Page 81 - 2019_07 resto del Mondo-web

Post-lenalidomide immunomodulation in del(5q) MDS
Results
No disease-specific gene usage or shared individual B- or T-cell clones in immune environments of patients with del(5q) myelodysplastic syndromes
In search of a repertoire signature of immune environ- ments in del(5q) MDS or a signature of treatment response to lenalidomide, we globally compared B- and T-cell spaces of our cohort of del(5q) MDS patients (characteris- tics shown in Table 1) with those of healthy, age-matched controls. To this end, next-generation immunosequencing was performed on the T lineage TRB locus and the B line- age IGH locus in all subjects at baseline as well as 6-42 months after initiation of lenalidomide treatment. As regards TRB and IGH gene usage, del(5q) MDS samples did not differ from samples from age-matched healthy donors and no systematic biases in gene usage were observed upon lenalidomide treatment (Figure 1A-D). Moreover, no disease-specific shared B- or T-cell clones were discernable (Figure 2). Interestingly, PB and BM sam- ples as well as follow-up samples from the same patient showed significant overlapping for TRB (Figure 2B), but not for IGH (Figure 2A).
The clonal bone marrow B-cell space diversifies upon lenalidomide treatment in del(5q) myelodysplastic syndromes
Next, we explored B-cell architectural changes upon lenalidomide treatment by comparing pre- and post- lenalidomide IGH repertoires. The pre-lenalidomide BM B-cell space in del(5q) MDS was comparable to the B-cell space of age-matched healthy donors in terms of IGH clonality, but showed a significantly higher somatic hyper- mutation rate of IGH clones, indicating an extended num- ber of antigen-experienced B lineage cells (Figure 3A,C). BM (but not PB) B lineage clonality decreased significantly even below the levels in the age-matched healthy donor group and somatic hypermutation rates of IGH clones normalized in BM and PB upon lenalidomide treatment (Figure 3A-D).
TRB clonality is increased in myelodysplastic syndrome del(5q) bone marrow and GLIPH analysis reveals the presence of myelodysplastic syndrome-specific and lenalidomide-induced T-cell clusters
The BM T-cell space of patients with del(5q) MDS showed a higher TRB clonality compared to that of healthy control BM both before and after lenalidomide treatment, while there were no differences in PB T cells suggesting antigen-driven clonal T-cell selection only in the local BM environment (Figure 4A,B).
To further define antigen-driven T-cell dynamics in the BM of del(5q) MDS patients undergoing lenalidomide treatment, we performed GLIPH analysis27 on our TRB NGS dataset. The GLIPH algorithm clusters T-cell recep- tors according to their presumed antigen specificity based on conserved motif analysis.26 Since our global analysis of T-cell clonality suggested antigen-driven selection in the BM but not in PB, we exclusively used TRB repertoires from the local MDS microenvironment for this analysis. In a first step, T-cell clusters were determined by GLIPH analysis in BM from healthy controls as well as del(5q) MDS patients before and after lenalidomide treatment. When looking at MDS-exclusive T-cell clusters, we found a higher number of newly emerging clusters that were
shared between individual patients in the samples after lenalidomide treatment compared to the clusters that were present only at the pre-treatment time point (n=534 vs. 240 clusters, respectively) (Figure 5A). This indicated new T- cell cluster generation in response to lenalidomide. Of all clusters that persisted in the BM across the two sampling time points (n=1,242), the majority (n=813) decreased after treatment, while only fewer clusters clon- ally expanded after treatment (Figure 5A,B). The few MDS-exclusive T-cell clusters that were present before exposure to lenalidomide and that expanded upon treat- ment were mostly clusters with high generation probabil- ity and were therefore less likely to constitute a true T-cell response directed against the MDS clone. In contrast, the T-cell clusters newly generated upon lenalidomide treat- ment showed much lower generation probabilities, fur- ther supporting the hypothesis that the newly emerging clusters (in contrast to the preexisting ones) are specifically generated in response to the MDS clone (Figure 5B). Together, this indicates that despite large overlaps in the pre- and post-therapeutic TRB repertoire of individual patients, lenalidomide-induced T-cell immunity may be significantly driven by newly generated T-cell clusters in the local MDS del(5q) BM microenvironment which are shared between individual MDS patients. Table 2 summa- rizes the presumably most specific top 25 lenalidomide- induced T-cell clusters (low generation probability) that were shared between two or more patients.
Discussion
The immunomodulatory drug lenalidomide is effective in the treatment of del(5q) MDS. While lenalidomide has undebatable direct effects on the del(5q)-clone mediated by CRBN, the immunomodulatory effects on the microenvironment are less well defined to date. In this study we used state-of-the-art NGS technology to gain insight into local BM immune environments in del(5q) MDS and their modulation upon treatment. NGS technol- ogy makes it possible to characterize large immune reper- toires in depth and at high throughput, to monitor individ- ual immune clones over time and to integrate all informa- tion to derive quantitative and reliable repertoire metrics data such as clonality indices, gene usage, clonal space dis- tribution and somatic mutational frequencies. Thereby, the immune repertoire determined by NGS not only trans- forms our ability to detect imprints of antigenic drive,23,30 but also provides valuable insight into immunological drug effects.24, 31-35
Our analyses showed that lenalidomide exerts system- atic effects on the local B- and T-cell compartments in the BM of patients with del(5q) MDS. Since no significant clonal B-cell overlap between pre- and post-treatment samples was detectable in BM, the small but highly fre- quent clones detected after initiation of lenalidomide treatment most likely represent newly generated clones, given that our sequencing depth of around 17,000 success- fully aligned productive IGH reads per sample exceeds the greatest possible number of B lineage cells in the sample (1,136-5,682 per PCR, assuming a percentage of B lineage cells with completed VDJ recombination of 3-15% of BM mononuclear cells). Collectively, the changes observed in the IGH repertoire reflect the proliferative effect on healthy B-cell precursors previously described for this
haematologica | 2019; 104(7)
1359