Here we present a simple and sensitive flow cytometric—based assay to assess T cell proliferation. Given the critical role STAT5A phosphorylation in T cell proliferation, we decided to evaluate phosphorylation of STAT5A as an indicator of T cell proliferation. We determined pSTAT5A in T cell treated with either CD3/CD28 or PHA. After stimulation, T cells from adult healthy donors displayed a strong long-lasting phosphorylation of STAT5A, reaching a peak value after 24 h. The median fluorescence intensity (MFI) of pSTAT5A increased from 112 ± 17 to 512 ± 278 (CD3/CD28) (24 h) and to 413 ± 123 (PHA) (24 h), the IL-2 receptor-α (CD25) expression was greatly enhanced and after 72 h T cell proliferation amounted to 52.3 ± 10.3% (CD3/CD28) and to 48.4 ± 9.7% (PHA). Treatment with specific JAK3 and STAT5 inhibitors resulted in a complete blockage of phosphorylation of STAT5A, CD25 expression, and suppression of T cell proliferation. Compared with currently available methods, STAT5A phosphorylation is well-suited to predict T cell proliferation. Moreover, the method presented here is not very time consuming (several hours) and delivers functional information from which conclusions about T cell proliferation can be drawn.

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The prognosis of IDH1 wild-type MGMT promoter-unmethylated GBM patients remains poor. Addition of Temozolomide (TMZ) to first-line local treatment shifted the median overall survival (OS) from 11.8 to 12.6 months. We retrospectively analyzed the value of individualized multimodal immunotherapy (IMI) to improve OS in these patients. All adults meeting the criteria and treated 06/2015–06/2021 were selected. Thirty-two patients (12f, 20m) had a median age of 47 y (range 18–69) and a KPI of 70 (50–100). Extent of resection was complete (11), <complete (12) or not documented (9). Seven patients were treated with surgery/radio(chemo)therapy and subsequent IMI (Group-1); 25 patients were treated with radiochemotherapy followed by maintenance TMZ plus IMI during and after TMZ (Group-2). Age, KPI and extent of resection were not different amongst both groups. The median OS of group-1 patients was 11 m (2 y OS: 0%). Surprisingly the median OS of group-2 patients was 22 m with 2 y OS of 36% (CI95%: 16–57), which was significantly (Log-rank: p = 0.0001) different from group-1. The data suggest that addition of IMI after local therapy on its own has no relevant effect on OS in these GBM patients, similar to maintenance TMZ. However, the combination of both TMZ + IMI significantly improved OS.

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