Development of an 18F-labeled anti-human CD8 VHH for same-day immunoPET imaging.

PURPOSE: Cancer immunotherapies (CITs) have revolutionized the treatment of certain cancers, but many patients fail to respond or relapse from current therapies, prompting the need for new CIT agents. CD8+ T cells play a central role in the activity of many CITs, and thus, the rapid imaging of CD8+ cells could provide a critical biomarker for new CIT agents. However, existing 89Zr-labeled CD8 PET imaging reagents exhibit a long circulatory half-life and high radiation burden that limit potential applications such as same-day and longitudinal imaging. METHODS: To this end, we discovered and developed a 13-kDa single-domain antibody (VHH5v2) against human CD8 to enable high-quality, same-day imaging with a reduced radiation burden. To enable sensitive and rapid imaging, we employed a site-specific conjugation strategy to introduce an 18F radiolabel to the VHH. RESULTS: The anti-CD8 VHH, VHH5v2, demonstrated binding to a membrane distal epitope of human CD8 with a binding affinity (KD) of 500 pM. Subsequent imaging experiments in several xenografts that express varying levels of CD8 demonstrated rapid tumor uptake and fast clearance from the blood. High-quality images were obtained within 1 h post-injection and could quantitatively differentiate the tumor models based on CD8 expression level. CONCLUSION: Our work reveals the potential of this anti-human CD8 VHH [18F]F-VHH5v2 to enable rapid and specific imaging of CD8+ cells in the clinic.

The immune factors driving DNA methylation variation in human blood.

Epigenetic changes are required for normal development, yet the nature and respective contribution of factors that drive epigenetic variation in humans remain to be fully characterized. Here, we assessed how the blood DNA methylome of 884 adults is affected by DNA sequence variation, age, sex and 139 factors relating to life habits and immunity. Furthermore, we investigated whether these effects are mediated or not by changes in cellular composition, measured by deep immunophenotyping. We show that DNA methylation differs substantially between naïve and memory T cells, supporting the need for adjustment on these cell-types. By doing so, we find that latent cytomegalovirus infection drives DNA methylation variation and provide further support that the increased dispersion of DNA methylation with aging is due to epigenetic drift. Finally, our results indicate that cellular composition and DNA sequence variation are the strongest predictors of DNA methylation, highlighting critical factors for medical epigenomics studies.

Human thymopoiesis is influenced by a common genetic variant within the TCRA-TCRD locus.

The thymus is the primary lymphoid organ where naïve T cells are generated; however, with the exception of age, the parameters that govern its function in healthy humans remain unknown. We characterized the variability of thymic function among 1000 age- and sex-stratified healthy adults of the Milieu Intérieur cohort, using quantification of T cell receptor excision circles (TRECs) in peripheral blood T cells as a surrogate marker of thymopoiesis. Age and sex were the only nonheritable factors identified that affect thymic function. TREC amounts decreased with age and were higher in women compared to men. In addition, a genome-wide association study revealed a common variant (rs2204985) within the T cell receptor TCRA-TCRD locus, between the DD2 and DD3 gene segments, which associated with TREC amounts. Strikingly, transplantation of human hematopoietic stem cells with the rs2204985 GG genotype into immunodeficient mice led to thymopoiesis with higher TRECs, increased thymocyte counts, and a higher TCR repertoire diversity. Our population immunology approach revealed a genetic locus that influences thymopoiesis in healthy adults, with potentially broad implications in precision medicine.

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.

Distinctive roles of age, sex, and genetics in shaping transcriptional variation of human immune responses to microbial challenges.

