Mass Cytometry Reveals PD1 Upregulation Is an Early Step in MDS Disease Progression

Abstract

Background MDS is characterized by ineffective haematopoiesis and a propensity to leukaemia transformation, with increasing evidence linking immune exhaustion to disease progression. Immune checkpoints are known to be upregulated on T cells in cancer, and inhibitors of CTLA-4 and PD1/PLD1 axis have demonstrated efficacy with likely clinical benefit in MDS. We have previously shown profound changes in both the number and function of components of the adaptive immune system, particularly Tregs, in MDS (Kordasti, Blood 2007). In order to characterise the immune signature in a wider range of T cell subsets simultaneously, with particular emphasis on cells likely to be affected by checkpoint inhibitor therapy (CPI), we analyse CTLA-4 and PD1 expression in MDS by cytometry by time-of-flight (CyTOF). Additionally, we aim to explore whether these differences are accentuated by the absence or presence of somatic mutations, or in morphologically more advanced disease. Materials and Methods MDS patients (n=56) and age-matched healthy donors (HD, n=6) were stained with two panels of 35 and 34 antibodies for unstimulated and PMA/Ionomycin-stimulated PBMCs, respectively. Samples were run on CyTOF and data analysed using visual stochastic neighbour embedding (viSNE, Cytobank) to generate t-distributed SNE scores by unsupervised multi-dimensional reduction of T cells. Spanning-tree progression analysis of density-normalized events (SPADE), was performed and T cell subsets identified from heat maps based on typical phenotypic markers (Regulatory, Naive, Memory, Effector Memory (EM) Central Memory (CM), Effector, Terminal Effector (TE)). T cells were also clustered based on cytokine secretion (IFN-ϒ, TNF-α, IL-17, IL-2 and IL-10). Somatic mutation analysis was performed on 48 of the MDS patients using our established targeted panel of 24 genes known to be mutated in MDS, for subgroup analysis (Mohamedali, Leukaemia 2015) Results and Discussion Demographics and subgroups are outlined in figure 1. The number of Tregs was significantly higher in RAEB than non-RAEB MDS (9.6% of total CD4+ cells vs 7.5% p=0.02) and in RAEB versus HD (9.6% vs 5.8% p=0.01). There was a significantly higher proportion of Tregs in MDS patients with somatic mutations compared to those without (8.7% vs 7.06% p<0.05) and HD (8.7% vs 5.9% p<0.05), confirming our previous findings. Amongst two subpopulations of Tregs previously identified (Kordasti, Blood 2016) there was no difference between HD and MDS in terms of their number. However, Tregs A and B have an increased PD1 expression in MDS vs HD (p=0.03 & p=0.003). In addition, CD4+EM and CD4+Memory, CD4+/CD8+ Na•ve T cells and TNF-α secreting cells all expressed more PD1 when compared to HD (p<0.05), see figure 2. However, there were no differences in T cell PD1 expression when comparing low and high risk MDS patients, suggesting immune exhaustion to tumour antigens is a common founder event in MDS. By contrast, CTLA-4 expression was not significantly different in T cell subsets between MDS and HD. Sub-groups based on specific somatic mutations did reveal that CD4+ Memory, CD4+TE and CD8+CM subsets in SF3B1-mutated MDS cases had less CTLA-4 expression compared to MDS cases with other mutations (p<0.05). Furthermore, SF3B1-mutant cases also had less CTLA-4 expression when compared to MDS cases without any detected mutations in CD4+ Memory, CD4+EM and CD8+CM subsets (p<0.05). Taken together this work suggests a possible 3 hit-model for disease progression in MDS. The establishment of MDS is accompanied by increased expression of PD1 most likely in response to increased PDL1 on tumour cells, which argues for early introduction of PD1 inhibitor in the disease course. Increased CTLA-4 expression is likely a later step, dependent on the acquisition of specific mutations by the malignant clone, that further alters the relationship between tumour and immune surveillance. Finally, in disease progression through from non-RAEB to RAEB/AML there is an expansion of suppressive Tregs facilitating tumour outgrowth. Furthermore, pro-inflammatory IL17 secreting T helper cells (Th17) have been shown to be increased in low risk MDS and are known to be associated with autoimmune disease. The trend towards increased PD1 expression in Th17 cells in a group of low risk patients harbouring SF3B1 mutations (p=0.09) is suggestive that acquisition of increased PD1 expression is a key branch point in disease outcome. Disclosures Irish: Incyte: Research Funding; Janssen: Research Funding; Cytobank, Inc.: Equity Ownership, Membership on an entity's Board of Directors or advisory committees.

