TLR9 plus STING Agonist Adjuvant Combination Induces Potent Neopeptide T Cell Immunity and Improves Immune Checkpoint Blockade Efficacy in a Tumor Model.

Immune checkpoint blockade (ICB) immunotherapies have emerged as promising strategies for the treatment of cancer; however, there remains a need to improve their efficacy. Determinants of ICB efficacy are the frequency of tumor mutations, the associated neoantigens, and the T cell response against them. Therefore, it is expected that neoantigen vaccinations that boost the antitumor T cell response would improve ICB therapy efficacy. The aim of this study was to develop a highly immunogenic vaccine using pattern recognition receptor agonists in combination with synthetic long peptides to induce potent neoantigen-specific T cell responses. We determined that the combination of the TLR9 agonist K-type CpG oligodeoxynucleotides (K3 CpG) with the STING agonist c-di-AMP (K3/c-di-AMP combination) significantly increased dendritic cell activation. We found that immunizing mice with 20-mer of either an OVA peptide, low-affinity OVA peptides, or neopeptides identified from mouse melanoma or lung mesothelioma, together with K3/c-di-AMP, induced potent Ag-specific T cell responses. The combined K3/c-di-AMP adjuvant formulation induced 10 times higher T cell responses against neopeptides than the TLR3 agonist polyinosinic:polycytidylic acid, a derivative of which is the leading adjuvant in clinical trials of neoantigen peptide vaccines. Moreover, we demonstrated that our K3/c-di-AMP vaccine formulation with 20-mer OVA peptide was capable of controlling tumor growth and improving survival in B16-F10-OVA tumor-bearing C57BL/6 mice and synergized with anti-PD-1 treatment. Together, our findings demonstrate that the K3/c-di-AMP vaccine formulation induces potent T cell immunity against synthetic long peptides and is a promising candidate to improve neoantigen vaccine platform.

The microRNA miR-155 controls CD8(+) T cell responses by regulating interferon signaling.

We found upregulation of expression of the microRNA miR-155 in primary effector and effector memory CD8(+) T cells, but low miR-155 expression in naive and central memory cells. Antiviral CD8(+) T cell responses and viral clearance were impaired in miR-155-deficient mice, and this defect was intrinsic to CD8(+) T cells, as miR-155-deficient CD8(+) T cells mounted greatly diminished primary and memory responses. Conversely, miR-155 overexpression augmented antiviral CD8(+) T cell responses in vivo. Gene-expression profiling showed that miR-155-deficient CD8(+) T cells had enhanced type I interferon signaling and were more susceptible to interferon's antiproliferative effect. Inhibition of the type I interferon-associated transcription factors STAT1 or IRF7 resulted in enhanced responses of miR-155-deficient CD8(+) T cells in vivo. We have thus identified a previously unknown role for miR-155 in regulating responsiveness to interferon and CD8(+) T cell responses to pathogens in vivo.

MicroRNA-139 Expression Is Dispensable for the Generation of Influenza-Specific CD8+ T Cell Responses.

MicroRNAs (miRNAs/miRs) are small, endogenous noncoding RNAs that are important post-transcriptional regulators with clear roles in the development of the immune system and immune responses. Using miRNA microarray profiling, we characterized the expression profile of naive and in vivo generated murine effector antiviral CD8+ T cells. We observed that out of 362 measurable mature miRNAs, 120 were differentially expressed by at least 2-fold in influenza-specific effector CD8+ CTLs compared with naive CD8+ T cells. One miRNA found to be highly downregulated on both strands in effector CTLs was miR-139. Because previous studies have indicated a role for miR-139-mediated regulation of CTL effector responses, we hypothesized that deletion of miR-139 would enhance antiviral CTL responses during influenza virus infection. We generated miR-139-/- mice or overexpressed miR-139 in T cells to assess the functional contribution of miR-139 expression in CD8+ T cell responses. Our study demonstrates that the development of naive T cells and generation or differentiation of effector or memory CD8+ T cell responses to influenza virus infection are not impacted by miR-139 deficiency or overexpression; yet, miR-139-/- CD8+ T cells are outcompeted by wild-type CD8+ T cells in a competition setting and demonstrate reduced responses to Listeria monocytogenes Using an in vitro model of T cell exhaustion, we confirmed that miR-139 expression similarly does not impact the development of T cell exhaustion. We conclude that despite significant downregulation of miR-139 following in vivo and in vitro activation, miR-139 expression is dispensable for influenza-specific CTL responses.

