Page 240 - 2021_10-Haematologica-web

Letters to the Editor
Single-cell profiling reveals the dynamics of cytomegalovirus-specific T cells in haploidentical hematopoietic stem cell transplantation
Human leukocyte antigen (HLA)-haploidentical hematopoietic stem cell transplantation (haplo-HSCT) with post-transplant cyclophosphamide (pt-cy) is a valid alternative to HLA-identical HSCT,1 but many patients still suffer from viral infections, mostly cytomegalovirus (CMV) reactivation.2 CMV-specific T cells contribute to control of CMV reactivation post-transplant, but their evaluation has been limited to a handful of immunophe- notypic parameters. In particular, the dynamics and qual- ity of CMV-specific T cells in relation to immune recon- stitution and control of CMV viremia following haplo- HSCT with pt-cy are poorly known. To these aims, we employed high-dimensional flow cytometry simultane- ously investigating four effector functions and markers of T-cell differentiation, inhibitory molecules, and metabolic and activation markers, along with computational analy- sis of single-cell data.
We performed longitudinal analysis of high-dimension- al T-cell immunophenotypes in blood samples of 21 recipients of haplo-HSCT with pt-cy treated at our insti- tution for hematological diseases (Online Supplementary Table S1). A median of seven samples per patient were analyzed, including graft (n=13) and peripheral blood (PB) samples ranging from day +21 to day +386 post- transplant (n=111). As control, we included PB samples from donors (n=9) and healthy individuals with detectable CMV pp65-specific T cells (n=17). The cluster- ing tool PhenoGraph3 identified 13 CD8+ and 11 CD4+ T-cell clusters (Figure 1A). Principle component analysis (PCA) of cluster frequencies identified which of the 24 T-cell clusters were co-varying in a time-dependent man- ner. A variable plot of the first two principle components revealed four major groups of T-cell phenotypes (Figure 1B). Median PCA coordinates of all samples plotted at defined intervals indicated a clear pattern in T-cell dynamics: loss of naïve and CD73+ memory clusters and emergence of proliferating clusters at 3-4 weeks post- transplant, dominance of non-proliferating, activated clusters at month 2 and accumulation of effector and ter- minal effector (TTE) clusters from month 3 onwards (Figure 1B).
Pt-cy interferes with highly-proliferating alloreactive T cells, and alloreactivity resides preferentially in the naïve pool.4-6 Furthermore, naïve cells that escape pt-cy may initially acquire a stem cell memory phenotype to later give rise to effector cells.6,7 These processes may explain the rapid decline in naïve T cells early after trans- plant. By week 3-4, Ki-67+HLA–DR+ proliferating CD8+ cluster 10 and CD4+ cluster 9 expanded (Figure 1A), likely in response to a combination of exogenous or alloreactive antigens, homeostatic cytokines and inflammation.6,7 The frequency of proliferating cells declined by month 2 and was accompanied by an increase in activated CD8+ and CD4+ Ki-67–HLA-DR+ T cells (cluster 6 and 4, respectively) that persisted for several months. CD4+ cluster 8 of regulatory T cells (TREG) and CD8+ cluster 13 of TIM-3highPD-1highTIGIThigh cells resembling exhausted cells, displayed dynamics similar to that of proliferating cells (Figure 1A). Transient TREG expansion following haplo-HSCT with pt-cy corroborates previous findings6,8 and seems critical for the prevention of graft-versus-host disease (GvHD) by pt-cy.8,9 From month 3 onwards, the T-cell compartment became dominated by TTE or effector
memory re-expressing CD45RA (TEMRA) clusters, express- ing T-bet, 2B4, CD45RA and/or senescence marker CD57. One year after transplant, cluster distribution within the CD4+ compartment resembled that of the donor, including partial recovery of the naïve pool, while that within the CD8+ compartment showed a persistent defect in this regard (Figure 1A).
CMV infection is a major event following haplo-HSCT with pt-cy.2 In our cohort, 19 out of 21 patients experi- enced CMV viremia, with a median onset of 39 days post-transplant. In order to determine the effect of CMV viral load on T-cell reconstitution, we divided patients experiencing post-transplant CMV viremia into two groups using a viremia threshold above which antiviral therapy was given: subclinical CMV viremia (any viremia with peak ≤4,000 IU/mL; n=6) and clinical CMV viremia (peak viremia >4,000 IU/mL; n=13). PCA of T-cell immunophenotypes indicated an accelerated acquisition of T-bet+2B4+CD45RA+/-CD57+/- effector/terminal effec- tor cells in patients with clinical CMV viremia (group IV of CD8 clusters 2, 4, 5, 9 and CD4 clusters 6, 10; Figure 1B). These cells originated from both the CD8+ and CD4+ compartment, reaching statistically significant differences for TEMRA cells in the former (Figure 1C and D). Furthermore, patients with subclinical viremia showed slightly improved recovery of naïve CD8+ T cells. Although limited to two individuals in our cohort, those patients who did not experience CMV viremia lacked TTE clusters and instead showed strong recovery of naïve subsets (Figure 1B). Accordingly, Suessmuth et al. found a decrease in naïve CD8+ T cells in CMV-reactivating patients receiving unmanipulated unrelated allografts, suggesting a link between CMV reactivation and a defect in thymopoiesis.10 Occurrence of clinically significant grade II-IV acute GvHD (aGvHD) and/or its treatment with corticosteroids could be a confounding factor and indeed tended to associate with worse CMV control in our cohort (Online Supplementary Figure S1A). However, hierarchical clustering indicated that patients developing aGvHD or receiving corticosteroids displayed overlap- ping T-cell cluster dynamics with aGvHD-negative patients (Figure 1E). These data suggest that CMV reacti- vation has a more prominent effect on T-cell reconstitu- tion than does aGvHD, which is in line with findings at the clonal level in the HLA-matched setting.11 Collectively, our data suggest that high CMV viral load drives premature senescence of T cells and delays recov- ery of naïve T cells following haplo-HSCT with pt-cy.
We next analyzed the functional and phenotypic pro- file of CMV-specific T-cells, identified through effector cytokines produced in response to CMV pp65 peptide library stimulation. Although likely underestimating the full extent of the CMV-directed T-cell response, which involves a broad range of antigens, pp65-specific T-cell responses are largely representative of the total response against CMV.12 Uniform manifold approximation and projection (UMAP) revealed dynamic changes in CMV- specific T-cell phenotypes during reconstitution (Figure 2A). PhenoGraph analysis of CMV-specific T cells gener- ated 15 CD8+ and 14 CD4+ T-cell clusters (Figure 2B). At week 3-4 post-haplo-HSCT, CMV-specific T cells were undetectable in most patients. By month 2, in which CMV viremia emerged in the majority of patients, both CD8+ and CD4+ CMV-specific T cells expanded and dis- played a proliferating phenotype, featuring high levels of Ki-67, HLA-DR, CD71, PD-1, CD95 and CD98 (Figure 2B; Online Supplementary Figures S1B and S2A). From month 3 onwards, these phenotypes were replaced by
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haematologica | 2021; 106(10)