|
|||||||||
|
|
|
|
|
|
|
|
|
|
|
Blood, Vol. 93 No. 10 (May 15), 1999:
pp. 3550-3557
By
From the Laboratorio di Immunologia e Unità di Trapianto di
Midollo Osseo, Dipartimento di Scienze Pediatriche, Università
degli Studi di Pavia, IRCCS Policlinico San Matteo, Pavia, Italy; and
E.T.S and INSERM Unité 429, Hôpital Necker-Enfant Malades,
Paris, France.
The success of bone marrow transplantation (BMT) from HLA-disparate
donors depends on the development of new strategies able, on one hand,
to efficiently prevent graft-versus-host disease (GVHD) and, on the
other hand, to protect leukemic patients from relapse and infections.
Using an immunotoxin (IT) directed against the
HEMATOPOIETIC STEM cell transplantation
(HSCT) is a commonly used therapy for malignant diseases and inborn
errors. Unfortunately, less than one third of patients eligible for
this procedure have an HLA-identical related donor. The feasibility of
HSCT from related HLA-partially matched donors has been demonstrated in
pediatric patients receiving HSCT for inborn errors, while worse
results have been obtained in patients with malignancy.1 Graft-versus-host disease (GVHD) remains a major problem with these
transplants. Even though T-cell depletion of stem cells reduces the
incidence and severity of this complication,2-4 it increases the risk of graft rejection and plays a major role in prolonging the period of immunodeficiency after transplantation. The
incidence of rejection can be reduced either by increasing the number
of stem cells infused through mobilization with granulocyte colony-stimulating factor (G-CSF)5 or by the administration of immunosuppressive agents to the recipients. In particular, anti-thymocyte globulin (ATG), Campath-1G,6 or monoclonal
antibodies (MoAbs) directed against adhesion molecules such as
anti-LFA-1 plus anti-CD27,8 proved to be of value in
increasing the recipient's immunosuppression. For the time being, the
significant delay in immune reconstitution, due to removal of mature T
cells from donor marrow, in vivo immunosuppression, and HLA disparity between donor and recipient, remains the major problem of HSCT from
HLA-disparate donors, because it contributes to the dramatic incidence
of life-threatening viral and fungal infections observed after this
type of HSCT and, in leukemia patients, to disease recurrence.9,10 The development of new strategies able, on one hand, to efficiently prevent GVHD and, on the other hand, to
protect the patients from relapse and infection by accelerating their
immunological reconstitution could both extend the applicability of
HSCT and increase the success of this procedure. In the last few years,
several groups have attempted to develop different methods of T-cell
manipulation to block and/or to kill mature T cells activated by HLA
antigens. In particular, two different ex vivo approaches have been
recently proposed for this aim. The first approach tries to induce host
alloantigen-specific anergy in human donor T cells before allogeneic
HSCT by using CTLA4-Ig either alone11 or in combination
with cyclosporine A.12 The second approach tries to
eliminate alloreactive T cells after specific activation through their
killing13,14 or fluorescence-activated cell
sorting,15,16 while sparing T cells with other functions. In a previous human preclinical study, we demonstrated that
allospecific T-cell depletion by using an immunotoxin (IT), directed
against the p55 chain of interleukin-2 (IL-2) receptor, was feasible, reproducible, and specific.14 Even though the proliferative response to alloantigens does not predict with absolute reliability the
development of GVHD, the marked inhibition of alloreactivity through
this IT provided a rational basis for considering the use of this
approach in humans. The spared T cells were still able to proliferate
against third-party cells, Candida, and cytomegalovirus (CMV)
antigens.17 Moreover, in vivo studies in a murine animal model showed that this particular T-cell depletion was efficient, at
least partially, in preventing both graft rejection and GVHD in a
complete haplotype-mismatched combination.18 However, so far, few data are available regarding the possibility to maintain antileukemia reactivity after elimination of alloreactive T
cells.19
The clinical and experimental data reported in the literature on the
possibility of separating GVHD and graft-versus-leukemia reaction
(GVLR) still remain controversial.20-22 We previously reported23 that cytotoxic T-cell clones reactive to
autologous leukemic blasts (LB) but not to autologous bone marrow
remission cells (BMRC) can be obtained from peripheral blood
mononuclear cells (PBMC) of leukemic children by in vitro lymphocyte
stimulation with autologous tumor cells and recombinant IL-2
(rIL-2).23 Moreover, we showed that cytotoxic T-lymphocyte
precursors (CTLp), specifically reactive towards recipient LB, were
undetectable or low in donor peripheral blood, although their frequency
reached high values in the recipients after
transplantation.24 This provided evidence for an in vivo
expansion of donor T cells able to recognize leukemic recipient cells.