Immunogenicity of an Additional mRNA-1273 SARS-CoV-2 Vaccination in People With HIV With Hyporesponse After Primary Vaccination.

BACKGROUND: The COVIH study is a prospective coronavirus disease 2019 (COVID-19) vaccination study in 1154 people with HIV (PWH), of whom 14% showed reduced antibody levels after primary vaccination. We evaluated whether an additional vaccination boosts immune responses in these hyporesponders. METHODS: The primary end point was the increase in antibodies 28 days after additional mRNA-1273 vaccination. Secondary end points included neutralizing antibodies, S-specific T-cell and B-cell responses, and reactogenicity. RESULTS: Of the 66 participants, 40 previously received 2 doses ChAdOx1-S, 22 received 2 doses BNT162b2, and 4 received a single dose Ad26.COV2.S. The median age was 63 years (interquartile range [IQR], 60-66), 86% were male, and median CD4+ T-cell count was 650/µL (IQR, 423-941). The mean S1-specific antibody level increased from 35 binding antibody units (BAU)/mL (95% confidence interval [CI], 24-46) to 4317 BAU/mL (95% CI, 3275-5360) (P < .0001). Of all participants, 97% showed an adequate response and the 45 antibody-negative participants all seroconverted. A significant increase in the proportion of PWH with ancestral S-specific CD4+ T cells (P = .04) and S-specific B cells (P = .02) was observed. CONCLUSIONS: An additional mRNA-1273 vaccination induced a robust serological response in 97% of PWH with a hyporesponse after primary vaccination. Clinical Trials Registration. EUCTR2021-001054-57-N.

Correction: Immunogenicity and reactogenicity of SARS-CoV-2 vaccines in people living with HIV in the Netherlands: A nationwide prospective cohort study.

[This corrects the article DOI: 10.1371/journal.pmed.1003979.].

Immunogenicity and reactogenicity of SARS-CoV-2 vaccines in people living with HIV in the Netherlands: A nationwide prospective cohort study.

