From November 2016 until March 2019, a total of 19 eligible patients (median age, 11 years; IQR = 4.5 years; 53% female, 47% male) were enrolled in stratum A and 10 eligi- ble patients (median age, 13 years; IQR = 4.25 years; 60% female, Table 1. Patients’ baseline characteristics Characteristics Whole cohort (n = 29) DIPG (stratum A; n = 19) Other DMG (stratum B; n = 10) Median age, yr (range) 11 (5–18) 11 (5–17) 12 (7–18) Female, no. (%) 16 (55%) 10 (53%) 6 (60%) Male, no. (%) 13 (45%) 9 (47%) 4 (40%) Location Pons 21 19 2 (with cerebellar/spinal cord) Thalamic 6 0 7 Spinal cord 2 0 1 Race White 18 11 7 African American 2 1 1 Other (not available) 8 7 1 Declined to identify 1 0 1 Ethnicity Non-Hispanic 23w 14 9 Hispanic 3 3 0 Not available/declined 3 2 1 Median no. of days from biopsy to start of RT (range)A16 (2–49) 17 (4–25) 22 (2–49) Median no. of days from end of RT to first vaccine (range) 40 (22–71) 39 (27–71) 39 (22–53) Median no. of vaccines (range) 6 (1–11) 7 (2–11) 5 (1–11) Positive H3.3K27M detection in plasma (% of available samples) 41/60 (68.3%) 29/42 (69.0%) 12/18 (66.7%) PBMC immune profiling, median no. of samples per patient (range) 2 (0–5)B2 (1–5) 2 (0–3) Steroid use, no. (%) 13 (45) 9 (47) 4 (40) Temozolomide use during RT, no. (%) 4 (13.8%) 3 (15.8%) 1 (10%) Off-study treatment criteria Progression 27 18 9 Lost to follow-up; withdrawal of consent 1 0 1 Toxicity 1 1 0 AExcluding 1 patient who had biopsy after start of radiation therapy. BOne patient sample (PNOC007-16) lost due to technical difficulties during data acquisition. The Journal of Clinical Investigation CLINICAL MEDICINE 6327 jci.org Volume 130 Number 12 December 2020 lected at multiple time points (n = 18), we analyzed longitudinal H3.3K27M-reactive CD8+ T cell frequencies (Supplemental Fig - ure 4), which revealed that 7 (39%) patients met the criteria for an immunological response (Figure 1E). Among these patients, the median OS of overall immunological responders was 16.3 months (n = 7 , 95% CI, 12.6–NA) compared with 9.9 months (n = 11, 95% CI, 9.0–NA) for nonresponders (P = 0.05, log-rank test) (Figure 1F), with an estimated multivariate HR of 0.04 (95% CI, 0.004– 0.045, P < 0.01) (Supplemental Table 1). Further, immunological responders exhibited prolonged PFS (P = 0.05, log-rank test) com- pared with nonresponders (Figure 1G). Patients with Tem (n = 6, P = 0.02, log-rank test) expansions also exhibited trends toward prolonged OS (Figure 1H and Supplemental Figure 5) and PFS (P = 0.06, log-rank test) compared with their counterparts (Figure 1I and Supplemental Figure 6). These trends persisted when con- trolling for stratum, as patients with DIPG showing overall immu- nological responses had a median OS of 16.1 months (n = 6, 95% CI, 12.4–NA) compared with 10 months (n = 6, 95% CI, 9.5–NA) for nonresponders (P = 0.11, log-rank) (Supplemental Figure 7), with an estimated multivariate HR of 0.03 (95% CI, 0.001 to 1.960, P = 0.10) (Supplemental Table 1). Prolonged OS among patients with DIPG with Tem expansions (n = 6, P = 0.03, log-rank) further strengthened this association, while also being substantiated by prolonged PFS (P < 0.01, log-rank) relative to nonresponders (Sup - plemental Figure 8). These corroborating survival trends did not persist when using the same longitudinal immunological response threshold for bulk CD8+ T cell phenotypic subtypes (Supplemental Clinical efficacy . The primary efficacy endpoint was the over- all survival at 12 months (OS12) of patients in stratum A. With a median follow-up time of 17 .6 months, the OS12 rate was 44% (95% CI, 27%–75%) for patients in stratum A and 39% (95% CI, 16%–93%) for those in stratum B (Figure 1A). The median progres- sion-free survival (PFS) was 4.9 months (95% CI, 3–8.3 months) for stratum A and 3.5 months (95% CI, 3–NA) for stratum B (Fig - ure 1B). One patient in stratum A had a positive response (partial response; PNOC007-25). Detection, validation, clustering, and phenotyping