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Blood, Vol. 93 No. 10 (May 15), 1999:
pp. 3550-3557
Depletion of Alloreactive T Cells by a Specific Anti-Interleukin-2
Receptor p55 Chain Immunotoxin Does Not Impair In
Vitro Antileukemia and Antiviral Activity
By
Daniela Montagna,
Eric Yvon,
Valeria Calcaterra,
Patrizia Comoli,
Franco Locatelli,
Rita Maccario,
Alain Fisher, and
Marina Cavazzana-Calvo
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.
 |
ABSTRACT |
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  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.
© 1999 by The American Society of Hematology.
| |
INTRODUCTION |
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  chain IT
(RFT5-SMPT-dgA),25,26 and then tested for antileukemia activity. This new IT has been previously used in a murine
model27 and in a Food and Drug Administration-approved
clinical phase I trial on patients with refractory Hodgkin's
lymphoma.28 Reactivity against virus-infected cells was
evaluated in PBMC of healthy controls after allodepleted MLC. We showed
that allodepletion using RFT5-SMPT-dgA efficiently removed alloreactive
T cells but did not significantly affect in vitro antileukemia and
antivirus responses.
| |
MATERIALS AND METHODS |
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--methyl-(2-pyridyldithio) toluene
(SMPT)29 and chemically deglycosylated ricin chain (dgA) according to published reports.30
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*.
After in vitro activation, the cells were harvested, washed twice,
resuspended at 10 × 106 cells/mL, and treated
overnight at 37°C with the IT in RPMI 1640 containing 20 mmol/L
NH4Cl (pH 7.8) to increase the cytotoxicity of
RFT5-SMPT-dgA. After two washes, cells were resuspended in RPMI-HS at
2.5 × 106 cells/mL and used immediately for
proliferation and LDAs.
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.
One hundred microliters of the same cell mixtures was also cocultured
in duplicate with 100 µL of irradiated (3,000 rads) PBMC of a
third-party HLA nonidentical subject referred as C* and cultured for 6 days (ie, day 8 from the start of each experiment). The cultures were
pulsed with 1 µCi of (3H) thymidine (Amersham, Amersham,
UK) for 18 hours, harvested, and counted in a liquid
scintillation counter (MINAXI-TRICARB 4000; Packard Instruments,
Zürich, Switzerland).
The MLC results (mean cpm of duplicate cultures) were expressed as the
percentage of the respective controls according to the formula: (AB*
[IT-treated]  AA* [IT-treated])/(AB* [untreated] AA* [untreated]) × 100. Similarly, the proliferation against third-party cells (C*) was calculated as the percentage of the mock-treated sample.
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  -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.
| |
RESULTS |
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+/CD8+ cells, 1 TCC
was CD3+/TCR+/CD8+, 1 TCC was
CD3+/TCR+/CD4/CD8 double negative (DN),
and the last was CD3+/TCR+/CD4/CD8DN
cells. Six TCC were obtained from cultures after depletion of
alloreactive T cells: 4 of them were
CD3+/TCR+/CD8+ lymphocytes, 1 TCC was CD3+/TCR+/CD8+, and 1 displayed CD3+/TCR+/CD4/CD8DN phenotype.
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| Fig 1.
LB-reactive CTLp frequency of PBMC ( ), 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.
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Some LB-reactive TCC derived from either untreated and IT-treated
cultures could be expanded sufficiently to be tested in blocking
experiments with anti-CD3 and anti-HLA class I MoAbs (Table 2). The killing
capacity of the +/DN TCC, +/DN TCC,
and +/CD8+ TCC was not affected by the
addition of the two MoAbs. In contrast, the addition of these MoAbs
strongly reduced cytolytic activity of
+/CD8+ TCC.
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Table 2.
Effect of the Addition of Anti-CD3 and Anti-HLA Class I
MoAbs on the Cytolytic Activity of LB-Reactive TCC Against Autologous
Pretransplant LB
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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.
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| Fig 2.
CMV-specific CTLp frequency of PBMC (), IT-treated
(), and mock-treated () cultures of the 2 donors. 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.
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| Fig 3.
EBV-specific CTLp frequency of PBMC (), IT-treated
(), and mock-treated () cultures of the 2 donors. 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.
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DISCUSSION |
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.