BACKGROUND: Vaccines can be less immunogenic in people living with HIV (PLWH), but for SARS-CoV-2 vaccinations this is unknown. In this study we set out to investigate, for the vaccines currently approved in the Netherlands, the immunogenicity and reactogenicity of SARS-CoV-2 vaccinations in PLWH. METHODS AND FINDINGS: We conducted a prospective cohort study to examine the immunogenicity of BNT162b2, mRNA-1273, ChAdOx1-S, and Ad26.COV2.S vaccines in adult PLWH without prior COVID-19, and compared to HIV-negative controls. The primary endpoint was the anti-spike SARS-CoV-2 IgG response after mRNA vaccination. Secondary endpoints included the serological response after vector vaccination, anti-SARS-CoV-2 T-cell response, and reactogenicity. Between 14 February and 7 September 2021, 1,154 PLWH (median age 53 [IQR 44-60] years, 85.5% male) and 440 controls (median age 43 [IQR 33-53] years, 28.6% male) were included in the final analysis. Of the PLWH, 884 received BNT162b2, 100 received mRNA-1273, 150 received ChAdOx1-S, and 20 received Ad26.COV2.S. In the group of PLWH, 99% were on antiretroviral therapy, 97.7% were virally suppressed, and the median CD4+ T-cell count was 710 cells/µL (IQR 520-913). Of the controls, 247 received mRNA-1273, 94 received BNT162b2, 26 received ChAdOx1-S, and 73 received Ad26.COV2.S. After mRNA vaccination, geometric mean antibody concentration was 1,418 BAU/mL in PLWH (95% CI 1322-1523), and after adjustment for age, sex, and vaccine type, HIV status remained associated with a decreased response (0.607, 95% CI 0.508-0.725, p < 0.001). All controls receiving an mRNA vaccine had an adequate response, defined as >300 BAU/mL, whilst in PLWH this response rate was 93.6%. In PLWH vaccinated with mRNA-based vaccines, higher antibody responses were predicted by CD4+ T-cell count 250-500 cells/µL (2.845, 95% CI 1.876-4.314, p < 0.001) or >500 cells/µL (2.936, 95% CI 1.961-4.394, p < 0.001), whilst a viral load > 50 copies/mL was associated with a reduced response (0.454, 95% CI 0.286-0.720, p = 0.001). Increased IFN-γ, CD4+ T-cell, and CD8+ T-cell responses were observed after stimulation with SARS-CoV-2 spike peptides in ELISpot and activation-induced marker assays, comparable to controls. Reactogenicity was generally mild, without vaccine-related serious adverse events. Due to the control of vaccine provision by the Dutch National Institute for Public Health and the Environment, there were some differences between vaccine groups in the age, sex, and CD4+ T-cell counts of recipients. CONCLUSIONS: After vaccination with BNT162b2 or mRNA-1273, anti-spike SARS-CoV-2 antibody levels were reduced in PLWH compared to HIV-negative controls. To reach and maintain the same serological responses as HIV-negative controls, additional vaccinations are probably required. TRIAL REGISTRATION: The trial was registered in the Netherlands Trial Register (NL9214). https://www.trialregister.nl/trial/9214.

Rapid in vitro generation of bona fide exhausted CD8+ T cells is accompanied by Tcf7 promotor methylation.

Exhaustion is a dysfunctional state of cytotoxic CD8+ T cells (CTL) observed in chronic infection and cancer. Current in vivo models of CTL exhaustion using chronic viral infections or cancer yield very few exhausted CTL, limiting the analysis that can be done on these cells. Establishing an in vitro system that rapidly induces CTL exhaustion would therefore greatly facilitate the study of this phenotype, identify the truly exhaustion-associated changes and allow the testing of novel approaches to reverse or prevent exhaustion. Here we show that repeat stimulation of purified TCR transgenic OT-I CTL with their specific peptide induces all the functional (reduced cytokine production and polyfunctionality, decreased in vivo expansion capacity) and phenotypic (increased inhibitory receptors expression and transcription factor changes) characteristics of exhaustion. Importantly, in vitro exhausted cells shared the transcriptomic characteristics of the gold standard of exhaustion, CTL from LCMV cl13 infections. Gene expression of both in vitro and in vivo exhausted CTL was distinct from T cell anergy. Using this system, we show that Tcf7 promoter DNA methylation contributes to TCF1 downregulation in exhausted CTL. Thus this novel in vitro system can be used to identify genes and signaling pathways involved in exhaustion and will facilitate the screening of reagents that prevent/reverse CTL exhaustion.

The miR-200c/141-ZEB2-TGFß axis is aberrant in human T-cell prolymphocytic leukemia.