of H3.3K27M- reactive CD8+ T cells . CyTOF-based immune analyses were con- ducted on 28 of the total 29 patients enrolled in the study, in which H3.3K27M-reactive CD8+ T cells were detected using a conven- tional CD8+ gating strategy (Supplemental Figure 1; supplemental material available online with this article; https:/ /doi.org/10.1172/ JCI140378DS1). H3.3K27M dextramer staining revealed negligible nonspecific staining on healthy donor–derived PBMCs (Figure 1C). CyTOF-based H3.3K27M-reactive CD8+ T cell detection was deter- mined to be as sensitive as flow cytometry (Supplemental Figure 2). We observed that H3.3K27M-reactive CD8+ T cells were clustered on a t-distributed stochastic neighbor embedding (t-SNE) plot and grouped into 5 subpopulations (Supplemental Figure 3): early acti- vated (Tea), stem cell memory (Tscm), effector (Teff), effector mem- ory (Tem), and exhausted (Tex) phenotypes based on their aggregate phenotypic profiles (Figure 1D and Supplemental Figure 3). Expansion of H3.3K27M-reactive CD8+ T cells is associated with a better prognosis. Of the patients from whom PBMCs were col-Table 2. H3.3K27M/TT-related AEsA Stratum A (n = 19) Stratum B (n = 10) Any AE 416 182 Any grade ≥Grade 3 Any grade ≥Grade 3 Injection site reaction 27 1 8 0 Gastrointestinal disorders (nausea, vomiting/diarrhea) 12 0 3 0 Fatigue 6 0 6 0 Fever 5 0 0 0 Nervous system related Gait disturbance 4 1 1 0 Muscle weakness 4 0 0 0 Abducens paresis 3 0 0 0 Ataxia 2 0 0 0 Glossopharyngeal nerve paresis 4 0 0 0 Headache 9 0 2 0 Paresthesia 3 0 1 0 Anorexia/weight loss 3 0 2 0 Blood disorders Neutropenia 3 1 1 0 Lymphopenia 12 0 16 1 Leukopenia 8 0 12 0 Anemia 1 0 0 0 Laboratory results Hypokalemia 2 0 2 0 ALT increase 5 0 0 0 AST increase 4 0 0 0 AOccurring in more than 1 subject. The Journal of Clinical Investigation CLINICAL MEDICINE 6328 jci.org Volume 130 Number 12 December 2020 The Journal of Clinical Investigation CLINICAL MEDICINE 6329 jci.org Volume 130 Number 12 December 2020 (Supplemental Figure 17). However, we found that baseline MDSC frequencies did not correlate with tumor volume or age (Supple - mental Figure 18). With regard to other immunoregulatory cell populations, CD4+ Tregs showed negligible associations with patient outcomes in the DIPG cohort (Supplemental Figure 19). Dexamethasone administration is associated with lower rates of vaccine-specific CD8+ T cell responses and high MDSC levels among patients with DIPG. A total of 9 (47%) patients with DIPG received oral dexamethasone while in the study. Six of these patients com- menced dexamethasone at least 3 days before baseline PBMC collection and exhibited a trend toward higher levels of baseline circulatory MDSCs, with 4 (67%) of the 6 patients on dexametha - sone exhibiting circulatory MDSC frequencies above 5%, whereas 3 (23%) of the 13 patients not receiving dexamethasone surpassed the same threshold (n = 19) (Figure 4A). Among patients receiv- ing dexamethasone who were analyzed longitudinally, 3 (50% of 6 total) exhibited an overall decrease in H3.3K27M-reactive CD8+ T cells at the final collection time point relative to baseline, whereas only 1 (17% of 6 total) showed an overall expansion (Fig - ure 4B and Supplemental Figure 20). Furthermore, patients with DIPG treated with dexamethasone at baseline had a median OS of 9.0 months (n = 6, 95% CI, 7 .7–NA) compared with a median OS of 13.7 months (n = 13, 95% CI, 9.8–NA) for untreated patients (P < 0.01, log-rank test) (Figure 4C). Although not statistically signifi- cant, patients with DIPG treated with dexamethasone had a medi- an PFS of 3.0 months (n = 6, 95% CI, 2.3–NA) compared with 5.3 (n = 13, 95% CI, 4.1–NA) for untreated patients (P = 0.09, log-rank test) (Figure 4D). These trends persisted when combining strata A and B (Supplemental Figure 21). Circulating tumor DNA analysis is not predictive of outcome. A cutoff threshold for positive circulating tumor DNA (ctDNA) was set at 0.001% on the basis of our