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Keywords:

  • immune reconstitution;
  • haploidentical;
  • CD34+ stem cell transplantation;
  • T-cell repertoire diversity;
  • CDR3 size spectratyping

Abstract

  1. Top of page
  2. Abstract
  3. Patients and methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. References

Transplantation of haematopoietic stem cells from human leucocyte antigen (HLA)-disparate parental donors presents a promising new approach for the treatment of patients lacking a HLA-matched donor. Success against major obstacles such as graft-versus-host disease (GvHD) and graft rejection has recently been demonstrated, so that immune reconstitution is one of the prime factors that determines the long-term prognosis following transplantation. Twenty children transplanted with megadoses of highly purified CD34+ haematopoietic stem cells after rigorous T-cell depletion were prospectively monitored for their immune reconstitution during the first post-transplant year. Natural killer (NK) cells showed a marked increase on d +30. T and B cells began to reconstitute on d +72 and +68 respectively. During extended follow-up, their numbers and proliferative capacity upon mitogen stimulation continually increased. Early reconstituting T cells were predominantly of a primed, activated phenotype with severely skewed T-cell receptor (TCR)-repertoire complexity. Naive T cells emerged 6 months post transplantation, paralleled by an increase in TCR-repertoire diversity. All patients self-maintained sufficient immunoglobulin levels after d +200. This study demonstrates that paediatric recipients of highly purified, haploidentical stem cells are able to reconstitute functioning T-, B- and NK-cell compartments within the first post-transplant year. This, together with the absence of significant GvHD, provides a strong indication for this approach to be considered in children who lack a HLA-matched donor.

Haematopoietic stem cell transplantation (SCT) offers the only curative approach for patients with advanced haematological malignancies. However, the need for an human leucocyte antigen (HLA)-identical donor has presented major limitations to this therapeutic modality, necessitating a search for alternative donors. Recent advances have revolutionized criteria for donor selection (Aversa et al, 1998), with the development of novel immunosuppressive drugs (Chen et al, 2000), approaches towards anergizing antigen-specific T cells (Blazar et al, 1998; Guinan et al, 1999), and techniques for in vitro and in vivo T-cell depletion (Aversa et al, 1994; Handgretinger et al, 1999). Together, these have pushed the limitations of donor selection beyond HLA barriers.

T-cell depletion affects both the incidence and severity of graft-versus-host disease (GvHD) after HLA-mismatched transplantation with a reduction in morbidity rates and even prevention of its onset (Anasetti, 1998; Martin et al, 1999). However, T-cell depletion of marrow grafts has been generally associated with higher rates of graft rejection (Anasetti et al, 1989) and delayed restoration of the immune system (Roux et al, 1996; Small et al, 1999), thus compromising patient response to post-transplant infectious complications.

‘Megadose’usage of highly purified peripheral blood stem cells obtained from a haploidentical (parental) donor with up to three antigen mismatches on different HLA loci is now one of the major bases for alternative donor selection in patients with advanced haematological malignancies (Reisner & Martelli, 1999). Haploidentical SCT was initially used in the treatment of paediatric patients with severe combined immunodeficiency (Friedrich et al, 1985). In immunocompetent recipients, transplantation of T cell-depleted grafts has been associated with higher rejection rates (Martin et al, 1999). Studies in murine models have shown that by increasing stem cell dosage, host alloresistance can be overcome with sustained engraftment of HLA-disparate stem cells (Wang et al, 1997; Reisner & Martelli, 2000). Additionally, doses of > 20 × 106 CD34+ cells have also been shown to promote lymphoid recovery (Handgretinger et al, 1999). Results from the first clinical trial using megadoses of peripheral blood stem cells in adult high-risk leukaemia patients demonstrated safe engraftment but a high mortality rate in the first post-transplant year, owing to infectious complications (Aversa et al, 1994). A second trial with a paediatric cohort confirmed safe engraftment, but with a surprisingly low incidence of infectious complications (Handgretinger et al, 1999). This may be as a result of a more efficient and faster restoration of the immune system in paediatric patients, but data on the pattern of immune reconstitution in children following haploidentical SCT remains scarce.