T-cell prolymphocytic leukemia (T-PLL) is mostly characterized by aberrant expansion of small- to medium-sized prolymphocytes with a mature post-thymic phenotype, high aggressiveness of the disease and poor prognosis. However, T-PLL is more heterogeneous with a wide range of clinical, morphological, and molecular features, which occasionally impedes the diagnosis. We hypothesized that T-PLL consists of phenotypic and/or genotypic subgroups that may explain the heterogeneity of the disease. Multi-dimensional immuno-phenotyping and gene expression profiling did not reveal clear T-PLL subgroups, and no clear T-cell receptor a or ß CDR3 skewing was observed between different T-PLL cases. We revealed that the expression of microRNA (miRNA) is aberrant and often heterogeneous in T-PLL. We identified 35 miRNA that were aberrantly expressed in T-PLL with miR-200c/141 as the most differentially expressed cluster. High miR- 200c/141 and miR-181a/181b expression was significantly correlated with increased white blood cell counts and poor survival. Furthermore, we found that overexpression of miR-200c/141 correlated with downregulation of their targets ZEB2 and TGFßR3 and aberrant TGFß1- induced phosphorylated SMAD2 (p-SMAD2) and p-SMAD3, indicating that the TGFß pathway is affected in T-PLL. Our results thus highlight the potential role for aberrantly expressed oncogenic miRNA in T-PLL and pave the way for new therapeutic targets in this disease.

Transcriptome Analysis of Infected and Bystander Type 2 Alveolar Epithelial Cells during Influenza A Virus Infection Reveals In Vivo Wnt Pathway Downregulation.

Influenza virus outbreaks remain a serious threat to public health. A greater understanding of how cells targeted by the virus respond to the infection can provide insight into the pathogenesis of disease. Here we examined the transcriptional profile of in vivo-infected and uninfected type 2 alveolar epithelial cells (AEC) in the lungs of influenza virus-infected mice. We show for the first time the unique gene expression profiles induced by the in vivo infection of AEC as well as the transcriptional response of uninfected bystander cells. This work allows us to distinguish the direct and indirect effects of infection at the cellular level. Transcriptome analysis revealed that although directly infected and bystander AEC from infected animals shared many transcriptome changes compared to AEC from uninfected animals, directly infected cells produce more interferon and express lower levels of Wnt signaling-associated transcripts, while concurrently expressing more transcripts associated with cell death pathways, than bystander uninfected AEC. The Wnt signaling pathway was downregulated in both in vivo-infected AEC and in vitro-infected human lung epithelial A549 cells. Wnt signaling did not affect type I and III interferon production by infected A549 cells. Our results reveal unique transcriptional changes that occur within infected AEC and show that influenza virus downregulates Wnt signaling. In light of recent findings that Wnt signaling is essential for lung epithelial stem cells, our findings reveal a mechanism by which influenza virus may affect host lung repair.IMPORTANCE Influenza virus infection remains a major public health problem. Utilizing a recombinant green fluorescent protein-expressing influenza virus, we compared the in vivo transcriptomes of directly infected and uninfected bystander cells from infected mouse lungs and discovered many pathways uniquely regulated in each population. The Wnt signaling pathway was downregulated in directly infected cells and was shown to affect virus but not interferon production. Our study is the first to discern the in vivo transcriptome changes induced by direct viral infection compared to mere exposure to the lung inflammatory milieu and highlight the downregulation of Wnt signaling. This downregulation has important implications for understanding influenza virus pathogenesis, as Wnt signaling is critical for lung epithelial stem cells and lung epithelial cell differentiation. Our findings reveal a mechanism by which influenza virus may affect host lung repair and suggest interventions that prevent damage or accelerate recovery of the lung.

T cell toxicity of HIV latency reversing agents.

