Lymphocyte reconstitution following allogeneic hematopoietic stem cell transplantation: a retrospective study including 148 patients

Here we investigated the influence of parameters known before hematopoietic stem cell transplantation (HSCT) as well as the relevance of graft-versus-host disease (GvHD) and cytomegalovirus (CMV) reactivation on post transplant lymphocyte reconstitution in 148 patients treated in our institution between 1996 and 2003. Median patient age was 42 (19-68) years, HSCT followed standard high dose (n=91) or reduced-intensity conditioning regimens (n=57) with bone marrow (BM, n=67) or peripheral blood stem cells (PBSC, n=81) from related (n=71) or unrelated (n=77) donors. In the first months, we observed a partially faster reconstitution of CD3+4+, CD3+8+ and CD4+45RA+ T cells in patients following peripheral blood stem cell transplantation when compared to bone marrow transplantation. Prolonged CD3+4+ and CD4+45RA+ lymphopenia was noted after unrelated donor HSCT and GvHD prophylaxis containing anti-T-lymphocyte globulin. Lymphocyte subset counts in patients older than the median age were comparable to those in patients transplanted at a younger age and not influenced by the conditioning regimen. CD3+8+ T cell reconstitution was strongly correlated with CMV reactivation, but not significantly affected by CMV serostatus before HSCT. Incidence or extent of GvHD did not significantly influence lymphocyte reconstitution. Therefore, the source of graft is the most predictive parameter in early lymphocyte reconstitution, but the differences in lymphocyte recovery completely resolved within the first year after HSCT.     

Immune Reconstitution and Early Infectious Complications Following Nonmyeloablative Hematopoietic Stem Cell Transplantation

Non-myeloablative stem cell transplantation (NMT) has been increasingly used in compromised patients who would otherwise have been unable to undergo allotransplant. There is little understanding of the kinetics of immune reconstitution and its influence on infective complications following NMT.

The aim of present study was to evaluate lymphocyte subset reconstitution over the first 12 months post-transplant in 15 adult patients receiving NMT with comparison to that of 30 patients grafted with a conventional hemopoietic stem cell transplantation (HSCT). NMT recipients were conditioned with fludarabine-based conditioning regimens. Peripheral blood stem cell (PBSC) was the source of stem cells in 13 NMT recipients and in 24 conventional HSCT recipients.

Absolute numbers of helper (CD4+) T cells, naive (CD4+ CD45RA+) and memory (CD4+ CD45RO+) T cells as well as suppressor (CD8+) T cells, CD19+ B cells and NK cells were comparable in the two groups at all time points after transplantation. A median value of 200 CD4+ T cells/μl was achieved at 2 months post-transplant by the NMT and HSCT recipients. The CD4:CD8 ratio remained severely depressed throughout the study period. Almost all CD4+ lymphocytes expressed CD45RO antigen in the both groups of patients B lymphocytes showed low counts throughout the entire study period in both groups.

Bacteremia and CMV antigenemia occurred respectively in 13 and 36% of the patients in the NMT group and in 15 and 39% of the patients in the HSCT group. Our preliminary data indicate that patients receiving a NMT have a lymphocyte reconstitution similar to that observed in patients who received a conventional HSCT. The incidence of bacteremia and CMV infection were not significantly different between the groups. Nevertheless, due to the small sample size, these results should be considered suggestive rather than definitive.     

Immune reconstitution and early infectious complications following nonmyeloablative hematopoietic stem cell transplantation