The aim of this study was to assess whether in vitro alloreactive
T-cell depletion could affect the capacity of spared T cells to kill LB
or virus-infected cells. To address these issues, we established a
limiting dilution assay (LDA) to measure the frequency of CTLp directed
against autologous LB, CMV-infected fibroblasts, and Epstein-Barr virus
lymphoblastoid cell lines (EBV-LCL) before and after depletion of
alloreactive T cells. Antileukemia activity was assessed in PBMC of 3 patients with acute myeloid leukemia (AML) who had developed a high
frequency of LB-reactive CTLp after either autologous or allogeneic
HSCT. PBMC of patients were activated in vitro in a one-way mixed
lymphocyte culture (MLC) against PBMC of their father, depleted of
activated T cells by an anti-CD25-ricin Isolation of PBMC and BM cells from patients and healthy subjects.
Three children with AML were studied to evaluate antileukemia activity
after elimination of alloreactive T cells activated against paternal
HLA-antigens. One patient (BP) had been treated with autologous BM
transplantation (BMT), whereas patients ER and GA had received
allogeneic BMT from HLA-identical sibling donors. Patients were
analyzed 6 months after BMT, when the presence of sizeable values of
LB-directed CTLp was documented. Persistence of antiviral activity was
evaluated in PBMC of 4 healthy donors.
Cell preparation and cell line establishment.
Heparinized BM aspirate containing greater than 90% LB was obtained
from patients at the time of diagnosis and BMRC were collected after
demonstration of complete hematological remission. PBMC of patients
were collected 6 months after either allogeneic or autologous BMT,
while patients were in remission. BMRC and PBMC were isolated by
Ficoll-hypaque density gradient of anticoagulated whole blood,
cryopreserved in fetal calf serum (FCS; GIBCO Ltd, Paisley, UK)
supplemented with 10% dimethyl sulfoxide (DMSO), and stored in liquid nitrogen.
IT.
RFT5-SMPT-dgA. RFT5 is a murine anti-CD25 MoAb (IgG1) selected from a
group of 25 antibodies based on its ability to form a potent IT and to
stain only activated T cells in a panel of 28 normal human
tissues.25 This IT was prepared by using the hindered
heterobifunctional crosslinker
N-succinimidyloxycarbonyl- Cell activation and in vitro treatment with RFT5-SMPT-dgA.
The experimental system to activate T lymphocytes was a one-way MLC, as
previously described.14 Briefly, 25 × 106
PBMC from patients or healthy donors (A) were incubated at 37°C for
2 days with 25 × 106 irradiated (3,000 rads)
stimulating PBMC from haploidentical parents referred as B*. Th medium
used was RPMI 1640 (GIBCO) supplemented with 2 mmol/L L-glutamine, 50 µg/mL gentamicine, and 10% human AB serum (RPMI-HS). All cultures
were performed in 25-cm2 flasks (Corning, Corning,
NY) in a final volume of 20 mL. The unstimulated control
was performed with 25 × 106 irradiated autologous
PBMC referred as A*.
Study of MLC inhibition.
Two hundred microliters of treated and mock-treated cell mixtures (AB*
and AA*) were plated in duplicate in 96-well, round-bottomed microtiter
plates (Limbro; Flow Lab, Scotland) and incubated at 37°C in 5% CO2 until day 6.
LDA for evaluation of LB-reactive CTLp.
To evaluate the persistence of antileukemia activity after
allodepletion, fresh PBMC or cultured-treated and mock-treated patient
cells were seeded as responder cells with autologous LB used as
stimulator cells in 96-well round-bottom microplates. Briefly,
decreasing numbers of responder cells (4 × 104,
104, 5 × 103, 2.5 × 103, and 1.25 × 103) were seeded together
with 2 × 104 irradiated LB (7,000 rads). When IT- or
mock-treated cells were used as responders, 2 × 104
irradiated autologous PBL were added to the cultures as feeder cells.
Control wells contained irradiated autologous LB without responder
cells. The medium used was RPMI-HS supplemented with 100 U/mL of rIL-2
(Hoffman-La Roche, Basel, Switzerland). The cultures were
incubated at 37°C in 5% CO2. On day 10, wells were accurately resuspended, and 100 µL of each well was transferred for
split experiments. Cultures were then tested for cytolytic activity
against both LB and autologous BMRC.
Production of EBV-B-lymphoblastoid cell line (B-LCL).
