Graft Rejection

Successful treatment of incipient graft rejection with donor leukocyte infusions, further proof of a graft versus host lymphohaemopoietic effect

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Summary:

Graft rejection is a major cause of treatment failure after T-cell-depleted stem cell transplantation (TCD-SCT) and remains a therapeutic challenge. Donor leukocyte infusions (DLIs) have an efficient graft versus host effect, which has been successfully used to treat recipient relapses. We hypothesized that this effect could be exploited to counteract the host versus graft reactions responsible for graft rejection. We report two adult patients with haematological malignancies who underwent sex-mismatched TCD-SCT from HLA-identical sibling donors. Peripheral blood (PB) counts and bone marrow (BM) cellularity were studied on a serial basis. Sequential chimaerism and minimal residual disease analysis were performed by FISH on PB and BM samples as well as on leukocyte lineages (T and B lymphocytes and myeloid cells) purified from PB using immunomagnetic technology. Both patients were diagnosed with incipient graft rejection 2–3 months after engraftment, based on persistently decreasing PB counts and BM cellularity together with the observation of decreasing mixed chimaerism (increasing percentage of recipient cells), mostly in whole PB and T lymphocytes. Both patients were successfully treated with a single DLI (1 × 107 CD3+ cells/kg), thereafter achieving normal PB counts and BM cellularity as well as complete chimaerism. Interestingly, the only side effect observed was mild graft versus host disease that did not require treatment. In conclusion, provided that an early diagnosis is made, the graft versus host lymphohaemopoietic effect harboured by immunocompetent donor cells can be successfully used for the treatment of incipient graft rejection.

Main

Allogeneic stem cell transplantation (SCT), using either peripheral blood (PB) or bone marrow (BM), is nowadays the therapy of choice for an increasing number of patients with haematological and nonhaematological diseases.1 Graft versus host disease (GVHD), infections and relapse are major factors causing morbidity and mortality after SCT. T-cell depletion of the graft has proven useful in reducing the incidence and severity of GVHD.2 However, since T-cell-depleted (TCD) grafts are less immunocompetent than unmanipulated grafts, they show a reduced graft versus leukemia (GVL) effect, and are therefore associated with an increased risk of relapse.3 Moreover, T-cell depletion increases the incidence of graft failure and graft rejection.4 Other factors that contribute to graft rejection include HLA disparity between donor and recipient, severe chronic GVHD (cGVHD) or associated viral infections.5 Once graft rejection is detected, the different possible causes should be carefully reviewed and, if identified, appropriately treated. Effective treatments for overt graft rejection are scarce and mainly based on second SCT,6 which has been successfully used in a few selected patients. The objective of the present investigation was to show the usefulness of early diagnosis of graft rejection on the basis of persistently decreasing PB and BM cellularity together with decreasing mixed chimaerism (MC; increasing percentage of recipient cells) as well as the efficacy and safety of prompt therapy with donor leukocyte infusions (DLIs).

Patients and methods

We report two patients with haematological malignancies who underwent TCD-SCT (Table 1). Haemopoietic stem cells were harvested, after G-CSF mobilization (subcutaneous administration of 10 μg/kg/day × 5 days), from the PB of HLA and ABO identical sibling donors. The grafts were partially TCD by positive immunomagnetic selection of CD34+ cells (Isolex 300i, Baxter in patient 1; CliniMACS, Miltenyi Biotec in patient 2). Table 1 shows patient characteristics, number of CD34+ and CD3+ cells infused, engraftment and acute GVHD (aGVHD) development. GVHD prophylaxis included cyclosporine A from day −1 in both patients. Donor leukocytes for DLI were obtained by apheresis procedures without prior G-CSF mobilization and infused without further GVHD prophylaxis.

