Ex Vivo Expansion of Human CD8+ T Cells Using Autologous CD4+ T Cell Help

Background: Using in vivo mouse models, the mechanisms of CD4+ T cell help have been intensively investigated. However, a mechanistic analysis of human CD4+ T cell help is largely lacking. Our goal was to elucidate the mechanisms of human CD4+ T cell help of CD8+ T cell proliferation using a novel in vitro model. Methods/Principal Findings: We developed a genetically engineered novel human cell-based artificial APC, aAPC/mOKT3, which expresses a membranous form of the anti-CD3 monoclonal antibody OKT3 as well as other immune accessory molecules. Without requiring the addition of allogeneic feeder cells, aAPC/mOKT3 enabled the expansion of both peripheral and tumor-infiltrating T cells, regardless of HLA-restriction. Stimulation with aAPC/mOKT3 did not expand Foxp3+ regulatory T cells, and expanded tumor infiltrating lymphocytes predominantly secreted Th1-type cytokines, interferon-γ and IL-2. In this aAPC-based system, the presence of autologous CD4+ T cells was associated with significantly improved CD8+ T cell expansion in vitro. The CD4+ T cell derived cytokines IL-2 and IL-21 were necessary but not sufficient for this effect. However, CD4+ T cell help of CD8+ T cell proliferation was partially recapitulated by both adding IL-2/IL-21 and by upregulation of IL-21 receptor on CD8+ T cells. Conclusions: We have developed an in vitro model that advances our understanding of the immunobiology of human CD4+ T cell help of CD8+ T cells. Our data suggests that human CD4+ T cell help can be leveraged to expand CD8+ T cells in vitro

aAPC/mOKT3 expands both CD4⁺ and CD8⁺ T cells without using allogeneic feeder PBMC.

<p>(<b>A</b>) CD3⁺ T cells were stimulated twice with aAPC/mOKT3 and supplemented with IL-2 between stimulations. Fold expansion of CD3⁺ T cells over one month is shown for three donors. Shading shows the proportion of expanded CD4⁺ (white) and CD8⁺ (black) T cells, and percent CD8⁺ T cells is indicated. (<b>B</b>) CD3⁺ T cells were stimulated twice with aAPC/mOKT3 or beads (Dynabeads CD3/CD28) and supplemented with IL-2 between stimulations. Fold expansion of CD3⁺ T cells over one month is shown for three donors. Shading shows the proportion of expanded CD4⁺ (white) and CD8⁺ (black) T cells, and percent CD8⁺ T cells is indicated. (<b>C</b>) CD3⁺ T cells were expanded as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0030229#pone-0030229-g002" target="_blank">Figure 2A</a>. Expression of surface molecules on gated CD4⁺ and CD8⁺ T cells is shown (open). Isotype mAb staining was used as a control (shaded). (<b>D</b>) CD4⁺ CD25⁺ Foxp3⁺ Treg cells, present pre-expansion, were absent in expanded cultures. CD4⁺ CD25⁺ cells, pre- and post-expansion, were stained intracellularly with anti-Foxp3 mAb (open) and isotype control (shaded).</p

IL-2/IL-21 and upregulation of IL-21R expression replace CD4⁺ T cell help of CD8⁺ T cell expansion <i>in vitro.</i>

<p>(<b>A</b>) IL-21R expression on CD8⁺ T cells stimulated with aAPC/mOKT3 in the presence or absence of CD4⁺ T cells was studied by flow cytometry. On the left, histogram plots for 1 donor is shown and, on the right, IL-21R expression on day 4 is displayed for 5 donors. (<b>B</b>) IL-21R expression on CD8⁺ T cells ectopically transduced with mock or IL-21R is shown (left). Expansion of transduced CD8⁺ T cells stimulated twice by aAPC/mOKT3 with or without IL-21 is compared (right). Percent expansion was calculated by dividing the number of expanded transduced CD8⁺ T cells by that of CD8⁺ T cells stimulated in the presence of CD4⁺ T cells. Values indicate mean of four independent experiments; error bars show s.d. *<i>P</i><0.005.</p

Generation of aAPC/mOKT3.

<p>Surface expression of a transduced membranous form of anti-CD3 mAb, and transduced CD80, CD83, and endogenous HLA class I, class II, CD54, and CD58 on aAPC/mOKT3 is shown. A membranous form of anti-CD3 mAb on aAPC/mOKT3 (open) and wild type K562 (shaded) was stained using goat anti-mouse IgG (H+L). Other surface molecules were stained with each specific mAb (open) and isotype control (shaded) and analyzed by flow cytometry. Note the lack of endogenous expression of HLA class I and II on aAPC/mOKT3.</p

Autologous CD4⁺ T cell secretion of IL-2/IL-21 is necessary but not sufficient to help CD8⁺ T cells proliferate.

