Salah Mansour

Cholesteryl esters stabilize human CD1c conformations for recognition by self-reactive T cells

Significance T cells autoreactive to cluster of differentiation 1c (CD1c) are abundant in human blood but lipid antigens recognized by these T cells remained poorly understood. A new 2.4-Å structure of CD1c and computational simulations thereof indicated substantial conformational plasticity of CD1c with ligand-induced formation of an F′ roof and G′ portal, as well as the potential of CD1c to present acylated sterols. Confirming these predictions we demonstrated CD1c loading and biophysical interaction of CD1c–lipid complexes with self-reactive human T-cell receptors for two lipid classes: cholesteryl esters similar to those accumulating in foamy macrophages (e.g., in atherosclerosis) and acylated steryl glycosides from Borrelia burgdorferi. These findings differentiate CD1c from other CD1 isoforms and open up new avenues for research into the role of CD1c in human immunity. Cluster of differentiation 1c (CD1c)-dependent self-reactive T cells are abundant in human blood, but self-antigens presented by CD1c to the T-cell receptors of these cells are poorly understood. Here we present a crystal structure of CD1c determined at 2.4 Å revealing an extended ligand binding potential of the antigen groove and a substantially different conformation compared with known CD1c structures. Computational simulations exploring different occupancy states of the groove reenacted these different CD1c conformations and suggested cholesteryl esters (CE) and acylated steryl glycosides (ASG) as new ligand classes for CD1c. Confirming this, we show that binding of CE and ASG to CD1c enables the binding of human CD1c self-reactive T-cell receptors. Hence, human CD1c adopts different conformations dependent on ligand occupancy of its groove, with CE and ASG stabilizing CD1c conformations that provide a footprint for binding of CD1c self-reactive T-cell receptors.

CD1d protein structure determines species-selective antigenicity of isoglobotrihexosylceramide (iGb3) to invariant NKT cells

Isoglobotrihexosylceramide (iGb3) has been identified as a potent CD1d‐presented self‐antigen for mouse invariant natural killer T (iNKT) cells. The role of iGb3 in humans remains unresolved, however, as there have been conflicting reports about iGb3‐dependent human iNKT‐cell activation, and humans lack iGb3 synthase, a key enzyme for iGb3 synthesis. Given the importance of human immune responses, we conducted a human–mouse cross‐species analysis of iNKT‐cell activation by iGb3‐CD1d. Here we show that human and mouse iNKT cells were both able to recognise iGb3 presented by mouse CD1d (mCD1d), but not human CD1d (hCD1d), as iGb3‐hCD1d was unable to support cognate interactions with the iNKT‐cell TCRs tested in this study. The structural basis for this discrepancy was identified as a single amino acid variation between hCD1d and mCD1d, a glycine‐to‐tryptophan modification within the α2‐helix that prevents flattening of the iGb3 headgroup upon TCR ligation. Mutation of the human residue, Trp153, to the mouse ortholog, Gly155, therefore allowed iGb3‐hCD1d to stimulate human iNKT cells. In conclusion, our data indicate that iGb3 is unlikely to be a major antigen in human iNKT‐cell biology.

Structural and Functional Changes of the Invariant NKT Clonal Repertoire in Early Rheumatoid Arthritis.

Invariant NKT cells (iNKT) are potent immunoregulatory T cells that recognize CD1d via a semi-invariant TCR (iNKT-TCR). Despite the knowledge of a defective iNKT pool in several autoimmune conditions, including rheumatoid arthritis (RA), a clear understanding of the intrinsic mechanisms, including qualitative and structural changes of the human iNKT repertoire at the earlier stages of autoimmune disease, is lacking. In this study, we compared the structure and function of the iNKT repertoire in early RA patients with age- and gender-matched controls. We analyzed the phenotype and function of the ex vivo iNKT repertoire as well as CD1d Ag presentation, combined with analyses of a large panel of ex vivo sorted iNKT clones. We show that circulating iNKTs were reduced in early RA, and their frequency was inversely correlated to disease activity score 28. Proliferative iNKT responses were defective in early RA, independent of CD1d function. Functional iNKT alterations were associated with a skewed iNKT-TCR repertoire with a selective reduction of high-affinity iNKT clones in early RA. Furthermore, high-affinity iNKTs in early RA exhibited an altered functional Th profile with Th1- or Th2-like phenotype, in treatment-naive and treated patients, respectively, compared with Th0-like Th profiles exhibited by high-affinity iNKTs in controls. To our knowledge, this is the first study to provide a mechanism for the intrinsic qualitative defects of the circulating iNKT clonal repertoire in early RA, demonstrating defects of iNKTs bearing high-affinity TCRs. These defects may contribute to immune dysregulation, and our findings could be exploited for future therapeutic intervention.

