Frontiers in Immunology

Ivermectin (IVM), an antiparasitic drug, was repurposed to treat COVID-19 based on its in vitro antiviral effects. However, it was abandoned after multiple clinical trials reported a lack of efficacy. Immunomodulatory effects have been proposed but remain unclear, yet they may be relevant given IVM use for other infections. We assessed the IVM immunomodulatory effect in 24 participants from a clinical trial evaluating its potential to reduce COVID-19 transmission in mild cases within 48 hours of symptoms onset. The IVM-treated patients showed non-significant lower viral loads, and a significantly shorter duration of hyposmia/anosmia. We measured IgG, IgA, and IgM against five SARS-CoV-2 antigens, and 30 cytokines by Luminex, alongside whole blood RNA sequencing, pan-leukocyte immunophenotyping, and SARS-CoV-2-specific T cell analysis by flow cytometry from day 1 to day 28 post-treatment. All antibody responses increased from day 4, while 13 cytokines significantly decreased over time (adjusted p<0.05). IVM-treated patients had only significantly higher anti-nucleocapsid IgG levels at day 4 (adjusted p=0.041) and 7 (adjusted p=0.045) compared to placebo. SARS-CoV-2-specific CD4+ and CD8+ T cells increased over time, with significantly higher effector memory CD4+ T cells at day 7 compared to day 1 (p=0.027) and the only difference between groups was lower frequencies of spike-specific naive CD4+ T cells at day 7 in IVM-treated participants (0.006% vs 0.036% p=0.02). Transcriptomic data showed downregulation of innate and antiviral blood transcriptional modules (BTMs) over time, with an increase in adaptive immune related BTMs. While no differential gene expression was detected, the IVM-treated had upregulated innate and downregulated T cell and cell cycle BTMs compared to placebo. Overall, our comprehensive longitudinal analysis of early immune responses in mild COVID-19 revealed no robust immunological effects of IVM, consistent with clinical trials results and suggesting a lack of efficacy of IVM in COVID-19 treatment. AUTHOR SUMMARYIvermectin (IVM), an antiparasitic drug, was tested in clinical trials as a potential treatment for COVID-19 due to its in vitro antiviral properties and hypothetical immunomodulatory effects. In this study, we explored the immunomodulatory effects of IVM in a clinical trial involving 24 mild COVID-19 patients who received IVM within 48 hours of symptoms onset. The IVM group showed a trend towards lower viral loads post-treatment, and a significantly shorter duration of hyposmia/anosmia. We comprehensively analyzed immune responses by measuring antibody levels, cytokine profiles, immune cell subsets and whole blood gene expression over 28 days. The IVM group only had increased levels of IgG against the SARS-CoV-2 nucleocapsid compared to the control group. IVM did not affect the kinetics of SARS-CoV-2 T cells, despite a slight decrease in naive CD4+ T cells. Additionally, gene expression analysis showed a decrease in innate and antiviral responses and an increase of adaptive responses over time that were slightly stronger in the placebo compared to the IVM group. In conclusion, despite some differences in IVM treated participants, our detailed analyses do not support significant immunomodulatory effects that could benefit disease progression.

Oxidative distress and inflammation are common biochemical disorders in individuals with cancer. The measurement of oxidative stress in oncology can be useful in clinical practice to monitor the effectiveness of therapy and unwanted effects of the treatment. Thus, the aim of the present study was to evaluate the redox status through the reactive oxygen metabolites (d-ROMs) and biological antioxidant potential (BAP) tests and investigate the correlations of these parameters with blood variables in cancer patients. This is an observational, retrospective study of analysis of medical records of patients evaluated the period from 2018 to 2020 in an integrative medicine center. The inclusion criteria were individuals of both sexes, over 18 years of age, diagnosed with cancer who performed the d-ROMs and BAP test in the same period of blood analysis. Following the inclusion criteria, the final sample of the study were 57 individuals, 60% were woman and 40% were men. The evaluation of redox state showed that the d-ROMs were high (420.2 {+/-} 112.1 U CARR) in total sample and higher in women compared to male (p < 0.01) and BAP tests were normal (2332 {+/-} 812 mol/l). The oxidative parameters, d-ROMs and OSI, was correlated positively with BAP, red cell distribution width (only d-ROMs), platelets (Plt), C-reactive protein (CRP), erythrocyte sedimentation (ESR) and negatively with hemoglobin (Hb) and mean corpuscular hemoglobin (MCH). Regarding the antioxidant potential index, BAP/dROMs, were correlated positively with Hb and serum albumin (HAS) and negatively correlated with Plt, CRP and ESR. The study shows that redox status of an individual with cancer is altered, and it is possible to monitor this system in clinical practice through d-ROMs and BAP test. These parameters, in addition to being suitable for assessing oxidative stress, were correlated with parameters predictors of inflammation.

