceptible patients. We conducted comprehensive cellular profiling on patient PBMCs, isolated during infection, by mass cytometry and a panel of 33 markers. We examined the frequency and activation of 40 immune cell subsets by unsupervised clustering analysis and discovered an expansion of CXCR3lo memory CD4T follicular helper cells (Tfh) in protected patients compared to susceptible patients. Importantly, with an in vitro antibody dependent opsonization assay we found increased phagocytosis of P. falciparum merozoites with plasma from protected versus susceptible patients, correlating CXCR3lo memory Tfh cell frequency with enhanced humoral responses. We speculate that CXCR3lo memory Tfh cells are critical for lasting protective responses against malaria in humans and may be essential for an efficacious future vaccine.
Contribution of the T cell receptor to the generation and maintenance of EBV epitopespecific CD8 T cell responses following primary infection
Authors: Kamga Larisa , Brody Robin , Gautam Anita , Ghersi Dario , Selin Liisa K , Luzuriaga Katherine
Published: January 2023

We have investigated the potential effect of TCRαβ usage on the maintenance and transcriptome of Epstein-Barr virus (EBV)-specific CD8T cells over the course of primary infection. Blood samples were collected over the course of a year from patients presenting with primary EBV infection. Peptide-MHC tetramers and FACS were used to sort CD8 T cells specific for two HLA-A*02-restricted EBV epitopes: BMLF1 (GLCTLVAML; GLC) and BRLF1 (YVLDHLIVV; YVL); single-cell sequencing and transcriptomics were used to evaluate TCRαβ usage with the expression of 47 immune genes. In 4 donors studied, we identified 16 and 4 public GLC- and YVL-specific CDR3αs; and 17 and 2 public GLC- and YVL-specific CDR3βs. The Morisita-Horn index was used to assess the degree of similarity of the TCRαβ repertoire between primary infection and convalescence. GLC-specific cells tended to have a higher similarity index than YVL-specific cells (mean similarity index: 0.55 vs. 0.27), suggesting that the TCRαβ repertoire GLC-specific cells tended to be more stable than that of YVL-specific cells. The similarity index correlated with the frequency of public CDR3α/β clones (Spearman r: 0.83; p = 0.0198), suggesting there may be a survival advantage to public CDR3αβ usage leading to T-cell persistence. 11 and 10 degenerate GLC- and YVL-specific CDR3αs; and 15 and 12 degenerate GLC- and YVL-specific CDR3βs were identified. In a donor, one of these CDR3αs was paired with up to 9 unique CDR3β. Future analyses will focus on single-cell gene expression to investigate whether there are specific gene patterns associated with public or degenerate TCR and T-cell persistence.
High specificity of human antibody response to nonstructural protein NS1 elicited by Zika virus infection.
Authors: Gao Xiujie , Wen Yingfen , Li Chunlin , Wang Jian , Zhang Fuchun , Yu Lei
Published: January 2023

The newly emerged Zika virus (ZIKV) belonging to the flavivirus family, may appear in the area dengue virus (DENV) infection occurred, raising the issue of diagnosis. The secreted NS1 protein from infected cell is not only an important diagnostic marker but also highly immunogenic. Here we investigated the cross-reaction of NS1 Ab responses between the two close related viruses. First we studied the binding of sera to the recombinant full length NS1 from three ZIKV infected patients (Samples collected at day 4, 7, 15, 106, 188 and 321 for patient 1; day 6, 12, and 66 for patient 2; day 2 for patient 3) by capture ELISA. ZIKV NS1 binding IgG markedly increased in convalescent sera from patient 1 and patient 2 with less cross-reaction to DENV 1–4 NS1. The serum from patient 3 did not bind to ZIKV NS1 and that may be explained by the early time point of serum collected on day 2 from the symptom onset. Specificity of ZIKV NS1 Ab response was evidenced by lack of reactivity to DENV 1–4 NS1proteins of memory B cell derived mAbs (4/5) isolated from the two ZIKV-infected. Only one mAb (1/5) cross-reacted to DENV 2 and DENV 4 NS1. Strikingly NS1 Ab responses of DENV1 infected (six samples) which serotype frequently circulates in our area, Guangdong, China with outbreak in 2014 showed strong cross-reactivity between DENV serotypes with mild or strong reaction to ZIKV NS1. Our data indicated that NS1 Ab response elicited by Zika virus infection was ZIKV-specific and the ZIKV specific mAb can be used for diagnostic tool.
Gut dysbiosis breaks immunological tolerance toward the central nervous system during young adulthood.
Authors: Yadav Sudhir Kumar , Boppana Sridhar , Ito Naoko , Mindur John E , Mathay Martin T , Patel Ankoor , Dhib-Jalbut Suhayl , Ito Kouichi
Published: January 2023

