Intracellular Cytokine Flow Cytometry Research Articles

Relative deficiency in interferon-γ-secreting CD4+ T cells is strongly associated with poorer COVID-19 vaccination responses in older adults.

Although the two-dose mRNA vaccination regime provides protection against SARS-CoV-2, older adults have been shown to exhibit poorer vaccination responses. In addition, the role of vaccine-induced T-cell responses is not well characterised. We aim to assess the impact of age on immune responses after two doses of the BNT162b2 mRNA vaccine, focussing on antigen-specific T-cells. A prospective 3-month study was conducted on 15 young (median age 31 years, interquartile range (IQR) 25-35 years) and 14 older adults (median age 72 years, IQR 70-73 years). We assessed functional, neutralising antibody responses against SARS-CoV-2 variants using ACE-2 inhibition assays, and changes in B and T-cell subsets by high-dimensional flow cytometry. Antigen-specific T-cell responses were also quantified by intracellular cytokine staining and flow cytometry. Older adults had attenuated T-helper (Th) response to vaccination, which was associated with weaker antibody responses and decreased SARS-CoV-2 neutralisation. Antigen-specific interferon-γ (IFNγ)-secreting CD4+ T-cells to wild-type and Omicron antigens increased in young adults, which was strongly positively correlated with their neutralising antibody responses. Conversely, this relationship was negative in older adults. Hence, older adults' relative IFNγ-secreting CD4+ T cell deficiency might explain their poorer COVID-19 vaccination responses. Further exploration into the aetiology is needed and would be integral in developing novel vaccination strategies and improving infection outcomes in older adults.

Distinct CRTH2+CD161+ (peTh2) memory CD4+ T-cell cytokine profiles in food allergy and eosinophilic gastrointestinal disorders.

Although IgE-mediated food allergy (FA) and eosinophilic gastrointestinal disorders (EGID) are clinically distinct and treated differently, pathogenic effector Th2 (peTh2) cells are implicated in the pathogenesis of both FA and EGID. The aim of this study was to better characterize peTh2 cells in the context of FA and EGID and the overlap between these two conditions. Peripheral blood peTh2 cells (CD3+CD4+CD27-CD49d+CRTH2+CD161+) were profiled by intracellular cytokine flow cytometry in the following patient cohorts: patients with FA alone (n = 8), FA and food-triggered EGID (EGID+FA+FT, n = 7), food-triggered EGID alone (EGID+FT, n = 7), EGID without FA or specific food triggers (ONLY_EGID, n = 9), and healthy volunteers (HV, n = 7). Overnight peripheral blood mononuclear cell (PBMC) culture supernatants were assessed for cytokine production by multiplex analysis. CRTH2+CD161+ (peTh2) memory CD4+ T cells were significantly increased in both patients with FA and those with ALL_EGID (inclusive of EGID+FA+FT, EGID+FT and ONLY_EGID) when compared to HV. However, ALL_EGID patients, particularly those with EGID+FA+FT, had significantly elevated IL-5+IL-13+ peTh2 cells, whereas FA patients had significantly elevated IFN-γ or IL-17A-expressing peTh2 cells. This finding was supported by increased spontaneous IL-5 and IL-13 production in overnight cultures of PBMC from EGID+FA+FT patients compared to spontaneous IL-10 and IFN-γ production by PBMC from FA patients. FA patients had increased IL-9, IL-10, IL-17A, and IFN-γ production in overnight cultures of stimulated PBMC. EGID and IgE-mediated FA share a common cell subtype defined by specific surface markers and termed CRTH2+CD161+ (peTh2) memory CD4+ T cells. However, the cytokine profiles of these CRTH2+CD161+ (peTh2) memory CD4+ T cells are markedly different between the two disorders.

Predominantly defective CD8+ T cell immunity to SARS-CoV-2 mRNA vaccination in lung transplant recipients

BackgroundAlthough mRNA vaccines have overall efficacy preventing morbidity/mortality from SARS-CoV-2 infection, immunocompromised persons remain at risk. Antibodies mostly prevent early symptomatic infection, but cellular immunity, particularly the virus-specific CD8+ T cell response, is protective against disease. Defects in T cell responses to vaccination have not been well characterized in immunocompromised hosts; persons with lung transplantation are particularly vulnerable to vaccine failure with severe illness.MethodsComparison groups included persons with lung transplantation and no history of COVID-19 (21 and 19 persons after initial mRNA vaccination and a third booster vaccination respectively), 8 lung transplantation participants recovered from COVID-19, and 22 non-immunocompromised healthy control individuals after initial mRNA vaccination (without history of COVID-19). Anti-spike T cell responses were assayed by stimulating peripheral blood mononuclear cells (PBMCs) with pooled small overlapping peptides spanning the SARS-CoV-2 spike protein, followed by intracellular cytokine staining (ICS) and flow cytometry for release of cytokines in response to stimulation, including negative controls (no peptide stimulation) and positive controls (phorbol myristate acetate [PMA] and ionomycin stimulation). To evaluate for low frequency memory responses, PBMCs were cultured in the presence of the mRNA-1273 vaccine for 14 days before this evaluation.ResultsIonophore stimulation of PBMCs revealed a less inflammatory milieu in terms of interleukin (IL)-2, IL-4, and IL-10 profiling in lung transplantation individuals, reflecting the effect of immunosuppressive treatments. Similar to what we previously reported in healthy vaccinees, spike-specific responses in lung transplantation recipients were undetectable (< 0.01%) when tested 2 weeks after vaccination or later, but were detectable after in vitro culture of PBMCs with mRNA-1273 vaccine to enrich memory T cell responses. This was also seen in COVID-19-recovered lung transplantation recipients. Comparison of their enriched memory responses to controls revealed relatively similar CD4+ T cell memory, but markedly reduced CD8+ T cell memory both after primary vaccination or a booster dose. These responses were not correlated to age or time after transplantation. The vaccine-induced CD4+ and CD8+ responses correlated well in the healthy control group, but poorly in the transplantation groups.ConclusionsThese results reveal a specific defect in CD8+ T cells, which have key roles both in transplanted organ rejection but also antiviral effector responses. Overcoming this defect will require strategies to enhance vaccine immunogenicity in immunocompromised persons.

