Allo-Reactive Lymphocytes

Antirecipient helper and cytotoxic T-cell frequencies in bone marrow transplantation

Summary:

We assayed helper T-lymphocyte precursor frequencies (HTLPf), interferon (IFN)-γ-producing cell frequencies (IFN-γPf) and CTL precursor frequencies (CTLPf) to see if they could predict the severity of acute graft-versus-host disease (aGVHD) and disease relapse after transplantation. In all, 48 bone marrow transplantation (BMT) patients and their HLA-identical sibling (n=29) or matched unrelated donors (MUD) (n=19) were recruited. HTLPf, IFN-γPf and CTLPf were measured using a limiting dilution assay (LDA). Patients were followed prospectively to assess the severity of aGVHD and the status of the primary disease after BMT. High (>5 × 10−6) HTLPf, CTLPf and IFN-γPf were significantly associated with the occurrence and severity of aGVHD in patients who received transplants from HLA-identical sibling. Among patients receiving BMT from MUD, HTLPf and CTLPf, but not IFN-γPf, were associated with aGVHD. Five patients had disease relapse post-BMT and the risk was not significantly associated with HTLPf, CTLPf or IFN-γPf. Patients with high (>5 × 10−6) HTLPf , IFN-γPf or CTLPf before BMT are at higher risk of developing aGVHD after transplantation from both matched sibling donors and MUD. Whether these parameters can predict disease relapse would have to be investigated with a larger cohort of patients.

Main

Allogeneic bone marrow transplantation (BMT) is becoming the treatment of choice for many patients with hematological malignancies or primary immunodeficiency.1, 2, 3 However, only 30% of patients have an HLA-identical sibling donor available and, for the rest, matched unrelated donors (MUD) or partially mismatched siblings become alternative sources of hematopoietic stem cells.4 BMT from these donors is, however, associated with an increased incidence and severity of graft-versus-host disease (GVHD) and graft failure.5, 6, 7, 8, 9, 10 We have previously shown that helper T-lymphocyte precursor frequencies (HTLPf) may predict the occurrence of GVHD in transplantation from HLA-identical sibling donors in a Chinese population.11 We report here an extended study of CTL precursor frequencies (CTLPf), HTLPf and interferon (IFN)-γ-producing cell frequencies (IFN-γPf) in BMT from both HLA-identical siblings or MUD.

Patients and methods

Patients

In all, 19 patients (17 adults and two children) receiving BMT from MUD and 29 adult patients receiving BMT from HLA-identical sibling donors, between July 1997 and December 2001, were included in this study. Their clinical characteristics are shown in Table 1. All patients received unmanipulated bone marrow or mobilized peripheral blood (PB) stem cell transplants. HLA-A, -B and -DR were identified by medium–high resolution molecular typing using polymerase chain reaction with sequence-specific primers. The HLA typing of the patients and the unrelated donors are shown in Table 2.

Table 1 Clinical characteristics of patients
Table 2 The HLA-A, -B and -DR molecular typing of the 19 patient-unrelated donor pairs

Post transplantation treatment

Prophylaxis against GVHD comprised methotrexate (15 mg/m2 on day 1, 10 mg/m2 on days 3, 6 and 11) and cyclosporin A (3 mg/kg/day intravenously or 9 mg/kg/day orally on days 1–50, tailed off at 6 months). The severity of GVHD was graded as described by Glucksberg et al12 and was classified into mild (overall grade <2) or severe (overall grade 2). Patients developing GVHD received additional immunosuppression according to the discretion of the attending physicians. This included intravenous methylprednisolone (2 mg/kg/day, increased to 4 mg/kg/day if no response within 48 h), and for patients with refractory GVHD, horse antithymocyte globulin (25 mg/kg/day for 5 days)

Measurement of HTLPf, CTLPf and INF-γPf using limiting dilution analysis

Collection and storage of peripheral blood mononuclear cells (PBMNC)

PB was collected from donors and recipients before BMT and MNC were separated by density-gradient centrifugation on Lymphoprep (Axis-Shield, Oslo, Norway), resuspended in 5 ml of the tissue culture medium RPMI 1640 containing 10% fetal calf serum (Invitrogen, USA) (FCS-RPMI) and diluted 10-fold with WBC solution. The cell density was determined and adjusted to 2 × 107 cells/ml and the cells were frozen in 1 ml aliquots in liquid nitrogen.

