Antiviral responses induced by Tdap-IPV vaccination are associated with persistent humoral immunity to Bordetella pertussis.

Many countries continue to experience pertussis epidemics despite widespread vaccination. Waning protection after booster vaccination has highlighted the need for a better understanding of the immunological factors that promote durable protection. Here we apply systems vaccinology to investigate antibody responses in adolescents in the Netherlands (N = 14; NL) and the United Kingdom (N = 12; UK) receiving a tetanus-diphtheria-acellular pertussis-inactivated poliovirus (Tdap-IPV) vaccine. We report that early antiviral and interferon gene expression signatures in blood correlate to persistence of pertussis-specific antibody responses. Single-cell analyses of the innate response identified monocytes and myeloid dendritic cells (MoDC) as principal responders that upregulate antiviral gene expression and type-I interferon cytokine production. With public data, we show that Tdap vaccination stimulates significantly lower antiviral/type-I interferon responses than Tdap-IPV, suggesting that IPV may promote antiviral gene expression. Subsequent in vitro stimulation experiments demonstrate TLR-dependent, IPV-specific activation of the pro-inflammatory p38 MAP kinase pathway in MoDCs. Together, our data provide insights into the molecular host response to pertussis booster vaccination and demonstrate that IPV enhances innate immune activity associated with persistent, pertussis-specific antibody responses.

CXCL12 and CXCL13 Cytokine Serum Levels Are Associated with the Magnitude and the Quality of SARS-CoV-2 Humoral Responses.

A better understanding of the immunological markers associated with long-lasting immune responses to SARS-CoV-2 infection is of paramount importance. In the present study, we characterized SARS-CoV-2-specific humoral responses in hospitalized (ICU and non-ICU) and non-hospitalized individuals at six months post-onset of symptoms (POS) (N = 95). We showed that the proportion of individuals with detectable anti-SARS-CoV-2 IgG or neutralizing (NAb) responses and the titers of antibodies were significantly reduced in non-hospitalized individuals, compared to ICU- or non-ICU-hospitalized individuals at 6 months POS. Interestingly, SARS-CoV-2-specific memory B cells persist at 6 months POS in both ICU and non-ICU patients and were enriched in cells harboring an activated and/or exhausted phenotype. The frequency/phenotype of SARS-CoV-2-specific memory B cells and the magnitude of IgG or NAb responses at 6 months POS correlated with the serum immune signature detected at patient admission. In particular, the serum levels of CXCL13, IL-1RA, and G-CSF directly correlated with the frequency of Spike-specific B cells and the magnitude of Spike-specific IgG or NAb, while the serum levels of CXCL12 showed an antagonizing effect. Our results indicate that the balance between CXCL12 and CXCL13 is an early marker associated with the magnitude and the quality of the SARS-CoV-2 humoral memory.

The deficiency in Th2-like Tfh cells affects the maturation and quality of HIV-specific B cell response in viremic infection.

Optimal T follicular helper (Tfh) cells function is important to promote the development of germinal centers and maturation of high affinity antigen-specific B cells. We have found that the expression of CXCR3 defines distinct Tfh subsets: CXCR3+ Th1-like Tfh cells mainly producing single IFN-γ and dual IL-21/IFN-γ and CXCR3- Th2-like Tfh cells mainly producing single IL-4 and dual IL-21/IL-4 cytokines. CXCR3- Th2-like Tfhs are significantly reduced during ongoing HIV replication. While the percentage of Th2-like Tfh cells correlates with that of total and cycling HIV-specific B cells, the percentage of CXCR3+ Th1-like Tfhs correlates with HIV-specific B cells expressing T-bet and CXCR3. Of note, only IL-4 and IL-21 cytokines boosted efficient maturation of HIV-specific B cells while IFN-γ induced expression of T-bet and CXCR3 in B cells. Interestingly, total and HIV-specific CXCR3+ B cells showed lower rate of somatic hypermutation, as compared to CXCR3- B cells. Therefore, the imbalance in Th2/Th1-like Tfhs affects B cell responses in viremic HIV infection.

SLAMF Receptor Expression Identifies an Immune Signature That Characterizes Systemic Lupus Erythematosus.

