| Background and ObjectiveGenerally, hematopoietic stem cell transplantation (HSCT) is the most effectiveoption in the treatment of leukemia, lymphoma, severe aplastic anemia and otherhematopoietic diseases. However, only around30%patients could receiveHLA-identical transplantation due to the strict limit of HLA compatibility. Meanwhile,haplo-identical HSCT (Haplo-HSCT) is suitable for nearly almost all patients inconsideration of the immediate availability of donors.Intensive investigations have been clinically performed since Haplo-HSCT wasfirstly reported by Powles and his colleagues in1983. However, the initial clinicaltrials were not successful, and the majority of the patients died of graft rejection andsevere graft-versus-host disease (GVHD). In recent years, the protocols ofHaplo-HSCT have been improved greatly and the two-year disease free survival ratehas reached closely to that of HLA-identical HSCT. In the meantime, Haplo-HSCThas been developed and used in larege scale in China. Our group has been performedHaplo-HSCT in early1990s. An acceptable outcome has been achieved by theestablishment and application of regiments for preconditioning and aGVHDprophylaxis.Even though the great progress during recent years, immune reconstitution issereiously retarded in Haplo-HSCT. Sustained low cellular immunity contributes tothe occurrence of infections and relapse, which are the main causes of mortality afterHaplo-HSCT.T lymphocytes are not only the components of cellular immunity but also theregulators for the humoral immunity. Therefore, the reconstruction of T cells is thecritical part of immune reconstitution in HSCT. The recovery of amounts andfunctionality of T cells after Haplo-HSCT depends on two pathways. The firt one isnominated as peripheral reconstruction, which is thymus-independent and indicatesthe expansion process of T cells from the grafts in the peripheral lymphoid organs ofthe recipients. The second pathway is thymus-dependent, indicating the process of themigration of T lymphocyte progenitors from the donor into the thymus of the recipient, the development of these progenitors in the thymus environment, and the productionof na ve T lymphocytes with a variety of T cell receptors, resulting in the definiteimmunity. In Haplo-HSCT, T cell depletion is usually performed in vitro or in vivo(ATG) to prevent the occurrence of severe GVHD, the thymus-dependent pathwaymight predominate in the immune reconstitution.With the development of molecular and cellular immunity, some novel methodsto monitor the immune status have been emerged in recent years. The thymus outputfunctionality can be evaluated by detecting the frequency of T-cell receptor excisioncircles (TRECs) and diversity of T-cell receptor β chain variable regions (TCR-Vβ) ofthe periperhal T cells. ImmuKnow technique, a method to assess T cell function bydetecting ATP production of stimulated CD4~+T cells, has been widely used in thetransplantation of solid organs. Recently, the usage of ImmuKnow technique in HSCThas also been reported.Till now, few investigations have been reported about the patterns of T cellreconstitution and their association with clinical parameters in Haplo-HSCT. In thisstudy, thirty-one patients who had received Haplo-HSCT were enrolled.Preconditioning and GVHD prophylaxis were performed according to the regimensroutinely described in our previous reports. The parameters of the outcome includedhematopoietic recovery, the occurrence of aGVHD, pulmonary infections and relapsesix months after transplantation. The numbers of T cell subsets and sjTREC, thediversity of TCR-Vβ, and ATP amounts produced by stimulated CD4~+T cells wereinvestigated. The relationship between the clinical parameters and the laboratory datawere statistically analyzed. The data here might provide some instructive references inthe clinical settings of the reduction or withdrawal of immunosuppressive agents andthe application of donor lymphocyte infusion.Methods1. HLA typing was performed by sequence-specific primers PCR (PCR-SSP).2. The enrolled31patients received the regimens for preconditioning and aGVHDprophylaxis that are routinely used in our center.3. Surface and cytoplasmic antigens were detected by flow cytometry, after the cellswere reacted with FITC, PE, APC or PerCP-conjugated monoclonal antibodies. Theproportion of CD34~+cells in the grafts and the T lymphocyte subsets in theperipheral blood before and post tranplant were assessed and the absolute totalnumbers of the cells were calculated according to the counts of the leukocytes in the grafts and the peripheral blood. CD34-positivity was recognized as the markerof hematopoietic stem/progenitor cells, while positivity for CD4, CD45RA andCD62L or CD8, CD45RA, and CD62L was defined as the markers of na ve T cells.Regulatory T cells were those CD4~+/CD25~+/FOXP3~+cells.4. TREC evaluation. Peripheral blood was harvested and mononuclear cells wereisolated. Genomic DNA was extracted and quantitative PCR was performed todetect signal-joint TRECs with the primers reported previously.5. TCR-Vβ diversity analysis. The peripheral blood from six patients who hadexperienced aGVHD post-transplant was harvested and total cellular RNA wasextracted from the mononucleated cells. RT-RCR was performed to amplify thetargeted sequences of the24subfamilies of TCR-Vβ family, followed bygene-scanning to analyze the gene spectromes.6. The evaluation of T cell function. T cell function was assessed by ImmuKnowtechnique according to the manufacture’s instructions. CD4~+T cells weremagnetically isolated and stimulated by phytohem agglutinin (PHA). Theintracellular contents of ATP were detected18hours after incubation. Three levelsof immune status were defined as high-level (>525ng/mL), medium-level(226–524ng/mL), and low-level (<225ng/mL) respectively.7. Statistics. The data were expressed mean±standard error. Mann-Whitney test andspearman rank correlation analysis were used to evaluate the differences of