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Keywords:

  • CML;
  • CD4+ T cells;
  • tumour immunology;
  • graft-versus-leukaemia effect;
  • transplantation

Abstract

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Generation of a CML-specific T-cell line
  6. Generation of a CML-specific T-cell clone
  7. DDII.4.4 specifically recognizes the CML of patient 1 (CML 1)
  8. The target cells recognized by DDII.4.4 include CD34+ CML progenitor cells
  9. The antigen recognized by DDII.4.4 is restricted by HLA-DRB1*16
  10. Discussion
  11. Acknowledgments
  12. References

Summary. The therapeutic effect of a human leucocyte antigen (HLA)-identical allogeneic stem cell transplantation (allo-SCT) for the treatment of haematological malignancies is mediated partly by the allogeneic T cells that are administered together with the stem cell graft. Chronic myeloid leukaemia (CML) is particularly sensitive to this graft-versus-leukaemia (GVL) effect. Several studies have shown that in allogeneic responses both CD4 and CD8 cells are capable of strong antigen-specific growth inhibition of leukaemic progenitor cells, but that CD4 cells mainly exert the GVL effect against CML. Efficient activation of allogeneic CD4 cells, as well as CD8 cells, may explain the sensitivity of CML cells to elimination by allogeneic T cells. Identification of the antigens recognized by CD4 cells is crucial in understanding the mechanism through which CML cells are so successful in activating allogeneic T cells. In the present report, we describe the characterization of an allogeneic CD4 T-cell clone, DDII.4.4. This clone was found to react against an antigen that is specifically expressed in myeloid cells, including CD34+ CML cells. The antigen recognition is restricted by HLA-DRB1*16. To our knowledge, this is only the second report on an allogeneic CD4 T-cell clone that reacts with early CD34+ myeloid progenitor cells.

The therapeutic effect of a human leucocyte antigen (HLA)-identical allogeneic stem cell transplantation (allo-SCT) for the treatment of haematological malignancies is mediated partly by the allogeneic T cells that are administered together with the stem cell graft (Drobyski et al, 1998a,b; Jiang et al, 1998; Leber et al, 1998; Mackinnon et al, 1998; van Rhee et al, 1998; Theobald, 1998). This graft-versus-leukaemia (GVL) effect results in the elimination of residual leukaemic cells. The allo-reactivity of donor T cells is not restricted to leukaemic cells, but often extends to non-hematopoietic tissues, resulting in graft-versus-host disease (GVHD), a major complication of allo-SCT. The GVL effect of allogeneic T cells is further exploited in donor lymphocyte infusions (DLI) for the treatment of relapsed leukaemia, and in reduced-intensity conditioning transplants (Kolb et al, 1990, 1995; Bar et al, 1993; Ferster et al, 1994; Tricot et al, 1996; Verdonck et al, 1996; Bertz et al, 1997; Giralt et al, 1997; Lokhorst et al, 1997; Khouri et al, 1998; Slavin et al, 1998; Szer et al, 1998; McSweeney et al, 2001).

Pioneering work by Goulmy's group has demonstrated that, in HLA-identical patient/donor combinations, the allogeneic T cells respond to minor histocompatibility antigens (miHa) (Tekolf & Shaw, 1984; Korngold & Wettstei, 1990; Wallny & Rammensee, 1990; Goulmy, 1997). Recently, the molecular identity of miHa has been elucidated (Brichard et al, 1993; den Haan et al, 1995, 1998; Wang et al, 1995; Guilloux et al, 1996; Mandruzzato et al, 1998; Simpson et al, 1998; Vogt et al, 2000a,b). The operationally defined miHa are made up of short peptides, derived from endogenous proteins, which are presented by HLA molecules. In essence, these peptides are polymorphic, resulting in differences in amino acid (AA) composition between host and donor. Apparently, these small differences in AA composition are sufficient to evoke powerful immune responses in situations of complete HLA matching. The function and expression pattern of the proteins from which the peptides are derived are diverse; they can be encoded by housekeeping genes and be broadly expressed, but they can also be expressed in an exquisite tissue-specific fashion, e.g. on haematopoietic cells or on B-lineage cells only (Dolstra et al, 1997; Mutis et al, 1998a). The identification of miHa that are expressed in haematopoietic cells, including leukaemic cells, but not in fibroblasts and other tissue types, has suggested that such tissue-restricted antigens could potentially serve as targets for T cell-mediated therapy to enhance GVL activity without inducing GVHD (de Bueger et al, 1992; Dolstra et al, 1997; van der Harst et al, 1998; Warren et al, 1998). This proposition was recently substantiated in a mouse model (Fontaine et al, 2001).

