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Keywords:

  • haematopoietic stem cell transplantation;
  • malignant disease;
  • lymphocytes;
  • dendritic cells;
  • alemtuzumab

Summary

  1. Top of page
  2. Summary
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. Author contributions
  8. Funding
  9. Conflict of interest
  10. References

Graft-versus-leukaemia (GvL) and graft-versus-host disease (GvHD) are both caused by alloreactive lymphocytes. We previously reported that GvHD correlated with higher numbers of effector CD4 T cells and Natural Killer cells early after allogeneic transplantation using a regimen comprising fludarabine, busulphan and alemtuzumab. Here, we assessed immune cell subset recovery in these patients in the context of early myeloid malignant disease relapse. Despite the close relationship between the GvL and GvHD immune responses, rapid recovery of lymphocyte subsets was not associated with protection from disease relapse. These results indicated that GvL may be weak in this treatment setting for patients with myelodysplastic syndromes and acute myeloid leukaemia. Consistent with low GvL activity, we previously reported that mixed T cell chimaerism had no detrimental effect on relapse in this treatment setting and instead correlated with better outcome because of reduced GvHD incidence. We now report that patients with significantly higher lymphocyte numbers prior to transplantation subsequently maintained the mixed T cell chimaeric state. This pre-transplant profile, together with absence of the early post-transplant signature indicative of GvHD predisposition, could potentially be used to identify patients suitable for early withdrawal of immunosuppression and prophylactic donor leucocyte infusion to boost GvL activity.

Although allogeneic haematopoietic stem cell transplantation (HSCT) is a curative therapy for haematological malignancies, disease relapse and graft-versus-host disease (GvHD) are major causes of morbidity and mortality following treatment. Two aspects of the procedure contribute to the elimination of malignant cells. They are ablative therapy immediately prior to transplant and the post-transplant donor immune response to residual malignant cells known as the graft versus leukaemia (GvL) effect (Horowitz et al, 1990). Both the beneficial GvL and the detrimental immune response that causes GvHD result from donor lymphocyte recognition of allogeneic patient cells. Control of clinical GvHD is achieved by immunosuppression, which curbs all forms of alloreactivity. Therefore the desire to preserve GvL has to be balanced with the need to prevent GvHD (Barber & Madrigal, 2006).

Myeloid malignancies predominantly affect the elderly. Dose-attenuated HSCT protocols, known as reduced intensity conditioning (RIC) regimens, have been developed to enable treatment of these patients (Slavin et al, 1998). The protocols do not aim for complete ablation of the patient’s haematopoietic cells prior to transplant. Instead, donor cell engraftment is dependent on immunosuppression, which also serves to control GvHD. However, there is greater dependence on GvL for eradication of residual malignant cells (McSweeney et al, 2001) so judicious use of immunosuppression is essential.

RIC regimens frequently result in a prolonged period of mixed donor and patient chimaerism after transplantation. In some settings, slow attainment of full donor chimaerism within the T cell population is associated with disease relapse (Childs et al, 1999), (Baron et al, 2005), (Mohty et al, 2007) attributed to inadequate GvL activity. A prolonged state of mixed T cell chimaerism is therefore used to indicate a need for prophylactic donor leucocyte infusion (DLI) in order to boost GvL (Dey et al, 2003), (Peggs et al, 2004) despite the attendant risk of promoting GvHD. However, some studies have not found an association between rapid full donor T cell chimaerism and disease remission (Mattsson et al, 2001), (Montero et al, 2005), (Lim et al, 2007). This ambiguity complicates decisions about use of prophylactic DLI. Variable correlation of mixed T cell chimaerism with disease relapse probably reflects differences in RIC regimens and immunosuppression used and differing susceptibility of malignancy types to GvL activity. The study of homogeneous cohorts of patients is therefore required to understand the relative role of disease type, induction ablative therapy prior to transplantation and the post-transplant GvL effect on disease relapse.