The contribution of host genetic and nongenetic factors to immunological differences in humans remains largely undefined. Here, we generated bacterial-, fungal-, and viral-induced immune transcriptional profiles in an age- and sex-balanced cohort of 1,000 healthy individuals and searched for the determinants of immune response variation. We found that age and sex affected the transcriptional response of most immune-related genes, with age effects being more stimulus-specific relative to sex effects, which were largely shared across conditions. Although specific cell populations mediated the effects of age and sex on gene expression, including CD8+ T cells for age and CD4+ T cells and monocytes for sex, we detected a direct effect of these intrinsic factors for the majority of immune genes. The mapping of expression quantitative trait loci (eQTLs) revealed that genetic factors had a stronger effect on immune gene regulation than age and sex, yet they affected a smaller number of genes. Importantly, we identified numerous genetic variants that manifested their regulatory effects exclusively on immune stimulation, including a Candida albicans-specific master regulator at the CR1 locus. These response eQTLs were enriched in disease-associated variants, particularly for autoimmune and inflammatory disorders, indicating that differences in disease risk may result from regulatory variants exerting their effects only in the presence of immune stress. Together, this study quantifies the respective effects of age, sex, genetics, and cellular heterogeneity on the interindividual variability of immune responses and constitutes a valuable resource for further exploration in the context of different infection risks or disease outcomes.

Triggering of TLR-3, -4, NOD2, and DC-SIGN reduces viral replication and increases T-cell activation capacity of HIV-infected human dendritic cells.

A variety of signals influence the capacity of dendritic cells (DCs) to mount potent antiviral cytotoxic T-cell (CTL) responses. In particular, innate immune sensing by pathogen recognition receptors, such as TLR and C-type lectines, influences DC biology and affects their susceptibility to HIV infection. Yet, whether the combined effects of PPRs triggering and HIV infection influence HIV-specific (HS) CTL responses remain enigmatic. Here, we dissect the impact of innate immune sensing by pathogen recognition receptors on DC maturation, HIV infection, and on the quality of HS CTL activation. Remarkably, ligand-driven triggering of TLR-3, -4, NOD2, and DC-SIGN, despite reducing viral replication, markedly increased the capacity of infected DCs to stimulate HS CTLs. This was exemplified by the diversity and the quantity of cytokines produced by HS CTLs primed by these DCs. Infecting DCs with viruses harboring members of the APOBEC family of antiviral factors enhanced the antigen-presenting skills of infected DCs. Our results highlight the tight interplay between innate and adaptive immunity and may help develop innovative immunotherapies against viral infections.

Standardized Whole-Blood Transcriptional Profiling Enables the Deconvolution of Complex Induced Immune Responses.

Systems approaches for the study of immune signaling pathways have been traditionally based on purified cells or cultured lines. However, in vivo responses involve the coordinated action of multiple cell types, which interact to establish an inflammatory microenvironment. We employed standardized whole-blood stimulation systems to test the hypothesis that responses to Toll-like receptor ligands or whole microbes can be defined by the transcriptional signatures of key cytokines. We found 44 genes, identified using Support Vector Machine learning, that captured the diversity of complex innate immune responses with improved segregation between distinct stimuli. Furthermore, we used donor variability to identify shared inter-cellular pathways and trace cytokine loops involved in gene expression. This provides strategies for dimension reduction of large datasets and deconvolution of innate immune responses applicable for characterizing immunomodulatory molecules. Moreover, we provide an interactive R-Shiny application with healthy donor reference values for induced inflammatory genes.

HIV-Infected Dendritic Cells Present Endogenous MHC Class II-Restricted Antigens to HIV-Specific CD4+ T Cells.

It is widely assumed that CD4(+) T cells recognize antigenic peptides (epitopes) derived solely from incoming, exogenous, viral particles or proteins. However, alternative sources of MHC class II (MHC-II)-restricted Ags have been described, in particular epitopes derived from newly synthesized proteins (so-called endogenous). In this study, we show that HIV-infected dendritic cells (DC) present MHC-II-restricted endogenous viral Ags to HIV-specific (HS) CD4(+) T cells. This endogenous pathway functions independently of the exogenous route for HIV Ag presentation and offers a distinct possibility for the immune system to activate HS CD4(+) T cells. We examined the implication of autophagy, which plays a crucial role in endogenous viral Ag presentation and thymic selection of CD4(+) T cells, in HIV endogenous presentation. We show that infected DC do not use autophagy to process MHC-II-restricted HIV Ags. This is unlikely to correspond to a viral escape from autophagic degradation, as infecting DC with Nef- or Env-deficient HIV strains did not impact HS T cell activation. However, we demonstrate that, in DC, specific targeting of HIV Ags to autophagosomes using a microtubule-associated protein L chain 3 (LC3) fusion protein effectively enhances and broadens HS CD4(+) T cell responses, thus favoring an endogenous MHC-II-restricted presentation. In summary, in DC, multiple endogenous presentation pathways lead to the activation of HS CD4(+) T cell responses. These findings will help in designing novel strategies to activate HS CD4(+) T cells that are required for CTL activation/maintenance and B cell maturation.

