Human CD4⁺CD25⁺ Cells in Combination with CD34⁺ Cells and Thymoglobulin to Prevent Anti-hematopoietic Stem Cell T Cell Alloreactivity

Tregs isolation and effect on CD34⁺ cell allostimulatory activity. (A) Purity of CD4⁺CD25⁺ cells after immunomagnetic isolation (Treg isolation kit, Miltenyi) and (B) expression of CD25, CD127, and FoxP3 in CD4⁺CD25⁺ (filled histogram) or CD4⁺CD25⁻ (open histogram) cell fractions. (C) Tregs or CD4⁺CD25⁻ T cells isolated from allogeneic peripheral blood (ALLO PB), or autologous cord blood (AUTO CB), or autologous PBSC (AUTO PBSC) were added to an MLC with irradiated CD34⁺ cells and allo-responder T cells. CD34: allo-responder, and responder: Treg or CD4⁺CD25⁻ cell ratios were: 1:2 and 1:1, respectively (n = 3 experiments for each condition). ³H-thymidine uptake assay was utilized to measure T cell responses and mean ± SD cpm are shown (C, left). Addition of Tregs resulted in reduced alloreactivity compared to CTR experiments in each condition (ALLO PB: P = .2; Auto CB: P = .05, Auto PBSC: P = .05). Analysis of T cell inhibition (C, right) showed that Tregs obtained from the same stem cell product as CD34⁺ cells (AUTO CB or AUTO PBSC) had a greater suppressive activity (P = .04 by ANOVA test). Suppression activity is indicated by the percentage of inhibition of responder cells.

Unmodified suppressive activity of Tregs primed with allogeneic CD34⁺ cells. Allogeneic PB Tregs that were initially cultured with irradiated CD34⁺ cells for 6 days maintained their suppressive activity when added to a third party MLC at a 1:1 ratio with responder cells, as opposed to control CD4⁺CD25⁻ cells, which instead increased T cell alloreactivity (n = 3 experiments). The effect of Tregs or CD4⁺CD25⁻ cells is shown as percentage (%) of T cell proliferation or suppression compared with control MLC where allo-T cells are stimulated by CD34⁺ cells. Results are shown as average ± SD of 3 separate experiments.

Clonogenic activity of CD34⁺ cells in the presence of Tregs. (A) CD34⁺ cells were mixed with CD4⁺CD25⁺ or CD25⁻ T cells at a 1:1 ratio and then tested for CFU-C in semisolid media (n = 5 experiments). Differences were not statistically significant by ANOVA test. (B) Human CD34⁺ cells were transplanted into NOD/SCID mice w/wo allogeneic Tregs at a 1:1 or 1:2 ratio, and 6 weeks later the marrow was harvested and tested by flow cytometry to assess human hematopoietic stem cell engraftment. The percentage of human CD45⁺ cells was comparable in each group (n = 5 mice per group). Analysis of subsets of huCD45⁺ cells did not show differences in myeloid or erythroid lineages in animals transplanted w/wo Tregs (not shown).

Effect of thymoglobulin on T cell proliferation in vitro. (A) Thymoglobulin was tested in vitro at 0 (control), 50, 100, 300, or 500 μg/mL, in primary MLC with MNC stimulator cells and allogeneic T cell responders at a 1:2 ratio in media containing human AB serum that was not inactivated. T cell alloreactivity was measured by ³H-thymidine incorporation assay after 6 days of culture. Results are shown as mean S.I. ± SD of 5 separate experiments. T cell alloresponses in the presence of thymoglobulin at 50, 100, or 300 μg/mL were greater than in control MLC (P = .02), whereas thymoglobulin at 500 μg/mL completely inhibited T cell response (P = .03). (B) Thymoglobulin was added to liquid cultures of purified T cells at the physiologic dose of 100 μg/mL on day 1 only (B, left), days 1 and 3 (B, center), or days 1, 3, and 6 (B, right). T cell proliferation was measured daily for 12 days by ³H-thymidine incorporation assay, and cpm values of 1 experiment representative of 3 are shown.

Thymoglobulin does not affect Tregs in vitro . (A) Liquid cultures of CD4⁺CD25⁺ or CD4⁺CD25⁻ cells were performed in the presence of thymoglobulin at 10 or 100 μg/mL. Dotted line represents the baseline number of cells plated. After 3 and 6 days of culture, cells were harvested and counted by trypan blue exclusion to assess viable cells. Results are shown as average ± SD of 5 separate experiments. (B) Expression of CCR7, GITR, CD152, CD62L, and intracytoplasmic FoxP3 before (Pre) and after a 3-day culture with thymoglobulin (Post-thymo) was analyzed by flow cytometry in PB CD4⁺CD25⁺ Tregs. Histograms shown are representative of one of 3 separate experiments; (C) Allogeneic CD4⁺CD25⁺ cells, or CD4⁺CD25⁻ cells as control, were exposed to thymoglobulin for 3 days in vitro and then were added back to a primary MLC with irradiated CD34⁺ stimulator cells and third-party T cells (n = 3 experiments). Thymoglobulin-exposed Tregs (Thymo-CD4⁺CD25⁺) suppressed the anti-HSC T cell alloreactivity as opposed to thymoglobulin-exposed CD4⁺CD25⁻ cells. Suppression activity is indicated by the percentage of inhibition of responder cells.

Thymoglobulin-induced IL10 production by Tregs. CD4⁺CD25⁺ Tregs were cultured with CD34⁺ cells at a 1:1 ratio, with CD34 and thymoglobulin at 100 μg/mL, or with thymoglobulin alone. After 3 days supernatants were harvested and cytokine levels were measured by Bio-Plex Cytokine assay (n = 3 experiments). In the presence of thymoglobulin, Tregs produced mostly IL-10 and IL-13 independently of the allostimulatory effect of CD34⁺ cells.