Immune reconstitution following umbilical cord blood transplantation: IRES, a study of UK paediatric patients

Abstract

To obtain a qualitative as well as quantitative view immune reconstitution following umbilical cord blood (UCB) transplantation of paediatric patients, we utilised a broad panel of flow cytometry markers to monitor the phenotypes of lymphoid and myeloid cells at 1-12 months post-transplant. Samples were received from 46 patients with a median age of 3.3 years and survival was 76% at 1 year. Monocytes were at similar or higher median levels than in adult controls at all times tested, with a high CD16+ proportion in the first 3 months. NK cells were also within adult ranges, with a CD56++ high proportion in the first 6 months. B cell recovery was seen from 2 months in most patients and T cells from 3 months, both were delayed with anti-thymocyte globulin (ATG) treatment. CD4:CD8 ratios were high in the first 6 months, and the proportion of T cells with recent thymic emigrant and naïve phenotypes rose from 3 months. NK and plasmacytoid dendritic cell numbers remained at reduced levels in patients not surviving to 1 year. Our results can serve as a useful reference for detailed monitoring of immune reconstitution in paediatric recipients of UCB.

Keywords: cord blood; immunophenotyping; stem cell transplantation.

FIGURE 1

A, Overall survival of paediatric cohort. MD solid line (73%: 24 alive at one year out of 33 patients), non‐MD dotted line (85%: 11/13). B, Relapse‐free survival of patients treated for MD. C, Neutrophil engraftment (93%: 43/46), median 20.5 days

FIGURE 2

Absolute cell counts for the main white blood cell populations. Flow cytometry was performed on whole blood with counting beads to determine the number of cells /mL for (A) monocytes (CD14+), (B) granulocytes (FSC/SSC), (C) T cells (CD3+), (D) B cells (CD19+), (E) NK cells (CD56++ and CD56+/16+), (F) NKT cells (CD3+ CD56+), (G) Myeloid dendritic cells (HLA‐DR+ CD19‐ CD1c+ CD11c+), and (H) plasmacytoid dendritic cells (PDC: HLA‐DR+ CD123+ CD304+). The Y‐axes are 10⁶ cells/mL for A‐E, 10⁴ cells/mL for F, and 10³ cells/mL for G‐H and the X‐axes are months after transplant. The ‘>18’ group includes eight patients sampled at 18‐36 months. Values for umbilical cord blood (C) and adult peripheral blood (A) controls are provided for reference. The FACS files for outlying samples were reanalysed for confirmation. Horizontal bars represent means with standard error. Key: The boxes below the plots represent t‐test comparisons against the values for UCB (upper row) and AB (bottom row): white P > 0.5; grey P ≤ .05; black P ≤ .005. UCB and AB differed significantly (P < .05) for cell types in (A‐E). Further statistical information in Table S1

FIGURE 3

ATG use. (A) T cell and (B) B cell numbers (10⁶ /mL) at 1‐12 months post‐transplant according to ATG use. Percentage of patients grades of aGvHD (C) and development of cGvHD (D) by ATG use. Asterisks indicate P < .05. Further statistical information for A and B in Table S2

FIGURE 4

T Cell stages. RTE analysis of (A) CD3+ CD4+, (B) CD3+ CD4‐ cells: Relative proportions of CD45RA+ CD31+ (RTE, black); +/– (white), –/+ (grey), –/– (stripes). Maturation state analysis of (C) CD3+ CD4, (D) CD3+ CD8 cells: CD45RA+ CCR7+ (naïve, black), +/– (effector memory RA+, white), –/+ (central memory, grey), –/– (effector memory, stripes). ‘C’ cord blood and ‘A’ adult blood, horizontal axis numbers indicate months after transplant. The boxes below the plots represent t‐test comparisons against UCB and AB (as in Figure 2) for black bar sub‐populations: RTE (CD45RA+ CD31+) and naïve cells (CD45RA+ CCR7+). The number (n) of samples analysed for each set of markers is indicated above the bars in Figure 4A

FIGURE 5

Functional T cell markers. Proportion of (A) CD4 and (B) CD8 cells expressing co‐stimulatory factors CD27 and CD28: CD27+CD28+ (black), +/– (white), –/+ (grey), –/– (stripes). The number (n) of samples analysed for each set of markers is indicated above the bars in Figure 5A. Percentage of (C) CD3+CD4+ and (D) CD3+CD4‐cells expressing CD161. (E) Total CD3 population expressing CD57. (F) T regulatory cells (CD3+4+25+127low). ‘C’ cord blood and ‘A’ adult blood; horizontal axis numbers indicate months after transplant. The boxes below the plots represent t‐tests against UCB (upper) and AB (lower), for A and B, the comparisons are for CD27+CD28+ (black bars). Further statistical information for (C‐F) in Table S3

FIGURE 6

B Lymphocytes. The percentages of CD19+ B cells that have a (A) TrB (CD24hiCD38hi) or (B) memory (CD27+) phenotype. Further statistical information in Table S4

FIGURE 7

Innate immune cells. A, CD16+ monocytes as % of total CD14+ monocytes. B, CD56++ proportion of total NK cells. C, CD62L+ NK cells as a proportion of CD56+CD16+ cells. D, CD57+ NK cells as a proportion of CD56+CD16+ cells. Further statistical information in Table S5

FIGURE 8

Age‐related differences in lymphocyte sub‐populations. A, CD3+ CD4– CD45RA+ 31+ cells as a proportion of CD3+ 4– by median age (– below, + above median). B, CD4:CD8 ratio. (C) TrB by median age as a proportion of total CD19+ lymphocytes. (*P < .05; **P < .005). Further statistical information in Table S6