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Prepublished online as a Blood First Edition Paper on September 12, 2002; DOI 10.1182/blood-2002-02-0525.
IMMUNOBIOLOGY
From the Departments of Haematology and Immunology,
Imperial College School of Medicine, London, United
Kingdom; and the Center for Cell and Gene Therapy, Texas
Children's Cancer Center, Houston.
Recent advances have made haploidentical transplantation for
leukemia feasible, but the rigorous T-cell depletion used contributes to the high relapse rates observed. We have attempted to improve the
graft-versus-leukemia (GVL) effect by generating allorestricted cytotoxic T lymphocytes (CTLs) directed against human CD45. Such CTLs
should recognize patient hematopoietic cells including leukemia, enhancing donor cell engraftment and improving the GVL effect, but they
should not recognize host nonhematopoietic tissues or donor cells from
the graft. Using the T2 binding assay, 4 CD45-derived peptides were
found to bind HLA-A2 molecules. These peptides were used to generate
cytotoxic T-cell lines from HLA-A2 Recent advances have made hematopoietic stem cell
transplantation (HSCT) from haploidentical donors feasible in terms of
reliable engraftment and acceptable rates of graft-versus-host disease (GVHD),1,2 though morbidity and mortality rates related to posttransplantation immunodeficiency remain problematic. A number of
lines of evidence demonstrate that donor T lymphocytes play a critical
role in eradicating residual leukemia after HSCT. These include
experimental studies in vitro and in animal models,3-5 the
higher relapse rate seen after T-cell-depleted HSCT,6 and the efficacy of donor lymphocyte infusions in restoring remission in
patients who experience relapse after HSCT.7 However,
after HLA-mismatched and haploidentical HSCT, this
graft-versus-leukemia (GVL) effect is likely to be lost because of the
rigorous T-cell depletion used, contributing to the high relapse rates
seen in these patients. Several groups have investigated the potential use of ex vivo-generated antigen-specific cytotoxic T lymphocytes (CTLs), recognizing antigens that differ in their expression between patient and donor for adoptive immunotherapy to restore the GVL effect.4,5,8 Candidate target antigens for such an
approach include tumor-specific antigens, those that are overexpressed in tumor cells, and mismatched minor histocompatibility antigens. In contrast, we have investigated the possibility of taking advantage of the disparity in HLA molecules needed to present shared antigens rather than relying on a difference in antigenic expression between host and donor. We have studied the antileukemic activity of such allorestricted CTLs in patients who have undergone HLA-mismatched HSCT.
The concept of this approach has previously been
described9 and is summarized in Figure
1. Autologous T cells recognizing self-peptides derived from cellular proteins are usually clonally deleted, leading to self-tolerance. In contrast, in the absence of the
appropriate HLA molecule, antigen-presenting cells (APCs) from an
HLA-mismatched donor may not present such a peptide, so that the donor
may have T cells recognizing epitopes to which the patient is tolerant.
In patients who undergo HLA-mismatched or haploidentical HSCT, this
could be exploited by stimulating donor T cells with APCs carrying the
host HLA restriction (eg, HLA A201), pulsed with epitopes from a
hematopoietic protein, to generate donor T cells that recognize
hematopoietic antigens in the context of host HLA molecules. It could
be predicted that such allorestricted CTLs would kill patient leukemic
cells, which express the hematopoietic antigen and the appropriate HLA
restriction, but they would not kill host nonhematopoietic tissues,
which do not express the antigen (averting GVHD), or donor
hematopoietic cells, which do not have the appropriate HLA restriction
to present the antigen (averting toxicity to the graft).
