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I. Veletic et al.
To investigate dynamic changes in T-cell surface marker expression over the course of ruxolitinib treatment, consecutive cell surface marker profiles were analyzed using mixed linear models with repeated measures. To account for the progressive nature of MF, our final model also included spleen size, grade of BM fibrosis, and JAK2V617F allele burden (Online Supplementary Tables S3 and S4). After correcting for these variables, we observed no significant change in CD4+ and CD8+ T cells over the course of ruxolitinib treatment (Figure 3Ci), suggesting that the increase in cytotoxic T cells that we observed over the whole treatment period is a result of disease progression rather than an effect of JAK inhibition. In contrast, after correction we still observed time-dependent shifts from effector to resting T- cell subsets (Figure 3Cii-iii), confirming our hypothesis that long-term ruxolitinib treatment mitigates T-cell overactivation. Whereas significant TN and TCM cell increases were observed in the second, third and fifth years of therapy, in both CD4+ and CD8+ subsets TEFF cells consistently decreased over the same period. Similar changes were also observed in the fourth year of treat- ment, although they reached statistical significance only in the TCM and CD4+ TEFF subsets. Remarkably, both TEM subsets showed no significant change during treatment, except for the CD8+ sub- set during the fifth year of therapy, suggesting that long-term ruxolitinib treatment prevents terminal activation of T cells in MF, but has little effect on the effector memory arm of T-cell activation.
To determine whether baseline distributions of T-cell dif- ferentiation and activation subsets affect the overall survival, datasets were further analyzed using the Kaplan-Meier method and no significant differences were found (Online Supplementary Figure S1). In summary, these data suggest that ruxolitinib treatment shifts the activation state of T-cell subsets from terminal effector towards resting phenotype in a time- dependent manner.
PD1-expressing fractions within the T-cell subsets of myelofibrosis patients
Because it was recently reported that MF myeloid cells express high levels of PDL1,19 we sought to evaluate PD1- expressing fractions within T-cell subsets of MF patients. The proportion of cells co-expressing PD1 in CD4+ and CD8+ T cells of MF patients (n=35) was higher by 55.9% (P=0.028) and 86.8% (P=0.001), respectively, compared to T cells of healthy controls (n=28) (Figure 4Ai and Bi). Specifically, PD1+ fractions were increased within both CD4+ and CD8+ TCM, TEFF, and TEM cells (mean fold-changes, 1.49, 2.97, and 3.05 in CD4+ cells; 1.77, 2.64, and 2.83 in CD8+ cells, respectively; P=0.013 in CD4+ TEFF, P<0.001 in the rest), and within CD8+ TN cells (mean fold- change, 1.74; P=0.007) (Figure 4Aii-iii and Bii-iii). Importantly, most PD1+ fractions correlated positively between one another (Figure 4C) while no significant correlation was observed with any of the T-cell subsets, suggesting that PD1+ cells are prevalent among MF T cells irrespective of their differentiation or activa- tion state. In addition, we analyzed how ruxolitinib affects PD1+ CD4/CD8 and activation subsets over the whole follow-up peri- od and in each year of treatment, corrected for the parameters of disease progression (spleen size, BM fibrosis grade, and JAK2V617F allele burden). Overall, no significant differences were observed in PD1+ fractions over the course of ruxolitinib treatment (Online Supplementary Figure S2).
Association between T-cell subsets, PD1+ fractions, and disease progression
Because MF is a progressive myeloproliferative neoplasm,28 and T cells are known to interact with clonal neoplastic cells,29 we ana- lyzed the correlation between T-cell subsets and PB cell counts of untreated MF patients (n=41). We found that the number of CD8+
cells correlated positively with monocyte counts and negatively with platelet counts (r=0.317 and r=-.335; P=0.043 and P=0.032, respectively); however, CD4+ cell subsets had a negative and pos- itive correlation with monocyte and platelet counts (r=-0.371 and r=0.375, respectively; P=0.017 and P=0.016, respectively) (Figure 5Ai). Given that both monocytosis and thrombocytopenia are
30 + associated with disease progression, it is likely that CD8 cells
expand with disease propagation in untreated MF patients. Conversely, increased PD1 levels of both CD4+ and CD8+ cells correlated with total leukocyte counts (r=0.628 and r=0.547, respectively; P<0.001 for both) and palpable spleen size (r=0.435 and r=0.465; P=0.005 and P=0.002, respectively Figure 5Aii), sug- gesting that the increase in PD1+ T-cell fractions, typically associ- ated with T-cell exhaustion, correlates with disease progression, regardless of PD1 distribution across those T-cell subsets.
To investigate the effect of disease progression on subset levels at baseline and following treatment with ruxolitinib, we stratified patients based on spleen size, BM fibrosis grade, and JAK2V617F allele burden, and compared their total, TEM and PD1+ subsets, using healthy controls as a reference (Figure 5B). Although we found a 29.5% larger CD8+ T-cell population in MF patients with a palpable spleen larges than 20 cm at treatment baseline (n=11), this effect did not reach statistical significance (P=0.087). Interestingly, however, we also found 17.1% fewer CD4+ cells in this group of patients than in the control group (P=0.027). Moreover, MF patients with advanced-stage disease prior to treat- ment did not exhibit the significant repolarization of CD4/CD8 populations over time shown by patients with early-stage disease, further indicating that CD8 predominance is not a ruxolitinib effect but a result of disease progression. MF patients with splenomegaly greater than 20 cm had 1.4-fold larger baseline CD4+ TEM subsets and CD4+ PD1+ fractions (P=0.045 and P=0.029, respectively) compared to patients with smaller spleens. Of note, both PD1+ CD4+ and CD8+ subsets of these patients were signifi- cantly higher than normal (P=0.003 and P=0.013, respectively), similartopatientswithMF-3gradefibrosis(P=0.042andP<0.001, respectively) and patients with mutant JAK2 allele burden above 50% (P=0.038 and P=0.002, respectively). Overall, the CD8+ TEM subset showed little difference based on the analyzed parameters of disease progression, supporting the idea that CD8+ resting cells in MF rapidly transit to T-effectors as they become activated. Remarkably, patients with high mutant JAK2 allele burden had significantly lower numbers of CD8+ TEM (P=0.022).
Association between T-cell subsets, PD1+ fractions, and clinical response to ruxolitinib
Because a reduction in spleen size is typically associated with a good response to ruxolitinib and favorable treatment outcome,24,26,27 we tested the association between pretreatment T-cell subsets and spleen size 6 months into therapy. We found that complete reso- lution of palpable splenomegaly was associated with an increased percent of CD4+ cells and a decreased percent of CD8+ cells (mean differences 14.4% and -23%; P=0.038 and P=0.049, respectively) (Figure 6A). Furthermore, complete resolution of palpable splenomegaly was associated with a low percent of PD1+ fractions in both CD4+ and CD8+ cell subsets (mean differences, -30.7% and -31.7%; P=0.012 and P=0.036, respectively) (Figure 6B), suggesting that MF patients with low levels of exhausted (PD1+) T cells likely respond favorably to ruxolitinib treatment.
Effect of PD1+ T-cell fractions on survival rates of myelofibrosis patients
Data from 41 MF patients were further analyzed using the Kaplan-Meier method (34 patients [82.9%] had died) to deter- mine whether the distribution of PD1+ T-cell fractions affects
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haematologica | 2021; 106(9)