The COVID-19 vaccinations have played a significant role in controlling the pandemic. To elucidate their impact on the immune system, a COVID-19 vaccination atlas was developed through an integrative systems vaccinology approach. The atlas includes both healthy individuals and those infected with or without prior vaccination, and covers the administration of five vaccines in different regimens: Covilo(R), Zifivax(R), Vaxzebria(R) or Covishield(R), Spikevax(R), and Comirnaty(R). Critical markers were identified to discriminate the different types of vaccines and infection, in which infection was associated with GATA3, ZNF3, KMT2A, ASXL1, SP100, and GZMM, and vaccine types were marked by ITGAM, ACTG1, LGALS3, and STAT5B. Additionally, the immunological signatures of heterologous vaccination and infection were described, and it was also shown how a full vaccination regimen markedly limited the shift of immune responses during natural infection, thereby constraining disease progression. Finally, the common transcripts shared across COVID-19 vaccines and vaccines against other pathogens were described.

BackgroundThe worldwide pandemic caused by SARS-CoV-2 has claimed millions of lives and has had a profound effect on global life. Understanding the pathogenicity of the virus and the bodys response to infection is crucial in improving patient management, prognosis, and therapeutic strategies. To address this, we performed functional transcriptomic profiling to better understand the generic and specific effects of SARS-CoV-2 infection. MethodsWhole blood RNA sequencing was used to profile a well characterised cohort of patients hospitalised with COVID-19, during the first wave of the pandemic prior to the availability of approved COVID-19 treatments and who went on to survive or die of COVID-19, and patients hospitalised with influenza virus infection between 2017 and 2019. Clinical parameters between patient groups were compared, and several bioinformatic tools were used to assess differences in transcript abundances and cellular composition. ResultsThe analyses revealed contrasting innate and adaptive immune programmes, with transcripts and cell subsets associated with the innate immune response elevated in patients with influenza, and those involved in the adaptive immune response elevated in patients with COVID-19. Topological analysis identified additional gene signatures that differentiated patients with COVID-19 from patients with influenza, including insulin resistance, mitochondrial oxidative stress and interferon signalling. An efficient adaptive immune response was furthermore associated with patient survival, while an inflammatory response predicted death in patients with COVID-19. A potential prognostic signature was found based on a selection of transcript abundances, associated with circulating immunoglobulins, nucleosome assembly, cytokine production and T cell activation, in the blood transcriptome of COVID-19 patients, upon admission to hospital, which can be used to stratify patients likely to survive or die. ConclusionsThe results identified distinct immunological signatures between SARS-CoV-2 and influenza, prognostic of disease progression and indicative of different targeted therapies. The altered transcript abundances associated with COVID-19 survivors can be used to predict more severe outcomes in patients with COVID-19.