Combined HTLPf, CTLPf and IFN-γPf assays

Cryopreserved PBMNC were thawed and washed, counted and diluted to a concentration of 106 cells/ml. Recipient cells were the stimulators and donor cells the responders. HTLPf, CTLPf and INF-γPf were determined in the graft-versus-host direction from a single limiting dilution assay (LDA).13 In brief, a LDA was setup by culturing limiting numbers of responder cells (5 × 104–1250 cells/well) with constant numbers (5 × 104) of irradiated (50 Gy) stimulator cells in 96-well U-bottomed plates. Per dilution 24 wells were set up. As a reference, irradiated stimulator cells were incubated in the culture medium alone. In all, 24 replicate wells with stimulator cells alone (5 × 104) and responder cells alone (5 × 104) were plated as the negative controls. Completely mismatched third-party cells were used either as effector or stimulator to determine stimulator capacity of the irradiated stimulator cells or the responsive capacity of the effector cells, respectively. After 3 days, the plates were centrifuged at 200 g for 1 min and 100 μl of each supernatant was transferred onto new plates and frozen at –70°C until use in the HTLPf assay. On day 6, the steps were repeated to obtain another 100 μl of each supernatant for IFN-γPf assay. The fresh medium supplemented with human rIL2 (20 U/ml 0.01 μg/ml) (Boehringer Mannheim) was added to the wells of the LDA culture on days 3 and 6. On day 10, CTLPf was determined. HTLPf, IFN-γPf and CTLPf estimations were calculated using the computer program developed by Strijbosch et al.14 The frequency of the responding cells was determined by the maximum-likelihood estimation using a computer program as described by Fazekas de St Groth.15

HTLPf assay

Proliferation of the IL-2-dependent cell line CTLL-2 was used as the readout for the detection of IL-2 secretion by alloreactive T cells. This cell line was maintained in RPMI 1640 in the presence of L-glutamate (2 mmol/ml), pyruvate (100 U/ml), penicillin and streptomycin (100 μg/ml) (Gibco, USA), FCS (10%) (Invitrogen, USA) and human rIL2 (20 U/ml) (Boehringer Mannheim) at 37°C in a humidified atmosphere containing 5% CO2. Before use, the CTLL-2 cells were washed three times with phosphate-buffered saline and then resuspended in the culture medium without IL-2.

The HTLPf assay was performed as described previously,16 with some modifications. Briefly, frozen plates containing supernatant as described above were thawed, and 1 × 103 CTLL-2 cells in 50 μl culture medium were added to each well. After 48 h of incubation at 37°C, 0.5 μCi [3H]TdR was added to each well and the plates were harvested the following day and counted. A dose–response curve was setup to determine the cutoff point for scoring positive and negative wells in the HTLPf assay. The cutoff value for the assay was set at the beginning of the linear part of the dose–response curve. HTLPf were determined from the proportion of wells negative for IL-2 production at each responder (donor) cell dilution. Wells were scored positive for IL-2 production if the [3H]TdR retention was more than three standard deviations (s.d.) above the mean of the control culture containing supernatant of the stimulator cells alone and the CTLL-2 cells.

IFN-γPf assay

The concentration of IFN-γ in supernatants of 6-day LDA cultures was measured by sandwich enzyme-linked immunosorbent assay (ELISA) according to the supplier's instructions (OptEIA Human IFN-γ set, PharMingen). The results were measured photometrically at 450 nm. IFN-γPf were determined from the proportion of wells negative for IFN-γ production at each responder (donor) cell dilution. Wells were scored positive for IFN-γ if the amount of IFN-γ produced was higher than three s.d. above the mean of the control culture containing only the stimulator cells.

CTLPf assay

The target cells were generated from the original stimulator or third-party PBMC by incubating the cells (106/ml) with purified PHA-P (Difco, 5 μg/ml) from days 0 to 6, and with IL-2 (20 U/ml) from days 3 to 10. On day 8, target cells were incubated at 37°C in a humidified atmosphere containing 5% CO2 for 48 h in the presence of 10 μCi [3H]TdR. Cells were washed and resuspended in the culture medium. [3H]TdR-labeled target cells were added to each well of the LDA culture. After 4 h of incubation at 37°C, the wells were harvested and counted. A well was scored positive if the [3H]TdR retention was less than the mean minus three s.d. of the wells containing irradiated stimulator cells alone.

Outcome definitions

Neutrophil engraftment was defined as the first of 3 consecutive days with an absolute neutrophil count (ANC) of >0.5 × 109/l. Primary graft failure occurred if ANC remained less than 0.5 × 109/l 28 days after transplantation. Secondary graft failure was defined as the disappearance of myeloid engraftment in patients in whom neutrophils had previously engrafted. Development of acute graft-versus-host disease (aGVHD) was established by the standard criteria.17 Patients who died within 28 days of BMT or patients with primary graft failure were not evaluated for aGVHD.

Statistical analysis

Comparisons between groups of data were performed using the χ2 test (categoric data). The probability of aGVHD was calculated by the Kaplan–Meier method.18 P-values less than 0.05 were considered statistically significant.