Although such an approach could be used with any HLA mismatch, we
focused on HLA-A0201 because it is expressed in approximately 50% of the white population; mismatches of this molecule were relatively common in haploidentical patient-donor pairs and in 1 antigen-mismatched patient-donor pair. We have previously
demonstrated that HLA-A0201 allorestricted CTLs directed against an
epitope from the Wilm tumor transcription factor (WT-1) kill leukemic cell lines and inhibit hematopoietic colony formation from progenitor cells from patients with chronic myeloid leukemia (CML).10
WT-1 is overexpressed in myeloid leukemias and is thus an excellent target for such an approach in these diseases, but it may not be so
useful for lymphoid malignancies that do not express WT-1 (eg, chronic
lymphoid leukemia). Here we have investigated CD45 as an alternative
target for allorestricted CTLs. CD45 is a 180- to 220-kDa plasma
membrane-associated tyrosine phosphatase critical for T- and B-cell
receptor-mediated activation.11,12 A number of features
make it an excellent target for the immunotherapy of leukemias. It is
abundantly expressed on most leucocytes, including leukemic blasts, so
that CTLs directed against CD45 epitopes would likely be active in
myeloid and lymphoid malignancies. Furthermore, allorestricted CTLs
directed against CD45 should recognize normal host T cells and
myelopoiesis and might thus contribute to immunosuppression and
myeloablation of the host as an immunologic arm to conditioning. Finally, CD45 expression is restricted to hematopoietic cells; hence,
CTLs recognizing CD45 epitopes should not target nonhematopoietic cells
and would not be expected to cause GVHD. In this study we have explored
the possibility of exploiting CD45 as a target molecule for
allorestricted CTLs in patients with HLA-A0201 mismatch, and we have
demonstrated that such CTLs show significant antileukemic activity in vitro.
Cell lines, generation of primary fibroblasts, and purification
of CD34+ cells
Synthetic peptides, peptide-binding assays, and intracytoplasmic
flow cytometry
Intracytoplasmic flow cytometry for IFN- Generation of allo-HLA-restricted CTL lines PBMCs were separated from buffy-coat packs using Ficoll-Hypaque density gradient centrifugation and were stained with monoclonal antibodies HB54 (anti-HLA-A2, B17) and HB117 (anti-HLA-A2, A28). A2-negative PBMCs were used as responders. T2 cells that had been loaded with 100 µM peptide for 6 hours and then irradiated (60 Gy) were used as initial stimulators. Each well of a 24-well plate received 2 × 106 responder PBMCs and 2 × 105 irradiated, peptide-loaded T2 stimulators in 2 mL RF10 (RPMI with 10% fetal calf serum) with 2.5 ng/mL recombinant human IL-7 (huIL-7) and 500 nM peptide. Drosophila A2 cells were used for the initial restimulation. These were induced in 100 mM CuSO4 for 48 hours, washed 3 times with medium, and loaded with 100 µM peptide for 4 hours. On day 6, T cells were harvested, plated at a density of 5 × 105 per well, and restimulated with 2 × 105 peptide-coated Drosophila A2 cells with the addition of 2 × 106 autologous irradiated (30 Gy) PBMCs as feeders, in fresh medium containing 10% QS4120 culture supernatant (containing anti-CD4 antibodies), 10 U/mL recombinant huIL-2 (Roche Diagnostics, Lewes, United Kingdom), 2.5 ng/mL recombinant huIL-7, and 500 nM peptide. On day 18, T cells were harvested and restimulated in a similar fashion but using RMA/S-A2 cells that had been incubated at 25°C to induce HLA-A0201 expression, loaded with 100 µM peptide, and irradiated (80 Gy) as stimulators. On day 26, after 3 cycles of stimulation, bulk cultures were seeded in 96-well plates at densities of 1, 20, 50, and 100 cells per well; 2 × 104 peptide-coated irradiated T2 cells and 2 × 105 irradiated HLA-A2-negative PBMC feeders were added to each well in RF10 with 10 U/mL huIL-2 and 2.5 ng/mL huIL-7. Cultures received additional stimulator and feeder cells on day 40. Six days later, the cytotoxicity of each well was tested against T2 target cells coated with the immunizing peptide or a control HLA-A0201-binding peptide. Peptide-specific microcultures were expanded and restimulated weekly in 24-well plates by adding 2 × 106 irradiated feeders and 2 × 105 peptide-loaded, irradiated T2 cells as stimulators in medium containing 10 U/mL IL-2 and 2.5 ng/mL IL-7.Cytotoxicity and colony-inhibition assays Cytotoxic T-lymphocyte assays were performed as described. Briefly, 3 × 106 targets were labeled with 100 µCi (3.7 MBq) chromium Cr 51 for 2 hours, washed 3 times, and added to serial dilutions of effector cells in triplicate, round-bottomed, 96-well plates to obtain a total volume of 200 