We have recently reported analysis of the relationship between immune cell subset reconstitution and incidence of GvHD in a group of 25 patients presenting with acute myeloid leukaemia (AML) or myelodysplastic syndromes (MDS) and treated with a uniform allogeneic RIC regimen (Matthews et al, 2009). The protocol comprises the alkylating agent busulphan, lympho-depletion with fludarabine plus the anti-CD52 monoclonal antibody alemtuzumab (also known as Campath 1H) and post-transplant GvHD prophylaxis with ciclosporin for 2 months (Ho et al, 2004). Prolonged mixed T cell chimaerism is common after treatment but is not associated with increased relapse (Lim et al, 2007). GvHD break-through was found to correlate with increased numbers of donor-derived effector CD4 T cells and a relative deficit of regulatory CD4 T cells early after transplant (Matthews et al, 2009). We now report analysis of the recovery of immune cell subsets for these patients in the context of disease relapse.

Materials and methods

  1. Top of page
  2. Summary
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. Author contributions
  8. Funding
  9. Conflict of interest
  10. References

Patients and transplantation protocol

Twenty-five patients with myeloid malignancies that underwent allogeneic HSCT between September 2005 and September 2006 at King’s College Hospital were studied. Preparation for transplantation comprised fludarabine (30 mg/m2 intravenous daily from day -9 to day -5), busulphan (3·2 mg/kg intravenous in four divided doses daily from day -3 to day -2), and alemtuzumab (20 mg/day intravenous on days -8 to day -4). All patients received unselected allogeneic peripheral blood progenitor cells that were infused on day 0. Intravenous ciclosporin was started from day -1 at a dose adjusted to achieve plasma trough levels of 150–200 ng/l for all patients. Oral ciclosporin was substituted when a good oral intake was achieved and rapidly tapered to discontinuation from day 60 in the absence of GvHD. Recombinant granulocyte-colony stimulating factor was administered subcutaneously or intravenously from day +7 to time of neutrophil engraftment. Therapy for GvHD involved ciclosporin, steroids (prednisolone initiated at a dose of 1 mg/kg) or psoralen ultra violet A (PUVA). Clinical data was censored at May 2007. Peripheral blood samples were collected immediately prior to preparation for transplant and at days 30, 60, 90, 180, 270 and 360 after transplantation. Samples of peripheral blood were also collected from 11 healthy age-matched individuals (median age 51 years, range 41–56 years). King’s College Hospital Research Ethics Committee approved the study and written informed consent was obtained from all participants.

Immunophenotypic analysis

Lymphocyte subsets and dendritic cells were enumerated in whole peripheral blood using fluorochrome-labelled monoclonal antibodies to CD4 (clone SK3), CD8 (SK1), CD25 (2A3), CD27 (M-T271), CD45RO (UCHL1), CD56 (B159), HLA-DR (L243) CD11c (B-ly6) (BD Biosciences, San Jose, CA, USA) and CD3 (OKT3), CD19 (HIB19) CD123 (6H6) (eBioscience Inc., San Diego, CA, USA) and CD1c (BDCA-1), CD303 (BDCA-2) (Miltenyi Biotec, Bergish Gladbach, Germany). Cells in 200 μl peripheral blood were stained for surface markers and erythrocytes were removed using FACS lysing solution (BD Biosciences). Eight-colour analysis was performed by flow cytometry using a BD FACSCanto II (BD Biosciences) and results analyzed with FlowJo software (Tree Star Inc., Ashland, OR, USA). Natural killer (NK) cells were defined as CD3 CD56+. B cells were defined as CD19+. CD3+ CD4+ and CD3+ CD8+ T cells subsets were defined as CD45RO CD27+ naïve, CD45RO+ CD27+ memory, CD45RO+ CD27 effectors and CD45RO CD27 terminal effectors. Myeloid dendritic cells were defined as CD1c+ CD11c+ HLA-DR+ CD19. Plasmacytoid dendritic cells were defined as CD303+ CD123+ HLA-DR+. Cell subset numbers were calculated from percentage values based on absolute lymphocyte and monocyte counts of the blood sample obtained using an automated leucocyte counter.