PBMC were incubated with EBV-containing supernatant from the B95.8 cell
line (American Type Culture Collection, Rockville, MD) in
the presence of 800 ng/mL of cyclosporin A in RPMI 1640 medium
supplemented with 2 mmol/L L-glutamine, 50 µg/mL gentamicin, and 10%
FCS (RPMI-FCS). Cells were continuously incubated at 37°C, 5%
CO2 for 3 to 4 weeks. Each week, 2 mL of culture medium was removed and 2 mL of fresh medium was added until growth of B-LCL was established.
LDA for evaluation of CMV and EBV-specific CTLp.
To evaluate CTLp frequency to CMV-infected fibroblasts, fresh PBMC or
cultured IT-treated and mock-treated cells obtained from healthy
controls were seeded in a final volume of 200 µL in 96-well
round-bottom microplates. Autologous fibroblast were infected with
AD169 strain CMV following a previously described method.31
A decreasing number of responder cells (104, 5 × 103, 2.5 × 103, 1.25 × 103, and 0.6 × 103) were stimulated with
2 × 103 autologous infected fibroblasts. When the IT-
or mock-treated cells were used as responders, 4 × 104 irradiated (3,000 rads) were added as feeder cells. On
days 4 and 9, 40 U/mL rIL-2 was added to the cultures. On day 12, the plates were accurately splitted as described above and tested against
CMV-infected and mock-infected fibroblasts. For evaluation of
EBV-specific CTLp, responder cells were stimulated with autologous irradiated B-LCL following a previously described method.32
Cytotoxicity assay.
At the end of the cultures, effector cells were assayed for cytolytic
activity against 51Cr-labeled targets. Target cells
included autologous LB, autologous BMRC, CMV-infected or mock-infected
fibroblasts, and EBV B-LCL. Briefly, 2 to 3 × 106 LB,
autologous BMRC, and EBV B-LCL or 5 × 105 fibroblasts
were labeled with 100 to 200 µCi 51Cr for 2 hours, washed
four times, and added to the wells. Plates were incubated for 5 hours
at 37° and then centrifuged at 200g for 10 minutes.
Finally, 100 µL of supernatant was collected from each well and
counted for 1 minute in a gamma-counter. To provide necessary controls,
spontaneous and total 51Cr release from target cells were
also determined. Spontaneous release from all types of target cells was
consistently less than 20%.
Calculation of CTLp frequency.
Assay wells were defined as positive when 51Cr release
exceeded the average plus 3 sD of control wells. The
frequency of responding cells was determined by maximum likelihood
estimation using a statistical program and the variance by the use of
95% confidence limits.33
Generation of leukemia-reactive T-cell clones.
LB-reactive effector cells of patient ER were recovered from pooled
positive wells obtained from 10-day LDA of both untreated and
allodepleted cultures and seeded into Terasaki trays at 0.3 cells/well
in the presence of rIL-2 (200 U/mL), phytohemagglutinin (PHA; GIBCO; M-form 1:100), 2 × 105/mL
irradiated (7,000 rads) autologous LB, and 5 × 105/mL
of allogeneic irradiated (3,000 rads) feeder cells. After 12 to 14 days
of culture, all growing wells were harvested and expanded by
cultivation in 96-well flat-bottom plates with 106/mL
irradiated allogeneic feeder cells in HS-RPMI containing 200 U/mL rIL-2
and PHA. Conditions for maintenance of T-cell clones (TCC) have been
described elsewhere.34 The clones thus obtained were
screened for their capacity to lyse recipient LB in a 51Cr
release assay. Thereafter, LB-reactive TCC were further characterized for their surface phenotype and specificity.
MoAbs for phenotyping and blocking experiments.
MoAbs used in this study included anti-Leu4(CD3)-fluorescein
isothiocyanate (FITC) or -phycoerythrin (PE), anti-Leu 3a(CD4)-PE, anti-Leu-2a(CD8)-PE, anti-Leu28 (CD28)-PE, anti-BB-1:B7 (CD80)-FITC, anti-TCR Specific inhibition of an MLC (AB*).
IT-induced specific inhibition of MLC was verified before assaying each
limiting dilution experiment for evaluation of leukemia-specific or
virus-specific CTLp frequency. The functional depletion of A anti-B
reactive cells was tested in a 2-day AB* MLC. The proliferative capacity of treated T cells towards third-party cells (C*), unrelated to B*, was assessed to evaluate the spared T-cell reactivity towards unrelated HLA antigens. The residual proliferative capacity of responder cells versus B* ranged between 0% and 1.5% of the control. By contrast, the alloreactive response against C* was almost completely preserved (Table 1).
