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Keywords:

  • GVL;
  • GVH;
  • T cell;
  • TCR repertoire;
  • stem cell transplant

Abstract

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

We studied oligoclonal T-cell expansions of 24 T-cell receptor (TCR) Vβ families in normal donor lymphocytes stimulated with patient's cells and in recipient blood after transplant, using a polymerase chain reaction-based assay (spectratyping). T cells from donor blood were incubated with separated myeloid leukaemia cells or T cells from the HLA-identical sibling recipient. In five of the six patients tested, the T-cell Vβ skewing pattern observed in vitro was seen in vivo after transplant. After transplant, the myeloid-specific Vβ skewing coincided with the disappearance of residual disease in three patients and in one patient skewing was lost at the time of leukaemic relapse. In functional tests, T cells generated against leukaemic cells in vitro produced interferon γ in response to the leukaemia. Removal of the leukaemia-expanded skewed Vβ families significantly decreased cytotoxic killing of the leukaemia. However, while there was a general concordance in the Vβ family exhibiting clonal expansion in vitro and in vivo, the exact clonotype expanded in vitro and in vivo differed. These findings suggest that alloresponses involve multiple T-cell clones within a restricted TCR Vβ repertoire that undergo different selection pressures in vitro and in vivo.

The outcome of allogeneic marrow stem cell transplants (SCT) for patients with haematological malignancies is strongly influenced by alloresponses of donor lymphocytes, provoking both undesirable complications from graft-versus-host (GVH) disease and a favourable graft-versus leukaemia (GVL) effect. In human leucocyte antigen (HLA)-identical sibling transplants, the T-lymphocyte alloresponse of the donor is directed against minor histocompatibility antigens (mHA). These antigens can be either ubiquitously expressed, eliciting both GVH and GVL reactions, or tissue-restricted (Barrett & Malkovska, 1996). Alloresponding T cells can be identified as clonal expansions within a specific T cell receptor (TCR) Vβ family. Using polymerase chain reaction (PCR) techniques to study SCT recipients, several investigators have found a skewed distribution of T-cell clones within individual TCR Vβ families, temporally linked to the onset of GVH (Dietrich et al, 1994; Liu et al, 1996) or to leukaemic regression (without GVHD) following donor lymphocyte infusion (DLI) (Claret et al, 1997). These results suggest that the alloresponse to leukaemia and other host tissues can be distinct.

In vitro data supports the existence of haemopoietic lineage-restricted alloresponses within TCR Vβ families. We previously found that donor CD4+ T-cell clones, generated against a single HLA-A locus mismatched recipient with chronic myelogenous leukaemia (CML), had distinct GVL- or GVH-like reactivity against the leukaemia or against the recipient's lymphocytes. Furthermore, GVL and GVH responding clones had different TCR Vβ specificities (Jiang et al, 1997).

In order to assign a functional significance to clonal T-cell expansions occurring in an HLA-identical setting after allogeneic SCT, we cultured donor lymphocytes with leukaemia cells or normal lymphocytes from the recipient. We then used a TCR Vβ repertoire analysis technique (spectratyping) (Maslanka et al, 1995) to identify GVL- or GVH-like clonal expansions of donor lymphocytes. In vitro skewed Vβ patterns were compared with those obtained in vivo following allogeneic SCT. Here, we report concordance of in vitro with in vivo findings and a functional relationship between skewed Vβ families and cytotoxicity in vitro. These results provide further evidence of lineage-restricted alloresponses following HLA-identical SCT.

Patients and methods

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

Patients and blood samples Patients described in this study gave written informed consent and were entered into National Institutes of Health (NIH) Institutional Review Board approved protocols 97-H-0097 and 99-H-0046. They received 1360 cGy total body irradiation in eight fractions and cyclophosphamide 60 mg/kg given on two successive days, followed by a granulocyte colony-stimulating factor (G-CSF)-mobilized, T cell-depleted peripheral blood SCT from an HLA-identical family donor. On d 45 and d 100 they received 1 × 107 and 5 × 107 CD3+ cells/kg, respectively, to reconstitute the immune repertoire and provide a GVL effect. Cyclosporine in standard doses was given from d −4 to d 100–180 to prevent GVH. Patients were studied at a time when they were 100% donor lymphocyte chimaeric by lineage-specific minisatellite assay (Childs et al, 1999). Blood samples for culture and spectratyping were obtained before transplant from patient and donor by leukapheresis and after transplant at various time points to study TCR Vβ spectratype. Mononuclear cells were separated on Ficoll-Hypaque (Organon Teknika, Durham, NC, USA) and stored in 10% dimethyl sulphoxide (DMSO) in liquid nitrogen until use.