The regulation of follicular (F) and germinal center (GC) immune reactivity in human lymph nodes (LNs), particularly during the early stages of viral infections, remains poorly understood. We have analyzed lung-draining lymph nodes (LD-LNs) from COVID-19 autopsies using multiplex imaging and spatial transcriptomics to examine the immune landscape and with respect to the aging. We identified three subgroups of Reactive Follicles (RFs) based on Bcl6 prevalence, RF-Bcl6no/low, RF-Bcl6int and RF-Bcl6high. RF-Bcl6high tissues express a distinct B/TFH immune landscape associated with increased prevalence of proliferating B- and TFH-cell subsets. Comparison between LD-LNs and matched subdiaphragmatic LNs revealed a disconnected Bcl6 reactivity between the two anatomical sites. LD-LNs Bcl6 reactivity was associated with a distinct spatial transcriptomic profile. TH1-associated genes/pathways (e.g. CXCR3, STAT5, TNF signaling) were significantly upregulated in RF-Bcl6no/low tissues while the RF-Bcl6high tissues exhibited significant upregulation of GC-promoting genes/pathways (e.g. CXCL13, B cell receptor signaling). Despite the similar prevalence, the in-situ transcriptome profiling indicates a higher monocyte/macrophage functionality in "Aged" compared to "Young" follicles from donors with comparable Bcl6 reactivity. Our findings reveal a heterogeneous F/GC landscape in COVID-19 LD-LNs and highlight specific molecular targets and pathways that could regulate human F/GC immune dynamics during early viral infections.

BackgroundIn recent studies, up to half of immunocompromised (IC) subject populations fail to develop antibodies after COVID-19 vaccination. Purpose and MethodsHere, we explore whether T-cells which respond to the spike (S) antigenic sequence and its less conserved S1, and the conserved S2 component are present in serial samples before and after each dose of mRNA1273 or BNT162b2 vaccines in 20 healthy immunocompetent subjects. Single samples from 7 vaccinated IC subjects were also tested. Simultaneously, we measured IgG antibodies to the receptor binding domain (RBD) of S1, and anti-S IgG, and frequencies of monocytic CD14+HLA-DR-(M-MDSC) and polymorphonuclear CD14-CD15+CD11b+ (PMN-MDSC) myeloid-derived suppressor cells. ResultsIn healthy subjects, S1-, S2-, and S-reactive CD4 and CD8 T-cell frequencies showed a numeric but not statistically significant decrease after the first vaccine dose and were accompanied by increased MDSC frequencies (p<0.05). After the second dose, S2-and S-reactive CD4 and CD8 cells and MDSC approached pre-vaccination levels. In healthy subjects, a) S1-reactive CD8 frequencies were significantly higher after the second dose compared with pre-vaccination levels (p=0.015), b) anti-RBD and anti-S IgG were present in all after the second dose. Among seven IC subjects, anti-RBD and anti-S IgG were absent in 4 and 3 subjects, respectively. S1-reactive CD8 cells were identified in 2 of 4 anti-RBD negative subjects. S-reactive CD4 or CD8 cells were identified in all three anti-S negative subjects. ConclusionsIn healthy immunocompetent subjects, mRNA vaccines induce antibodies to the spike antigenic sequences and augment CD8 cells reactive to the S1 spike sequence, which is more specific for the SARS-CoV-2 virus. In this exploratory cohort of vaccinated immunocompromised subjects, S1-reactive CD8 cells can be detected in some who are negative for RBD antibody, and S-reactive T-cells are present in all who are negative for spike antibody.

IntroductionWe have reanalyzed the genomic data from the International Collaboration for the Genomics of HIV (ICGH), focusing on HIV-1 Elite Controllers (EC). MethodsA genome-wide association study (GWAS) was performed, comparing 543 HIV-1 EC individuals with 3,272 uninfected controls (CTR) of European ancestry. 8 million single nucleotide polymorphisms (SNPs) and HLA class I and class II gene alleles were imputed to compare EC and CTR. Results2,626 SNPs were associated with EC (p<5.10-8), all located within the Major Histocompatibility Complex (MHC) region. Stepwise regression analysis narrowed this list to 17 SNPs. In parallel, 22 HLA class I and II alleles were associated with EC. Through meticulous mapping of the LD between all identified signals and employing reciprocal covariate analyses, we delineated a final set of 6 independent SNPs and 3 HLA class I gene alleles that accounted for most of the associations observed with EC. Our study revealed the presence of cumulative haploblock effects (SNP rs9264942 contributing to the HLA-B*57:01 effect) and that several HLA allele associations were in fact caused by SNPs in linkage disequilibrium (LD). Upon investigating SNPs in LD with the selected 6 SNPs and 3 HLA class I alleles for their impact on protein function (either damaging or differential expression), we identified several compelling mechanisms potentially explaining EC among which: a multi-action mechanism of HLA-B*57:01 involving MICA mutations and MICB differential expression overcoming the HIV-1 blockade of NK cell response, and overexpression of ZBTB12 with a possible anti-HIV-1 effect through HERV-K interference; a deleterious mutation in PPP1R18 favoring viral budding associated with rs1233396. ConclusionOur results show that MHC influence on EC likely extends beyond traditional HLA class I or class II allele associations, encompassing other MHC SNPs with various biological impacts. They point to the key role of NK cells in preventing HIV-1 infection. Our analysis shows that HLA-B*57:01 is indeed associated with a partially functional NK cell response which could also explain this markers involvement in other diseases such as psoriasis. More broadly, our findings suggest that within any HLA class I and II association in diseases, there may exist distinct causal SNPs within this crucial, gene-rich, and LD-rich MHC region.