Combination antiretroviral therapy reduces morbidity and mortality in HIV infected patients. However, the cure of HIV infection is hindered by the persistence of the latent HIV reservoir. Latency reversing agents (LRAs) are developed to target the HIV latently infected cells for HIV reactivation. In addition to reversal of HIV latency, the eradication of HIV latently infected cells will require effector HIV-specific CD8+ T cells. Therefore it is imperative we understand how LRAs affect immune cells. We have performed a comparative in depth analysis of the cytotoxicity of several compounds belonging to four LRA classes on T cells, B cells, and NK cells. In addition, the effect of these LRAs on activation and inhibitory receptor expression of CD8+ T cells was examined. We show that the HDAC inhibitors romidepsin and panobinostat are highly cytotoxic for CD4+ and CD8+ T cells, whereas the PKC agonists bryostatin and prostratin and BET inhibitors JQ1 and OXT-015 were less cytotoxic. The BAF inhibitors CAPE and pyrimethamine exhibit no cytotoxicity. Drug-specific cytotoxicity on CD8+ T cells was comparable between healthy controls and cART-treated HIV-infected patients. Bryostatin and both BET inhibitors downregulated the expression of CD279 on CD8+ T cells without affecting their activation. Our comparison of LRAs identified differences in cytotoxicity between LRA classes and members within a class and suggests that some LRAs such as bryostatin and BET inhibitors may also downregulate inhibitory receptors on activated HIV-specific CD8+ T cells. These findings may guide the use of LRAs that have the capacity to preserve or restore CD8+ T cell immunity.

Rapid Evolution of the CD8+ TCR Repertoire in Neonatal Mice.

Currently, there is little consensus regarding the most appropriate animal model to study acute infection and the virus-specific CD8(+) T cell (CTL) responses in neonates. TCRß high-throughput sequencing in naive CTL of differently aged neonatal mice was performed, which demonstrated differential Vß family gene usage. Using an acute influenza infection model, we examined the TCR repertoire of the CTL response in neonatal and adult mice infected with influenza type A virus. Three-day-old mice mounted a greatly reduced primary NP(366-374)-specific CTL response when compared with 7-d-old and adult mice, whereas secondary CTL responses were normal. Analysis of NP(366-374)-specific CTL TCR repertoire revealed different Vß gene usage and greatly reduced public clonotypes in 3-d-old neonates. This could underlie the impaired CTL response in these neonates. To directly test this, we examined whether controlling the TCR would restore neonatal CTL responses. We performed adoptive transfers of both nontransgenic and TCR-transgenic OVA(257-264)-specific (OT-I) CD8(+) T cells into influenza-infected hosts, which revealed that naive neonatal and adult OT-I cells expand equally well in neonatal and adult hosts. In contrast, nontransgenic neonatal CD8(+) T cells when transferred into adults failed to expand. We further demonstrate that differences in TCR avidity may contribute to decreased expansion of the endogenous neonatal CTL. These studies highlight the rapid evolution of the neonatal TCR repertoire during the first week of life and show that impaired neonatal CTL immunity results from an immature TCR repertoire, rather than intrinsic signaling defects or a suppressive environment.

Phosphatidylinositol 3-Kinase p110δ Isoform Regulates CD8+ T Cell Responses during Acute Viral and Intracellular Bacterial Infections.

The p110δ isoform of PI3K is known to play an important role in immunity, yet its contribution to CTL responses has not been fully elucidated. Using murine p110δ-deficient CD8(+) T cells, we demonstrated a critical role for the p110δ subunit in the generation of optimal primary and memory CD8(+) T cell responses. This was demonstrated in both acute viral and intracellular bacterial infections in mice. We show that p110δ signaling is required for CD8(+) T cell activation, proliferation and effector cytokine production. We provide evidence that the effects of p110δ signaling are mediated via Akt activation and through the regulation of TCR-activated oxidative phosphorylation and aerobic glycolysis. In light of recent clinical trials that employ drugs targeting p110δ in certain cancers and other diseases, our study suggests caution in using these drugs in patients, as they could potentially increase susceptibility to infectious diseases. These studies therefore reveal a novel and direct role for p110δ signaling in in vivo CD8(+) T cell immunity to microbial pathogens.

Type I interferon upregulates Bak and contributes to T cell loss during human immunodeficiency virus (HIV) infection.