µL/well. In some experiments T2 cells were peptide loaded by preincubation with 100 µM synthetic peptide before labeling. In antibody-blocking experiments, 103 chromium-labeled target cells were preincubated with 10 µL blocking antibodies to HLA class 1 (clone W6/32; DAKO, Carpinteria, CA), HLA class 2 (clone CR3/43; DAKO), or isotype control for 30 minutes at 37°C before the addition of CTLs. In some experiments a 50-fold excess of cold K562 cells or a 20-fold excess of peptide-loaded T2 cells was added to cultures to assess specificity. Assay plates were incubated for 6 hours at 37°C, 5% CO2, and 100 µL supernatant was harvested and counted using a Wallac gamma counter (Wallac, Milton Keynes, United Kingdom). Specific lysis was calculated by the equation (experimental release spontaneous release)/maximum release spontaneous release) × 100%. Colony-inhibition assays were performed by culturing CD34+ PBMCs and BMMCs in the
presence and absence of CTLs at an effector-target ratio of 10:1 for 6 hours. CTL-treated and mock-treated CD34+ cells were then
plated in duplicate plates in methylcellulose supplemented with 100 ng/mL granulocyte-colony-stimulating factor (G-CSF), 1 ng/mL
granulocyte macrophage-colony-stimulating factor (GM-CSF), 5 ng/mL
recombinant human IL-3 (rhIL-3), and 20 ng/mL recombinant human stem
cell factor (rhSCF). Granulocyte macrophage-colony-forming units
(CFU-GM) with more than 100 cells were counted after 10 to 12 days.
Percentage inhibition of colony formation was determined as (no. CFU-GM
in the presence of CTL/no. CFU-GM in the absence of
CTL) × 100%.
Identification of CD45 peptides binding HLA A201 and generation of CD45-specific CTLs As shown in Figure 2A, we confirmed high levels of CD45 expression on leukemic blasts from patients with acute lymphoblastic leukemia (ALL) and AML and on PBMCs from patients with CML, indicating that CTLs directed against CD45 are likely to have activity against myeloid and lymphoid leukemias. The level of expression was higher in normal and CML PBMCs than in AML and ALL blasts. To determine the expression pattern of CD45 in a variety of human tissues, we performed Northern blot analysis. Figure 2B shows that the expression of CD45 is restricted to hematopoietic tissues, with high levels of mRNA expression in the thymus, spleen, and peripheral blood. The apparent low level of CD45 RNA seen in heart and liver probably resulted from the presence of contaminating hematopoietic cells. To identify CTL epitopes in CD45, we synthesized 16 peptides that were predicted by the BIMAS computer model to bind to HLA-A0201. We investigated the relative ability of these 16 peptides to bind HLA-A0201 by assaying the ability of exogenously pulsed peptide to up-regulate HLA-A0201 expression on the cell surface in the TAP-deficient cell line T2. Figure 3 shows that 4 peptides (P292, P684, P737, P1218) showed significant binding to HLA-A0201, whereas the remaining peptides did not bind.
We then attempted to generate allorestricted CTLs with 4 of these
peptides by stimulation of PBMCs from healthy HLA A0201-negative responders with a variety of HLA A0201+ APCs exogenously
pulsed with peptide, followed by limiting-dilution cultures to isolate
peptide-specific CTL lines. No peptide-specific lines were generated
using P292, P684, or P737. However, peptide-specific CTL lines directed
against P1218 were generated from 3 of 7 donors. This peptide (sequence
FLYDVIAST) is located in the second phosphotyrosine phosphatase domain
of the cytoplasmic tail of CD45 and was the best binder to HLA A0201 in
peptide-binding assays. As illustrated in Figure
4A, all 3 of these CTL lines showed
exquisite peptide specificity in cytotoxicity assays, with almost total
killing of T2 targets pulsed with P1218 at low effector-target ratios and little cytotoxicity against T2 cells alone or pulsed with a control
peptide known to bind HLA A0201. Little major histocompatibility complex (MHC)-unrestricted cytotoxicity against K562 targets was observed, and cytotoxicity against T2 cells pulsed with P1218 was not
abrogated by a 50-fold excess of cold K562 cells (Figure 4C),
demonstrating the observed cytotoxicity was specific. All 3 CTL lines
were initially 60% to 70% CD8+ and 30% to 40%
CD4+, but with prolonged culture CD8+ cells
increased to 90% to 95% and the CD4+ population decreased
to 3% to 5%. To test the avidity of our T-cell lines, we performed
cytotoxicity assays against T2 targets titrating the concentration of
exogenously pulsed P1218. As shown in Figure 4B, line 2 showed the
highest avidity with significant cytotoxicity at nanomolar levels of
peptide. Line 2 was therefore selected for further experiments.