Salmonella Typhimurium is a major zoonotic pathogen, with pigs acting as important subclinical carriers. To explore the specific intestinal immune response at the cellular level, we used Single-cell RNA sequencing (scRNA-seq), enabling detailed analysis of immune cell types and gene expression profiles during infection. In addition to enterocytes, our results revealed the presence of diverse immune populations, including monocytes/macrophages, dendritic cells, innate lymphoid cells (ILCs), thirteen T cell subtypes and five B cell populations were identified, revealing pronounced infection-driven alterations in cellular composition and transcriptional states. Among T cells, naive and follicular CD4+/CD8+ {beta} T cells and NK T cells were expanded, whereas effector CD8+ T cells and CD2- and SELLhi {gamma}{delta} T cells were decreased. B-cell populations shifted toward activated and cycling states, with decreased antibody-secreting, resting, and transitioning cells. Dendritic cells and monocyte/macrophage populations were expanded, and group 3 ILCs and enterocytes were markedly reduced. Pathway analyses revealed robust cell type-specific immunometabolic remodeling, including enhanced protein-folding and stress-adaptive pathways in T and B cells, heightened inflammatory, interferon, and cytokine signaling in myeloid populations, and coordinated metabolic and immune adjustments in epithelial cells, highlighting the complexity of host responses to Salmonella infection. This study provides the first scRNA-seq landscape of the porcine ileum during S. Typhimurium infection, offering insight into host immune cell dynamics and immunometabolic responses.

