The complex and specific pMHC interactions with diverse HIV-1 TCR clonotypes reveal a structural basis for alterations in CTL function

Immune control of viral infections is modulated by diverse T cell receptor (TCR) clonotypes engaging peptide-MHC class I complexes on infected cells, but the relationship between TCR structure and antiviral function is unclear. Here we apply in silico molecular modeling with in vivo mutagenesis studies to investigate TCR-pMHC interactions from multiple CTL clonotypes specific for a well-defined HIV-1 epitope. Our molecular dynamics simulations of viral peptide-HLA-TCR complexes, based on two independent co-crystal structure templates, reveal that effective and ineffective clonotypes bind to the terminal portions of the peptide-MHC through similar salt bridges, but their hydrophobic side-chain packings can be very different, which accounts for the major part of the differences among these clonotypes. Non-specific hydrogen bonding to viral peptide also accommodates greater epitope variants. Furthermore, free energy perturbation calculations for point mutations on the viral peptide KK10 show excellent agreement with in vivo mutagenesis assays, with new predictions confirmed by additional experiments. These findings indicate a direct structural basis for heterogeneous CTL antiviral function

T cell receptors for the HIV KK10 epitope from patients with differential immunologic control are functionally indistinguishable

HIV controllers (HCs) are individuals who can naturally control HIV infection, partially due to potent HIV-specific CD8+ T cell responses. Here, we examined the hypothesis that superior function of CD8+ T cells from HCs is encoded by their T cell receptors (TCRs). We compared the functional properties of immunodominant HIV-specific TCRs obtained from HLA-B*2705 HCs and chronic progressors (CPs) following expression in primary T cells. T cells transduced with TCRs from HCs and CPs showed equivalent induction of epitope-specific cytotoxicity, cytokine secretion, and antigen-binding properties. Transduced T cells comparably, albeit modestly, also suppressed HIV infection in vitro and in humanized mice. We also performed extensive molecular dynamics simulations that provided a structural basis for similarities in cytotoxicity and epitope cross-reactivity. These results demonstrate that the differential abilities of HIV-specific CD8+ T cells from HCs and CPs are not genetically encoded in the TCRs alone and must depend on additional factors

Antiviral CD8(+) T Cells Restricted by Human Leukocyte Antigen Class II Exist during Natural HIV Infection and Exhibit Clonal Expansion.

CD8(+) T cell recognition of virus-infected cells is characteristically restricted by major histocompatibility complex (MHC) class I, although rare examples of MHC class II restriction have been reported in Cd4-deficient mice and a macaque SIV vaccine trial using a recombinant cytomegalovirus vector. Here, we demonstrate the presence of human leukocyte antigen (HLA) class II-restricted CD8(+) T cell responses with antiviral properties in a small subset of HIV-infected individuals. In these individuals, T cell receptor β (TCRβ) analysis revealed that class II-restricted CD8(+) T cells underwent clonal expansion and mediated killing of HIV-infected cells. In one case, these cells comprised 12% of circulating CD8(+) T cells, and TCRα analysis revealed two distinct co-expressed TCRα chains, with only one contributing to binding of the class II HLA-peptide complex. These data indicate that class II-restricted CD8(+) T cell responses can exist in a chronic human viral infection, and may contribute to immune control

T cell receptors for the HIV KK10 epitope from patients with differential immunologic control are functionally indistinguishable

HIV controllers (HCs) are individuals who can naturally control HIV infection, partially due to potent HIV-specific CD8+ T cell responses. Here, we examined the hypothesis that superior function of CD8+ T cells from HCs is encoded by their T cell receptors (TCRs). We compared the functional properties of immunodominant HIV-specific TCRs obtained from HLA-B*2705 HCs and chronic progressors (CPs) following expression in primary T cells. T cells transduced with TCRs from HCs and CPs showed equivalent induction of epitope-specific cytotoxicity, cytokine secretion, and antigen-binding properties. Transduced T cells comparably, albeit modestly, also suppressed HIV infection in vitro and in humanized mice. We also performed extensive molecular dynamics simulations that provided a structural basis for similarities in cytotoxicity and epitope cross-reactivity. These results demonstrate that the differential abilities of HIV-specific CD8+ T cells from HCs and CPs are not genetically encoded in the TCRs alone and must depend on additional factors

A genome-wide CRISPR screen identifies a restricted set of HIV host dependency factors

Host proteins are essential for HIV entry and replication and can be important nonviral therapeutic targets. Large-scale RNA interference (RNAi)-based screens have identified nearly a thousand candidate host factors, but there is little agreement among studies and few factors have been validated. Here we demonstrate that a genome-wide CRISPR-based screen identifies host factors in a physiologically relevant cell system. We identify five factors, including the HIV co-receptors CD4 and CCR5, that are required for HIV infection yet are dispensable for cellular proliferation and viability. Tyrosylprotein sulfotransferase 2 (TPST2) and solute carrier family 35 member B2 (SLC35B2) function in a common pathway to sulfate CCR5 on extracellular tyrosine residues, facilitating CCR5 recognition by the HIV envelope. Activated leukocyte cell adhesion molecule (ALCAM) mediates cell aggregation, which is required for cell-to-cell HIV transmission. We validated these pathways in primary human CD4 + T cells through Cas9-mediated knockout and antibody blockade. Our findings indicate that HIV infection and replication rely on a limited set of host-dispensable genes and suggest that these pathways can be studied for therapeutic intervention

