DISCUSSION
This study analyzed lymphocyte recovery after allo-HSCT in pediatric patients with malignant and non-malignant diseases up to 180 days after HSCT. Over this 6-month period, lymphocyte reconstitution was gradual, and each subtype showed different reconstitution rates on D+100. Our study reiterates that lymphocyte count after HSCT impacts patient survival and is associated with pre-transplant factors such as age, source of stem cells, donor, serotherapy, conditioning, disease, and clinical outcomes like CMV and aGvHD. 3,6–8
On D+100, 89% of patients achieved ALC recovery while CD3+CD8+ and CD16+CD56+ lymphocytes showed the highest recovery rates (68% and 47%, respectively). As natural killer (NK) lymphocytes and TCD8+ clonal expansion propel lymphocyte reconstitution in the first 3 months after HSCT, ALC recovery in our cohort occurred possibly due to these subtypes. 3,17 Kim et al (2013) also obtained this pattern of reconstitution in children and similar median counts for the same period, except for the higher percentage of NK recovered patients owing to a lower cut-off value based on the age-matched reference values. 18
In this study, patients <8 years showed better recovery for both ALC on D+30 and CD3+CD4+ on D+100. This finding contrast with those of Bartelink et al (2013), which indicated no association between IR and age in a cohort that also included umbilical cord blood transplants.19 Previous studies have showed an earlier recovery trend in younger patients, through rapid recovery of CD3+CD4+, CD19+ and ALC in patients <5 years and CD3+CD8+, CD19+ and CD16+CD56+ in patients <10 years. 7,18 A consensus on this matter is hampered because published studies have used distinct lymphocyte recovery criteria according to stem cell sources and age-matched reference values. Our results are inconclusive due to the small number of patients. However, there is evidence that children rapidly recover naïve TCD4+ compared with adults, supporting the association between age and greater pre-HSCT thymic activity as an influencing factor in IR. 20
The potential deleterious effect on thymus caused by MAC regimen was attenuated owing to higher ALC recovery on D+30 when compared with that in the RIC regimen. In children, this impact might be reduced because the thymus is fully functional until puberty, after which thymopoiesis progressively diminishes. 21 Although MAC is related to a better CD19+ recovery compared with RIC regimen in adults, our study showed no association between the type of conditioning regimen and lymphocyte subtype recovery. 22 Patient and transplant heterogeneity led to a variety of preparatory regimens, masking possible correlations between IR and the intensity of these regimens. Moreover, individual conditions (age and weight) may have also altered pharmacokinetics and pharmacodynamics of regimens leading to different exposures and effects on IR. 22,23
Patients who received BM grafts achieved higher ALC recovery on D+30. Although PBSC mobilized by granulocyte-colony stimulating factor contain more cells contributing to lymphocyte count (CD34+, T lymphocytes and NK) and promote CD3+CD4+ rapid recovery, 93% of the PBSC recipients in this cohort were haplo-HSCT. 24–26 Therefore, donor type may have an impact on early lymphocyte recovery, regardless of the stem cell source.