In the present study, we have carried out a prospective analysis examining the pattern of immune reconstitution in 20 paediatric patients during the first year after haploidentical SCT. Reconstitution has been monitored by: (i) phenotypic analysis of lymphocyte subsets; (ii) functional proliferative capacity of peripheral blood lymphocytes to mitogenic stimulation; and (iii) analysis of the T-cell receptor (TCR)-repertoire diversity using complementarity-determining region 3 (CDR3)-size spectratyping. All patients received megadoses of rigorously T cell-depleted, peripheral CD34+ stem cells after myeloablative therapy. Our data demonstrated that, in children, this innovative approach results in excellent engraftment rates, together with rapid and sustained reconstitution of T-, B- and natural killer (NK)-cell compartments and a complete absence of significant GvHD. These findings indicate that transplantation of haploidentical stem cells is an attractive option for high-risk paediatric patients who lack a conventional HLA-matched donor.

Patients and methods

  1. Top of page
  2. Abstract
  3. Patients and methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. References

Clinical methods All patients eligible to receive a transplant of highly purified stem cells obtained from a parental donor from February 1997 to August 1999 were included in this study of immune reconstitution. Informed consent was obtained in accordance with institutional regulations. HLA-typing for class I (A and B) was carried out serologically; for class II alleles (DRB1) molecular methods were routinely used. All patients received a graft of positively selected CD34+ peripheral stem cells. CD34+ cells were separated using a modified MACS-technique, as previously described (Handgretinger et al, 1999). Granulocyte colony-stimulating factor (G-CSF) (5 μg/kg/d) was administered intravenously to all patients starting on d +4 until engraftment. No post-transplant GvHD prophylaxis was given. Details of the patients are given in Table I.

Table I.  Patient characteristics.
 Age    Transplanted cellsANCOutcome, deaths 
UPN(years) /Sex Diagnosis DonorHLA mismatchConditioning regimenCD34+ (/kg bw × 106)CD3+ (/kg bw × 103)> 0·5 × 109/l (d)indicated by =, survival in dDLI (which day given)
  • *

    Patient requiring a second transplant from the same donor owing to primary non-engraftment.

  • Patients requiring a second transplant from the same donor owing to graft rejection.

  • Parental donor without HLA-disparity owing to consanguinity. For compliance with our inclusion criteria, this transplant was included in the analysis.

  • AIHA, autoimmune haemolytic anaemia; ALG (60), α-lymphocyteglobulin (horse) (3 × 20 mg/kg bw); ALL, acute lymphoid leukaemia; AML, acute myeloid leukaemia; ANC, absolute neutrophil count; ATG (30), α-thymocyte globulin (rabbit) (3 × 10 mg/kg bw); Bu (16), busulphan (4 × 4 mg/kg bw); CML, chronic myeloid leukaemia; CP, chronic phase; CR, complete remission; Cyc (120), cyclophosphamide (2 × 60 mg/kg bw); DLI, donor leucocyte infusion; FTBI (12), fractionated total body irradiation (12 Gray); GvHD, graft-versus-host disease; ID, immunodeficiency (unclassified, functional T-cell deficiency, hypogammaglobulinaemia, chronic cytomegalovirus infection); MDS, myelodysplastic syndrome; NHL, non-Hodgkin's lymphoma; OKT3, α-CD3 antibody; PR, partial remission; TT (10), thiotepa (10 mg/kg bw); OP, osteopetrosis; SCID, severe combined immunodeficiency; VP16 (40), etoposide (40 mg/kg bw); UPN, unique patient number (first four digits).