previously described method (8), where sensitivity and specificity were fully described using spec- imens from a large cohort of patients. At baseline, we collected plasma from 27 of 29 patients for quantification of H3.3K27M mutation (H3F3A c.83A>T) allelic frequency (MAF) (8–10). We detected circulating H3F3A c.83A>T tumor DNA H3.3K27M- specific ctDNA in 70.4% (n = 19 of 27) of the patients’ samples, but its presence at baseline or longitudinal fluctuation in abundance did not correlate with clinical outcomes (Supplemental Figures 22 and 23) or immunological responses (unpublished observations). Longitudinal assessment of immunological profiles in correlation with the clinical outcome of a patient with DIPG exhibiting a strong CD8+ response to vaccination . One patient enrolled in stratum A demonstrated a partial radiographic response at weeks 12 and 24 (Figure 5A). Following a total of 9 vaccinations, the patient was taken off the study at week 36 because of clinical worsening but only a minor increase in tumor size, which did not meet radio - graphic progression criteria based on a central imaging review. The patient underwent reirradiation 10 months after the first vaccine and never received dexamethasone. The patient has not received additional therapies and remains alive 19 months after the initial diagnosis (March 2020 cutoff). A longitudinal immu- nological assessment revealed the time course–dependent expan- sion and persistence of H3.3K27M-reactive CD8+ T cells (Figure 5B). The continuously mounting T cell responses, along with the minor increase in tumor size by week 36, suggest a possibility of an Figures 9–12), suggesting that the survival benefit associated with the expansion of H3.3K27M-reactive CD8+ T cells is independent of overall T cell status. No association between H3.3K27M-reactive Tex expansion and patient outcomes was established (Supplemen- tal Figures 5–8). The number of vaccines administered correlated with the longitudinal change in the percentage of H3.3K27M- reactive CD8+ T cells at the final time point analyzed relative to the percentage at baseline (Supplemental Figure 13). However, the median number of vaccines preceding immunological responses was 6 (IQR = 2) among responders, whereas the median number of total vaccines was also 6 (IQR = 4) among the nonresponders (Figure 2), suggesting that the number of vaccines is not the sole factor for separating responders from nonresponders. Circulatory MDSC abundance at baseline functions as a prog - nostic indicator for patients with DIPG. We identified myeloid cells using a conventional myeloid gating strategy (Figure 3A). Of these clusters, early myeloid-derived suppressor cells (E-MDSCs) (CD33+CD11b+HLA-DRloLinlo) and monocytic MDSCs (M-MD - SCs) (CD33+CD11b+CD14+HLA-DRlo) could be distinguished using their phenotypic profiles (Supplemental Figure 14 and Supplemental Table 2). Patients were stratified into MDSClo and MDSChi cohorts (Figure 3B) for each subpopulation. Although baseline E-MDSC abundance did not correlate with improved outcomes (Supplemental Figures 15 and 16), M-MDSClo patients with DIPG had prolonged OS (P = 0.05, log-rank test) relative to OS for the M-MDSChi cohort of patients with DIPG (Supplemental Figure 16). MDSClo patients with DIPG had a median OS of 13.7 months (95% CI, 9.8–NA) compared with 9.0 months (95% CI, 7 .7–NA) for MDSChi patients with DIPG (P < 0.01, log-rank test) (Figure 3C), with an estimated multivariate HR of 0.09 (95% CI, 0.01–0.92, P = 0.04) (Supplemental Table 1). Further, MDSChi patients had a median PFS of 2.9 months (95% CI, 2.8–NA) com- pared with 5.3 months (95% CI, 3.2–NA) for their counterparts (P = 0.03, log-rank test) (Figure 3D), with an estimated univariate HR of 3.1 (95% CI, 1.1–8.9, P = 0.04). Analyses incorporating both strata A and B did not yield significant findings (Supplemental Fig - ure 15), whereas the limited sample size of stratum B prevented a robust cohort-specific survival