DOP04 Single cell RNA sequencing reveals a potential role for IFNγ in priming circulating monocytes for intestinal inflammatory function in Crohn’s Disease.

Abstract Background Hyper-inflammatory monocyte-derived cells accumulate in the intestine and contribute to pathology in inflammatory bowel disease (IBD). Our previous work has shown that intestinal monocyte-derived cells from Crohn’s Disease patients have a reduced response to the immunoregulatory cytokine IL-10 and that this property is already established in circulating monocytes prior to their arrival in the intestine. Here, we have used scRNAseq and functional experiments to define the scope of IBD-associated changes in blood monocytes and explore the role of inflammatory cytokines in shaping these. Methods Total monocytes were isolated from the peripheral blood of 12 newly diagnosed, treatment naive IBD patients with active endoscopic disease (6 Crohn’s Disease [CD] and 6 Ulcerative Colitis [UC]) and 10 healthy controls (HC) by negative magnetic selection. scRNAseq was performed using the 10x Genomics Chromium 3’ assay at a target capture rate of 1000 cells/sample and sequencing at ~50,000 reads/cell. Data were analysed using Cell Ranger, Seurat, Metascape and Gene Set Enrichment Analysis. Blood CD14+ monocytes from healthy donors were pre-incubated in medium alone or with IFNγ (100U/ml) for 24 hours. Inhibition of LPS-induced TNFα production by IL-10 was measured by intracellular cytokine staining and flow cytometry. Results UMAP clustering identified six transcriptionally distinct monocyte clusters: clusters 0, 1 and 2 mapped to classical monocytes; cluster 3 to non-classical monocytes; cluster 4 to intermediate monocytes; and cluster 5 contained platelet signals. The proportion of cluster 4 was increased in CD patients compared to HC. Differential gene expression analysis of CD and UC monocyte clusters compared to healthy controls identified a total of 246 differentially expressed genes (DEGs) in CD and 149 in UC. Only 39 of these DEGs were significantly affected in both diseases. Both CD and UC DEGs were significantly enriched for pathways related to cytokine signalling and inflammation (Figure 1). IFNγ and TNFα -response gene sets were positively enriched in CD, but negatively enriched in UC (Figure 2). Pre-treatment of healthy monocytes with IFNγ in vitro reduced their response to IL-10 as measured by inhibition of LPS-induced TNFα (Figure 3). Conclusion Expression of genes associated with inflammation and cytokine signaling are altered in blood monocytes from IBD patients implying their modification by systemic signals prior to arrival in the intestinal mucosa. Cytokine- (IFNγ and TNFα) stimulated genes are differentially affected in CD and UC monocytes. In CD, systemic IFNγ may reduce the responsiveness of monocytes to IL-10 to prime them for inflammatory function.

Characterization of peripheral cytokine-secreting cells responses in HIV/TB co-infection.

Currently the responses of peripheral cytokine-secreting cells in the natural course of human immunodeficiency virus (HIV) and tuberculosis (TB) co-infection haven't been fully elucidated. The function of peripheral proinflammatory, regulatory and cytotoxic cytokine-secreting cells were investigated by direct intracellular cytokine staining (ICS) and flow cytometry, additionally, the absolute numbers of different cytokine-secreting cells were measured among patients with HIV/TB co-infection (HT group), and compared them with the healthy controls (HC group), patients with TB (TB group) and patients with HIV infection (HIV group). After one week's anti-TB treatment, the changes of the percentages of cytokine-secreting cells were further evaluated in TB and HT groups. Totally 26 individuals in the HC group, 51 in the TB group, 26 in the HIV group and 29 in the HT group were enrolled. The HT. HT group exhibited significantly lower absolute numbers of IFN-γ+CD4+, IFN-γ+CD8+, TNF-α+CD4+, IL17A+CD4+ T cells and TNF-α+CD14+ monocytes than the TB and HIV groups. Compared with the TB group, the percentages of CD8+ T cells secreting IFN-γ and perforin (p=0.010; p=0.043) were significantly lower among the HT group. Compared with the HIV group, the percentages of CD4+, CD8+ T cells and CD14+ monocytes secreting TNF-α (p=0.013; p=0.001; p<0.001) were significantly decreased, and the percentage of CD8+ T cells secreting IL-17A (p=0.015) was significantly increased among the HT group. Both the percentages of CD4+ T cells secreting TGF-β (p<0.001; p=0.001), and CD4+ and CD8+ T cells secreting granzyme A (all p<0.001), were significantly higher among the HT group than among the TB group and HIV group. After one week's anti-TB treatment, an increased percentage of CD4+ T cells secreting TNF-α (p=0.003) was found in the TB group, and an increased percentage of CD8+ T cells secreting TNF-α (p=0.029) was found in the HT group. Significantly different functional profiles of peripheral proinflammatory, regulatory, and cytotoxic cytokine-secreting cells were observed in the natural course of HIV/TB co-infection compared to TB and HIV infection alone, even though the absolute numbers of those cells were significantly lower in HIV/TB co-infection. TNF-α-secreting CD8+ T cells may be a more sensitive marker for early evaluation of anti-TB treatment efficacy in patients with HIV/TB co-infection.