HIV-Infected Spleens Present Altered Follicular Helper T Cell (Tfh) Subsets and Skewed B Cell Maturation.

Follicular helper T (Tfh) cells within secondary lymphoid organs control multiple steps of B cell maturation and antibody (Ab) production. HIV-1 infection is associated with an altered B cell differentiation and Tfh isolated from lymph nodes of HIV-infected (HIV+) individuals provide inadequate B cell help in vitro. However, the mechanisms underlying this impairment of Tfh function are not fully defined. Using a unique collection of splenocytes, we compared the frequency, phenotype and transcriptome of Tfh subsets in spleens from HIV negative (HIV-) and HIV+ subjects. We observed an increase of CXCR5+PD-1highCD57-Tfh and germinal center (GC) CD57+ Tfh in HIV+ spleens. Both subsets showed a reduced mRNA expression of the transcription factor STAT-3, co-stimulatory, regulatory and signal transduction molecules as compared to HIV- spleens. Similarly, Foxp3 expressing follicular regulatory T (Tfr) cells were increased, suggesting sustained GC reactions in chronically HIV+ spleens. As a consequence, GC B cell populations were expanded, however, complete maturation into memory B cells was reduced in HIV+ spleens where we evidenced a compromised production of B cell-activating cytokines such as IL-4 and IL-10. Collectively our data indicate that, although Tfh proliferation and GC reactions seem to be ongoing in HIV-infected spleens, Tfh "differentiation" and expression of costimulatory molecules is skewed with a profound effect on B cell maturation.

Semi-automated and standardized cytometric procedures for multi-panel and multi-parametric whole blood immunophenotyping.

Immunophenotyping by multi-parametric flow cytometry is the cornerstone technology for enumeration and characterization of immune cell populations in health and disease. Standardized procedures are essential to allow for inter-individual comparisons in the context of population based or clinical studies. Herein we report the approach taken by the Milieu Intérieur Consortium, highlighting the standardized and automated procedures used for immunophenotyping of human whole blood samples. We optimized eight-color antibody panels and procedures for staining and lysis of whole blood samples, and implemented pre-analytic steps with a semi-automated workflow using a robotic system. We report on four panels that were designed to enumerate and phenotype major immune cell populations (PMN, T, B, NK cells, monocytes and DC). This work establishes a foundation for defining reference values in healthy donors. Our approach provides robust protocols for affordable, semi-automated eight-color cytometric immunophenotyping that can be used in population-based studies and clinical trial settings.

Automated flow cytometric analysis across large numbers of samples and cell types.

Multi-parametric flow cytometry is a key technology for characterization of immune cell phenotypes. However, robust high-dimensional post-analytic strategies for automated data analysis in large numbers of donors are still lacking. Here, we report a computational pipeline, called FlowGM, which minimizes operator input, is insensitive to compensation settings, and can be adapted to different analytic panels. A Gaussian Mixture Model (GMM)-based approach was utilized for initial clustering, with the number of clusters determined using Bayesian Information Criterion. Meta-clustering in a reference donor permitted automated identification of 24 cell types across four panels. Cluster labels were integrated into FCS files, thus permitting comparisons to manual gating. Cell numbers and coefficient of variation (CV) were similar between FlowGM and conventional gating for lymphocyte populations, but notably FlowGM provided improved discrimination of "hard-to-gate" monocyte and dendritic cell (DC) subsets. FlowGM thus provides rapid high-dimensional analysis of cell phenotypes and is amenable to cohort studies.