Cell culture Cells from patients, HLA-identical sibling donors and third party controls were cultured in Roswell Park Memorial Institute (RPMI; Biofluids, Rockville, MD, USA) medium containing 15% normal AB-human serum (Gemini, Calabasas, CA, USA), 5 mmol/l HEPES (Gibco/BRL, Gaithersburg, MD, USA) and 62·5 mg/l gentamycin (Biofluids). Control cells were obtained from a leukapheresis product by Ficoll-Hypaque separation and subsequent depletion of CD14+ (monocyte) and CD19+ (B) cells by magnetic bead selection (Dynal, Oslo, Norway). Cells from the patient's leukapheresis product were depleted of CD3+ cells by magnetic bead separation, while the remaining products, enriched for leukaemia cells, were used as stimulator cells in the in vitro assays. The patient CD3+ cells bound to magnetic beads were cultured with 100 U/ml recombinant human interleukin-2 (IL-2) (Hoffman-LaRoche, Nutley, NJ, USA) and expanded. Before use, the magnetic beads were dissociated from the T cells and removed.

In vitro culture conditions Donor T cells were incubated alone or with irradiated (5000 cGy) patient leukaemia cells, patient T cells or third party T cells in a 1:1 ratio for 10–12 d, with the addition of 100 U/ml IL-2 on d 5 and 8. The T cells were then cryopreserved in 10% DMSO and stored at −70°C until RNA was extracted. To confirm reproducibility of skewing patterns, several donor/patient pairs were tested up to five times.

Spectratype analysis RNA was isolated according to the manufacturer's recommendations (Trizol, Gibco Life Sciences) and 1 μg of total RNA was converted to cDNA using oligo (dT) as the primer for reverse transcription (first strand synthesis kit, Boeringer Manheim, Indianapolis, IN, USA). Spectratyping analysis was performed according to Maslanka et al (1995). Briefly, cDNA was amplified for 30 cycles under non-saturating PCR conditions with a panel of TCR Vβ family specific primers and a β-chain constant primer in duplex or simplex (for Vβ 6·1 and 6·2) reactions using 24 TCR Vβ family specific primers and pairings as described. The Vβ specific primers screened families 1, 2, 3, 4, 5·1, 5·2, 6·1, 6·2, 7, 8, 9, 11, 12, 13, 14, 15, 16, 17, 18, 20, 21, 22, 23, and 24. The common constant primer was labelled at the 5′ end with 6′ carboxyfluorescein (6-FAM). In addition to the TCR Vβ family specific and constant primers, each reaction contained an actin-specific primer pair producing a 6-FAM-labelled 230-bp product as an internal PCR control for verification of cDNA integrity and the fidelity of the PCR reactions, as described by Bacsi et al (1999).

TCR spectratyping TCR spectratyping was performed as described by Maslanka et al (1995). Briefly, an equivalent volume of PCR-labelled product was mixed with formamide dye-loading buffer, heated at 94°C for 2 min and applied to a prerun 5% acrylamide-urea sequencing gel. Gels were run for 110 min at 40 W. After resolution on the gel, the labelled PCR product was analysed with a Molecular Dynamics FluorImager 575 (Sunnyvale, CA, USA). TCR spectratyping of a healthy peripheral blood mononuclear cell (PBMC) repertoire typically results in a banding pattern composed of between seven and eight bands at three-nucleotide base intervals, reflecting the correct ‘in frame’ nature of functionally rearranged β-chain TCR gene products. The limited number of PCR cycles used (30) leads to the generation of PCR products with a distribution representative of the starting material, i.e. a Gaussian distribution. Skewing was defined as a band intensity twofold or greater than expected.

PCR product cloning and DNA sequencing The TCR-chain PCR products corresponding to specific spectratype bands of interest were cloned using a T/A cloning kit (Invitrogen, San Diego, CA, USA) for PCR fragment cloning as described by Bacsi et al (1999). Entire PCR reactions were purified using a QIAquick PCR purification kit (Qiagen, Chatsworth, CA, USA) before cloning. This approach produced a spectrum of clones representative of all band species and their relative frequencies within a particular spectratype PCR reaction. DNA sequence analysis of clones was performed using a Taq dye deoxy-terminator cycle sequencing kit (Applied Biosystems, Foster City, CA, USA).