analysis for these patients. These associations persisted when analyzing the outcomes of patients with the top and bottom quartile of baseline MDSC abundances Figure 1. Mass cytometry–based phenotyping of H3.3K27M-reactive CD8+ T cell subpopulations reveals associations between immunological responses and prolonged OS or PFS. ( A and B ) Kaplan-Meier survival curves contrasting the (A ) OS and (B ) PFS of patients enrolled in stratum A (red) and stratum B (blue) using log-rank tests. (C ) Left: PBMCs derived from a healthy donor showed an absence of H3.3K27M dextramer staining. Right: The H3.3K27M dextramer exhibited sensitive detection of H3.3K27M- specific T cell receptor–transduced (TCR-transduced) CD8+ T cells. (D ) Heatmap visualizing the relative expression (Z score) of subpopulation markers in patient-derived H3.3K27M-reactive CD8+ T cells. (E ) Heatmap visualizing the patient-specific presence (blue) or absence (red) of an expansion of H3.3K27M-reactive CD8+ T cells in a subpopulation-specific manner. (F and G ) Kaplan-Meier survival curves contrasting the (F ) OS and (G) PFS of patients who had an immunological response (blue) compared with patients who did not (red) using log-rank tests. (H and I ) Kaplan-Meier survival curves contrasting the (H ) OS and (I ) PFS of patients who had an expansion of effector memory H3.3K27M-reactive CD8+ T cells (blue) com- pared with patients who did not (red) using log-rank tests. The Journal of Clinical Investigation CLINICAL MEDICINE 6330 jci.org Volume 130 Number 12 December 2020 inflammation-associated imaging change in this case. This patient was classified as MDSClo among DIPG patients with total MDSC percentages of 2.6% (median = 4.2%), indicative of an improved prognosis among patients with DIPG, as our findings have reflected (Figure 3, C and D). Furthermore, this patient’s MDSC abundance did not increase above median levels until disease progression was identified at 36 weeks (Figure 5C). Discussion To our knowledge, this is the first multicenter trial assessing the safety and clinical efficacy of a vaccine directed toward the novel, shared neoantigen H3.3K27M(27–36) in HLA-A *02:01+ patients with H3.3K27M+ DIPG or DMGs. The study demon- strated safety of the regimen and supports the significance of immunological monitoring for the identification of predictive biomarkers. Moreover, clinical and immune data suggest that concurrent corticosteroid administration is associated with increased MDSC levels and lower vaccine efficacy. The PNOC007 trial regimen demonstrated safety in regard to expected TRAEs. One child developed meningitis of unclear etiol- ogy and recovered completely with standard treatment. It remains uncertain if this event was related to the vaccine, since no addi- tional cases were identified. The PNOC007 treatment strategy did not improve the overall outcome of H3.3K27M+ patients with DMG, representing the worst prognostic group among patients with DMG (11). A recent analysis from the International and Euro - pean DIPG registries reported a similar OS12 of 42.3% (95% CI, 38.1%–44.1%) (1). To our knowledge, this is the first trial in which individuals with DMG were enrolled on the basis of their mutation status rather than tumor location. Outcome measures for these tumors remain a topic of ongoing prospective studies, but retrospective reviews show poor outcomes for these tumor location–specific sub - groups. For example, a retrospective analysis of H3.3K27M+ DMGs enrolled in the German HIT-HGG registry revealed a median survival of 13 months among patients with thalamic tumors and 4.8 months among those with tumors within the spine (11), simi- lar to our results. To date, very few peptide vaccine–based strate - gies have been reported for DIPGs or DMGs. We have previously reported a median survival of 12.7 months in patients with DIPG using a peptide vaccine strategy against IL-13Rα, EphA2, and sur- vivin in combination with poly-ICLC (12). This trial used a