Systemic lupus erythematosus (SLE) is a chronic autoimmune disease of unknown etiology, linked to alterations in both the innate and the adaptive immune system. Due to the heterogeneity of the clinical presentation, the diagnosis of SLE remains complicated and is often made years after the first symptoms manifest, delaying treatment, and worsening the prognosis. Several studies have shown that signaling lymphocytic activation molecule family (SLAMF) receptors are aberrantly expressed and dysfunctional in SLE immune cells, contributing to the altered cellular function observed in these patients. The aim of this study was to determine whether altered co-/expression of SLAMF receptors on peripheral blood mononuclear cells (PBMC) identifies SLE characteristic cell populations. To this end, single cell mass cytometry and bioinformatic analysis were exploited to compare SLE patients to healthy and autoimmune diseases controls. First, the expression of each SLAMF receptor on all PBMC populations was investigated. We observed that SLAMF1+ B cells (referred to as SLEB1) were increased in SLE compared to controls. Furthermore, the frequency of SLAMF4+ monocytes and SLAMF4+ NK were inversely correlated with disease activity, whereas the frequency SLAMF1+ CD4+ TDEM cells were directly correlated with disease activity. Consensus clustering analysis identified two cell clusters that presented significantly increased frequency in SLE compared to controls: switch memory B cells expressing SLAMF1, SLAMF3, SLAMF5, SLAMF6 (referred to as SLESMB) and circulating T follicular helper cells expressing the same SLAMF receptors (referred to as SLEcTFH). Finally, the robustness of the identified cell populations as biomarkers for SLE was evaluated through ROC curve analysis. The combined measurement of SLEcTFH and SLEB1 or SLESMB cells identified SLE patients in 90% of cases. In conclusion, this study identified an immune signature for SLE based on the expression of SLAMF receptors on PBMC, further highlighting the involvement of SLAMF receptors in the pathogenesis of SLE.

Humoral Responses Against Variants of Concern by COVID-19 mRNA Vaccines in Immunocompromised Patients.

Importance: There are limited comparative data on the durability of neutralizing antibody (nAb) responses elicited by messenger RNA (mRNA) vaccines against the SARS-CoV-2 variants of concern (VOCs) in immunocompromised patients and healthy controls. Objective: To assess the humoral responses after vaccination with BNT162b2 (Pfizer-BioNTech) or mRNA-1273 (Moderna) vaccines. Design, Setting, and Participants: In this prospective, longitudinal monocentric comparative effectiveness study conducted at the Lausanne University Hospital, binding IgG anti-spike antibody and nAb levels were measured at 1 week, 1 month, 3 months, and 6 months after vaccination with mRNA-1273 (24.6% of participants) or BNT162b2 (75.3% of participants). Interventions: All participants received 2 doses of either mRNA-1273 or BNT162b2 vaccines 4 to 6 weeks apart. Main Outcomes and Measures: The primary outcome of the study was the persistence of nAb responses against the original, nonvariant SARS-CoV-2 (2019-nCoV) and different VOCs at 6 months after vaccination. Key secondary outcomes were associations of the type of mRNA vaccine, the underlying disease, and the treatment with the response to vaccination. Results: Among the 841 participants enrolled between January 14 and August 8, 2021, the patient population comprised 637 participants (mean [SD] age, 61.8 [13.7] years; 386 [60.6%] female), and the healthy control population comprised 204 participants (mean [SD] age, 45.9 [12.0] years; 144 [70.6%] female). There were 399 patients with solid cancers, 101 with hematologic cancers, 38 with solid organ transplants, 99 with autoimmune diseases, and 204 healthy controls. More than 15 000 nAb determinations were performed against the original, nonvariant 2019-nCoV and the Alpha, Beta, Gamma, and Delta variants. The proportions of nAbs and their titers decreased in all study groups at 6 months after vaccination, with the greatest decreases for the Beta and Delta variants. For Beta, the proportion decreased to a median (SE) of 39.2% (5.5%) in those with hematologic cancers, 44.8% (2.7%) in those with solid cancers, 23.1% (8.3%) in those with solid organ transplants, and 22.7% (4.8%) in those with autoimmune diseases compared with 52.1% (4.2%) in healthy controls. For Delta, the proportions decreased to 41.8% (5.6%) in participants with hematologic cancer, 51.9% (2.7%) in those with solid cancers, 26.9% (8.7%) in those with solid organ transplants, and 30.7% (5.3%) in those with autoimmune diseases compared with 56.9% (4.1%) healthy controls. Neutralizing antibody titers decreased 3.5- to 5-fold between month 1 and month 6, and the estimated duration of response was greater and more durable among those participants vaccinated with mRNA-1273. In participants with solid cancers, the estimated duration of nAbs against the Beta variant was 221 days with mRNA-1273 and 146 days with BNT162b2, and against the Delta variant, it was 226 days with mRNA-1273 and 161 with BNT162b2. The estimated duration of nAbs in participants with hematologic cancers was 113 and 127 days against Beta and Delta variants, respectively. Conclusions and Relevance: This comparative effectiveness study suggests that approximately half of patients with hematologic cancers and solid cancers, about 70% of patients with solid organ transplants or autoimmune diseases, and 40% of healthy controls have lost nAbs against the circulating VOCs at 6 months after vaccination. These findings may be helpful for developing the best boosting vaccination schedule especially in immunocompromised patients.

