Blood, Vol. 94 No. 12 (December 15), 1999:
pp. 4374-4376
CORRESPONDENCE
Induction of Minor Histocompatibility Antigen HA-1-Specific
Cytotoxic T Cells for the Treatment of Leukemia After Allogeneic
Stem Cell Transplantation
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LETTER |
To the Editor:
Disparities in the polymorphic minor histocompatibility antigens (mHag)
between donor and recipient are associated with the development of
graft-versus-host disease (GVHD) and graft-versus-leukemia (GVL)
reactions after allogeneic stem cell transplantation. The mHag HA-1 has
a restricted tissue distribution with an exclusive expression in human
hematopoietic cells. HA-1 is recognized by alloreactive donor-derived
major histocompatibility complex (MHC) class I-restricted
cytotoxic T cells (CTL), making this epitope an interesting target for
the development of specific immunotherapies against leukemia after
allogeneic stem cell transplantation with a low risk of
GVHD.1 Recently, the peptide sequence of the HLA-A*0201-restricted HA-1 mHag (HA-1H) has been
identified and shown to differ in only 1 amino acid in position 3 from
the HA-1-negative allelic counterpart
(HA-1R).2 Moreover, Mutis et al3
have demonstrated that HA-1H peptide-pulsed dendritic cells
(DC) generated from bone marrow CD34+ cells induce
HA-1-specific CTL in vitro that efficiently lyse leukemic cells
without lytic activity against nonhematopoietic cells, confirming the
restricted expression of this epitope. The in vitro expansion kinetics
of the HA-1-specific CTL were slow, although it was estimated that
109 HA-1-specific CTL can be obtained after 5 weeks of
culture for adoptive immunotherapy purposes.3
In this report, we present additional data providing evidence that
HA-1H peptide-specific CTL induced in vitro using
HA-1H peptide-pulsed monocyte-derived DC as
antigen-presenting cells from unprimed HA-1-negative healthy donors
are not only able to lyse primary leukemic blasts or immortalized B
cells naturally expressing the HA-1H/H phenotype (Fig
1), but also recognize
heterozygous leukemic cells with the HA-1H/R phenotype,
showing that these HA-1-specific CTL are of high affinity to the
peptide/MHC complex. This finding was also confirmed in peptide
titration assays (Fig 2). Furthermore, the
HA-1H peptide-specific CTL did not cross-react with the
HA-1R peptide (Fig 1C), because HA-1-negative
(HA-1R/R) U266 cells or HA-1R peptide-pulsed
U266 cells were not lysed by HA-1H-specific CTL, whereas
U266 cells pulsed with the cognate HA-1H peptide were
efficiently lysed by HA-1H-specific CTL. Interestingly, we
were also able to induce T cells specific for the HA-1R
peptide in 2 individuals; however, these cells were not able to lyse
targets with the HA-1R/R phenotype, demonstrating either
that this epitope is not presented or that the CTL are of low affinity
(data not shown).
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| Fig 1.
(A and B) Lysis of homozygous or heterozygous
HA-1-positive primary leukemic blasts by HA-1-specific CTL.
HA-1H peptide-specific CTL were induced in vitro from 2 different healthy HLA-A2-positive, HA-1-negative blood donors by
monocyte-derived DC pulsed with synthetic HA-1H
peptide.2 DC were generated from adherent blood monocytes
in the presence of GM-CSF, IL-4, and tumor necrosis factor 
(TNF), as described.6 Leukemic HA-1-positive acute
myelogenous leukemia (AML) or acute lymphoblastic leukemia (ALL) blasts
from 6 different patients were used as target cells in a classical
51Cr release assay.6 The HA-1-negative cell
line "RR" pulsed with an irrelevant human immunodeficiency virus
(HIV)-peptide was used as a negative control. Cell line "RR"
(line 6574803) was kindly provided by Dr E. Goulmy (University of
Leiden, Leiden, The Netherlands). (C) HA-1H
peptide-specific CTL do not cross-react with the HA-1R
peptide. CROFT is an HLA-A2+,
HA-1H/H-positive immortalized B-cell line, whereas U266 is
an HLA-A2+, HA-1-negative (HA-1R/R)
myeloma cell line.
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| Fig 2.
Peptide titration assay. U266 cells were pulsed with
titrating amounts of the HA-1H peptide (P2) or an
irrelevant HLA-A2 binding peptide derived from HIV and used as targets
in a 51Cr release assay with HA-1H-specific
CTL.
