Molecular and cellular features of CTLA-4 blockade for relapsed myeloid malignancies after transplantation

Abstract

Relapsed myeloid disease after allogeneic stem cell transplantation (HSCT) remains largely incurable. We previously demonstrated the potent activity of immune checkpoint blockade in this clinical setting with ipilimumab or nivolumab. To define the molecular and cellular pathways by which CTLA-4 blockade with ipilimumab can reinvigorate an effective graft-versus-leukemia (GVL) response, we integrated transcriptomic analysis of leukemic biopsies with immunophenotypic profiling of matched peripheral blood samples collected from patients treated with ipilimumab following HSCT on the Experimental Therapeutics Clinical Trials Network 9204 trial. Response to ipilimumab was associated with transcriptomic evidence of increased local CD8+ T-cell infiltration and activation. Systemically, ipilimumab decreased naïve and increased memory T-cell populations and increased expression of markers of T-cell activation and costimulation such as PD-1, HLA-DR, and ICOS, irrespective of response. However, responding patients were characterized by higher turnover of T-cell receptor sequences in peripheral blood and showed increased expression of proinflammatory chemokines in plasma that was further amplified by ipilimumab. Altogether, these data highlight the compositional T-cell shifts and inflammatory pathways induced by ipilimumab both locally and systemically that associate with successful GVL outcomes. This trial was registered at www.clinicaltrials.gov as #NCT01822509.

Keywords: CHEMOKINES/chemokines; FFPE RNA-seq; IMMUNOBIOLOGY/tumor immunology; MARROW AND STEM CELL TRANSPLANTATION/basic biology; NEOPLASIA/myeloid leukemias and dysplasias: immunotherapeutic approaches; allogeneic stem cell transplantation; graft-versus-leukemia; ipilimumab; myeloid disease.

Figures

Graphical abstract

Figure 1.

Response to ipilimumab is characterized by transcriptional evidence of T-cell infiltration and activation. (A) RNA sequencing on FFPE disease-site biopsies (♦, n = 33) from 3 patients with CR (17, 21, 26; dark blue), 3 patients with TR (6, 14, 28; light blue) and 7 patients with NR (5, 11, 22, 24, 29, 31, 33; red) pre-ipi or post-ipi ipilimumab treatment. Disease sites: bone marrow (light), extramedullary (dark), or isolated skin (white). Peripheral blood samples (●, n = 28) used for TCR repertoire sequencing. (B) DGEA between 4 site-matched biopsies from CR patients (top) and between unmatched biopsies from NR patients pre- (n = 7) vs post-ipi (n = 8) (bottom). Genes that are part of the Gene Ontology term “leukocyte activation” are labeled and those associated with T-cell activation are highlighted. (C) Gene ontology enrichment analysis of the differentially expressed genes. (D) PCA based on expression of the differentially expressed genes. Biopsies from all CR pre-ipi (n = 4, gray) and post-ipi (n = 7, blue), NR pre-ipi (n = 7, salmon), and post-ipi (n = 8, red) samples, and biopsies from sites of GVHD or immune-related toxicities (n = 9, black). (E,F) Cell type abundance estimation of CD8+ T cells with (E) CIBERSORTx and (F) clonotypes per million reads assembled using TRUST. Relapse biopsies post-ipi were sampled at time of relapse.

Figure 2.

Systemic effects of ipilimumab. (A) Peripheral blood T-cell subsets in patients with myeloid (n = 14) and nonmyeloid (n = 6) disease pre-ipi and after 1 (C2D1) or 3 (C4D1) cycles of ipilimumab (post-ipi) quantified using flow cytometry as percentage of CD4+ or CD8+ T cells. (B) MDS plot calculated from CyTOF data of peripheral blood T cells of 10 NR patients pre-ipi (baseline, n = 10), 1 cycle post-ipi (C2D1, n = 10), or a later timepoint (follow-up, n = 8). Numbers next to dots refer to the patient identifiers used throughout the study (supplemental Tables 4-7). (C) Mean metal intensity (CyTOF) on CD4+ and CD8+ T cells pre-ipi (blue) or 1 cycle post-ipi (yellow) from samples shown in panel B. (D) Frequency of CDR3α and CDR3β sequences in peripheral blood pre-ipi (baseline) and post-ipi (C2D1) (n = 572 017; TCR repertoire sequencing). Dynamic CDR3 sequences with significant changes in abundance (adjusted P value < .01) in purple (expanded, novel = not detectable pre-ipi) or green (contracted, disappeared = not detectable post-ipi). (E) Percentage of CDR3 sequences from disease biopsies detectable in peripheral blood pre-ipi and post-ipi. (F) Absolute number of dynamic CDR3 sequences in patients with response to ipilimumab (CR/TR) (blue, n = 4) and NR (red, n = 5). (G) Differential protein expression post-ipi in patients with response (n = 4) vs patients without response (n = 8) to ipilimumab measured with proximity extension assay (PEA, Olink). (H) Heatmap of protein expression measured with PEA in NR pre-ipi (n = 8), NR post-ipi (n = 16), CR/TR pre-ipi (n = 4), CR/TR post-ipi (n = 8). C2D1/C4D1, second/fourth cycle of ipilimumab; EM, effector memory T cell; MDS, multidimensional scaling; MMI, mean metal intensity; naive, naive T cell; PB, peripheral blood.