Regulatory T cells expanded from HIV-1-infected individuals maintain phenotype, TCR repertoire and suppressive capacity

Abstract

While modulation of regulatory T cell (Treg) function and adoptive Treg transfer are being explored as therapeutic modalities in the context of autoimmune diseases, transplantation and cancer, their role in HIV-1 pathogenesis remains less well defined. Controversy persists regarding their beneficial or detrimental effects in HIV-1 disease, which warrants further detailed exploration. Our objectives were to investigate if functional CD4(+) Tregs can be isolated and expanded from HIV-1-infected individuals for experimental or potential future therapeutic use and to determine phenotype and suppressive capacity of expanded Tregs from HIV-1 positive blood and tissue. Tregs and conventional T cell controls were isolated from blood and gut-associated lymphoid tissue of individuals with HIV-1 infection and healthy donors using flow-based cell-sorting. The phenotype of expanded Tregs was assessed by flow-cytometry and quantitative PCR. T-cell receptor ß-chain (TCR-β) repertoire diversity was investigated by deep sequencing. Flow-based T-cell proliferation and chromium release cytotoxicity assays were used to determine Treg suppressive function. Tregs from HIV-1 positive individuals, including infants, were successfully expanded from PBMC and GALT. Expanded Tregs expressed high levels of FOXP3, CTLA4, CD39 and HELIOS and exhibited a highly demethylated TSDR (Treg-specific demethylated region), characteristic of Treg lineage. The TCRß repertoire was maintained following Treg expansion and expanded Tregs remained highly suppressive in vitro. Our data demonstrate that Tregs can be expanded from blood and tissue compartments of HIV-1+ donors with preservation of Treg phenotype, function and TCR repertoire. These results are highly relevant for the investigation of potential future therapeutic use, as currently investigated for other disease states and hold great promise for detailed studies on the role of Tregs in HIV-1 infection.

Figure 1. Cell sorting, FOXP3 TSDR and gene expression.

A. Flow-cytometry gating strategy used to isolate CD25⁺CD127^low regulatory T cells (Tregs) and CD25⁻CD127⁺ conventional T cells (Tconv) from CD4⁺ T cells (Left Panel). Expansion fold change of Tregs isolated from HIV-1-infected (dark grey) (n = 8 controllers + 13 chronic untreated) and healthy (light grey) (n = 4) individuals during 7 days of cell culture (right Panel). B. Relative mRNA expression in arbitrary units (A.U.) of FOXP3 and IL-10 quantified by real time PCR in expanded Tregs (n = 3 controllers+2 chronic untreated) and Tconvs (n = 2 controllers+2 chronic untreated) isolated from HIV-1 infected individuals after 7 days of culture. C. Frequency of demethylation of the Treg Specific Demethylation (TSDR) region of the FOXP3 gene in expanded Tregs and Tconvs after 7 days of culture as assayed by real time PCR. Empty symbols represent HIV-1 controllers and solid symbols HIV-1 chronic untreated individuals.

Figure 2. Phenotyping of expanded Tregs by flow cytometry.

A. Representative examples of gating strategy used for CD25⁺FOXP3⁺ staining by flow-cytometry of ex vivo PBMC (upper panel) isolated from a HIV-1 controller and matched expanded Tregs (lower panel) at day 7 of expansion. B. Expression of different Tregs markers quantified by flow-cytometry of expanded (day 7) and ex vivo unexpanded Tregs and Tconvs. MFI = Mean Fluorescence intensity. Empty symbols represent HIV-1 controllers and solid symbols HIV-1 chronic untreated individuals. C. Representative example of flow-cytometry gating strategy used to phenotype Tregs, Tconvs (n = 3 controllers+9 chronic untreated) and ex vivo CD4 T cells (n = 3 controllers+3 chronic untreated) isolated from HIV-1 positive individuals based on their CD45RA and FOXP3 expression profiles . The left dot plot shows ex vivo CD4⁺ T cells from PBMC, the middle dot plot represents an example of expanded Tregs (black dots) and Tconvs (light grey dots). The right histogram graph quantifies the different Treg subsets in HIV-1 positive individuals. Gate 1 and white columns represent “resting” CD45RA⁺FOXP3^low Tregs, gate 2 and grey columns represent “non-suppressive cytokine-secreting” CD45RA⁻FOXP3^low T cells and gate 3 and black columns represent “activated” CD45RA⁻FOXP3^high Tregs.

Figure 3. The TCR repertoire is not altered after in vitro expansion of Tregs.

A. Degree of expansion of the TCRß repertoire (i.e. number of TCRs in a sample that belongs to an individual clone and expressed as percentage of total reads) from 2×10⁴ ex vivo sorted unexpanded (light grey) and 2×10⁴ in vitro expanded (Day 14; dark grey) Tregs isolated from the same original PBMC specimen. B. Distribution of variable-gene (Vß-gene) variants from 2×10⁴ ex vivo sorted unexpanded (light grey) and 2×10⁴ in vitro expanded (Day 14; dark grey) Treg TCR-β clones isolated from the same PBMC specimen. C. Distribution of joining-gene (Jß-gene) variants from 2×10⁴ ex vivo sorted unexpanded (light grey) and 2×10⁴ in vitro expanded (Day 14; dark grey) Treg TCR-β clones isolated from the same PBMC specimen.

Figure 4. Suppressive function of expanded Tregs.

A. Suppressive activity of expanded Tregs from HIV-1+ (n = 7 controllers +11 chronic untreated individuals) and healthy controls (n = 4) on activated CD8⁺ T cells (left) and CD4⁺ T cells (right). Columns represent activated T cells (white) co-cultured with autologous expanded Tregs (black) or Tconvs (grey). A suppressive activity of 100% indicates that the proliferation of activated T cells was completely inhibited and a negative suppressive activity signifies that the proliferation of T cells was higher than in the condition “T cells alone”. B. Representative example of a flow-based Treg suppressive assay after 4 days of co-culture. CFSE dilution of activated CD8⁺ T cells (upper panel) and CD4⁺ T cells (lower panel) are represented as histograms. Left columns show CFSE dilution of bead-activated T cells from frozen PBMC, the other columns represent activated T cells co-cultured with autologous expanded Tregs (middle) or Tconvs (right). C. ⁵¹Chromium release assay. Representation of the cytotoxic function (% lysis) of a HIV-1 specific CTL clone (effector) using a HIV-1-peptide-loaded B cell line labeled with [⁵¹Cr] as a target with or without expanded Tregs at a 1 Effector:1 Treg :1 Target ratio. D. Example of gating strategy used to isolate Tregs from the peripheral blood of an HIV-1-infected infant (Left). Numbers of cells counted during the expansion of these Tregs (Middle). Percentage of suppression of the expanded Tregs or expanded Tconvs on activated CD4⁺ T cells when co-cultured with autologous CFSE loaded PBMC at a 1∶1 ratio (Right). E. Example of gating strategy used to isolate Tregs from the colon of an HIV-1-infected individual (Left). The middle panel represents the numbers of cells counted during the expansion of these Tregs (Middle). Percentage of suppression of the expanded Tregs (n = 1 HIV-1-negative sample +4 HIV-1-positive samples) or expanded Tconvs (n = 1 HIV-1-negative sample +4 HIV-1-positive samples) isolated from the GALT on activated CD8⁺ T cells when co-cultured with CFSE loaded PBMC at a 1∶1 ratio (Right).