Brain illustration taken from Adobe Stock Standard under License ID 222738500. Nature | Vol 592 | 15 April 2021 | 465Patients with PsPD had higher maximal levels of peripheral IDH1-vac induced T cell immune responses than patients who had progressive disease (Fig. 3e). Retrospective assessment of prognostic molecular markers in pre-treatment astrocytoma tissues (Supplementary Table 9, Extended Data Fig. 8) enabled further subgrouping of 24 out of 32 (75.0%) patients in the SDS. PsPD was not associated with any of the assessed tumour-intrinsic molecular markers, such as copy number variation load (CNV-L), methylation class, CDKN2A or CDKN2B deletion status, frequencies of peripheral immune cell subsets, or alterations in top peripheral T cell clonotypes (Supplementary Table 9, Extended Data Figs. 7, 9, 10). During followup, four out of ten patients (40%) with methylation class high grade glioma experienced progressive disease. Of these, patients with an MSS that stayed below median had a 2-year progression-free rate of 0.4 (95% CI 0.052–0.753) compared to 0.8 (95% CI 0.204–0.969) for patients with an MSS that reached above median, despite an equal distribution of unfavourable molecular markers (Fig. 3f, g). Seven out of 12 (58.3%) of the patients with PsPD, including patient ID08, still have stable disease with a median followup time of 53.1 months (95% CI 45.8–58.2 months). Specific T cell receptor in PsPD Among patients with PsPD, only patient ID08 underwent resection of the lesion (Supplementary Table 8). An ex vivo IFNγ ELISpot assay with lesion-infiltrating leukocytes (LILs) showed IDH1(R132H)-reactive T cells (Fig. 4a). On the basis of preclinical data4,5,8 and the obser - vations that neither actively cytotoxic cytokine-producing ex vivo CD8+ LILs nor selected CD8+ T cell clonotype-retrieved T cell recep - tor (TCR)-transgenic cells reacted to IDH1(R132H) (Extended Data Fig. 11), we focused on CD4+ T cells. Single-cell RNA sequencing (scRNA-seq) identified three clusters of CD4+ T cells within the PsPD lesion of patient ID08: regulatory T cells, activated CD40LG+CD4+ T cells, and CXCL13+CD4+ T cells (Fig. 4b, c , Extended Data Fig. 12). CXCL13+CD4+ T cells have been reported to be important for antitu- mour immunity9. By combining scRNA-seq and TCR sequencing, we found that both the CD40LG+CD4+ and CXCL13+CD4+ T cell clusters were dominated by one TCR (TCR14; Fig. 4d). In total, TCR14 was the fourth most abundant TCR within the CD4+ single T cell repertoire, whereas the top three abundant CD4+ TCRs (TCR11–13) were expressed on regulatory T cells that largely lacked TCR14. TCR14 was enriched 50.6-fold in the PsPD lesion of patient ID08 compared to peripheral blood of this patient after administration of IDH1-vac (Extended Data Figs. 10, 12). Transgenic TCR expression in a TCR-deficient human T cell line co-cultured with autologous antigen-presenting cells from patient ID08 showed that TCR14 reacted to IDH1(R132H) (Fig. 4e, Extended Data Fig. 12). These results indicate that IDH1-vac induced clonal expansion of IDH1(R132H)-specific TH cells that infiltrated into the resected lesion.ID01 ID03 ID04 ID06 ID07 ID08 ID09 ID10 ID11 ID12 ID13 ID15 ID17 ID19 ID20 ID21 ID22 ID23 ID24 ID25 ID26 ID27ID28ID31 ID32 ID33 ID02 ID05 ID14ID18 ID29 ID30V03 V05 V07 V10V12 V13<49 50–99 100–400 IDH1(R132H) (p122–136, 15-mer peptide) IDH1(R132H) (p123–142, 20-mer peptide) <4.995.00-29.9930.00–100.00ID01 ID03 ID04 ID06 ID07 ID08 ID09 ID10 ID11ID12 ID13 ID15 ID17 ID19 ID20 ID21 ID22 ID23 ID24 ID25 ID26 ID27ID28ID31 ID32 ID33 ID02 ID05 ID14ID18 ID29 ID30V03 V05V07 V10 V12 V13a bT cell responder T cell non-responder IR positive IR strongIR below threshold for positivity IR not evaluatedELISpot assay ELISA Spots (negative control subtracted) Relative optical density (IDH1(R132H)/negative control) 05 01 00 1502 0000.51.01.52.02.5 MSSRelative value (PLA signals per nucleus)c ID12 ID01 ID05ID07 ID26ID10 ID14ID20ID25 ID31 ID32ID22 ID28r = 0.799 (95% CI 0.443–0.937) P = 0.001n = 13V03 V05 V07 V10 V12V13 dID03 ID04 ID06 ID11 ID27TNF+ IFN+ IL-17+ IL-4+ CD25+CD127–IL-10+ 5101520253035CD4+CD8+TNF+ IFN+ IDH1(R132H)/negative control FC Fig. 2 | Cellular and humoral immunogenicity of IDH1-vac. a, b, Semi-quantitative analysis of T cell ( a) and B cell ( b) immune responses (IR) in all patients in the IDS measured by IFNγ enzyme-linked immunosorbent spot (ELISpot) assay ( a) or IDH1 peptide enzyme-linked immunosorbent assay (ELISA) ( b) (n = 30 patients). Patients are classified as T cell responders ( n = 24 patients) and non-responders ( n = 6 patients) on the basis of specific spot count cut-off of 50 as defined in the study protocol. Response for each visit (V) is shown. c, Flow cytometric effector phenotyping of peripheral IDH1-vac- induced T cells (single live CD3 + cells) from available patient samples with high MSS ( n = 5 patient samples). Relative values after re-stimulation with IDH1(R132H) peptide compared to negative control peptide (myelin oligodendrocyte glycoprotein; MOG) are shown. FC, fold-change. Gating strategy is shown in Extended Data Fig. 4c. d , Correlation of intratumoral IDH1(R132H) peptide presentation at baseline (quantified by PLA signal) with the magnitude and sustainability of specific peripheral T cell responses (quantified by the MSS; see Extended Data Fig. 5). r , Pearson correlation coefficient. Patient ID numbers are shown in a –d. 466 | Nature | Vol 592 | 15 April 2021 ArticleConclusions NOA16 met its primary endpoints by demonstrating the safety and immunogenicity of IDH1-vac in patients with newly diagnosed WHO grade 3 and 4 IDH1(R132H)+ astrocytomas without further positive prognostic factors. Immunogenicity, irrespective of HLA type, and the high rate of PsPD warrant further clinical investigation of IDH1-vac. Patients who did not mount an IDH1-vac induced immune response showed reduced efficacy of the vaccine and disease progression within two years (Extended Data Fig. 3) compared to patients who did mount an immune response (Fig. 3b). IDH1-vac was immunogenic across mul - tiple HLA alleles, supporting the concept of promiscuity of presenta- tion on MHCII4 and justifying patient inclusion independent of HLA alleles. To characterize the specificity and dynamics of vaccine-induced peripheral immune responses, we used deep TCR sequencing from most of the patient samples, in addition to central imaging review, molecular pathology and immune monitoring. Single-cell sequenc - ing of T cells from post-vaccine peripheral blood and a tissue sample provided important insights into vaccine-induced systemic and local immune responses and the underlying biological mechanisms of vaccine-induced PsPD, which was associated with a favourable clinical course in some patients. Although this study provides strong circum- stantial evidence of de novo induction of cytotoxic T cell responses by IDH1(R132H)-reactive TH cells within the CNS10 (Fig. 2c, Extended Data Figs.