Publisher Correction: Natural variation in the parameters of innate immune cells is preferentially driven by genetic factors.

In the version of this article initially published, the name of one author was incorrect (James P. Santo). The correct name is James P. Di Santo. The error has been corrected in the HTML and PDF versions of the article.

Natural variation in the parameters of innate immune cells is preferentially driven by genetic factors.

The quantification and characterization of circulating immune cells provide key indicators of human health and disease. To identify the relative effects of environmental and genetic factors on variation in the parameters of innate and adaptive immune cells in homeostatic conditions, we combined standardized flow cytometry of blood leukocytes and genome-wide DNA genotyping of 1,000 healthy, unrelated people of Western European ancestry. We found that smoking, together with age, sex and latent infection with cytomegalovirus, were the main non-genetic factors that affected variation in parameters of human immune cells. Genome-wide association studies of 166 immunophenotypes identified 15 loci that showed enrichment for disease-associated variants. Finally, we demonstrated that the parameters of innate cells were more strongly controlled by genetic variation than were those of adaptive cells, which were driven by mainly environmental exposure. Our data establish a resource that will generate new hypotheses in immunology and highlight the role of innate immunity in susceptibility to common autoimmune diseases.

Antimicrobial activity of mucosal-associated invariant T cells.

Mucosal-associated invariant T lymphocytes (MAIT lymphocytes) are characterized by two evolutionarily conserved features: an invariant T cell antigen receptor (TCR) alpha-chain and restriction by the major histocompatibility complex (MHC)-related protein MR1. Here we show that MAIT cells were activated by cells infected with various strains of bacteria and yeast, but not cells infected with virus, in both humans and mice. This activation required cognate interaction between the invariant TCR and MR1, which can present a bacteria-derived ligand. In humans, we observed considerably fewer MAIT cells in blood from patients with bacterial infections such as tuberculosis. In the mouse, MAIT cells protected against infection by Mycobacterium abscessus or Escherichia coli. Thus, MAIT cells are evolutionarily conserved innate-like lymphocytes that sense and help fight off microbial infection.

Blood monocytes sample MelanA/MART1 antigen for long-lasting cross-presentation to CD8+ T cells after differentiation into dendritic cells.

Human blood monocytes are very potent to take up antigens. Like macrophages in tissue, they efficiently degrade exogenous protein and are less efficient than dendritic cells (DCs) at cross-presenting antigens to CD8+ T cells. Although it is generally accepted that DCs take up tissue antigens and then migrate to lymph nodes to prime T cells, the mechanisms of presentation of antigens taken up by monocytes are poorly documented so far. In the present work, we show that monocytes loaded in vitro with MelanA long peptides retain the capacity to stimulate antigen-specific CD8+ T cell clones after 5 days of differentiation into monocytes-derived dendritic cells (MoDCs). Tagged-long peptides can be visualized in electron-dense endocytic compartments distinct from lysosomes, suggesting that antigens can be protected from degradation for extended periods of time. To address the pathophysiological relevance of these findings, we screened blood monocytes from 18 metastatic melanoma patients and found that CD14+ monocytes from two patients effectively activate a MelanA-specific CD8 T cell clone after in vitro differentiation into MoDCs. This in vivo sampling of tumor antigen by circulating monocytes might alter the tumor-specific immune response and should be taken into account for cancer immunotherapy.

Analysis of APC types involved in CD4 tolerance and regulatory T cell generation using reaggregated thymic organ cultures.

Tolerance to self-Ags is generated in the thymus. Both epithelial and hematopoietic thymic stromal cells play an active and essential role in this process. However, the role of each of the various stromal cell types remains unresolved. To our knowledge, we describe the first comparative analysis of several types of thymic hematopoietic stromal cells (THSCs) for their ability to induce CD4 tolerance to self, in parallel with the thymic epithelium. The THSCs--two types of conventional dendritic cells (cDCs), plasmacytoid dendritic cells, macrophages (MΦs), B lymphocytes, and eosinophils--were first characterized and quantified in adult mouse thymus. They were then examined in reaggregated thymic organ cultures containing mixtures of monoclonal and polyclonal thymocytes. This thymocyte mixture allows for the analysis of Ag-specific events while avoiding the extreme skewing frequently seen in purely monoclonal systems. Our data indicate that thymic epithelium alone is capable of promoting self-tolerance by eliminating autoreactive CD4 single-positive thymocytes and by supporting regulatory T cell (Treg) development. We also show that both non-Treg CD4 single-positive thymocytes and Tregs are efficiently deleted by the two populations of cDCs present in the thymus, as well as to a lesser extent by MΦs. Plasmacytoid dendritic cells, B lymphocytes, and eosinophils were not able to do so. Finally, cDCs were also the most efficient THSCs at supporting Treg development in the thymus, suggesting that although they may share some characteristics required for negative selection with MΦs, they do not share those required for the support of Treg development, making cDCs a unique cell subset in the thymus.