Malignancies of diverse origin exhibit varying sensitivities towards the GVL effect. While acute leukaemias generally show a poor response to the administration of donor lymphocytes, chronic myeloid leukaemia (CML) appears to be particularly sensitive to the GVL effect, resulting in remission in 85% of patients following DLI (Porter et al, 1994; Kolb et al, 1995). The underlying properties of leukaemic cells that govern their sensitivity towards a GVL effect have not been understood. A recent report suggests that lack of expression of the appropriate co-stimulatory molecules may be one of the factors involved (Mutis et al, 1998b).

A number of studies have shown that both CD4+ T cells (CD4 cells) and CD8+ T cells (CD8 cells) are capable of strong antigen-specific growth inhibition of leukaemic progenitor cells, but the GVL effect against CML is mainly exerted by CD4 cells (Falkenburg et al, 1991; Verfaillie et al, 1992; Leemhuis et al, 1993; Faber et al, 1995a,b; Mutis et al, 1997; Serrano et al, 1999). Efficient activation of allogeneic CD4 cells, as well as CD8 cells, may explain the sensitivity of CML cells for elimination by allogeneic T cells. This is in line with the observed predominant CD4+ phenotype of cytotoxic T lymphocytes (CTL) generated in vitro through stimulation of allogeneic T cells with CML cells (Serody et al, 1997; Falkenburg et al, 1999). Identification of miHa recognized by CD4 cells is, therefore, crucial in understanding the mechanism through which CML cells are so successful in activating allogeneic T cells.

In the present report, we describe an allogeneic CD4 T-cell clone, DDII.4.4. We show that it strongly reacted with patient's CML cells, including the CD34+ subpopulation, but not T and B cells, and was restricted by HLA-DRB1*16. Moderate reactivity was seen against donor monocytes and other HLA-DRB1*16-expressing leukaemic and normal myeloid cells. This pattern of reactivity indicates that the cognate antigen is either a miHa, or derived from a mutated or over-expressed gene product.

Cells.  Philadelphia-chromosome-positive CML cells were collected from a patient (Patient 1) in chronic phase, separated via Fycoll–Hypaque (Pharmacia Biotech, Uppsala, Sweden) and stored in liquid nitrogen until use. Peripheral blood mononuclear cells (PBMC) were collected from the patient's HLA-identical brother [HLA-A1, A24(9)/B8, B48/DRB1*16, DRB1*03]. Phytohaemagglutinin (PHA) blasts were generated by incubating PBMC with 1 µg/ml PHA and interleukin 2 (IL-2; 300 IU/ml; Proleukin, Chiron, Amsterdam, the Netherlands). Epstein–Barr virus (EBV)-transformed B-cell lines were generated through incubation of 5 × 106 PBMC in culture medium from an EBV-producing marmoset cell line for 1 h at 37°C. Cells were then plated in 24-well plates in the presence of 1 µg/ml PHA. Monocytes were purified through GαM-labelled immunomagnetic bead selection of CD14-labelled PBMC (Miltenyi Biotec, Bergisch Gladbach, Germany). CD34+ cells were selected with CD34-specific immunomagnetic beads (Miltenyi Biotec).

Culture of CML cells.  CML cells were thawed and treated with DNAse I for 30 min at 37°C, then peletted and cultured for 7 d in T25 flasks in Roswell Park Memorial Institute (RPMI)-1640 medium and 10% human serum (together termed human medium), to which was added a mixture of four human growth factors (4HGF): IL3 10 ng/ml (Peprotech, Rocky Hill, NJ, USA), IL6 20 ng/ml, granulocyte colony stimulating factor (G-CSF) 100 ng/ml (Amgen Europe, Breda, the Netherlands) and stem cell factor (SCF) 50 ng/ml (4HGF medium, Moore et al, 1998). Cells thus cultured (4HGF cells) were stored in liquid nitrogen until use.