Chimaerism analysis

The relative proportion of donor and recipient chimaerism in the lymphoid and myeloid lineages was performed using isolated peripheral blood CD3+ T cells and CD15+ granulocytes, respectively, and determined as part of routine clinical care using methods described previously (Lim et al, 2007). To assess chimaerism in dendritic cell populations, peripheral blood mononuclear cells were purified by density gradient centrifugation on Lympholyte-H (Cedarlane Laboratories, Burlington, ON, Canada) and populations were isolated using a FACSAria sorter after surface staining with CD1c CD11c HLA-DR CD19 to identify myeloid dendritic cells or CD303 CD123 HLA-DR to identify plasmacytoid dendritic cells. Purity of the populations was >95%. Cells were lysed with proteinase K (0·2 mg/ml in 1 mmol/l EDTA, 20 mmol/l Tris-HCl pH 8·0, 1% Tween-20) to release DNA. Polymerase chain reaction amplification of informative alleles from 15 polymorphic short tandem repeat loci and the sex-determining amelogenin loci (Powerplex®; Promega Corp, Madison, WI, USA) was performed. Products were separated by capillary electrophoresis using an ABI 3130XL DNA sequencer. Results were analyzed using Genemapper 4.0 software (Applied Biosystems, Foster City, CA, USA) and donor and recipient composition was quantified based on area under peaks. The sensitivity of this methodology was previously shown to be 5% by cell dilution studies (Lim et al, 2007). therefore patients were considered full donor chimaeric if the percentage of donor cells was >95%.

Statistical analysis

Statistical analysis was performed using the Statistical Package for the Social Science (spss) for Windows software, version 14.0 (SPSS Inc. Chicago, IL, USA). Categorical data was evaluated using the Chi-square test. Non-parametric numerical data was evaluated using the Mann-Whitney U-test. Differences were considered statistically significant when P < 0·05.

Results

  1. Top of page
  2. Summary
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. Author contributions
  8. Funding
  9. Conflict of interest
  10. References

Twenty-five patients (median age 53; range 34–70 years) with myeloid malignancies were studied. All patients had achieved complete morphological remission of disease, defined as <5% blasts in the bone marrow prior to transplantation. Eight patients (32%) experienced disease relapse (>5% blasts in the bone marrow) at a median of 10 months (range 6–16 months). Disease presentation was refractory anaemia with excess blasts (RAEB) (three patients), AML (three patients), and myeloproliferative disorder (MPD) (two patients). Patient characteristics are shown in Table I. A comparison of immune cell subset composition and tempo of recovery in peripheral blood after transplantation with disease relapse was performed.

Table I.   Characteristics of patient cohort segregated by disease relapse status.
 No Relapse (n = 17)Relapse (n = 8)P value
  1. GvHD, graft- versus -host disease; NS, not significant.

  2. *HLA (human leucocyte antigen) match indicates identity at 10 alleles (HLA-DP type not determined).

  3. †HLA mismatched transplants were n = 1 HLA-A, n = 1 HLA-C, n = 1 HLA-A&C, n = 1 DRB1, n = 2 DQB1.

Patient
 Age, median years (range)58 (43–70)50 (34–66)NS
 Sex (male/female)11/6 4/4NS
Donor
 Unrelated HLA matched* 9 3NS
 Unrelated HLA mismatched† 3 3 
 Sibling HLA matched 5 2 
 Sex mismatched 7 5NS
Diagnosis
 Myelodysplastic syndrome 8 3NS
 Acute myeloid leukaemia 7 3 
 Myeloproliferative disorders 1 2 
 Chronic myeloid leukaemia 1 0 
Disease stage
 Early10 10.04
 Advanced 7 7 
Disease status at transplant
 Complete remission17 8 
CD34 cell dose × 106/kg, median (range) 7 (2–20) 6 (2–17)NS
GvHD (acute and/or chronic) 6 3NS
 Acute (Grade II–IV) 5 2 
 Chronic 1 3 
Cytomegalovirus reactivation after transplantation11 4NS
Full donor T cell chimaerism by day 90 6 5NS
Mixed T cell chimaerism > day 9011 3 

Correlation of immune cell subset recovery early after transplantation with relapse