Integrating the “Immunome” in the Stratification of Myelodysplastic Syndromes and Future Clinical Trial Design

“…122,123 CyTOF has also been already successfully adopted for immunophenotypic analysis of clinical samples in MDS, 124 for prospective immune monitoring of patients with chronic myeloid leukemia (CML), 125 and to further characterize the immune signature in a wider range of T-cell subsets in MDS. 126 There are, however, two important questions to be addressed: (1) Which immunologic markers to use? and (2) How will we define an immunoscore?…”

Targeting PD-1/PD-L1 pathway in myelodysplastic syndromes and acute myeloid leukemia

“…In general, lower-risk MDS is often linked to increased intramedullary apoptosis and pyroptosis, accompanied by elevated interferon-γ (IFN-γ), tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), S100A8/S100A9 and NLRP3 inflammasome levels [ 17 – 19 ]. While in higher-risk MDS and AML, the milieu in BM is relatively more immunosuppressive, as effector T cells and NKs are exhausted and functionally impaired, accompanied by elevated frequencies of hyperfunctional regulatory T cells (Tregs) [ 20 – 26 ]. Furthermore, immune checkpoint molecules, including PD-1, cytotoxic T-lymphocyte-associated-protein 4 (CTLA4), T-cell immunoglobulin mucin-3 (TIM-3), T cell immunoglobulin and ITIM domain (TIGIT), lymphocyte activation gene-3 (LAG-3), also have critical functions in this process by protecting blasts from host immune surveillance [ 27 – 32 ].…”

“…Recent studies have demonstrated that these markers are aberrantly expressed in MDS and AML patients. Detailed results noted that PD-L1 was mainly upregulated in CD34 + blasts, while PD-1 was increased in effectors T cells and Tregs [ 23 , 25 , 26 , 30 , 31 , 62 , 63 , 66 – 79 ]. Table 1 summarizes current studies focusing on the dysregulated PD-L1 and PD-1 axis in MDS and AML as assessed by flow cytometry.…”

Epigenetic treatment‑mediated modulation of PD‑L1 predicts potential therapy resistance over response markers in myeloid malignancies: A molecular mechanism involving effectors of PD‑L1 reverse signaling

“…PD-L1, programmed death-ligand 1; PBMC, peripheral blood mononuclear cells; EGT, epigenetic therapy; AML, acute myeloid leukemia; MDS, myelodysplastic syndrome.. response to EGT, NUR77 suggesting enhanced PD-L1 expression and associated downstream molecular mechanisms may be a more potent phenotypic contributor to EGT outcomes than potential response biomarkers including NUR77. Whilst previous studies have demonstrated EGT is able to increase PD-L1 expression in MDS (16)(17)(18) and may correlate with poor responses to treatment (18) our study is the first to identify this effect independent of potential response marker expression. Whilst our in vivo observations support our in vitro findings a potential limitation of our study exists due to logistical constraints in obtaining chronologically identical patient blood samples for analysis during cycle 1.…”

“…EGT. PD-L1 reverse signaling has recently been identified as a potential mechanism of EGT resistance in the setting of MDS treatment (8,(16)(17)(18)(19). We investigated the in vitro and in vivo effect of EGT on potential down-stream effector molecules of reverse PD-L1 signaling.…”