Interferon γ cytokine production assay Donor T cells isolated with a negative selection T-cell isolation kit (Dynal) were stimulated twice with patient leukaemic monocytes on d 1 and d 7 and skews tested before the cytokine assay started on d 16. T cells were incubated for 18 h in the presence of IL-2 (10 U/ml) and IL-12 (20 ng/ml) either alone, with patient CD3+ lymphocytes or leukaemia cells. Brefeldin A (25 ng/ml) was added for the last 4 h of culture to inhibit cytokine release from the cells. The FastImmune assay system (Becton Dickenson, San Jose, CA, USA) was utilized to detect IFNγ production by individual cells. The cells were fixed, stained with antibodies against CD3 and made permeable before staining with IFNγ. The results were analysed on a FacsCalibur (Becton Dickenson). Cells were gated on a CD3+ lymphocyte population to avoid detection of the stimulating monocytes.

Cytotoxicity assay Donor T cells isolated by negative selection (Dynal) were stimulated twice with patient leukaemic mononuclear cells on d 1 and d 8. On d 21, donor T cells were separated into two fractions, one sham-depleted with sheep anti-mouse magnetic beads alone and the other depleted with a Vβ antibody [BioDesign International (Saco, ME, USA); Coulter (Fullerton, CA, USA)] corresponding to the Vβ family skewed in culture. The cells were washed and incubated with sheep anti-mouse magnetic beads. In both conditions, non-adhering cells were collected and incubated overnight at 37°C, 5% CO2. Cytotoxicity was measured using the technique of Hensel et al (1999). Briefly, leukaemia cells (thawed and maintained in culture for 24 h) were loaded with Calcien-AM (Molecular Probes, Eugene, OR, USA), washed and plated in 40 μl Terasaki plates at 1000 cells/well. T cells were added to targets at ratios of 30:1, 10:1 and 3:1. Cells were incubated for 6 h before measuring cytotoxicity on a Lambda Scan Plus II (One Lambda, Canoga Park, CA, USA). Replicates of 12 were performed for each assay condition. Percentage cytotoxicity was determined using the equation: [1 − (test well-media alone)/(targets alone-media alone)] × 100.

Results

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

Spectratyping in vitro and in vivo

Vβ spectratyping was performed for all 24 Vβ families in cultures from eight donor recipient pairs (Table I). In vitro skewing of donor lymphocytes stimulated by patient's leukaemia cells or lymphocytes were found in seven out of eight pairs, involving nine leukaemia-restricted, five lymphoid-restricted and one myeloid plus lymphoid skewed Vβ pattern. Spectratype analysis was carried out at various time points post transplant in five patients. Five out of 10 skewed Vβ patterns identified in culture were also found in recipient blood at various times post transplant. Post-transplant skewing, shown in vitro to be leukaemia-induced, correlated temporally with persistence of leukaemia in three out of four informative cases. In patient 114, post-transplant skewing, found in vitro to be lymphoid-specific, was temporally associated with acute GVHD.

Table I.  Clinical outcome and spectratyping results in eight patients transplanted for myeloid leukaemia.
Patient in vitro expansion*  Survival
numberDiagnosisLeukaemiaLymphocytein vivo Vβ skewingPost-transplant events(months)
  1. *See Patients and Methods.

  2. CML CP, chronic phase chronic myeloid leukaemia; AML, acute myeloid leukaemia; NT, not tested; cGVHD, chronic graft-vs.-host disease; aGVHD, acute graft-vs.-host disease; DLI, donor lymphocyte infusion; PCR, polymerase chain reaction.

19CML CP1818None at 1,4,13,15,21 monthsPCR+ until 20 months Grade III GVHD from DLI 17 months> 65
101CML CP5·33 and 5 monthsPCR+ until 7 months> 22
18None at 3 and 5 monthsLimited cGVHD 5 months 
84CML CP15None d 93,105,189PCR+ d 93 PCR– d 180> 27
5·1d 93,105aGVHD II d 120 
20d 93,105,189  
114CML CP11NTPCR+ until 12 months, no GVHD> 19
127CML CP311NTPCR+ until 7 months, no GVHD> 16
13   
20   
90AML5·3d 43Leukaemic relapse d 1004
20None d 43No GVHD 
14None d 43  
64AML1215 monthsLeukaemic relapse 15 months16
117AMLNTLeukaemic relapse d 783