Evaluation of LB-reactive CTLp frequency.
The frequency of CTLp directed against recipient LB in peripheral blood
of the patients was investigated immediately after treatment with the
IT. Baseline LB-reactive CTLp frequency was 1/14.296, 1/10.911, and
1/36.156 in the 3 patients tested, respectively. After allospecific
T-cell depletion, an apparent increase in LB-reactive CTLp frequency
was observed in all patients analyzed, with frequencies being 1/2.480,
1/5.141, and 1/24.374, respectively (Fig 1). Undetectable frequencies
of CTLp were found against autologous BMRC both before and after
treatment with IT (data not shown).
Surface phenotype and cytolytic activity of LB-reactive TCC.
Effector subsets of cytotoxic activity against recipient LB before and
after T-cell-specific allodepletion were characterized by
cloning in limiting dilution pooled positive wells from patient ER.
The growing clones were
expanded and then screened and selected for their capacity to lyse
recipient LB but not BMRC in a cytotoxicity assay. Different subsets of
LB-reactive TCC could be defined on the basis of the surface phenotype.
In untreated cultures (when effector cells were fresh PBMC), we were
able to isolate 5 LB-reactive TCC. Two TCC were
CD3+/TCR
Evaluation of virus-specific CTLp frequency.
The capacity to kill autologous CMV-infected fibroblasts was evaluated
in PBMC from 2 healthy controls. As shown in
Fig 2, we observed that allodepletion did
not affect the frequency of CMV-specific precursors, because in the
first subject it was 1/19.834 in untreated cells versus 1/15.212 after
allodepletion and in the second subject frequencies were 1/5.052 and
1/4.566, respectively. Reactivity against mock-infected fibroblasts was
always undetectable. Similar results were obtained against EBV-LCL
(Fig 3). In particular, CTLp
frequencies were 1/25.899 and 1/17.719 in PBMC of the 2 donors tested,
and they reached values of 1/3.799 and 1/26.442 after T-cell
allodepletion, respectively.
The present study demonstrates that allospecific T-cell depletion
obtained by using RFT5-SMPT-dgA does not affect the frequency of CTLp
directed against autologous LB in transplanted patients who had
developed a sizeable frequency of LB-directed CTLp after transplantation. Moreover, the spared T cells maintained a high frequency of CTLp directed against EBV- and CMV-infected cells. Treatment with RFT5-SMPT-dgA results in strong inhibition of primary MLC response from PBMC of subjects used to evaluate antileukemia and
antiviral activities, whereas residual T cells maintain the capacity to
proliferate in the presence of third-party stimulating cells.
Submitted October 19, 1998; accepted December 31, 1998.
Supported in part by grants from Associazione Italiana Ricerca sul
Cancro (AIRC) to F.L. and to R.M. and by Grants No. 261RFM95/01, 390RFM96/01, and 010RCR97/01 from IRCCS Policlinico San Matteo to F.L.
and R.M.
The publication costs of this
article were defrayed in part by
page charge payment. This article
must therefore be hereby marked
"advertisement"
in accordance with 18 U.S.C. section
1734 solely to indicate this fact.
Address reprint requests to Daniela Montagna, PhD, Laboratorio di
Immunologia, Dipartimento di Scienze Pediatriche, IRCCS Policlinico San
Matteo, P.le Golgi 2, 27100 Pavia, Italia.
1.
Fischer A, Landais P, Friedrich W, Gerritsen B, Blanche S, Fasth A, Porta F, Vellodi A, Benkerrou M, Jais JP, Cavazzana-Calvo M, Souillet G, Bordigoni P, Morgan G, Van Dijken P, Vossen J, Locatelli F, Di Bartolomeo P:
Bone marrow transplantation in Europe for primary immunodeficiencies other than severe combined immunodeficiency, EBMT/EGID report.
Blood
83:1149, 1994
2.
Ash RC, Casper JT, Chitambar CR, Hansen R, Bunin N, Truitt R, Lauton P, Murray K, Hunter J, Baxter-Lowe LA, Gottschall J, Oldham K, Anderson T, Camitta B, Menitove J:
Successful allogeneic transplantation of T-cell depleted bone marrow from closely HLA-matched unrelated donors.
N Engl J Med
8:485, 1990
3.
Henslee-Downey PJ, Abhyankar SH, Parrish RS, Pati AR, Godder KT, Neglia WJ, Goon-Johnson KS, Geier SS, Lee CG, Gee AP:
Use of partially mismatched related donors extends access to allogeneic marrow transplant.
Blood
89:3864, 1997
4.
