Type I interferons and Mycobacterium tuberculosis whole cell lysate induce distinct transcriptional responses in M. tuberculosis infection.

Type I interferon (IFN)-induced genes have the potential for distinguishing active tuberculosis (ATB) from latent TB infection (LTBI) and healthy controls (HC), monitoring treatment, and detection of individuals at risk of progression to active disease. We examined the differential effects of IFN-α, IFN-ß and Mycobacterium tuberculosis whole cell lysate (Mtb WCL) stimulation on the expression of selected IFN-stimulated genes in peripheral blood mononuclear cells from individuals with either LTBI, ATB, and healthy controls. Stimulation with IFN-α and IFN-ß induced a higher expression of the interrogated genes while Mtb WCL stimulation induced expression similar to that observed at baseline, with the exception of IL-1A and IL-1B genes that were downregulated. The expression of IFN-α-induced FCGR1A gene, IFN-ß-induced FCGR1A, FCGR1B, and SOCS3 genes, and Mtb WCL-induced IFI44, IFI44L, IFIT1, and IFITM3 genes differed significantly between LTBI and ATB. These findings suggest stimulation-driven gene expression patterns could potentially discriminate LTBI and ATB. Mechanistic studies are necessary to define the processes through which distinct type I IFNs and downstream ISGs determine infection outcomes and identify potential host-directed therapeutic strategies.

Expansion of cytotoxic tissue-resident CD8+ T cells and CCR6+CD161+ CD4+ T cells in the nasal mucosa following mRNA COVID-19 vaccination.

Vaccines against SARS-CoV-2 have shown high efficacy in clinical trials, yet a full immunologic characterization of these vaccines, particularly within the human upper respiratory tract, is less well known. Here, we enumerate and phenotype T cells in nasal mucosa and blood using flow cytometry before and after vaccination with the Pfizer-BioNTech COVID-19 vaccine (n = 21). Tissue-resident memory (Trm) CD8+ T cells expressing CD69+CD103+ increase in number ~12 days following the first and second doses, by 0.31 and 0.43 log10 cells per swab respectively (p = 0.058 and p = 0.009 in adjusted linear mixed models). CD69+CD103+CD8+ T cells in the blood decrease post-vaccination. Similar increases in nasal CD8+CD69+CD103- T cells are observed, particularly following the second dose. CD4+ cells co-expressing CCR6 and CD161 are also increased in abundance following both doses. Stimulation of nasal CD8+ T cells with SARS-CoV-2 spike peptides elevates expression of CD107a at 2- and 6-months (p = 0.0096) post second vaccine dose, with a subset of donors also expressing increased cytokines. These data suggest that nasal T cells may be induced and contribute to the protective immunity afforded by this vaccine.

Serological and cellular inflammatory signatures in end-stage kidney disease and latent tuberculosis.

OBJECTIVES: Tuberculosis comorbidity with chronic diseases including diabetes, HIV and chronic kidney disease is of rising concern. In particular, latent tuberculosis infection (LTBI) comorbidity with end-stage kidney disease (ESKD) is associated with up to 52.5-fold increased risk of TB reactivation to active tuberculosis infection (ATBI). The immunological mechanisms driving this significant rise in TB reactivation are poorly understood. To contribute to this understanding, we performed a comprehensive assessment of soluble and cellular immune features amongst a unique cohort of patients comorbid with ESKD and LTBI. METHODS: We assessed the plasma and cellular immune profiles from patients with and without ESKD and/or LTBI (N = 40). We characterised antibody glycosylation, serum complement and cytokine levels. We also assessed classical and non-classical monocytes and T cells with flow cytometry. Using a systems-based approach, we identified key immunological features that discriminate between the different disease states. RESULTS: Individuals with ESKD exhibited a highly inflammatory plasma profile and an activated cellular state compared with those without ESKD, including higher levels of inflammatory antibody Fc glycosylation structures and activated CX3CR1+ monocytes that correlate with increased inflammatory plasma cytokines. Similar elevated inflammatory signatures were also observed in ESKD+/LTBI+ compared with ESKD-/LTBI+, suggesting that ESKD induces an overwhelming inflammatory immune state. In contrast, no significant inflammatory differences were observed when comparing LTBI+ and LTBI- individuals. CONCLUSION: Our study highlights the highly inflammatory state induced by ESKD. We hypothesise that this inflammatory state could contribute to the increased risk of TB reactivation in ESKD patients.

