Page 146 - Haematologica Vol. 110 - January 2025

ARTICLE - Immune microenvironment in nodal PTCL
P. Stephan et al.
tions, we split our 18-sample cohort into CD39 low, in- termediate and high groups (Online Supplementary Figure S8B). No correlation with major immune subtypes could be found (Online Supplementary Figure S8C). Interest- ingly, increase in CD39 expression was associated with enhanced TIM-3/PD-1 and CTLA-4+CD8+ T-cell subsets, although this did not reach significance (Online Supple- mentary Figure S8D). Conversely, high CD39 expression was correlated with lower GZMB expression, likely to be indicative of immune dysfunction sustaining tumor pro- gression. We thus wondered whether CD39 expression impacted PTCL clinical outcome. First, we investigated the putative prognostic value of CD39 at the RNA level in bulk samples by examining the association between ENTPD1 expression and OS in the TENOMIC cohort, that consists of a dataset of microarray-defined gene expres- sion profiles of 85 LN samples of AITL patients. In this cohort, the level of ENTPD1 expression in the whole bulk of cells was not significantly associated with a prognos- tic value (Online Supplementary Figure S8E). To further explore the expression of CD39 at the protein level in T-cell subsets, we used a multi-immunofluorescence (IF) approach on tissue microarrays (TMA) comprising 43 LN samples from AITL patients at diagnosis, with a 10-year clinical history (Online Supplementary Tables S3 and S4). PFS and OS in the cohort matched previously reported data (Online Supplementary Figure S8F). Slides were stained for CD3, CD8, CD39, and DAPI, and the proportion of each population was determined. CD3+ T cells, in particular the CD8+ subset, exhibited higher CD39 expression compared to non-T cells. As in FACS analyses, we observed an important variability in CD39 expression between patients (Figure 5C, D). We first measured the impact of CD39 expression in different subsets by splitting the samples based on the median expression of CD39. Although the proportion of CD39+ cells among all cells was not associated with prognostic significance, patients harboring high CD39 expression among T cells (regard- less of CD8 expression) showed a trend toward poorer prognosis (Online Supplementary Figure S8G). To refine this analysis, we analyzed the data following Receiver Operating Characteristic (ROC) curve-based separation of patients. We found that patients harboring the highest expression of CD39 in T cells (N=15, 35% of the entire cohort) exhibited a significantly poorer prognosis, both at the PFS and OS levels (Figure 5E). Similar conclusions were reached when analyzing CD39 expression in the CD3+CD8+ and CD3+CD8- subsets (Online Supplementary Figure S8H). The prognostic value of CD39 expression by T cells was further confirmed through multivariate Cox models including Prognostic Index for AITL (PIAI), sex, and age (Figure 5F). Together, our data suggest CD39 expression by T cells may represent a novel independent prognostic factor in AITL and should be explored as a putative target.
Discussion
To date, and in contrast to many solid tumors and other hematologic malignancies, there is a clear lack of knowledge in the distribution and phenotype of immune cells in the context of PTCL. Our study documents an accumulation of highly activated and heterogeneous T-cell populations, whereas B-cell populations were less abundant in this dis- ease. Although this confirms recently reported data,18 this does not preclude the therapeutic targeting of B cells, as these can support tumor cells.22,23
One striking observation that can be made based on our data is the large phenotypic heterogeneity of Tconv cells between PTCL patients. Although most Tconv cells ex- pressed at least one checkpoint surface receptor, only about half of them expressed PD-1 or ICOS, suggesting that the phenotype and biology of AITL cells extend beyond these 2 single markers. This merits further exploration; for example, by combining high-dimensional cytometry to single-cell TCR-seq that allows the identification of clonal cell populations. Most CD8+ T cells in both AITL and PTCL, NOS expressed at least one inhibitory checkpoint and a prominent PD-1+TIM3+ population could be detected, sug- gesting that an active exhaustion process might be taking place in PTCL tissues. This is in accordance with observa- tions made in patients with cutaneous T-cell lymphoma and other non-Hodgkin lymphomas,24-26 and confirms the potential benefit of checkpoint-based immunotherapies in PTCL.
In this regard, we aimed to identify surface receptors expressed by cytotoxic T cells and/or NK cells but not by Tconv cells (that included most malignant cells) and Treg cells, or vice versa. Similar to the large spectrum of PD-1 expression (that may explain the hyperprogression detected in some PTCL patients following PD-1 blockade), we observed that TIGIT and TIM-3 were largely expressed by both tumor-promoting (tumor cells, Treg cells) and tumor-inhibiting (CD8+ T cells, NK cells) populations. This broad expression may negate the therapeutic potential of blocking Ab, that are currently under clinical development in solid and hematologic malignancies.27 In contrast, other surface markers, such as OX-40, or, to a lesser extent, CT- LA-4 or ICOS, displayed a much higher expression on CD4+ (comprising tumor cells and Treg cells) than other subsets. These may thus represent attractive therapeutic targets for the treatment of PTCL. In line with this, mRNA expression of CTLA4 was shown to be associated with poor prognosis in a meta-analysis of PTCL transcriptomes.28 Specifically, this would require the use of depleting monoclonal Ab (mAb) through the use of Ab-dependent cellular cytotox- icity and phagocytosis-optimized IgG1 molecules.29 To date, and to our knowledge, OX-40 mAb under development have agonistic functions and should not be recommended for the treatment of PTCL. The depleting effect of current anti-CTLA-4 mAb, such as ipilimumab, is still a subject of
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