strategy Figure 2. Individual patient study course. Depicted within this swimmer plot is the patient-specific timing of vaccine administration, dexamethasone treatment, detection of H3.3K27M-reactive T cell responses, tumor progression, and vital status (patients who withdrew consent were censored). The Journal of Clinical Investigation CLINICAL MEDICINE 6331 jci.org Volume 130 Number 12 December 2020 similar to that for PNOC007 , however, H3.3K27M status was not available and could have influenced outcomes. Our cytometry by time of flight–based (CyTOF-based) anal- yses of multiple immune subsets (13, 14) provided an opportunity to explore associations between expansion of H3.3K27M-reactive CD8+ T cell subsets and prolonged OS and PFS. Furthermore, this approach identified baseline circulatory MDSCs as a poten- tial negative prognostic indicator for patients with DIPG. To our knowledge, this is the first CyTOF-based immunomonitoring platform used to evaluate epitope-specific CD8+ T cell responses in patients with cancer treated with peptide-based vaccines. Detection of this cell population was accomplished through HLA dextramer–based staining (15–17), as dextramers exhibit higher affinity and specificity for epitope-specific CD8+ T cell popula -tions in comparison with conventional HLA tetramers (15, 17). Furthermore, an increased quantity of fluorophores conjugated to each dextramer allowed for increased binding to metal-conju- gated secondary antibodies, resulting in higher resolution on both fluorophore- and lanthanide-based analytical platforms. This approach maximized sensitivity on the CyTOF platform, allowing for the comparable detection of H3.3K27M-reactive CD8+ T cells between flow and mass cytometry. This study validates CyTOF as a powerful tool for high-definition immunomonitoring of CD8+ T cell–based immunotherapies. While other immune cell populations are the focus of inter- est (18, 19) across the broad spectrum of cancer vaccines (20), CD8+ T cells remain the primary target population for develop - ing an efficacious antitumor immunotherapy (21). Consistent Figure 3. Mass cytometry–based analysis of circulatory MDSCs reveals an association between baseline levels of MDSCs and shortened OS and PFS. (A) Representative gating strategy to identify myeloid cells from patient-derived PBMCs by mass cytometry. Cells that stained double-positive with iridium intercalator were identified as intact cells (far left). Cells with low cisplatin staining were identified as live cells (second from left). Cells that stained negative for CD45 or positive for CD3 were excluded (middle). Cells that stained positive for CD19 or positive for CD56 were excluded (second from right). Cells that stained positive for either CD11b or CD11c were identified as myeloid cells (far right). (B) Box plot shows the range of abundance of circulatory total MDSCs among patients with DIPG classified as MDSClo or MDSChi. MDSC classifications were established on the basis of a median threshold. (C and D) Kaplan-Meier survival curves contrasting (C) OS and (D) PFS of DIPG patients with baseline circulatory MDSC levels above (red) and below (blue) the median threshold, as determined by log-rank tests. The Journal of Clinical Investigation CLINICAL MEDICINE 6332 jci.org Volume 130 Number 12 December 2020 Figure 4. Administration of dexamethasone is positively associated with increased baseline MDSC levels and inversely associated with H3.3K27M- reactive CD8+ T cell responses and survival. ( A) Box plot demonstrates the abundance of circulatory MDSCs at baseline among patients with DIPG treated with dexamethasone (n = 6) for at least 3 days prior to treatment relative to patients who did not receive dexamethasone (n = 13). (B ) Waterfall plot depicts the overall longitudinal percentage change of H3.3K27M-reactive CD8+ T cells at the final time point analyzed relative to baseline for each patient with DIPG who was treated with dexamethasone. Yellow bars indicate