To determine whether the response against P1218 peptide-pulsed T2 cells
was mediated by CD4 or CD8+ cells, intracytoplasmic flow
cytometry was used to study the expression of IFN-
CD45-specific CTLs kill leukemic cell lines To determine whether P1218-specific CTLs were able to recognize endogenously processed CD45, we analyzed cytotoxicity against a panel of leukemic cell lines. As can be seen in Figure 6, P1218-specific CTLs were able to efficiently lyse the B-lymphoblastoid cell line, CIR-A2, which expresses CD45 and HLA-A0201, but they did not kill a similar lymphoblastoid cell line, WS29, which expresses CD45 but is HLA-A0201 . Likewise there was no significant cytotoxicity
against the breast cancer epithelial cell line T4D7, which is
HLA-A0201 and CD45 , or against
HLA-A0201 K562 cells, a target for natural killer (NK)
cells. These results indicate that the observed cytotoxicity was
HLA-A0201 restricted and independent of NK activity. Cytotoxicity
against C1R-A2 cells was partially abrogated in blocking experiments
with an anti-HLA class 1 antibody but not with an anti-HLA class 2 antibody (data not shown). P1218-specific CTLs were unable to lyse
BV173, a leukemia cell line that expresses HLA-A0201 but has lost
expression of CD45 or 293 cells, an embryonic kidney cell line
expressing HLA-A0201. These results demonstrate that HLA-A0201
expression alone is insufficient for recognition by this CTL line and
suggest that coexpression of CD45 is required. Surprisingly, at high
effector-target ratios, 2 nonhematopoietic targets, MCF-7, an
HLA-A0201+ breast cancer cell line, and HeLa-A2, a cervical
epithelial cell line transfected with HLA-A0201, were also killed by
P1218-specific CTLs (though less well than CIR-A2 targets), despite the
fact that neither of these cell lines expressed detectable CD45 by FACS
staining. The cytotoxicity of P1218-specific CTL against both these
targets was only partially inhibited by a 50-fold excess of cold K562
cells (data not shown), indicating that recognition of these
HLA-A0201+, CD45 targets is not primarily
caused by NK activity.
The observed cytotoxicity against CD45 P1218-specific CTLs recognize endogenously presented CD45 in malignant hematopoietic cell lines To determine whether P1218-specific CTLs are capable of recognizing endogenously processed CD45 in malignant hematopoietic cells, we took advantage of the observation that the myeloma cell line U-266 is naturally biphenotypic with respect to CD45 expression. This cell line was FACS sorted into CD45 and
CD45+ fractions (Figure 7Aii,iv), and these were
used as targets in cytotoxicity assays. As shown in Figure
7Av, P1218-specific CTLs were able to
lyse CD45+ U-266 cells efficiently, whereas the
CD45 U-266 cells were killed poorly. These results
demonstrate that endogenously expressed CD45 was sufficient for
recognition of U-266 cells by the CTLs. The low-level cytotoxicity
observed against the CD45 U-266 fraction may reflect
lysis of contaminating CD45+ cells because a minority of
cells show phenotypic conversion to CD45+ after prolonged
culture (Figure 7Ai-iv).