42104/MaleALL, CR3FatherA, B, DRFTBI(12)/TT(10)/VP16(40)/OKT333·812·58Alive, 3830
401317/FemaleALL, CR2MotherDRFTBI(12)/TT(10)/Cyc(120)/ATG(30)5·78·212Alive, 8360
41188/MaleALL, CR2FatherA, B, DRBu(16)/TT(10)/Cyc(120)/ALG(60)23·210·612Alive, 6760
42035/FemaleALL, CR2FatherDRBu(16)/TT(10)/Cyc(120)/ALG(60)39149= relapse, 3872 (56, 84)
41044/FemaleALL, PR2FatherA, B, DRBu(16)/TT(10)/Cyc(200)/ATG(30)/OKT350·410·111= relapse, 1163 (29, 56, 84)
42369/MaleALL, CR1MotherA, BFTBI(12)/TT(10)/VP-16(40)/OKT38·914·910= relapse, 1170
40649/MaleALL, PR1FatherA, B, DRBu(16)/TT(10)/Cyc(200)/ATG(30)/OKT317·46·19Alive, 8461 (56)
4001*9/FemaleAML/MDS, CR2FatherA, DRBu(16)/TT(10)/Cyc(120)/ALG(60)13·617·114Alive, 12471 (3)
40112/FemaleAML, PR2FatherA, B, DRBu(20)/TT(10)/Cyc(120)/ALG(60)28·714·410= relapse, 701 (19)
40049/MaleAML, PR2MotherA, B, DRBu(16)/TT(10)/Cyc(200)/ALG(60)14·2111= relapse, 1091 (56)
405310/MaleAML, PR1FatherA, B, DRBu(16)/TT(10)/Cyc(200)/ATG(15)24·63·710= relapse, 1463 (14, 56, 84)
40868/MaleAML, CR1MotherBBu(16)/TT(10)/Cyc(200)/ATG(30)11·613·612= AIHA,aspergillosis,3382 (28, 56)
40243/FemaleCML, CPFatherB, DRBu(16)/TT(10)/Cyc(200)/ATG(15)26710Alive, 8712 (56,84)
418112/FemaleCML, CPFatherB, DRFTBI(12)/TT(10)/Cyc(120)/OKT316·53·910= varicellosis, 1250
414712/MaleCML, CPMotherA, B, DRBu(16)/TT(10)/Cyc(200)/ATG(30)/OKT317·56·49= GvHD, 1954 (28,56,84,112)
412812/MaleNHL, PR2FatherA, B, DRFTBI(12)/VP16(40)/Cyc(120)/ATG(30)/OKT325·511715= relapse, 921 (56)
42180·75/MaleSCIDFatherA, B, DRnone32·213·2 Alive, 4020
40017/MaleIDMotherA, BBu(16)/TT(10)/Cyc(200)/ALG(60)37·518·710= pneumonia, 1700
41010·5/MaleOPFatherNoneBu(20)/TT(10)/Cyc(200)/ATG(30)34·61410Alive, 7690
40153/FemaleOPFatherB, DRFTBI/(8)/TT(10)/Cyc(120)/OKT32915·39Alive, 4310
Median4    23·212·510  
(range)(0·5–17·5)    (5·7–50·4)(1–117)(8–14)  

Flow cytometry Peripheral blood was monitored for lymphocyte subsets by flow cytometry at weekly intervals during the first month post transplant and monthly thereafter. Anti-CD3-phycoerythrin (PE), anti-CD19-fluorescein isothiocyanate (FITC) and anti-CD16/CD56-FITC monoclonal antibodies were used to identify T, B and NK cells respectively. B- and T-cell subsets were further characterized using three-colour flow cytometry to evaluate the expression of CD10, CD23 and CD40 on B cells, and CD4, CD8, TCRαβ, TCRγδ, CD11a, CD25, CD27, CD28, CD29, CD45RA, CD45RO, CD62L, CD69 and HLA-DR on T cells. All monoclonal antibodies were purchased from Becton Dickinson (Heidelberg, Germany).

Proliferation assays Peripheral blood mononuclear cells (PBMCs) were isolated and resuspended at a final concentration of 2 × 106 cells/ml in Roswell Park Memorial Institute (RPMI)-1640 medium supplemented with 10% human AB-serum (PAA, Linz, Austria), 1 mmol/l L-glutamine, 100 U/ml penicillin and 100 μg/ml streptomycin (Gibco, Eggenstein, Germany). PBMCs (1 × 105) (57·9 ± 19·8% versus 62·1 ± 20% lymphocytes in patients and controls respectively), in a final volume of 200 μl/well, were plated in 96-well flat-bottomed plates and incubated for 72 h (37°C, 5% CO2) in triplicate with each of seven different mitogens using the following concentrations: phytohaemagglutinin (PHA) 5 μg/ml, concanavalin A (Con A) 5 μg/ml, phorbol-12-myristate 13-acetate (PMA) 10 ng/ml, ionomycin 1 nmol/ml, pokeweed mitogen (PWM) 5 μg/ml, staphylococcus enterotoxin A (SEA) 5 μg/ml (Sigma, Deisenhofen, Germany), interleukin 2 (IL-2) 105 U/ml (Chiron-Therapeutics, Ratingen, Germany), and anti-CD3 50 μg/ml (OKT3; Janssen-Cilag, Neuss, Germany). Cells were then pulsed with 37 kBq [3H]-thymidine (Amersham Pharmacia, Germany) per well and, after an additional 18 h incubation (37°C, 5% CO2), harvested on filtermats in a Tomtec harvester (Wallac, Freiburg, Germany), as previously described (Schlegel et al, 1996). Counts were analysed on a beta scintillation counter (Microbeta Wallac, Freiburg, Germany).