68Ga-DOTATOC PET/CT to detect immune checkpoint inhibitor-related myocarditis.

BACKGROUND: Immune checkpoint inhibitor (ICI)-related myocarditis is a rare but potentially fatal adverse event that can occur following ICI exposure. Early diagnosis and treatment are key to improve patient outcomes. Somatostatin receptor-based positron emission tomography-CT (PET/CT) showed promising results for the assessment of myocardial inflammation, yet information regarding its value for the diagnosis of ICI-related myocarditis, especially at the early stage, is limited. Thus, we investigated the value of 68Ga-DOTA(0)-Phe(1)-Tyr(3)-octreotide (68Ga-DOTATOC) PET/CT for the early detection and diagnosis of ICI-related myocarditis. METHODS: Consecutive patients with clinically suspected ICI-related myocarditis from July 2018 to February 2021 were retrospectively evaluated in this single-center study. All patients underwent imaging for the detection of ICI-related myocarditis using either cardiac magnetic resonance (CMR) imaging or 68Ga-DOTATOC PET/CT. PET/CT images were acquired 90 min after the injection of 2 MBq/kg 68Ga-DOTATOC with pathological myocardial uptake in the left ventricle (LV) suggestive of myocarditis defined using a myocardium-to-background ratio of peak standard uptake value to mean intracavitary LV standard uptake (MBRpeak) value above 1.6. Patients had a full cardiological work-up including ECG, echocardiography, serum cardiac troponin I (cTnI), cardiac troponin T and creatine kinase (CK), CK-MB. Endomyocardial biopsy and inflammatory cytokine markers were also analyzed. The detection rate of ICI-related myocarditis using 68Ga-DOTATOC PET/CT and CMR was assessed. RESULTS: A total of 11 patients had clinically suspected ICI-related myocarditis; 9 underwent 68Ga -DOTATOC PET/CT. All nine (100%) patients with 68Ga-DOTATOC PET/CT presented with pathological myocardial uptake in the LV that was suggestive of myocarditis (MBRpeak of 3.2±0.8, range 2.2-4.4). Eight patients had CMR imaging and 3/8 (38%) patients had lesions evocative of myocarditis. All PET-positive patients were previously treated with a high dose of steroids and intravenous immunoglobulin prior to PET/CT had elevated serum cTnI except for one patient for whom PET/CT was delayed several days. Interestingly, in 5/6 (83%) patients who presented with concomitant myositis, pathological uptake was seen on whole-body 68Ga-DOTATOC PET/CT images in the skeletal muscles, suggesting an additional advantage of this method to assess the full extent of the disease. In contrast, four patients with CMR imaging had negative findings despite having elevated serum cTnI levels (range 20.5-5896.1 ng/mL), thus defining possible myocarditis. Newly identified immune correlates could provide specific biomarkers for the diagnosis of ICI-related myocarditis. Most tested patients (six of seven patients) had serum increases in the inflammatory cytokine interleukin (IL)-6 and in the chemokines CXCL9, CXCL10, and CXCL13, and the mass cytometry phenotypes of immune cell populations in the blood also showed correlations with myocardial inflammation. Four of five patients with myocarditis exhibited a Th1/Th2 imbalance favoring a pronounced inflammatory Th1, Th1/Th17, and Th17 CD4 memory T-cell response. The high proportion of non-classical monocytes and significantly reduced levels of CD31 in four to five patients was also consistent with an inflammatory disease. CONCLUSION: The use of 68Ga-DOTATOC PET/CT along with immune correlates is a highly sensitive method to detect ICI-related myocarditis especially in the early stage of myocardial inflammation, as patients with elevated cTnI may present normal CMR imaging results. 68Ga-DOTATOC PET/CT is also useful for detecting concomitant myositis. These results need to be confirmed in a larger population of patients and validated against a histological gold standard if available.