Chronic inflammation plays an obscure role in cancer initiation, with broad references implicating immune exhaustion (IEX) or free radical mediated cell damage. Chronic inflammation is, however, paradoxically synonymous with the term "immune tolerance" which in other cases presents itself as a therapeutic limitation to the efficacy of tumor immune therapies particularly those involving microbial -associated molecular patterns (MAMPs) in regimens. As "tolerance" remains to this day a "phenomenon" there is a pressing need to fully understand every biological aspect of this apparent negative feedback response, because doing so will serve to guide development of targeted immune therapies. In this work, we employ a rudimentary model, which can be adopted in which it is possible to provoke negative feedback through sustained antigen stimulation in immunocompetent cells, as we monitor, define and characterize phenotype evolution using next generation whole transcriptome sequencing + validation studies. This model can be used to study /create in vitro the "M2" phenotype, which is itself involved in aggressive tumors with synergistic rapid expansion of myeloid-derived suppressor cells (MDSCs), dysfunctional CD8+ T cytotoxic (CTL)/ and dysfunctional natural killer (NK) cells. Briefly, the data in this work, shows negative feedback dominates at 7 to 11 days, after acute being associated with phase specific (time dependent) elevated checkpoints; e.g. PDL1+/MSN, HAVCR2/TIM-3+, SPP1+, C3ar1+, CD73+, IL1RN+, LILRbs+, glycoproteins, integrins etc. Here we report on phase specific patterns and bidirectional changes aligning with immune escape, much centered around loss of host defense against viral infection and malignancy. Negative feedback is associated with rampant induction of degradative proteases, SOCS/JAK/STAT/IL-10, the CCL2/7 axis tantamount to sustained loss of MHC1/2 antigen recognition systems, Type I interferon response, NOD signaling, antiviral/antibacterial defense, p62/SQSTM also aligning with a disturbed metabolic signature. The data in this work demonstrates that the colloquial terms "tolerance and IEX" are somewhat flawed terminology, because this negative feedback is a potent, intentional and formidable immune offense to eradicate the active arm of immune defense. Abstract Truncated /Removed due to word countThe data show that "tolerance" aligns with six distinct chronological differential gene (DEG) expression patterns that circumscribe extensive immune suppression which dominate during chronic inflammation. These involve the following time dependent patterns: 1) transcripts overexpressed, e.g. checkpoint receptors; PDL1+/MSN, HAVCR2/TIM-3+, SPP1+, C3ar1+, CD73+, IL1RN+, LILRbs+, glycoproteins, etc.; 2) transcripts overexpressed only in chronic; e.g cytokine suppressor signaling (SOC3/Jak/Stat), cyto/chemokines (IL-10, CCL2, CCL7), proteases (cathepsins L, D, K, Adam 8, PIM2), and adhesives (TSPAN3, QSOX1, PDPN, ITGA5), (PLK2, ADGRE1, CALM1, PCNA, etc.); 3) transcripts downregulated in acute and chronic; e.g. a severe collective loss in MHC1/II antigen presentation capacity (CD74, H2-Q4, H2-Q6, etc.), NOD signaling, and interferon (IFN) type I signaling systems; 4) transcripts downregulated in chronic only, including OXPHOS/metabolic genes (Aldo A, C, Eno2, Gpi1, etc.), antiviral/antibacterial defense genes (Lyz1, Lyz2, Card19, Ninj1), and autophagy-related genes (p62/SQSTM1). In the category of Tolerance were: 5) transcripts induced sharply in acute, no longer responsive in chronic; IFN Type 1 antiviral response genes (OAS, BST2, ISG15, ISG20, IRF7, RSAD2/Viperin), TLR2, antibacterial defense genes (SAA3, SP140), and proinflammatory cytokines (CCL5, TNF, IL1a, and IL1b) along with the IL-1/TLR signaling axis. Last, 6) reverse tolerance, corresponded to restored baseline levels to maintain cell mitotic and thymosin homeostasis. In conclusion, these data suggest chronic inflammation precipitates negative feedback, aligning with the same checkpoint targets being sought after today in tumor immune therapies.

Unbiased classification of equine dendritic cells (DC) is necessary to address various research questions such as the role of DC subsets in immune-mediated diseases of horses. We applied single-cell RNA sequencing (scRNA-seq) on DC enriched from the blood of two horses. All main DC subsets were detected by key gene expression, including conventional DC type 1 (cDC1; XCR1) and type 2 (cDC2; FCER1A, CD1E) as well as plasmacytoid DC (pDC; TCF4). In addition, we detected a small cluster of hematopoietic progenitors, as well as transitional DC (tDC; FCER1A, TCF4) and putative DC type 3 (DC3; FLT3, CD163). Our data confirms the previously reported phenotype of equine pDC (Flt3+MHC-IIlowCADM1lowCD172aint), cDC1 (Flt3+MHC-IIhighCADM1highCD172alow-int) and cDC2 (Flt3+MHC-IIhighCADM1intCD172ahigh), while also highlighting considerable CD14 expression for cDC2. Two subclusters of equine cDC2 were found to be enriched in FCER1A or CX3CR1 transcripts (cDC2.1 and cDC2.2, respectively), with suggested enhanced extravasation and T-cell stimulatory capacities of the latter. Conservation of DC subsets across species (horse, pig, human, mouse) was illustrated by enrichment analyses with subset-specific gene signatures and by cross-species data integration with publicly available scRNA-seq datasets. Our atlas of equine blood DC is a valuable resource for comparative analyses, and it forms the foundation for understanding the involvement of distinct DC subsets in infections and immune-mediated pathologies.