Generation of T-cell line DDII.4.  CML cells were thawed and treated with DNAse I for 30 min at 37C. Cells were pelleted, counted, irradiated at 5000 cGy and plated in RPMI-1640 medium with 10% human serum (HS) in a 96-well round-bottomed microtiter plate (Nunc, Denmark) at a concentration of 6 × 103 cells/well. Donor PBMC were thawed and T cells were isolated using Lymfokwik (One Lambda, USA), resuspended in RPMI-1640 medium + 10% HS and added to the wells at a concentration of 6 × 103 cells/well. IL-6 and IL-12 were added to final concentrations of 1000 IU/ml IL-6 and 10 ng/ml IL-12 (Peprotech). After 1 week, the T cells were restimulated with 6 × 103 4HGF cells/well + 5 ng/ml IL-7 (Peprotech) + 10 IU/ml IL-2. The third and fourth stimulations were similarly performed with either CML cells or 4HGF cells. Thereafter, cells were restimulated in 96-well round-bottomed microtiter plates (0·5 × 106 T cells/plate) with 1·0 × 106 CML cells or 4HGF cells in the presence of 50 IU IL-2/ml, resulting in the CTL line DDII.4.

Generation of T-cell clones.  T-cell clones were generated by plating DDII.4 under limiting dilution condition in human medium in 96-well round-bottomed microtiter plates in the presence of 0·5 × 106 CML cells/plate + 50 IU IL-2/ml. Weekly restimulations consisted of the addition of 1·0 × 106 CML cells or 4HGF cells/plate + 50 IU IL-2/ml. After 2 months, proliferation of the clones decreased. Thereafter, they were cultured in human medium in the presence of PBMC from three different donors (2·0 × 106 cells/plate), 1·0 × 106 EBV-transformed B cells from patient 1/plate, 1 µg PHA/ml and 50 IU IL-2/ml. The next week, the cells were transferred to a T25 flask and cultured in human medium with 50 IU IL-2/ml at 0·5 × 106 cells/ml.

Sequence analysis of T-cell receptor TCR V regions.  Expression of TCRβ variable (BV) genes was analysed by reverse transcription polymerase chain reaction (RT-PCR). Total RNA was isolated using Trizol (Life Technologies, Gaithersburg, MD, USA). Two micrograms of RNA were used for cDNA synthesis using random primers in a total reaction volume of 50 µl. One microlitres of cDNA were subjected to PCR using forward primers specific for different Vβ gene families. As a reverse primer, an oligonucleotide specific for Cβ1 was used. The primers used for amplification of TCR Vβ gene families were as follows: BV1, GCA AAA GGA AAC ATT CTT GAA CG; BV2, AGA GTC TCA TGC TGA TGG C; BV3, AGG ACG GGA GAG AAA GTT TTT C; BV4, GGC CAC TAT GAG AGT GGA TTT GTC; BV5, CCC TAA CTA TAG CTC TGA GCT G; BV6, GGG GCA GGG CCC AGA GTT TCT AAT; BV7, CTT AAA CCT TCA CCT ACA CGC; BV8, CTT TAA CAA CAA CGT TCC G; BV9, CAG TTC CAA ATC GCT TCT CAC; BV10, GGA TTG TGT TCC TAT AAA AGC; BV11, CCA CTA TTC CTA TGG AGT TAA TTC C; BV12, GTC ACC AGA CTG AGA ACC ACC GCT A; BV13, GCA TGA CAC TGC AGT GTG CCC; BV14, ACC CAA GAT ACC TCA TCA CAG; BV15, TCT CAG ACT AAG GGT CAT GAT AGA; BV16, CTG TTA CAT TTT GTG AAA GAG TC; BV17, CTC ACA GAT AGT AAA TGA CTT TC; BV18, AGC CCA ATG AAA GGA CAC AGT CAT; BV19, ACC CCC GAA AAA GGA CAT ACT TTT; BV20, GAG GGA ACA TCA AAC CCC AAC CTA; BV21, TCC AGC CTG CAG AGC TTG GGG GAC T; BV22, AAG TGA TCT TGC GCT GTG TCC CCA; BV23, GCA GGG TCC AGG TCA GGA CCC CCA; BV24, CCC AGT TTG GAA AGC CAG TGA CCC; BV25, ATG TCT TTG ATG AAA CAG GTA; BV26, TCC AGT ACC AAA ACA TTG CAG; CB1, GTG TTT GAG CCA TCA GAA GC. The nomenclature is according to Arden et al (1995). PCR was carried out with Taq polymerase (Life Technologies, Gaithersburg, MD, USA), according to standard procedures, in 35 cycles consisting of 30 s at 95°C, 30 s at 55°C and 1 min at 72°C. PCR products were separated on a 1·5% agarose gel and analysed by ethidium bromide staining. The expressed Vα genes were analysed in a similar fashion. The primers used were the following: AV1, CAG CTT CTC CTC AAG TAC; AV2, TGG AAG GTT TAC AGC ACA; AV3, TCC ACC TAA TTT TAA TAC GT; AV4, TGC CTT GTA ACC ACT CCA; AV5, CAC CCT TAA CCA GAG TTT G; AV6, AAC CTT GTC ATC TCC GCT; AV7, ACC CAC ATT TCT GTC TTA CA; AV8, TTA TTA TAG ACA TTC GTT CAA AT; AV9, GAG ACA CAT CTC TAG AGA G; AV10, CCT CCT GGT GAC AGT AGT TAC; AV11, GTG TCC AAT GCT TAC AAC TT; AV12, CAG TTC CTT CAA CTT CAC C; AV13, CTG TCG CTA CGG AAC GCT; AV14, GAG AAT CGT TTC TCT GTG AA; AV15, CCA GTT GCT GAC GTA TAT TTT; AV16, CCT ATT CAG TCT CTG GAA AC; AV17, TAC GTC CAG ATG TGA GTG; AV18, CCA AAA CAC CCG AGG CCT; AV19, ACA ATA AAC ATA CAG GAA AAG; AV20, CAA GGA TAC AAG ACA AAA GTT; AV21, CTG AAG GTC CTA CAT TCC; AV22, ACA TAC CGT AAA GAA ACC ACT; AV23, GCT ATT TAC AAC CTC CAG; AV24, CCC CTT CAG CAA CTT AAG; AV25, GCT GGT GAA TTG ACC TCA; AV26, ACT GCC AAG TTG GAT GAG; AV27, TGA TAC CAA AGC CCG TCT; AV28, GGA AAA GAA AGC TCC CAC; AV29, GAA AAA ATA TCT GCT TCA TTT A; AV30, CTA AAA GCC ACA TTA ACA AAG; AV31, CAC GGG GTA CCC TAC C; AV32, AGG AGA GGA CTT CAC CAC; CaCONF, CAG CAT TAT TCC AGA AGA CAC; CaCONR, CCT CAG CTG GAC CAC AGC. For determination of the nucleotide sequence of the TCR α and β chain variable regions, specific PCR products were isolated with the Qiaquick gel extraction kit (Qiagen, Hilden, Germany). Cycle sequence reactions were carried out with the Cβ− and Cα-specific primers using the big dye terminator cycle sequencing ready reaction kit [Applied Biosystems (ABI), Warrington, UK]. Reaction mixtures were analysed on an ABI Prism 3100 genetic analyser.