Non-myeloablative stem cell transplantation (NMT) has been increasingly used in compromised patients who would otherwise have been unable to undergo allotransplant. There is little understanding of the kinetics of immune reconstitution and its influence on infective complications following NMT. The aim of present study was to evaluate lymphocyte subset reconstitution over the first 12 months post-transplant in 15 adult patients receiving NMT with comparison to that of 30 patients grafted with a conventional hemopoietic stem cell transplantation (HSCT). NMT recipients were conditioned with fludarabine-based conditioning regimens. Peripheral blood stem cell (PBSC) was the source of stem cells in 13 NMT recipients and in 24 conventional HSCT recipients. Absolute numbers of helper (CD4+) T cells, naive (CD4+ CD45RA+) and memory (CD4+ CD45RO+) T cells as well as suppressor (CD8+) T cells, CD19+ B cells and NK cells were comparable in the two groups at all time points after transplantation. A median value of 200 CD4+ T cells/microl was achieved at 2 months post-transplant by the NMT and HSCT recipients. The CD4:CD8 ratio remained severely depressed throughout the study period. Almost all CD4+ lymphocytes expressed CD45RO antigen in the both groups of patients B lymphocytes showed low counts throughout the entire study period in both groups. Bacteremia and CMV antigenemia occurred respectively in 13 and 36% of the patients in the NMT group and in 15 and 39% of the patients in the HSCT group. Our preliminary data indicate that patients receiving a NMT have a lymphocyte reconstitution similar to that observed in patients who received a conventional HSCT. The incidence of bacteremia and CMV infection were not significantly different between the groups. Nevertheless, due to the small sample size, these results should be considered suggestive rather than definitive.     

Immune reconstitution after purified autologous and allogeneic blood stem cell transplantation compared with unmanipulated bone marrow transplantation in children

Immune reconstitution is critical for the long-term success of haematopoietic stem cell transplantation (HSCT). We prospectively analysed immune reconstitution parameters after transplantation of autologous (group 1; n = 10) and allogeneic (group 2; n = 12) highly purified CD34+ peripheral blood stem cells (PBSC) and unmanipulated allogeneic bone marrow (BM) (group 3; n = 9) in children. Median follow-up after HSCT was 56 (group 1), 61 (group 2), and 40.5 months (group 3). Median CD34-cell dose transplanted in the three groups was 9.4 x 10(6)/kg, 20.3 x 10(6)/kg, and 4.25 x 10(6)/kg recipient's body weight (BW) respectively. Complete haematopoietic engraftment was seen in all patients without any significant differences between the three groups. T-cell reconstitution at 6 months was significantly delayed in autologous peripheral blood stem cell transplantation (PBSCT) compared with allogeneic BM transplantation (P < 0.028) and allogeneic PBSCT (P < 0.034). At 3 months after transplantation numbers of CD56+/3- natural killer cells were higher in the allogeneic PBSC group (P < 0.01) compared with the BM group. The numbers of proven bacterial and viral infections were equally distributed between the three groups. In conclusion, recipients of allogeneic highly purified CD34+ PBSC or unmanipulated BM have higher lymphocyte subset counts at 6 months after transplantation than recipients of autologous CD34-selected PBSC. Infection rates and outcome, however, were not significantly different.     

Immunologic effects of prophylactic donor lymphocyte infusion after allogeneic marrow transplantation for multiple myeloma

Reconstitution of T-cell immunity after bone marrow transplantation (BMT) is often delayed, resulting in a prolonged period of immunodeficiency. Donor lymphocyte infusion (DLI) has been used to enhance graft-versus-leukemia activity after BMT, but the effects of DLI on immune reconstitution have not been established. We studied 9 patients with multiple myeloma who received myeloablative therapy and T-cell-depleted allogeneic BMT followed 6 months later by infusion of lymphocytes from the same donor. DLI consisted of 3 x 10(7) CD4(+) donor T cells per kilogram obtained after in vitro depletion of CD8(+) cells. Cell surface phenotype of peripheral lymphocytes, T-cell receptor (TCR) V beta repertoire, TCR rearrangement excision circles (TRECs), and hematopoietic chimerism were studied in the first 6 months after BMT and for 1 year after DLI. These studies were also performed in 7 patients who received similar myeloablative therapy and BMT but without DLI. Phenotypic reconstitution of T and natural killer cells was similar in both groups, but patients who received CD4(+) DLI developed increased numbers of CD20(+) B cells. TCR V beta repertoire complexity was decreased at 3 and 6 months after BMT but improved more rapidly in patients who received DLI (P =.01). CD4(+) DLI was also associated with increased numbers of TRECs in CD3(+) T cells (P <.001) and with conversion to complete donor hematopoiesis (P =.05). These results provide evidence that prophylactic infusion of CD4(+) donor lymphocytes 6 months after BMT enhances reconstitution of donor T cells and conversion to donor hematopoiesis as well as promoting antitumor immunity.     