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To estimate the applicability of HA-1-specific CTL for the treatment
of relapsed HA-1H/H- or HA-1H/R-positive
leukemias after allogeneic transplantation, we analyzed the frequency
of HA-1 gene expression in 65 HLA-A*0201-positive healthy individuals
using reverse transcription-polymerase chain reaction
(RT-PCR) with HA-1 allele specific primers (Fig
3). We found that 16% of this
population is homozygous for HA-1 (HA-1H/H) and that 54%
of this population is heterozygous (HA-1H/R), whereas
30% is HA-1-negative (HA-1R/R), suggesting that
approximately 70% of the HLA-A2-positive patient population could be
candidates for such a treatment if they have an
HA-1R/R-negative stem cell donor. Taken together, these
results open new possibilities for the treatment of HA-1-positive
leukemia patients after allogeneic stem cell transplantation from
HA-1R/R-negative donors by adoptive transfer of
HA-1-specific CTL, as suggested by Mutis et al,3 or by
immunizing the donor with HA-1H peptide-pulsed autologous
DC before transplantation. The quantitation and isolation of in
vivo-induced HA-1-specific CTL in HA-1-negative stem cell donors
after vaccination would be performed using tetrameric HLA-peptide
complexes4,5 and thus would probably facilitate the
clinical application of donor-derived mHag-specific CTL.
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| Fig 3.
HA-1 gene expression in 65 HLA-A2-positive healthy blood
donors, hematopoietic cell lines, and primary leukemic
blasts by allele-specific RT-PCR. PCR using HA-1 allele-specific
primers was performed as previously described,2 resulting
in a 255-bp fragment. Results from representative samples from
homozygous (HA-1H/H) (including CROFT and cell line HH
[6574805; kindly provided by Dr E. Goulmy]) and heterozygous
(HA-1H/R) HA-1-positive cell lines (OPM2 and KG1) or
primary leukemic blasts (1, 2, and 3) as well as HA-1-negative cell
lines (U266, RR [line 6574803]) are demonstrated.
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ACKNOWLEDGMENT |
Supported in part by grants from Deutsche Forschungsgemeinschaft (SFB
510) and Deutsche Krebshilfe.
Peter Brossart
Brigitte Spahlinger
Frank Grünebach
Gernot Stuhler
Volker L. Reichardt
Lothar Kanz
Wolfram Brugger
Department of Hematology, Oncology and
Immunology
University of Tübingen
Tübingen,
Germany
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REFERENCES |
1.
Goulmy E, Schipper R, Pool J, Blokland W, Falkenburg JHF, Vossen J, Gratwohl A, Vogelsang GB, van Houwelingen H, van Rood JJ:
Mismatches of minor histocompatibility antigens between HLA-identical donors and recipients and the development of graft-versus-host disease after bone marrow transplantation.
N Engl J Med
334:281, 1996
[Abstract/Free Full Text]
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Den Haan JMM, Meadows LM, Wang W, Pool J, Blokland E, Bishop TL, Reinhardus C, Shabanowitz J, Offringa R, Hunt D, Engelhard VH, Goulmy E:
The minor histocompatibility antigen HA-1: A diallelic gene with a single amino acid polymorphism.
Science
279:1054, 1998
[Abstract/Free Full Text]
3.
Mutis T, Verdijk R, Schrama E, Esendam B, Brand A, Goulmy E:
Feasibility of immunotherapy of relapsed leukemia with ex vivo-generated cytotoxic T lymphocytes specific for hematopoietic system-restricted minor histocompatibility antigens.
Blood
93:2336, 1999
[Abstract/Free Full Text]
4.
Mutis T, Gillespoie G, Schrama E, Falkenburg JHF, Moss P, Goulmy E:
Tetrameric HLA-class I-minor histocompatibility antigen peptide-complexes demonstrate minor histocompatibility antigen-specific cytotoxic T lymphocytes in patients with graft-versus-host disease.
Nat Med
5:839, 1999
[Medline]
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5.
Callan MF, Tan L, Annels N, Ogg GS, Wilson JD, O'Callaghan CA, Steven N, McMichael AJ, Rickinson AB:
Direct visualization of antigen-specific CD8+ T cells during the primary immune response to Epstein-Barr virus in vivo.
J Exp Med
187:1395, 1998
[Abstract/Free Full Text]
6.