Findings at the early day 30 and day 60 time points are shown in Table II. We have previously reported that numbers of effector CD4 T cells and NK cells were significantly higher early (day 30) after transplantation in patients that subsequently developed GvHD (Matthews et al, 2009). In contrast, analysis of immune recovery in the context of disease relapse showed no significant differences within the lymphocyte subsets at day 30 in patients that relapsed and those that did not. Numbers of myeloid dendritic cells were significantly lower at day 30 in patients that relapsed (P = 0·01). This was not a general feature of the myeloid lineage because monocyte numbers were very similar in both groups of patients (Table II). There was also a trend towards lower numbers of plasmacytoid dendritic cells at day 30 in the relapse group (P = 0·07) indicating a relative deficiency of dendritic cell types early after transplantation in patients that subsequently experienced disease relapse. Analysis of chimaerism in dendritic cell populations showed these cells were predominantly of donor origin by day 30. Dendritic cells from four patients that did not experience disease relapse were studied. Both myeloid and plasmacytoid dendritic cells were 100% donor for three patients and one patient was 79% donor within the myeloid dendritic cell population and 100% donor within the plasmacytoid dendritic cell population. Due to low dendritic cell numbers at day 30 among patients in the relapse group, chimaerism analysis was only successful for one individual (Patient 6) who was 100% donor in both dendritic cell populations.

Table II.   Univariate analysis of cell subset numbers and disease relapse at day 30 and 60 after transplantation.
Cell TypeDay 30 after transplantationDay 60 after transplantation
Relapse (n = 6)No relapse (n = 13)P valueRelapse (n = 7)No relapse (n = 11)P value
  1. Median cell number × 109/l blood (range).

  2. Bold values indicate P < 0·05.

Lymphocytes0·315 (0·1–0·62)0·23 (0·06–0·81)0·520·77 (0·29–2·08)0·41 (0·06–1·82)0·04
CD4 T cells0·033 (0·003–0·15)0·013 (0·001–0·17)0·280·06 (0·005–0·298)0·024 (0·002–0·24)0·43
CD4 naiveNone detectedNone detected 0 (0–0·0007)0 (0–0·0005)0·72
CD4 memory0·008 (0·001–0·11)0·007 (0·0006–0·089)0·700·026 (0·003–0·156)0·017 (0·001–0·081)0·54
CD4 effector0·024 (0·002–0·04)0·006 (0·0004–0·084)0·150·028 (0·001–0·142)0·009 (0·001–0·141)0·48
CD8 T cells0·052 (0·0003–0·293)0·001 (0·0003–0·184)0·110·156 (0·022–0·917)0·027 (0·0006–0·144)0·02
CD8 naiveNone detectedNone detected 0·0045 (0·001–0·024)0·0001 (0–0·0025)0·00
CD8 memory0·017 (0·0001–0·074)0·003 (0·0002–0·096)0·570·09 (0·007–0·271)0·009 (0·0005–0·106)0·06
CD8 effector0·034 (0·0002–0·189)0·001 (0·0001–0·086)0·180·055 (0·01–0·438)0·006 (0–0·047)0·01
NK cells0·085 (0·022–0·219)0·075 (0·005–0·186)0·470·17 (0·046–0·35)0·094 (0·0005–0·67)0·60
B cells0·0006 (0–0·118)0·0009 (0–0·03)0·830·049 (0·003–0·632)0·002 (0–0·371)0·06
Plasmacytoid dendritic cells0·0005 (0·0001–0·0019)0·0019 (0–0·0065)0·070·00046 (0–0·0017)0·00005 (0–0·0053)0·25
Myeloid dendritic cells0·0001 (0–0·0014)0·0011 (0·0003–0·0096)0·010·0025 (0–0·0042)0·0004 (0–0·0069)0·32
Monocytes0·38 (0·15–1·93)0·395 (0·01–1·05)0·890·62 (0·08–0·75)0·505 (0·09–0·85)1·00

By day 60 after transplantation, correlations between immune cell subsets and disease relapse were detected in populations of the lymphoid lineage (Table II). Almost twice as many lymphocytes were present in peripheral blood of patients in the relapse group at a median of 0·77 × 109/l compared to 0·41 × 109/l for patients that did not relapse (P = 0·04). This was particularly striking for the CD8 T cell population (P = 0·02) and was evident in all subsets (naïve, memory and effector). The CD8 T cells had an activated phenotype with a median of 0·118 × 109/l HLA-DR+ CD8 T cells (range 0·021–0·863) in the relapse group compared to 0·022 × 109/l (range 0·0005–0·142) in the no relapse group (P = 0·02). Cell numbers within other lymphocyte subsets were also increased in the relapse group (Table II) and approached significance for the B cell population (P = 0·06). No differences in monocyte or dendritic cell numbers at day 60 were detected between the patient groups.