TH1/Treg ratio may be a marker of autism in children with immune dysfunction

Evidence suggests a link between autism spectrum disorder (ASD), behavioral symptoms in the context of ASD, and presence of an altered immune function. Several studies have highlighted differences in T-lymphocyte subpopulations, their activation status, and their response to stimulation in children and adults with ASD. These T cell abnormalities have often been associated with more impaired behaviors. However, few studies have attempted to address whether T cell subsets have the potential to serve as biomarkers in ASD. Moreover, although many studies have been performed in Western populations, few (if any) have been performed in Asian populations in mainland China. In this study we used intracellular cytokine flow cytometry to assess the frequencies of CD4+ T-cell subpopulations (T-helper (TH) 1, TH2, TH17, Foxp3+ regulatory T cells (Treg) as well as CD8+, subpopulations of T cytotoxic (TC) 1, TC2, and TC17 in 82 children with ASD and 50 healthy typical developing children from the Second Affiliated Hospital of Kunming Medical University of Yunnan province. To further elucidate immune status cytokine levels were also measured in the plasma and serum using a bead-based cytokine assay. Our results showed that the frequency of circulating Treg cells and the levels of active TGF-β1 in plasma were lower in children with ASD than in healthy controls. In contrast, the frequencies of TH1, TH2, TH17 and TC1 cells were increased. Proinflammatory cytokine levels of TNF-α, IL-4, IL-5 and IL-17A were higher in the plasma of children with ASD compared to typical controls. We also found an association between the severity of behavior impairments in ASD children and altered immune responses as measured using the effector T cell responses and regulatory responses (using Teff/Treg ratios). Higher the Teff/Treg ratios, were associated with more severe problematic behavioral symptoms. Further, the potential biomarker relevance of Teff/Treg ratio was evaluated by the receiver operating characteristics curve. Data suggests that high TH1/Treg cell ratios could also be used as a potential marker for the diagnosis of children with ASD. Overall, our data suggest an imbalance in inflammatory and regulatory immune responses in ASD. Ratios of inflammatory/regulatory cells or cell frequencies such as Teff/Treg cells may be useful biomarkers for children with ASD with immune dysfunction.

Graft Versus Leukaemia (GvL): Identification & Characterisation of Alloreactive Antigens and Cognate T Cell Responses in Acute Myeloid Leukemia

Allogeneic stem cell transplantation (allo-SCT) is the most established, curative T cell cancer immunotherapy. The curative effect of allo-SCT relies on a graft-versus-leukemia (GvL) effect; but this can be difficult to dissociate from graft-versus-host disease (GvHD). For decades, the key unsolved challenge has been to identify common alloreactive peptide-human leukocyte antigen (HLA) complexes targeted by T cells that mediate GvL but not GvHD. We developed a workflow for unbiased identification of alloreactive antigens and analysis of cognate donor alloreactive T cells and their T cell receptors (TCRs; Fig 1). We studied sequential bone marrow and peripheral blood samples, collected over 15 years, from 12 high-risk AML patients who remain in continuous complete remission following allo-SCT (all >2 years). All patients had relapsed at least once pre-allo-SCT and/or were AML flow cytometry minimal residual disease (MRD)+ pre-allo-SCT. 7 had received donor lymphocyte infusions. No patients developed >1 grade GvHD. We performed whole exome sequencing of DNA from isolated patient T cells (patient germline), AML blasts and donor DNA to identify: (i) AML-specific coding variants; and (ii) germline coding single nucleotide polymorphisms (SNPs), mismatched between patient and donor. 98.7% of the mismatched variants were germline. More variants were identified in unrelated compared with sibling donors (mean: 5744 vs 3253 variants). To narrow the list of variants taken forward for functional studies, we applied two sequential filtering steps: (i) we restricted analysis to variants expressed in the patients' AML cells (determined by RNA-Seq). 28.7% of all mismatched coding variants were expressed in AML cells; and (ii) we used in silico prediction of HLA-binding affinities of peptides spanning variant amino acids to patient-specific HLA-I and -II using NetMHCpan 4.0 and NetMHCIIpan 3.0. To functionally identify alloreactive antigens, we then screened ~200-900 peptides per patient-donor pair (total 5300 peptides) for recognition by T cells present in patient peripheral blood collected 2-7 years after allo-SCT. This enriches for the identification of long-lived anti-AML responses at a time when patients were both infection- and GvHD- free. Response screening, using IFNγ ELISPOT assays, identified T cell responses to 24 germline, mismatched epitopes. In all cases, responses were only observed when the amino acid encoded by the patient SNP, but not the donor allele, was present in the peptide. The phenotype of the epitope-specific T cells was established by intracellular cytokine staining (ICS) and flow cytometry analysis, and the HLA restriction of each T cell response was determined using HLA-I, -DR, -DP and -DQ blocking antibodies. Of the 24 alloreactive peptides,19 elicited CD4+ HLA class II restricted responses and 3 were targeted by CD8+ class I restricted T cells. Two remain to be characterised (Fig 2). The population allele frequency of the germline variant SNPs targeted varies from 0.5% to 72% in the Caucasian population. Likewise, some of the restricting HLA alleles are very common (e.g. a HLA-DP allele presenting 3 epitope peptides is present in ~66% of the Caucasian population). ICS showed that epitope-specific CD4+ T cells produce Th1 cytokines (IFNg, TNFa) and degranulate (express CD107a) in response to peptide stimulation (Fig 2). Next, we performed single cell 5'RNA-seq with TCR-seq of epitope-restricted T cells (isolated via IFN-g catch assay) for all CD4+ epitope-specific T cells. Alloreactive CD4+ T cells have cytokine-producing and cytotoxic effector potential, expressing MIP1a and b, IFNg, granzyme B and FasL (Fig 2). Finally, lentiviral-based gene transfer of one of the candidate alloreactive epitope-specific TCRs into third party primary T cells confers the capacity to generate IFNg and TNFa in response to peptide stimulation, and to recognise primary AML cell-derived antigen in the presence of monocyte-derived dendritic cells (Fig 2). In vitro and in vivo killing assays are in progress. The high proportion of CD4+ T Cell responses identified by our pipeline is concordant with downregulation of HLA II expression on AML blasts at relapse post-allo-SCT, which is a common mechanism of immune evasion, found in one-third of patients. Our approach dissects the biology of alloreactive anti-AML GvL and GvHD responses and to manipulate them for therapeutic purposes. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Dynamic analysis of tuberculosis specific mononuclear cells in peripheral blood of HIV patients with tuberculosis