TCR-repertoire analysis The complexity of the peripheral T-cell repertoire was analysed every 3 months post transplant using the spectratyping approach previously described in detail by Gorski et al (1994). A median of 9·3 × 105 isolated CD3+ cells (range 2 × 104−10·6 × 106) were used for analysis and only five samples contained T-cell numbers below 1 × 105 (2–9·1 × 104). RNA was isolated on a CsCl gradient and reverse transcribed into cDNA. To ensure equal amounts of T cell-specific cDNA for each spectratype, cDNAs were titrated and amplified in a polymerase chain reaction (PCR) using a Cβ-specific primer pair. PCR products were analysed by gel electrophoresis on 8% polyacrylamide gels. cDNA from signals equivalent in intensity were used for subsequent spectratyping (Gorski et al, 1994). Using this approach, titration experiments with decreasing T-cell numbers (range 107−103 CD3+ cells) from healthy controls did not show skewing of the repertoire when ≥ 104 T cells were used in the assay. cDNA was amplified with Vβ1–24 sense primers and a fluorescence-labelled (IRD800) Cβ-region antisense primer. Products were denatured and visualized on a denaturing polyacrylamide gel using the LiCor4200 automated DNA sequencer (MWG Biotech, Ebersberg, Germany). Each Vβ family was scored for the number of detectable bands per lane. As a normal spectratype has been shown to consist of 5–8 bands per family with a Gaussian size distribution of TCR fragments (Gorski et al, 1994; Zeng et al, 1999; Wu et al, 2000), scoring and size distribution were used to assess the normalization of TCR repertoire.

Results

  1. Top of page
  2. Abstract
  3. Patients and methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. References

Clinical outcome

One patient (UPN 4181) developed GvHD grade I immediately following engraftment, necessitating a short course of methylprednisolone. Three patients required a second transplant from the same donor, two owing to early graft rejection and one owing to graft failure (see Table I). All three patients engrafted successfully after an OKT3-based reconditioning regimen followed by a second stem cell graft from the same donor (Schlegel et al, 2000). Full donor chimaerism was achieved after a median of 17 d (range 11–142 d). In one patient with severe combined immunodeficiency (SCID) (UPN 4218), a stable mixed chimaerism with 40–60% autologous signals was established. Seven out of 20 patients died during relapse after a median of 120 d (range 70–387). Eleven out of 20 patients received 1–4 T-cell addbacks (2·5 × 104 CD3+cells/kg bw) during follow-up for an increasing mixed chimaerism or because they were not in remission at the time of transplant. Of these, one (UPN 4147) subsequently developed fatal GvHD grade IV. Two others showed limited GvHD grade I (UPN 4104) and grade II (UPN 4011) in response to T-cell addback, which resolved during follow-up. Three patients died with infectious complications: one of pulmonary infection on d +170 post transplant and one of fatal disseminated varicella infection on d +125. One boy (UPN 4086) developed severe autoimmune haemolytic anaemia necessitating profound immunosuppression and died with cerebral aspergillosis on d +338.

NK-cell reconstitution

NK cells (CD16+/56+) were the first lymphoid cells to emerge following transplantation of highly purified stem cells. Reconstitution was rapid and reached a median of 305 cells/μl (range 30–1200) as early as d +30 post transplant (Fig 1). At this time point, NK cells represented up to 74% of the total lymphoid count. This early predominance of NK cells was reflected by the proliferative response of PBMCs when challenged with IL-2. All other mitogens in our panel were unable to elicit a proliferative response at this timepoint (Table II).

image

Figure 1. Increase of CD16+/56+ natural killer (NK) cells in the peripheral blood mononuclear cell (PBMC) fraction after haploidentical SCT (median values, n indicates the number of patients available at each time point of analysis). On d +30 post transplant, the median NK-cell count was 305 CD16+/56+ cells/μl (range 30–1200).

