Dynamics of cytotoxic T cell subsets during immunotherapy predicts outcome in acute myeloid leukemia

Abstract

Preventing relapse after chemotherapy remains a challenge in acute myeloid leukemia (AML). Eighty-four non-transplanted AML patients in first complete remission received relapse-preventive immunotherapy with histamine dihydrochloride and low-dose interleukin-2 in an international phase IV trial (ClinicalTrials.gov; NCT01347996). Blood samples were drawn during cycles of immunotherapy and analyzed for CD8+ (cytotoxic) T cell phenotypes in blood. During the first cycle of therapy, a re-distribution of cytotoxic T cells was observed comprising a reduction of T effector memory cells and a concomitant increase of T effector cells. The dynamics of T cell subtypes during immunotherapy prognosticated relapse and survival, in particular among older patients and remained significantly predictive of clinical outcome after correction for potential confounders. Presence of CD8+ T cells with specificity for leukemia-associated antigens identified patients with low relapse risk. Our results point to novel aspects of T cell-mediated immunosurveillance in AML and provide conceivable biomarkers in relapse-preventive immunotherapy.

Keywords: Immune response; Immunity; Immunology and Microbiology Section; acute myeloid leukemia; antigen-specific T cells; cytotoxic T cells; immunotherapy.

Conflict of interest statement

CONFLICTS OF INTEREST

Authors MB and KH are past or present consultants to the study sponsor (Meda Pharma). Author KH holds patents protecting the use of histamine dihydrochloride in cancer immunotherapy. Authors AM, RF and FBT have received honoraria and/or travel grants from the study sponsor. The other authors declare no conflict of interest.

Figures

Figure 1. Overview of the Re:Mission phase IV trial

Eligible AML patients in first complete remission (CR) received ten 3-week cycles of HDC/IL-2 over 18 months. Peripheral blood mononuclear cells (PBMC) were isolated from blood collected before and after cycles 1 and 3. Patients were followed-up for 6 months after completing the last treatment cycle.

Figure 2. Distribution of CD8+ subsets in non-relapsing and relapsing AML patients during immunotherapy with HDC/IL-2

A. Blood counts of CD8+ T cells before (D1) and after (D21) the first and third cycles of HDC/IL-2 treatment (C1D1 n = 62; C1D21 n = 54; C3D1 n = 52; C3D21 n = 51). B. Gating strategy for determining naïve (TN; CD45RA+CCR7+), central memory (TCM; CD45RO+CCR7+), effector memory (TEM; CD45RO+CCR7−) and effector (Teff; CD45RA+CCR7−) cells within the CD8+ T cell compartment. C-F. Frequency of the CD8+ subpopulations TNC., TCMD., TEME. and TeffF. cells in non-relapsing (n = 18) and relapsing (n = 26) patients at the onset (C1D1) or end of (C1D21) the first cycle of immunotherapy. Statistical analysis was performed by Student's paired t-test.

Figure 3. Impact of altered distribution of CD8+subsets on the clinical outcome of patients receiving HDC/IL-2

In A-D. all patients, and in F-I. patients ≥ 60 years old, were dichotomized based on induction or reduction of the frequency of CD8+ T cell subsets during the first treatment cycle, followed by analyses of LFS and OS by the logrank test. In E. all patients, and in J. patients≥ 60 years old, were dichotomized based on transition (trans) or no transition from TEM to Teff cells and LFS and OS were analyzed by the logrank test. A patient was considered transition-positive by the occurrence of a reduction of TEM cells (%) and a simultaneous induction of Teff cells (%) during the first treatment cycle.

Figure 4. Impact of HLA-DR expression and leukemia-specific CD8+T cells on LFS in patients receiving HDC/IL-2

A. Patients were dichotomized by the median HLA-DR expression on CD3+CD8+ T cells at onset of therapy (C1D1; n = 44) or after the first treatment cycle (C1D21; n = 47). LFS and OS were analyzed by the logrank test. B-C. Blood samples from patients undergoing HDC/IL-2 treatment were stimulated with a pool of peptides from leukemia-associated antigens (AML-peptides) or a pool of peptides from CMV, EBV and influenza viruses (CEF-peptides), or no peptides (negative control). The percentage of IFN-γ producing CD8+ T cells was determined by flow cytometry. In B. representative dot plots show IFN-γ production in samples without stimulation and samples stimulated with AML- or CEF-peptides. In C. patients were dichotomized based on the presence or absence of AML-specific or CEF-specific CD8+ T cells, followed by analysis of LFS by the logrank test. Only patients with no events occurring before the last time point of analysis of antigen-specific T cells (C3D21; 105 days) were considered in the latter analyses.

Figure 5. Impact of TEM to Teff cell transition and NK cell NKp46 expression on clinical outcome

A.-B. Patients were regarded as transition-positive when showing a reduction of TEM cells (%) and a simultaneous induction of Teff cells (%) during the first treatment cycle, and were considered NKp46high when their CD16+ NK cells expressed above median levels of NKp46 after the first cycle of immunotherapy (C1D21). Data show the LFS and OS (analyzed by the logrank test for trend) of patients with transition TEM-Teff and NKp46high (both), transition only, NKp46high only, no transition TEM-Teff and low NKp46 expression (neither).