Objective: To evaluate the response of peripheral blood mononuclear cells (PBMCs) in patients with human immunodeficiency virus (HIV) combined with active tuberculosis (TB) to TB-specific antigen stimulation. Methods: From January to December, 2018, individuals infected with both HIV and TB (HIV/TB group) were taken as the study subjects. Individuals infected with HIV alone (HIV group), individuals infected with TB alone (TB group) and healthy people (Health control group, HC group) were taken as the control groups. PBMCs were isolated and stimulated with purified protein derivative of bacillus calmette-guerin (BCG-PPD). The expression of surface molecules in T cells (CD3+, CD4+, CD8+) and monocytes (CD14+) and the percentages of Interferon (IFN)-γ and tumor necrosis factor (TNF)-α were detected by cell surface molecular staining, direct intracellular cytokine staining and flow cytometry (CD3- lymphocytes were mainly B lymphocytes and NK cells). Analysis of non-parametric data was used to compare the data between the two groups, and paired t-test was used to compare the data before and after PPD stimulation in each group. Results: Before PPD stimulation, the percentage of IFN-γ+ CD8+ cells in the peripheral blood of HIV/TB group(mean 0.52%) was significantly lower than that in TB group(mean 0.94%, P=0.010). The TNF-α+cell percentages in CD3+, CD4+, CD8+, or CD14+ cells in the HIV/TB group(mean 19.2%) were significantly lower than those in the HIV group(mean 31.9%, P=0.002). The percentage of TNF-α secreted by monocytes in the HIV group was significantly lower than that in the HC group. The percentages of IFN-γ+ CD8+ and IFN-γ+ CD3- cells in the peripheral blood of the TB group (mean 0.94%) were significantly higher than thoset in the HC group(mean 0.51%, P=0.020), while the percentages of TNF-α+ cells in each subsets of PBMCs were significantly lower than those in the HC group. After PPD stimulation, the percentage of IFN-γ+ CD8+ cells in the HIV/TB group was significantly lower than that in the TB group(P=0.008), and the change was more marked than that before stimulation. The percentage of IFN-γ+ CD8+ cells in the HIV group(mean 0.20%) was lower than that in the HC group (mean 0.52%, P=0.044). The percentage of IFN-γ+ CD3- in the TB group was significantly higher than in the HC group. There were no significant differences in TNF-α+ cell percentages in the 3 groups compared with the control group after PPD stimulation. The percentages of IFN-γ+ CD4+ cells in the HC and the TB groups were significantly increased after PPD stimulation in each group (P=0.002, P=0.001, respectively). However, there were no significant differences of IFN-γ+ CD4+ cell percentages in the HIV/TB group and the HIV group. The percentages of TNF-α production by monocytes were significantly increased after PPD stimulation in all groups. Conclusions: Chronic Mycobacterium tuberculosis (MTB) infection reduced the ability of PBMCs to produce TNF-α. For patients with TB infection, the production of TNF-α was reduced when combined with HIV infection. The capacity of CD8+ and CD3- lymphocytes to produce IFN-γ was increased in TB patients, while the capacity of CD8+ T cells to produce IFN-γ was decreased with co-infection of HIV. Infection of HIV weakened the immune response to MTB infection, which made the clinical diagnosis and treatment of TB more difficult.

Comparison between enzyme-linked immunospot assay and intracellular cytokine flow cytometry assays for the evaluation of T cell response to SARS-CoV-2 after symptomatic COVID-19.

