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

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

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

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

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

Suppression of HIV and viral evolution in the plasma of NSG-CTL mice.

<p><b>A.</b> Blood plasma from the same HIV-1_HSA-HA infected mice as described in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002649#ppat-1002649-g003" target="_blank">Figure 3</a> was collected 2 weeks and 6 weeks post infection. Viral RNA (vRNA) levels per sample (typically 50 µl of plasma per mouse) were determined by quatitative reverse transcriptase (RT)-PCR and results were multiplied by a standard factor to yield copies of vRNA per milliliter (ml) of blood. The points represent the copies of HIV vRNA per milliliter (ml) of blood and the solid line represents mean per group (+/− SEM). Statistical comparison was performed between SL9-specific TCR containing mice and non-specific TCR-containing mice and p values are provided (Student's t test). The dotted line indicates the limit of detection of the assay. The data are representative of 3 separate experiments, with a minimum of 3 mice per experimental condition, and utilized the same mice depicted in the experiment described in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002649#ppat-1002649-g003" target="_blank">Figure 3</a>. <b>B.</b> Evolution of the SL9 epitope in infected mice. Viral stock of the input virus, and virus from plasma of mice containing the control TCR or virus from plasma of mice containing the SL9-specific TCR 6 weeks following infection was sequenced utilizing a RT-PCR technique. The translated sequence is provided for each sample, with the SL9 epitope highlighted in bold.</p

Effector cell differentiation and control of viral replication.

<p><b>A.</b> Peripheral blood from uninfected (left column) and HIV infected (right column) mice was analyzed six weeks post infection with HIV for expression of CD8, the transgenic HIV-specific TCR (SL-9 Tetramer), and the CD45RA and CCR7 differentiation markers. The top row displays gated CD8+ T cells expressing the transgenic HIV-specific TCR. CD8+ cells expressing the transgenic TCR are indicated in the box gate and the percentage of total CD8+ cells expressing the transgenic TCR is provided in the gate. The bottom row displays the CD45RA versus CCR7 staining profile of the CD8+, SL-9 tetramer+cells in the gates indicated in the top row with the percentage of cells in each quadrant provided in their respective quadrants. <b>B.</b> Levels of transgenic HIV-specific TCR+, CD8+ T cells reconstituting mice 2 weeks prior to HIV infection versus viral load 6 weeks following HIV infection. The levels of SL-9 tetramer+cells of the CD8+ T cell population in individual mice were assessed in peripheral blood 2 weeks (week -2) prior to HIV infection by flow cytometry and are provided on the y-axis. The x-axis indicates the serum viral loads of these individual mice 6 weeks following infection. The data significantly correlate as determined by the SRCT and the p value is provided. Note that initially high levels of immune reconstitution correlate with lower viral loads at the 6 week time point. The dotted line indicates the limit of detection of the assay. <b>C.</b> Levels of transgenic HIV-specific TCR+, CD8+ T cells versus viral load 6 weeks following HIV infection. Data were analyzed as described above 6 weeks post HIV infection. The data significantly correlate as determined by the SRCT and the p value is provided. Note that at this time point, higher levels of CTL in the blood are found in animals with higher viral load. The dotted line indicates the limit of detection of the assay. <b>D.</b> Antigen-driven expansion of CD8+, HIV-specific TCR expressing cells in HIV infected mice. Levels of SL9 tetramer staining, CD8+ T cells were assessed in the peripheral blood of infected animals (solid lines) or uninfected animals (dashed lines) two weeks prior to infection (week -2), and 4 and 6 weeks post infection. Data is expressed as the percentages of tetramer+cells of total CD8+ T cells. Note that the level of HIV-specific cells in animals showing initially low levels of reconstitution are considerably higher at the late time point, suggesting proliferative response to the high levels of antigen. The data are representative of 1 of 3 separate experiments with a minimum of 3 mice per experimental condition.</p

Construction and multilineage reconstitution of NSG-CTL mice.

<p><b>A.</b> Schematic illustrating the construction of NSG-CTL mice: CD34+ cells are isolated from fetal liver by cell sorting (1). then are transduced with lentiviral vector containing the SL9-specific TCR (2). A fraction of these cells are then implanted under the kidney capsule in NSG mice flowing combination with fetal liver stromal elements and fetal thymus in matrigel (3a). Another fraction of these transduced cells are viably frozen in liquid nitrogen (LN2) (3b). Three weeks following implantation, the engrafted mice are then sublethally irradiated (3 Gy) and previously frozen cells are thawed and injected intravenously into these mice, where the cells home to and engraft in the bone marrow (4). 6–12 weeks following injection of cells, TCR expression was analyzed and mice were infected with HIV (5). Mouse blood is then assessed for HIV infection 2 and 6 weeks following infection (6). <b>B.</b> Multilineage hematopoietic reconstitution of NSG mice receiving genetically modified HSCs. Peripheral blood from these mice were assessed by flow cytometry and gated for CD45+ human leukocytes (top panel). These cells were assessed for the denoted cell surface marker expression including HLA-DR+CD11c+myeloid cells, CD3-CD56+ NK cells, CD3+ T cell, CD19+ B cells. CD3+ cells were gated (lower left panel) and assessed for CD4 and CD8 expression (lower right panel). The numbers indicate the percentage of each population of cells in the mouse peripheral blood. <b>C.</b> Repopulation of HSCs in mouse bone marrow. Mouse bone marrow was assessed for the presence of CD34+ human HSCs (left panel) and CD3+ T cell and CD19+ B cell engraftment 6 weeks following CD34+ cell injection by flow cytometry. <b>D.</b> Reconstitution of NSG-CTL mice with cells expressing the HIV-specific TCR transgene. Cells were isolated from the indicated organ in NSG-CTL mice 6 weeks following CD34+ cell injection and analyzed by flow cytometry for CD3+ T cells binding SL9-containing tetramers. The numbers indicate the percentage of T cells within the indicated organ expressing the transgenic TCR. The data are representative of mice receiving human tissue and HIV-specific TCR transduced CD34+ cells in the same experiment identified above (n = 12).</p

Suppression of HIV replication by HIV-TCR containing T cells.

<p><b>A.</b> Suppression of HIV-1 induced CD4 cell depletion by cells containing HIV-specific TCR. Mice containing cells derived from HSC transduced with either HIV Gag SL9-epitope specific TCR (n = 6 mice) or a non-specific control TCR (n = 7 mice) were infected with HIV-1_HSA-HA or left uninfected (n = 4 mice)(SL9-specific TCR containing mice) and assessed 2 weeks and 6 weeks following infection for peripheral human CD45+, CD4+ cells. Statistical comparison of CD4 cell depletion to uninfected controls was performed using the Student's t test, p values are provided for each indicated comparison. The solid lines represent the mean +/− the standard error of the mean (SEM). <b>B.</b> Suppression of HIV expressing cells. Mice treated as described in (A) were assessed for human CD45+ cells expressing HIV by flow cytometry for the HSA-HA marker gene. Comparison of HIV expression levels between SL-9 containing and control TCR-containing mice are provided at week 2 and week 6 post infection (Student's t test). The data represent 1 experiment of 3, with a minimum of 3 mice per experimental condition, and is a separate experiment than that depicted in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002649#ppat-1002649-g002" target="_blank">Figure 2</a>.</p