HIV-specific CD8+ T cells from HIV+ individuals receiving HAART can be expanded ex vivo to augment systemic and mucosal immunity in vivo

Abstract

Most HIV+ individuals require lifelong highly active antiretroviral therapy (HAART) to suppress HIV replication, but fail to eliminate the virus in part because of residual replication in gut-associated lymphoid tissues (GALT). Naturally elicited HIV-specific CD8+ T cells generated in the acute and chronic infectious phases exhibit antiviral activity, but decrease in number after HAART. Therapeutic vaccines represent a potential strategy to expand cellular responses, although previous efforts have been largely unsuccessful, conceivably because of a lack of responding HIV-specific central-memory CD8+ T cells (Tcm). To determine whether patients receiving HAART possess CD8+ T cells with Tcm qualities that are amenable to augmentation, HIV-specific CD8+ T-cell clones were derived from HIV-reactive CD28+CD8+ T-cell lines isolated from 7 HIV+ HAART-treated patients, expanded ex vivo, and reinfused into their autologous host. Tracking of the cells in vivo revealed that clones could persist for ≥ 84 days, maintain expression and/or re-express CD28, up-regulate CD62L, secrete IL-2, proliferate on cognate Ag encounter and localize to the rectal mucosa. These results suggest some infused cells exhibited phenotypic and functional characteristics shared with Tcm in vivo, and imply that more effective therapeutic vaccination strategies targeting CD8+ Tcm in patients on HAART might provide hosts with expanded, long-lasting immune responses not only systemically but also in GALT.

Trial registration: ClinicalTrials.gov NCT00110578.

Figures

Figure 1

Phenotypic and functional characterization of HIV-specific CD8+ T-cell lines and clones isolated and expanded for infusion. (A) Percentages of HIV-specific CD8+ T cells in each cell line (left column) from which each infused clone was ultimately derived. Specific pentamer binding (y-axis) and CD8 (x-axis) are shown. Expression of CD28 (right column) on gated HIV-specific pentamer+CD8+ T cells (bold line) compared with isotype control (gray line). Inset values represent percentages of CD28+CD8+ T cells. (B) Data for patient 7705 is shown (HLA B*0702/IPRRIRQGL-HIV-ENV) and is representative of HIV-specific CD8+ T-cell clones used for adoptive transfer. Lytic activity (top panel) of the HIV-specific CD8+ T-cell clone to autologous B-LCL pulsed with decreasing concentrations of peptide, IFNγ secretion (bottom left panel), and binding to the corresponding MHC-peptide pentamer (bottom right panel) is shown. (C) Expression of CD28 on clonal HIV-specific CD8+ T cells infused (bold line) for each patient compared with isotype control (gray line). Inset values represent percentages of CD28+CD8+ T cells. (D) Expression of CD45RA, CD45RO, CD62L, CCR7, and CD127 (bold line) compared with isotype control (gray line) on a representative CD8+ T-cell clone used for infusion (7705). (E) Expression of CD103, CCR9, CD11a, CD11b, CD11c, integrin α4 and integrin β7 (bold line) compared with isotype control (gray line). The bottom right panel shows coexpression of integrin α4 and integrin β7 on the clone (black dots) compared with PBMC (gray dots).

Figure 2

In vivo persistence of HIV-specific CD8+ T-cell clones isolated from CD28-expressing CD8+ T-cell lines. (A shaded area) The percentage of pentamer+CD8+ T cells (x-axis) detected in PBMCs collected −7 days (+/−2 days), immediately before and after infusions are shown for 5 (7701, 7702, 7703, 7705, and 7707) of 7 infused HIV-specific CD8+ T-cell clones that showed persistence for ≥ 21 days after the first infusion. Persistence was calculated as the last time point at which pentamer+CD8+ T cells were 2 times background levels or > 0.1%. (A nonshaded area) The percentage of pentamer+CD8+ T cells after each infusion and up to 112 days after the first infusion is shown for the same patients. (B) Two patients (7704 and 7709) that showed a rapid disappearance of the infused clones within 3 days of both infusions are shown.

Figure 3

Adoptively transferred HIV-specific CD8+ T cells show phenotypic characteristics associated with CD8+ Tcm over time after transfer in vivo. (A) Expression of CD28 (x-axis) and CD62L (y-axis) on pentamer+ cells from patient 7707 before and after transfer. The phenotype of pentamer+ T cells (A*0301/QVPLRPMTYK-HIV-NEF) analyzed by flow cytometry 3, 14, 28, and 84 days after the second infusion are shown. Percentages of pentamer+CD8+ T cells that express CD28 and CD62L in each gated quadrant are shown in the top right aspect of each plot. (B top panel) Expression of CD28 (x-axis) and CD62L (y-axis) on pentamer+CD8+ T cells (B*0801/GEIYKRWII-HIV-GAG) from patient 7702 obtained 3, 7, 42, and 84 days after the second infusion. (B bottom panel) Pentamer+CD8+ T cells (HLA B*0702/IPRRIRQGL-HIV-ENV) from patient 7705 obtained 3, 14, 28, and 84 days after the second infusion.