The cytokines HGF and CXCL13 predict the severity and the mortality in COVID-19 patients.

The objective of the present study was to identify biological signatures of severe coronavirus disease 2019 (COVID-19) predictive of admission in the intensive care unit (ICU). Over 170 immunological markers were investigated in a 'discovery' cohort (n = 98 patients) of the Lausanne University Hospital (LUH-1). Here we report that 13 out of 49 cytokines were significantly associated with ICU admission in the three cohorts (P < 0.05 to P < 0.001), while cellular immunological markers lacked power in discriminating between ICU and non-ICU patients. The cytokine results were confirmed in two 'validation' cohorts, i.e. the French COVID-19 Study (FCS; n = 62) and a second LUH-2 cohort (n = 47). The combination of hepatocyte growth factor (HGF) and C-X-C motif chemokine ligand 13 (CXCL13) was the best predictor of ICU admission (positive and negative predictive values ranging from 81.8% to 93.1% and 85.2% to 94.4% in the 3 cohorts) and occurrence of death during patient follow-up (8.8 fold higher likelihood of death when both cytokines were increased). Of note, HGF is a pleiotropic cytokine with anti-inflammatory properties playing a fundamental role in lung tissue repair, and CXCL13, a pro-inflammatory chemokine associated with pulmonary fibrosis and regulating the maturation of B cell response. Up-regulation of HGF reflects the most powerful counter-regulatory mechanism of the host immune response to antagonize the pro-inflammatory cytokines including CXCL13 and to prevent lung fibrosis in COVID-19 patients.

Restoration of NK Cell Cytotoxic Function With Elotuzumab and Daratumumab Promotes Elimination of Circulating Plasma Cells in Patients With SLE.

Systemic lupus erythematosus (SLE) is a multisystem autoimmune disease characterized by multiple cellular and molecular dysfunctions of the innate and adaptive immunity. Cytotoxic function of NK cells is compromised in patients with SLE. Herein, we characterized the phenotypic alterations of SLE NK cells in a comprehensive manner to further delineate the mechanisms underlying the cytotoxic dysfunction of SLE NK cells and identify novel potential therapeutic targets. Therefore, we examined PBMC from SLE patients and matched healthy controls by single-cell mass cytometry to assess the phenotype of NK cells. In addition, we evaluated the cell function of NK cells (degranulation and cytokine production) and the killing of B cell subpopulations in a B cell-NK cell in vitro co-culture model. We found that SLE NK cells expressed higher levels of CD38 and were not able to adequately upregulate SLAMF1 and SLAMF7 following activation. In addition, ligation of SLAMF7 with elotuzumab or of CD38 with daratumumab on SLE NK cells enhanced degranulation of both healthy and SLE NK cells and primed them to kill circulating plasma cells in an in vitro co-culture system. Overall, our data indicated that dysregulated expression of CD38, SLAMF1 and SLAMF7 on SLE NK cells is associated with an altered interplay between SLE NK cells and plasma cells, thus suggesting their contribution to the accumulation of (auto)antibody producing cells. Accordingly, targeting SLAMF7 and CD38 may represent novel therapeutic approaches in SLE by enhancing NK cell function and promoting elimination of circulating plasma cell.