Evaluation of a collaborative model for successful implementation of a National CD4 enumeration EQA program in Cameroon.

Participation in an EQA program is critical to the quality assurance process. Reliable and precise CD4 T-cells enumeration are essential to improve the clinical management of patients by evaluating the disease progression and by monitoring the effectiveness of ART in HIV-patients. The CIRCB, CD4 reference laboratory, in collaboration with the Canadian QASI-program, recruited sites, distributed and analyzed CD4-panels in 61 sites across Cameroon. A trend and performance analysis in the pre-analytical, analytical and post-analytical phases was performed. Continuous training and corrective actions carried out from 2014 to 2018 increased the number of participating sites from 15 to 61 sites, the number of unacceptable results decreased from 50 to 10%. Specific challenges included errors in pre analytic (17.5%), analytic (77.0%) and post-analytic (5.5%) phases. This EQA requires the application of good laboratory practices, fluidic communication between all the stakeholders, continuous training, application of specific on-site corrective measures, and timely equipment maintenance in order to avoid repetitive errors and to increase laboratory performance. It could be extended to other HIV-1 testing like viral load and EID point-of-care. Partnership with QASI serve as a model for implementation of a successful EQA model for resource limited countries wanting to implement EQA for HIV testing and monitoring in alignment with 90-90-90 targets.

Recommendations for sample pooling on the Cepheid GeneXpert® system using the Cepheid Xpert® Xpress SARS-CoV-2 assay.

The coronavirus disease 2019 (Covid-19) pandemic, caused by SARS-CoV-2, has resulted in a global testing supply shortage. In response, pooled testing has emerged as a promising strategy that can immediately increase testing capacity. In pooled sample testing, multiple samples are combined (or pooled) together and tested as a single unit. If the pool is positive, the individual samples can then be individually tested to identify the positive case(s). Here, we provide support for the adoption of sample pooling with the point-of-care Cepheid Xpert® Xpress SARS-CoV-2 molecular assay. Corroborating previous findings, the limit of detection of this assay was comparable to laboratory-developed reverse-transcription quantitative PCR SARS-CoV-2 tests, with observed detection below 100 copies/mL. The Xpert® Xpress assay detected SARS-CoV-2 after samples with minimum viral loads of 461 copies/mL were pooled in groups of six. Based on these data, we recommend the adoption of pooled testing with the Xpert® Xpress SARS-CoV-2 assay where warranted based on public health needs. The suggested number of samples per pool, or the pooling depth, is unique for each point-of-care testing site and can be determined by the positive test rates. To statistically determine appropriate pooling depth, we have calculated the pooling efficiency for numerous combinations of pool sizes and test rates. This information is included as a supplemental dataset that we encourage public health authorities to use as a guide to make recommendations that will maximize testing capacity and resource conservation.

Mucosal-Associated Invariant T Cells Are Depleted and Exhibit Altered Chemokine Receptor Expression and Elevated Granulocyte Macrophage-Colony Stimulating Factor Production During End-Stage Renal Disease.

Background: End-stage renal disease (ESRD) is associated with an increased susceptibility to infectious diseases, including infection with Mycobacterium tuberculosis (Mtb). Mucosal-associated invariant T (MAIT) cells recognize vitamin B metabolites produced by many bacterial species, including Mtb, and may play an important role in providing protective immunity against tuberculosis infection in the lung. To date, little is known about MAIT cell frequency, phenotype, or function in ESRD patients. Methods: MAIT cells, identified by surface marker expression or MR1 tetramer binding, were characterized in 20 ESRD and 20 healthy control participants by multicolor flow cytometry. Ex vivo MAIT cell phenotype and cytokine production following PMA/ionomycin, IL-12/IL-18, or Escherichia coli stimulation were determined. Monocyte phenotype and plasma C-reactive protein/inflammatory cytokine levels were quantified by flow cytometry, ELISA, and multiplex bead array. Results: Peripheral blood MAIT cells were significantly depleted among ESRD patients compared to controls by both phenotypic and tetramer analysis and exhibited a loss of CXCR3 expression coupled to increased expression of CCR6 and CXCR6. ESRD was also associated with a shift in MAIT PMA-induced cytokine production away from IFNγ production and toward granulocyte macrophage-colony stimulating factor (GM-CSF) secretion, and a loss of E. coli-stimulated tumor necrosis factor α expression. Loss of IFNγ expression was associated with a combination of age, alterations in Tbet and Eomes expression, and inflammatory plasma cytokine levels. Conclusion: The loss of peripheral blood MAIT cells and associated shifts in tissue homing receptor expression and GM-CSF production may contribute to an immune environment that is permissive to bacterial replication, particularly in the lungs.