51Cr-release assay. Target cells were resuspended in 3·7 MBq 51Cr (Amersham, Buckinghamshire, UK) per 1 × 106 cells and incubated for 1 h at 37°C. Cells were washed three times in RPMI-1640 medium with 10% FCS (RPMI/FCS). Labelled cells were seeded in triplicate in 96-well round-bottomed microtiter plates in RPMI/FCS at a concentration of 3 × 104 cells/well. T cells were added at different effector:target (E:T) ratios in a total volume of 200 µl. After centrifugation of the plates at 1400 r.p.m. for 5 min, cells were incubated at 37°C for 4 h. Then supernatants were harvested using the Skatron system (Molecular Devices Corporation, Sunnyvale, CA, USA), and radioactivity was measured with a Cobra autogamma betaplate reader (Packard, Groningen, the Netherlands). Wells containing RPMI/FCS only were used to measure the background radioactivity. For determination of the maximum c.p.m. values, a sample of labelled cells was lysed with Trition X100. The specific lysis was determined using the following calculation:

  • image

Interferon-γ (IFNγ) assay.  T cells were stimulated overnight with stimulator cells in 96-well round-bottomed microtiter plates in a total volume of 100 µl. Supernatant was harvested the following day and either stored at −20°C until IFNγ measurement or assayed directly. The IFNγ concentration in supernatants was determined using an IFN-γ-specific enzyme-linked immunosorbent assay (PeliPair reagent set; CLB, Amsterdam, the Netherlands).

Proliferation assay.  T cells were stimulated overnight with stimulator cells in 96-well round-bottomed microtiter plates in a total volume of 100 µl. The following day 3H-thymidine (Amersham, Warrington, UK) was added (37 kBq/well). The incorporated radioactivity was measured after 18 h in a β-scintillation counter (Wallac 1410; Gaithersburg, MD, USA).