O'Reilly RJ, Keever CA, Small TN, Brochstein J:
The use of HLA-non-identical T-cell depleted marrow transplants for correction of severe combined immunodeficiency disease.
Immunodeficiency Rev
1:273, 1989[Medline]
[Order article via Infotrieve]
5.
Aversa F, Tabilio A, Terenzi A, Velardi A, Falzetti F, Giannoni C, Iacucci R, Zei T, Martelli MP, Gambelunghe C, Rossetti M, Caputo PF, Latini P, Aristei C, Raymondi C, Reisner Y, Martelli MF:
Successful engraftment of T-cell-depleted haploidentical "three loci" incompatible transplants in leukemia patients by adding recombinant human granulocyte colony stimulating factor-mobilized peripheral blood progenitor cells to bone marrow inoculum.
Blood
84:3948, 1994
6.
Hale G, Waldmann H:
Recent results using CAMPATH-1 antibodies to control GVHD and graft rejection.
Bone Marrow Transplant
17:305, 1996[Medline]
[Order article via Infotrieve]
7.
Jabado N, Le Deist F, Cant A, De Graeff G, Meeders ER, Fasth A, Morgan G, Vellodi A, Hale G, Bujan W, Thomas C, Cavazzana-Calvo M, Widijenes J, Fischer A:
Bone marrow transplantation from genetically HLA-non identical donors in children with fatal inherited disorders excluding severe combined immunodeficiencies: Use of two monoclonal antibodies to prevent graft-rejection.
Pediatrics
98:420, 1996
8.
Cavazzana-Calvo M, Bordigoni P, Michel G, Esperou H, Souillet G, Leblanc T, Stephan JL, Vannier JP, Mechinaud F, Reiffers J, Vilmer E, Landman-Parker J, Benkerrou M, Baruchel A, Pico J, Bernaudin F, Bergeron C, Plouvier E, Thomas C, Widjienes J, Lacour B, Blanche S, Fischer A:
A phase II trial of partially incompatible bone marrow transplantation for high-risk acute lymphoblastic leukaemia in children: Prevention of graft rejection with anti-LFA-1 and anti-CD2 antibodies.
Br J Haematol
93:131, 1996[Medline]
[Order article via Infotrieve]
9.
Haddad E, Landais P, Friedrich W, Gerritsen B, Cavazzana-Calvo M, Morgan G, Bertrand Y, Fasth A, Porta F, Cant A, Espanol T, Müller S, Veys P, Vossen J, Fischer A:
Long term immune reconstitution and outcome after HLA-non identical T-cell depleted bone marrow transplantation for severe combined immunodeficiency: A European retrospective study of 116 patients.
Blood
91:3646, 1998
10.
Cavazzana-Calvo M, Bensoussan D, Jabado N, Haddad E, Yvon E, Moskwa M, Tachet-Descombes A, Buisson M, Morand P, Virion JM, Le Deist F, Fischer A:
Prevention of EBV induced B-lymphoproliferative disorder by ex vivo marrow B-cell depletion in HLA phenoidentical or non identical T-depleted bone marrow transplantation.
Br J Haematol
103:543, 1998[Medline]
[Order article via Infotrieve]
11.
Gribben JG, Guinan EC, Boussiotis VA, Ke XY, Linsley L, Sieff C, Gray GS, Freeman GJ, Nadler LM:
Complete blockade of B7 family-mediated costimulation is necessary to induce human alloantigen-specific anergy: A method to ameliorate graft-versus-host disease and extend the donor pool.
Blood
87:4887, 1996
12.
Comoli P, Locatelli F, Montagna D, Calcaterra V, Moretta A, Giorgiani G, Bonetti F, Zecca M, Maccario R:
Induction of alloantigen-specific anergy does not impair cytolytic activity of leukemia-reactive human T cells.
Bone Marrow Transplant
21:5132, 1998 (suppl 1)
13.
Mavroudis DA, Jiang YZ, Hensel N, Lewalle P, Cocoriel D, Kreitman RJ, Pastan I, Barrett AJ:
Specific depletion of alloreactivity against haplotype mismatched related individuals by a recombinant immunotoxin: A new approach to graft-versus-host disease prophylaxis in haploidentical bone marrow transplantation.
Bone Marrow Transplant
17:793, 1996[Medline]
[Order article via Infotrieve]
14.
Cavazzana-Calvo M, Fromont C, Le Deist F, Lusardi M, Coulombel L, Derocq JM, Gerota I, Griscelli C, Fischer A:
Specific elimination of alloreactive T cells by an anti-interleukin-2 receptor B chain-specific immunotoxin.
