Tumor-reactive T cell batches were generated in MLTC by weekly stimulation of PBMC with autologous tumor cells. Sufficient cell numbers for infusion could be reached after one MLTC of 4 weeks for some patients, while for others multiple MLTC were needed to reach the required cell numbers for infusion. The expansion rates of T cells were highest in the second half of the MLTC (week 2–week 4). Analysis of the T cell batches that were infused into the patients in our ongoing clinical protocol [
5] showed that they contain CD4
+CD25
hiFoxP3
+ T cells (Supplementary Figure S1a). Importantly, while there were no overt differences between the frequencies of CD4
+CD25
hiFoxP3
+ T cells in the PBMC used for MLTC, it became clear that higher frequencies of these cells were observed after the MLTC culture period in T cell batches used for treatment of non-responder patients (Fig.
1a). This suggests that the relatively high frequencies of CD4
+CD25
hiFoxP3
+ T cells observed in 3 out of 5 infusion products from non-responders accumulated during culture. Subsequently, the expansion of CD4
+CD25
hiFoxP3
+ T cells was analyzed during the MLTC cultures. There was a peak in CD4
+CD25
hiFoxP3
+ T cells frequency at day 14 of the MLTC (Fig.
1b, c), and there was a direct inverse correlation between CD4
+CD25
hiFoxP3
+ T cell frequencies and the final expansion of T cells at the end of the MLTC (Spearman’s rho,
r = −0.700,
p = 0.04) (Fig.
1d). Since we previously found that the expansion of tumor-specific T cells became visible after 2 weeks of culture, our data suggested that the presence of high numbers of CD4
+CD25
hiFoxP3
+ T cells at this time point had a negative impact on overall T cell proliferation.
To functionally assess if the co-expanded CD4
+CD25
hiFoxP3
+ T cells were responsible for the low expansion capacity of tumor-specific T cells, we first depleted the CD4
+CD25
hiFoxP3
+ T cells from a T cell infusion product from a non-responder patient with a high CD4
+CD25
hiFoxP3
+ T cell frequency and compared the proliferation of the CD4
+CD25
hiFoxP3
+ T cell-depleted T cell batch to the non-CD4
+CD25
hiFoxP3
+ T cell-depleted T cells during MLTC. CD4
+CD25
hiFoxP3
+ T cell depletion resulted in increased proliferation (Fig.
1e) and tumor-specific IFNγ secretion (Fig.
1f) of the T cells, indicating that the CD4
+CD25
hiFoxP3
+ T cells were capable of suppressing the expansion of tumor-reactive T cells. Based on this result, a series of experiments was performed to improve the expansion of T cells in the MLTC by elimination of CD4
+CD25
hiFoxP3
+ T cells at week 2 of the MLTC using a GMP-compliant MACS procedure for CD25-depletion. Only when this procedure resulted in the selective depletion of CD25
hi cells (Fig.
2a, left panel), it was associated with an improved expansion of T cells (Fig.
2a, middle panel), and an increased number of CD8
+ tumor-reactive cells (Fig.
2a, right panel), similar to our initial experiment. However, in cases where this method not only led to the depletion of CD25
hi T cells but also in that of CD25
+ effector T cells (Fig.
2b, left panel), the CD25-depleted fraction hardly expanded and lower numbers of CD8
+ tumor-reactive cells were detected (Fig.
2b, middle and right panel). We repeated this protocol eight times using autologous PBMC and tumor cells from several patients but the variability in outcome remained and apparently was associated with the quality of the separation between CD25
hi T cells and CD25
+ T effector cells. If only CD25
hi T cells and not CD25
+ T effector cells were depleted, it resulted in an improved expansion of tumor-reactive T cells in three out of four experiments (Supplementary Figure S1b). As an alternative for the depletion of CD4
+CD25
hiFoxP3
+ T cells, we depleted the complete CD4
+ fraction from the PBMC at the start of the MLTC (Supplementary Figure S2). CD4
+ T cell depletion was very consistent and almost complete, although low numbers of CD4
+ T cells were still detectable during the culture. Although CD4
+ T cell depletion did not always result in increased expansion rates of the T cells (Fig.
2c, upper panels), the number of tumor-reactive CD8
+ T cells was often increased (Fig.
2c, lower panels). In four out of six MLTC using the PBMC and melanoma cells of different patients, CD4
+ T cell depletion increased the absolute number of tumor-reactive CD8
+ T cells obtained after 4 weeks of MLTC. However, the opposite effect was observed for the two other MLTC (Fig.
2d). Hence, also CD4
+ T cell depletion does not guarantee a more pronounced expansion of tumor-specific CD8
+ T cells.