Multiple sclerosis (MS) is an immune mediated disease targeting the central nervous system (CNS) mainly in young adults, and a breakage of immune tolerance to CNS antigens has been suggested to initiate CNS autoimmunity. Recent studies have suggested that gut microbiota affects the development of autoimmune diseases and alterations in the gut microbiota, termed gut dysbiosis, are associated with MS. However, it is still unknown how gut dysbiosis affects the onset and/or progression of CNS autoimmunity. In this study, we investigated the effects of gut dysbiosis on the development of CNS autoimmunity in humanized transgenic mice expressing the MS- associated HLA-DR gene, DR2a, and the T cell receptor (TCR) gene specific for MBP87-99/DR2a that were derived from an MS patient. We show here that the induction of gut dysbiosis triggers the development of spontaneous experimental autoimmune encephalomyelitis (EAE) during adolescence and early adulthood, while increase in immunological tolerance with aging suppresses the disease onset after late young adulthood. Furthermore, gut dysbiosis induces the expression of complement C3 and production of the anaphylatoxin C3a, and down-regulates the expression of the Foxp3 gene in Tregs and anergy-related E3-ubiquitin ligase genes in CD4+CD25− T cells. Consequently, gut dysbiosis was able to trigger the development of encephalitogenic T cells and promote the induction of EAE during the young adulthood age-window. This data suggests that gut dysbiosis increases the risk for CNS autoimmunity in young adulthood through the up-regulation of the complement C3 gene and down-regulation of Foxp3 and E3-ubiquitin ligase genes.
Could Connexin 43 dependent ATP release represent a new therapeutic target for sepsis?
Authors: Dosch Michel Ernest , Salamanca Tamara , Djonova Djulia , Keogh Adrian , Stroka Deborah , Candinas Daniel , Beldi Guido
Published: January 2023

During sepsis, ATP is released into the extracellular space where it modulates immune response via specific receptors and ectonucleotidases present on various immune cells. ATP can be released by hemichannel Connexin 43 (Cx43). Here, we examine the importance of Cx43 mediated ATP release in macrophages and the role of this pathway in modulating innate immune responses in the context of sepsis.

For murine sepsis model, caecal ligation and puncture (CLP) was used. Mice were treated with Gap27, a Cx43 inhibitor. Liver, lung, spleen, bone marrow, peritoneal fluid and blood cells expressing Cx43 were characterized (IHC, FACS). Macrophages were isolated from peritoneum and liver of wild-type mice and stimulated with PAMPs. ATP was quantified using luciferin-luciferase assay. Cytokine levels (ELISA), gene expression (qPCR) and protein expression (immunoblot) were assessed.

Blocking of Cx43 during CLP prolonged survival up to 48 hours in comparison with 24 hours for controls. In addition, it decreased systemic levels of inflammatory cytokines (TNFa, IL1b and IL6) and chemokines (CCL2) and lowered bacterial load (CFU/ml) in the blood and peritoneal fluid. Cx43 was not constitutively expressed in the liver, but was induced following CLP. Cells expressing Cx43 in septic livers were identified as infiltrating M1 macrophages and neutrophils. Ex vivo, Cx43 expression was upregulated in primary macrophages upon stimulation with LPS. Macrophages were actively releasing ATP in response to TLR 4 and 2 agonists, and Cx43 inhibition reduced extracellular ATP levels.

We observed improved survival to sepsis by blocking Cx43. By mediating release of ATP, and potentially other DAMP, Cx43 might overactivate macrophages and exacerbate the immune response.
TCR recognition is a mediator in the control of HIV infection by CD8 T cells
Authors: Nicodemos Rodrigo A Matus , Casazza Joseph , Price David , Koup Richard , Douek Daniel
Published: January 2023

CD8 T cell responses to HIV-specific MHC class I epitopes are critical for the control of HIV infection. The precise mechanisms that govern the control of HIV by CD8 T cells are largely unknown. Certain protective HLA alleles, such as, B*5701, are associated with long-term control of HIV-1. However, other mechanisms by CD8 T cells might be contributing to the control of HIV, because non-controllers with these protective alleles have been reported.

