Figure 1.
Clonal expansion of CD8+ lymphocytes temporally associated with immunization and graft dysfunction in a patient with AA. (A) Time course of platelet count showing decreasing platelet counts postimmunization and stabilization of thrombocytopenia after an increase in cyclosporine (CsA) dose. (B) Time course of absolute reticulocyte count, demonstrating a precipitous decline in the immediate postimmunization period, with an improvement in reticulocytopenia following an increase in cyclosporine dose. (C) Time course of CD4+ and CD8+ lymphocyte counts showing a selective expansion of CD8+ T lymphocytes postvaccination. (D) Time course of the percentage of donor chimerism in total PB and CD3+ T lymphocytes, showing poor mixed CD3 chimerism, with improved chimerism after treatment with donor lymphocyte infusion (DLI). (E) Frequency histograms of the 10 top productive TCRβ rearrangements, as determined by bulk NGS sequencing of the TCR Vβ gene in the patient’s bone marrow (BM) at diagnosis before stem cell transplantation (pre BMT), at day +104 posttransplant prior to vaccination (Vacc.), at day +250 posttransplant after vaccination, and in sorted PB CD8+ T lymphocytes at day +354 posttransplant after vaccination. The patient was subsequently treated with donor lymphocyte infusion (DLI), with improvement in the percentage of donor chimerism, as shown in panel D, and diminution of the top expanded clones, as shown in PB CD8+ lymphocytes on day +750 posttransplant. The pie charts illustrate the proportion of the top 2 expanded clones (red) relative to all of the remaining clones (blue). The total number of remaining productive TCR Vβ gene rearrangements identified by bulk NGS are listed (n). (F) The 2 dominant CD8+ T lymphocyte clonotypes, with their corresponding frequencies pre- and postvaccination. Frequencies in BM at diagnosis, at day +104 posttransplant prevaccination, at day +250 posttransplant after vaccination, and in sorted PB CD8+ T lymphocytes at day +354 posttransplant were obtained by bulk TCRβ sequencing. TCRα pairing was determined through 10× genomics single-cell immune profiling of sorted PB CD8+ T lymphocytes on day +354 posttransplant.

Clonal expansion of CD8+ lymphocytes temporally associated with immunization and graft dysfunction in a patient with AA. (A) Time course of platelet count showing decreasing platelet counts postimmunization and stabilization of thrombocytopenia after an increase in cyclosporine (CsA) dose. (B) Time course of absolute reticulocyte count, demonstrating a precipitous decline in the immediate postimmunization period, with an improvement in reticulocytopenia following an increase in cyclosporine dose. (C) Time course of CD4+ and CD8+ lymphocyte counts showing a selective expansion of CD8+ T lymphocytes postvaccination. (D) Time course of the percentage of donor chimerism in total PB and CD3+ T lymphocytes, showing poor mixed CD3 chimerism, with improved chimerism after treatment with donor lymphocyte infusion (DLI). (E) Frequency histograms of the 10 top productive TCRβ rearrangements, as determined by bulk NGS sequencing of the TCR Vβ gene in the patient’s bone marrow (BM) at diagnosis before stem cell transplantation (pre BMT), at day +104 posttransplant prior to vaccination (Vacc.), at day +250 posttransplant after vaccination, and in sorted PB CD8+ T lymphocytes at day +354 posttransplant after vaccination. The patient was subsequently treated with donor lymphocyte infusion (DLI), with improvement in the percentage of donor chimerism, as shown in panel D, and diminution of the top expanded clones, as shown in PB CD8+ lymphocytes on day +750 posttransplant. The pie charts illustrate the proportion of the top 2 expanded clones (red) relative to all of the remaining clones (blue). The total number of remaining productive TCR Vβ gene rearrangements identified by bulk NGS are listed (n). (F) The 2 dominant CD8+ T lymphocyte clonotypes, with their corresponding frequencies pre- and postvaccination. Frequencies in BM at diagnosis, at day +104 posttransplant prevaccination, at day +250 posttransplant after vaccination, and in sorted PB CD8+ T lymphocytes at day +354 posttransplant were obtained by bulk TCRβ sequencing. TCRα pairing was determined through 10× genomics single-cell immune profiling of sorted PB CD8+ T lymphocytes on day +354 posttransplant.

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