IntroductionEvaluation of different cell‐based assays for the study of adaptive immune responses against SARS‐CoV‐2 is crucial for studying long‐term and vaccine‐induced immunity.MethodsEnzyme‐linked immunospot assay (ELISpot) and intracellular cytokine staining (ICS) using peptide pools spanning the spike protein and nucleoprotein of SARS‐CoV‐2 were performed in 25 patients who recovered from paucisymptomatic (n = 19) or severe COVID‐19 (n = 6).ResultsThe proportion of paucisymptomatic patients with detectable SARS‐CoV‐2 T cells was low, as only 44% exhibit a positive T cell response with the ICS and 67% with the ELISpot. The magnitude of SARS‐CoV‐2 T cell responses was low, both with ICS (median at 0.12% among total T cells) and ELISpot (median at 61 SFCs/million peripheral blood mononuclear cells [PBMC]) assays. Moreover, T cell responses in paucisymptomatic patients seemed lower than among patients with severe disease. In the paucisymptomatic patients, the two assays were well correlated with 76% of concordant responses and a Cohen's kappa of 55. Furthermore, in four patients SARS‐CoV‐2 T cells were detected by ELISpot but not with ICS. Short‐term culture could improve the detection of specific T cells.ConclusionsIn patients who recovered from paucisymptomatic COVID‐19, the proportion of detectable anti‐SARS‐CoV‐2 responses and their magnitude seemed lower than in patients with more severe symptoms. The ELISpot appeared to be more sensitive than the ICS assay. Short‐term culture revealed that paucisymptomatic patients had nonetheless few SARS‐CoV‐2 T cells at a very low rate in peripheral blood. These data indicate that various ex‐vivo assays may lead to different conclusions about the presence or absence of SARS‐CoV‐2 T cell immunity.

600 Microarray patch-based adenovirus vaccines enable effective skin immunization

Recombinant live adenovirus (Ad) vectors represent a readily modular vaccine construct that can be engineered to include any antigen of interest, thereby enabling rapid vaccine development against a myriad of infectious pathogens, including coronaviruses. However, current approaches used for delivery, storage, and distribution of Ad vaccines hinder their full potential for effective global immunization campaigns. Here, we developed simple, effective, practical, and needle-free Ad vaccines based on microarray patches (MAPs) to enable sustainable mass vaccination against SARS-CoV-2. Rational formulation of live Ads with nonreducing sugars and mechanically strong carbohydrates into dissolvable MAPs enabled effective skin-targeted delivery of Ads and efficient cutaneous transduction as determined by in vivo live imaging of mice following skin application of MAPs integrating Ad vectors with a reporter gene. Immunogenicity assessment of dissolving MAP-based Ad vectors encoding SARS-CoV-2 proteins in mice demonstrated that skin immunization via MAP delivery of Ad-vectored COVID-19 vaccines elicited robust antigen-specific antibody responses, and these antibodies led to virus-specific neutralization activities, which were enhanced compared to those obtained with traditional intramuscular immunization. Furthermore, MAP delivery of Ad-vectored vaccines elicited potent cell-mediated immune responses, including polyfunctional virus-specific CD8+ and CD4+ T-cell responses in spleens and lungs of immunized mice, as determined by intracellular cytokine staining and flow cytometry, as well as antigen-specific cytotoxic T-cell responses in spleens of mice, as determined by lytic assay. Collectively, our results suggest that dissolvable MAPs could enable the development of skin-targeted Ad-based vaccines that may increase the effectiveness of global immunization programs for SARS-CoV-2 and other existing or future pathogens.

T cell responses to Mycobacterium indicus pranii immunotherapy and adjunctive glucocorticoid therapy in tuberculous pericarditis

BackgroundIn the Investigation of the Management of Pericarditis (IMPI) randomized control, 2x2 factorial trial, Mycobacterium indicus pranii (MIP) immunotherapy, adjunctive corticosteroids or MIP combined with corticosteroids was compared to standard tuberculosis (TB) therapy for tuberculous pericarditis (TBP). While MIP and/or the combination of MIP and corticosteroids had no impact on all-cause mortality or pericarditis related outcomes, corticosteroids reduced the incidence of constrictive pericarditis at 12 months. Data suggests that both adjunctive therapies modulate the immune and inflammatory responses to pulmonary TB. Whether they affect systemic antigen-specific T cell responses, key immune mediators of Mycobacterium tuberculosis control, in patients with TBP is unknown. MethodsParticipants with definite or probable TBP were randomly assigned to receive five injections of MIP or placebo at 2-week intervals and either 6 weeks of oral prednisolone or placebo. Frequencies of CD4 and CD8 T cells expressing IFN-γ, IL-2 or TNF in response to MIP or purified protein derivative stimulation were measured by intracellular cytokine staining and flow cytometry up to 24 weeks post treatment. ResultsImmunotherapy with MIP did not significantly modulate frequencies of Th1 CD4 and CD8 T cells compared to placebo. Adjunctive prednisolone also did not change mycobacteria-specific CD4 or CD8 T cell responses. By contrast, combinatorial therapy with MIP and prednisolone was associated with a modest increase in frequencies of multifunctional and single cytokine-expressing CD4 T cell responses at 6 and 24 weeks post treatment. ConclusionsConsistent with the lack of a significant clinical effect in the IMPI trial, MIP immunotherapy did not significantly modulate mycobacteria-specific T cell responses. Despite the positive effect of prednisolone on hospitalizations and constrictive pericarditis in the IMPI trial, prednisolone did not significantly reduce pro-inflammatory T cell responses in this sub-study. The modest improvement of mycobacteria-specific T cell upon combinatorial therapy with MIP and prednisolone requires further investigation.