Correlation of immune cell subset recovery beyond day 100 after transplantation with relapse

Given the variation in time of disease relapse (range 6–14 months), lymphocyte, CD8 T cell and myeloid dendritic cell population dynamics is shown for individual patients for the first year after transplantation (Fig 1). With the exception of Patient 7, where engraftment was poor, immune cell subset recovery was robust until at least day 90, with numbers tracking and often surpassing those for the no relapse patient group. Reasonable recovery was even seen within the myeloid dendritic cell population despite the significantly slower start at day 30. Populations typically began a marked decline from day 180 whereas expansion continued in the no relapse patient group, approaching the normal range by 1 year after transplantation. The fall in lymphocyte numbers was seen prior to diagnosis of relapse and before any chemotherapy treatment. A dramatic spike in CD8 T cell numbers was seen in Patients 10 and 12 preceded by cytomegalovirus (CMV) reactivation (Fig 1). To evaluate whether the correlation between higher CD8 T cell numbers at day 60 in the relapse patient group (Table II) is a consequence of skewing by these two patients, the day 60 statistical analyses was repeated excluding these individuals. A significant difference was retained with a median of 0·156 × 109/l CD8 T cells (range 0·022–0·733) in the relapse group compared to 0·027 × 109/l (range 0·0006–0·144) in the no relapse group (P = 0·04). There was no correlation between higher CD8 T cell numbers and CMV reactivation prior to day 90 (data not shown) or GvHD incidence (Matthews et al, 2009) in this patient cohort. Also there were no reported fungal, bacterial or viral (Epstein-Barr virus or adenovirus) infections for patients in the relapse group. Together, these observations support specific association of higher CD8 T cell numbers at day 60 with relapse.

imageimageimage

Figure 1.  Reconstitution of total lymphocytes (upper panels), CD8 T cells (middle panels) and myeloid dendritic cells (lower panels) in patients that had relapse of myeloid disease after allogeneic HSCT. Mean and SEM are shown for patients that did not relapse. Horizontal dotted lines enclosing grey boxes represent median and inter-quartile range for eleven age-matched healthy volunteers. Salient clinical events are indicated for each patient. T cell chimaerism status at day 90 is indicated by FDC (full donor chimaerism) or MC (mixed chimaerism). GvHD therapy with ciclosporin, steroids or PUVA is shown. Patient 6: male with HLA mismatched (HLA-DQB1) unrelated female donor. Patient 7: male with HLA-matched unrelated male donor. Patient 10: female with HLA-mismatched (HLA-A) unrelated male donor. Patient 11: male with HLA-matched sibling male donor. Patient 12: female with HLA-matched unrelated male donor. Patient 14: female with HLA matched sibling male donor. Patient 26: female with HLA-matched unrelated male donor. An HLA match for transplants involving unrelated donors indicates identity for at least 10 alleles because HLA-DP type was not determined. FDC, full donor chimaerism; MC, mixed chimaerism; DLI, donor lymphocyte infusion; CMV, cytomegalovirus; GvHD, graft versus host disease; PUVA, psoralen ultra violet A; AML, acute myeloid leukaemia; MPD, myeloproliferative disease; RAEB, refractory anaemia with excess blasts.