The AB-type toxin AIP56 is a key virulence factor of Photobacterium damselae subsp. piscicida (Phdp), inducing apoptosis in fish immune cells. The discovery of AIP56-like and AIP56-related toxins in diverse organisms, including human-associated Vibrio strains, highlights the evolutionary conservation of this toxin family, suggesting that AIP56 and its homologs may share conserved receptors across species. These toxins have potential for biotechnological applications, such as therapeutic protein delivery and immune modulation. Herein, the cell specificity of AIP56 for immune cells in sea bass, mice, and humans was characterized. Whether AIP56 interacts directly or indirectly with sea bass neutrophils was never investigated and it was shown that only a small population of sea bass neutrophils internalized AIP56, indicating that most of the neutrophilic destruction during Phdp infection and/or AIP56 intoxication does not result from the direct toxicity of the toxin. Moreover, the cellular tropism of AIP56 for myeloid cells was observed in the three species, including its preference for macrophages. Further, mouse and human M0 and M2-like macrophages internalized more toxin than M1-like macrophages. Despite the limited interaction of lymphoid cells with AIP56, mouse B1-cells were able to internalize the toxin, possibly due to its myeloid features. These findings are relevant for both pathogenicity and biomedical contexts.

Follicular lymphoma (FL) is the most common indolent form of non-Hodgkin lymphoma arising from malignant germinal center (GC) B-cells. The genetic hallmark that leads to the development of FL is the t(14:18) which occurs early in the bone marrow during B cell development, thereby placing the anti-apoptotic BCL2 gene under the direct control of the transcriptional enhancers in 3 of immunoglobulin heavy chain locus (IgH 3RR) and leading to the constitutive expression of the BCL2 protein. To assess the impact of the BCL2 deregulation on B-cell fate and try to reproduce FL development in mice, two models were designed: the Ig{kappa}-BCL2 (Knock in of the BCL2 in the light chain Ig kappa locus) and the 3RR-BCL2 (Transgene containing BCL2 and a micro-3RR), both containing the full BCL2 promoter region.

Sarcoidosis provides unique management challenges due to variable presentation and disease course. In this single-center analysis, we assess transcriptomic and epigenomic differentiators between severe and mild cases of sarcoidosis using peripheral blood. We showcase differences across multiple cell types that highlight decreased immunoregulation and increased inflammation in severe sarcoidosis. We provide an overall framework for the interactions between immune cells in severe sarcoidosis as a basis for future therapeutic research. Visual Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=133 SRC="FIGDIR/small/24313886v1_ufig1.gif" ALT="Figure 1"> View larger version (31K): org.highwire.dtl.DTLVardef@2e2517org.highwire.dtl.DTLVardef@1851a89org.highwire.dtl.DTLVardef@1911a2dorg.highwire.dtl.DTLVardef@14d8c71_HPS_FORMAT_FIGEXP M_FIG C_FIG

BackgroundThe role of cellular immunity in pathogenesis of COVID-19 is unclear and conflicting data points to insufficient or pathogenic immunity as drivers of COVID-19 progression. Here we aimed to delineate the phenotype and function of the immune system in patients with moderate, severe, and critical COVID-19. MethodsIn this prospective study, we included 53 patients with moderate (n=21), severe (n=18), and critical (n=14) COVID-19 manifestations. Using multiparametric flow cytometry we compared quantitative, phenotypic, and functional characteristics of circulating immune cells, SARS-CoV-2 antigen-reactive T-cells, and humoral immunity. ResultsDeep phenotypic profiling revealed a depletion of circulating bulk CD8+ T-cells, CD4+ and CD8+ T-cell subsets with activated memory/effector T-cells expressing CD57+, HLA-DR+, and the key activation and migration molecule CD11a++ in critical COVID-19. Importantly, survival from acute respiratory distress syndrome was accompanied by a recovery of the depleted CD11++ T-cell subsets including T-cells expressing CD28, CD57, HLA-DR activation/effector molecules. We further observed a stronger response of S-protein specific T-cells producing inflammatory cytokines in critical COVID-19 cases. This seemingly contradictory observation is in fact confirmation of the underlying immunopathogenesis in patients with critical COVID-19. ConclusionOur findings suggest a CD11a-based immune signature as a possible prognostic marker for disease development. Our data further reveal that increased rather than decreased SARS-CoV-2 specific T cell immunity is associated with adverse outcome in COVID-19. Tissue migration of activated effectors T-cells may constitute a crucial cornerstone in the immunopathogenesis of SARS-CoV-2 associated tissue injury. Trial registrationThis is a prospective observational study without a trial registration number. FundingThis work was supported by grants from Mercator Foundation, the BMBF e:KID (01ZX1612A), and BMBF NoChro (FKZ 13GW0338B). 25 Word summaryStronger S-protein reactivity and decreased frequency of activated memory/effector T-cells expressing CD11a++ suggests immunopathogenesis in critical COVID-19 mediated by tissue migration of activated effector T-cells.

