TCR BV spectratype profiles of CD8⁺ T cells expanded from peripheral blood and transplanted hand skin biopsy at day 573 after transplantation.

<p>CD8⁺ T cells were expanded from the blood (A) or biopsy (B) and harvested for RNA isolation and T cell receptor spectratyping as described in the Methods and Materials. “Relative units” refers to the ratio of each peak to the median concentration of all BV families.</p

Rejection status, immunosuppressive drug regimen, and T cell receptor clonal persistence after hand transplantation.

<p>The histopathologic Banff score (A), immunosuppressive drug doses (B), and identified persistent T cell receptor clones listed in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0136235#pone.0136235.t001" target="_blank">Table 1</a> (C) are plotted over time.</p

Correlation of CD8⁺ T cell spectratype profiles between duplicate samples and between time points.

<p>Left: All peaks in AV (triangles) and BV (circles) spectratypes from duplicate hand biopsies on day 751 after transplantation are plotted against each other (log₁₀ scale for peak size +1). The <i>p</i> value was <0.001 for the correlation. Right: Comparisons of BV spectratype profiles (Czekanowski Similarity Index) are given for each time point and the duplicate samples on day 751.</p

Histopathology of the transplanted hand.

<p>Examples of hematoxylin/eosin staining of biopsies from the transplanted hand are shown (20x). An increase in necrotic keratinocytes along the D-E junction (A) and epidermal spongiosis and lymphocyte exocytosis (B) seen with associated Banff criteria of 0 and III, respectively.</p

Persistent TCR clones infiltrating the transplanted hand.

<p>“</p><p>“>Dom” Indicates that the specified sequence was the dominant sequence revealed by bulk sequencing of the whole family</p><p>“ND” Not Done</p><p>*Obtained from first biopsy on Day 751</p><p>**Obtained from duplicate biopsy on Day 751</p><p>Any BV family demonstrating a peak expansion at more than one time point was subjected to bulk sequencing of the uncloned PCR product for the whole family. If no clearly dominant sequence was obtained, clonal sequencing (TOPO-TA) of at least 12 clones was performed for that family. For each listed peak and detected CDR3 sequence corresponding to that peak size, whether that sequence was the dominant sequence seen in bulk sequencing or the frequency of that sequence within clonal sequences is given for the indicated times after transplantation. The final column gives the minimum duration of each clonal sequence (time between first and last detection).</p

Spectratype BV peak expansions seen over time in CD8⁺ T cells from the transplanted hand.

<p>Spectratypes for BV families were performed (as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0136235#pone.0136235.g003" target="_blank">Fig 3</a>) at the indicated time points. A. All expanded peaks (>2 relative units magnitude) are indicated by black shading, according to BV family and peak size. B. The number of observed expansions is plotted against the Banff score at each time point. C. The magnitudes of observed expansions are plotted against the Banff score at each time point. *The results for duplicate sampling on day 751 were combined for panel A, and averaged for panel C.</p

HIV-1 Nef Sequence and Functional Compartmentalization in the Gut Is Not Due to Differential Cytotoxic T Lymphocyte Selective Pressure

<div><p>The gut is the largest lymphoid organ in the body and a site of active HIV-1 replication and immune surveillance. The gut is a reservoir of persistent infection in some individuals with fully suppressed plasma viremia on combination antiretroviral therapy (cART) although the cause of this persistence is unknown. The HIV-1 accessory protein Nef contributes to persistence through multiple functions including immune evasion and increasing infectivity. Previous studies showed that Nef’s function is shaped by cytotoxic T lymphocyte (CTL) responses and that there are distinct populations of Nef within tissue compartments. We asked whether Nef’s sequence and/or function are compartmentalized in the gut and how compartmentalization relates to local CTL immune responses. Primary <i>nef</i> quasispecies from paired plasma and sigmoid colon biopsies from chronically infected subjects not on therapy were sequenced and cloned into Env⁻ Vpu⁻ pseudotyped reporter viruses. CTL responses were mapped by IFN-γ ELISpot using expanded CD8+ cells from blood and gut with pools of overlapping peptides covering the entire HIV proteome. CD4 and MHC Class I Nef-mediated downregulation was measured by flow cytometry. Multiple tests indicated compartmentalization of <i>nef</i> sequences in 5 of 8 subjects. There was also compartmentalization of function with MHC Class I downregulation relatively well preserved, but significant loss of CD4 downregulation specifically by gut quasispecies in 5 of 7 subjects. There was no compartmentalization of CTL responses in 6 of 8 subjects, and the selective pressure on quasispecies correlated with the magnitude CTL response regardless of location. These results demonstrate that Nef adapts via diverse pathways to local selective pressures within gut mucosa, which may be predominated by factors other than CTL responses such as target cell availability. The finding of a functionally distinct population within gut mucosa offers some insight into how HIV-1 may persist in the gut despite fully suppressed plasma viremia on cART.</p></div

HIV-1 epitopes presented by MHC class I types associated with superior immune containment of viremia have highly constrained fitness landscapes

<div><p>Certain Major Histocompatibility-I (MHC-I) types are associated with superior immune containment of HIV-1 infection by CD8⁺ cytotoxic T lymphocytes (CTLs), but the mechanisms mediating this containment are difficult to elucidate <i>in vivo</i>. Here we provide controlled assessments of fitness landscapes and CTL-imposed constraints for immunodominant epitopes presented by two protective (B*57 and B*27) and one non-protective (A*02) MHC-I types. Libraries of HIV-1 with saturation mutagenesis of CTL epitopes are propagated with and without CTL selective pressure to define the fitness landscapes for epitope mutation and escape from CTLs via deep sequencing. Immunodominant B*57- and B*27- present epitopes are highly limited in options for fit mutations, with most viable variants recognizable by CTLs, whereas an immunodominant A*02 epitope-presented is highly permissive for mutation, with many options for CTL evasion without loss of viability. Generally, options for evasion overlap considerably between CTL clones despite highly distinct T cell receptors. Finally, patterns of variant recognition suggest population-wide CTL selection for the A*02-presented epitope. Overall, these findings indicate that these protective MHC-I types yield CTL targeting of highly constrained epitopes, and underscore the importance of blocking public escape pathways for CTL-based interventions against HIV-1.</p></div

Evidence of Compartmentalization.

a<p>Samples of just plasma or just gut isolates having >70% of 1000 bootstrap replicates supporting an independent cluster, NS  =  not significant.</p>b<p>Association Index. values <0.1 are highly significant for compartmentalization.</p>c<p>Slatkin-Maddison migration test. “1” indicates a single migration event consistent with compartmentalization, text following indicates the direction of the migration event between plasma and gut. “multiple” indicates multiple migrations or free exchange of sequences between plasma and gut consistent with lack of compartmentalization.</p

Measures of selective pressure on blood and gut <i>nef</i> quasispecies.

<p>A.) The global non-synonymous to synonymous substitution rate ratio (dN/dS) with 95% confidence intervals (CI) was calculated for all 234 sequences in the dataset, all gut-derived sequences, all plasma-derived sequences, and for all individual pairs of gut and plasma sequences. The range of the 95% CI for the dN/dS estimate for all sequences is highlighted in grey so that those estimates with a 95% CI entirely outside this range (542 gut, 648 gut and plasma, 650 gut) can be easily identified. B.) The diversity among all sequences from plasma and gut as well as among blood and gut isolates from each subject was calculated with a s.e.m. from 500 bootstrap replicates. The difference in diversity between plasma and gut sequences was evaluated with a two-tailed t test and pairs with significant differences are marked with a bar with the p-value indicated above it.</p