γδ T-cell function is inhibited in end-stage renal disease and impacted by latent tuberculosis infection.

Patients with end-stage renal disease (ESRD) are at elevated risk of acquiring infectious diseases, including tuberculosis (TB). Inflammation and uremia negatively impact immune function in this population, but specific pathways involved in TB immunity have not been identified. Although γδ T cells are known to contribute to protection from TB, their phenotype and function in patients with ESRD is relatively unknown. To determine this we recruited 20 patients with and 20 without ESRD (controls), with or without latent TB infection to assess γδ T cell frequency, surface phenotype, and cytokine production by flow cytometry in response to stimulation. γδ T cells derived from patients with ESRD exhibited significantly lower expression of CCR5, CXCR3, and CD26 compared to controls. Furthermore, patients with ESRD, particularly the group with latent TB infection, exhibited poor IFNγ, TNFα, and GMCSF responses to stimulation with either phosphoantigen HMB-PP, IL-12/IL-18, E. coli, or phorbol myristate acetate and ionomycin. Similar dysfunctional responses were observed in patients with active TB. Surprisingly, neither the γδ phenotype nor its function was associated with plasma markers of inflammation or microbial translocation. Thus, there is significant perturbation of the γδ T-cell population in patients with ESRD, particularly in those with latent TB infection.

Maintenance of Mycobacterium tuberculosis-specific T cell responses in End Stage Renal Disease (ESRD) and implications for diagnostic efficacy.

End-stage renal disease (ESRD) patients exhibit elevated risk of tuberculosis (TB) reactivation, but current diagnostics, including the interferon gamma release assay (IGRA), exhibit poor sensitivity in ESRD. We tested 80 ESRD patients and found an 18.75% prevalence of IGRA positivity. A subset of patients was assessed for Mtb-specific expression of 44 cytokines/chemokines, and CD4+ T cell phenotype and function. Similar to non-ESRD IGRA+ individuals, Mtb-specific IFNγ, IL-1RA, IP-10, MCP-3 and IL-2 responses were identified in the ESRD IGRA+ group. 27% of the ESRD IGRA- group exhibited MCP-3 or IL-2 Mtb-specific responses, which may identify cases of latent TB infection in ESRD. Stimulation of PBMC with PPD demonstrated similar CD4+ T cell production of IFNγ, TNFα and GM-CSF by ESRD patients. The reported low sensitivity of the IGRA in ESRD cohorts is therefore unlikely to be due to poor T cell cytokine secretion, and may instead reflect defects in antigen presentation.

Cytokine and chemokine expression profiles in response to Mycobacterium tuberculosis stimulation are altered in HIV-infected compared to HIV-uninfected subjects with active tuberculosis.

Mycobacterium tuberculosis (Mtb) infects nearly 2 million people annually and is the most common cause of death in HIV-infected individuals. Tuberculosis (TB) diagnostics cater to HIV-uninfected individuals in non-endemic countries, are expensive, slow, and lack sensitivity for those most affected. Patterns of soluble immune markers from Mtb-stimulated immune cells are not well defined in HIV co-infection. We assessed immune differences between HIV-infected and HIV-uninfected individuals with active TB utilizing IFNγ-based QuantiFERON®-TB Gold In-Tube (QFT) testing in Nairobi, Kenya. Excess QFT supernatants were used to measure cytokine and chemokine responses by a 17-plex bead array. Mtb/HIV co-infected participants were significantly less likely to be QFT+ (47.2% versus 84.2% in the HIV-uninfected group), and demonstrated lower expression of all cytokines except for IFNα2. Receiver operator characteristic analyses identified IL-1α as a potential marker of co-infection. Among HIV-infected individuals, CD4+ T cell count correlated weakly with the expression of several analytes. Co-expression analysis highlighted differences in immune profiles between the groups. These data suggest that there is a unique and detectable Mtb-specific immune response in co-infection. A better understanding of Mtb immunology can translate into much needed immunodiagnostics with enhanced sensitivity in HIV-infected individuals, facilitating their opportunity to obtain live-saving treatment.