Reduced circulating natural killer T cells and gamma/delta T cells in patients with systemic sclerosis

OBJECTIVE: To evaluate peripheral blood mononuclear cells (PBMC) expressing natural killer (NK) cell surface markers (CD16 and CD56, in both CD3- and CD3+ cells) and g/d T cell receptors (TCR) involved in non-MHC-restricted cytotoxicity, assessing their possible relationship with clinical and laboratory variables in patients with systemic sclerosis (SSc). METHODS: We submitted 50 patients with SSc to detailed clinical and laboratory assessment, and also performed PBMC subset analyses by direct dual immunofluorescence and flow cytometry. RESULTS: No statistically significant differences were found in the percentages or the absolute numbers of total lymphocytes, of B cells, and of CD4+ T cells. The absolute number of CD8+ cells was lower (p < 0.03), while HLA-DR+ elements were higher in frequency (p < 0.03) in SSc patients than in healthy controls. SSc patients had lower values (both percentage and absolute number) of NK-T cells (p < 0.01 and p < 0.003, respectively) and of T cells expressing g/d TCR (p < 0.01 and p < 0.005, respectively); whereas NK cells were marginally but not significantly decreased. The absolute number of NK-T cells showed an inverse correlation to erythrocyte sedimentation rate values (p < 0.03; rs = -0.306), percentage of g-globulins (p < 0.01; rs = -0.353), and serum concentrations of IgG (p < 0.02; rs = -0.334). CONCLUSION: Impairment of NK-T cells and of T cells expressing g/d TCR may lead to downregulation of normal immune response, and seems to be important for immunological and inflammatory aspects of SSc.     

Analysis of lymphocyte subpopulations in systemic sclerosis.

Systemic sclerosis (SSc) is a chronic inflammatory connective-tissue disease of unknown etiology, characterized by fibrosis and microvascular injury in affected organs. It has become clear that the activated cellular-immune system plays a central role in the pathogenesis of SSc. This study analyzes the numbers of lymphocyte subpopulations and their relations with clinical and laboratory manifestations. We studied a group of 42 patients with SSc and a group of 28 matched normal controls by flow cytometry using the lymphocyte cell-surface markers CD2, CD3, CD4, CD8, CD19, CD25, CD45RA, CD56, CD71, HLA-DR, TCR alpha/beta, and TCR gamma/delta. Patients with SSc had similar percentages of CD2+, CD3+, CD3+ CD4+, CD3+, CD8+, CD25+, CD4+ CD45RA+, CD8+ CD45RA+, CD71+ cells, and CD4+/CD8+ cell ratio when compared to normal controls. In contrast, the percentages of TCR gamma/delta cells were significantly lower in SSc patients with diffuse and late-stage disease with pulmonary involvement, muscle involvement, and the presence of anti-Scl-70 antibodies. Patients with diffuse SSc in early- and late-stage disease had significantly increased percentages of HLA-DR in CD4+ and CD8+ cells. Patients with late-stage disease had increased percentages of CD4+ CD45RA+ T-cells. Patients with limited and early-stage disease had smaller percentages of B-cells (CD19+). Patients with diffuse and late-stage disease had smaller percentages of NK-cells (CD56+). These results suggest that T-, B-, and NK-cell alterations may be involved in the onset of the disease, and/or in the perpetuation of disease, and may eventually be useful as a prognostic indicator in selected patient subgroups.     