Correlation of immune cell subset recovery with T cell chimaerism status

We have previously reported that a prolonged period of mixed T cell chimaerism after the fludarabine, busulphan, alemtuzumab RIC HSCT regimen was not associated with increased incidence of disease relapse (Lim et al, 2007). This finding was reiterated in the current study. Indeed five of the eight patients in the relapse group had full donor status by day 90 whereas mixed T cell chimaerism at day 90 was more prevalent within the no relapse group (11 of 17). Given that mixed T cell chimaerism does not have an adverse impact on risk of disease relapse in this setting but is associated with reduced incidence of detrimental GvHD, survival rates were better in these patients (Lim et al, 2007). To attempt early identification of patients likely to follow this favourable clinical course, a comparison of immune cell subset composition immediately prior to transplant and at day 30 after transplantation with T cell chimaerism status at day 90 was performed and the findings are shown in Table III. Prolonged mixed T cell chimaerism was associated with significantly higher numbers of lymphocytes in the peripheral blood of patients prior to transplantation (P = 0·04). Higher numbers were seen in all lymphocyte subsets. At day 30 after transplantation, the opposite association was seen, with significantly lower numbers of lymphocytes in patients with prolonged mixed T cell chimaerism (P = 0·001), and the trend was evident in all lymphocyte subsets. The mixed T cell chimaerism group included patients with regressive chimaerism due to poor engraftment. Statistical analysis was therefore repeated including only the 5 patients that maintained stable mixed T cell chimaerism, defined as >70% donor at day 90 (Lim et al, 2007). This subset also had significantly lower numbers of lymphocytes at day 30 with a median of 0·14 × 109/l lymphocytes (range 0·07–0·29) compared to 0·45 × 109/l (range 0·18–0·81) in the rapid full donor T cell chimaerism group (P = 0·03). Therefore those patients with mixed T cell chimaerism were characterized by better immune status prior to transplant and slower expansion of lymphocyte populations after transplantation.

Table III.   Univariate analysis of cell subset numbers and T cell chimaerism status.
Cell TypePatient prior to transplantationDay 30 after transplantation
Rapid full donor T cell chimaerism (n = 11)Prolonged mixed T cell chimaerism (n = 14)P valueRapid full donor T cell chimaerism (n = 8)Prolonged mixed T cell chimaerism (n = 11)P value
  1. Median cell number × 109/l blood (range).

  2. Bold values indicate P < 0·05.

Lymphocytes0·25 (0·03–1·11)0·9 (0·11–3·26)0·040·45 (0·18–0·81)0·12 (0·06–0·44)<0·01
CD4 T cells0·114 (0·008–0·455)0·257 (0·038–0·688)0·040·038 (0·013–0·17)0·007 (0·001–0·061)0·01
CD4 naive0·005 (0·0006–0·137)0·064 (0·001–0·332)0·02None detectedNone detected 
CD4 memory0·048 (0·004–0·283)0·108 (0·033–0·289)0·040·009 (0·007–0·11)0·004 (0·001–0·027)0·03
CD4 effector0·017 (0·001–0·081)0·039 (0·009–0·183)0·040·027 (0·006–0·084)0·005 (0–0·033)0·01
CD8 T cells0·089 (0·007–0·392)0·25 (0·014–1·393)0·050·039 (0·0007–0·293)0·001 (0·0003–0·041)0·05
CD8 naive0·008 (0·0008–0·125)0·032 (0·004–0·188)0·02None detectedNone detected 
CD8 memory0·03 (0·003–0·263)0·108 (0·006–0·589)0·040·014 (0·0002–0·096)0·002 (0·0001–0·02)0·13
CD8 effector0·011 (0·001–0·185)0·05 (0·001–0·511)0·090·05 (0·0005–0·189)0·001 (0·0001–0·018)0·05
NK cells0·026 (0·0002–0·134)0·07 (0·0004–0·236)0·080·131 (0·055–0·22)0·038 (0·005–0·114)<0·01
B cells0·002 (0·0003–0·18)0·051 (0·0006–0·35)0·180·006 (0–0·118)0·0005 (0–0·005)0·07
Plasmacytoid dendritic cells0·0005 (0–0·0024)0·0006 (0–0·0027)0·380·0009 (0–0·0054)0·0015 (0–0·0065)0·44
Myeloid dendritic cells0·0008 (0–0·0022)0·0019 (0–0·009)0·030·0006 (0–0·0023)0·001 (0–0·0094)0·31
Monocytes0·295 (0·09–0·8)0·52 (0·08–0·95)0·280·41 (0·01–1·93)0·365 (0·13–1·05)0·83

Discussion

  1. Top of page
  2. Summary
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. Author contributions
  8. Funding
  9. Conflict of interest
  10. References

In our previous study of these patients, we reported that higher numbers of effector CD4 T cells and NK cells in peripheral blood early after transplantation was associated with GvHD (Matthews et al, 2009). Despite the close relationship between GvHD and GvL alloresponses, our current study did not find a correlation between rapid recovery of any lymphocyte subsets and reduced incidence of malignant disease relapse. These results suggest that GvL immunity may be weak or ineffective in the context of patients that present with MDS or the related conditions AML or MDS/MPD and treated with a uniform regimen based on fludarabine and busulphan with alemtuzumab.