the pre-treatment period; red bars indicate patients who received dexamethasone after com- mencement of vaccination; and blue bars indicate patients who did not receive dexamethasone while in the study. (C and D ) Kaplan-Meier survival curves contrasting the (C ) OS and (D ) PFS of patients with DIPG based on pretreatment dexamethasone administration, as determined by log-rank tests. The Journal of Clinical Investigation CLINICAL MEDICINE 6333 jci.org Volume 130 Number 12 December 2020 Corticosteroids are often administered to patients with DIPG or DMG to treat neurological dysfunction caused by tumor- or treatment-related edema. However, steroid dependency has been associated with a worse prognosis (36) and may negatively affect responses to immunotherapies (5). Consistent with recent preclinical in vitro and in vivo studies (37 , 38), our data suggest a negative association between dexamethasone administration and the longitudinal expansion of vaccine-reactive CD8+ T cells, con- sistent with a recent report (5). It is conceivable that tumor size and growth rate, which typically necessitate the use of cortico - steroids, may confound our observations. Our data suggest a lack of association between the baseline tumor size and outcomes (Supplemental Figures 24 and 25), consistent with a recent study involving molecularly characterized DIPGs (39). However, given the small sample size, we cannot fully exclude that dexametha - sone use, independent of its impact on vaccine efficacy, leads to worse outcomes. Nonetheless, this association calls for additional corroborative studies. Key limitations of our study include the relatively small num- ber of patients and longitudinal samples for immune phenotypic analyses and classification of immunological responses. Some study participants were lost to follow-up because of clinical wors- ening of their disease before post-baseline PBMC collection, lim- iting the availability of longitudinal samples to 62% (18 of 29) of the patients enrolled. Systematic evaluation of pre- and post-treat - ment tumor microenvironment samples would have offered more insights on the vaccine’s effects, such as T cell infiltration as well as possible antigen loss and HLA downregulation. To date, post-treatment biopsy of DMGs remains a controversial topic in pediatric neuro-oncology (10, 40), and this limitation extended to our current study. At the time of submission, there were too few data to establish corroborations with CyTOF analyses (Supple - mental Figures 26 and 27). Given the clinical information gained by pathologic assessment of post-treatment tumor biopsies, these should be considered in future trials to better understand the effect of our therapies. In conclusion, our data reflect the safety of the regimen and highlight the encouraging roles of H3.3K27M-specific CD8+ T cell responses in these patients, warranting further studies targeting the H3.3K27M epitope. Such studies should focus on a vaccine regimen with anti–PD-1 therapy (based on expression of PD-1 on H3.3K27M-reactive CD8+ T cells) and adoptive transfer of T cells transduced with the H3.3K27M-reactive T cell receptor (4). Methods Study design. PNOC007 (ClinicalTrials.gov ID: NCT02960230) is a pilot trial assessing the safety, immunoreactivity, and efficacy of subcu- taneous administration of the H3.3K27M synthetic peptide combined with helper TT peptide (TetA830, PolyPeptide Group) (41, 42) emulsified in Montanide-ISA 51 VG (SEPPIC) and in combination with poly-ICLC (Hiltonol, Oncovir) in patients 3 to 21 years of age with newly diag - nosed DIPG (stratum A) or nonpontine DMG, including spinal cord DMGs (stratum B). Key inclusion criteria were the confirmation of HLA-A *02:01+ status and the H3.3K27M mutation by Clinical Labora - tory Improvement Amendments–approved (CLIA-approved) sequenc- ing of tumor tissue as well as adequate organ function. Exclusion crite - ria included known immune system disorders and treatment with other with