Generation of a CML-specific T-cell line

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Generation of a CML-specific T-cell line
  6. Generation of a CML-specific T-cell clone
  7. DDII.4.4 specifically recognizes the CML of patient 1 (CML 1)
  8. The target cells recognized by DDII.4.4 include CD34+ CML progenitor cells
  9. The antigen recognized by DDII.4.4 is restricted by HLA-DRB1*16
  10. Discussion
  11. Acknowledgments
  12. References

Through repetitive stimulation of donor T cells with HLA-identical CML cells and 4HGF cells from patient 1, we obtained a T-cell line, designated as DDII.4. Fluorescence-activated cell sorting (FACS) analysis demonstrated a homogeneous expression of CD4 on DDII.4 (data not shown). The specificity of DDII.4 for the patient's CML cells was tested in a 51Cr-release cytotoxicity assay. Up to 41 ± 15% of specific lysis was obtained at an E:T ratio of 30:1. In contrast, irrelevant CML cells or EBV-transformed B cells from donor or patient were not killed (Fig 1). We estimated the number of individual clones that made up DDII.4 through RT-PCR analysis of the expressed Vβ gene families. While virtually all Vβ gene families could be detected in PBMC from a healthy donor, only four Vβ gene families could be detected in DDII.4: BV2, BV9, BV13 and BV20 (data not shown). This indicated that DDII.4 was the result of a stringent selection pressure by the in-vitro stimulation procedure, and that it consisted of a minimum of four different clones.

image

Figure 1.  Specificity of T-cell line DDII.4. The CD4+ T-cell line DDII.4 was tested in triplicate for its specificity in a 51Cr-release assay at different E:T ratios. Target cells used were the HLA-identical CML cells of patient 1, HLA-mismatched CML cells from a second patient, and EBV-transformed B-cell lines of both patient (p) and donor (d).

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Generation of a CML-specific T-cell clone

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Generation of a CML-specific T-cell line
  6. Generation of a CML-specific T-cell clone
  7. DDII.4.4 specifically recognizes the CML of patient 1 (CML 1)
  8. The target cells recognized by DDII.4.4 include CD34+ CML progenitor cells
  9. The antigen recognized by DDII.4.4 is restricted by HLA-DRB1*16
  10. Discussion
  11. Acknowledgments
  12. References

Subsequently, DDII.4 was cultured under limiting dilution conditions in order to obtain the individual T-cell clones. Only two of the four clones were recovered by this method. One CD4+ clone, DDII.4.4, was recovered from independent cultures in multiple wells. RT-PCR analysis of Vβ gene expression demonstrated the clonality of this clone, as only BV13 could be detected (Fig 2). FACS analysis with antibodies specific for different Vβ gene families confirmed that BV13 was the only Vβ gene that was functionally expressed by DDII.4.4 (data not shown). Nucleotide sequence analysis revealed that the variable regions of the α and β chains were encoded by AV12S1, Jα27, and BV13S1, Dβ2·1, Jβ2–5, Cβ2 respectively (Fig 3; Toyonaga et al, 1985; Arden et al, 1995). The second clone that was recovered expressed BV20 only, but it did not kill the CML cells (data not shown).

image

Figure 2.  Analysis of Vβ gene expression of T-cell clone DDII.4.4. cDNA of normal PBMC and clone DDII.4.4 were subjected to separate PCR reactions using primers specific for the different Vβ gene families. The reaction mixtures were subsequently separated by agarose gel electrophoresis. Shown are photonegative pictures of ethidium-bromide-stained agarose gels. Above each lane, the identity of the Vβ gene is indicated. The nomenclature is according to Arden et al (1995). (A) Normal PBMC, (B) clone DDII.4.4. The fragment seen in lane 18 is an aspecific product of the PCR reaction. The 300 and 600 bp fragments of the size marker are indicated.

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image

Figure 3.  Nucleotide and amino acid sequences of complementarity-determining region 3 of the TCRα and β variable regions. (A) TCRα chain, (B) TCRβ chain. The various V, D, J and C regions are indicated by solid lines. The nomenclature of the J genes is according to the ImMunoGeneTics database (http://imgt.cines.fr). The nomenclature of the other segments is according to Arden et al (1995). The deduced amino acid sequence is indicated above each codon.