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Table II.  Phenotype of T cells and proliferative capacity of peripheral blood mononuclear cells (PBMCs) during the first year post transplant (n = 20; owing to fatal complications, the number of evaluable patients decreased during follow-up as detailed in Table I).
(A) Median percentage of expression on CD3+ T cells.
 2 months4 months6 months8 months10 months12 months
CD11a (αL-integrin)98·999·499·899·999·899·7
CD2845·548·957·461·943·875·9
CD29 (β1-integrin)91·388·191867069·5
CD62L (l-selectin)10·731·639·961·738·555·3
HLA-DR77·686·983·666·971·757·2
(B) Proliferative capacity of PBMCs (median values).
 2 months4 months6 months8 months10 months12 monthsControl
  1. Results of proliferation assays are given as a difference in counts per minute (Δcpm), i.e. mean of sample triplicate counts minus mean of background triplicate counts. Each assay included a positive control using PBMCs from a healthy donor.

PHA2307233937 186131 17848 96169 51895 602
ConA1209753490264 56621 46726 27547 485
PMA/Iono7081162111022 12313 93811 28743 138
PWM1006461432814 68018 30018 71219 306
IL-2815714 300994426 21721 26326 03223 750
OKT3188583116268347757737638 731
SEA1236512910 53826 08815 92337 27453 942

T-cell reconstitution

All 20 patients developed a functioning T-cell compartment with respect to phenotype, proliferative capacity and TCR diversity. The increase in overall T-cell numbers (CD3+) is shown in Fig 2A. No significant difference was observed for CD3+ cell counts on d +60, +120, +180 and +360 between patients with and without T-cell addback (Table III). Further phenotypic characterization of T-cell subsets revealed that early reconstituting T cells within the CD4+ subset were predominantly primed, activated CD45RO+ cells and that the number of naive CD45RA+ T cells started to increase after 180 d (Fig 2B). This early emergence of CD45RO+ cells was found for both CD4+ and CD8+ subsets. However, because the assignment of naive versus primed T cells in CD45RA/CD45RO+ is less defined in CD8+ (Okumura et al, 1993), a further characterization of the CD8+ subset into cytotoxic effector (cytotoxic T lymphocyte; CTL), memory and naive cells based on the CD27/CD45RA phenotype was performed, as previously described (Hamann et al, 1997). This subset analysis revealed that a majority of CD8+ T cells (57·6%) expressed a memory phenotype (CD45RA/CD27+) in the early post-transplant phase, compared with 1·9% of cytotoxic (CD45RA+/CD27) and 17·9% of naive (CD45RA+/CD27+) CD8+ cells (n = 5, Fig 3).

image

Figure 2. (A) Increase in peripheral T-cell numbers (CD3+, ▪) after haploidentical SCT (median values, n indicates the number of patients available at each time point of analysis). T-cell counts > 100/μl were present after a median of 72 d (range 14–123). Cytotoxic, suppressor T-cells (CD8+, ●) recovered faster than T-helper cells (CD4+, ▴), resulting in an inverted CD4/CD8 ratio. For CD4+ T cells, the median time to reach > 100 cells/μl was 82 d (range 13–131); for CD8+ T cells, the median time to reach > 100 cells/μl was 74 d (range 15–123). (B) Percentage of naive (CD45RA+, ▪) versus memory (CD45RO+, ●) T cells within the CD4+ compartment after haploidentical SCT (median values, n indicates the number of patients available at each time point of analysis).

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Table III.  Mean counts ± SEM of CD3+ T cells/μl on d +60, +120, +180 and +360 in patients with (n = 11) and without DLI (n = 9) (owing to fatal complications, the number of evaluable patients decreased during follow-up as detailed in Table I).
 d +60d +120d +180d +360
  • *

    The non-parametric Mann–Whitney-U-test was used for statistical comparison.

  • ns, P > 0·05 was considered not significant. No significant difference in T-cell numbers existed between the two groups.

T-cell addback466 ± 250761 ± 226697 ± 2261418 ± 564
No T-cell addback609 ± 483766 ± 424959 ± 6921720 ± 583
ns*ns*ns*ns*
image

Figure 3. Phenotypic characterization of CD8+ T cells post transplant. CD8+ naive (CD45RA/CD27 double positive), CD8+ memory (CD27 single positive) and CD8+ CTL-type (CD45RA single positive) subpopulations are identified by expression of CD45RA and CD27. Representative examples on d +100 (A) and d +462 (B) post transplantation.