<p>(<b>A</b>) CD8⁺ T cells were stimulated twice by aAPC/mOKT3 with or without CD4⁺ T cells and treated with IL-2 between stimulations. Fold expansion of CD8⁺ T cells over 28 days is shown for 3 donors. (<b>B</b>) CD8⁺ T cells were stimulated only once by aAPC/mOKT3 with or without CD4⁺ T cells in transwell plates. No IL-2 or other cytokines were given. Fold expansion of CD8⁺ T cells over 6 days is shown for 7 donors. (<b>C</b>) Culture supernatants were tested for a panel of soluble factors to identify mediators of CD4⁺ T cell help. Relative changes in cytokines, comparing mixed vs. separate cultures, are shown. Data is representative of two donors. Absolute values for two donors are shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0030229#pone.0030229.s003" target="_blank">Table S1</a>. (<b>D</b>) Suppression of CD8⁺ T cell expansion in the presence of CD4⁺ T cells by blocking reagents is presented as percent suppression relative to control. Values indicate mean of four independent experiments; error bars show s.d. *<i>P</i><0.005. (<b>E</b>) CD8⁺ T cells were stimulated twice with aAPC/mOKT3 in the presence or absence of CD4⁺ T cells. IL-2, IL-21, or both were added in each condition. Fold expansion of CD8⁺ T cells over 28 days is shown. Percent expansion was calculated by dividing the number of expanded CD8⁺ T cells by the number of CD8⁺ T cells expanded in the presence of CD4⁺ T cells. Values indicate mean of six independent experiments; error bars show s.d.</p

aAPC/mOKT3 expanded TIL are Foxp3 negative and secrete predominantly Th1 cytokines.

<p>(<b>A</b>) Expansion of TIL obtained from breast and ovarian cancer ascites and melanoma metastases is shown. Shading indicates the proportion of CD4⁺ (white) and CD8⁺ (black) T cells in expanded cultures. The percentage of CD8⁺ T cells in pre- and post-expansion cultures is shown. Note that in all samples tested, the percentage of CD8⁺ T cells increased even in those that initially contained a minimal percentage of CD8⁺ T cells. NT denotes not tested. (<b>B</b>) CD4⁺ CD25⁺ Foxp3⁺ Treg cells, present pre-expansion, were not detectable after one month of culture. CD4⁺ CD25⁺ cells were intracellularly stained with anti-Foxp3 mAb (open) and isotype control (shaded). (<b>C</b>) IFN-γ, IL-2, IL-4, and IL-10 secretion of expanded TIL was determined by ELISPOT assays. Cytokine secretion by TIL from the breast cancer ascites specimen prior to expansion is shown as a control. Pre-expansion samples from melanoma and ovarian cancer specimens were not studied because of low initial cell numbers.</p

Expansion with aAPC/mOKT3 does not induce skewing of the TCR Vβ repertoire.

<p>(<b>A</b>) TCR Vβ subfamily analysis before and after stimulation with aAPC/mOKT3 is shown. CD3⁺ T cells were stimulated with aAPC/mOKT3 on days 0 and 14 and were treated with IL-2 at 300 IU/ml between stimulations. TCR Vβ usage analysis was performed on days 0, 14, 28. Data shown is on gated CD4⁺ and CD8⁺ T cells. (<b>B, C</b>) A2⁺ CD3⁺ T cells were stimulated twice with aAPC/mOKT3 for one month. Subsequently, CD8⁺ T cells were purified from expanded CD3⁺ T cells and further stimulated with aAPC/A2 pulsed with Flu or MART1 peptide. (<b>B</b>) Flu specificity was demonstrated by multimer staining (left). Functional competence was demonstrated by antigen-specific cytotoxicity (middle) and IFN-γ secretion (right). T2 cells pulsed with Flu peptide (▪) or control peptide (•) were used as targets. (<b>C</b>) MART1 specificity was similarly demonstrated by multimer staining (left). The HLA-A2⁺/MART1⁺ melanoma line, Malme-3M (▪), and the HLA-A2⁺/MART1^- melanoma line, A375 (•), were used as targets in cytotoxicity (middle) and IFN-γ ELISPOT assays (right).</p