Therefore, to determine the molecular mechanisms that underlie HIV control by CD8 T cells we assembled a cohort of HIV positive individuals, which consisted of controllers (viral load less than 2000 copies per mL), ART-naive non-controllers, and non-controllers on ART-therapy. Using PBMCs from our cohort we first stained the cells with a panel of thirteen T cell markers and then performed polychromatic single cell index sorting of CD3+CD8+ MHC class I tetramer-binding cells. Our tetramers consisted of HIV epitopes bound to HLA-B*5701, A*0201, B*0702 or B*0801. We then determined the TCR diversity for each tetramer-binding population by single cell TCR sequencing. We also reconstructed HIV-specific TCRs and expressed them in a reporter T cell line to measure TCR avidity to the tetramer.

We found that HIV controllers have: (1) a diverse TCR repertoire for the KF11 epitope presented by B*5701, in contrast, to non-B*5701 alleles presenting HIV epitopes that are heavily skewed; (2) high avidity HIV-specific TCRs for HIV epitopes presented by B*5701; and (3) TCR-expanded clones with a novel CD27dimCD45ROneg T cell phenotype. Importantly, lack of any three of these parameters resulted in the inability to control HIV, indicating that no single parameter is sufficient for natural control.
A rapid single-cell RNA-seq approach to identify neoantigen-specific T-cell receptors targeting tumor-specific mutations for use in gene-engineered T-cell immunotherapy
Authors: Lu Yong-Chen William , Zheng Zhili , Robbins Paul F , Tran Eric , Prickett Todd D , Gartner Jared J , Li Yong F , Ray Satyajit , Bliskovsky Valery , Fitzgerald Peter C , Rosenberg Steven A
Published: January 2023

Accumulated evidence suggested that effective cancer immunotherapies were associated with neoantigen-reactive T cells, and adoptive transfer of neoantigen-reactive tumor infiltrating lymphocytes (TILs) could result in tumor regressions. To improve the efficacy of adoptive T-cell therapy targeting tumor-specific mutations, we have proposed a new therapeutic approach, which involves the genetic modification of autologous T cells with neoantigen-specific TCRs and the transfer of these T cells back to cancer patients. However, the current techniques to isolate neoantigen-specific TCRs are labor-intensive, time-consuming and technically challenging, not suitable for clinical applications. To facilitate this process, a new approach was developed, which included the co-culture of TILs with tandem minigene (TMG)-pulsed antigen-presenting cells, single-cell RNA-seq analysis of T cells and the identification of paired TCR sequences associated with cells expressing high levels of IFN-γ and IL-2 mRNA. In this study, TIL fragment cultures were grown from tumors resected from three patients with metastatic gastrointestinal cancers, and then were screened against TMG libraries encoding tumor-specific mutations identified by whole-exome sequencing. Following this new approach, three TCRs were identified, synthesized and cloned into a retroviral vector. These TCRs were then transduced into donor T cells, and these transduced T cells were shown to specifically recognize the following neoantigens encoded by the TMGs: USP8 (R667H), KRAS (G12D) and NBAS (C144S). In conclusion, this approach provides a rapid procedure to isolate neoantigen-specific TCRs for basic and translational research, as well as clinical applications.
GARP-TGFβ Axis on Activated Platelets Supports Tumor Progression
Authors: Metelli Alessandra , Wu Bill , Riesenberg Brian , Fugle Caroline Wallace , Sun Shaoli , Liu Bei , Li Zihai
Published: January 2023

Platelet-induced cancer progression is a well-known process driven by mitogenic factors released by activated platelets, among them the most pro-oncogenic is Latent Transforming Growth Factor Beta (LTGFβ). Glycoprotein-A Repetitions Predominant Protein (GARP), a receptor for LTGFβ that enhances its bioactivation, is expressed by regulatory T cells, platelets, and several human cancers. Upon activation platelets dramatically upregulate GARP, suggesting that GARP might play a role in activating LTGFβ released upon platelet degranulation and thus in platelet-induced cancer progression. To address this point we employed a novel mouse model based on the platelet-specific knock-out of the GARP gene. We show that in activated platelets GARP is critical for the expression of surface LTGFβ and for its conversion to the bioactive form. Lack of GARP on platelets, indeed, resulted in the complete absence of serum active TGFβ. To investigate whether GARP plays a role in platelet-induced cancer progression we tested multiple tumor models and observed that genetic deletion of GARP enhanced adoptive T cell therapy of B16 melanoma. We also found that in MC38 colon tumor model GARP on platelets contributes to cancer progression by increasing TGFβ bioavailability, promoting regulatory T lymphocytes and myeloid cells, and favoring the immune evasion of cancer cells. These results demonstrate that platelet-specific deletion of GARP blunted TGFβ activity in the tumor microenvironment and boosted protective immunity against pre-established cancers. We conclude that platelets constrain anti-tumor immunity via a GARP-TGFβ axis and we propose the combination of immunotherapy and platelet inhibitors as a novel treatment strategy against cancer.
Human cancer cells respond to cytosolic nucleic acids via RIG-I-MAVS-IRF3-mediated interferon-β production
Authors: Chen Jingtao , Qiao Yuan , Zhu Shan , Li Tete , Liu Yong-Jun
Published: January 2023