Patient 84 was studied in further detail (Fig 1): recipient leukaemia cells elicited skewing in Vβ 15 from the donor. This did not occur in cultures with recipient lymphocytes or third party lymphocytes, or when unstimulated donor T cells were cultured alone. Recipient lymphocytes elicited lymphocyte-restricted skewing of Vβ family 20, but no skewing was observed in Vβ 15 or any other Vβ family. This skewing was recipient lymphocyte-restricted because it did not occur after culture with recipient leukaemia or third party lymphocytes or in cultured unstimulated donor T cells. Peripheral blood spectratyping was performed on d 93, 105 and 189 following SCT. On d 93 and d 105, several skewed bands were found in Vβ 15, including the leukaemic cell-restricted band identified in vitro. This skewing was temporally associated with persisting minimal residual disease (PCR for bcr/abl was positive on d 93). After protocol withdrawal of cyclosporine, the patient became bcr/abl negative by d 180, and by d 189 the Vβ 15 band had disappeared. On d 93, 105 and 189 a skewed Vβ 20 band was found, identical in pattern to that found in vitro(Fig 2). This skewing was temporally associated with the development of grade II acute GVH disease on d 120, which persisted as limited chronic GVH disease by d 189. This pattern of skewing therefore appeared to be GVH-related. The in vitro- and in vivo-derived skewed Vβ 15 bands were then cut and sequenced with primers for the CDR3 region. The predominant in vitro CDR3 = 16 sequence was CATSDLAARGYEQFFG, while the in vivo CDR3 = 16 sequence on d 93 and 105 was CATSAVSRGYNEQFFG. Both sequences used Vβ 15–Jβ 2·1, however, while similar, they were not identical at the n-region.

image

Figure 1. Vβ 15 skewing in a GVL-like response in vitro and in vivo after transplant. Donor T cells were incubated with patient CML-enriched cells (lane 1), patient CD3+ cells (lane 2), HLA-mismatched third party cells (lane 3) or alone (lane 4). In vivo samples were collected pretransplant and at the time points indicated. Vβ families 15 and 17 were analysed, no skewing was detected in the Vβ 17 family. The donor did not show any skewing in Vβ pretransplant (data not shown).

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image

Figure 2. Vβ 20 skewing in a GVH-like response in vitro and in vivo after transplant. Donor T cells were incubated with patient CML-enriched cells (lane 1), patient CD3+ cells (lane 2), HLA-mismatched third party cells (lane 3) or alone (lane 4). The same Vβ families were analysed from peripheral blood samples taken from the patient before or after transplant. The Vβ family 20 was analysed.

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Patient 101 showed a leukaemia-restricted expansion of Vβ 5·3 and a bispecific skewing in Vβ 18 in vitro. After transplant, skewing was only seen in Vβ 5·3. At the time of testing 5 months after transplant, the patient had developed chronic GVH disease and remained in haematological remission but was positive for bcr/abl. Subsequently, the GVH disease resolved and the PCR became negative.

Patient 90, with acute myeloid leukaemia (AML), showed leukaemia-restricted skewing in Vβ 5·3 and 20 and lymphocyte-restricted skewing in Vβ 14 in vitro. A d 43 blood sample showed skewing in Vβ 20 but this was absent by d 112. This patient relapsed with leukaemia on d 100. Interestingly, the relapsed leukaemia failed to induce skewing of Vβ 20 in vitro and, at the time of relapse, the in vivo skewing was no longer detected. TCR sequencing of in vitro and in vivo Vβ 20 showed that there were different T-cell clonotypes present in these samples (in vitro sequence CALGVSYNEQFFG; in vivo sequence CAWRAERNTEAFFG, in which CA/CAWR represents the Vβ 20 segment and the bold, respectively, represents the J2·1 and JB1·1 segments. The intervening segments are the CDR3 regions).

To study the function of leukaemia-restricted Vβ T cells, donor T cells were incubated with leukaemic cells or patient lymphocytes for 21 d. After two stimulations, spectratype analysis showed continued skewing in Vβ 5·3 and 20 with leukaemic stimulators and Vβ 14 with lymphocyte stimulators. Only donor T cells cultured with leukaemia cells showed IFNγ production (18% positive CD3+ cells versus < 1% for cultures with lymphocytes or without stimulation) (Fig 3). Twenty-one day cultures of donor T cells with leukaemia cells were assayed for cytotoxicity after either sham depletion with sheep anti-mouse beads or with Vβ 5·3 and 20. As shown in Fig 4, at a 30:1 effector:target ratio, the sham-depleted T cells showed 39% specific cytotoxicity for leukaemia compared with 22% for T cells depleted of Vβ 5·3 and 20.

image

Figure 3. Donor T cells produce IFNγ against patient leukaemic but not non-leukaemic cells. Donor T cells were incubated alone (left), with patient leukaemia-enriched cells (middle) or with patient CD3+ blasts (right) and analysed by FACS. A CD3+ gate in the lymphocyte region was used for analysis.