Different immune reconstitution in multiple myeloma, chronic myeloid leukemia and acute myeloid leukemia patients after allogeneic transplantation of peripheral blood stem cells

In this study we compared the lymphocyte reconstitution in 13 multiple myeloma (MM), nine acute myeloid leukemia (AML) and 10 chronic myeloid leukemia (CML) patients after allogeneic G-CSF-mobilized PBSC transplantation from HLA-identical siblings. Conditioning regimens included standard total body irradiation + cyclophosphamide (CY), or busulphan + CY, whereas VP-16 was added in patients with advanced disease. Overall comparable numbers of mononuclear cells, CD34+ cells and CD3+ T cells were infused in each group. A significantly higher CD3+ T cell number was observed in MM and AML than in CML patients 1 month after transplant. However, MM patients showed a faster and better recovery of CD4+ T cells than both AML and CML patients at 3 months (P = 0.01 and P = 0.01, respectively) and 12 months (P = 0.01 vs AML, while P = NS vs CML) after transplant, and had a CD4:CD8 ratio > 1 with a median CD4+ T cell value > 400/microl 1 year after transplant. Development of acute graft-versus-host disease (GVHD) did not affect CD4:CD8 ratios but patients who experienced acute GVHD > grade I had lower CD4+ and CD8+ T cell numbers at all time points. However, after excluding patients with GVHD > grade I, MM patients still showed a significantly higher CD4+ T cell value than patients with myeloproliferative diseases 1 year after transplant. These findings suggest that although allogeneic PBSC transplantation induces rapid immune reconstitution, different kinetics may occur among patients with hematological malignancies. In particular, the rapid reconstitution of CD4+ T cells in MM patients may contribute to the low transplant-related mortality achieved in this disease.     

Characteristics and influencing factors of CD19+ B cell reconstitution in patients following haploidentical/mismatched hematopoietic stem cell transplantation

In patients undergoing hematopoietic stem cell transplantation (HSCT), B cells exert important, prolonged effects that provide protection from infection. In this study, we analyzed characteristics and influencing factors of CD19+ B cell reconstitution in 83 patients who underwent unmanipulated haploidentical/mismatched blood and bone marrow transplantation. Of these patients, 45?% showed a normal CD19+ B cell count at +360 days. Factors associated with lower CD19+ B cell levels were as follows: aGVHD grades II-IV had a trend to affect CD19+ B cell reconstitution at +180?days; clinically extensive cGVHD was significantly associated with CD19+ B cell deficiency at +360?days and serum IgG level at +180 and +360?days; cytomegalovirus (CMV) infection occurred after +38?days was correlated with lower B cell level at both +90 and +360?days, while those occurred before +38?days did not show this effect; glucocorticoids used around +90 and +180?days was associated with lower CD19+ B cell levels at +90 and +360?days, respectively, especially in patients that did not experience extensive cGVHD. In contrast, the number of HLA-mismatched locus positively correlated with CD19+ B cell at +90?days and serum IgG level at +180?days. In conclusion, CD19+ B cell recovery after haploidentical/mismatched HSCT was mainly influenced by GVHD and/or its treatment, CMV infection that occurred later (after +38?days) and use of glucocorticoids. Improvement of B cell recovery is likely to be achieved through effective prophylaxis of GVHD, minimized use of glucocorticoids, and preemptive treatment of CMV infection occurring after +38?days. More HLA-mismatched loci may initiate a stronger humoral response toward alloantigens >180?days after HSCT, which may be beneficial for the eradication of minimal residual disease and protection from infection.     

T cell regeneration in pediatric allogeneic stem cell transplantation

Delayed and/or insufficient T cell recovery post hematopoietic stem cell transplantation (HSCT) leads to an increased risk of morbidity and mortality. We evaluated thymic function and its association with T cell regeneration post HSCT and identified factors involved in the process among pediatric stem cell transplant recipients. T cell regeneration in 66 pediatric patients was prospectively followed by naive T cell phenotyping, measuring of T cell receptor excision circles (TRECs) and expression of Foxp3 by regulatory T cells for the first 18 months post HSCT. TRECs were lower pre-HSCT in children with a malignant than non-malignant primary disease or immunosuppressed controls (P=0.001). Naive T lymphocyte reconstitution and thymic recovery were slow in the recipients of allogeneic stem cell grafts post HSCT. Infections caused by herpesviruses had a prognostic impact on mortality. Children with low TRECs had a high mortality (P=0.05) and low TRECs were also associated with extensive chronic graft-versus-host disease from 6 months onwards. Low amount of Foxp3 pre-HSCT was associated with an increased mortality post HSCT (P=0.03). Our study indicates an association between impaired T cell regeneration and thymic dysfunction and the clinical post transplant complications in pediatric allogeneic stem cell transplantation.     

