PD-1 and PD-L1 Are Overexpressed in the "Intermediate CD14+CD16+" and "Non Classical CD14lowCD16+" but Not in the "Classical CD14+CD16-" Monocytes in the Peripheral Blood of Chronic Myelomonocytic Leukemia
Authors: Mya Hae Tha , Diez Campelo Maria , Valle Herrero Sandra , Díaz-Alvarez Agustin , Bustos Domingo , Vaquero Luis M , Pérez José J , González Verónica , Bastida Jose M , Aguilar Carlos , Bárez Abelardo , Puig Noemi , Martin-Sancho Alejandro , Jesús Garcia María , Aires Irene , Alonso Sara , González Marcos , Del Cañizo Consuelo , Vidriales Maria Belen 
Published: October 2019

Abstract
Chronic myelomonocytic Leukemia (CMML) is a heterogeneous clonal disorder highly resistant to the few therapies that are available nowadays.
There is increasing evidence to suggest that Programmed Death-1 (PD-1), and its major ligand Programmed Death Ligand-1 (PD-L1), are involved in immune suppression and disease progression, are highly expressed in many hematological malignancies, and can be involved in MDS pathogenesis and resistance mechanisms to hypomethylating agents. However, the expression of PD-1 and PD-L1 is not widely explored in CMML.
Different types of monocytes based on CD14 and CD16 expression show different genetic profiles and functions, having different distribution in several conditions, including malignancy.
In our study, we studied the expression of PD-1 and PD-L1 in the peripheral blood (PB) monocytic compartment of patients with CMML using flow cytometry , to better understand their potential role in the pathogenesis of the disease, and as a basis for the evaluation of this pathway for the development of future immunotherapy strategies.
Peripheral blood samples from 16 CMML, and age matched normal (n=10) and reactive (n=9) monocytosis (>1 x109 /L) were studied. Two hundred µl of each PB sample were stained with an 8-color panel of monoclonal antibodies (CD16-FITC, CD64-PE, PD1-PCP5.5, PDL1-PC7, CD300-APC, CD14-APCH7, HLADR-V450 and CD45-OC515). A minimum of 1x 106 events were acquired by FACSCanto II (BD Biosciences, San Jose, USA) and the data was analyzed with the Infinicyt software (Cytognos SL, Spain). Monocytic population was selected first on the automated population separator plot and confirmed by the expression of CD64 and HLADR expression. Lymphocytes were used as the internal control. Three types of monocytes were defined based on the CD14 and CD16 expression, as previously described.
As expected, CMML type 1 patients had higher absolute monocyte counts in PB than reactive and normal cases (p=0.001), and higher percentage of monocytic cells by flow (0.001).
The distribution (median) of the monocytic subpopulations based on CD14 and CD16 expression among the monocytic compartment in PB of CMML, reactive and normal cases, respectively, was as follows: "classical"(CD14+CD16-) were of 98%, 90% and 85% (p<0.000); "intermediate" (CD14+CD16+) 1.4%, 3.7% and 2.6% (p=0.01); and "non-classical" (CD14lowCD16+) monocytes 1%, 5% and 12% (p<0.001).
The expression of PD-1 in the major population ("classical" monocyte) was similar among CMML (Median MFI 370), reactive (Median MFI 403), and normal cases (Median MFI 265). However, in the "intermediate CD14+CD16+" and in the "non-classical CD14lowCD16+" monocytes, PD-1 was overexpressed in CMML and reactive cases, compared to normal controls. Reactive cases had even a higher overexpression of PD1 in both "intermediate" and "non-classical" monocytes compared to CMML (Median MFI of 312, 529, and 398 for "intermediate" and Median MFI of 185, 465, and 271 for "non- classical" in normal, reactive and CMML cases, respectively-p=0.01, and p<0.01).
For PDL1, we did not find differences in their expression in "classical" nor "intermediate" monocytes among CMML, reactive and normal cases (Median MFI in "classical" monocytes of 2415 vs 2086 vs 2003 for CMML, reactive and normal cases -p>0.05-; and Median MFI in "intermediate" monocytes of 3803 vs 2737 vs 3200 for CMML, reactive and normal cases -p>0.05-). However, in the "non-classical" monocytic population, PD-L1 was clearly overexpressed in CMML (Median MFI of 1782) compared to normal controls (Median MFI of 699), and this was also significantly higher than in reactive cases (Median MFI of 1040) (p=0.002).
We found that PD-1 and PD-L1 were overexpressed in CMML, but not in the main "classical" monocyte population of the PB, but in the less represented "intermediate" and "non classical" monocytic compartment. Interestingly, the CD16+ monocytes (intermediate and non-classical) were proposed to have a more important role in inflammation and immunomodulation. Therefore, these populations could have an important function in the pathogenesis of the CMML, and the overexpression of PD-1 and PD-L1 could be investigated as a target for immunotherapy in the development of new therapeutical strategies to improve the adverse prognosis of the CMML.

Disclosures
Diez Campelo: Novartis: Research Funding, Speakers Bureau; Janssen: Research Funding; Celgene: Research Funding, Speakers Bureau. Puig:The Binding Site: Consultancy; Janssen: Consultancy.