Human MAIT cells are xenobiotic-resistant, tissue-targeted, CD161hi IL-17-secreting T cells.

Mucosal-associated invariant T (MAIT) cells are very abundant in humans and have antimicrobial specificity, but their functions remain unclear. MAIT cells are CD161(hi)IL-18Rα(+) and either CD4(-)CD8(-) (DN) or CD8αß(int) T cells. We now show that they display an effector-memory phenotype (CD45RA(-)CD45RO(+)CD95(hi)CD62L(lo)), and their chemokine receptor expression pattern (CCR9(int)CCR7(-)CCR5(hi)CXCR6(hi)CCR6(hi)) indicates preferential homing to tissues and particularly the intestine and the liver. MAIT cells can represent up to 45% of the liver lymphocytes. They produce interferon-γ and Granzyme-B as well as high levels of interleukin-17 after phorbol myristate acetate + ionomycin stimulation. Most MAIT cells are noncycling cells (< 1% are Ki-67(+)) and express the multidrug resistance transporter (ABCB1). As expected from this phenotype, MAIT cells are more resistant to chemotherapy than other T-cell populations. These features might also allow MAIT cells to resist the xenobiotics potentially secreted by the gut bacteria. We also show that this population does not appear to have antiviral specificity and that CD8 MAIT cells include almost all the ABCB1(+)CD161(hi) CD8 T cells. Together with their already known abundance and antimicrobial specificity, the gut-liver homing characteristics, high expression of ABCB1, and ability to secrete interleukin-17 probably participate in the antibacterial properties of MAIT cells.

High content phenotypic cell-based visual screen identifies Mycobacterium tuberculosis acyltrehalose-containing glycolipids involved in phagosome remodeling.

The ability of the tubercle bacillus to arrest phagosome maturation is considered one major mechanism that allows its survival within host macrophages. To identify mycobacterial genes involved in this process, we developed a high throughput phenotypic cell-based assay enabling individual sub-cellular analysis of over 11,000 Mycobacterium tuberculosis mutants. This very stringent assay makes use of fluorescent staining for intracellular acidic compartments, and automated confocal microscopy to quantitatively determine the intracellular localization of M. tuberculosis. We characterised the ten mutants that traffic most frequently into acidified compartments early after phagocytosis, suggesting that they had lost their ability to arrest phagosomal maturation. Molecular analysis of these mutants revealed mainly disruptions in genes involved in cell envelope biogenesis (fadD28), the ESX-1 secretion system (espL/Rv3880), molybdopterin biosynthesis (moaC1 and moaD1), as well as in genes from a novel locus, Rv1503c-Rv1506c. Most interestingly, the mutants in Rv1503c and Rv1506c were perturbed in the biosynthesis of acyltrehalose-containing glycolipids. Our results suggest that such glycolipids indeed play a critical role in the early intracellular fate of the tubercle bacillus. The unbiased approach developed here can be easily adapted for functional genomics study of intracellular pathogens, together with focused discovery of new anti-microbials.

Escherichia coli-Specific CXCL13-Producing TFH Are Associated with Clinical Efficacy of Neoadjuvant PD-1 Blockade against Muscle-Invasive Bladder Cancer.

Biomarkers guiding the neoadjuvant use of immune-checkpoint blockers (ICB) are needed for patients with localized muscle-invasive bladder cancers (MIBC). Profiling tumor and blood samples, we found that follicular helper CD4+ T cells (TFH) are among the best therapeutic targets of pembrolizumab correlating with progression-free survival. TFH were associated with tumoral CD8 and PD-L1 expression at baseline and the induction of tertiary lymphoid structures after pembrolizumab. Blood central memory TFH accumulated in tumors where they produce CXCL13, a chemokine found in the plasma of responders only. IgG4+CD38+ TFH residing in bladder tissues correlated with clinical benefit. Finally, TFH and IgG directed against urothelium-invasive Escherichia coli dictated clinical responses to pembrolizumab in three independent cohorts. The links between tumor infection and success of ICB immunomodulation should be prospectively assessed at a larger scale. SIGNIFICANCE: In patients with bladder cancer treated with neoadjuvant pembrolizumab, E. coli-specific CXCL13 producing TFH and IgG constitute biomarkers that predict clinical benefit. Beyond its role as a biomarker, such immune responses against E. coli might be harnessed for future therapeutic strategies. This article is highlighted in the In This Issue feature, p. 2221.

