Donor CD4 T Cell Diversity Determines Virus Reactivation in Patients After HLA-Matched Allogeneic Stem Cell Transplantation

Abstract

Delayed reconstitution of the T cell compartment in recipients of allogeneic stem cell grafts is associated with an increase of reactivation of latent viruses. Thereby, the transplanted T cell repertoire appears to be one of the factors that affect T cell reconstitution. Therefore, we studied the T cell receptor beta (TCRβ) gene rearrangements of flow cytometry-sorted CD4(+) and CD8(+) T cells from the peripheral blood of 23 allogeneic donors before G-CSF administration and on the day of apheresis. For this purpose, TCRβ rearrangements were amplified by multiplex PCR followed by high-throughput amplicon sequencing. Overall, CD4(+) T cells displayed a significantly higher TCRβ diversity compared to CD8(+) T cells irrespective of G-CSF administration. In line, no significant impact of G-CSF treatment on the TCR Vβ repertoire usage was found. However, correlation of the donor T cell repertoire with clinical outcomes of the recipient revealed that a higher CD4(+) TCRβ diversity after G-CSF treatment is associated with lower reactivation of cytomegalovirus and Epstein-Barr virus. By contrast, no protecting correlation was observed for CD8(+) T cells. In essence, our deep TCRβ analysis identifies the importance of the CD4(+) T cell compartment for the control of latent viruses after allogeneic stem cell transplantation.

Keywords: Bone marrow; Epstein-Barr virus (EBV); T cell biology; cytomegalovirus (CMV); donors and donation; hematopoietic stem cell transplantation; infection and infectious agents; infectious disease; molecular biology; science; translational research.

Figure 1

TCRβ analysis in healthy control individuals. (A) Vβ subgroup usage of TCRs from CD4⁺ (left panel) or CD8⁺ (middle panel) T cells of six untreated healthy control individuals (designated as “C1” to “C6”). Seven of the most frequent Vβ subgroups were illustrated individually. The remaining subgroups were compiled. The nomenclature according to the international ImMunoGeneTics information system (IMGT) was used. The right panel shows mean frequencies of all six controls for CD4⁺ and CD8⁺ T cells. (B) HTS-derived CDR3 length distribution of CD4⁺ (left panel) or CD8⁺ (right panel) TCRβ sequences for each control donor. The right panel shows mean frequencies of all donors. TCRβ CDR3 size was defined as all amino acids (AAs) from the conserved cysteine in the Vβ segment to the conserved phenylalanine in the Jβ segment. (C) The left and middle panels show the individual clonotype frequency distribution of CD4⁺ or CD8⁺ T cells of each control donor. The right panel shows the mean values of CD4⁺ and CD8⁺ T cells of all control donors. Each dot represents the occurrence (percentage) of a distinct TCRβ clonotype. Only the clonotype frequencies for the first 250 clonotypes were illustrated. (D) Diversity dot plot of CD4⁺ and CD8⁺ T cells for six healthy control individuals. Each dot shows the number of unique clonotypes detected in one donor. The bars illustrate the mean value of the groups. (E) Overlaps of individual clonotypes in CD4⁺ or CD8⁺ T cells of one control donor sequenced twice out of one T cell preparation (5A) and of two separate cell sorts (5A/B). (F) Overlaps of individual clonotypes in CD4⁺ or CD8⁺ T cells of distinct control individuals.

Figure 2

TCR analysis in stem cell donors before and after G-CSF induced mobilization. (A) Mean Vβ subgroup usage of TCRs from CD4⁺ or CD8⁺ T cells of 23 donors before (pre) and after (post) G-CSF induced stem cell mobilization. The seven most frequent Vβ subgroups were illustrated individually; the remaining subgroups were grouped together. The nomenclature according to the international ImMunoGeneTics information system (IMGT) was used. (B) Mean percentage of overlaps of individual clonotypes in CD4⁺ or CD8⁺ T cells of the 23 donors pre– and post–G-CSF–induced stem cell mobilization. (C and D) Diversity dot plots of CD4⁺ and CD8⁺ T cells pre– and post–G-CSF mobilization. Each dot represents the number of unique clonotypes detected in individual donors. (C) The number of clonotypes in CD4⁺ T cells differs significantly before and after G-CSF mobilization from the number of clonotypes in the CD8⁺ compartment (****p < 0.0001; n = 44). Bars show mean values. (D) The number of clonotypes in CD4⁺ and CD8⁺ T cells is not affected by G-CSF administration. (E) Scatter plots of CD4⁺ and CD8⁺ clonotype numbers and the age for stem cell donors pre– and post–G-CSF mobilization. The number of individual CD8⁺ clonotypes decreases with increasing donor age irrespective of G-CSF mobilization (preMOB: r = −0.42; p = 0.052; postMOB: r = −0.42; p = 0.051; n = 22). For CD4⁺ T cells, no negative correlation was found. CD4⁺ T cells preMOB; CD4⁺ T cells postMOB; CD8⁺ T cells preMOB; CD8⁺ T cells postMOB.

Figure 3

Donor TCRβ diversity and clinical correlation. (A) Diversity dot plots of CD4⁺ and CD8⁺ T cells of cytomegalovirus (CMV)-seropositive or -seronegative donors before (pre) and after (post) G-CSF mobilization. Only for the CD4⁺ T cell compartment postmobilization a significant difference could be displayed between CMV-seropositive and -seronegative donors (*p < 0.05; neg. n = 14; pos. n = 8). (B) Diversity dot plots of CD4⁺ and CD8⁺ TCRβ rearrangements of G-CSF–mobilized donors premobilization and postmobilization according to patients either reactivated (“Yes”) or not (“No”) with CMV (“Yes”: CD4⁺ n = 10; CD8⁺ n = 11) or Epstein–Barr virus (EBV; “Yes”: CD4⁺ n = 7; CD8⁺ n = 7). Cases with CMV and/or EBV reactivation presented significantly lower numbers of unique clonotypes in CD4⁺ T cells post–G-CSF mobilization (*p < 0.05). In CD8⁺ T cells, no significant difference could be detected for virus reactivation. (C) Diversity dot plots of CD4⁺ and CD8⁺ T cells of donor T cells premobilization and postmobilization according to patients who either exhibited acute graft-versus-host disease (aGvHD; “Yes”) or not (“No”). No significant correlation could be detected between donor T cell diversity and aGvHD appearance in patients. CD4⁺ T cells preMOB; CD4⁺ T cells postMOB; CD8⁺ T cells preMOB; CD8⁺ T cells postMOB.