Tumor Microenvironment (TME) Regulation of Programmed Death-1 (PD1) Receptor and Its Ligands PDL1 and PDL2 in Chronic Lymphocytic Leukemia (CLL)

The PD1 pathway is involved in the inactivation of immune effectors and is potentially reactive against neoplastic cells in the TME. T cells from CLL patients exhibit defective immunity, decreased proliferative capacity, and impaired effector function leading to T cell exhaustion. These functional defects coincide with higher CD244, CD160 and PD1 expression on T cells. In the Eμ-TCL1 transgenic CLL model, PDL1 checkpoint blockade restores immune dysfunction and impairs leukemia growth. PD1/PDL1 ligation also affects BCR signaling, and because PD1 is also expressed on CLL cells, PD1 interference might directly influence tumor growth and proliferation directly.We studied the 1) expression the PD1 receptor and its ligands and their correlation with TFTT; 2) role of the TME in controlling PD1 axis and miRNA expression; 3) role of the BCRi ibrutinib (IB) in the expression of the receptor and ligands.Early-stage CLL patients were prospectively enrolled at diagnosis (n=211, O-CLL1: clinicaltrial.govID:NCT00917540). Gene expression (GE) profiling was performed using the GeneChipVR Gene1.0 STArray (Affymetrix) and validated by qPCR (Thermofisher) and flow-cytometry (FC, BD Biosciences) in an independent subset of patients. CLL lymph node samples underwent in situ immunolocalization analyses. Autologous T-cells (AAT) were obtained by in vitro exposure of patient T-cells with anti CD3/CD28 Dynabeads (Thermofisher) and IL2 in co-culture with CLL cells. Cultures were monitored until substantial clumping was observed and tested for PD1 axis expression. HS5 stromal cells were used in co-culture experiments with B-CLL (ratio 1:100). MiR-424 expression was evaluated by qPCR using validated TaqMan® MicroRNA Assays (Thermofisher). In selected experiments IB was added to cell culture. PD1 GEP of circulating B-CLL cells was significantly higher than of ligands. No differences in GEP of PD1 or its ligands were observed stratifying cases based on CD38, ZAP-70, or FISH markers. Significantly higher PDL1 GE was detected in IGHV-M cases. GEP results were validated by qPCR on 27 independent CLL samples. The impact of GE of the PD1 axis on clinical outcomes in CLL cases (median follow-up 39 months, range 6−82 months) indicated TTFT was significantly shorter in cases with higher levels of PDL2, while no significant differences were detected based on higher or lower PD1 or PDL1 levels. Multivariate analysis indicated higher PDL2 gene expression retained an independent prognostic power (HR=1.9, 95%CI 1.1-3.4, P=.022) in predicting TTFT together with IGHV-UM status, B-lymphocytosis≥5000/mm³ (P=.020), and CD38 expression (P=.021). In situ immunolocalization of CLL tissue infiltrates showed variable expression of PD1 that characterized small lymphoid elements, while PDL1 and PDL2 (PDL2>PDL1) characterized larger medium-sized elements within proliferation centers. Co-localization studies revealed PD1 co-expression of CD20+CLL cells and in scattered CD3+ cells. Both PDL1 and PDL2 were variably expressed in CD20+CLL cells; few T-cells and macrophages showed expression of both ligands within CLL infiltrates. FC analysis of CLL cells after AAT co-culture showed a higher percentage expression of circulating PD1 axis members (n=27, PDL1>>PDL2>PD1) compared to baseline indicating that the changes in PD1 axis expression may be due to events triggered by activation of the TME. In contrast, T cell populations had higher expression of CD3+, CD4+ and CD8+ cells bearing both PD1 and PDL1, while no substantial increment for either CD3+, CD4+ or CD8+/PDL2+ cells following AAT co-culture. GE analysis also showed a similar increase in PDLs (n=15, PDL2>>PDL1>PD1) expression with respect to non-activated B-CLL cells. Interestingly, a 48h co-culture of B-CLL with HS5 stromal cells (n=6) showed a similar increase in PD1 axis GE pattern as AAT cultures. Expression of miR-424 also decreased significantly (<80%) after AAT co-cultures (n=6), indicating its potential involvement in PDL1 regulation. IB reduced PD1/PDL1 gene and protein expression on CLL B-cells; the IB inhibitory effect was also observed only on CD8+ cells.We indicate that PDL2 expression characterizes a subset of high-risk early stage CLL patients. PD1 axis expression is characteristic of the CLL TME whereby modulatory co-stimulatory signals may be counteracted by IB. Further studies will examine the role of miR-424 regulation with this cellular context.