To further show that P1218-specific CTLs recognize endogenously processed CD45, we generated a stable transfectant of the HLA-A0201+ kidney cell line, 293, expressing CD45RO (Figure 7Bi-iv). As shown in Figure 7Bv, wild-type 293 cells were killed poorly, whereas after the introduction of CD45 this same cell line was efficiently lysed by P1218-specific CTLs, demonstrating that these CTLs recognized endogenously expressed CD45. CD45-specific CTLs are cytotoxic to primary normal hematopoietic and leukemic cells To analyze the activity of P1218-specific CTLs against primary leukemic targets, cytotoxicity assays were performed using CML PBMCs and leukemic blasts from HLA-A0201-positive patients with AML. As illustrated in Table 1, P1218-specific CTLs showed significant cytotoxicity against PBMCs from 4 of 4 patients with CML and against leukemic blasts from 4 of 4 patients with AML tested. No killing of HLA-A0201 normal or CML PBMCs was
observed, demonstrating that the observed cytotoxicity is HLA-A2
restricted. Leukemic blasts from 2 HLA-A0201+ patients with
ALL, both of which expressed CD45, were not killed (data not
shown).
To determine tissue specificity of P1218-specific CTLs against primary
targets, we performed cytotoxicity assays using primary fibroblasts
from HLA-A0201+ healthy donors as targets. After induction
with IFN- Given that CML PBMCs are a relatively heterogeneous population of
primitive and more mature leukemic cells, we then went on to test the
activity of P1218-specific CTLs against earlier normal and leukemic
clonogenic progenitors in colony-forming assays. As shown in Figure
8, preincubation of CD34+
BMMCs with P1218-specific CTLs significantly inhibited CFU-GM formation
in 4 of 4 healthy HLA-A2+ controls. Similarly, strong
inhibition of CFU-GM formation was observed in 4 of 5 HLA-A2+ patients with CML. The HLA-A2+ patient
in whom no inhibition was observed was only serologically typed and
might have had a subtype other than A0201. No inhibition of colony
formation was observed in HLA-A2
We have investigated a novel approach to adoptive immunotherapy to restore the GVL effect following haploidentical HSCT. Rather than relying on a disparity in antigenic expression between host and donor, we have exploited an HLA mismatch to generate CTLs directed against a hematopoietic antigen common to host and donor, recognized only in the context of host HLA molecules. Because it circumvents immunologic tolerance, this allorestricted approach is suitable for raising CTLs against any hematopoietic antigen. CD45 was chosen as a candidate because of its hematopoietic-restricted expression and because it is abundantly expressed in myeloid and lymphoid leukemia. The described P1218 epitope is located on the C-terminal second phosphotyrosine phosphatase domain and is not known to be polymorphic or to function as a minor histocompatibility antigen in patients with HLA match. It is anticipated that autologous and HLA-matched CTLs are tolerant of this epitope. The allorestricted CTL lines generated in this study contained predominantly CD8+ cells, particularly after prolonged culture, though some CD4+ cells persisted in all lines established. In previous experiments with allorestricted CTL lines directed against WT-1, CD4+ cells were also present and showed no proliferative response to peptide or T2 cells but did show strong interleukin-2 (IL-2)-dependent proliferation in the absence of antigen (H.J.S., unpublished data, 2000). Subcloning of these CTL lines yielded CD8+ CTL clones that showed the killing specificity displayed by the line, but the life span of these clones was limited to 6 to 8 weeks in the absence of CD4+ T cells. Similarly, we were unable to expand and maintain subclones of the P1218-specific CTL line. This in vitro observation is similar to what has been observed in clinical trials of adoptive immunotherapy with antiviral CTLs, suggesting that that the presence of helper T cells may increase the persistence of infused CTLs.17,18 Our observations that lysis of cell line targets, including U-266, by
P1218-specific CTL requires coexpression of HLA-A0201 and CD45,
together with our data from stable transfectants of 293 cells, show
that this CTL line is capable of recognizing endogenously expressed
CD45. Formal demonstration of endogenous presentation of the P1218
epitope, however, will require peptide elution from HLA-A0201+ hematopoietic cells. The activity of
