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. 2019 May 1;125(9):1470-1481.
doi: 10.1002/cncr.31896. Epub 2018 Nov 30.

The distribution of T-cell subsets and the expression of immune checkpoint receptors and ligands in patients with newly diagnosed and relapsed acute myeloid leukemia

Patrick Williams  1 , Sreyashi Basu  2 , Guillermo Garcia-Manero  3 , Christopher S Hourigan  4 , Karolyn A Oetjen  4 , Jorge E Cortes  3 , Farhad Ravandi  3 , Elias J Jabbour  1 , Zainab Al-Hamal  2 , Marina Konopleva  3 , Jing Ning  5 , Lianchun Xiao  5 , Juliana Hidalgo Lopez  6 , Steve M Kornblau  3 , Michael Andreeff  3 , Wilmer Flores  3 , Carlos Bueso-Ramos  6 , Jorge Blando  2 , Pallavi Galera  7 , Katherine R Calvo  7 , Gheath Al-Atrash  8 , James P Allison  2 , Hagop M Kantarjian  3 , Padmanee Sharma  2 , Naval G Daver  3
Affiliations

The distribution of T-cell subsets and the expression of immune checkpoint receptors and ligands in patients with newly diagnosed and relapsed acute myeloid leukemia

Patrick Williams et al. Cancer. .

Abstract

Background: Phenotypic characterization of immune cells in the bone marrow (BM) of patients with acute myeloid leukemia (AML) is lacking.

Methods: T-cell infiltration was quantified on BM biopsies from 13 patients with AML, and flow cytometry was performed on BM aspirates (BMAs) from 107 patients with AML who received treatment at The University of Texas MD Anderson Cancer Center. The authors evaluated the expression of inhibitory receptors (programmed cell death protein 1 [PD1], cytotoxic T-lymphocyte antigen 4 [CTLA4], lymphocyte-activation gene 3 [LAG3], T-cell immunoglobulin and mucin-domain containing-3 [TIM3]) and stimulatory receptors (glucocorticoid-induced tumor necrosis factor receptor-related protein [GITR], OX40, 41BB [a type 2 transmembrane glycoprotein receptor], inducible T-cell costimulatory [ICOS]) on T-cell subsets and the expression of their ligands (41BBL, B7-1, B7-2, ICOSL, PD-L1, PD-L2, and OX40L) on AML blasts. Expression of these markers was correlated with patient age, karyotype, baseline next-generation sequencing for 28 myeloid-associated genes (including P53), and DNA methylation proteins (DNA methyltransferase 3α, isocitrate dehydrogenase 1[IDH1], IDH2, Tet methylcytosine dioxygenase 2 [TET2], and Fms-related tyrosine kinase 3 [FLT3]).

Results: On histochemistry evaluation, the T-cell population in BM appeared to be preserved in patients who had AML compared with healthy donors. The proportion of T-regulatory cells (Tregs) in BMAs was higher in patients with AML than in healthy donors. PD1-positive/OX40-positive T cells were more frequent in AML BMAs, and a higher frequency of PD1-positive/cluster of differentiation 8 (CD8)-positive T cells coexpressed TIM3 or LAG3. PD1-positive/CD8-positive T cells were more frequent in BMAs from patients who had multiply relapsed AML than in BMAs from those who had first relapsed or newly diagnosed AML. Blasts in BMAs from patients who had TP53-mutated AML were more frequently positive for PD-L1.

Conclusions: The preserved T-cell population, the increased frequency of regulatory T cells, and the expression of targetable immune receptors in AML BMAs suggest a role for T-cell-harnessing therapies in AML.

Keywords: T cell; acute myeloid leukemia; flow cytometry; immune checkpoint; immunotherapy.

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Conflict of interest statement

Christopher S. Hourigan reports laboratory research funding from Merck and Sellas outside the submitted work. James P. Allison reports consulting fees from and stock ownership in Jounce, Kite Pharma, Neon, Amgen, Forty‐Seven, Apricity, Polaris, Marker Therapeutics, Codiak, BioAlta, ImaginAB, Tvardi Therapeutics, and TapImmune outside the submitted work; patents issued with The Regents of the University of California for “Blockade of Lymphocyte Down‐Regulation Associated With CTLA‐4 Signaling” (US5811097 A, US5855887 A, US6051227 A, and US7229628 B1), “Stimulation of T Cells Against Self Antigens Using CTLA‐4 Blocking Agents” (US20060034844 A1), “Diagnosis of Prostate Cancer with SPAS‐1 Cancer Antigen (US7704701 B2), and “Methods and Compositions for Localized Secretion of Anti‐CTLA‐4 Antibodies” (US9868961 B2); a patent issued with Biosante Pharmaceuticals and The Regents of the University of California for “Cancer Immunotherapy Compositions and Methods of Use” (US7919079 B2); a patient issued with the Icahn School of Medicine at Mount Sinai and Memorial Sloan Kettering Cancer Center for “Newcastle Disease Viruses and Uses Thereof” (US20160015760 A1); a patent issued with the Board of Regents of The University of Texas System and Memorial Sloan Kettering Cancer Center for “Combination Immunotherapy for the Treatment of Cancer (US9375475 B2); a patent issued with Albert Einstein College of Medicine Inc and the Sloan‐Kettering Institute for Cancer Research for “Antibodies to Human B7X for Treatment of Metastatic Cancer” (US9447186 B2); a patent issued for “SPAS‐1 Cancer Antigen (US20020150588 A1); a patent issued for “Compositions and Methods for Modulating Lymphocyte Activity (US20040175380 A1); a patent pending with The Regents of the University of California for “Stimulation of T Cells Against Self Antigens Using CTLA‐4 Blocking Agents” (US20090269353 A1); and a patent pending for “Multi‐Antigen Immunotherapy for Melanoma and Prostate Cancer.” Padmanee Sharma reports consulting fees from and stock ownership in Jounce, Neon, Constellation, Oncolytics, BioAlta, Forty‐Seven, Apricity, Polaris, Marker Therapeutics, and Codiak outside the submitted work and consulting fees from Kite Pharma, Pieris, Merck, and BioMx outside the submitted work. The remaining authors made no disclosures.

