Cord blood (CB) contains immature immune cells and is thought to be less active in inducing allogeneic immune reaction than other sources of stem cells. However, a high incidence of immune-mediated complications has been reported, such as pre-engraftment immune reaction (PIR) and haemophagocytic syndrome (HPS) early after cord blood transplantation (CBT) (Kishi et al, 2005; Narimatsu et al, 2007; Frangoul et al, 2009; Takagi et al, 2009; Patel et al, 2010). In addition, we reported that human leucocyte antigen (HLA) disparity in the graft-versus-host (GVH) direction adversely affected engraftment kinetics when single calcineurin inhibitors were used for GVH disease (GVHD) prophylaxis (Matsuno et al, 2009). These observations suggested that the GVH reaction plays a critical role in engraftment. Here, we report the engraftment kinetics of donor-derived T cells using a multicolour flow cytometry-based method (HLA-Flow method) (Watanabe et al, 2008) and also describe the results of naïve/memory T-cell phenotype analyses early after CBT.
Between November 2009 and September 2010, 73 adult patients underwent single-unit CBT at Toranomon hospital. This study reports 41 patients who were eligible for chimerism analysis using the HLA-Flow method and survived more than 14 d after CBT. Characteristics of the patients and CB are summarized in Table SI. All patients provided written informed consent, and the study was conducted in accordance with institutional review board requirements. Peripheral blood was collected at 1, 2, 3, 4, and 8 weeks after CBT. Anti-HLA monoclonal antibodies in combination with lineage-specific antibodies were used to analyse the lineage-specific chimerism as previously reported (Watanabe et al, 2008). Anti-HLA antibodies specific for donor and recipient HLA in all patients are summarized in Table SII. At 2, 4, and 8 weeks after CBT, T-cell subsets were analysed using the following monoclonal antibodies: peridinin-chlorophyll-protein – cyanin 5·5 (PerCP-Cy5·5)-CD8, phycoerythrin – cyanin 7 (PE-Cy7)-CCR7, allophycocyanin (APC)-CD4, APC-Cy7-CD3 (BD Pharmingen, San Jose, CA, USA), and Pacific Blue-CD45RA (CALTAG, Carlsbad, CA, USA). Absolute numbers of CD4+ T cells (CD3+CD4+), CD8+ T cells (CD3+CD8+), and naïve (CD45RA+CCR7+) and memory (CD45RA−CCR7+/−) T cells were calculated by multiplying the peripheral lymphocyte counts by the percentage of positive cells. PIR was characterized by non-infectious high-grade fever (>38·5°C) coexisting with skin eruption, diarrhoea, jaundice and/or body weight gain greater than 5% of baseline, developing 6 or more days before engraftment (Kishi et al, 2005; Uchida et al, 2011). Cumulative incidence of neutrophil engraftment, PIR, and GVHD were calculated using Gray's method. Intergroup comparisons were performed using the Mann–Whitney U-test.
We analysed lineage-specific chimerism for 32, 40, 40, 34, and 34 patients at a median of 8 (range, 7–11; week 1), 15 (14–20; week 2), 22 (21–25; week 3), 29 (28–36; week 4), and 57 (56–62; week 8) days post-transplant, respectively. Fig 1A shows representative results for CD4+ T-cell chimerism. CD4+ and CD8+ T-cell chimerism results in all patients are shown in Fig 1B. Of 41 enrolled patients, 37 achieved neutrophil engraftment at a median of 19 d (range, 13–38 d). Thirty-nine patients achieved donor-dominant T-cell chimerism (>90%) by 3 weeks after CBT, whereas the remaining two patients, with recipient-dominant T-cell chimerism (>90%) at every point tested, developed graft failure because of early relapse (day 14 post-transplant) and rejection, respectively. Among the 39 patients who achieved donor-dominant T-cell chimerism, two died before engraftment due to non-relapse causes on day 28 (infection) and day 25 (diffuse alveolar haemorrhage), respectively. Among those with donor-dominant chimerism, 24 (63%) of 38 evaluable patients developed PIR at a median of 8 (6–11) days after CBT. Patients who achieved donor-dominant T-cell chimerism (>90%) at 1 week had a higher incidence of PIR compared to those who did not (P = 0·017, Fig 1C). In a representative patient at 2 weeks after CBT, rapid conversion from naïve to memory phenotype was observed in both CD4+ and CD8+ T cells (Fig 2A). Fig 2B shows the relative proportion of naïve CD4+ and CD8+ T cells at 2, 4, and 8 weeks after CBT in 37 evaluable patients who achieved donor-dominant T-cell chimerism. Patients who developed PIR had significantly more lymphocytes, CD4+ T cells, CD8+ T cells, CD4+ memory T cells, and CD8+ memory T cells at 2 weeks after CBT compared with those without PIR (Fig 2C and data not shown).
Our data confirmed that a majority of patients achieved donor-dominant T-cell chimerism around 2 weeks after CBT. We also found that early recipient-type T-cell chimerism was closely associated with graft rejection. A remarkable finding was that a rapid recipient-to donor-dominant switch of T-cell chimerism at 1 week post-transplant was associated with a higher incidence of PIR, supporting a hypothesis that PIR could be an early variant form of GVH reaction caused by donor-derived T cells. CB T cells are naïve and do not include pathogen-specific effector T cells. Grindebacke et al (2009) demonstrated that about 80% of CD4+ T cells kept the naïve phenotype during the first 18 months after birth. In contrast, we found a rapid conversion from naïve to memory phenotype at 2 weeks after CBT. In addition, PIR could be associated with peripheral expansion of donor-derived memory T cells. Recently, Gutman et al (2010) reported that CD8+ T cells predominately expressed effector memory or effector phenotype early after double-unit CBT, reflecting an immune response of the dominant unit against the non-engrafting unit. These findings suggest that donor-derived naïve T cells will be activated by alloantigens and differentiate into mature cells early after CBT. Most of the present patients with PIR responded promptly after a short course of steroid treatment, and none experienced graft failure due to HPS. This observation could be attributed to more intensive immunosuppression from adding mycophenolate mofetil to tacrolimus in the majority of patients (Uchida et al, 2011). Although neither the T-cell chimerism nor the memory T-cell counts affected the incidence of acute GVHD, steroid treatment for PIR could suppress the onset of acute GVHD. In conclusion, rapid T-cell chimerism switch and donor-derived memory T-cell expansion were associated with PIR, supporting a significant role of donor-derived T cells in the pathogenesis of the early immune reaction after CBT.