P1218-specific CTLs against the HLA-A0201+ nonhematopoietic
cell lines HeLa-A2 and MCF-7 in the absence of CD45 has been explored
in cold-target inhibition experiments. Results suggest that the
unexpected killing activity is not caused by MHC-unrestricted NK
activity but rather is most likely caused by the presence of a small
number of alloreactive CTLs capable of recognizing A0201 molecules
presenting irrelevant peptide epitopes. It is interesting to note that
though our CTL line showed significant killing of TAP-deficient T2
cells without peptide pulsing (Figure 2A), and of HeLa-A2 and MCF-7
cells (Figure 6A), little killing was observed against other
A0201-positive targets such as BV173, 293, and IFN- Allorestricted CTLs directed against P1218 show significant
cytotoxicity against primary leukemic targets from
HLA-A0201+ patients with AML and CML. Leukemic blasts from
2 HLA-A0201+ patients with ALL were not lysed, and further
studies will be needed to determine whether this is generally true in
ALL. In this regard it is interesting to note that ALL blasts are also less susceptible to lysis by alloreactive NK clones, perhaps because of
their lack of LFA-1 expression,20 and they also appear
resistant to anti-CD45 antibody-mediated lysis (M.K.B., personal
communication, June 2002). Additionally, P1218-specific CTLs
strongly inhibit colony formation from CD34+ CML
progenitors in an HLA-A0201-restricted fashion. These results suggest
that P1218-specific CTLs may have significant antileukemic activity in
the context of an HLA-A0201+ patient undergoing
transplantation from an HLA-A0201 Sufficient numbers of P1218-specific CTLs can be generated for clinical protocols using our current methodology; once the CTL lines are established, they are easily expanded. Clearly, however, if such an approach is to be adapted to the clinical setting, our protocol for the initial generation of allorestricted CTLs will have to be simplified greatly. Selection of P1218-specific CTLs from bulk cultures with HLA-A0201 tetramers loaded with P1218 peptide may allow rapid isolation of peptide-specific CTLs without resorting to cloning. Alternatively, T-cell receptor gene transfer from existing CTL lines could be used to redirect the specificity of circulating T cells, obviating the need to generate allorestricted CTLs for each patient.23
Submitted February 15, 2002; accepted September 3, 2002.
Prepublished online as Blood First Edition Paper, September 12, 2002; DOI 10.1182/blood-2002-02-0525.
Supported by the Medical Research Council of the United Kingdom and the Leukemia Research Fund.
The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked "advertisement" in accordance with 18 U.S.C. section 1734.
Reprints: Persis J. Amrolia, Department of Bone Marrow Transplantation, Great Ormond St Children's Hospital, Great Ormond St, London WC1, United Kingdom; e-mail: amrolp1{at}gosh.nhs.uk.
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 M. H. M. Heemskerk, M. Hoogeboom, R. A. de Paus, M. G. D. Kester, M. A. W. G. van der Hoorn, E. Goulmy, R. Willemze, and J. H. F. Falkenburg Redirection of antileukemic reactivity of peripheral T lymphocytes using gene transfer of minor histocompatibility antigen HA-2-specific T-cell receptor complexes expressing a conserved alpha joining region Blood, November 15, 2003; 102(10): 3530 - 3540. [Abstract] [Full Text] [PDF]
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 H. E. Heslop, F. K. Stevenson, and J. J. Molldrem Immunotherapy of Hematologic Malignancy Hematology, January 1, 2003; 2003(1): 331 - 349. [Abstract] [Full Text] [PDF]
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Abstract
Introduction
-2
microglobulin, B7.1, and intracellular adhesion molecule 1 (ICAM-1)
were the kind gift of Dr M. Jackson. RMA/S-A2 is a mouse lymphoma line
transfected with a human genomic HLA-A0201 clone including the
endogenous promoter. C1R-A2 is an HLA-A0201-transfected Epstein-Barr
virus (EBV)-transformed lymphoblastoid cell line, and WS29 cell is an
EBV-transformed lymphoblastoid cell line from an HLA-A0201-negative
patient. BV173 is an HLA-A0201-positive cell line derived from a
patient with CML in lymphoid blast crisis that lost expression of CD45.
HeLa-A2 cells were made by transfecting the human cervical epithelial
cell line HeLa with a genomic HLA-A0201 clone, including the endogenous
promoter. T4D7 and MCF-7 are HLA-A2
(IFN-