Evaluation of host genetics on outcome of tuberculosis infection due to differences in killer immunoglobulin-like receptor gene frequencies and haplotypes.

BACKGROUND: Outcome of Mycobacterium tuberculosis (Mtb) infection is affected by virulence of the infecting strain of Mtb, host environment, co-morbidities, and the genetic composition of the host, specifically the presence or absence of genes involved in immune responses/regulation. It is hypothesized that specific killer immunoglobulin-like receptor (KIR) genes may be associated with Mtb infection and clinical outcome. This cross-sectional study examined the KIR gene frequencies, profiles, and haplotypes of individuals with active tuberculosis, latent tuberculosis infection, compared to TB and HIV negative healthy controls. RESULTS: Analysis of KIR gene frequencies revealed differences among disease status groups, suggesting that enrichment or depletion of specific KIR genes may direct the disease outcome. Mtb infected individuals were more likely to have a centromeric-AA haplotype compared to controls. CONCLUSION: The differences in KIR gene frequencies and haplotypes may result in differential cytokine expression, contributing to different disease outcomes, and suggest a genetic influence on Mtb susceptibility and pathogenesis.

The role of G protein gene GNB3 C825T polymorphism in HIV-1 acquisition, progression and immune activation.

BACKGROUND: The GNB3 C825T polymorphism is associated with increased G protein-mediated signal transduction, SDF-1α-mediated lymphocyte chemotaxis, accelerated HIV-1 progression, and altered responses to antiretroviral therapy among Caucasian subjects. The GNB3 825T allele is highly prevalent in African populations, and as such any impact on HIV-1 acquisition or progression rates could have a dramatic impact. This study examines the association of the 825T polymorphism with HIV-1 acquisition, disease progression and immune activation in two African cohorts. GNB3 825 genotyping was performed for enrolees in both a commercial sex worker cohort and a perinatal HIV transmission (PHT) cohort in Nairobi, Kenya. Ex vivo immune activation was quantified by flow cytometry, and plasma chemokine levels were assessed by cytokine bead array. RESULTS: GNB3 genotype was not associated with sexual or vertical HIV-1 acquisition within these cohorts. Within the Pumwani cohort, GNB3 genotype did not affect HIV-1 disease progression among seroconverters or among HIV-1-positive individuals after adjustment for baseline CD4 count. Maternal CD4 decline and viral load increase in the PHT cohort did not differ between genotypes. Multi-parametric flow cytometry assessment of T cell activation (CD69, HLA-DR, CD38) and Treg frequency (CD25(+)FOXP3(+)) found no differences between genotype groups. Plasma SDF-1α, MIP-1ß and TRAIL levels quantified by cytokine bead array were also similar between groups. CONCLUSIONS: In contrast to previous reports, we were unable to provide evidence to suggest that the GNB3 C825T polymorphism affects HIV-1 acquisition or disease progression within African populations. Ex vivo immune activation and plasma chemokine levels were similarly unaffected by GNB3 genotype in both HIV-1-negative and HIV-1-positive individuals. The paucity of studies investigating the impact of GNB3 polymorphism among African populations and the lack of mechanistic studies make it difficult to assess the true biological significance of this polymorphism in HIV-1 infection.

For protection from HIV-1 infection, more might not be better: a systematic analysis of HIV Gag epitopes of two alleles associated with different outcomes of HIV-1 infection.