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The expression of integrins CD11a (αL-integrin, LFA-1 subunit), CD28, CD29 (β1-integrin), CD62L (l-selectin) and HLA-DR on T cells and the proliferative response of PBMCs to the panel of selected mitogens during the first post-transplant year are summarized in Table II.

T-cell repertoire complexity

T-cells were further analysed for expression of either the αβ- or γδ-T cell receptor (TCR) using flow cytometry. It was found that > 90% of T cells in the periphery expressed the αβ-TCR, although a transient expansion of γδ+ T cells (> 15%) was observed during the first 6 months in 9 out of 20 patients, followed by a return to normal levels within 30 d.

As both phenotypic and functional analysis of the CD3+ pool (Fig 2 and Table II) provide no information about the TCR diversity of the peripheral T-cell pool, we prospectively analysed TCR-repertoire complexity in 15 out of 20 patients using the spectratyping approach of Gorski et al (1994). Each Vβ family (Vβ1−24) was scored for the number of detectable bands per lane. During the first 100 d after transplant, all patients had a markedly skewed repertoire with a median of only three bands per Vβ family (Fig 4A). Oligoclonality was the predominant pattern with no preference indicated for smaller or larger fragment sizes. During d +100 to +200, scoring of the Vβ families marginally increased to a median of four bands per family. However, after d +200, in parallel with the increase in naive T-cell counts (Fig 2B), the score rapidly increased to a median of six bands per family with a normalization in fragment size distribution. After this marked expansion of the TCR repertoire, later follow-up revealed only minor improvements. Differences in the TCR-repertoire diversity in patient UPN 4013 between d +100 and +302 post transplantation are provided as a representative example in Fig 4B and C.

image

Figure 4. Development of the peripheral T-cell repertoire. (A) Box plot of number of bands per Vβ family (Vβ-1 through Vβ-24) within the first post-transplant year (n = 15 patients analysed). Line = 50th percentile (median), box = 75th and 25th percentile, error bars = 90th and 10th percentile, ▪ = mean value. (B,C) Representative example of the development of the peripheral T-cell repertoire in the first year after haploidentical SCT. Differences in the TCR-repertoire diversity in patient UPN4013 are shown between d +100 (B) and d +302 (C) post transplantation.

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B-cell reconstitution

Reconstitution of the B-cell compartment was seen in all patients following transplantation and paralleled T-cell reconstitution (Fig 5). Expression of CD10 was found to be high on the early emerging B cells, but decreased with time. Patients with relapse of a CD10+ leukaemia were excluded from this analysis. B cells expressed CD23 at high levels throughout the observation period in contrast to CD40 expression which increased with time. Increase in the peripheral B-cell count was mirrored by an increase in the proliferative response of PBMCs to PWM (Table II). All patients received intravenous immunoglobulins up to d +200 post transplantation, after which no supplementation was required. Immunoglobulin levels were 9·13 ± 4·21 between d 200–300, 10·32 ± 6·44 between d 300–400, and 10·05 ± 4·77 g/l after d 400 (mean ± SD).

image

Figure 5. Increase in peripheral B-cell numbers (CD19+, ▪) after haploidentical SCT (median values, n indicates the number of patients available at each time point of analysis). The median time to reach > 100 CD19+ cells/μl was 68 d (range 13–340).

Discussion

  1. Top of page
  2. Abstract
  3. Patients and methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. References

Immune reconstitution has been extensively studied following allogeneic bone marrow transplantation in both adult and paediatric patients (Lum, 1987; Foot et al, 1993; Godthelp et al, 1999), and after chemotherapy-induced myelosuppression (Mackall et al, 1995). In haploidentical SCT, however, three major factors may influence the pattern of immune reconstitution. First, HLA disparity of up to three major HLA-alleles between donor and host; second, the high number of stem cells infused to allow for stable engraftment; and third, the profound T-cell depletion by positive selection of donor peripheral stem cells to prevent GvHD.

Following reconstitution of granulocytes, NK cells were the first lymphoid cells to emerge during the first 4 weeks post transplantation. Recent evidence suggests that these early NK cells may play an important role in graft facilitation and graft-versus-leukaemia mediation without causing GvHD (Ruggeri et al, 1999). Thus, in the HLA-disparate transplant setting, NK cells may represent prime candidates for post-transplantation immunotherapy and are currently the subject of intense investigation.