A comparison of T-, B- and NK-cell reconstitution following conventional or nonmyeloablative conditioning and transplantation with bone marrow or peripheral blood stem cells from human leucocyte antigen identical sibling donors

This retrospective study compares the reconstitution of T, B and NK cells in three groups of patients transplanted for haematological malignancies with grafts from their HLA-identical sibling donors. In all, 15 patients received PBSC after a nonmyeloablative conditioning regimen consisting of fludarabine and 200 cGy TBI, 13 patients received PBSC after myeloablative conditioning and 37 patients received BM after myeloablative conditioning. In the nonmyeloablative group, the NK cells normalised after 1 month, the CD8+ T cells normalised after 3 months, the CD4+ T cells reached near normal values after 9 months and the B cell values were reduced until 12 months after transplant. In the two myeloablative groups, recipients of PBSC had a significantly higher number of CD4+ T cells after 4 months (P=0.004) and after 12 months (P=0.001), than recipients of BM. We found no differences in the T cell reconstitution between the two PBSC groups. This was of interest as the recipients of nonmyeloablative conditioning were older (P<0.001) and had a higher occurrence of chronic GVHD (P<0.05) than the recipients of myeloablative conditioning. In contrast, the recipients of nonmyeloablative conditioning had a delayed B cell recovery when compared to the patients who received myeloablative conditioning (P=0.04).     

Immune reconstitution and production of intracellular cytokines in T lymphocyte populations following autologous peripheral blood stem cell transplantation

For the better understanding of engraftment properties after autologous peripheral blood stem cell transplantation (PBSCT), hematopoietic recovery, immune reconstitution and functional capacity of cytokine production in different lymphocyte populations were examined. In a prospective study, we examined 24 patients suffering from different malignancies after autologous PBSCT. The examination intervals were 1, 3, 6 and 12 months after PBSCT. T cells, B cells and NK cells were analyzed using flow cytometry. The expression and kinetics of cytokines in T lymphocytes were evaluated in 10 patients by intracellular staining of cytokines after PMA/ionomycin stimulation. We observed rapid hematopoietic engraftment proceeding to stable long-term reconstitution. For CD3(+) lymphocytes, a consistent reconstitution associated with an increase in CD3(+)CD8(+) cytotoxic T cells was observed, whereas the CD3(+)CD4(+) helper/inducer T cells remained low (< 200/microl). Impaired B lymphopoiesis with severe depression (<1%) was detected 1 month after PBSCT but recovered thereafter (12.8% after 3 months). The percentages of cytokine-producing T cells and the mean fluorescence intensity (MFI) shifts suggested an insufficient capacity for producing IFNgamma, in particular for CD3(+)CD4(+) T cells, compared to healthy volunteers early after PBSCT. Rapid hematopoietic recovery and partly impaired immune reconstitution, especially regarding the regeneration of B lymphocytes and T helper cells, was observed. The CD4(+) subpopulation remained low throughout the period of examination, whereas the B cells showed a delayed recovery after 3 months. Cytokine production proved to be sufficient after in vitro stimulation in T cell populations with the exception of IFNgamma synthesis.     

Rapid helper T-cell recovery above 200 x 10 6/l at 3 months correlates to successful transplant outcomes after allogeneic stem cell transplantation

The current study evaluates the role of quantitative measurement of peripheral lymphocyte subsets, especially CD4+ helper T-cell recovery, in predicting transplant outcomes including overall survival (OS) and non-relapse mortality (NRM) after allogeneic stem cell transplantation. A total of 69 allogeneic recipients were included with following diagnoses: acute myeloid leukemia 42, acute lymphoblastic leukemia 5, chronic myeloid leukemia 15, non-Hodgkin's lymphoma 5 and high-risk myelodysplastic syndrome 2. The peripheral lymphocyte subset counts (CD3+ T cells, CD3+4+ helper T cells, CD3+8+ cytotoxic T cells, CD19+ B cells, and CD56+ natural killer cells) were measured at 3, 6 and 12 months. The CD4+ helper T-cell reconstitution at 3 months was strongly correlated with OS (P<0.0001), NRM (P=0.0007), and opportunistic infections (P=0.0108) at the cutoff value of 200 x 10(6)/l CD4(+) helper T cells. Rapid CD4+ helper T-cell recovery was also associated with a higher CD4+ helper T-cell transplant dose (P=0.006) and donor type (P<0.001). An early CD4+ helper T-cell recovery at 3 months correlated with a subsequent faster helper T-cell recovery until 12 months, yet not with B-cell recovery. In a multivariate analysis, rapid recovery of CD4+ helper T cells at 3 months was a favorable prognostic factor together with higher CD34+ cell transplant dose in terms of OS (P=0.001) and NRM (P=0.005).     