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DDII.4.4 specifically recognizes the CML of patient 1 (CML 1)

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Generation of a CML-specific T-cell line
  6. Generation of a CML-specific T-cell clone
  7. DDII.4.4 specifically recognizes the CML of patient 1 (CML 1)
  8. The target cells recognized by DDII.4.4 include CD34+ CML progenitor cells
  9. The antigen recognized by DDII.4.4 is restricted by HLA-DRB1*16
  10. Discussion
  11. Acknowledgments
  12. References

The specificity of DDII.4.4 for the CML cells of patient 1 was tested in several assays. In a 51Cr-release assay, DDII.4.4 was able to lyse up to 70% of allogeneic CML cells, but not EBV-transformed B cells or PHA-stimulated T cells from the same patient, suggesting that DDII.4.4 recognized an antigen with a leukaemia- or myeloid-restricted expression pattern. DDII.4.4. also did not kill EBV-transformed B cells or PHA-stimulated T cells from the donor (Fig 4A). The specific recognition of the CML cells of patient 1 by DDII.4.4 was confirmed in proliferation and IFNγ secretion assays (Fig 4B and C). As bone marrow cells of the donor were not available, we included purified monocytes with the closest resemblance to myeloid cells from the donor as target cells in these assays. Similar to the cytotoxicity assay, no reactivity of DDII.4.4 was detected against EBV-transformed B cells from the donor or patient, nor against PHA-stimulated T cells. A low level of reactivity was detected towards the donor monocytes, leading to a significant production of 608 ± 71 pg IFNγ/ml. Nevertheless, this was far less than the 4333 ± 457 pg/ml IFNγ that was produced in response to the patient's CML cells. A similar pattern of reactivity was seen in the proliferation assay. To determine the restriction element of the antigen recognized by DDII.4.4, we performed a blocking experiment with different neutralizing monoclonal antibodies (mAbs). As shown in Fig 5, the cytotoxic activity of DDII.4.4 was inhibited significantly by a mAb that was directed against HLA-DR. In contrast, a class-I-specific mAb was not able to inhibit the specific lysis of CML cells.

image

Figure 4.  Specificity of DDII.4.4. DDII.4·4 was tested against EBV-transformed B-cell lines of both patient (p) and donor (d) PHA-stimulated T cells, and donor-derived monocytes. (A) 51Cr-release assay: DDII.4.4 was incubated overnight in triplicate with 51Cr-labelled target cells at different E:T ratios, after which the released radioactivity was measured in the culture supernatant. (B) IFNγ secretion assay: DDII.4.4 was incubated overnight in triplicate with target cells at an E:T ratio of 30:1. (C) Proliferation assay: DDII.4.4 was incubated overnight in triplicate with target cells at an E:T ratio of 30:1. After incubation with 3H-thymidine (3Tdr) for 18 h, incorporated radioactivity was measured.

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image

Figure 5.  HLA-restriction of DDII.4.4. DDII.4.4 was incubated overnight in triplicate with 51Cr-labelled CML cells from patient 1 in the presence or absence of neutralizing αHLA-DR or HLA-ABC mAb, after which the released radioactivity was measured.

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The target cells recognized by DDII.4.4 include CD34+ CML progenitor cells

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Generation of a CML-specific T-cell line
  6. Generation of a CML-specific T-cell clone
  7. DDII.4.4 specifically recognizes the CML of patient 1 (CML 1)
  8. The target cells recognized by DDII.4.4 include CD34+ CML progenitor cells
  9. The antigen recognized by DDII.4.4 is restricted by HLA-DRB1*16
  10. Discussion
  11. Acknowledgments
  12. References

We then tested whether DDII.4.4 was able to kill CD34+ CML progenitor cells. Thawed CML cells were cultured for 1 week in 4HGF medium, which allows outgrowth of CD34+ cells only. Figure 6 shows that incubation with DDII.4.4 during this period completely blocked growth of cells from CML 1 in this culture system, but not of an unrelated sample of irrelevant CML cells (CML 3).

image

Figure 6.  Reactivity of DDII.4.4 against CD34+ CML progenitor cells. CML cells were cultured in 4HGF either in the presence or absence of T cells. Cell counts were performed daily. (A) Culture of CML cells of patient 1, either with (•) or without (▪) DDII.4.4. (B) Culture of irrelevant CML cells 3 with (•) or without (▪) DDII.4.4.

