Page 142 - Haematologica Vol. 110 - January 2025

ARTICLE - Immune microenvironment in nodal PTCL
of surface CD3 (sCD3), we also looked for aberrant cell phenotypes in the sCD3-negative compartment. Among the 23 clusters identified within these cells, we found, as expected, B cells, NK cells or DC (Online Supplementary Figure S4A, B). Interestingly, several other cell populations were more abundant in PTCL samples (Online Supple- mentary Figure S4C). These included cluster 5, retrieved in 2 patients, that matched a CD4+ checkpoint+ TFH-like phenotype, similar to that found in sCD3+ Tconv cells; these were likely to be malignant. The cytotoxic pheno- type of patient 11 was also found within a sCD3-negative cluster (cluster 3). In addition to this, we found 2 aberrant CD4-Foxp3+CD39+ clusters (clusters 9 and 15) only found in patients 9 and 25, but which did not match the phe- notype of their sCD3+ counterpart (Figure 2B, D).
To further harness the complexity of these tumor cell phenotypes, we next analyzed each sample individually in order to ascertain that no rare cell cluster was overlooked. We specifically looked for clusters of cells presenting a non-classical phenotype, i.e., T cells harboring loss of CD7, gain of CD10, or aberrant expression of surface or intracellular proteins. This strategy led to the identification of putative neoplastic phenotypes in most, but not all, samples (Online Supplementary Table S8). Interestingly, in 3 PTCL, NOS samples, cell clusters exhibiting co-expres- sion of PD-1 and ICOS could be found; thus, a diagnosis of nodal follicular helper T-cell lymphoma NOS could not be fully excluded in these cases. In line with our previous observations, we detected a very high heterogeneity be- tween samples. Importantly, in about half of the cases, we found more than one aberrant phenotype, indicating that, as shown in many other cancers, PTCL are also prone to intra-tumor heterogeneity.
The data thus illustrate both the complexity of proper tumor-cell identification in nodal PTCL, and the pertur- bations of immune homeostasis in these diseases. This also warrants the use of additional tools (TRBC1 staining, TCR clonality at the single-cell level) to better understand the biology of nodal PTCL.
Foxp3+ Treg cells are critical inhibitors of anti-tumor immunity. We detected a significant increase in the pro- portion of Treg cells among CD4+ T cells in both AITL and PTCL, NOS compared to tonsil and LN samples (Online Supplementary Figure S5A). Based on classical activation markers and Treg cell hallmarks, 6 cell clusters were iden- tified through FlowSOM, corresponding to CD45RA+ naïve (cluster 6), PD1+ICOS+ TFR-like (cluster 4), CD39brightTIGIThigh (cluster 1) cells, as well as a subset with high expression of multiple Treg markers (cluster 2) (Online Supplementa- ry Figure S5B, C). Cluster 5 was specific to patient PTCL, NOS 11. Of those populations, we only found a decrease in naïve-like TCF1+CD45RA+ cells in AITL compared to ton- sils, but not LN; this was confirmed through supervised analysis (Online Supplementary Figure S5D). Collectively, Treg cells were abundant in PTCL and exhibited a high
P. Stephan et al. activation profile, suggesting active immunosuppression.
Natural killer cell phenotype is not impaired in peripheral T-cell lymphoma
Natural killer cells are important effectors of the anti-tu- mor response; nevertheless, their activation and function are often dampened in cancer, constituting an important mechanism of immune escape. We analyzed the distribu- tion and activation / maturation levels of CD56+ NK cells. Unsupervised analyses revealed 6 clusters that could be generally defined as CD56bright cytokine-producing cells, CD57+ cytotoxic cells, and Ki67+ proliferating cells (Online Supplementary Figure S6A, B). There was no significant difference in these different populations between control and PTCL samples, with the exception of a slight increase in total GZMB+ cells in AITL, detected through supervised analysis and manual gating (Online Supplementary Figure S6B-E). Thus, the PTCL TME did not seem to significantly impact NK-cell distribution.
CD8+ T cells display features of exhaustion
Next, we examined the phenotype of CD8+ T cells in lym- phoid tissues. FlowSOM analysis on 17 markers of activa- tion highlighted 18 distinct cell clusters that corresponded to: i) CD45RA+ TCF1+/- naïve-like cells, with low expres- sion of checkpoint molecules; ii) Ki67+ proliferating cells; iii) GZMB+ cells devoid of checkpoint expression; and iv) CD45RA- activated / memory cells with the expression of at least 2 checkpoint receptors (Figure 3A, B). There were clear differences between tissues (Figure 3C). Differential expression analyses between subgroups of samples, high- lighted the loss of naïve-like cells (CD45RA+TCF1+GZMB-) in both AITL and PTCL, NOS (clusters 12, 16 and 17) compared to non-malignant tissues (Figure 3B, D). In contrast, Ki67+ cells increased (Figure 1D), and clusters 1, 4, 6 and 7, cor- responding to cell subsets with very strong co-expression of PD-1 and TIM-3 among other inhibitory receptors, were significantly enriched in tumor samples. Although variable, the total number of PD-1+TIM-3+ cells was higher in tumor cells compared to tonsils and reactive LN (Figure 3E). Un- supervised clustering efficiently distinguished PTCL from tonsils, but not reactive LN samples, based exclusively on the phenotype of CD8+ T cells (Online Supplementary Figure S7). Of note, there were no striking differences in CD8+ T-cell profiles between AITL and PTCL, NOS patients, revealing common features of T-cell immunity between the 2 lymphoma subtypes. We further investigated the phenotype of these cells by performing Boolean analysis following manual gating, and observed a strong increase in cells expressing at least 3 inhibitory receptors in tumor samples compared to healthy tonsils and LN (Figure 3F). This exhausted phenotype, together with the expansion of Treg cells, suggests that strong immune suppression takes place in PTCL lymph nodes, emphasizing the need for im- munotherapeutic strategies in these conditions.
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