For many diseases, including cancer, viral infections such as COVID-19, bacterial infections, and auto-immune diseases, the immune response is a major determinant of progression, response to therapy, and clinical outcome. Innate and adaptive immune response are controlled by coordinated activity of multiple immune cell types. The functional activity state of immune cells is determined by cellular signal transduction pathways (STPs). A novel mRNA-based signaling pathway assay platform has been developed to quantitatively measure relevant STP activities in all types of immune cells and mixed immune cell samples for experimental and diagnostic purposes. We generated a STP activity profile, termed Immune-Pathway Activity Profile (I-PAP), for a variety of immune cell types in resting and activated state, and provide a first example for use in patient samples. MethodsThe technology to measure STP activity has been described for androgen and estrogen receptor, PI3K, MAPK, TGF{beta}, Notch, NF{kappa}B, JAK-STAT1/2, and JAK-STAT3 pathways. STP activity was measured on Affymetrix expression microarray data from preclinical studies containing public data from different types of immune cells, resting/naive or immune-activated in vitro, to establish I-PAPs. Subsequently data from a clinical study on rheumatoid arthritis were analyzed. ResultsI-PAPs of naive/resting and immune-activated CD4+ and CD8+ T cells, T helper cells, B cells, NK cells, monocytes, macrophages, and dendritic cells were established and in agreement with known experimental immunobiology. In whole blood samples of rheumatoid arthritis patients TGF{beta} pathway activity was increased; JAK-STAT3 pathway activity was selectively increased in female patients. In naive CD4+ Tregs TGF{beta} pathway activity was increased, while in memory T effector cells JAK-STAT3 pathway activity tended to increase, suggesting that these immune cell types contributed to whole blood analysis results. ConclusionSTP assay technology (currently being converted to qPCR-based assays) makes it possible to directly measure functional activity of cells of the innate and adaptive immune response enabling quantitative assessment of the immune response of an individual patient. Envisioned utility lies in (1) prediction and monitoring of response to immunomodulatory treatments for a variety of immune-mediated diseases, including RA; (2) uncovering novel treatment targets; (3) improvement and standardization of in vitro immunology research and drug development.

The host immune response determines the clinical course of a viral infection, for example in case of COVID-19 infection. The effectiveness of vaccination also depends on the induced immune response. Currently there is no method to measure the cellular immune response in blood samples. The functional activity of cells of innate and adaptive immune system is determined by coordinated activity of signaling pathways, especially the JAK-STAT pathways. Using a previously described approach we developed mRNA-based tests to measure activity of these signaling pathways, and show that they can be used to measure in a quantitative manner the cellular innate and adaptive immune response to a viral infection or vaccine in whole blood, PBMC, and specific immune cell type samples. Pathway activity level and range in healthy individuals was established, enabling interpretation of a pathway activity score on a patient sample without the need for a reference sample. Evidence is presented that the pathway activity analysis may also be useful for in vitro vaccine development and assessment of vaccine immunogenicity. Other envisioned applications lie in development of immunomodulatory drugs and drug response prediction and monitoring. Tests are expected to be of value in the COVID-19 crisis. In addition to the described Affymetrix microarray-based pathway tests for measuring host immune response, qPCR-based versions are in development; the latter can in principle be performed within three hours in routine hospital labs.