Transplantation
50:1, 1990[Medline]
[Order article via Infotrieve]
15.
Mickey B, Kohn C, Lowdell MW, Prentice HG:
Selective removal of alloreactive lymphocytes from peripheral blood mononuclear cell preparations.
Blood
88:253a, 1996 (suppl 1)
16.
Reucher SD, Houston JA, Lockey TD, Hurwitz JL:
Eliminating graft-versus-host potential from T cell immunotherapeutic populations.
Bone Marrow Transplant
18:415, 1996[Medline]
[Order article via Infotrieve]
17.
Valteau-Couanet D, Cavazzana-Calvo M, Le Deist F, Fromont C, Fisher A:
Functional study of residual T lymphocytes after specific elimination of alloreactive T cells by a specific anti-interleukin-2 receptor B chain immunotoxin.
Transplantation
56:1574, 1993[Medline]
[Order article via Infotrieve]
18.
Cavazzana-Calvo M, Stephan JL, Sarnacki S, Chevret S, Fromont C, de Coene C, Le Deist F, Guy-Grand D, Fisher A:
Attenuation of graft-versus-host disease and graft rejection by ex vivo immunotoxin elimination of alloreactive T cells in an H-2 haplotype disparate mouse combination.
Blood
83:288, 1994
19.
Mavroudis DA, Dermime S, Molldrem J, Jiang YZ, Raptis A, van Rhee F, Hensel N, Felllowes V, Eliopoulos G, Barrett AJ:
Specific depletion of alloreactive T cells in HLA-identical siblings: A method for separating graft-versus-host and graft-versus-leukemia reactions.
Br J Haematol
101:565, 1998[Medline]
[Order article via Infotrieve]
20.
Weiden PL, Flournoy N, Thomas ED, Prentige R, Fefer A, Buckner D, Storb R:
Antileukemic effect of graft-versus-host disease in human recipients of allogeneic-marrow grafts.
N Engl J Med
300:1068, 1979[Abstract]
21.
Horowitz MM, Gale RP, Sondel PM, Goldman JM, Kersey J, Kolb HJ, Rimm AA, Ringden O, Rozman C, Speck B, Truit RL, Zwaan FE, Bortin MM:
Graft-versus-leukemia reactions after bone marrow transplantation.
Blood
75:555, 1990
22.
Marmont AM, Horowitz MM, Gale RP, Sobocinski K, Ash RC, van Bekkum DW, Champlin RE, Dicke KA, Goldman JM, Good RA, Herzig RH, Hong R, Masaoka T, Rimm AA, Ringden O, Speck B, Weiner RS, Bortin MM:
T-cell depletion of HLA-identical transplants in leukemia.
Blood
78:2120, 1991
23.
Montagna D, Aricò M, Montini E, De Benedetti F, Maccario R:
Identification of HLA-unrestricted CD8+/CD28
24.
Montagna D, Locatelli F, Calcaterra V, Comoli P, Moretta A, Giorgiani G, Zecca M, Bonetti F, Giraldi E, Rondini G, Maccario R:
Does the emergence and persistence of donor derived leukemia-reactive cytotoxic T-lymphocytes protect patients given an allogeneic BMT from recurrence? Results of a preliminary study.
Bone Marrow Transplant
22:743, 1998[Medline]
[Order article via Infotrieve]
25.
Engert A, Martin G, Amlot P, Wijdenes J, Diehl J, Thorpe P:
Immunotoxins constructed with anti-CD25 monoclonal antibodies and deglycosylated ricin A-chain have potent anti-tumour effects against human Hodgkin cells in vitro and solid Hodgkin tumours in mice.
Int J Cancer
94:450, 1991
26.
Bell KD, Ramilo O, Vitetta ES:
Combined use of an immunotoxin and cyclosporine to prevent both activated and quiescent peripheral blood T cells from producing type 1 human immunodeficiency virus.
Proc Natl Acad Sci USA
90:1411, 1993
27.
Winkler U, Gottstein C, Schön G, Kapp U, Wolf J, Hansmann ML, Bohlen H, Thorpe P, Diehl V, Engert A:
Successful treatment of disseminated human Hodgkin's disease in SCID mice with deglycosylated ricin A-chain immunotoxins.
Blood
83:466, 1994
28.
Engert A, Diehl V, Schnell R, Radszuhn A, Hatwig MT, Drillich S, Schön G, Bohlen H, Tesch H, Hansmann ML, Barth S, Schindler J, Ghetie V, Uhr J, Vitetta E:
A phase-1 study of an anti-CD25 ricin A-chain immunotoxin (RFT5-SMPT-dgA) in patients with refractory Hodgkin's lymphoma.