twogroups and the relationships of laboratory and clinical parameters with the softwareof SPSS.18respectively. Survival curve was analyzed with log rank test. A P valueless than0.05was considered as significance.Results1. Haplo-HSCT was performed with the use of intravenous injection of ATG todeplete T cells in vivo. Complete hematopoietic engraftment occurred in27patients.The mean time for neutrophil recovery was17.16±4.75days (n=31) and that forplatelet recovery was30.59±18.05days (n=27). Failure of pletlet recovery occurredin3patients with high-risk and1patient with stand-risk leukemia and thesepatients died of early transplantation-associated complications. Acute GVHD(grade II-IV) occurred in thirteen patients (42%) within100days aftertransplantation and3of them were grade III-IV (9.7%). Early post-transplantation,infections occurred in all the patients and13cases were pulmonary infections(42%). Twelve patients died and seven died of relapse, two died of infections and2of aGVHD. 2. The CD3~+cell absolute number was710±286.5/μL before Haplo-HSCT,372.85±313.52/μL at1month,1071.36±664.30/μL at3months, and reached up to1393.32±857.89/μL at6months post-transplant. The CD4~+cell absolute valuewas277.24±124.49/μL before HSCT,57.67±49.11/μL at1month,178.04±148.93/μL at3months, and210.19±130.74/μL at6months. The CD8~+cell absolute value was396.36±210.23/μL before Haplo-HSCT,258.87±229.65/μL at1month,701.73±492.82/μL at3months, and reached to879.11±566.40/μLat6months. The CD4~+CD45RO~+memory T cell absolute value was203.61±101.44/μL before transplant,46.56±42.08/μL at1month,171.67±141.64/μL at3months,176.73±104.53/μL at6months. The CD8~+CD45RO~+memory Tcell absolute value was165.94±102.67/μL/μl before Haplo-HSCT,177.10±167.71/μL at1month,523.23±415.34/μL at3months,437.63±410.09/μLat6months. CD4~+CD45RA~+CD62L~+na ve T cell number was55.261±44.17/μLbefore Haplo-HSCT,1.95±2.72/μL at1month,4.69±5.62/μL at3months,23.08±37.04/μL at6months post transplantation. The CD8~+CD45RA~+CD62L~+na ve T cell absolute value was135.74±42.31/μL before Haplo-HSCT, decreasedto15.95±16.90/μL at1month,70.57±47.61/μL，at3months, and reachedto176.09±222.18/μL at6months.3. The absolute numbers of CD4-and CD8-positive cells at any time points of theobservation were lower in the peripheral blood from patients with aGVHD thanthose of patients without aGVHD, but the difference did not reach significant. Thenumbers of memory T and na ve T cells one month post-transplant weresignificantly higher than those of patients without aGVHD (P=0.0097). Meanwhile,the differences were not significant at any other observed time points. The numbersof Treg cells between patients with and without GVHD were not different (P>0.05).4. The sjTREC frequency was78.72149±135.0016copies/10~5PBMC in22patientsbefore Haplo-HSCT, which was comparable with that from8donors(699.4644±1221.6469copies/10~5PBMC, P<0.05). One month post-transplant,sjTREC of the peripheral blood from the recipients decreased to20.0129±26.700copies/10~5PBMC, and those were25.4031±29.7042copoies/10~5PBMC2monthslater and21.5883±17.4021copies/10~5PBMC5months later. The sjTREC numbersbefore transplantation were not correlated to those post-transplantation; thecorrelation indexes were0.278,0.467and0.714for1month (P=0.279),3months(P=0.205), and6months (P=0.111), respectively. Furthermore, there was no difference between patients with and without aGVHD (P>0.05for all observedtime-points).5. TCR-Vβ analysis in one healthy donor showed that all the24subfamilies existedand the peaks in the gene scanning paragraphs corresponded to the Gaussiandistribution. Meanwhile, loss of some of the subfamilies was observed in6patientsbefore transplantation, and double-peaks and oligoclonal peaks existed in some ofthe subfamilies. The frequency of oligoclonal peaks increased graduallypost-transplantation.6. The ATP value in CD4~+T cell decreased to210.19±119.37ng/mL at1month postHaplo-HSCT and increased to280.62±110.03ng/mL by3months,and357.28±76.18ng/mL by6months. The ATP contents prior to transplantationwere significantly lower in patients who had experienced pulmonary infectionspost-transplant than those who had not suffered from pulmonary infections. Thelower levels were sustained in patients with pulmonary infections. The ATPcontents prior to and post transplantation were significantly higher in patients withaGVHD than those without aGVHD. Further, the ATP levels were lower in patientswho relapsed than those without leukemia relapse at any observed time points.Conclusions1. The outcome of Haplo-HSCT is generally acceptable. Hematopoietic recovery israpid and the occurrence of aGVHD is comparable to that reported by other groups.Relapse and infection are the main causes of death.2. In Haplo-HSCT, the recovery of CD8-positive T cells occurs earlier than that ofCD4-positive cells. The recovery might mainly be attributed to the expansion ofmemory T cells other than the emergence of na ve T cells. The number of Treg cellsdecreases greatly one month post-transplantation, and reaches up to50%of thenormal range6months post-transplant.3. The thymus output dysfunction occurs before Haplo-HSCT and has not recovered6months post transplantation.4. The loss of some of the TCR-Vβ subfamilies and the existence of oligoclonal peakssuggest that the immune status of the patients before transplantation is abnormal. Insix patients with aGVHD, these abnormalities were sustained for at least threemonths, indicating the reconstitution of na ve T lymphocyte is not cccured or the aGVHD and the usage of immunosuppressive agents delayed the immuneconstruction.5. T cell function restores three months after Haplo-HSCT, though it is dramaticallydecreased at one month. T cell functionality is strongly associated with theoccurrences of pulmonary infections, leukemia relapse and aGVHD. The resultsindicate that ImmuKnow could be used clinically to predict the main complicationsin Haplo-HSCT. |
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