We observed that serotherapy interfered with ALC recovery on D+180. The same was noted in a pediatric study that indicated inadequate ALC recovery up to 1 year after HSCT. 18 In our cohort, lymphocyte subtypes were not associated with serotherapy. In a large pediatric cohort of malignant and non-malignant diseases, Admiraal et al (2015) reported that only high concentrations of ATG after BM or PBSC transplant reduced TCD4+ reconstitution, whereas in umbilical cord blood transplants even low concentrations of ATG exerted a deleterious effect. Therefore, CD3+CD4+ and serotherapy revealed no association in our study possibly because of the inclusion of only BM and PBSC transplants, the reduced number of patients who used serotherapy, and the inter-patient variability of doses and periods of ATG administration.27
Viral infections and lymphocyte reconstitution have a complex correlation as opportunistic infections are related to both the cause and the effect of delayed IR. 28,29 Patients who recovered ALC and CD3+CD8+ on D+100 had a higher CI of CMV infection, which occurred on a median 37 days after transplant in our patients. Literature shows that impairment of CD3+CD8+ early reconstitution contributes to CMV reactivation, while high counts of CD3+CD4+ and CD16+CD56+ represent protective factors in adults.30,31 But after infection, a clonal expansion of TCD8+ lymphocytes occurs in response to the CMV antigenic stimulus, resulting in an oligoclonal repertoire of memory T cells. 32 The high CD3+CD8+ count observed in patients with CMV infection, described also by Janecsko et al (2016), possibly denotes its specific CMV expansion, which could be confirmed in a larger cohort using molecular tests. 30
The development of aGvHD ≥grade 2 resulted in lower CD3+CD4+ reconstitution on D+100. Previous reports showed that patients with GvHD had low counts of CD3+CD4+ between the 1st and 3rd months after HSCT, especially naïve TCD4+ cells.33,34 The intense immunodepression is due to the damage that the disease causes in thymic function and BM microenvironment – essential factors for lymphopoiesis – but can be aggravated by GvHD treatment with corticosteroids.35 Considering the early development of aGvHD in this cohort (median day +28), the poor CD3+CD4+ recovery on D+100 may reflect the disease itself and its treatment.
In our study, haploidentical transplants had a higher CI of CMV infection and aGvHD but OS was similar to other types of transplants. The inadequate ALC recovery on D+30 and the higher CMV infection risk in haplo-HSCT may have resulted from the immunosuppressive effect of GvHD prophylaxis with PT-Cy, CsA and MMF 36–38 We also observed that CD19+ counts on D+100 were lower in haplo-HSCT than in MSD and unrelated transplants. Although major outcomes after haplo-HSCT are comparable to those of MSD and matched unrelated donors, more studies are needed to better understand IR in the haplo-HSCT settings, especially for pediatric patients with non-malignant diseases39.
In accordance with a previous study, our data suggest that ALC≥500/µL on D+100 is a good predictor of OS for pediatric patients undergoing allo-HSCT. 12 Several cell subsets play a crucial role in protecting from infections, however sophisticated exams are not readily available in resource-limited countries. These countries need simple biomarkers to demonstrate if transplanted patients have achieved an acceptable T cell reconstitution. In our study, we observed a trend towards inadequate CD3+CD4+ recovery on D+100 in patients dying from severe infections, but the reduced number of patients was insufficient to reveal a significant effect on OS. However, there is robust evidence in the literature suggesting that this is the most relevant parameter for survival as it is related to mortality from infections.33,40
Our retrospective study has important limitations related to the irregularity in immune surveillance by flow cytometry after HSCT. Moreover, the relatively small number of patients and disease heterogeneity did not allow us to reach any definitive conclusions. Nevertheless, these real-world data can be extrapolated to other pediatric HSCT centers and also form the basis for future collaborative studies.
This study exposes the reality in developing countries, ranging from limited financial resources for laboratory tests, to the failure to follow-up due to long travel distances to the reference center. Concurrently, as developed countries move towards predictive medicine in IR monitoring, through greater regularity and specificity of tests, herein we still endeavor to implement cost-effective strategies that may help our patients. To better estimate the time to reach immunocompetence after allo-HSCT in resource-limited countries, we need to standardize IR monitoring with the available tests. Although only studies compiling all data in a prospective way will be able to address this question, monitoring ALC counts on a monthly basis after transplant and measuring lymphocyte subtypes on D+100 and D+180 are easy to perform and could be implemented in clinical practice to identify patients at risk for inadequate IR.
In conclusion, this study summarized the results of IR after HSCT for malignant and non-malignant diseases in one of the main pediatric transplant centers in Latin America. Lymphocyte reconstitution was progressive up to D+180, mainly due to CD3+CD8+ counts. Overall, younger age, BM grafts, MAC regimen, non-malignant disease, non-use of serotherapy, and non-haploidentical donor were associated to adequate lymphocyte recovery. Multiple factors influence IR and our study suggests that ALC≥500/µL on D+100 may be a predictor of pediatric survival after HSCT, preferably concomitant with lymphocyte subpopulations monitoring.