Innate immune receptors that form complexes with secondary receptors, activating multiple signalling pathways, modulate cellular activation and play essential roles in regulating homeostasis and immunity. We have previously identified a variety of bovine C-type lectin-like receptors that possess similar functionality than their human orthologues. Mincle (CLEC4E), a heavily glycosylated monomer, is involved in the recognition of the mycobacterial component Cord factor (trehalose 6,6'-dimycolate). Here we characterise the bovine homologue of Mincle (boMincle), and demonstrate that the receptor is structurally and functionally similar to the human orthologue (huMincle), although there are some notable differences. In the absence of cross-reacting antibodies, boMincle-specific antibodies were created and used to demonstrate that, like the human receptor, boMincle is predominantly expressed by myeloid cells. BoMincle surface expression increases during the maturation of monocytes to macrophages. However, boMincle mRNA transcripts were also detected in granulocytes, B cells, and T cells. Finally, we show that boMincle binds to isolated bovine CD4+ T cells in a specific manner, indicating the potential to recognize endogenous ligands. This suggests that the receptor might also play a role in homeostasis in cattle.

Long-COVID affects at least 10% of COVID-19 survivors, displaying debilitating symptoms across multiple organ systems. Despite the increasing prevalence, the underlying causes remain unclear. This study presents a unique analysis of the PBMC transcriptomic landscape of COVID-19 and Long-COVID patients at a single-cell resolution. We reconstructed the cell state and communication using differentially expressed gene profiling and ligand-receptor interaction analyses. Our results reveal altered T and NK cell subset proportions, diminished proliferating lymphocyte and B cell signalling capacity, and the expression of exhaustion and cytotoxicity associated genes 1.5 - 2 years post-infection, suggesting incomplete immune recovery. Collectively, these findings provide insights into the immune processes underlying the progression of COVID-19 into a chronic Long-COVID state.

IL-12p70 is crucial for T helper 1 polarization and the generation of type 1 immunity that is required to fight cancer and intracellular pathogens. Therefore, strategies to optimize the production of IL-12p70 by dendritic cells (DCs) may significantly improve the efficacy of vaccines and immunotherapies for cancer. However, the rules governing the production of IL-12p70 remain obscure. Here, we stimulated pattern recognition receptors (PRRs) representing all five families of PRRs, to evaluate their ability to elicit high production of IL-12p70 by human DCs. We used ten well-characterized agonists and stimulated human monocyte-derived DCs in vitro with either single agonists or 26 different combinations. We found that poly(I:C), which engages the RNA-sensing PRRs TLR3 and/or MDA5, was the only agonist that could elicit IL-12p70 production when used alone. Combinations of agonists of cell surface and intracellular PRRs were found to synergize to induce high IL-12p70 production, given that the combination included poly(I:C) or resiquimod, which both are agonists of intracellular, RNA sensing PRRs (TLR3/MDA5 and TLR7/8, respectively). Our data show that production of high IL-12p70 is strictly controlled, which is different from what we observed for IFN{beta}, whose production could be elicited by several intracellular PRRs. In conclusion, we identified six different combinations of PRR ligands able to induce high IL-12p70 production by human DCs. The identified synergistic PRR ligand combinations may represent strong adjuvant candidates in particular for therapeutic cancer vaccines.

Antibodies produced by B cells aid in recognition and clearance of pathogens and is the cornerstone of vaccination strategies. Humans produce nine different antibody isotypes and their effector functions differ according to the type of antigen and route of exposure. Phenotypic variation between isotype-switched B cell subsets is expected but not studied in detail. To obtain a molecular definition of isotype-defined cell identity, we performed proteomics and transcriptomics on isotype-defined populations of human naive and memory B cells (MBCs): CD27-IgM+IgD+, CD27+CD38lo/-IgM+IgD+, CD27+CD38lo/-IgM+IgD-, and IgA1, IgA2, IgG1, IgG2, IgG3, and IgG4 MBCs (CD27+CD38lo/-Ig+). Combined proteome and transcriptome analysis revealed that mRNA and protein expression profiles separate isotype-defined B cell subsets according to their differentiation status. mRNA and protein expression levels correlated reasonably well for many genes. IgG4-switched B cells were most distinct from naive B cells in terms of mRNA as well as protein expression profiles. Besides a distinct expression profile of cytokine and Fc receptors, we identified a high expression of IgE-coding mRNA in IgG4-switched B cells. SDR16C5 was identified as uniquely upregulated in IgG4-switched B cells. Taken together, this study highlights the distinct phenotypic profile of IgG4-switched B cells.