Table 1 Patient characteristics and the transplants performed

BM and PB samples were obtained as close to 1, 3, 6 and 12 months post-transplant as possible, for chimaerism and minimal residual disease (MRD) studies.7 Additionally, PB samples were obtained at shorter intervals whenever it was considered necessary. FISH was performed on routine BM and PB smears and on cytospin preparations of leukocyte lineages (T lymphocytes (CD3+), both helper (CD4+) and cytotoxic (CD8+), B lymphocytes (CD19+) and myeloid cells (CD15+)) isolated by immunomagnetic procedures (MiniMACS, Miltenyi Biotec).8 The purity of enriched samples, as measured by flow cytometry, was higher than 95%. Chimaerism was quantified by FISH for the sex chromosomes (CEP X SpectrumOrange/Y SpectrumGreen; Vysis, Inc.).8 The sensitivity of the analysis, scoring 500 nuclei per slide, is 1%, as calculated using the upper level of a one-sided 95% confidence interval for observing the maximum number of opposite sex cells (One in 500 nuclei) in control specimens using the binomial distribution. The sensitivity of FISH assays performed on leukocyte lineages is 5%, considering a minimum purity of 95% in enriched samples. MRD was quantified by FISH to identify trisomy 6 in patient 1 (CEP 6 SpectrumOrange, (Vysis, Inc.) combined with CEP 7 SpectrumGreen (Vysis, Inc.) as control probe; sensitivity 3% scoring 500 nuclei) and t(11;14) in patient 2 (LSI IGH/CCND1 dual colour–dual fusion translocation probe Vysis Inc.; sensitivity 1% scoring 500 nuclei).8

Definitions

Engraftment

PB counts above 0.5 × 109 neutrophils/l and 20 × 109 platelets/l during 3 consecutive days unsupported by transfusions.

Incipient graft rejection

Persistent decrease in PB counts and BM cellularity after documented haematologic engraftment, in the absence of leukemic relapse, drug toxicity or infection, together with progressive disappearance of the allogeneic cells (ie decreasing MC).9,10

Mixed chimerism (MC)

Presence of 1% of recipient cells in BM or PB samples (sensitivity of the FISH assay), and of 5% of recipient cells in purified leukocyte lineages (purity of samples >95%).

Complete chimaerism (CC)

Presence of <1% of recipient cells in BM or PB samples and of <5% of recipient cells in purified leukocyte lineages.

Results

The two patients reported here received TCD-SCT from sex-mismatched HLA identical sibling donors for the treatment of haematologic malignancies (Table 1). In order to avoid rejection, the conditioning regimen included ATG reinforcement and the graft was supplemented with donor T lymphocytes, after TCD, to contain 0.12 and 0.15 × 106 CD3+ cells/kg, respectively. Both patients engrafted and showed mild/moderate aGVHD (Table 1, Figure 1), which required a short course of steroid therapy in patient 1. PB counts and BM cellularity, which had shown increasing values early after SCT (see results on day +35 for patient 1 and on day +68 for patient 2 in Figure 1), persistently decreased in both patients (reaching 20% of BM cellularity), in the absence of disease relapse (absence of MRD), drug toxicity or infection (Figure 1). Poor red blood cell engraftment observed in patient 2 was unsuccessfully treated with subcutaneous erythropoietin (10 000 U/week from day +80 after SCT). Concomitantly, serial chimaerism quantification showed decreasing MC (increasing percentage of recipient cells) in both patients (after initial CC in patient 1; Figure 1). The percentage of recipient cells was higher in PB than in BM samples, as well as in T lymphocytes (CD3+), mainly cytotoxic (CD8+, data not shown), than in B lymphocytes or myeloid cells, which showed CC in most samples (Figure 1). On the basis of all these observations, incipient graft rejection was diagnosed and immunosuppression was withdrawn in both patients. Unfortunately, no response was observed in terms of increasing cellularity and chimaerism. PB counts further decreased to critical values with maintenance of chimaerism levels in patient 1 after 40 days and with a 2-fold increase in the percentage of recipient cells in patient 2 after 1 week. Within this scenario, DLI (1 × 107 CD3+/kg) was administered seeking a graft versus recipient lymphohaemopoietic effect. Response, in terms of improving cell counts, was observed early after DLI (ca. 50 days, Figure 1). Patient 2 became red blood cell transfusion independent. PB counts and BM cellularity recovered to normal levels 3–4 months after DLI in both patients. Furthermore, chimaerism showed increasing values after DLI in both patients, achieving CC 3 and 8 months after DLI respectively (Figure 1). Of note, the side effects of DLI were mild, including only limited (skin) cGVHD that did not require treatment. Both patients remain alive in complete remission and CC 50 and 38 months after transplant, respectively.

Figure 1
figure1

Results of the sequential chimaerism testing in the different samples analyzed (BM, PB, and CD3+, CD19+, CD15+ cells). Day of onset and finish of acute and chronic GVHD, day of initiation and withdrawal of immunosuppressive therapy, number of leukocytes (Leu.), neutrophils (Neu.), platelets (Plt.), reticulocytes (Ret.), haemoglobin (Hb) and bone marrow cellularity (BM cel.) are also shown. GVHD=graft versus host disease; aGVHD=acute GVHD, cGVHD=chronic GVHD; CsA=cyclosporine A; Ster=steroids; BM=bone marrow; PB, peripheral blood; DLI contained 1 × 107 CD3+ cells/kg. *Transfusion dependent.