Reconstitution of the T- and B-cell compartment was seen to occur after NK-cell reconstitution. B cells showed a continuous increase in numbers starting around d +60. On these early B cells, expression of CD10 represents an early differentiation marker. Functionality was demonstrated by a progressive increase in proliferative response to PWM and by the ability to self-maintain sufficient IgG levels after d +200.

T-cell reconstitution is the prime determinant in the successful outcome of SCT. Previous studies have shown two separate pathways of T-cell regeneration that contribute to the reconstitution of the T-cell compartment. First, mature T cells inoculated in the graft may, under antigenic stimulation, expand in the host (Mackall et al, 1996). The TCR diversity of these T cells is skewed and limited by the number of T cells in the initial inoculum. This pathway is generally referred to as peripheral expansion and predominates in the early phase following conventional bone marrow transplantation. This has been observed to be a persistent phenomenon in thymectomized hosts (Heitger et al, 1997) and in hosts with decreased thymic function, namely in adults or patients with intensive pretransplant chemotherapy (Mackall et al, 1995). Second, de novo maturation of naive T cells from bone marrow emigrants via passage through the thymus has been shown to occur after 6 months in children (Small et al, 1999). This pathway allows for de novo generation of T-cell responses and may play a part in correcting the initially skewed repertoire (Godthelp et al, 1999). Although it is a prerequisite for the restitutio ad integrum of the T-cell compartment, this pathway is limited to patients with residual thymic function and is therefore dependent upon a variety of factors, notably age and pretransplant chemotherapy (Mackall et al, 1997).

In our study, reconstitution of the T-cell compartment following SCT could be divided into two distinct phases. In the early phase, which was found to start as early as 2 months post transplant, expanded mature T cells of a predominantly CD45RO+ phenotype with limited TCR diversity were found to dominate the T-cell pool. As the minimal number of T cells in our spectratyping assays was > 5–10 times higher than that used by other investigators (Verfuerth et al, 2000), we argue that the limited TCR diversity observed in the early post-transplant phase is not caused by low numbers of T cells available for analysis. Furthermore, in titration experiments with decreasing numbers of T cells (range: 107−103 CD3+ cells) from healthy controls, we had not been able to detect skewing of the TCR repertoire when ≥ 104 T cells were used for the assay (data not shown). The early appearing T cells showed high expression of the activation marker HLA-DR and integrin subunits CD11a and CD29, but only a low capacity to proliferate upon stimulation with various mitogens. The majority of CD8+ T cells in this phase could be identified as memory cells. These early T cells were of donor origin, as demonstrated by chimaerism analysis of the magnetic-activated cell sorted (MACS) CD3+ T cells (data not shown). On the basis of their phenotype, these early T cells are presumably derived from graft-contaminating T cells that have expanded in the antigenic milieu of the host. It should be stressed that the number of co-transplanted T cells in this study was minimal (median 12·5 × 103 CD3+ cells/kg bw, range 1–117 × 103) and that 12 out of 20 patients had received OKT3 as part of their conditioning regimen for prevention of graft rejection. However, although improbable, it is still possible that these T cells may represent new T cells that have rapidly matured from the haematopoietic stem cell and, following antigenic stimulus in the host, acquired a memory phenotype (Murali Krishna & Ahmed, 2000). Although these mature, activated T cells were derived from a HLA-mismatched donor, they did not cause GvHD ≥ grade II and lacked a proliferative response to host cells upon in vitro rechallenge, but proliferated in response to third party cells (data not shown).

Donor leucocyte infusion (DLI) was given in 11 out of 20 patients. Although mixed chimaerism could be converted back to full donor chimaerism in 3 out of 11 patients (Bader et al, 1997), no significant difference in T-cell numbers on d +60, +120, +180 and +360 could be observed in patients with or without T-cell addback (Table III). This is in contrast to results from patients receiving DLI after partial T cell-depleted transplantations from related or unrelated donors (Small et al, 1999). It should be taken into account that the number of infused T cells was more than one log lower (2·5 × 104/kg bw) in our cohort. Despite the low T-cell dose used, one patient (UPN 4147) developed fatal GvHD IV after four T-cell addbacks. Thus, in the haploidentical setting, caution is warranted even with low numbers of unmanipulated donor T cells. The influence of DLI on TCR diversity is currently unknown. A recent study found a variable impact of DLI on T cell-repertoire diversity (Verfuerth et al, 2000). The authors reported a beneficial effect of DLI on TCR-repertoire diversity in only 3 out of 18 patients, whereas spectratypes became even more restricted post DLI in 6 out of 18 patients. In our study, the effect of DLI on TCR diversity was difficult to assess owing to the small number of patients and the fact that most DLIs were given before sufficient levels of peripheral T cells were detectable. Further studies, including comparison of sequence information of T-cell clones in donor and host, are needed to address this question.