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The antigen recognized by DDII.4.4 is restricted by HLA-DRB1*16

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Generation of a CML-specific T-cell line
  6. Generation of a CML-specific T-cell clone
  7. DDII.4.4 specifically recognizes the CML of patient 1 (CML 1)
  8. The target cells recognized by DDII.4.4 include CD34+ CML progenitor cells
  9. The antigen recognized by DDII.4.4 is restricted by HLA-DRB1*16
  10. Discussion
  11. Acknowledgments
  12. References

To test whether the antigen recognized by DDII.4.4 is unique or shared by other normal or malignant myeloid cells, we tested DDII.4.4 reactivity against a panel of samples in the IFNγ secretion assay. First, we tested DDII.4.4 against other CML and acute myeloid leukaemia (AML) samples that shared expression of either HLA-DR2 (DRB1*15, DRB1*16) or HLA-DR3 (DRB1*03, Table I). Out of five HLA-DR3+ samples, none was recognized by DDII.4.4. Of 12 HLA-DR2+ samples, two were recognized by DDII.4.4. Interestingly, the level of IFNγ synthesized was similar to the level produced in response to donor-derived monocytes (116 and 412 pg/ml IFNγ). Of note, both samples expressed HLA-DRB1*16. The same pattern of reactivity was observed in the proliferation assay (data not shown). In the next experiment, a collection of normal bone marrow samples was separated into CD34+ and CD34 fractions with immunomagnetic beads. Both fractions were used to stimulate DDII.4.4. Only cells of the single donor that expressed HLA-DRB1*16 were recognized by DDII.4.4. Both CD34+ and CD34 fractions were recognized (486 and 300 pg/ml IFNγ respectively), though it should be noted that the CD34 fraction still contained 10·6% CD34+ cells (Table II).

Table I.   Reactivity of DDII.4.4 against CML and AML samples.
PatientTypeHLAIFNγ (pg/ml)
1CMLA1, A24(9)/B8, B48/DRB1*16, DRB1*034333
2CMLA11, A26/B8/DRB1*15, DRB1*07  0
3CMLA2, A28/B51, B8/DRB1*03, DRB1*12  0
4AMLA9, A31/B51, B62/DRB1*16, DRB1*11 116
5AML/MDSA2/B44(12), B62/DRB1*16, DRB1*04 412
6AMLA2, A28/B27, B14/DRB1*15, DRB1*16  0
7AMLA2, A19/B44(12), B7/DRB1*03, DRB1*07  0
8AMLA2/B51(5), B7/DRB1*15  0
9CMLA1, A2/B15(62), B7/DRB1*15, DR1*06  0
10MDSA1, A31 (19)/B7, B40(60)/DRB1*15  0
11AMLA2, A11/B88, B62(15)/DRB1*03, DRB1*13  0
12AMLA2, A3/B7, B39(16)/DRB1*03, DRB1*08  0
13AMLA2, A3/B7, B40/DRB1*15, DRB1*13  0
14AMLA1, A24(9)/B8, B27/DRB1*03  0
15AMLA1, A24(9)/B39(16), B35/DRB1*01, DRB1*15  0
16AMLA2, A33 (19)/B40(60), B22/DRB1*15, DRB1*12  0
17AMLA2, A3/B7, B48/DRB1*01, DRB1*15  0
Table II.   Reactivity of DDII.4.4 against normal CD34+ and CD34 bone marrow samples.
DonorHLA%CD34+ cellsIFNγ (pg/ml)
Positive fractionNegative fractionPositive fractionNegative fraction
1A2, A3/B7, B17/DRB1*01, DRB1*1599·7n.d.00
2A24(9), A28/B57(17), B51(5)/DRB1*01,DRB1*1592·99·900
3A2, A11/B15, B51(5)/DRB1*16, DRB1*1155·310·6486300
4A1/B8/DRB1*0348·212·200
5A1, A2/B8, B62(15)/DRB1*15, DRB1*0365·12·000
6A2, A31 (19)/B7, B60(40)/DRB1*15, DRB1*0386·411·400
7A1/B7, B37/DRB1*10, DRB1*1560·25·200
8A1/B13, B52(5)/DRB1*13, DRB1*1578·21·100
9A2, A68(28)/B44(12), B7/DRB1*15, DRB1*1330·12·400
10A1, A24(9)/B60(40), B7/DRB1*0103, DRB1*1587·78·500
11A2, A3/B7, B27/DRB1*14, DRB1*1580·43·100
12A2, A3/B7, B14/DRB1*15, DRB1*0777·42·900
13A1, A28/B8, B60(40)/DRB1*03, DRB1*0979·14·800
14A1/B8, B44(12)/DRB1*03, DRB1*1379·35·700
15A2, A11/B51(5), B8/DRB1*03, DRB1*1425·70·900
16A3, A11/B7, B39(16)/DRB1*15, DRB1*1386·01·100
17A1, A2/B7, B37/DRB1*15, DRB1*1085·50·800