The innate immune system is the first line of defense against invading microbes via the action of pattern recognition receptors (PRR). Recently, human cancer cells were reported to express PRRs and respond to cytosolic nucleic acids by producing type I interferon. However, whether all nucleic acid-sensing pathways are intact and whether these pathways function in all types of cancer cells remain unclear. In this study, we investigated the effects of nucleic acids in eight types of cancer cells transfected with 5′ppp-dsRNA, poly(A:U), poly(I:C)-HMW, poly(I:C)-LMW, ISD, HSV60, VACV-70, poly(dG:dC), 3′-3′cGAMP, and poly(dA:dT). Interferon-β (IFN-β) secretion was increased following stimulation with 5′ppp-dsRNA, poly(I:C)-HMW, poly(I:C)-LMW, or poly(dA:dT) in most cells. Further, we evaluated the role of TLR3 and RIG-I/MDA-5 signaling in the pancreatic cancer cell line PANC-1. The expression of RIG-I, MDA5, and LGP2 was up-regulated by cytosolic poly(I:C) and poly(dA:dT). Knockdown experiments confirmed that IFN-β production was mediated by the RIG-I-IPS-1-IRF3 signaling pathway in response to cytosolic poly(I:C) and poly(dA:dT). Therefore, these potent adjuvant activities may be useful for drug and vaccine design for cancer immunotherapy.
Macrophage migration inhibitory factor promotes interplay between colorectal cancer, macrophages and T cells to regulate cancer progression.
Authors: Rodriguez-Sosa Miriam , Juarez-Avelar Imelda , Olguin Jonadab Efrain , Illescas Oscar , Terrazas Luis I , Cuellar María Sandra , Pacheco-Fernandez Thalia
Published: January 2023

Colitis-Associated Colorectal Cancer (CAC) is triggered by chronic inflammation. Macrophage migration inhibitory factor (MIF) is a pro-inflammatory cytokine promoting angiogenesis, metastasis, proliferation and survival in malignant cells. When MIF binds to CXCR2 and CXCR4 attracts macrophages and T cells, respectively. As MIF promotes inflammation and recruit immune cells, we investigated the role of MIF in murine model of CAC. Mif−/− and WT BALB/c mice received intraperitoneally azoxymethane and later were exposed to 3 cycles of Dextran Sodium Sulfate (DSS, 2%) in drinking water. After the first DSS cycle WT mice received a synthetic MIF’s inhibitor (CPSI-156). All mice were killed 68 days after CAC induction. Greater burden of tumors were observed in both Mif−/− (25.38±1.772) and CPSI-156-treated mice (18.83±2.43) compared to CAC-WT mice (13.00±1.862). Tumors from Mif−/− mice were histologically-identified as more aggressive. Lower percentages of tumor-associated macrophages were found in both, Mif−/− (0.6577 ± 0.117) and CPSI-156-treated mice (1.2 ± 0.14) compared to CAC-WT mice (2.044±0.39). Similar percentage of T-cells was observed in all groups. RT-PCR of tumors was performed, relative expression (RE) was compared to β-actin gene. CAC-WT mice increased T-regs (foxp3 RE= 0.49), M2 (arginase-1 RE=1.53) and IL-10 (il-10 RE=0.52) and reduces expression of IL-17 (il-17 RE=3.14). Conversely, CAC-Mif−/− mice developed higher IL-17 (il-17 RE=17.15) and lower foxp3 (RE=0.32), arginase-1 (RE=1.12) and il-10 (RE=0.47) expression. Our results suggest that MIF plays a role in regulating the tumor microenvironment attracting macrophages at the tumor site, condition that may favor antitumor responses.