大强度运动对大鼠脾脏淋巴细胞、巨噬细胞、T淋巴细胞亚群的影响

目的探讨大强度运动对大鼠脾脏淋巴细胞、巨噬细胞、T淋巴细胞亚的影响。方法40只SD大鼠按体重随机分为安静组(A组,n=10)、运动组(B组,n=30),在最后一次力竭运动后,将B组大鼠分为运动后即刻组(B 1组,n=10)、运动后12 h组(B 2组,n=10),运动后24 h组(B 3组,n=10)。A组不运动,B组大鼠进行为期9 w的大强度训练,末次训练后,A组、B 1组摘取脾脏,制作脾细胞悬液,噻唑蓝(MTT)法分别测T淋巴细胞增殖能力、B淋巴细胞增殖能力,流式细胞仪测CD4^+、CD8^+细胞数。中性红比色法测巨噬细胞吞噬能力。B 2组、B 3组分别在运动后12 h、运动后24 h按上述方法测试。结果与A组相比,B 1组T淋巴细胞增殖、B淋巴细胞增殖、CD4^+、CD8^+、CD4^+/CD8^+、巨噬细胞吞噬能力显著降低,B 2组T淋巴细胞增殖、B淋巴细胞增殖、CD4^+、CD4^+/CD8^+显著降低,巨噬细胞吞噬能力差异无统计学意义,B 3组T淋巴细胞、B淋巴细胞增殖能力、CD4^+、CD8^+、CD4^+/CD8^+、巨噬细胞吞噬能力差异无统计学意义;与B 1相比,B 2组、B 3组T淋巴细胞增殖、B淋巴细胞增殖、CD4^+、CD8^+、CD4^+/CD8^+、巨噬细胞吞噬能力显著升高;与B 2相比,B 3组T淋巴细胞增殖、B淋巴细胞增殖、CD4^+均显著升高,CD8^+、CD4^+/CD8^+、巨噬细胞吞噬能力无显著差异。结论大强度运动后,大鼠脾脏、B淋巴细胞、T淋巴增殖能力降低、巨噬细胞吞噬能力降低、CD3^+、CD4^+、CD8^+、CD4^+/CD8^+降低,恢复期间逐步上升,其中CD8^+、CD4^+/CD8^+、巨噬细胞吞噬能力在恢复期12 h基本达到正常状态,T淋巴细胞增殖能力、B淋巴细胞增殖能力、CD4^+在恢复期24 h达到正常状态。     

Presence of CD4+CD8+ double-positive T cells with very high interleukin-4 production potential in lesional skin of patients with systemic sclerosis

OBJECTIVE: Fibrotic skin changes in systemic sclerosis (SSc) are preceded by the appearance of an inflammatory infiltrate rich in T cells. Since no direct comparison with T cells in normal skin has been performed previously, this study was undertaken to functionally characterize T cells in the skin of patients with early active SSc and in normal skin. METHODS: We characterized coreceptor expression, T cell receptor (TCR) usage, cytokine production, and helper and cytolytic activity of T cell lines and clones established from skin biopsy specimens from 6 SSc patients and 4 healthy individuals. Immunofluorescence analysis of skin biopsy and peripheral blood samples was performed to confirm the presence of specific subsets in vivo. RESULTS: A distinct subset expressing both CD4 and CD8alpha/beta coreceptors at high levels (double-positive [DP]) was present in T cell lines from SSc and normal skin. DP T cells actively transcribed both accessory molecules, exerted clonally distributed cytolytic and helper activity, and expressed TCR clonotypes distinct from those in CD4+ or CD8+ single-positive (SP) T cells. In SSc skin, DP T cells produced very high levels of interleukin-4 (IL-4) compared with CD4+ SP T cells. Furthermore, DP T cells were directly identified in SSc skin, thus providing evidence that they are a distinct subset in vivo. CONCLUSION: The present findings show that T cells with the unusual CD4+CD8+ DP phenotype are present in the skin. Their very high level of IL-4 production in early active SSc may contribute to enhanced extracellular matrix deposition by fibroblasts.     