Discussion

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Generation of a CML-specific T-cell line
  6. Generation of a CML-specific T-cell clone
  7. DDII.4.4 specifically recognizes the CML of patient 1 (CML 1)
  8. The target cells recognized by DDII.4.4 include CD34+ CML progenitor cells
  9. The antigen recognized by DDII.4.4 is restricted by HLA-DRB1*16
  10. Discussion
  11. Acknowledgments
  12. References

Of all the haematological malignancies, CML is perhaps the one that displays the highest sensitivity for the GVL effect of allogeneic T cells. This is exemplified by the high cure rate of CML patients upon allo-SCT or DLI for the treatment of relapse. The underlying properties of CML cells that enable them to evoke this strong allogeneic immune response have not been identified yet. Both the slow proliferation rate of CML cells as well as the proper expression of co-stimulatory molecules have been proposed to explain the superior response of CML to allogeneic T cells compared with, e.g., AML. The observation that the GVL effect against CML cells is mainly exerted by CD4 cells is in line with the latter possibility (Falkenburg et al, 1991; Verfaillie et al, 1992; Leemhuis et al, 1993; Faber et al, 1995a,b; Mutis et al, 1997).

In the present study, we have shown that the CD4 clone DDII.4.4 strongly reacts against a DRB1*16-restricted antigen expressed by the CML cells of the HLA-identical patient 1. Of note, the target cells recognized include the CD34+ CML progenitor cells. Of all other cell populations tested, none evoked a similar response. A moderate but significant reaction was only seen against donor monocytes, two AML samples and a single sample from normal bone marrow. All samples expressed DRB1*16, strongly suggesting that DRB1*16 is the restriction element. Intriguingly, the response against these other samples was of the same order of magnitude and approximately 7–40-fold lower than that against CML 1. This peculiar recognition pattern may be explained in several ways. Firstly, the antigen expressed by CML 1 may be a miHa, evoking a strong response from DDII.4.4. Other alleles, expressed by the other positive samples, would be recognized with lower avidity. Secondly, CML 1 may express a mutated gene, and the wild-type gene may similarly be recognized with lower avidity. An obvious candidate gene in CML is, of course, the BCR-ABL fusion product, a hallmark of CML. Cells from CML 1 do express the BCR-ABL gene. However, this would not explain the moderate response against the other samples. HLA-identical cells not expressing BCR-ABL, such as EBV-transformed B cells of patient origin (data not shown), did not evoke the slightest response. Thirdly, the antigen expressed by CML 1 may have resulted from a gene that was over-expressed by CML 1 cells, but not by the other positive cell populations. The paucity of individuals expressing the DRB1*16 allele prevents extensive testing of samples from other patients and normal donors in order to discriminate between these possibilities. A final answer is expected with the molecular identification of the exact peptide that is recognized. The pattern of reactivity is also consistent with the notion that, in myeloid development, HLA-DR is expressed in the early stages of development and in the monocytic lineage. The latter is an important finding, as CD34+ reactive T cells were found to mediate the antileukaemic effect in vivo (Smit et al, 1998). As for the tissue specificity of the antigen, the recognition pattern, which includes CD34+ progenitor cells from patient 1 and excludes B and T cells, also indicates that the cognate antigen is derived from a gene which, within the haematopoietic lineage, is expressed in myeloid cells only.

In conclusion, we have isolated a CD4 clone that reacts strongly with allogeneic CML cells, including CD34+ CML progenitor cells and, to a lesser extent, with other myeloid cells sharing expression of the HLA-DRB1*16-restriction element. To our knowledge, this is only the second CD4+ T-cell clone described that recognizes CD34+ cells, though not entirely in an exclusive fashion (Mutis et al, 1997). Molecular identification of the cognate antigen will shed light on the antigen-processing mechanisms that are operational in both normal and CML CD34+ cells. In addition, identification of the antigen may allow targeting of clonogenic CD34+ CML progenitor cells.

References

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Generation of a CML-specific T-cell line
  6. Generation of a CML-specific T-cell clone
  7. DDII.4.4 specifically recognizes the CML of patient 1 (CML 1)
  8. The target cells recognized by DDII.4.4 include CD34+ CML progenitor cells
  9. The antigen recognized by DDII.4.4 is restricted by HLA-DRB1*16
  10. Discussion
  11. Acknowledgments
  12. References
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