Single-Dose Intranasal Administration of AdCOVID Elicits Systemic and Mucosal Immunity against SARS-CoV-2 and Fully Protects Mice from Lethal Challenge.

The coronavirus disease 2019 (COVID-19) pandemic has highlighted the urgent need for effective prophylactic vaccination to prevent the spread of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Intranasal vaccination is an attractive strategy to prevent COVID-19 as the nasal mucosa represents the first-line barrier to SARS-CoV-2 entry. The current intramuscular vaccines elicit systemic immunity but not necessarily high-level mucosal immunity. Here, we tested a single intranasal dose of our candidate adenovirus type 5-vectored vaccine encoding the receptor-binding domain (RBD) of the SARS-CoV-2 spike protein (AdCOVID) in inbred, outbred, and transgenic mice. A single intranasal vaccination with AdCOVID elicited a strong and focused immune response against RBD through the induction of mucosal IgA in the respiratory tract, serum neutralizing antibodies, and CD4+ and CD8+ T cells with a Th1-like cytokine expression profile. A single AdCOVID dose resulted in immunity that was sustained for over six months. Moreover, a single intranasal dose completely protected K18-hACE2 mice from lethal SARS-CoV-2 challenge, preventing weight loss and mortality. These data show that AdCOVID promotes concomitant systemic and mucosal immunity and represents a promising vaccine candidate.

Single-dose intranasal administration of AdCOVID elicits systemic and mucosal immunity against SARS-CoV-2 in mice.

The coronavirus disease 2019 (COVID-19) pandemic has highlighted the urgent need for effective preventive vaccination to reduce burden and spread of severe acute respiratory syndrome (SARS) coronavirus 2 (SARS-CoV-2) in humans. Intranasal vaccination is an attractive strategy to prevent COVID-19 as the nasal mucosa represents the first-line barrier to SARS-CoV-2 entry before viral spread to the lung. Although SARS-CoV-2 vaccine development is rapidly progressing, the current intramuscular vaccines are designed to elicit systemic immunity without conferring mucosal immunity. Here, we show that AdCOVID, an intranasal adenovirus type 5 (Ad5)-vectored vaccine encoding the receptor binding domain (RBD) of the SARS-CoV-2 spike protein, elicits a strong and focused immune response against RBD through the induction of mucosal IgA, serum neutralizing antibodies and CD4+ and CD8+ T cells with a Th1-like cytokine expression profile. Therefore, AdCOVID, which promotes concomitant systemic and local mucosal immunity, represents a promising COVID-19 vaccine candidate.

A phase I clinical study of vaccination of melanoma patients with dendritic cells loaded with allogeneic apoptotic/necrotic melanoma cells. Analysis of toxicity and immune response to the vaccine and of IL-10 -1082 promoter genotype as predictor of disease progression.

BACKGROUND: Sixteen melanoma patients (1 stage IIC, 8 stage III, and 7 stage IV) were treated in a Phase I study with a vaccine (DC/Apo-Nec) composed of autologous dendritic cells (DCs) loaded with a mixture of apoptotic/necrotic allogeneic melanoma cell lines (Apo-Nec), to evaluate toxicity and immune responses. Also, IL-10 1082 genotype was analyzed in an effort to predict disease progression. METHODS: PBMC were obtained after leukapheresis and DCs were generated from monocytes cultured in the presence of GM-CSF and IL-4 in serum-free medium. Immature DCs were loaded with gamma-irradiated Apo-Nec cells and injected id without adjuvant. Cohorts of four patients were given four vaccines each with 5, 10, 15, or 20 x 106 DC/Apo-Nec cell per vaccine, two weeks apart. Immune responses were measured by ELISpot and tetramer analysis. Il-10 genotype was measured by PCR and corroborated by IL-10 production by stimulated PBMC. RESULTS: Immature DCs efficiently phagocytosed melanoma Apo-Nec cells and matured after phagocytosis as evidenced by increased expression of CD83, CD80, CD86, HLA class I and II, and 75.2 +/- 16% reduction in Dextran-FITC endocytosis. CCR7 was also up-regulated upon Apo-Nec uptake in DCs from all patients, and accordingly DC/Apo-Nec cells were able to migrate in vitro toward MIP-3 beta. The vaccine was well tolerated in all patients. The DTH score increased significantly in all patients after the first vaccination (Mann-Whitney Test, p < 0.05). The presence of CD8+T lymphocytes specific to gp100 and Melan A/MART-1 Ags was determined by ELISpot and tetramer analysis in five HLA-A*0201 patients before and after vaccination; one patient had stable elevated levels before and after vaccination; two increased their CD8 + levels, one had stable moderate and one had negligible levels. The analysis of IL-10 promoter -1082 polymorphism in the sixteen patients showed a positive correlation between AA genotype, accompanied by lower in vitro IL-10 production by stimulated PBMC, and faster melanoma progression after lymph nodes surgery (p = 0.04). With a mean follow-up of 49.5 months post-surgery, one stage IIC patient and 7/8 stage III patients remain NED but 7/7 stage IV patients have progressed. CONCLUSION: We conclude that DC/Apo-Nec vaccine is safe, well tolerated and it may induce specific immunity against melanoma Ags. Patients with a low-producing IL-10 polymorphism appear to have a worst prognosis. TRIAL REGISTRATION: Clinicaltrials.gov (NHI) NCT00515983.