this paradigm, the expansion of H3.3K27M-reactive CD8+ T cells, but not bulk CD8+ T cells, is suggestive of prolonged OS. Although this expansion provides insight into patient-specific immunological outcomes, it does not account for phenotypic com- position or functionality. As peripheral Tex expansion has been correlated with poor prognoses in several cancer types (22, 23), the discrimination of this cell population, among others, is nec- essary for a contextual assessment of immunological responses. Our data demonstrated no association between Tex expansion and favorable outcomes for patients. However, the expansion of H3.3K27M-reactive Tem cell subpopulations, which exhibit cyto - lytic and tissue localization capabilities (24), was associated with clinical benefits among patients with DMG, consistent with pre - vious reports (25–27). One could raise a concern about whether the observed H3.3K27M-reactive CD8+ T cell responses merely reflect over- all immune competency, which is often compromised in these patients. Our multivariate analysis on bulk CD8+ T cells suggested that this was not the case. Another line of concern is the possibil- ity that patients with intrinsically slow-growing tumors may have received more vaccines than those with fast-growing tumors, thereby mounting higher levels of vaccine-reactive responses. Based on our analyses, this does not appear to be the case, as the median numbers of vaccines preceding immunological responses among responders was the same as the median number of total vaccines among nonresponders. To date, there are no vaccine regimens that have been shown to be efficacious in CNS malignancies, as shown by randomized studies with adequate statistical powers. As we discussed in our recent review articles (28, 29), we recognize that underlying these failures are a number of mechanistic challenges, such as antigen-heterogeneity, local and systemic immunosuppression, and lack of adequate homing of effector immune cells. Although most tumor-specific antigens are expressed heterogeneous- ly within the tumor (29), H3.3K27M appears to be uniformly expressed in the tumor tissue (7). In terms of relevant immunosuppressive mechanisms, MDSCs have attracted particular interest not only as tumor promoters (30) but as entities antagonistic to peptide vaccine efficacy (31–33) in preclinical models. Our data reflected this, as MDSClo DIPG patients exhibited improved outcomes relative to their counter- parts. It is also noteworthy that our data set showed an absence of associations between relative circulatory MDSC abundance and both the patient’s age and tumor volume. Although Tregs are important immunosuppressive cells, our analyses did not detect any association between Tregs and patient outcomes in the DIPG cohort, corroborating recent high-throughput analyses of PBMC samples in patients with glioblastoma (30). However, these results warrant caution. As cryopreservation and paraformaldehyde fixation are known to reduce polymorphonuclear MDSC viabil- ity (34), E-MDSCs and M-MDSCs were used as proxies for total MDSC quantification. Our data did not establish a robust correla - tion between baseline MDSC levels and longitudinal H3.3K27M- reactive CD8+ T cell frequencies, which may have been affected by the limited longitudinal samples available. Nevertheless, MDSCs represent an enticing target for combinatorial therapies (35) that maximize vaccine efficacy. The Journal of Clinical Investigation CLINICAL MEDICINE 6334 jci.org Volume 130 Number 12 December 2020 of RT, and given every 3 weeks for the first 8 doses followed by every 6 weeks (Supplemental Figure 28) on an outpatient basis for a maximum treatment period of 96 weeks. Vaccine was administered at the same location unless prevented by an adverse skin reaction. Concurrently, the adjuvant poly-ICLC was administered (30 μg/kg) intramuscularly within 2 cm of the vaccine injection site (43, 44). Follow-up data were collected through February 2020.