A second wave of T-cell regeneration was found to start after 6 months resulting in T cells that expressed a naive phenotype (CD45RA+ for CD4+ or CD45RA+/CD27+ for CD8+ cells). This increase in the number of naive T cells encompassed both CD8+ and CD4+ fractions. A chronic deficiency of naive CD4+ T cells was not seen after haploidentical SCT in our paediatric cohort. These findings are in marked contrast to those from adult recipients of haploidentical grafts whose CD4+ counts remained below 100 and 200/μl for 10 and 16 months respectively (Aversa et al, 1998).

In parallel with the increase in naive T cells, the TCR-repertoire complexity and proliferative responses to mitogens improved. Although normal counts of peripheral T cells were not achieved by the end of the first post-transplant year, CDR3 size distribution of the TCR Vβ-chain had normalized. HLA-disparity with up to three HLA-mismatches did not seem to result in a major long-term skewing of the TCR repertoire. However, it is possible that minor alterations may exist that cannot be detected using primers spanning the Vβ–Cβ region. Further studies using high resolution PCR for analysis of the Vβ–Jβ junction are in progress.

The findings presented in this study have important implications for post-transplant immunotherapy. Caution is warranted using unmanipulated donor T cells for adoptive immunotherapy, as the threshold for induction of severe GvHD appears to be lower than in recipients of HLA-identical or partially mismatched stem cell grafts. Cellular strategies aimed at augmenting the graft-versus-leukaemia effect will potentially focus on the role of NK cells during the immediate post-transplant phase. Alternatively, vaccination-based approaches in the HLA-disparate setting may not be successful if applied before 6 months post transplant. An improvement during this 6-month period in the specific T-cell immunity may be achieved by approaches designed to increase the repertoire diversity of the peripheral T-cell pool. These strategies may include adoptive transfer of either ex vivo-generated T cells with low alloreactivity (Koh et al, 1999) or antigen-specific T cells (Reusser et al, 1997), or T cells that have been engineered to carry a suicide gene as a safety switch (Lal et al, 2000). The striking difference in reconstitution of naive T cells between children and adults (Aversa et al, 1998) after haploidentical SCT underlines the importance of thymic function for rapid and complete restoration of immune function (Weissman & Shizuru, 1999). Although residual thymic function seems to be present even in elderly patients (Douek et al, 1998), thymic output clearly needs improvement in stem cell recipients, particularly in adults. Thymic maturation may be enhanced by the administration of specific cytokines (Abdul-Hai et al, 1996). Alternatively, recent advances in transplantation of thymic tissue have raised new possibilities, providing this could be accomplished in tandem with SCT (Markert et al, 1999).

In summary, the data show that the transplantation of megadoses of haploidentical, haematopoietic stem cells after myeloablative therapy resulted in a rapid and sustained engraftment followed by a fast recovery of the NK-cell compartment around d +30. T and B cells started to reconstitute as early as d +70. The surprisingly low incidence of fatal infectious complications in our paediatric cohort indicates that the early emerging NK-cell compartment, together with the limited T-cell repertoire, seem to have sufficient functional capacity for controlling infections in a majority of patients. Results from this study are highly suggestive that haploidentical peripheral SCT should be considered in paediatric patients in urgent need of a transplantation, but who lack a conventional HLA-matched donor.

Acknowledgments

  1. Top of page
  2. Abstract
  3. Patients and methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. References

We would like to thank the staff of the BMT unit and outpatient department, as well as the technical staff of the stem cell processing unit, at the University Children's Hospital, Tuebingen, for their excellent and dedicated patient care.

M.E. is the recipient of a post-doctoral fellowship awarded by the Else Uebelmesser Foundation for Cancer Research (1.3–0415.221.18–03/97). This work was supported by a grant from the fortuene program of the University of Tuebingen (#587/1999) and by the Deutsche Forschungsgemeinschaft SFB 510-C4 (to P.G.S. & D.N., 1997).

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  6. Acknowledgments
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