CD56 as a marker of an ILC1-like population with NK cell properties that is functionally impaired in AML.

An understanding of natural killer (NK) cell physiology in acute myeloid leukemia (AML) has led to the use of NK cell transfer in patients, demonstrating promising clinical results. However, AML is still characterized by a high relapse rate and poor overall survival. In addition to conventional NKs that can be considered the innate counterparts of CD8 T cells, another family of innate lymphocytes has been recently described with phenotypes and functions mirroring those of helper CD4 T cells. Here, in blood and tissues, we identified a CD56+ innate cell population harboring mixed transcriptional and phenotypic attributes of conventional helper innate lymphoid cells (ILCs) and lytic NK cells. These CD56+ ILC1-like cells possess strong cytotoxic capacities that are impaired in AML patients at diagnosis but are restored upon remission. Their cytotoxicity is KIR independent and relies on the expression of TRAIL, NKp30, NKp80, and NKG2A. However, the presence of leukemic blasts, HLA-E-positive cells, and/or transforming growth factor-ß1 (TGF-ß1) strongly affect their cytotoxic potential, at least partially by reducing the expression of cytotoxic-related molecules. Notably, CD56+ ILC1-like cells are also present in the NK cell preparations used in NK transfer-based clinical trials. Overall, we identified an NK cell-related CD56+ ILC population involved in tumor immunosurveillance in humans, and we propose that restoring their functions with anti-NKG2A antibodies and/or small molecules inhibiting TGF-ß1 might represent a novel strategy for improving current immunotherapies.

Tumor suppression of novel anti-PD-1 antibodies mediated through CD28 costimulatory pathway.

Classical antagonistic antibodies (Abs) targeting PD-1, such as pembrolizumab and nivolumab, act through blockade of the PD-1-PDL-1 interaction. Here, we have identified novel antagonistic anti-PD-1 Abs not blocking the PD-1-PDL-1 interaction. The nonblocking Abs recognize epitopes on PD-1 located on the opposing face of the PDL-1 interaction and overlap with a newly identified evolutionarily conserved patch. These nonblocking Abs act predominantly through the CD28 coreceptor. Importantly, a combination of blocking and nonblocking Abs synergize in the functional recovery of antigen-specific exhausted CD8 T cells. Interestingly, nonblocking anti-PD-1 Abs have equivalent antitumor activity compared with blocker Abs in two mouse tumor models, and combination therapy using both classes of Abs enhanced tumor suppression in the mouse immunogenic tumor model. The identification of the novel nonblocker anti-PD-1 Abs and their synergy with classical blocker Abs may be instrumental in potentiating immunotherapy strategies and antitumor activity.

CD32+ and PD-1+ Lymph Node CD4 T Cells Support Persistent HIV-1 Transcription in Treated Aviremic Individuals.