Skin carcinoma arising from donor cells in a kidney transplant recipient.

The incidence of skin cancer is increased in transplant recipients. UV radiation, papillomaviruses, and immunosuppression participate in the pathogenesis of these tumors. In addition, donor cells may leave the grafted organ, reach peripheral tissues and either induce immune phenomena or possibly take part in tissue remodeling. Herein, we investigated the possible involvement of donor cells in the development of skin tumors in kidney allograft recipients. We analyzed a series of 48 malignant and benign cutaneous tumors developing in 14 females who had been grafted with a male kidney. The number of male cells was measured on microdissected material by quantitative PCR for Y chromosome. In the samples with high levels of male cells, fluorescent in situ hybridization (FISH) with X and Y probes and/or immuno-FISH with anticytokeratin antibodies were carried out. Male cells were detected in 5/15 squamous cell carcinomas and Bowen disease (range 4-180 copies), 3/5 basal cell carcinomas (91-645), 6/11 actinic keratosis (7-102), 2/4 keratoacanthoma (22-41), and 2/5 benign cutaneous lesions (14-55). In a basal cell carcinoma specimen with a high number of male cells, FISH showed that most cells within the tumoral buds were XY. In this lesion, immuno-FISH showed the presence of XY cytokeratin-positive cells indicating that the tumor nests contained male keratinocytes. In contrast, in other female transplants, male cells present in the tumors were not epithelial. In conclusion, stem cells originating from a grafted kidney may migrate to the skin, differentiate, or fuse as keratinocytes that could, rarely, undergo cancer transformation.

Dendritic cell-derived exosomes as maintenance immunotherapy after first line chemotherapy in NSCLC.

Dendritic cell-derived exosomes (Dex) are small extracellular vesicles secreted by viable dendritic cells. In the two phase-I trials that we conducted using the first generation of Dex (IFN-γ-free) in end-stage cancer, we reported that Dex exerted natural killer (NK) cell effector functions in patients. A second generation of Dex (IFN-γ-Dex) was manufactured with the aim of boosting NK and T cell immune responses. We carried out a phase II clinical trial testing the clinical benefit of IFN-γ-Dex loaded with MHC class I- and class II-restricted cancer antigens as maintenance immunotherapy after induction chemotherapy in patients bearing inoperable non-small cell lung cancer (NSCLC) without tumor progression. The primary endpoint was to observe at least 50% of patients with progression-free survival (PFS) at 4 mo after chemotherapy cessation. Twenty-two patients received IFN-γ-Dex. One patient exhibited a grade three hepatotoxicity. The median time to progression was 2.2 mo and median overall survival (OS) was 15 mo. Seven patients (32%) experienced stabilization of >4 mo. The primary endpoint was not reached. An increase in NKp30-dependent NK cell functions were evidenced in a fraction of these NSCLC patients presenting with defective NKp30 expression. Importantly, MHC class II expression levels of the final IFN-γ-Dex product correlated with expression levels of the NKp30 ligand BAG6 on Dex, and with NKp30-dependent NK functions, the latter being associated with longer progression-free survival. This phase II trial confirmed the capacity of Dex to boost the NK cell arm of antitumor immunity in patients with advanced NSCLC.

High numbers of differentiated effector CD4 T cells are found in patients with cancer and correlate with clinical response after neoadjuvant therapy of breast cancer.