Blood
89:403, 1997
29.
Thorpe PE, Wallace M, Knowles PP, Relf MG, Brown ANF, Watson GJ, Knyba R, Wawrzynczak EJ, Blakey DC:
New XXX agents for the synthesis of immunotoxins containing a kindered disulfide bond with improved stability in vivo.
Cancer Res
47:5924, 1987
30.
Ghetie V, Thorpe PE, Ghetie MA, Knowles P, Uhr JW, Vitetta ES:
The GLP large scale preparation of immunotoxins containing deglycosylated ricin A chain and hindered disulfide bond.
J Immunol Methods
142:223, 1991[Medline]
[Order article via Infotrieve]
31.
McLaughlin-Taylor E, Pande E, Forman SJ, Tanamachi B, Li C, Zaia JA, Greenberg PD, Riddell SR:
Identification of the major late human cytomegalovirus matrix protein pp65 as a target antigen for CD8+ virus-specific cytotoxic T lymphocytes.
J Med Virol
43:103, 1994[Medline]
[Order article via Infotrieve]
32.
Moretta A, Comoli P, Montagna D, Gasparoni A, Percivalle E, Carena I, Revello MG, Gerna G, Mingrat G, Locatelli F, Rondini G, Maccario R:
High frequency of Epstein-Barr virus (EBV) lymphoblastoid cell line-reactive lymphocytes in cord blood: Evaluation of cytolytic activity and IL-2 production.
Clin Exp Immunol
312:107, 1997
33.
Sharrock CEM, Kaminski E, Man E:
Limiting dilution analysis of human T cells: A useful clinical tool.
Immunol Today
11:281, 1990[Medline]
[Order article via Infotrieve]
34.
Abrignani S, Montagna D, Jeannet M, Wintsch J, Haigwood NL, Shuster JR, Steimer KS, Cruchaud A, Staehelin T:
Priming of CD4+ T cells specific for conserved regions of human immunodeficiency virus glycoprotein gp 120 in human immunized with a recombinant envelope protein.
Proc Natl Acad Sci USA
87:6136, 1990
35.
Falkenburg JHF, Smit WM, Willemze R:
Cytotoxic T-lymphocyte (CTL) responses against acute or chronic myeloid leukemia.
Immunol Rev
157:223, 1997[Medline]
[Order article via Infotrieve]
36.
Goulmy E:
Human minor histocompatibility antigens: New concepts for marrow transplantation and adoptive immunotherapy.
Immunol Rev
157:125, 1997[Medline]
[Order article via Infotrieve]
37.
Faber LM, van der Hoeven J, Goulmy E, Hooftman-den Otter AL, van Luxemburg-Heijs SAP, Willemze R, Falkenburg JHF:
Recognition of clonogenic leukemic cells, remission bone marrow and HLA identical donor bone marrow by CD8+ or CD4+ minor histocompatibility antigen-specific cytotoxic T lymphocytes.
J Clin Invest
96:877, 1995
38.
Faber LM, van Luxemburg-Heijs SAP, Veenhof WFJ, Willemze R, Falkenburg JHF:
Generation of CD4+ cytotoxic T-lymphocyte clones from a patient with severe graft-versus-host disease after allogeneic bone marrow transplantation: Implications for graft-versus-leukemia reactivity.
Blood
86:2821, 1995
39.
Weiss L, Lubin I, Factorowich I, Lapidot Z, Reich Y, Reisner Y, Slavin S:
Effective graft-versus-leukemia effect independent of graft-versus-host disease after T cell depleted allogeneic bone marrow transplantation in a murine model of B cell leukemia/lymphoma.
J Immunol
153:2562, 1994[Abstract]
40.
Jiang JYZ, Mavroudis DA, Dermine S, Molldrem J, Hensel NF, Barrett AJ:
Preferential usage of T cell receptor (TCR) V by allogeneic T cells recognizing myeloid leukemia cells: Implications for separating graft-versus-leukemia effect from graft-versus-host disease.
Bone Marrow Transplant
19:899, 1997[Medline]
[Order article via Infotrieve]
41.
Slavin S, Ackerstein A, Naparstek E, Or R, Weiss L:
The graft-versus-leukemia (GVL) phenomenon: Is GVL separable from GVHD?
Bone Marrow Transplant
6:155, 1990[Medline]
[Order article via Infotrieve]
42.
van Lochem E, de Gast B, Goulmy E:
In vitro separation of host specific graft-versus-host and graft-versus-leukemia cytotoxic T cell activities.