A recent study conducted in blood has proposed CD32 as the marker identifying the "elusive" HIV reservoir. We have investigated the distribution of CD32+ CD4 T cells in blood and lymph nodes (LNs) of HIV-1-uninfected subjects and viremic untreated and long-term-treated HIV-1-infected individuals and their relationship with PD-1+ CD4 T cells. The frequency of CD32+ CD4 T cells was increased in viremic compared to treated individuals in LNs, and a large proportion (up to 50%) of CD32+ cells coexpressed PD-1 and were enriched within T follicular helper (Tfh) cells. We next investigated the role of LN CD32+ CD4 T cells in the HIV reservoir. Total HIV DNA was enriched in CD32+ and PD-1+ CD4 T cells compared to CD32- and PD-1- cells in both viremic and treated individuals, but there was no difference between CD32+ and PD-1+ cells. There was no enrichment of latently infected cells with inducible HIV-1 in CD32+ versus PD-1+ cells in antiretroviral therapy (ART)-treated individuals. HIV-1 transcription was then analyzed in LN memory CD4 T cell populations sorted on the basis of CD32 and PD-1 expression. CD32+ PD-1+ CD4 T cells were significantly enriched in cell-associated HIV RNA compared to CD32- PD-1- (averages of 5.2-fold in treated individuals and 86.6-fold in viremics), CD32+ PD-1- (2.2-fold in treated individuals and 4.3-fold in viremics), and CD32- PD-1+ (2.2-fold in ART-treated individuals and 4.6-fold in viremics) cell populations. Similar levels of HIV-1 transcription were found in CD32+ PD-1- and CD32- PD-1+ CD4 T cells. Interestingly, the proportion of CD32+ and PD-1+ CD4 T cells negatively correlated with CD4 T cell counts and length of therapy. Therefore, the expression of CD32 identifies, independently of PD-1, a CD4 T cell population with persistent HIV-1 transcription and coexpression of CD32 and PD-1, the CD4 T cell population with the highest levels of HIV-1 transcription in both viremic and treated individuals.IMPORTANCE The existence of long-lived latently infected resting memory CD4 T cells represents a major obstacle to the eradication of HIV infection. Identifying cell markers defining latently infected cells containing replication-competent virus is important in order to determine the mechanisms of HIV persistence and to develop novel therapeutic strategies to cure HIV infection. We provide evidence that PD-1 and CD32 may have a complementary role in better defining CD4 T cell populations infected with HIV-1. Furthermore, CD4 T cells coexpressing CD32 and PD-1 identify a CD4 T cell population with high levels of persistent HIV-1 transcription.

Identification of innate lymphoid cells in single-cell RNA-Seq data.

Innate lymphoid cells (ILCs) consist of natural killer (NK) cells and non-cytotoxic ILCs that are broadly classified into ILC1, ILC2, and ILC3 subtypes. These cells recently emerged as important early effectors of innate immunity for their roles in tissue homeostasis and inflammation. Over the last few years, ILCs have been extensively studied in mouse and human at the functional and molecular level, including gene expression profiling. However, sorting ILCs with flow cytometry for gene expression analysis is a delicate and time-consuming process. Here we propose and validate a novel framework for studying ILCs at the transcriptomic level using single-cell RNA-Seq data. Our approach combines unsupervised clustering and a new cell type classifier trained on mouse ILC gene expression data. We show that this approach can accurately identify different ILCs, especially ILC2 cells, in human lymphocyte single-cell RNA-Seq data. Our new model relies only on genes conserved across vertebrates, thereby making it in principle applicable in any vertebrate species. Considering the rapid increase in throughput of single-cell RNA-Seq technology, our work provides a computational framework for studying ILC2 cells in single-cell transcriptomic data and may help exploring their conservation in distant vertebrate species.

Expression profiling of budding cells in colorectal cancer reveals an EMT-like phenotype and molecular subtype switching.

BACKGROUND: Tumour budding, described as the presence of single cells or small clusters of up to five tumour cells at the invasive margin, is established as a prognostic marker in colorectal carcinoma. In the present study, we aimed to investigate the molecular signature of tumour budding cells and the corresponding tumour bulk. METHODS: Tumour bulk and budding areas were microdissected and processed for RNA-sequencing. As little RNA was obtained from budding cells, a special low-input mRNA library preparation protocol was used. Gene expression profiles of budding as compared with tumour bulk were investigated for established EMT signatures, consensus molecular subtype (CMS), gene set enrichment and pathway analysis. RESULTS: A total of 296 genes were differentially expressed with an FDR <0.05 and a twofold change between tumour bulk and budding regions. Genes that were upregulated in the budding signature were mainly involved in cell migration and survival while downregulated genes were important for cell proliferation. Supervised clustering according to an established EMT gene signature categorised budding regions as EMT-positive, whereas tumour bulk was considered EMT-negative. Furthermore, a shift from CMS2 (epithelial) to CMS4 (mesenchymal) was observed as tumour cells transit from the tumour bulk to the budding regions. CONCLUSIONS: Tumour budding regions are characterised by a phenotype switch compared with the tumour bulk, involving the acquisition of migratory characteristics and a decrease in cell proliferation. In particular, most tumour budding signatures were EMT-positive and switched from an epithelial subtype (CMS2) in the tumour bulk to a mesenchymal subtype (CMS4) in budding cells.