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Multiple measurements of lymphocyte subsets in 91 children treated with steroid-free Tacrolimus, and rabbit anti-human thymocyte globulin induction demonstrate early reconstitution of T-cytotoxic cells, and gradual reconstitution of all other subsets, which is complete after 1 year. Rejection-prone children demonstrate significantly higher counts of lymphocytes and all subsets prior to liver transplantation, and may exemplify one basis for enhanced baseline immunocompetence.
Induction immunosuppression regimens which eliminate steroids, and minimize calcineurin inhibitor usage are appealing for children who receive liver transplantation (LTx) (1-3). Potentially, steroid-induced impairment of growth and development, and the high incidence of post-transplant lymphoproliferative disorder (PTLD) associated with calcineurin inhibitors can be minimized (4, 5). Wider adoption is limited by concerns about the long-term effects of induction with lymphocyte depleting antibodies, on lymphocyte reconstitution in an age-group characterized by a maturing immune system. Among the published reports describing outcomes after anti-thymocyte globulin in pediatric recipients of solid organ allografts (6,9-15), only two single-center reports document serial changes in lymphocyte subsets in pediatric kidney recipients (6,9). No data is available from pediatric LTx.
Since 2001, over 100 children have received steroid-free Tacrolimus, after induction with a total dose of 5 mg/kg rabbit anti-human thymocyte globulin (rATG, Genzyme, Cambridge, MA.). rATG was preceded by premedications in order to minimize cytokine release syndrome. These premedications were diphenhydramine 1 mg/kg, acetaminophen 10 mg/kg, and methylprednisolone 1 mg/kg. Target Tacrolimus whole blood concentrations were maintained at 12-15 ng/ml in the first month, and brought down to 8-10 ng/ml after the third month. By the end of month 12, target levels are 5-7 ng/ml. If acute cellular rejection occurs, these targets are delayed by 3-6 months. Multiple measurements (Total n=210, 2.3 time points per patient) of lymphocyte counts, as well as their CD3+ (T-cell), CD3+CD4+ (T-helper cells), CD3+CD8+ (T-cytotoxic cells), CD19+ (B-cell) and CD16+/56+ (NK cell) subsets were recorded for 91 children after approval by the University of Pittsburgh Institutional Review Board. Subjects were grouped as Rejectors (n=47) or Non-rejectors (n=44) based on the presence or absence of biopsy-proven acute cellular rejection during the first 60 days after LTx. Whole blood samples 0.1 milliliter each, were obtained with clinically indicated complete blood count (CBC) with differential, prior to LTx, and post-LTx at 1 month, between 31-100 days, 101-365 days, 12-24 months, and 24-30 months. Subset frequencies were measured by the IMK kit (BD Biosciences, San Jose, CA.), and multiplied by the total lymphocyte count to determine absolute count per cubic milliliter for each subset. For the pre-LTx time-point, Rejectors and Non-Rejectors were compared using two-tailed t-tests for two samples assuming unequal variance for all 91 children. To estimate longitudinal changes, pair-wise comparisons were performed using Pre-LTx subset data from corresponding children as reference.
Rejectors were similar to Non-rejectors with respect to median (range) age [6.4 (0.4 to 21.7) vs. 5.4 (0.1 to 18.4) years, p=NS], gender (male: female= 20:27 vs. 21:23, p=NS) and race (Caucasian: African-American: Others= 40:5:2 vs. 42:1:1, p=NS). Steroids were not used initially in either group. Steroids were initiated to treat rejection, and weaned gradually thereafter among Rejectors. Also, tacrolimus doses were increased to achieve targets similar to the first post-LTx month during episodes of acute cellular rejection. Time to first rejection was (median±SEM) 30±4.4 days (mean=36.1 days). Therefore, whole blood concentrations of tacrolimus (Table 1) were higher among Rejectors, when compared with Non-Rejectors, during 31 to 100 days after LTx (p=0.01). Also, during this time period, steroids were initiated for the first time, to treat rejection episodes in all Rejectors. Rejection episodes were graded as mild (n=18), moderate (n=6) and severe (n=0); 23 rejectors were not graded. Rejection episodes were also classified as steroid-sensitive (n=37) and steroid-resistant (n=10). Steroid-resistant rejection episodes were treated with anti-lymphocyte therapy [rATG (n= 5), OKT3 (n= 3) and both (n= 2)]. There were no significant differences among the Rejectors and Non-rejectors with regard to use of other drugs (tacrolimus, ganciclovir, acyclovir, TMP-SMX, amoxicillin and intravenous immunoglobulin; p= NS), use of cadaveric vs. living graft (42:5 vs. 35:9, P=NS) and primary diagnoses leading to liver failure (table 4). There was no ABO incompatibility and there were no infection ongoing at the time of subset monitoring. Patient and graft survival is 89/91 (97.8%) and 88/91, (96.7%), respectively. Two deaths occurred among 91 children, the first due to recurrent hepatocellular carcinoma and the second due to recurrent disseminated hepatoblastoma. In a third child, re-transplantation was needed for thrombotic graft loss due to a hypercoagulable state. The incidence of EBV-PTLD in this series was 3/91 (3.3%). Median (±SEM) follow up was 200±35 days (R vs NR= 240±51 vs 174±46).