Brossart P, Heinrich K, Stuhler G, Behnke L, Reichardt VL, Stevanovic S, Muhm A, Rammensee HG, Kanz L, Brugger W:
Identification of HLA-A2 restricted T-cell epitopes derived from the MUC-1 tumor antigen for broadly applicable tumor vaccines.
Blood
93:4309, 1999
[Abstract/Free Full Text]
Response
We appreciate the letter of Brossart et al confirming our recent
results of ex vivo generation of cytotoxic T cells specific for HA-1
minor histocompatibility antigen (mHag) for adoptive immunotherapy of
relapsed leukemia. Brossart et al have generated HA-1-specific CTL
from unprimed, HLA-A2-positive healthy donors using monocyte-derived
dendritic cells (moDC) as antigen-presenting cells (APC),
whereas we used peripheral blood dendritic cells (PBDC) or DC cultured
from CD34+ progenitor cells (BMDC).1 The usage
of moDC as primary APC is convenient and generally applicable. The
drawback in the protocol of Brossart et al is the addition of fetal
calf serum (FCS) in their moDC culture.2
Xenogenic material should be avoided in the clinical work, but FCS
removal from the protocol may affect the outcome. For instance,
xeno-antigens of FCS may activate helper T cells, which in turn can
provide nonspecific help to HA-1-specific CTL. We have also observed
that maturation of DC is easier if they are cultured in FCS as compared
with autologous plasma (unpublished observations). We have
recently addressed this latter issue and developed a clinically
applicable moDC culture protocol in which we show the stable and full
maturation of moDC cultured in autologous plasma by the usage of
clinically applicable poly I:C, a double-stranded RNA that can be
safely used in vivo.3
Identical to our results, Brossart et al show that ex vivo-generated
HA-1-specific CTLs exhibit potent and antigen-specific lytic activity
against HA-1-positive leukemic cells. Unfortunately, Brossart et al
did not show the discrimination of hematopoietic cells from
nonhematopoietic cells by HA-1-specific CTL, which is crucial for
clinical application. Yet, we expect the hematopoietic specificity
based on our results in which ex vivo-generated HA-1-specific CTL do
not lyse nonhematopoietic cells.1
Regarding the number of candidates who are eligible for the therapy, we
find the remark of Brossart et al that 70% of the HLA-A2-positive
individuals would be candidates if they have HA-1-negative donors
misleading. Based on population frequency studies, combined with
segregation analyses, we and others have calculated that only 13% of
HA-1-positive patients will have an HA-1-negative sibling.4-6 Evidently, the number of candidates for the
therapy will increase if HA-1R-specific CTL can also be
generated. We therefore appreciate the attempts of Brossart et al to
generate HA-1R-specific CTL.
T. Mutis
E. Goulmy
Department of Immunohematology and Blood Bank
Leiden
University Medical Center
Leiden, The Netherlands
| |
REFERENCES |
1.
Mutis T, Verdijk R, Schrama E, Esendam B, Brand A, Goulmy E:
Feasibility of immunotherapy of relapsed leukemia with ex vivo-generated cytotoxic T lymphocytes specific for hematopoietic system-restricted minor histocompatibility antigens.
Blood
93:2336, 1999
2.
Brossart P, Heinrich KS, Stuhler G, Behnke L, Reichardt VL, Stevanovic S, Muhm A, Rammensee HG, Kanz L, Brugger W:
Identification of HLA-A2-restricted T-cell epitopes derived from the MUC1 tumor antigen for broadly applicable vaccine therapies.
Blood
93:4309, 1999
3.
Verdijk RM, Mutis T, Esendam B, Kamp J, Melief CJ, Brand A, Goulmy E:
Polyriboinosinic polyribocytidylic acid (poly(I:C)) induces stable maturation of functionally active human dendritic cells.
J Immunol
163:57, 1999
[Abstract/Free Full Text]
4.
van Els CA, D'Amaro J, Pool J, Blokland E, Bakker A, van Elsen PJ, van Rood JJ, Goulmy E:
Immunogenetics of human minor histocompatibility antigens: Their polymorphism and immunodominance.
Immunogenetics
35:161, 1992
[Medline]
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5.
Schreuder GM, Pool J, Blokland E, van Els C, Bakker A, van Rood JJ, Goulmy E:
A genetic analysis of human minor histocompatibility antigens demonstrates Mendelian segregation independent of HLA.
Immunogenetics
38:98, 1993
[Medline]
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6.
Martin PJ:
How much benefit can be expected from matching for minor antigens in allogeneic marrow transplantation?
Bone Marrow Transplant
20:97, 1997
[Medline]
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