Immunological Assessment of Lung Responses to Inhalational Lipoprotein Vaccines Against Bacterial Pathogens.

Lipopeptides or lipoproteins show potential as safe and effective subunit vaccines for protection against bacterial pathogens. Provided suitable adjuvants are selected, such as the TLR2-stimulating molecules Pam2Cys and Pam3Cys, these may be formulated as inhalational vaccines to optimize localized pulmonary immune responses. Here, we present methods to assess antigen-specific memory lymphocyte responses to novel vaccines, with a focus on immune responses in the lung tissue and bronchoalveolar space. We describe detection of T-cell responses via leukocyte restimulation, followed by intracellular cytokine staining and flow cytometry, enzyme-linked immunosorbent spot assay (ELISpot), and sustained leukocyte restimulation for detection of antigen-specific memory responses. We also detail assessment of antibody responses to vaccine antigens, via enzyme-linked immunosorbent assay (ELISA)-based detection. These methods are suitable for testing a wide range of pulmonary vaccines.

Detection of IFNγ-Secreting CD4+ and CD8+ Memory T Cells in COVID-19 Convalescents after Stimulation of Peripheral Blood Mononuclear Cells with Live SARS-CoV-2.

Background: New coronavirus SARS-CoV-2, a causative agent of the COVID-19 pandemic, has been circulating among humans since November 2019. Multiple studies have assessed the qualitative and quantitative characteristics of virus-specific immunity in COVID-19 convalescents, however, some aspects of the development of memory T-cell responses after natural SARS-CoV-2 infection remain uncovered. Methods: In most of published studies T-cell immunity to the new coronavirus is assessed using peptides corresponding to SARS-CoV-1 or SARS-CoV-2 T-cell epitopes, or with peptide pools covering various parts of the viral proteins. Here, we determined the level of CD4+ and CD8+ memory T-cell responses in COVID-19 convalescents by stimulating PBMCs collected 1 to 6 months after recovery with sucrose gradient-purified live SARS-CoV-2. IFNγ production by the central and effector memory helper and cytotoxic T cells was assessed by intracellular cytokine staining assay and flow cytometry. Results: Stimulation of PBMCs with live SARS-CoV-2 revealed IFNγ-producing T-helper effector memory cells with CD4+CD45RA−CCR7− phenotype, which persisted in circulation for up to 6 month after COVID-19. In contrast, SARS-CoV-2-specific IFNγ-secreting cytotoxic effector memory T cells were found at significant levels only shortly after the disease, but rapidly decreased over time. Conclusion: The stimulation of immune cells with live SARS-CoV-2 revealed a rapid decline in the pool of effector memory CD8+, but not CD4+, T cells after recovery from COVID-19. These data provide additional information on the development and persistence of cellular immune responses after natural infection, and can inform further development of T cell-based SARS-CoV-2 vaccines.

Innate Immune Cytokine Profiling and Biomarker Identification for Outcome in Dengue Patients.

BackgroundEarly biomarkers of progression to severe dengue are urgently required to enable effective patient management and control treatment costs. Innate immune cells, which comprise the earliest responders to infection and along with the cytokines and chemokines they secrete, play a vital role in orchestrating the subsequent adaptive immune response and have been implicated in the enhancement of infection and “cytokine storm” associated with dengue severity. We investigated the early innate immune cytokine profile of dengue patients during acute phase of disease in a prospective blinded study that included subjects with acute dengue and febrile controls from four major hospitals in Bengaluru, India along with healthy controls. We used intracellular cytokine staining and flow cytometry to identify innate immune biomarkers that can predict progression to severe dengue.ResultsDengue infection resulted in enhanced secretion of multiple cytokines by all queried innate immune cell subsets, dominated by TNF-α from CD56+CD3+ NKT cells, monocyte subsets, and granulocytes along with IFN-γ from CD56+CD3+ NKT cells. Of note, significantly higher proportions of TNF-α secreting granulocytes and monocyte subsets at admission were associated with mild dengue and minimal symptoms. Dengue NS1 antigenemia used as a surrogate of viral load directly correlated with proportion of cytokine-secreting innate immune cells and was significantly higher in those who went on to recover with minimal symptoms. In patients with secondary dengue or those with bleeding or elevated liver enzymes who revealed predisposition to severe outcomes, early activation as well as efficient downregulation of innate responses were compromised.ConclusionOur findings suggested that faulty/delayed kinetics of innate immune activation and downregulation was a driver of disease severity. We identified IFN-γ +CD56+CD3+ NKT cells and IL-6+ granulocytes at admission as novel early biomarkers that can predict the risk of progression to severity (composite AUC = 0.85–0.9). Strong correlations among multiple cytokine-secreting innate cell subsets revealed that coordinated early activation of the entire innate immune system in response to dengue virus infection contributed to resolution of infection and speedy recovery.