Figure 4

CD28 expression by adoptively transferred HIV-specific CD8+ T-cell clones is associated with IL-2 secretion after stimulation by specific Ag. (A) Expression of CD28 on the HIV-specific CD8+ T-cell clone before infusion (left histogram) for patient 7707 (A*0301/QVPLRPMTYK-HIV-NEF). Whole PBMCs were obtained at day 3 after the 2nd infusion and stained with specific pentamer (A*0301/QVPLRPMTYK-HIV-NEF). The expression of CD28 ex vivo on pentamer+ T cells is shown (plot to the right of the histogram). Cells were stimulated with specific peptide (QVPLRPMTYK) and pentamer+CD8+ T cells analyzed for the surface expression of CD28 and intracellular production of IFNγ, TNFα, and IL-2 (rightmost plots). (B) The same analysis was performed on whole PBMCs obtained at day 112 after transfer of the HIV-specific clone into patient 7707. (C) Data on PBMCs that were obtained on day 21 after the first infusion from patient 7703 (B*0801/GEIYKRWII-HIV-GAG) are shown. Analysis for pentamer+CD28+ and CD28− subpopulations are shown individually. (D) Data for patients 7701 and 7702 (B*0801/GEIYKRWII-HIV-GAG) are shown on PBMC that were obtained 14 days after the first infusion (7701), 7 and 112 days after the second infusion (7702), after stimulation with the cognate peptide (GEIYKRWII). Pentamer−CD8+ T cells in the same assays did not produce IFNγ, TNFα, or IL-2 (not shown).

Figure 5

Adoptively transferred HIV-specific CD8+ T cells that express CD28 are capable of proliferation after specific Ag exposure in the absence of supplemental exogenous cytokines. All plots are gated on pentamer+ T cells or as otherwise indicated. (A-B) PBMCs from patients 7701 and 7702 collected, respectively, 14 and 3 days after the first infusion and PBMCs from patients 7703 and 7705 collected, respectively, 21 and 3 days after the first infusion were stained with CFSE and incubated in the presence of cognate peptide (B*0801/GEIYKRWII-HIV-GAG for 7701-7703 and B*0702/IPRRIRQGL-HIV-ENV for 7705). Expression of CD28 on pentamer+CD8+ T cells (plots in the top row) is shown. Plots in the second and third rows show CFSE dilution after 5 and 7 days ex vivo (x-axis) relative to CD28 expression (y-axis). Dilution of CFSE after 7 days ex vivo is shown (histogram, bottom row). (C) Pentamer+CD8+ T cells in PBMCs from patient 7707 collected 3 days after the second infusion and after 7 days of ex vivo stimulation with cognate peptide (A*0301/QVPLRPMTYK-HIV-NEF; left panel). The same experiment performed with the addition of exogenous IL-2 50 IU/mL (right panel). (D) CFSE dilution of detected pentamer+CD8+ T cells PBMC from patient 7707 collected 84 days after the second infusion and after 7 days of ex vivo stimulation with cognate peptide with (right panel) or without IL-2 (left panel).

Figure 6

Infused HIV-specific CD8+ T-cell clones survey the rectal mucosa. (A-B) Fluorescence images of fresh rectal tissue sections (30 μm) from patient 7707 (A) and 7709 (B) stained with DAPI in blue, CD8 in green and Qdot655 multimer A*0301/QVPLRPMTYK-HIV-NEF for patient 7707 and Qdot655 multimer B*2705/KRWIILGLNK-HIV-GAG for patient 7709 in red. Arrows indicate Qdot655+CD8+cells (yellow). Inset percentage values reflect Qdot655+CD8+ T cells as a percent of total CD8+ T cells. (C) Cryopreserved rectal tissue sections stained concurrently with CD8 and the corresponding HIV-specific Qdot655 multimer B*0801/GEIYKRWII-HIV-GAG for 7702 and 7703; B*0702/IPRRIRQGL-HIV-ENV for 7705. Ten fields of 1 mm2 spanning 2 rectal biopsies per time point were enumerated for CD8+Qdot655+ T cells and results expressed as a percentage of total CD8+ T cells. (D) Fluorescence images of cryopreserved rectal tissue sections (7 μm) from patient 7702 stained with DAPI in blue, CD8 in green, and Qdot 655 multimer. B*0801/GEIYKRWII-HIV-GAG in red. Inset values represent the average of 10 counted fields. (E) Frequencies of HIV-specific T cells in rectal biopsies for patients 7701, 7704, and 7707 expressed as a percent HIV-specific Vβ copies relative to CD8β copies. Results are an average of 2 independent rectal samples taken at each time point. (F) CMV-specific CD8+ T cell clones quantified in rectal biopsies from patients 7701 and 7707 (TCR-specific RT-PCR) and CMV-specific CD8+ T-cell clones in rectal biopsies from patients 7702, 7703, and 7707 (specific Qdot655 multimers). One-tailed, paired Student t tests were used for statistical analysis.