Functional analysis via standardized whole-blood stimulation systems defines the boundaries of a healthy immune response to complex stimuli.

Standardization of immunophenotyping procedures has become a high priority. We have developed a suite of whole-blood, syringe-based assay systems that can be used to reproducibly assess induced innate or adaptive immune responses. By eliminating preanalytical errors associated with immune monitoring, we have defined the protein signatures induced by (1) medically relevant bacteria, fungi, and viruses; (2) agonists specific for defined host sensors; (3) clinically employed cytokines; and (4) activators of T cell immunity. Our results provide an initial assessment of healthy donor reference values for induced cytokines and chemokines and we report the failure to release interleukin-1α as a common immunological phenotype. The observed naturally occurring variation of the immune response may help to explain differential susceptibility to disease or response to therapeutic intervention. The implementation of a general solution for assessment of functional immune responses will help support harmonization of clinical studies and data sharing.

Differential impact of age and cytomegalovirus infection on the γδ T cell compartment.

γδ T cells represent a subset of unconventional T lymphocytes that are known for their reactivity against different pathogens and considered as intermediate mediators between adaptive and innate immunity. We provide in this paper further insights underlying the changes that affect the γδ T cell compartment with advanced age in humans. We show that both aging and CMV infection impact independently on the γδ T cell compartment. Most γδ T cells are significantly affected by age and present a decreased frequency in the elderly. The decline of the γδ T cell pool appears to be independent from the activity of the thymus, arguing in favor of an extrathymic site of γδ T cell production in humans. Of note, CMV infection, which is directly associated with the activation of the pool of Vδ2(-) γδ T cells, promotes nonetheless the inflation of this compartment throughout life. CMV seropositivity accentuates further the accumulation of highly differentiated lymphocytes in Vδ2(-) γδ T cell subsets with time, in contrast to Vδ2(+) γδ T cells, which maintain a less differentiated phenotype. This is similar to the effect of CMV on αß T cells and suggests that γδ T cells may vary in differentiation phenotype according to distinct stimuli or pathogens.

HIV-1 capture and antigen presentation by dendritic cells: enhanced viral capture does not correlate with better T cell activation.

During HIV-1 infection, dendritic cells (DC) facilitate dissemination of HIV-1 while trying to trigger adaptive antiviral immune responses. We examined whether increased HIV-1 capture in DC matured with LPS results in more efficient Ag presentation to HIV-1-specific CD4(+) and CD8(+) T cells. To block the DC-mediated trans-infection of HIV-1 and maximize Ag loading, we also evaluated a noninfectious integrase-deficient HIV-1 isolate, HIV(NL4-3ΔIN). We showed that higher viral capture of DC did not guarantee better Ag presentation or T cell activation. Greater HIV(NL4-3) uptake by fully LPS-matured DC resulted in higher viral transmission to target cells but poorer stimulation of HIV-1-specific CD4(+) and CD8(+) T cells. Conversely, maturation of DC with LPS during, but not before, viral loading enhanced both HLA-I and HLA-II HIV-1-derived Ag presentation. In contrast, DC maturation with the clinical-grade mixture consisting of IL-1ß, TNF-α, IL-6, and PGE(2) during viral uptake only stimulated HIV-1-specific CD8(+) T cells. Hence, DC maturation state, activation stimulus, and time lag between DC maturation and Ag loading impact HIV-1 capture and virus Ag presentation. Our results demonstrate a dissociation between the capacity to capture HIV-1 and to present viral Ags. Integrase-deficient HIV(NL4-3ΔIN) was also efficiently captured and presented by DC through the HLA-I and HLA-II pathways but in the absence of viral dissemination. HIV(NL4-3ΔIN) seems to be an attractive candidate to be explored. These results provide new insights into DC biology and have implications in the optimization of DC-based immunotherapy against HIV-1 infection.

Escape from highly effective public CD8+ T-cell clonotypes by HIV.