New data on the chronology of the Vale do Forno sedimentary sequence (Lower Tejo River terrace staircase) and its relevance as a fluvial archive of the Middle Pleistocene in western Iberia

The Vale do Forno archaeological sites (Alpiar\ue7a, central Portugal) document the earliest human occupation in the Lower Tejo River, well established in geomorphological and environmental terms, within the Middle Pleistocene. In a staircase of six fluvial terraces, the Palaeolithic sites were found on the T4 terrace (\ufe24 m, above river bed) which is made of a basal Lower Gravels unit (LG) and an overlying Upper Sands unit (US). Geomorphological mapping, coupled with lithostratigraphy, sedimentology and luminescence dating (quartz-OSL and K-feldspar post-IRIR290) were used in this study. The oldest artefacts found in the LG unit show crude bifacial forms that can be attributed to the Acheulian. In contrast, the US unit has archaeological sites stratigraphically documenting successive phases of an evolved Acheulian. Luminescence dating and correlation with the Marine Isotopic Stages suggest that the LG unit has a probable age of ca. 335 to 325 ka and the US unit an age of ca. 325 to 155 ka. This is in contrast to previous interpretations ascribing this terrace (and lithic industries) to the Last Interglacial and early phases of the Last Glacial. The VF3 site (Milharos), containing Micoquian (Final Acheulian) industries (with fine and elaborated bifaces), found in a stratigraphic level located between the T4 terrace deposits and a colluvium associated with Late Pleistocene aeolian sands, is younger than 155 ka but much older than 32 ka

Role of T cell receptors in suppressing HIV infection in elite controllers

Virtually all HIV-infected patients progress to AIDS if not treated. A group of patients, called Elite Controllers (ECs), can suppress HIV infection without treatment. This ability of ECs is, in part, due to potent CD8+ T cell (CTL) responses against HIV epitopes presented by human leukocyte antigen (HLA) molecules. In this study, we focused on CTL responses restricted to HLA-B27, an allele that is enriched in ECs, but is not sufficient for protective ability. In HLA-B27+ ECs or non-controllers (NCs), immunodominant CTL responses specific to the KK10 epitope in Gag are found. Previous studies have shown that unlike NCs, B27-KK10-specific CTLs from ECs are efficient at eliminating HIV-infected or KK10-peptide pulsed cells. As the potency of CTLs can be modulated by their T cell receptor (TCR), we hypothesized that B27-KK10-specific TCRs play a significant role in suppressing HIV in ECs. Therefore, we cloned B27-KK10-specific TCRs from ECs or NCs, expressed them in primary T cells, and tested their cytotoxicity. We incubated TCR-transduced T cells with either HIV-infected or KK10-peptide pulsed target cells and measured target cell lysis. Unexpectedly, EC-TCRs were not significantly different than NC-TCRs in their cytotoxicity. We then tested the ability of these cells to suppress HIV in humanized mice and found no significant difference between EC- and NC-TCRs. We are currently investigating various aspects of these TCRs such as cross-reactivity to variants of KK10. The result that TCRs from ECs and non-controllers do not recapitulate the phenotype of CTLs is intriguing and warrants further studies to uncover the mechanism of immunologic control of HIV. Our studies also have implications in immunotherapy and vaccine approaches for HIV

K-Feldspar IRSL dating of a Pleistocene river terrace staircase sequence of the Lower Tejo River (Portugal, western Iberia)

We present the results of K-feldspar IRSL dating of the four lower terraces (T3–T6) of the Portuguese Tejo River, in the Arripiado-Chamusca area. Terrace correlation was based upon: a) analysis of aerial photographs, geomorphological mapping and field topographic survey; b) sedimentology of the deposits; and c) luminescence dating. Sediment sampled for luminescence dating gave unusually high dose rates, of between 3.4 and 6.2 Gy/ka and, as a result, quartz OSL was often found to be in saturation. We therefore used the IRSL signal from K-feldspar as the principal luminescence technique. The K-feldspar age results support sometimes complex geomorphic correlations, as fluvial terraces have been vertically displaced by faults (known from previous studies). Integration of these new ages with those obtained previously in the more upstream reaches of the Tejo River in Portugal indicates that the corrected K-feldspar IRSL ages are stratigraphically and geomorphologically consistent over a distance of 120 km along the Tejo valley. However, we are sceptical of the accuracy of the K-feldspar ages of samples from the T3 and T4 terraces (with uncorrected De values >500 Gy). In these cases the Dose Rate Correction (DRC) model puts the natural signals close to luminescence saturation, giving a minimum corrected De of about 1000 Gy, and thus minimum terrace ages; this may even be true for those doses >200 Gy. Luminescence dating results suggest that: T3 is older than 300 ka, probably ca. 420–360 ka (wMarine Isotope Stage [MIS]11); T4 is ca. 340–150 ka (wMIS9-6); T5 is 136–75 ka (wMIS5); T6 is 60–30 ka (MIS3); an aeolian sand unit that blankets T6 and some of the older terraces is 30– 12 ka. Collectively, the luminescence ages seem to indicate that regional river downcutting events may be coincident with periods of low sea level (associated, respectively, with the MIS10, MIS6, MIS4 and MIS2)