Graft Versus Host Disease Activity of Donor T Cells Is Regulated By Vasoactive Intestinal Polypeptide Synthesized By Donor Plasmacytoid Dendritic Cells

Introduction: Increased numbers of plasmacytoid dendritic cells (pDC) in the bone marrow graft improve clinical outcomes with decreased graft-versus-host disease (GvHD) and improved survival in previous pre-clinical murine experiments (Lu Y, Blood. 2012;119:1075-1085) and in clinic data from BMT CTN 201 (Waller JCO 2014; 32:2365-72). The mechanism by which donor pDC limit severe GvHD and improve survival after allo-BMT is unknown. We have previously described higher levels of donor T cell activation and expansion (measured as bio-luminescent radiance) among recipients of VIP-KO pDC (Li, J.X Blood 2016 128:2154). We hypothesize that blocking VIP signaling augment the magnitude of T cell activation and expansion, and that modulating the VIP pathway may be a potential therapeutic target for regulating GvHD in allo-HSCT.Methods:We used a MHC mismatched C57BL/6 àB10BR allo-BMT model, transplanting purified populations of FACS sorted Lin-Sca-1+C-kit+ (LSK) stem cells combined with FACS purified BM pDC and MACS purified splenic T cells. Lethally irradiated B10BR recipients received 5,000 HSC, 50,000 pDC from wild type (WT) or VIP-KO donor and 1×106 T cells from WT C57BL6 donor mice. Survival and GvHD were measured based on fur texture, posture, activity, skin integrity and weight body loss. To test VIP production by pDC cells, we purified donor pDC from VIP-GFP C57 BL/6 mice, in which GFP transcription is under the control of the VIP gene promotor, and transplanted them into lethally irradiated MHC mis-matched B10.BR recipients. VIP expression in mice receiving pDC cells from a VIP-GFP transgenic donor was detected by fluorescence confocal microscopy. Immune polarization of donor T cells was accessed by intracellular cytokine staining and flow cytometry. Mice transplanted with VIP-GFP pDC cells were sacrificed on D10 and spleens were collected in OCT.Results: Survival of recipients receiving HSC, T cells and WT pDC was 71% on day100 post-transplant while survival of those receiving VIP-KO pDC was 31% (Fig 1A). Splenocytes were obtained from recipients on day 3 post-transplant. IFN-γexpression was higher and IL-10 epression was significantly lower in donor CD8+ T cells in mice receiving VIP-KO pDC compared with WT pDC (Fig 1B). Confocal microscopy showed VIP expression in donor pDC among recipients of VIP-GFP pDC (Fig 2)Conclusion:These data indicated VIP signaling occurs via interactions between donor T cells and donor pDC. Local production of VIP inhibits activation and Th1 immune polarization of donor T cells. In murine allo-BMT, absence of VIP expression in pDC leads to increased T cell activation, expansion, allo-reactivity and Th1 polarization resulting in worse survival. Thus VIP is a critical pathway in regulating GvHD and GvL action of donor T cells and an attractive pharmacological target for GvHD prevention. [Display omitted] DisclosuresWaller:AMGEN: Consultancy; PRA: Consultancy; Cambium Medical Technologies: Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties; Celldex: Consultancy; Novartis Pharmaceuticals Corporation: Consultancy, Honoraria, Research Funding; Helocyte: Consultancy; Cerus: Equity Ownership; National Institutes of Health: Research Funding; Chimerix: Equity Ownership; Coulter Foundation: Research Funding; Katz Foundation: Research Funding.

Pathogenic effector TH2 cells discriminate between allergic and eosinophilic inflammation in food allergy and eosinophilic gastrointestinal disease

Abstract IgE-mediated food allergies (FA) and delayed food-triggered (FT) gastrointestinal tissue eosinophilia (EGID) have distinct clinical manifestations and are treated differently. Pathogenic effector TH2 (peTH2) cells are implicated in inappropriate TH2 responses in both of these diseases. Furthermore, EGID patients can have concomitant FA, and some EGID patients do not report any food triggers. To better understand the role of peTH2 cells in the context of FA and EGID and the overlap between these two conditions, peTH2 cells (CD3+CD4+CD27-CD49d+CRTH2+CD161+) from purified peripheral blood mononuclear cells (PBMC) were profiled by intracellular cytokine flow cytometry in these deeply-characterized patient cohorts: patients with FA alone (FA, n=5), EGID with FA and FT (EGID+FA+FT, n=7), EGID with FT (EGID+FT, n=7) and EGID without FA or FT (EGID, n=9). Patients with any food allergy or food trigger (FA, EGID+FA+FT, and EGID+FT) have increased peTH2 cells compared to healthy volunteers (HV). Interestingly, a greater percentage of peTH2 cells in patients with food triggers (EGID+FA+FT, EGID+FT) exhibit a trend toward IL5+IL13+ double positivity (P = 0.0556) than those from patients without food triggers (FA, EGID). In contrast, peTH2 cells from patients with FA had a greater percentage that were IL17A+ (P=0.009) or IFNγ+ (P=0.0444) than patients with eosinophilic disease (EGID+FA+FT, EGID+FT, EGID). Our studies indicate that while elevated levels of peTH2 cells are indicative of allergic inflammation of both immediate hypersensitivity and eosinophilic disease, the cytokine profile of each disorder may be distinct, where IL17A+ and IFNγ+ predominate in those without eosinophilic disease.

Is the New Interferon-Gamma Releasing Assay Beneficial for the Diagnosis of Latent and Active Mycobacterium tuberculosis Infections in Tertiary Care Setting?