Discussion

Graft rejection is an ominous and frequently fatal complication observed in 10–30% (3/10 in our hands) of patients after TCD-SCT.10,11 In order to prevent graft rejection, several strategies have been developed such as the use of high intensity conditioning regimes,12 infusion of large amounts of stem cells13 or, more recently, infusion of a fixed number of T cells.10 However, these approaches to reinforce the donor graft may result in an increased risk of GVHD. Interestingly, establishment of aGVHD in our patients did not protect against subsequent incipient graft rejection. Thus, graft rejection still remains an important complication in this transplant setting, and therefore further strategies for prevention and treatment are needed.

After TCD-SCT, the observation of persistently decreasing PB and BM cellularity obliges exclusion of graft rejection from other possible causes such as relapse, drug toxicity or infection.5 Moreover, early diagnosis is of key importance, since the treatment of overt rejection with second SCT has a high mortality rate and is seldom successful.6,14 The patients reported here show that increasing mixed chimaerism in whole PB and CD3+ cells (Figure 1) is of great help in establishing both a differential and an early diagnosis of incipient graft rejection. Although mixed chimaerism has been classically associated with the development of donor–recipient tolerance and therefore with a lower incidence of GVHD,15,16 patient 2 developed mild aGVHD in the context of mixed chimaerism, suggesting that the two biological situations are not mutually exclusive.

Postgraft immunosuppression has proven useful to facilitate engraftment and retain the graft after nonmyeloablative conditioning.15 However, once incipient graft rejection is diagnosed, immunosuppression withdrawal can be considered as the first choice to reinforce donor lymphohaemopoiesis particularly after myeloablative conditioning, since it is associated with few side effects.17 However, this approach did not show any benefit in our patients (Figure 1) and alternative approaches had to be considered. We hypothesized that the graft versus host lymphohaemopoietic effect of DLI could be exploited, before the onset of overt rejection, as a pre-emptive therapy to counteract host versus graft reactions responsible for graft rejection.11 In animal models, DLIs have proven useful to convert established mixed chimaeras into full chimaeras, through their graft versus host effect, without causing GVHD.15,18 This immunotherapeutic effect has been clinically used as a GVL effect in the management of relapse after SCT19 and as a graft versus host lymphohaemopoietic effect to improve graft function and to convert MC into CC after nonmyeloablative SCT,16,20 after engineered SCT (pre-emptive ‘add backs’)20,21 and after SCT for haemoglobinopathies.22,23 More recently, DLIs have also been used to prevent imminent graft rejection after nonmyeloablative SCT in two paediatric patients with nonmalignant diseases.24 In the two adult patients reported here, diagnosed with haematological malignancies and treated with TCD-SCT, a markedly hypocellular BM and low PB counts, together with lineage-specific chimaerism and MRD studies, allowed relapse to be ruled out and the incipient graft rejection to be accurately identified. Within this scenario, both patients were promptly treated with a single DLI (1 × 107 CD3+/kg, 104 and 142 days after TCD-SCT, respectively). Early after DLI (Figure 1) both patients showed increasing cellularity, and normal PB counts and BM cellularity within 3–4 months. The percentage of recipient cells also increased after DLI and CC was observed first in the BM and later in PB (within 3–8 months after DLI), mostly due to the slow dynamics of the CD3+ compartment (Figure 1). Interestingly, both patients developed only limited (skin) cGVHD that did not require treatment.

In the context of increasing MC, the early use of DLI is crucial in order to obtain the greatest benefit at the lowest cost. DLIs administered once the percentage of recipient cells is high show less efficacy25 and have an increased risk of inducing secondary severe bone marrow aplasia.26 Additionally, this approach, which combines early diagnosis of graft rejection and prompt therapeutic DLI, allows the infusion of a small to moderate amount of T cells, which reduces the risk of severe GVHD24,27 and shows less side effects than the scheduled DLI ‘add backs’,20,21 while maintaining a similar efficacy.

We would therefore like to emphasize that if an early diagnosis is made, on the basis of persistently decreasing PB and BM cellularity together with decreasing MC, DLI can be considered as an efficient and safe approach for the treatment of incipient graft rejection.

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Acknowledgements

This work was partially supported by Grants FIS01-3035 and FIS02-1177 from the Fondo de Investigación Sanitaria (Spain).

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Correspondence to J L Díez-Martín.

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Keywords

  • graft rejection
  • T-cell depletion
  • donor leukocyte infusion
  • chimaerism
  • FISH

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