In a previous study,18 we demonstrated that specific
depletion of donor T cells reactive against the host led to a reduction in the incidence and severity of GVHD after haploidentical HSCT in
mice. The persistence of antileukemia activity after removing host-specific reactive T cells is crucial for successful allogeneic HSCT in patients with malignancy. A number of clinical and experimental data indicate that mature adoptively transferred donor lymphocytes are
involved in the generation of both GVHD and GVLR after HSCT. In
particular, clinical evidence suggests the existence of a strong relationship between GVHD and GVLR.20-22 After allogeneic
HSCT from HLA-matched sibling donors, it is believed that
non-MHC-encoded minor histocompatibility antigens (mHAg) are involved
in both GVHD and GVLR activities. Although widely distributed mHAg
account for the GVLR associated to GVHD, tissue-restricted or
leukemia-specific antigens can elicit a specific GVLR,35-42
and it has been demonstrated that both CD4+ and
CD8+ CTL recognizing mHAg in a classical MHC-restricted
fashion can be generated in vitro.35,36 In particular,
mHAg-specific CD8+ CTL can display strong lysis of mature
leukemia cells as well as suppress, together with CD4+
mHAg-specific CTL, the growth of clonogenic leukemia precursor cells.37,38
We have previously described how CTLp reactive towards recipient LB can
be detected in PBMC of children receiving allogeneic HSCT.24 LB-reactive CTLp developed in the first months
after transplant and persisted for at least up to 18 months in patients in hematological remission.24 The GVLR we observed in vitro seems not to be strictly correlated with GVHD, given that the emergence
of a high frequency of LB-reactive CTLp was not related to the
occurrence and severity of clinical GVHD. Results of the present work,
obtained in patients who have developed in vivo antileukemia activity,
demonstrate that, notwithstanding depletion of alloreactive T cells, a
high frequency of precursor cells able to kill LB is still present
among spared lymphocytes. Our data confirm results recently reported by
Mavroudis et al,19 who demonstrated the maintenance of
antileukemia reactivity after depletion of alloreactive T cells through
an IT targeting the -chain of IL-2 receptor. Moreover, evaluation of
LB-reactive TCC documents that clones derived from allodepleted
cultures exhibit surface phenotype and functional characteristics
similar to clones derived from untreated cultures. In particular, we
demonstrate that LB-reactive, CD3+/CD8+, or
CD3+/CD4-CD8 DN TCC expressing either TCR or
are involved in the generation of in vitro antileukemia activity
obtained from untreated and T-cell allodepleted cultures. These results
confirm the data on LB-directed TCC previously obtained in children who have received allogeneic HSCT. They demonstrate that several types of
CTL clones can contribute to antileukemia activity in vitro, because we
also isolated, together with
CD3+/+/CD8+ MHC-restricted
TCC, TCC expressing TCR or , whose cytolytic activity is
not affected by the addition of MoAb directed against HLA class I
antigens and the CD3 complex. The isolation of a number of
HLA-unrestricted TCC that use a TCR/CD3-independent recognition pathway
suggests that some T-lymphocyte subsets may use alternative mechanisms
to kill malignant cells. These nonconventional recognition and
activation pathways could be due to the variable expression of surface
molecules on LB used as stimulator and target cells. In particular, LB
of patients used in this study express neither CD80 nor CD86, whereas
LB-directed TCC displayed a CD28 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.43-45
The inefficient delivery of crucial costimulatory signals may allow the
development of alternative pathways of target cell recognition. We can,
therefore, hypothesize that both HLA-restricted CTL directed towards
mHAg and other CTL subsets, such as those expressing / TCR
and using a TCR/CD3-independent activation pathway, are involved in mediating GVLR in vitro and in sustaining in vivo anticancer
immunosurveillance.46,47
Persistence of a high frequency of LB-directed CTLp after removal of
alloreactive T cells in patients receiving HSCT provides further
support to the possibility that GVLR and GVHD can be at least partially
separated and that some lymphocyte subsets are able to selectively
recognize leukemia-specific antigens, heat-shock proteins, or
overexpressed tissue-restricted molecules on tumor cells. Whether these
lymphocytes can contribute in vivo to the eradication or immune control
of clonogenic malignant cells, as well as the capacity of this approach
to prevent GVHD occurrence in human beings, remains to be proven in a
clinical study.
In a previous work, we have shown that the proliferative response of
IT-treated cells toward candidin and CMV, as well as cytotoxic activity
against EBV, was conserved.17 In the present study, we
demonstrate that the number of cytotoxic precursor cells evaluated by
LDA and directed against both CMV-infected fibroblasts and EBV-LCL are
not affected by such T-allospecific depletion. It has been previously
reported that (1) among the population of T cells specific for
allogeneic MHC, there is a mix of virgin and experienced cells; (2)
TCRs are broadly cross-reactive; and (3) many T cells specific for
environmental antigens also cross-react on foreign
MHC.48,49 On the basis of these data, it could be theoretically hypothesized that T-cell allodepletion be associated with
reduction of the number of T cells specific for viral antigens. On the
contrary, present data demonstrate that virus-specific precursor cells
are not impaired by the strategy of T-cell depletion here described.
In conclusion, our data indicate that treatment with RFT5-SMPT-dgA is
able to delete alloantigen specific T cells while sparing in vitro
antiviral and antileukemia activity. Use of this IT is now under
evaluation in a phase I clinical trial aimed at testing the feasibility
of this approach for extending the applicability of allogeneic HSCT
from HLA-disparate donors.
| |
FOOTNOTES |
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.
| |
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Bone marrow transplantation in Europe for primary immunodeficiencies other than severe combined immunodeficiency, EBMT/EGID report.
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ABSTRACT
INTRODUCTION