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image

Figure 4. Cytotoxic function of Vβ depleted T cells. Donor T cells were either mock depleted (▪) or depleted of previously determined skewed Vβ families (◆). Replicates of 12 were performed. Error = SEM < 0·5% for all data points.

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Discussion

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

In these experiments, we extended previous observations of oligoclonal expansions of donor T cells following SCT (Jiang et al, 1997) detecting in vitro tissue-restricted oligoclonal Vβ expansion similar to that found in vivo in the same donor–recipient pairs. These results suggest that only a few immunodominant antigens are responsible for eliciting dominant T-cell responses within a single Vβ family. Furthermore, the ability to expand donor T cells with recipient cells in short-term culture, without the help of professional antigen presentation from dendritic cells, suggests that such alloreactive cells are of memory phenotype. The finding of similar expansions of donor T cells after transplantation suggests that the oligoclonal T-cell expansions that occur in the first few months after transplantation are also derived from T cells expanded from an initial memory cell precursor. The pattern of T-cell clonal expansion revealed by spectratyping was, however, more complex after transplant than that of T cells expanded in vitro. For example, in patient 94, one GVL-specific band was seen in vitro, whereas in vivo several bands were skewed during leukaemic regression (Fig 1). Furthermore, while showing strong similarity, the CDR3 sequences of in vitro and in vivo clones in patient 94 and patient 119 were not identical, suggesting that in vitro and in vivo conditions favoured the expansion of different T-cell clones recognizing the same antigen. This possibility is supported by other observations suggesting that identical CDR3 sequences are not strictly necessary for two TCRs to recognize the same antigen/MHC complex in aplastic anaemia (Zeng et al, 1999) and multiple sclerosis (Oksenberg et al, 1993; Musette et al, 1996). Changes in the antigens presented or loss of antigen presentation by the leukaemia at relapse might also be responsible for differences between in vitro and in vivo findings. For example, in patient 5, TCR Vβ 20 skewing in vitro and in vivo was only observed early after transplant. By d 112 when the leukaemia relapsed, no TCR Vβ expansion was seen in either condition.

Our results provide further evidence that alloresponding donor cells can exhibit tissue restriction. The finding of leukaemia-restricted alloresponses should not, however, be assumed to indicate leukaemia-specific T-cell responses. The antigens recognized by T cells on myeloid leukaemia cells are not known but presumably include tissue-restricted minor histocompatibility antigens (Goulmy, 1997) or overexpressed myeloid-specific proteins such as proteinase-3 (Molldrem et al, 1997). The responses observed may therefore have been myeloid-, rather than leukaemia-specific. Further definition of specificity would require testing donor T cells against normal myeloid cells and a large panel of cells from different tissues. Interestingly, the majority of the Vβ skewing observed was either leukaemia- or lymphoid-specific and few Vβ expansions had both specificities. Again, precise elucidation of the tissue restriction of expanded Vβ families would require extended screening against other cells, such as fibroblasts, B cells, keratinocytes and normal (non-leukaemic) myeloid cells.

Spectratyping analysis of donor-derived lymphocytes after SCT confirmed the observations of others that oligoclonal expansions of Vβ families are frequent occurrences, often correlating with clinical events of GVH disease, leukaemic regression or relapse (Dietrich et al, 1994; Liu et al, 1996; Claret et al, 1997). Caution must be used in ascribing functional properties to clonally expanded T cells on the basis of a temporal association between emergence of the clone and a clinical event. However, the correlation of in vitro and in vivo TCR Vβ family skewing adds support for a functional interpretation of clonal expansions temporally related to either GVH or GVL reactions. In one patient we were able to confirm that T cells of the Vβ family, expanded in the presence of leukaemia, were the source of leukaemia-specific IFNγ production and anti-leukaemic cytotoxicity. These experiments were complicated by the difficulty of demonstrating T-cell function in cells positively selected using Vβ antibodies that rapidly downregulate their TCR (Lanzaveccia et al, 1999), We therefore used negatively selected T cells depleted of leukaemia-reactive Vβ families to demonstrate the loss of cytotoxicity to leukaemia (Figs 3 and 4).

These findings show that in vitro spectratyping assays may be useful in predicting functional behaviour of T-cell expansions occurring post transplant at the level of individual Vβ families. Further confirmation of the functional similarity of in vitro and in vivo expanded Vβ families would make it possible to develop predictive tests to identify GVH-reacting Vβ families so as to eliminate them from the graft inoculum to prevent GVH disease while conserving GVL responses.

Acknowledgments

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

David A. Margolis received grant support from The Midwest Athletes Against Childhood Cancer (MACC) Fund, the Children's Hospital Foundation and NIH grant K08 CA77330-01A1.

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