Mapping the precise determinants of T-cell efficacy against viruses in humans is a public health priority with crucial implications for vaccine design. To inform this effort, we performed a comprehensive analysis of the effective CD8(+) T-cell clonotypes that constitute responses specific for the HIV p24 Gag-derived KK10 epitope (KRWIILGLNK; residues 263-272) restricted by HLA-B*2705, which are known to confer superior control of viral replication in HIV-infected individuals. Particular KK10-specific CD8(+) T-cell clonotypes, characterized by TRBV4-3/TRBJ1-3 gene rearrangements, were found to be preferentially selected in vivo and shared between individuals. These "public" clonotypes exhibit high levels of TCR avidity and Ag sensitivity, which impart functional advantages and enable effective suppression of HIV replication. The early L(268)M mutation at position 6 of the KK10 epitope enables the virus to avoid recognition by these highly effective CD8(+) T-cell clonotypes. However, alternative clonotypes with variant reactivity provide flexibility within the overall KK10-specific response. These findings provide refined mechanistic insights into the workings of an effective CD8(+) T-cell response against HIV.

CTL escape mediated by proteasomal destruction of an HIV-1 cryptic epitope.

Cytotoxic CD8+ T cells (CTLs) play a critical role in controlling viral infections. HIV-infected individuals develop CTL responses against epitopes derived from viral proteins, but also against cryptic epitopes encoded by viral alternative reading frames (ARF). We studied here the mechanisms of HIV-1 escape from CTLs targeting one such cryptic epitope, Q9VF, encoded by an HIVgag ARF and presented by HLA-B*07. Using PBMCs of HIV-infected patients, we first cloned and sequenced proviral DNA encoding for Q9VF. We identified several polymorphisms with a minority of proviruses encoding at position 5 an aspartic acid (Q9VF/5D) and a majority encoding an asparagine (Q9VF/5N). We compared the prevalence of each variant in PBMCs of HLA-B*07+ and HLA-B*07- patients. Proviruses encoding Q9VF/5D were significantly less represented in HLA-B*07+ than in HLA-B*07- patients, suggesting that Q9FV/5D encoding viruses might be under selective pressure in HLA-B*07+ individuals. We thus analyzed ex vivo CTL responses directed against Q9VF/5D and Q9VF/5N. Around 16% of HLA-B*07+ patients exhibited CTL responses targeting Q9VF epitopes. The frequency and the magnitude of CTL responses induced with Q9VF/5D or Q9VF/5N peptides were almost equal indicating a possible cross-reactivity of the same CTLs on the two peptides. We then dissected the cellular mechanisms involved in the presentation of Q9VF variants. As expected, cells infected with HIV strains encoding for Q9VF/5D were recognized by Q9VF/5D-specific CTLs. In contrast, Q9VF/5N-encoding strains were neither recognized by Q9VF/5N- nor by Q9VF/5D-specific CTLs. Using in vitro proteasomal digestions and MS/MS analysis, we demonstrate that the 5N variation introduces a strong proteasomal cleavage site within the epitope, leading to a dramatic reduction of Q9VF epitope production. Our results strongly suggest that HIV-1 escapes CTL surveillance by introducing mutations leading to HIV ARF-epitope destruction by proteasomes.

Increased CD127 expression on activated FOXP3+CD4+ regulatory T cells.

Regulatory T cells (Treg) are commonly identified by CD25 (IL-2R alpha) surface expression and/or intracellular expression of the FOXP3 transcription factor. In addition, Treg are also characterized by low CD127 (IL-7R alpha) expression when compared to conventional T cells and their biology in the periphery is considered essentially independent of IL-7. We further investigated CD127 expression on Treg and we demonstrated differential CD127 expression depending on Treg subsets considered. Notably, we observed high CD127 expression on inducible costimulatory molecule (ICOS)- and CD103-expressing Treg subsets. Since these two markers reflect activation status, we addressed whether Treg activation modulated CD127 expression. We demonstrated that in contrast to conventional T cells, Treg significantly upregulated CD127 expression during in vitro and in vivo activation using adoptive transfer and contact dermatitis models. High CD127 expression on Treg was also predominantly detected ex vivo in some specific sites, notably bone marrow and skin. Importantly, higher CD127 expression on Treg correlated with higher phosphorylation of STAT5 upon IL-7 exposure. High CD127 expression on Treg also provided survival advantage upon in vitro incubation with IL-7. We thus demonstrated that low CD127 expression is not an intrinsic characteristic of Treg and we identified activated Treg as a potential target of endogenous or therapeutic IL-7.