The role of Type I interferon (IFN) during pathogenic HIV and SIV infections remains unclear, with conflicting observations suggesting protective versus immunopathological effects. We therefore examined the effect of IFNα/ß on T cell death and viremia in HIV infection. Ex vivo analysis of eight pro- and anti-apoptotic molecules in chronic HIV-1 infection revealed that pro-apoptotic Bak was increased in CD4+ T cells and correlated directly with sensitivity to CD95/Fas-mediated apoptosis and inversely with CD4+ T cell counts. Apoptosis sensitivity and Bak expression were primarily increased in effector memory T cells. Knockdown of Bak by RNA interference inhibited CD95/Fas-induced death of T cells from HIV-1-infected individuals. In HIV-1-infected patients, IFNα-stimulated gene expression correlated positively with ex vivo T cell Bak levels, CD95/Fas-mediated apoptosis and viremia and negatively with CD4+ T cell counts. In vitro IFNα/ß stimulation enhanced Bak expression, CD95/Fas expression and CD95/Fas-mediated apoptosis in healthy donor T cells and induced death of HIV-specific CD8+ T cells from HIV-1-infected patients. HIV-1 in vitro sensitized T cells to CD95/Fas-induced apoptosis and this was Toll-like receptor (TLR)7/9- and Type I IFN-dependent. This sensitization by HIV-1 was due to an indirect effect on T cells, as it occurred in peripheral blood mononuclear cell cultures but not purified CD4+ T cells. Finally, peak IFNα levels and viral loads correlated negatively during acute SIV infection suggesting a potential antiviral effect, but positively during chronic SIV infection indicating that either the virus drives IFNα production or IFNα may facilitate loss of viral control. The above findings indicate stage-specific opposing effects of Type I IFNs during HIV-1 infection and suggest a novel mechanism by which these cytokines contribute to T cell depletion, dysregulation of cellular immunity and disease progression.

Abasic phosphorothioate oligomers inhibit HIV-1 reverse transcription and block virus transmission across polarized ectocervical organ cultures.

In the absence of universally available antiretroviral (ARV) drugs or a vaccine against HIV-1, microbicides may offer the most immediate hope for controlling the AIDS pandemic. The most advanced and clinically effective microbicides are based on ARV agents that interfere with the earliest stages of HIV-1 replication. Our objective was to identify and characterize novel ARV-like inhibitors, as well as demonstrate their efficacy at blocking HIV-1 transmission. Abasic phosphorothioate 2' deoxyribose backbone (PDB) oligomers were evaluated in a variety of mechanistic assays and for their ability to inhibit HIV-1 infection and virus transmission through primary human cervical mucosa. Cellular and biochemical assays were used to elucidate the antiviral mechanisms of action of PDB oligomers against both lab-adapted and primary CCR5- and CXCR4-utilizing HIV-1 strains, including a multidrug-resistant isolate. A polarized cervical organ culture was used to test the ability of PDB compounds to block HIV-1 transmission to primary immune cell populations across ectocervical tissue. The antiviral activity and mechanisms of action of PDB-based compounds were dependent on oligomer size, with smaller molecules preventing reverse transcription and larger oligomers blocking viral entry. Importantly, irrespective of molecular size, PDBs potently inhibited virus infection and transmission within genital tissue samples. Furthermore, the PDB inhibitors exhibited excellent toxicity and stability profiles and were found to be safe for vaginal application in vivo. These results, coupled with the previously reported intrinsic anti-inflammatory properties of PDBs, support further investigations in the development of PDB-based topical microbicides for preventing the global spread of HIV-1.

SARS-CoV-2-specific immune responses converge in kidney disease patients and controls with hybrid immunity.