RESULTS

Baseline characteristics

The clinical characteristics of patients are shown in Table 1. Their median age was 35 years (range: 4–57). The median duration of follow-up was 8 months (range: 2–26). The median time of neutrophil engraftment for all patients was 22 days (range: 15–28 days). Two patients were excluded from analysis because they died of transplant-related toxicity.

Correlation between HTLPf, CTLPf and IFN-γPf and clinical outcomes (Tables 3a–d)

Table 3 (a)HTLPf and GVHD, (b) CTLPf and GVHD, (c) IFN-γPf and GVHD and (d) HTLPf, CTLPf and IFN-γPf and GVHD in BMT with sibling and unrelated donors

When using an arbitrary cutoff of 5 × 10−6, HTLPf was significantly associated with the severity of aGVHD in patients who received BMT from HLA-identical sibling donors and MUD, both when they were separately analyzed or grouped together (Table 3a). The same results were obtained when CTLPf was assayed (Table 3b). IFN-γPf, however, was significantly associated with aGVHD when BMT from matched sibling donors was considered (Table 3c). When only BMT from MUD was analyzed, no significant association between IFN-γPf and aGVHD could be demonstrated. We further investigated whether the combination of these parameters can better identify patients who are at risk of aGVHD (Table 3d). In all patients studied (either HLA-identical siblings or MUD analyzed separately or together), a low frequency (5 × 10−6) of all three parameters was associated with a low risk of aGVHD. On the other hand, a high frequency (>5 × 10−6) of all three parameters was distinctly associated with a high risk of aGVHD.

In this cohort of 48 patients, only five patients had disease relapse post-BMT and the risk was not significantly associated with HTLPf, CTLPf or IFN-γPf. These parameters were not associated with neutrophil engraftment in our patients.

Discussion

We have evaluated alloimmune reactive donor CTLPf, HTLPf and IFN-γPf using a LDA in 19 patients who received BMT from MUD and 29 patients who received BMT from HLA-identical siblings. In both groups of patients, we demonstrated a significant association between CTLPf and HTLPf and the occurrence of aGVHD. IFN-γPf was also associated with aGVHD in patients receiving BMT from HLA-identical siblings, but not in those receiving BMT from MUD. Similar results have been reported by others19, 20, 21, 22, 23, 24, 25, 26 and our group,27 and the present study demonstrated some interesting findings that might be of clinical importance. First, although HTLPf/CTLPf were associated with aGVHD in general, some patients with high HTLPf/CTLPf did not develop severe aGVHD, whereas others who develop aGVHD had low HTLPf/CTLPf levels. It could be that some minor histocompatibility antigens are expressed in a tissue-specific manner, and those expressed by peripheral blood mononuclear cells may be different from those expressed by the target tissues for GVHD. Along the same argument, those antigens that present at higher frequencies on stimulator PBMCs may indicate a graft-versus-hematopoietic cell or graft-versus-leukemia reaction, rather than a GVHD reaction. Another explanation for ‘unexpectedly’ high HTLPf in some patients is related to the polymorphic proteins derived from the fetal calf or human AB sera used for cryopreservation of cells or in the assay culture medium. Some foreign serum proteins may be processed and on occasion presented in vitro as antigenic peptides.28, 29, 30, 31

IFN-γ production has been reported to be sensitive to HLA class II differences,32 but this finding was not confirmed in this study. Not all donors and recipients in the present study were tested at the allelic level for HLA-DRB1. Moreover, HLA-DQ and HLA-DP matching was not taken into consideration in this study. Whether allelic differences at these loci played any part in the observed results remains to be seen. The ethnic background in this Chinese patient–donor cohort also needs to be considered as genetic differences in reactivity related to racial origin may also play a role. We also demonstrated that using a combination of CTLPf/HTLPf/IFN-γPf assay may better predict the occurrence of aGVHD in both sibling and MUD. The clinical significance of this finding would have to be further studied with a larger cohort of patients.

In conclusion, CTLPf and HTLPf are significantly associated with aGVHD in Chinese patients receiving BMT from HLA-identical siblings and MUD, while IFN-γPf is significantly associated with aGVHD in patients receiving BMT from HLA-identical siblings. A larger cohort of patients is needed to investigate the predictive role of CTLPf, HTLPf and IFN-γPf in aGVHD and disease relapse.

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Acknowledgements

This project was carried out under the sponsorship of the Ho Hung-Chiu Medical Education Foundation.

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Correspondence to R Liang.

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Kwok, J., Leung, A., Lie, A. et al. Antirecipient helper and cytotoxic T-cell frequencies in bone marrow transplantation. Bone Marrow Transplant 34, 207–213 (2004). https://doi.org/10.1038/sj.bmt.1704566

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Keywords

  • cytotoxic T-lymphocytes precursor frequency
  • helper T-cell precursor frequency
  • IFN-γ-producing cell frequency
  • limiting dilution assay
  • graft-versus-host disease

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