Antiretroviral therapy initiation during primary HIV infection enhances both CD127 expression and the proliferative capacity of HIV-specific CD8+ T cells.

OBJECTIVES: HIV-specific CD8+ T cells from patients with primary HIV infection (PHI) and after antiretroviral therapy initiation were evaluated for CD127 expression and proliferative capacity and were compared with cells from chronically-infected patients, including long-term nonprogressors and HIV controllers. METHODS: We studied 30 patients with PHI (from the Agence Nationale de Recherche sur le SIDA Primo-infection Cohort) and 33 patients with chronic HIV infection (including nonprogressor patients from the Agence Nationale de Recherche sur le SIDA ALT Cohort and the Agence Nationale de Recherche sur le SIDA HIV Controllers Study Group). HIV-specific CD8+ T cells were identified by costaining with HIV human leukocyte antigen class I pentamers. CD127 expression was assessed by flow cytometry and cell proliferation by carboxyfluorescein succinimidyl ester labeling. RESULTS: During PHI, most HIV-specific CD8+ T cells coexpressed CD27 and CD45RO, were highly activated, and showed weak Bcl-2 expression. Their CD127 expression was very low and correlated negatively both with HIV RNA and DNA levels and with expression of the activation marker CD38. CD127 expression correlated positively with CD4 cell count, Bcl-2 expression and proliferative capacity. Strong CD127 expression was observed in the two groups of chronically-infected nonprogressors. CD127 expression on HIV-specific CD8+ T cells increased in early-treated PHI patients, reaching levels similar to those observed in nonprogressors. In parallel, these cells acquired strong proliferative capacity. No change in CD127 expression or proliferative potential was observed in untreated patients. CONCLUSION: Early antiretroviral therapy initiation enhances CD127 expression on HIV-specific CD8+ T cells, reaching levels similar to those observed in aviremic nonprogressors, and restores their proliferative capacity.

Heterogeneity in HIV suppression by CD8 T cells from HIV controllers: association with Gag-specific CD8 T cell responses.

"HIV controllers" (HICs) are rare individuals in whom HIV-1 plasma viral load remains undetectable without antiretroviral treatment. This spontaneous viral control in HICs is usually associated to strong functional HIV-specific CD8(+) T cell responses. Accordingly, we have recently shown that CD8(+) T cells from HICs strongly suppress ex vivo HIV-1 infection of autologous CD4(+) T cells, suggesting a crucial role of this response in vivo. Knowledge of the mechanisms underlying the CD8(+) T cell antiviral activity might help to develop effective T cell-based vaccines. In the present work, we further characterized the HIV-suppressive capacity of CD8(+) T cells in 19 HICs. CD8(+) T cells from 14 of the 19 HICs showed strong HIV-suppressive capacity ex vivo. This capacity was stable over time and was partially effective even on other primate lentiviruses. HIV-suppressive capacity of CD8(+) T cells correlated strongly with the frequency of HIV-specific CD8(+) T cells, and in particular of Gag-specific CD8(+) T cells. We also identified five HICs who had weak HIV-suppressive CD8(+) T cell capacities and HIV-specific CD8(+) T cell responses. Among these five HICs, at least three had highly in vitro replicative viruses, suggesting that the control of viremia in these patients is not due to replication-defective viruses. These results, on the one hand, suggest the importance of Gag responses in the antiviral potency of CD8(+) T cells from HICs and, on the other hand, propose that other host mechanisms may contribute to restraining HIV infection in HICs.