High Amount of PD1+TIM3+ T Helper Cells May Predict Poor Outcome in Adult Acute Lymphoblastic Leukemia

Background and Aims

In solid tumors, increasing evidence suggests that the immune contexture of the tumor correlates with survival. However, in ALL the immunological status of the bone marrow (BM) niche has not been thoroughly studied. Especially with the emerging immunotherapies in sight, it is vital to chart the fundamentalities that could lie behind treatment success or failure. Therefore, we aimed to characterize the immunological microenvironment in B-cell ALL BM by multiplex immunohistochemistry (IHC), and to evaluate the results in correlation to treatment outcome parameters.

Methods

BM biopsies from the diagnosis stage included both Ph+ (n=25) and Ph-negative (n=19)adult B-cell ALL patients. BM biopsies from non-leukemic (NL) controls (n=14) were used as reference. Samples were hematopathologically evaluated and a tissue microarray (TMA) was constructed by selecting two BM cores with high leukemic cell infiltration per patient. The TMA sections were stained with both fluorescent and chromogenic dyes for six markers and nuclei simultaneously enabling cytometric analysis at cell-level resolution. Panels included T and B lymphoid cells, NK and dendritic cells (DCs), macrophages as well as myeloid-derived suppressor cells (MDSCs). Furthermore, we analyzed immune checkpoint molecules (PD1, LAG3, OX40, TIM3, CTLA4) and their ligands (PD-L1, PD-L2, HLA-G, HLA-ABC) alongside with various activation markers (granzyme B, CD45RO, CD25, CD57, CD27). After the staining, the cells were segmented and quantified with the image analysis software CellProfiler and the cell analysis software FlowJo.

All variables with P-value <0.10 in univariate Cox regression analysis were included in a L1-penalized elastic net Cox regression model. After model shrinkage, feature selection was performed with multivariate Cox regression model. The risk model was used to stratify patients into equally-sized groups. All patients included in the survival analysis were treated with similar cytostatic regimens. All Ph+ ALL patients received TKIs as part of their treatment protocol.

Results

In comparison to NL controls, ALL BM was mainly characterized by immunosuppression. The amount of NK cells (0.4 % vs. 0.9 % of all cells, P<0.0001) was decreased, as well as the number of CD27 expressing T cells (5.4 % vs. 21.5 % of CD4+ cells, P<0.0001 and 5.7 % vs. 34.6 % of CD8+ cells, P<0.0001). Further, the amount of MDSCs was increased (0.9 % vs. 0.04 % of all cells, P<0.0001), as well as the number of CD25 expressing T cells (46.5 % vs. 1.4 % of CD4+ cells, P<0.0001 and 56.7 % vs. 2.1 %, of CD8+ cells P<0.0001). The expression of inhibitory immune-checkpoint molecules PD1 (39.2 % vs. 7.7 %, of CD4+ cells, P<0.0001 and 33.5 % vs. 16.3 %, of CD8+ cells, P=0.0002) and CTLA4 (9.4 % vs. 0.9 %, of CD4+ cells, P<0.0001 and 8.7 % vs. 1.2 %, of CD8+ cells, P<0.0001) on T cells was elevated. However, the expression of LAG3 on T cells was decreased (7.7 % vs. 17.3 % of CD4+ cells, P<0.0001 and 5.9 % vs. 19.3 % of CD8+ cells, P<0.0001). The amount of TIM3 (0.12 % vs. 3.8 %, of all cells, P<0.0001) was decreased as well. Ph+ ALL BM did not differ significantly in comparison to Ph-negative B-cell ALL BM.

In the risk stratification model, high expression of BM PD1+TIM3+ T helper cells, high age and low platelet count at diagnosis differentiated a poor survival group. The hazard ratio (HR) for death in the high-risk group was 4.86 (P=0.00068) and the HR for relapse or death was 4.06 (P=0.0016) (Figure 1). In univariate analysis, high expression (>0.1 % of T cells) of PD1+TIM3+ double-positive T helper cells trended towards poor survival, but did not reach statistical significance.

Conclusions

Multiplex IHC enables ample cytometric evaluation of different immune cell subtypes in their original microenvironment of the bone marrow. B-cell ALL bone marrow is mainly characterized by immunosuppression, even though the heterogeneity is marked. In the multivariate model based on the amount of PD1+TIM3+ double-positive T helper cells, age and platelet count at diagnosis differentiated a poor survival group. In univariate analysis, high expression of PD1+TIM3+ double-positive T helper cells trended towards poor survival. PD1+TIM3+ double-positive T cells are associated with severe immune exhaustion, and high expression may predict poor survival in adult B-cell ALL patients. The results need yet to be validated in a larger data set.

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