Limited GvL efficacy provides an explanation for the lack of correlation between T cell chimaerism status and disease relapse in this treatment regimen that we reported previously (Lim et al, 2007). If donor lymphocytes do not produce an effective GvL response, there would be no benefit associated with rapid progression to full donor chimaerism. Instead, this course becomes disadvantageous because donor lymphocytes cause GvHD. Hence, in this setting, mixed T cell chimaerism correlates with improved outcome (Lim et al, 2007).

By definition, mixed chimaerism after transplantation is dependent upon the contribution of immune cells from the patient. Consequently we found that patients with prolonged mixed T cell chimaerism had significantly higher numbers of lymphocytes immediately prior to conditioning and transplantation. Patient immune competence will be determined by previous treatment and disease type. It is therefore not a factor that can be manipulated to promote the favourable mixed T cell chimaeric state. However, knowledge of patient immune status prior to transplantation may enable prediction of those likely to follow a good clinical course and those at risk of complications.

Some studies in other treatment settings have found early vigorous lymphocyte recovery is associated with reduced incidence of myeloid malignant disease relapse (Powles et al, 1998), (Kumar et al, 2001), (Kim et al, 2004), (Savani et al, 2007). In contrast, we found slower and steady lymphocyte recovery was best for the treatment regimen studied. We noted that slower reconstitution was also exhibited by patients with stable mixed T cell chimaerism. Studies using mouse models show maintenance of mixed haematopoietic chimaerism can occur when mutual donor and recipient immune tolerance exists (Sykes et al, 1997). However, mixed chimaerism may not always reflect a state of tolerance. Slower lymphocyte recovery in patients with mixed T cell chimaerism could be due to low levels of host versus graft and graft versus host activity that mutually restrain expansion. Presence of alloreactivity is consistent with our previous finding that patients with mixed T cell chimaerism can still experience GvHD (Matthews et al, 2009). Others have also reported that mixed T cell chimaerism does not necessarily provide absolute protection from GvHD (Baron et al, 2004). These observations illustrate the importance of understanding how immune reconstitution influences clinical outcome for individual treatment regimens.

Alemtuzumab has a major impact on immune recovery after transplantation. The antibody causes extensive depletion of all lymphocyte subsets which reduces the incidence and severity of GvHD (Chakraverty et al, 2002). This facilitates transplantation of patients previously considered ineligible for treatment due to inability to cope with GvHD. However, the use of alemtuzumab is associated with diminished immunity to infections (reviewed (Chakrabarti et al, 2004)) and our findings suggest that GvL activity is also reduced. This could be due to both the profound lymphopenia early after transplantation and deficiencies within the recovering lymphocyte population. We found that naïve T cells were absent in all patients until at least 6 months after transplantation using alemtuzumab (Matthews et al, 2009). Consequently the alloreactive T cell repertoire early after transplantation is limited to cross-reactivity by memory T cells specific for previously encountered pathogens, which may lack leukaemia specificities. A greater proportion of NK cells express high levels of CD56, consistently comprising 25–35% of the NK cell population during the first year after transplantation compared to 2·9% in healthy volunteers (data not shown). These NK cells are reportedly abundant producers of cytokines but have weak cytolytic activity (Cooper et al, 2001), which is an important potential GvL effector mechanism. Regulatory T cells can suppress alloreactivity and an association between increased frequencies of regulatory CD4 T cells and higher incidence of disease relapse after HSCT for chronic myeloid leukaemia has been reported (Nadal et al, 2007). Regulatory CD4 T cells may therefore cause suppression of GvL. However, we did not detect a correlation between these cells and disease relapse in our patient cohort (data not shown) despite finding a relative deficiency of regulatory CD4 T cells in the context of GvHD (Matthews et al, 2009).