Immune recovery in children undergoing allogeneic stem cell transplantation: absolute CD8+ CD3+ count reconstitution is associated with survival

To evaluate the correlation between kinetics of immune reconstitution and survival, we prospectively evaluated lymphocyte subsets in 32 paediatric patients undergoing allogeneic stem cell transplantation (SCT) for haematological malignancies. Four-colour flow cytometric analysis was performed at short intervals with a median follow-up of 4 years post SCT. A total of 50% of patients reached age-matched 5th percentile of natural killer, cytotoxic T, B and helper T cells 4, 9, 20 and 28 weeks after SCT, respectively, which increased to more than 80% within 1 year after SCT. Transplantation of peripheral blood stem cells (PBSC) seemed to elicit the fastest reconstitution of CD3+, CD4+ CD3+, CD8+ CD3+ and na?ve T cells compared to bone marrow (BM) or CD34-selected PBSC, which did not differ. Most importantly, we observed a significantly higher number of survivors among patients whose CD8+ CD3+ absolute counts rose above the 5th percentile of age-matched normal levels during the first year post SCT compared to patients who never reached these levels (19/25 vs 0/7, P<0.001). This was still present in both subgroups, BM- and CD34-selected grafts (P=0.03, 0.02). These results from a small patient sample underline the importance of particular lymphocyte subsets for the outcome of children undergoing SCT. A larger study with detailed subset analysis is underway.     

Optimized clonotypic analysis of T-cell receptor repertoire in immune reconstitution

OBJECTIVE: In recent years, T-cell receptor (TCR) sequencing analysis has proven an effective technique for the identification of T-cell populations of interest in cancer and autoimmunity, as well as for the characterization of peripheral immune repertoire reconstitution after hematopoietic stem cell transplantation (HSCT). However, despite its increased utilization, to our knowledge no group has investigated the minimum number of sequences necessary to accurately and efficiently describe the composition of TCR repertoire. The primary aim of this study was to optimize a procedure for clonotypic analysis of the TCR repertoire in patients undergoing autologous HSCT. MATERIALS AND METHODS: TCR beta-chain diversity was analyzed by DNA sequencing and CDR3 spectratyping CD8(+) T cells isolated from three patients with multiple sclerosis undergoing autologous HSCT. Samples were collected at baseline and 1 or 2 years post-HSCT. RESULTS: Using DNA cloning and high throughput sequencing, we analyzed over 1500 in-frame TCR sequences, allowing us to evaluate how our measures of TCR repertoire diversity change with increasing numbers of sequences included in the analysis. Our findings show that by analyzing 75 to 100 in-frame sequences, we are able to estimate TCR diversity within 5.0% to 7.4% of the values obtained at endpoint analysis (213-312 sequences per sample). CONCLUSIONS: This study confirms the use of TCR sequencing as an effective technique for the characterization of immune renewal after autologous HSCT. In addition, we demonstrate for the first time convincing evidence to support the use of moderate sample sizes to accurately and efficiently evaluate TCR repertoire diversity.     