CD4(+) T cells influence tumor immunity in complex ways that are not fully understood. In this study, we characterized a population of human differentiated effector CD4(+) T cells that is defined by low levels of the interleukin (IL)-2 and IL-7 receptors (CD25(-)CD127(-)). We found that this cell population expands in patients with various types of cancer, including breast cancer, to represent 2% to 20% of total CD4(+) blood T lymphocytes as compared with only 0.2% to 2% in healthy individuals. Notably, these CD25(-)CD127(-)CD4 T cells expressed effector markers such as CD244 and CD11b with low levels of CD27, contrasting with the memory phenotype dominating this population in healthy individuals. These cells did not cycle in patients, nor did they secrete IL-10 or IL-17, but instead displayed cytotoxic features. Moreover, they encompassed oligoclonal expansions paralleling an expansion of effector CD8(+) T cells that included tumor antigen-specific T cells. During neoadjuvant chemotherapy in patients with breast cancer, we found that the increase in CD25(-)CD127(-) CD4(+) T cells correlated with tumor regression. This observation suggested that CD4(+) T cells included tumor antigen-specific cells, which may be generated by or participate in tumor regressions during chemotherapy. In summary, our results lend support to the hypothesis that CD4(+) T cells are involved in human antitumor responses.

Age-related patterns in human myeloid dendritic cell populations in people exposed to Schistosoma haematobium infection.

BACKGROUND: Urogenital schistosomiasis is caused by the helminth parasite Schistosoma haematobium. In high transmission areas, children acquire schistosome infection early in life with infection levels peaking in early childhood and subsequently declining in late childhood. This age-related infection profile is thought to result from the gradual development of protective acquired immunity. Age-related differences in schistosome-specific humoral and cellular responses have been reported from several field studies. However there has not yet been a systematic study of the age-related changes in human dendritic cells, the drivers of T cell polarisation. METHODS: Peripheral blood mononuclear cells were obtained from a cohort of 61 Zimbabwean aged 5-45 years with a S. haematobium prevalence of 47.5%. Two subsets of dendritic cells, myeloid and plasmacytoid dendritic cells (mDCs and pDCs), were analyzed by flow cytometry. FINDINGS: In this population, schistosome infection levels peaked in the youngest age group (5-9 years), and declined in late childhood and adulthood (10+ years). The proportions of both mDCs and pDCs varied with age. However, for mDCs the age profile depended on host infection status. In the youngest age group infected people had enhanced proportions of mDCs as well as lower levels of HLA-DR on mDCs than un-infected people. In the older age groups (10-13 and 14-45 years) infected people had lower proportions of mDCs compared to un-infected individuals, but no infection status-related differences were observed in their levels of HLA-DR. Moreover mDC proportions correlated with levels of schistosome-specific IgG, which can be associated with protective immunity. In contrast proportions of pDCs varied with host age, but not with infection status. CONCLUSIONS: Our results show that dendritic cell proportions and activation in a human population living in schistosome-endemic areas vary with host age reflecting differences in cumulative history of exposure to schistosome infection.

Establishment and characterization of a panel of human uveal melanoma xenografts derived from primary and/or metastatic tumors.

PURPOSE: Uveal melanoma is the most common primary intraocular malignant tumor in adults and is defined by a poor natural outcome, as 50% of patients die from metastases. The aim of this study was to develop and characterize a panel of human uveal melanoma xenografts transplanted into immunodeficient mice. EXPERIMENTAL DESIGN: Ninety tumor specimens were grafted into severe combined immunodeficient mice, and 25 transplantable xenografts were then established (28%). Relationship between tumor graft and clinical, biological, and therapeutic features of the patients included were investigated. Characterization of 16 xenografts included histology, molecular analyses by immunohistochemistry, genetic alteration analysis (single-nucleotide polymorphism), and specific tumor antigen expression by quantitative reverse transcription-PCR. Pharmacologic characterization (chemosensitivity) was also done in four models using two drugs, temozolomide and fotemustine, currently used in the clinical management of uveal melanoma. RESULTS: Take rate of human uveal melanoma was 28% (25 of 90). Tumor take was independent of size, histologic parameters, or chromosome 3 monosomy but was significantly higher in metastatic tumors. Interestingly, in vivo tumor growth was prognostic for a lower metastasis-free survival in patients with primary tumors. A high concordance between the patients' tumors and their corresponding xenografts was found for all parameters tested (histology, genetic profile, and tumor antigen expression). Finally, the four xenografts studied displayed different response profiles to chemotherapeutic agents. CONCLUSIONS: Based on these results, this panel of 16 uveal melanoma xenografts represents a useful preclinical tool for both pharmacologic and biological assessments.