Dissection of the host-pathogen interaction in human tuberculosis using a bioengineered 3-dimensional model

Cell biology differs between traditional cell culture and 3-dimensional (3-D) systems, and is modulated by the extracellular matrix. Experimentation in 3-D presents challenges, especially with virulent pathogens. Mycobacterium tuberculosis (Mtb) kills more humans than any other infection and is characterised by a spatially organised immune response and extracellular matrix remodelling. We developed a 3-D system incorporating virulent mycobacteria, primary human blood mononuclear cells and collagen–alginate matrix to dissect the host-pathogen interaction. Infection in 3-D led to greater cellular survival and permitted longitudinal analysis over 21 days. Key features of human tuberculosis develop, and extracellular matrix integrity favours the host over the pathogen. We optimised multiparameter readouts to study emerging therapeutic interventions: cytokine supplementation, host-directed therapy and immunoaugmentation. Each intervention modulates the host-pathogen interaction, but has both beneficial and harmful effects. This methodology has wide applicability to investigate infectious, inflammatory and neoplastic diseases and develop novel drug regimes and vaccination approaches. DOI: http://dx.doi.org/10.7554/eLife.21283.001

KIR2DS2 recognizes conserved peptides derived from viral helicases in the context of HLA-C

KIR2DS2, an activating natural killer cell receptor, recognizes conserved peptides derived from the RNA helicases of pathogenic flaviviruses. Killing viral helicases Recognition of evolutionarily conserved pathogen-associated molecules drives innate immune responses. Naiyer et al. report that a killer cell immunoglobulin-like receptor (KIR), KIR2DS2, promotes activation of natural killer (NK) cells by recognizing conserved peptides from flaviviral RNA helicases when presented by a particular human leukocyte antigen (HLA) allele, HLA-C*0102. They have identified two distinct peptide motifs—LNPSVAATL and MCHAT—that are sensed by KIR2DS2. The former is conserved across hepatitis C virus isolates; the latter is conserved in a number of flaviviruses including dengue, Zika, and yellow fever viruses. The study illustrates that a single KIR receptor has evolved to activate NK cells in response to multiple pathogenic viruses. Killer cell immunoglobulin-like receptors (KIRs) are rapidly evolving species-specific natural killer (NK) cell receptors associated with protection against multiple different human viral infections. We report that the activating receptor KIR2DS2 directly recognizes viral peptides derived from conserved regions of flaviviral superfamily 2 RNA helicases in the context of major histocompatibility complex class I. We started by documenting that peptide LNPSVAATL from the hepatitis C virus (HCV) helicase binds HLA-C*0102, leading to NK cell activation through engagement of KIR2DS2. Although this region is highly conserved across HCV isolates, the sequence is not present in other flaviviral helicases. Embarking on a search for a conserved target of KIR2DS2, we show that HLA-C*0102 presents a different highly conserved peptide from the helicase motif 1b region of related flaviviruses, including dengue, Zika, yellow fever, and Japanese encephalitis viruses, to KIR2DS2. In contrast to LNPSVAATL from HCV, these flaviviral peptides all contain an “MCHAT” motif, which is present in 61 of 63 flaviviruses. Despite the difference in the peptide sequences, we show that KIR2DS2 recognizes endogenously presented helicase peptides and that KIR2DS2 is sufficient to inhibit HCV and dengue virus replication in the context of HLA-C*0102. Targeting short, but highly conserved, viral peptides provide nonrearranging innate immune receptors with an efficient mechanism to recognize multiple, highly variable, pathogenic RNA viruses.

Natural variations at position 93 of the invariant Vα24-Jα18 α chain of human iNKT-cell TCRs strongly impact on CD1d binding

Human invariant natural killer T (NKT) cell TCRs bind to CD1d via an “invariant” Vα24‐Jα18 chain (iNKTα) paired to semi‐invariant Vβ11 chains (iNKTβ). Single‐amino acid variations at position 93 (p93) of iNKTα, immediately upstream of the “invariant” CDR3α region, have been reported in a substantial proportion of human iNKT‐cell clones (4–30%). Although p93, a serine in most human iNKT‐cell TCRs, makes no contact with CD1d, it could affect CD1d binding by altering the conformation of the crucial CDR3α loop. By generating recombinant refolded iNKT‐cell TCRs, we show that natural single‐nucleotide variations in iNKTα, translating to serine, threonine, asparagine or isoleucine at p93, exert a powerful effect on CD1d binding, with up to 28‐fold differences in affinity between these variants. This effect was observed with CD1d loaded with either the artificial α‐galactosylceramide antigens KRN7000 or OCH, or the endogenous glycolipid β‐galactosylceramide, and its importance for autoreactive recognition of endogenous lipids was demonstrated by the binding of variant iNKT‐cell TCR tetramers to cell surface expressed CD1d. The serine‐containing variant showed the strongest CD1d binding, offering an explanation for its predominance in vivo. Complementary molecular dynamics modeling studies were consistent with an impact of p93 on the conformation of the CDR3α loop.