Prognostic value of pretransplantation host thymic function in HLA-identical sibling hematopoietic stem cell transplantation

Thymic function is critical for immune reconstitution after hematopoietic stem cell transplantation (HSCT). We evaluated recipient thymic function before HSCT by quantifying T-cell receptor excision circles (TRECs) in pretransplantation peripheral blood lymphocytes from 102 patients who received HSCs from an HLA-identical sibling for malignant (n = 87) or nonmalignant diseases (n = 15). Median TREC value before transplantation was 257 TRECs per 150,000 CD3+ cells (range, 0-42,746). We assessed 172 TRECs per 150,000 CD3+ cells as the most discriminating TREC value for survival in a first cohort of patients (n = 62). This cut-off was validated in a second independent prospective group of 40 patients. In the 102 patients, a TREC value greater than or equal to 172 was associated with a better survival (P < .000 01), a decreased incidence of grade II-IV acute graft-versus-host disease (GVHD; P = .017), chronic GVHD (P = .023), and bacterial (P = .003) and cytomegalovirus (CMV) infection (P = .024). In a multivariate analysis, low pretransplantation TREC values were associated with a higher incidence of CMV infection (hazard ratio [HR] = 2.0, P = .06) and severe bacterial infections (HR = 2.8, P = .036). Finally, high TREC values (HR = 6.6, P = .002) and ABO compatibility (HR = 2.7, P = .02) were associated with a better survival. Therefore, recipient host thymic function assessment could be helpful in predicting HSCT outcome and identifying patients who require a close immunologic monitoring.     

Autologous bone marrow transplantation in the treatment of refractory systemic sclerosis: early results from a French multicentre phase I-II study

Haematopoietic stem cell transplantation (HSCT) has been proposed for refractory autoimmune diseases, including systemic sclerosis (SSc). A sequential Bayesian phase I-II clinical trial was conducted in SSc patients to assess the feasibility, the tolerance and the efficacy of autologous HSCT. Peripheral blood stem cells (PBSC) were collected using cyclophosphamide (4 g/m2) and recombinant human granulocyte colony-stimulating factor (5 micro g/kg/d) and reinfused after positive CD34+ selection. Conditioning used cyclophosphamide (200 mg/kg) or melphalan (140 mg/m2) according to cardiac function. The main end-point was the failure of the procedure, defined by failure of either PBSC mobilization, CD34+ selection or intensification procedure, or by procedure-related death. Among the 12 enrolled patients, three failures occurred: one PBSC mobilization, one CD34+ selection and one CD34+ intensification. Probability of graft failure was estimated at 0.286 (95% confidence interval: 0.095-0.54). Autologous PBSC (n = 10) or bone marrow (n = 1) transplantation was actually performed in 11 patients with one procedure-related death. Median time to neutrophil (> 0.5 x 10(9)/l) and platelet (> 25 x 10(9)/l) haematopoietic reconstitution was 12 and 10 d respectively. After 18 months (range 1-26), eight out of 11 patients have shown major or partial response. Non-myeloablative conditioning, followed by a T cell-depleted autologous PBSC or bone marrow transplantation, appears feasible with low toxicity in severe SSc with short-term clinical benefits.     

Acute autoimmune hemolytic anemia following unrelated cord blood transplantation as an early manifestation of chronic graft-versus-host disease

A 16-month-old girl diagnosed with osteopetrosis underwent an unrelated, partially matched (with major mismatch at A locus) cord blood stem cell transplant. Twelve months later she developed severe acute autoimmune hemolytic anemia (AIHA). Immunophenotype analysis of lymphocyte subsets 8 months post transplant showed a low number of T lymphocytes, with normal subsets, and with NK cells and B lymphocytes within normal ranges. When the hemolytic anemia developed, the lymphocytes subsets changed and analysis showed higher numbers of B lymphocytes than previously, lower CD3+ T lymphocytes with inversion of the CD4/CD8 ratio and an abnormal proportion of T lymphocyte subsets. She was being treated with cyclosporine, and steroids and immunoglobulins were added. Initially the AIHA improved, but repeated infectious episodes led us to tail off the immunosuppressive treatment. The AIHA relapsed and cyclosporine was restarted. Currently, she is on cyclosporine and low-dose steroid treatment with no hemolytic features. During the 3 months when the AIHA was being treated, she developed extensive skin cGVHD and recurrent pneumothoraces. AIHA may be the first manifestation of abnormal reconstitution of immunity developing after a hematopoietic transplant. This abnormal reconstitution is also the basis of cGVHD. We suggest that aggressive immunosuppressive treatment with intensive measures against infection could give a better prognosis to such patients.