The spike of CD8 T cell numbers seen at day 60 in patients that subsequently relapsed may indicate there is an immune response to re-emerging malignant myeloid cells. However, any GvL activity evidently fails to control disease. It has recently been reported that T cell numbers are increased in newly diagnosed patients with AML, but their function is abnormal (Le Dieu et al, 2009). Leukaemia-specific T cells have been detected in patients after allogeneic HSCT for myeloid diseases (Molldrem et al, 2000), (Rezvani et al, 2003), (Melenhorst et al, 2009) but they can be susceptible to rapid replicative senescence (Beatty et al, 2009).

We found a correlation between higher numbers of myeloid dendritic cells in peripheral blood early after transplantation and no disease relapse. The association was probably not due to rapid engraftment of the myeloid lineage because there was no relationship between relapse and CD34 stem cell content of the graft (Table I) or monocyte recovery (Table II). Dendritic cells are heterogeneous antigen presenting cells that are essential for both induction and regulation of immune responses (Ueno et al, 2007). Blood dendritic cells of both myeloid origin and plasmacytoid dendritic cells were increased early after transplantation in patients that did not relapse, which suggests that rapid recovery of the antigen presenting cell population correlates with better outcome. Others have also reported association of higher numbers of dendritic cells early after transplantation with reduced incidence of disease relapse and better clinical course (Reddy et al, 2004), (Talarn et al, 2007). This may result from the promotion of GvL activity by dendritic cells. A mouse model showed this was most effective when the dendritic cells are of host origin (Reddy et al, 2005) which can directly present leukaemia antigens to T cells. However, we found that dendritic cells were predominantly of donor origin. Rapid replacement of dendritic cells in peripheral blood is common after HSCT (Auffermann-Gretzinger et al, 2002), particularly in treatment regimens that incorporate alemtuzumab (Klangsinsirikul et al, 2002), (Buggins et al, 2002). Although donor dendritic cells can cross present leukaemia antigens, murine studies indicate the GvL effect produced is weak and only effective at disease control when malignant cell burden is low (Reddy et al, 2005). An alternative explanation for our detection of an association between higher dendritic cell numbers early after transplantation and no relapse may be that these patients have a healthier immune milieu that is better able to sustain mature dendritic cells transferred with the graft in the period prior to recapitulation of dendritic cell myelopoiesis.

Our study showed no relationship between rapid recovery of donor lymphocytes and protection from disease relapse. The potential existence of qualitative differences in lymphocyte composition between patients that relapse and those that do not relapse needs to be examined. However, we have shown there are deficiencies within the recovering immune cell population, which suggests that patients may benefit from DLI to supplement the immune repertoire. This is particularly pertinent for patients with advanced disease that are at greater risk of relapse (Table I). Therapeutic use of DLI to treat relapsed MDS or AML is not very effective (Kolb, et al 1995), (Collins et al, 1997). This has led to prophylactic use of DLI (Schmid et al, 2006), which aims to boost GvL for disease control when leukaemia burden is low. Administration of prophylactic DLI in the treatment regimen we have studied was associated with reduced incidence of relapse, but at the expense of increased GvHD (Lim et al, 2007). Immune signatures indicative of lower inherent risk of GvHD could help guide clinical decisions. Absence of the early CD4 T cell signature indicative of GvHD predisposition (Matthews et al, 2009) combined with identification of patients likely to undergo slower and steady immune recovery based on immune competence before transplantation could perhaps be used to select patients for early discontinuation of immunosuppression and prophylactic DLI.

Acknowledgements

  1. Top of page
  2. Summary
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. Author contributions
  8. Funding
  9. Conflict of interest
  10. References

We thank Gary Warnes (Institute of Cellular and Molecular Medicine, The Royal London and Queen Mary’s Hospital, London) and Ayad Eddaoudi (Cancer Research UK, London Research Institute, London) for cell sorting by flow cytometry.

Author contributions

  1. Top of page
  2. Summary
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. Author contributions
  8. Funding
  9. Conflict of interest
  10. References

KM designed study, planned and performed experiments, analyzed data and edited manuscript. ZL designed study, collected patient samples and clinical data, and edited manuscript. LP planned and performed experiments and analyzed data. AP and JAM contributed to research discussion and reviewed manuscript. GJM designed study and contributed to research discussion. LDB designed study, planned and performed experiments, analyzed data and wrote manuscript.

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  6. Acknowledgements
  7. Author contributions
  8. Funding
  9. Conflict of interest
  10. References
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