Figures

Figure 1
Figure 1
Immunochemistry (IHC) was used to quantify T‐cell infiltration in bone marrow biopsies from healthy donors and from patients with acute myeloid leukemia (AML). (A) No significant difference was observed in the percentage of cluster of differentiation 3 (CD3)‐positive (CD3+) cells per medium‐power field (MPF) in bone marrow biopsies from patients with AML (blue circles) and from age‐matched, healthy donors (red squares). (B) There also was no difference in the absolute CD3‐positive cell infiltration per MPF (at 200 magnification; calculated by multiplying the average percentage of CD3‐positive cells infiltrating the biopsy per MPF by the cellularity of each sample) and the number of average total hematopoietic cells in a bone marrow that had 100% cellularity (see Materials and Methods). (C) Representative photomicrographs of bone marrow biopsies are from 2 healthy donors and 2 patients with AML. H&E indicates hematoxylin and eosin staining.
Figure 2
Figure 2
T‐cell subset distribution is illustrated in bone marrow aspirates from healthy donors, patients with newly diagnosed acute myeloid leukemia (AML), and patients with relapsed AML. (A) The flow‐cytometry gating strategy is illustrated. CD indicates cluster of differentiation; FoxP3, forkhead box P3; SCC‐A, side scatter area; Teff, Teff cells; Tregs, regulatory T cells. (B) T‐cell subsets are compared between healthy donors (blue circles), patients with newly diagnosed AML (red triangles), and patients with relapsed AML (green squares).When gating on CD45‐positive (CD43+) cells, there is an increase in the frequency of total T cells, CD4‐positive Teff cells, and Tregs in bone marrow aspirates from patients with AML compared with the aspirates from healthy donors.
Figure 3
Figure 3
The expression of immune checkpoints (programmed cell death protein 1 [PD1], OX40, inducible T‐cell costimulatory [ICOS]) is illustrated on T‐cell subsets in bone marrow aspirates from healthy donors (blue circles) and from patients with newly diagnosed AML (red triangles), first relapsed AML (green squares), and multiple relapsed AML (purple diamonds). (A) OX40‐positive and PD1‐positive cluster of differentiation 8 (CD8)‐positive (CD8+) T cells; (B) ICOS‐positive, OX40‐positive, and PD1‐positive/CD4+ T‐effector (Teff) cells; and (C) OX40‐positive T‐regulatory cells (Tregs) were noted more frequently in bone marrow aspirates from patients with AML compared with the aspirates from healthy donors.
Figure 4
Figure 4
The frequency of programed cell death 1 (PD1)/T‐cell immunoglobulin and mucin‐domain containing‐3(TIM3) (PD1TIM3) double‐positive T cells and of PD1/lymphocyte‐activation gene 3 (LAG3) (PD1LAG3) double‐positive T cells is illustrated in bone marrow aspirates from healthy donors (blue circles), patients with newly diagnosed acute myeloid leukemia (AML) (red triangles), and patients with relapsed AML (green squares). (A) There is an increased frequency of PD1/TIM3 double‐positive cluster of differentiation 8 (CD8)‐positive T cells and PD1/TIM3 double‐positive CD4‐positive T effector (Teff) cells in bone marrow aspirates from patients with AML compared with aspirates from healthy donors. (B) There is an increased frequency of PD1/LAG3 double‐positive CD8‐positive and CD4‐positive Teff cells in bone marrow aspirates from patients with AML compared with aspirates from healthy donors.
Figure 5
Figure 5
Programmed cell death 1 ligand (PD‐L1) expression is illustrated according to the percentage of positive blasts in patients with acute myeloid leukemia (AML). (A) PD‐L1–positive blasts were noted more frequently in bone marrow aspirates from patients who had tumor protein 53 (TP53)‐mutated AML versus those who had non‐TP53–mutated AML. (B) PD‐L1–positive blasts are compared between patients who had AML with adverse (Adv) cytogenetics versus those who had nonadverse cytogenetics.
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