With the exception of CD16+/56+ cells, Pre-LTx data for lymphocytes (lymp) and their subsets (Table 2) show significantly greater absolute counts. among Rejectors, when compared with Non-Rejectors. The CD4+/CD8+ ratio was similar between groups.
In paired comparisons, total lymphocytes and all subsets (Table 3 and Figure 1) decreased significantly below pre-LTx levels within the first month after LTx. At 31-100 days, which corresponded to the period of risk for rejection, all counts appeared to reconstitute to pre-LTx levels, while CD8+ counts increased significantly above pre-LTx level (p=0.024). This increase in CD8+ cells was significantly higher among Rejectors when compared with the Non-Rejectors (Fig.1a). CD4+/CD8+ ratio was reversed during this period for both the outcome groups. CD 19+ cells were also significantly higher (Fig.1a) during this period among Rejectors (p=0.011). Thereafter, CD8+ cell counts declined, but were similar to pre-LTx values throughout the follow-up period. For the total lymphocyte count as well as the remaining subsets, reconstitution to pre-LTx values was sustained after the 1st post-LTx year.
An intact immune system is vital for the normal development of anti-infectious and anti-tumor immunity in children. Therefore, after polyclonal depletion, reconstitution of T-, B-, and NK-cells to pre-LTx ranges, which begins within the first three months, but is sustained after the first post-LTx year, is reassuring. In this regard, the two deaths among children transplanted for malignancies have led us to omit induction agents in such patients. The incidence of EBV-PTLD in this series was 3/91 (3.3%) and comparable with the 2-10% incidences reported from our center previously (5). All children with PTLD had experienced early rejection, and had thus been exposed to higher amounts of immunosuppressive drugs early after LTx.
Our observations are noteworthy for the fact that children at risk for rejection had greater numbers of lymphocytes and all subsets, prior to LTx. Although age is known to influence lymphocyte counts in children, the Rejector and Non-rejector groups did not differ in mean age or in gender distribution, thereby making it less likely that our observed differences could be explained by age alone. Also, T-cytotoxic cell counts reconstituted to levels greater than pre-LTx values. Further, this increase in T-cytotoxic cell counts assumed statistical significance when Rejectors were compared with Non-Rejectors, during the period of greatest risk for rejection (31-100 days). These observations suggest that the rejection-prone child may express increased baseline immunocompetence in various ways. Support for this inference is also evident in our prior functional evaluation of sub cohorts of our study population. In these prior studies, children who experienced early rejection demonstrated enhanced resistance of T-cytotoxic cells and B-cells to immunosuppression (n=25) (7), and increased and persistent (n=36) donor-specific alloreactivity in mixed lymphocyte co cultures (8).
The impact of a relatively slower reconstitution of CD4+ cells, which manifests as a reversed CD4/CD8 ratio during the first 2 years after LTx is unknown. Because no opportunistic infections occurred after the first year, it is possible that any effect of the relatively lower CD4 counts was neutralized by earlier reconstitution of CD8 cells.
Our conclusions remain limited by the absence of longitudinal data on donor specific cellular and humoral alloreactivity in all 91 subjects. However, the numbers of children evaluated, demographic similarities between outcome groups, similarities with respect to use of drugs, and paired comparisons to evaluate time-dependent subset reconstitution strengthen our conclusions by minimizing potential confounders such as non-uniform observations from each patient, and age-dependent variation in normative subset data. At the very least, these observations suggest to us that enhanced baseline immunocompetence in the form of higher numbers of T- and B-cells represent another feature of predisposition to early rejection.
NIH-5RO1AI49156-05, Unrestricted educational grant-Genzyme, Cambridge, MA and Children's Hospital Research Advisory Committee, Pittsburgh, PA,
Talukdar A, AshokKumar C, Farrar J, Wilson P, Janakiramanan A, Tregaskes M, Sindhi R. Lymphocyte subset reconstitution in Pediatric liver recipients induced with steroid-free rabbit Anti-human Thymocyte Globulin.