Healthy individuals with hybrid immunity, due to a SARS-CoV-2 infection prior to first vaccination, have stronger immune responses compared to those who were exclusively vaccinated. However, little is known about the characteristics of antibody, B- and T-cell responses in kidney disease patients with hybrid immunity. Here, we explored differences between kidney disease patients and controls with hybrid immunity after asymptomatic or mild coronavirus disease-2019 (COVID-19). We studied the kinetics, magnitude, breadth and phenotype of SARS-CoV-2-specific immune responses against primary mRNA-1273 vaccination in patients with chronic kidney disease or on dialysis, kidney transplant recipients, and controls with hybrid immunity. Although vaccination alone is less immunogenic in kidney disease patients, mRNA-1273 induced a robust immune response in patients with prior SARS-CoV-2 infection. In contrast, kidney disease patients with hybrid immunity develop SARS-CoV-2 antibody, B- and T-cell responses that are equally strong or stronger than controls. Phenotypic analysis showed that Spike (S)-specific B-cells varied between groups in lymph node-homing and memory phenotypes, yet S-specific T-cell responses were phenotypically consistent across groups. The heterogeneity amongst immune responses in hybrid immune kidney patients warrants further studies in larger cohorts to unravel markers of long-term protection that can be used for the design of targeted vaccine regimens.

The cytokine network of acute HIV infection: a promising target for vaccines and therapy to reduce viral set-point?

Cytokines play a central role in the pathogenesis of many diseases, including HIV infection. However, the role of the cytokine network in early HIV infection is only now starting to be elucidated. A number of studies conducted in recent years have indicated that cytokines of the acute/early stages of HIV and SIV infection can impact viral set-point months later, and this is of critical importance since viral set-point during chronic HIV infection affects virus transmission and disease progression. This raises the question whether modulating the cytokine environment during acute/early HIV infection can be a target for novel approaches to develop a vaccine and therapeutics. In this review we focus on the kinetics and function of cytokines during acute HIV and SIV infection and how these may impact viral set-point. We also discuss unresolved questions that are essential for our understanding of the role of acute infection cytokines in HIV infection and that, if answered, may suggest novel therapeutic and vaccine strategies to control the worldwide HIV pandemic.

IL-15 delays suppression and fails to promote immune reconstitution in virally suppressed chronically SIV-infected macaques.

Human immunodeficiency virus (HIV) infection is characterized by a progressive loss of memory CD4(+) T cells in multiple tissues, especially at mucosal surfaces where most of these cells reside. Although antiretroviral therapy (ART) suppresses viral replication and promotes the recovery of peripheral CD4(+) T cells, HIV-infected patients fail to fully reconstitute the CD4(+) T-cell pool at mucosal sites. IL-15 has been shown to preferentially expand memory-phenotype T cells and promote their migration to nonlymphoid tissues. Here we examined IL-15 treatment in combination with highly active ART in chronically SIV-infected rhesus macaques and found that IL-15 delayed viral suppression and failed to enhance ART-induced total and antigen-specific CD4(+) T-cell reconstitution at mucosal and lymphoid sites. IL-15 was able to induce the transient proliferation of SIV-specific, CMV-specific, and total memory CD8(+) T cells, but not of SIV-specific or total CD4(+) T cells. Moreover, upon treatment interruption, macaques receiving combined IL-15+ART lost CD4(+) T cells faster than those receiving ART alone. These results suggest that the combination of IL-15 with highly active ART is not more efficient than ART alone in promoting CD4(+) T-cell recovery in HIV-infected individuals and may accelerate CD4+ T-cell loss after treatment interruption.

Dendritic cells and CD28 costimulation are required to sustain virus-specific CD8+ T cell responses during the effector phase in vivo.