Antigen-loaded ER microsomes from APC induce potent immune responses against viral infection.

Although matured DC are capable of inducing effective primary and secondary immune responses in vivo, it is difficult to control the maturation and antigen loading in vitro. In this study, we show that ER‐enriched microsomal membranes (microsomes) isolated from DC contain more peptide‐receptive MHC I and II molecules than, and a similar level of costimulatory molecules to, their parental DC. After loading with defined antigenic peptides, the microsomes deliver antigenic peptide–MHC complexes (pMHC) to both CD4 and CD8 T cells effectively in vivo. The peptide‐loaded microsomes accumulate in peripheral lymphoid organs and induce stronger immune responses than peptide‐pulsed DC. The microsomal vaccines protect against acute viral infection. Our data demonstrate that peptide–MHC complexes armed microsomes from DC can be an important alternative to DC‐based vaccines for protection from viral infection.

Tuberculosis: An Infection-Initiated Autoimmune Disease?

Tuberculosis (TB) is caused by Mycobacterium tuberculosis (Mtb) and provided original proof that an infectious agent can cause human disease. However, key steps in TB pathogenesis remain poorly understood. We propose that autoimmunity is a critical and overlooked process driving pathology in TB, and present clinical and experimental observations supporting this hypothesis.

Corticosteroids and infliximab impair the performance of interferon-γ release assays used for diagnosis of latent tuberculosis

The impact of immunosuppression on interferon-γ release assays and novel cytokine biomarkers of TB infection, mycobacteria-specific IL-2, IP-10 and TNF-α responses was investigated in an ex vivo model. Cytokine responses in standard QuantiFERON-TB Gold in-Tube (QFT-GIT) assays were compared with duplicate assays containing dexamethasone or infliximab. Dexamethasone converted QFT-GIT results from positive to negative in 30% of participants. Antigen-stimulated interferon-γ, IL-2 and TNF-α responses were markedly reduced, but IP-10 responses were preserved. Infliximab caused QFT-GIT result conversion in up to 30% of participants and substantial reductions in all cytokine responses. Therefore, corticosteroids and anti-TNF-α agents significantly impair interferon-γ release assay performance. IP-10 may be a more robust TB biomarker than interferon-γ in patients receiving corticosteroids.

CD1b-restricted GEM T cell responses are modulated by Mycobacterium tuberculosis mycolic acid meromycolate chains

Significance Tuberculosis is a major global pandemic responsible for more deaths than any other infectious disease, yet no effective vaccine exists. Here, we demonstrate CD1b expression within human tuberculous granulomas, supporting a role for CD1b lipid antigen presentation in host immunity to infection. CD1b presents mycolates, the dominant Mycobacterium tuberculosis (Mtb) cell wall lipid class and key virulence factors, to αβ T cells. We reveal that mycolate tail moieties, distal to the head group, are antigenic determinants for the conserved human germline-encoded mycolyl lipid-reactive (GEM) T cell receptors (TCRs). Computational simulations suggest a putative mechanism whereby lipid-ligand dynamics within CD1b regulate GEM-TCR activity. This work provides insights for the development of major histocompatibility complex (MHC)-independent Mtb lipid vaccines, including those that target GEM T cells. Tuberculosis (TB), caused by Mycobacterium tuberculosis, remains a major human pandemic. Germline-encoded mycolyl lipid-reactive (GEM) T cells are donor-unrestricted and recognize CD1b-presented mycobacterial mycolates. However, the molecular requirements governing mycolate antigenicity for the GEM T cell receptor (TCR) remain poorly understood. Here, we demonstrate CD1b expression in TB granulomas and reveal a central role for meromycolate chains in influencing GEM-TCR activity. Meromycolate fine structure influences T cell responses in TB-exposed individuals, and meromycolate alterations modulate functional responses by GEM-TCRs. Computational simulations suggest that meromycolate chain dynamics regulate mycolate head group movement, thereby modulating GEM-TCR activity. Our findings have significant implications for the design of future vaccines that target GEM T cells.