Interferon-Gamma Release Assays (IGRAs) are widely used in the laboratory diagnosis of Mycobacterium tuberculosis (MTB) infections, particularly in the latent form. We compared the performance of a newly developed IGRA, the Standard E TB-Feron ELISA (TBF) with the currently used QuantiFERON-TB Gold Plus assay (QFT-Plus) for the detection of latent tuberculosis infections (LTBIs) in tertiary care settings. We also investigated interferon-gamma (IFN-γ) released by T cell subsets via intracellular cytokine staining (ICS) and flow cytometry. A total of 335 subjects including 40 patients with active tuberculosis (ATB), 75 immunocompromised patients with LTBIs (P-LTBI), 70 health care workers with LTBIs (H-LTBI), and 150 healthy controls (HC) were studied. Overall, 168 subjects (50.1%) and 178 subjects (53.1%) displayed IGRA-positive results in the QFT-Plus and TBF, respectively. The overall concordance rate was 94.0%. The sensitivity and specificity of TBF were 88% and 95%, respectively, while the sensitivity and specificity of QFT-Plus were 90% and 100%, respectively. Twenty discordant results (6.0%) were observed in simultaneously performed QFT-Plus and TBF. Particularly, 13 LTBI subjects previously positive QFT-Plus showed negative results in QFT-Plus performed after enrollment. In TBF, six subjects showed positive results while five were negatively concordant with QFT-plus and two were indeterminate. The overall proportion of IFN-γ releasing CD8+ T lymphocytes was significantly higher in TBF compared to those of QFT-Plus TB1 and TB2 (0.21% vs. 0.01% and 0.02%; p-value < 0.05). The recombinant protein antigens in the TBF stimulated TB-specific CD8+ T cells more efficiently. Therefore, TBF would be a useful alternative to current IGRAs such as the QFT-Plus, particularly in tertiary care settings where the immunocompromised patients are subjected to IGRA tests to differentiate MTB infection. Further strategies to analyze the implications of the discrepancies, particularly near the cutoff values between different IGRAs, are needed.

058 [18F]FDG PET/CT-based imaging method to characterize the therapeutic effects of DMF in EAE

Data from clinical and preclinical studies have shown that fumarates like dimethylfumarate (DMF) - by suppressing the Th17 response - improve psoriasis and multiple sclerosis in human and experimental autoimmune encephalomyelitis (EAE) in mice. In our previous studies, we analyzed the anti-inflammatory effects of DMF on the immune response by methodologies like intracellular cytokine staining and flow cytometry or by performing quantitative mRNA expression from isolated cells. Here we aimed to establish an in vivo method to follow T cell activation and the anti-inflammatory properties of DMF in mice immunized for developing EAE.

Inhibition of mTOR suppresses IFNα production and the STING pathway in monocytes from systemic lupus erythematosus patients.

Increased IFNα is important in the pathogenesis of SLE. Plasmacytoid dendritic cells are considered the main producer of IFNα upon Toll-like receptor pathway activation. However, which cells produce IFNα following stimulation with cyclic GMP-AMP synthase (cGAS) and stimulator of IFN genes (STING) in SLE remains unknown. We investigated the IFNα producing capacity of myeloid cells under cGAS-STING pathway stimulation. IFNα levels in peripheral blood mononuclear cells from SLE patients and healthy controls stimulated with 2'3'c-GAMP, a stimulator of cGAS-STING, were measured by intracellular cytokine staining and flow cytometry. STING expression and its co-localization with TBK1 were examined by flow cytometry or confocal microscopy. The effects of in vitro exposure to IFNα on IFNα production and STING expression, and in vitro rapamycin treatment on IFNα production and STING, pTBK1 and IRF3 expression were examined. IFNα was produced by monocytes, conventional dendritic cells and plasmacytoid dendritic cells upon cGAS-STING pathway activation. The frequency of IFNα-producing monocytes positively correlated with SLE disease activity. STING expression and its co-localization with TBK1 were increased in lupus monocytes. Prior exposure to IFNα enhanced the IFNα-producing capacity of monocytes. Inhibition of the mechanistic target of the rapamycin (mTOR) pathway suppressed IFNα production from monocytes and downregulated enhanced STING expression and its downstream molecules. Enhanced IFNα from lupus monocytes induced by augmented STING pathway activation is associated with SLE pathogenesis. Suppression of the mTOR pathway downregulated the enhanced STING expression and the subsequent IFNα production by monocytes.

Association of mucosal-associated invariant T cells with different disease phases of polymyalgia rheumatica.

Although T cells are thought to be involved in the pathogenesis of PMR, whether innate-like T cells are involved in the process remains unknown. The serum levels of 27 cytokines/chemokines in patients with PMR were measured by a multiplex immunoassay (Bio-Plex Assay). The cytokine-producing capacity of T and innate-like T cells was assessed by intracellular cytokine staining and flow cytometry. The frequency and activated status of T and innate-like T cells were investigated by flow cytometry and their associations with clinical parameters were assessed. The levels of inflammatory cytokines were associated with disease activity in PMR. The cytokine-producing capacity by CD8+ T and innate-like T cells was associated with disease activity. The frequency of HLA-DR+ CD38+ cells among CD8+ T cells was increased in patients with active disease. The frequencies of HLA-DR+ CD38+ cells among CD4+ T, mucosal-associated invariant T (MAIT) and γδ T cells were higher in patients with inactive disease. The frequency of HLA-DR+ CD38+ MAIT cells was associated with the PMR activity score and CRP levels in patients in remission. The inflammatory cytokine-producing capacity and expression of activation markers of CD8+ T and innate-like T cells were associated with the disease activity of PMR. MAIT cell activation in patients in remission may contribute to the subclinical activity of the disease.