A subset of women in the Pumwani Sex Worker Cohort, established in 1985 in Nairobi, Kenya, remains uninfected despite repeated high-risk exposure (HIV-exposed, seronegative [HESN]) through active sex work. This HESN phenotype is associated with several alleles of human leukocyte antigens (HLAs) and specific CD8(+) and CD4(+) T cell responses to HIV-1. The associations of HLA alleles with differential HIV-1 infection are most likely due to their different abilities to present antigen and the different immune responses they induce. The characteristics of epitopes of HLA alleles associated with different outcomes of HIV-1 infection might therefore point to a vital clue for developing an effective vaccine. In this study, we systematically analyzed HIV-1 clade A and D Gag CD8(+) T cell epitopes of two HLA class I alleles associated with different outcomes of HIV-1 infection. Binding affinity and off-rates of the identified epitopes were determined. Gamma interferon (IFN-γ) enzyme-linked immunospot (ELISpot) assays with patient peripheral blood mononuclear cells (PBMCs) validated the epitopes. Epitope-specific CD8(+) T cells were further phenotyped for memory markers with tetramer staining. Our study showed that the protective allele A*01:01 recognizes only three Gag epitopes. By contrast, B*07:02, the allele associated with susceptibility, binds 30 epitope variants. These two alleles differ most importantly in the spectrum of Gag epitopes they can present and not in affinity, off-rates, the location of the epitopes, or epitope-specific Tem/Tcm frequencies. The binding of more epitopes and strong IFN-gamma ELISpot responses are associated with susceptibility to HIV-1 infection, while more focused antigen recognition of multiple subtypes is protective. Rational vaccine design should take these observations into account.

Epitope mapping of HIV-specific CD8+ T cell responses by multiple immunological readouts reveals distinct specificities defined by function.

The limited success of HIV vaccine candidates to date highlights our need to better characterize protective cell-mediated immunity (CMI). While HIV-specific CD8(+) T cell responses have been defined largely by measuring gamma interferon (IFN-γ), these responses are not always protective, and it is unclear whether the same epitopes would predominate if other functional parameters were examined. Here, we assessed the epitope specificity of HIV-specific CD8(+) T cell responses by multiparametric flow cytometry, measuring five CD8(+) T cell functions (IFN-γ, macrophage inflammatory protein 1ß [MIP-1ß], tumor necrosis factor alpha [TNF-α], interleukin-2 [IL-2], and proliferative capacity) in 24 chronically HIV-infected individuals. Sixty-nine epitope-specific responses to 50 epitopes within p24 were measured. Surprisingly, most epitope-specific responses were IFN-γ negative (50/69 responses). Many responses had polyfunctional (33%) and proliferative (19%) components. An inverse association between IL-2 and proliferation responses was also observed, contrary to what was described previously. We confirm that long-term nonprogressors (LTNP) have more polyfunctional responses and also have higher-magnitude and broader p24-specific proliferation and higher levels of IL-2 and TNF-α production than do progressing controls. Together, these data suggest that the specificity of CD8(+) T cell responses differs depending on the immunological readout, with a 3.5-fold increase in breadth detected by including multiple parameters. Furthermore, the identification of epitopes that elicit polyfunctional responses reinforces the need for the comprehensive evaluation of HIV vaccine candidates, and these epitopes may represent novel targets for CMI-based vaccines.

Loss of CD127 expression links immune activation and CD4(+) T cell loss in HIV infection.

Although chronic immune activation correlates with CD4(+) T cell loss in HIV infection, an understanding of the factors mediating T cell depletion remains incomplete. We propose that reduced expression of CD127 (IL-7 receptor alpha chain, IL-7Ralpha), induced by immune activation, contributes to CD4(+) T cell loss in HIV infection. In particular, loss of CD127 on central memory CD4(+) T cells (T(CM)) severely restrains the regenerative capacity of the memory component of the immune system, resulting in a limited ability to control T cell homeostasis. Studies from both pathogenic and controlled HIV infection indicate that the containment of immune activation and preservation of CD127 expression are critical to the stability of CD4(+) T cells in infection. A better understanding of the factors regulating CD127 expression in HIV disease, particularly on T(CM) cells, might unveil new approaches exploiting the IL-7/IL-7R receptor pathway to restore T cell homeostasis and promote immune reconstitution in HIV infection.