Bone Marrow Transplant
10:181, 1992[Medline]
[Order article via Infotrieve]
43.
Guinan EC, Gribben JG, Boussiotis VA, Freeman GJ, Nadler LM:
Pivotal role of the B7:CD28 pathway in transplantation tolerance and tumor immunity.
Blood
84:3261, 1994
44.
Boyer MW, Vallera DA, Taylor PA, Gray GS, Katsanis E, Gorden K, Orchard PJ, Blazar BR:
The role of B7 costimulation by murine acute myeloid leukemia in the generation and function of a CD8+ T-cell line with potent in vivo graft-versus-leukemia properties.
Blood
89:3477, 1997
45.
Lenschow DJ, Walunas TL, Bluestone JA:
CD28/B7 system of T cell costimulation.
Annu Rev Imunol
14:233, 1996[Medline]
[Order article via Infotrieve]
46.
Zocchi MR, Ferrarini M, Rugarli C:
Selective lysis of the autologous tumor by TCS1+ tumor infiltrating lymphocytes from human lung carcinoma.
Eur J Immunol
20:2685, 1990[Medline]
[Order article via Infotrieve]
47.
Douval M, Yotnda P, Benussan A, Oudhri N, Guidal C, Rohrlich P, Boumsel L, Grandchamp B, Vilmer E:
Potential antileukemic effect of
48.
Ashwell JD, Chen C, Schwartz RH:
High frequency and nonrandom distribution of alloreactivity in T cell clones selected for recognition of foreign antigen in association with self class II molecules.
J Immunol
136:389, 1986 |
TOP
ABSTRACT
INTRODUCTION
chain (p55) of the
human interleukin-2 receptor (RFT5-SMPT-dgA), we previously showed that
it is possible to kill mature T cells activated against a specific HLA
complex by a one-way mixed lymphocyte culture (MLC). The present study
was performed to investigate whether this protocol of allodepletion
affects the capacity of residual T cells to display antileukemia and
antiviral activity evaluated by limiting dilution assays (LDA),
measuring the frequency of cytotoxic T-lymphocyte precursors (CTLp)
directed against autologous leukemic blasts (LB) and cytomegalovirus
(CMV)- and Epstein-Barr virus (EBV)-infected target cells. Antileukemia
activity was evaluated in peripheral blood mononuclear cells (PBMC) of
3 patients treated for acute myeloid leukemia who had developed a high
frequency of LB-reactive CTLp after either autologous or allogeneic
BMT. Results demonstrate that (1) depletion with RFT5-SMPT-dgA
efficiently inhibited MLC; (2) fresh PBMC of patients yielded a high
frequency of LB-reactive CTLp comparable to that of the mock-treated
PBMC; and (3) effector cells obtained after allodepletion fully
retained the capacity to lyse pretransplant LB. By contrast, the
frequency of CTLp directed against patient's pretransplant BM
remission cells was always undetectable. Data obtained in 4 healthy
donors showed that specifically allodepleted T cells recognized and
killed autologous CMV-infected fibroblasts and autologous
EBV-B-lymphoblastoid cell lines. In conclusion, our data indicate that
allodepletion using RFT5-SMPT-dgA efficiently removed alloreactive
cells, while sparing in vitro antileukemic and antiviral cytotoxic responses.
chain IT
(RFT5-SMPT-dgA),
AA* [IT-treated])/(AB* [untreated] 
-FITC, anti-TCR 
-FITC (Becton Dickinson, Mountain View, CA), and anti-CD86-FITC (Pharmigen, San Diego, CA). Phenotypic analysis was performed by means of direct or indirect
immunofluorescence on a FACScan flow cytometer (Becton Dickinson).
Blocking experiments of cytolytic activity with TR66 anti-CD3 and with
anti-HLA class I MoAb (W6/32) were performed as follows. (1) Target
cells were previously incubated with anti-HLA (25 µg/mL) for 30 minutes at 4°C. (2) Effector cells were previously incubated with
anti-CD3 MoAb (1 µg/mL) for 30 minutes at 4°C. Subsequently, the
same concentration of each MoAb was added to the cultures in a 4-hour
standard cytotoxicity assay.
), IT-treated
(
), and mock-treated (
) cultures of the 3 patients (PB, ER, and
GA). The fraction of negative wells was plotted on a logarithmic scale
against the number of responder cells per well. Frequency and 95%
confidence limits are also reported.
or
CD28dim phenotype (data not shown). It is indeed well known
that CD80-CD86 costimulation pathway plays a critical role in
HLA-restricted, TCR-mediated activation of T cells.