Distinct Immunophenotypes of T Cells in Bronchoalveolar Lavage Fluid From Leukemia Patients With Immune Checkpoint Inhibitors-Related Pulmonary Complications

Abstract

Patients with acute myeloid leukemia (AML) and myelodysplastic syndrome (MDS) treated with immune checkpoint inhibitors (ICIs) are at risk of pneumonitis as well as pneumonia (combined henceforth as ICI-related pulmonary complications). Little is known about the cellular and molecular mechanisms underlying ICI-related pulmonary complications. We characterized lymphocytes from bronchoalveolar lavage (BAL) fluid and peripheral blood from seven AML/MDS patients with pulmonary symptoms after ICI-based therapy (ICI group) and four ICI-naïve AML/MDS patients with extracellular bacterial or fungal pneumonias (controls). BAL T cells in the ICI group were clonally expanded, and BAL IFNγ⁺ IL-17^- CD8⁺ T and CXCR3⁺ CCR6⁺ Th17/Th1 cells were enriched in the ICI group. Our data suggest that these cells may play a critical role in the pathophysiology of ICI-related pulmonary complications. Understanding of these cell populations may also provide predictive and diagnostic biomarkers of ICI-related pulmonary complications, eventually enabling differentiation of pneumonitis from pneumonia in AML/MDS patients receiving ICI-based therapies.

Keywords: Th17/Th1 cells; acute myeloid leukemia; checkpoint inhibitor; immune-related adverse event; pneumonitis.

Figure 1

Selection process for patients in the immune checkpoint inhibitor (ICI) group and the control group. AML, acute myeloid leukemia; MDS, myelodysplastic syndrome; SCT, stem cell transplantation; BAL, bronchoalveolar lavage.

Figure 2

Proportion of major bronchoalveolar lavage (BAL) cell subsets with manual differential tests. Bars indicate the mean and the SEM. One-way ANOVA. *P < 0.05, **P < 0.01, ***P < 0.001, n.s., not significant. ICI, immune checkpoint inhibitor.

Figure 3

Characterization of lymphoid immune cell subsets in bronchoalveolar lavage (BAL) fluid and peripheral blood (PB). (A) Proportions of major BAL immune cell subsets within live lymphocytes and absolute cell numbers in 1 ml BAL fluid. NK, natural killer cells; NK T, natural killer T cells; B, B cells. Bars indicate the mean and the SEM. Mann-Whitney U test. *P<0.05, **P<0.01. (B) Proportions of CD8⁺ T cell subsets within BAL CD8⁺ T cells. Tn, naïve T cells; Tcm, central memory T cells; Tem, effector memory T cells; Temra, terminally differentiated T cells. Bars indicate the mean and the SEM. One-way ANOVA. *P<0.05, **P<0.01, ***P<0.001. (C) Proportions of major PB immune cell subsets within live lymphocytes and absolute cell numbers in 1 ml PB. Bars indicate the mean and the SEM. (D) Proportions of CD4⁺ T cell subsets within CD4⁺ T cells and absolute cell numbers in 1 ml BAL fluid. Treg, regulatory T cells; Tfh, follicular helper T cells. Bars indicate the mean and the SEM. Mann-Whitney U test. *P<0.05. (E) Proportion of BAL CD4⁺ T cells expressing indicated transcription factors (left), transcription factors and surface molecules (middle and right). Bars indicate the mean and the SEM. Mann-Whitney U test. *P<0.05. (F) PD-1 on BAL naïve CD4⁺ T cells and BAL CXCR3⁺ CCR6⁺ Th17/Th1 cells. Left panel shows one of the most representative plots and right panel shows quantification. Wilcoxon paired rank test. *P<0.05. (G) Proportions and absolute numbers of IFNγ- and/or IL-17-producing CD4⁺ and CD8⁺ T cells in BAL fluid. Bars indicate the mean and the SEM. Mann-Whitney U test. *P<0.05. (H) Levels of IFNγ, IL-6, and IL-17A in BAL fluid measured by multiplex ELISA. Bars indicate the mean and the SEM.

Figure 4

(A) Clonality and diversity of T cells in the bronchoalveolar lavage (BAL) fluid and peripheral blood (PB). Bars indicate the mean and the SEM. One-way ANOVA. *P<0.05, **P<0.01, n.s., not significant. (B) Quantification of overlapped T-cell receptor sequences between BAL and PB. Bars indicate the mean and the SEM. One-way ANOVA.