In a phase 2 trial, local-regionally advanced HPV-positive oropharyngeal carcinoma patients (N = 35) received ipilimumab (anti-CTLA-4) and nivolumab (anti-PD-1) as induction immunotherapy and concurrently with radiotherapy (NCT03799445). Co-primary endpoints included 6-month complete metabolic response rate (94%) and 2-year progression-free survival (84%). Induction yielded a 46% major histopathologic response rate. Single-cell profiling revealed responders had higher baseline intratumoral CD8+ T cells with a tumor-reactive, tissue-resident memory (TRM) phenotype and a treatment-related decrease in effector regulatory T (eTreg) cells. The eTreg decrease correlated with CD8+ T-cell clonotype transitioning from TRM to effector memory and IFNG+ effector cells. In nonresponders, clonotypes transitioned to exhausted TRM and proliferating cells. Multivariable regression modeling determined the baseline feature most associated with reduction in tumor viability was the proportion of FCGR3A-expressing NK cells, which are capable of ipilimumab-dependent depletion of CTLA4high eTregs. eTreg depletion may be critical for major response to induction dual immune checkpoint blockade.

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First page of Depletion of effector regulatory T cells associates with major response to induction dual immune checkpoint blockade<alt-title alt-title-type="left-running">Effector Treg depletion in HPV-positive oropharyngeal cancer</alt-title>

Supplementary data

Supplementary Tables S1-S6

Table S1. Patient and tumor characteristics. Table S2. Representativeness of study participants. Table S3. Clinical and histopathologic characteristics of individual patients. Table S4. Treatment related adverse events that occurred after first dose. Table S5. Adverse events related to immune checkpoint blockade. Table S6. Serious adverse events.

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Supplementary Table S7

Single-cell RNA and TCR sequencing information and metrics

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Supplementary Table S8

Statistical test for proportions of cell types in the tumor immune microenvironment stratified by histological response and timepoint

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Supplementary Table S9

Sample-level cell counts of CD4 clusters

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Supplementary Table S10

Differentially expressed genes of CD4 clusters

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Supplementary Table S11

Statistical test for proportions of CD4+ T cell clusters in the tumor immune microenvironment stratified by histological response and timepoint

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Supplementary Table S12

Differentially expressed genes of CD8+ T cells from MHRs vs non-MHRs at baseline

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Supplementary Table S13

Sample-level cell counts of CD8 clusters

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Supplementary Table S14

Differentially expressed genes of CD8 clusters

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Supplementary Table S15

Statistical test for proportions of CD8+ T cell clusters in the tumor immune microenvironment stratified by histological response and timepoint

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Supplementary Table S16

CD8+ T-cell clonotypes with significant frequency changes in response to treatment

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Supplementary Table S17

Alpha- and beta-chain sequence data of validated anti-HPV16 TCRs

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Supplementary Table S18

Differentially expressed genes of FCGR3A-positive vs FCGR3A-negative NK cells

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Supplementary Table S19

Statistical test for association of lymphocyte cluster proportion with progression

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Supplementary Table S20

Clinical and single-cell features used in multivariable regression models

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Supplementary Figures S1-S18

Fig. S1. Patient disposition and characteristics. Fig. S2. Timing of OI biopsy collection did not correlate with response. Fig. S3. Major cell types identified in the tumor immune microenvironment. Fig. S4. CD4+ T cells identified in the tumor immune microenvironment. Fig. S5. Effector Treg cells in the HPV-positive OPC TIME highly express activation markers. Fig. S6. Treatment-induced changes in Treg proportions and clonotype frequencies. Fig. S7. CD8+ T cells in MHRs have different transcriptional programs vs those in nMHRs. Fig. S8. CD8+ T cells identified in the tumor immune microenvironment. Fig. S9. T cell clusters did not vary by anatomic site. Fig. S10. TRM CD8+ T cells had high expression of tumor-reactive, cytotoxic and checkpoint genes. Fig. S11. Fluorescent imaging of antigen-experienced, memory CD8+ T cells in baseline tumors. Fig. S12. Clonality of CD4+ T-cell TCR repertoire. Fig. S13. CD8+ T-cell TCR repertoire clonality and dynamics. Fig. S14. Phenotypes of treatment-altered CD8 clonotypes varied by response. Fig. S15. Predominant phenotypes of treatment-responsive CD8+ T-cell clonotypes. Fig. S16. Identification of HPV-specific TCRs. Fig. S17. NK cells that express Fc gamma receptor IIIa (CD16) are more activated than those that do not. Fig. S18. Increase in proportion of Effector1(IFNG+) cells associated with reduction in tumor viability.

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