Although much is known about the initiation of immune responses, much less is known about what controls the effector phase. CD8(+) T cell responses are believed to be programmed in lymph nodes during priming without any further contribution by dendritic cells (DCs) and Ag. In this study, we report the requirement for DCs, Ag, and CD28 costimulation during the effector phase of the CD8(+) T cell response. Depleting DCs or blocking CD28 after day 6 of primary influenza A virus infection decreases the virus-specific CD8(+) T cell response by inducing apoptosis, and this results in decreased viral clearance. Furthermore, effector CD8(+) T cells adoptively transferred during the effector phase fail to expand without DC, CD28 costimulation, and cognate Ag. The absence of costimulation also leads to reduced survival of virus-specific effector cells as they undergo apoptosis mediated by the proapoptotic molecule Bim. Finally, IL-2 treatment restored the effector response in the absence of CD28 costimulation. Thus, in contrast to naive CD8(+) T cells, which undergo an initial Ag-independent proliferation, effector CD8(+) T cells expanding in the lungs during the effector phase require Ag, CD28 costimulation, and DCs for survival and expansion. These requirements would greatly impair effector responses against viruses and tumors that are known to inhibit DC maturation and in chronic infections and aging where CD28(-/-) CD8(+) T cells accumulate.

Targeted multiomics in childhood-onset SLE reveal distinct biological phenotypes associated with disease activity: results from an explorative study.

OBJECTIVE: To combine targeted transcriptomic and proteomic data in an unsupervised hierarchical clustering method to stratify patients with childhood-onset SLE (cSLE) into similar biological phenotypes, and study the immunological cellular landscape that characterises the clusters. METHODS: Targeted whole blood gene expression and serum cytokines were determined in patients with cSLE, preselected on disease activity state (at diagnosis, Low Lupus Disease Activity State (LLDAS), flare). Unsupervised hierarchical clustering, agnostic to disease characteristics, was used to identify clusters with distinct biological phenotypes. Disease activity was scored by clinical SELENA-SLEDAI (Safety of Estrogens in Systemic Lupus Erythematosus National Assessment-Systemic Lupus Erythematosus Disease Activity Index). High-dimensional 40-colour flow cytometry was used to identify immune cell subsets. RESULTS: Three unique clusters were identified, each characterised by a set of differentially expressed genes and cytokines, and by disease activity state: cluster 1 contained primarily patients in LLDAS, cluster 2 contained mainly treatment-naïve patients at diagnosis and cluster 3 contained a mixed group of patients, namely in LLDAS, at diagnosis and disease flare. The biological phenotypes did not reflect previous organ system involvement and over time, patients could move from one cluster to another. Healthy controls clustered together in cluster 1. Specific immune cell subsets, including CD11c+ B cells, conventional dendritic cells, plasmablasts and early effector CD4+ T cells, differed between the clusters. CONCLUSION: Using a targeted multiomic approach, we clustered patients into distinct biological phenotypes that are related to disease activity state but not to organ system involvement. This supports a new concept where choice of treatment and tapering strategies are not solely based on clinical phenotype but includes measuring novel biological parameters.

Classification of Inflammation of Unknown Origin patients based on RNA-seq and SomaScan data.

A preliminary analysis was conducted on data acquired from RNA sequencing and SomaScan platforms, for the classification of patients with Inflammation of Unknown Origin. To this end, a multimodal data integration approach was designed, by combining the two platforms, in order to assess the potentiality of learning estimators, using the differentially expressed features from the independent profiling experiments of both platforms. The classification framing was the differentiation of Inflammation of Unknown Origin patients against a multitude of Systemic Autoinflammatory disease patients. Separate false discovery rate analyses were performed on each dataset to extract statistically significant features between the two designated sample groups. Genomic analysis managed higher overall classification metrics compared to proteomic analysis, averaging an ~19% increase overall metrics and classifiers, with a ~0.07% increase in standard error. The multimodal data integration approach achieved similar results to the individual platforms' analyses. More specifically, it managed the same classification accuracy, sensitivity, and specificity scores as the best individual analysis, with the simple Logistic Regression estimator.Clinical Relevance- This study highlights the advantage of exploiting RNA sequencing data to identify potential Inflammation of Unknown Origin disease specific biomarkers, even against other Systemic Autoinflammatory diseases. These findings are further emphasized given the non-apparent clinical discrepancy between Inflammation of Unknown Origin and other Systemic Autoinflammatory diseases.