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IMMUNOBIOLOGY
From the University Children's Hospital Innsbruck;
Department of Urology, University Innsbruck, Austria; Children's
Cancer Research Institute, St Anna Children's Hospital Vienna,
Austria.
Impaired T-cell function after T-cell- depleting (TCD) therapy
has been hypothesized to be related to a transient predominance of
extrathymically expanding memory T cells. To test whether after TCD
therapy the naive T-helper cell population is functionally intact, the
in vitro immune response of CD4+CD45RA+ (naive)
and of CD4+CD45RA Severe immune dysfunction is a predictable
consequence of high-dose anticancer chemotherapy with or without
autologous or allogeneic stem cell support and results in significant
morbidity and mortality in potentially curable
diseases.1-3 One obvious explanation for such immune
dysfunction is a depletion of immune cells by the cytotoxic effects of
anticancer drugs.4,5 However, even after quantitative
regeneration immune cells do not regain full immune competence for a
prolonged period of time.6-10
T cells play a key role in multiple immune responses. Therefore, both a
depletion and a disturbed function caused by cytotoxic anticancer
treatment are potentially harmful to the immune surveillance of the
host, including an increased susceptibility to infections with
otherwise less harmful pathogens.11-15 T-cell
reconstitution after cytotoxic therapy does not repeat fetal T-cell
ontogeny.16-19 In fact, the first T cells that repopulate
the host after T-cell depleting (TCD) therapy expand extrathymically
from peripheral residual T cells whereas a thymus-dependent T-cell
regeneration is delayed and may be even incomplete in adult patients in
whom thymic function has declined.20-22 Extrathymically
expanded T cells bear a memory phenotype,23 as their
expansion is determined by antigenic stimulation.24 Their
capacity to restore full T-cell function is believed to be limited,
because of (1) skewing of the T-cell repertoire25-27 and
(2) an increased susceptibility to apoptosis, including
activation-induced apoptosis.28 In contrast, T cells with
a naive phenotype presumably represent recent thymic emigrants,23,29-31 having matured from precursors via
thymopoiesis, and thus may be spared from the postchemotherapy immune dysfunction.
To test this hypothesis we investigated by flow cytometry the in vitro
immune function of distinct naive (CD4+CD45RA+)
and memory (CD4+CD45RA Upon stimulation with polyclonal mitogens in vitro we found a
reduced up-regulation of the early activation-induced cell surface molecule, CD69, detectable in CD4+ T cells even in the
presence of exogenous interleukin 2 (IL-2). Strikingly,
CD4+CD45RA+ cells were similarly affected as
CD4+CD45RA Patients
Lymphocyte culture
Lymphocyte proliferation assays PBMCs (1 × 105) were cultured in 100 µL medium per well in 96-well flat-bottom tissue culture plates (Falcon, Becton Dickinson Labware, Franklin Lakes, NJ) in humidified air containing 5% CO2 at 37°C in the absence (negative control) or presence of PHA or anti-CD3. The lymphoproliferative response was measured by adding 1 µL [3H]thymidine (1 µCi = 37 kBq/well; ICN Biomedicals, Costa Mesa, CA) per well for the last 18 hours of culture. Cells were then harvested on glass fiber filters (Skatron Instruments, Lier, Norway) and analyzed on a liquid scintillation counter. Net stimulation of triplicate cultures (mean ± SEM) was calculated by subtraction of the counts per minute of nonstimulated cultures from that of stimulated cultures. The in vitro proliferative responses of patients after TCD therapy were compared with that of a healthy control in each individual experiment. Initial studies (data not shown) showed that the in vitro proliferative response and the up-regulation of cell surface activation molecules (see below) as induced by polyclonal mitogens of healthy children were similar to healthy adults, thus allowing the use of adult volunteers of the hospital staff as controls.Cell surface marker analysis PBMCs (1 × 106) were cultured in 12-well plates (Falcon) in 1 mL medium with and without the above-mentioned polyclonal T-cell stimulants and were stained after time periods of 4 to 72 hours, as indicated. The following monoclonal antibodies (mabs) all purchased from Becton Dickinson (Mountain View, CA) were used: simultest control (IgG fluorescein isothiocyanate [FITC; FL1], IgG2a phycoerythrin [PE; FL2], IgG1 peridinin chlorophyll protein [PerCP; FL3]), anti-CD4 (PerCP), anti-CD45 (FITC), anti-CD14 (PE), anti-CD45RA (FITC), anti-CD45RO (FITC), anti-CD8 (PerCP), anti-CD69 (PE), anti-CD25 (PE), anti-HLA-DR (PE). The binding of cell surface phosphatidyl-serine structures by annexin V (PE; Pharmingen, San Diego CA) was used to detect apoptosis. Aliquots of 3 × 105 PBMCs were first incubated with MOPC 21 (mouse IgG1 ; Sigma) on ice for 20 minutes to block
nonspecific binding. The cells were then incubated with the appropriate
mab on ice for 20 minutes in the dark, and washed and immediately
analyzed on a FACScan flow cytometer (Becton Dickinson). List
mode data were analyzed using Cell Quest software (Becton Dickinson).
To examine stimulation-induced activation of T cells and of individual
T-cell subsets by flow cytometry, the following algorithm was devised:
lymphocytes were identified by forward scatter (FSC) versus side
scatter (SSC) and gated. To exclude a contamination with residual
monocytes, the lymphocytes as gated were examined for the expression of
CD45 (FITC) versus CD14 (PE). The proportion of
CD45+CD14+ cells contained in the lymphocyte
gate was less than 1%. CD4+ or CD8+ cells were
quantified by quadrant analysis (FL3 versus SSC). To detect cellular
activation, cells as gated were examined for the expression of the
activation markers CD69 or CD25 or HLA-DR. In experiments exploring
potential differences of naive and memory T-helper cells,
CD4+ cells (FL3 versus SSC) were gated and assessed for the
expression of CD45RA (FL1). Both the CD45RA+ and
CD45RA Statistical analysis Statistical analyses were performed using SSPS for Windows software. The Student t test (2-tailed) was used to compare results obtained from patient and control samples. Differences at P < .05 were considered significant. Linear regressions (Pearson correlation coefficient) were used to determine a correlation between the lymphoproliferative response ([3H]thymidine incorporation) and the density of expression of CD69+ cells upon in vitro stimulation.
Impaired induction of cell-surface activation molecules in T cells after TCD therapy Measuring lymphoproliferative responses to polyclonal stimulation has been a standard approach to estimate a depressed T-cell function after cytotoxic chemotherapy.1,34 In the present study, PBMCs of patients after TCD therapy showed reduced in vitro lymphoproliferative responses to polyclonal stimulation with immobilized anti-CD3 or PHA (mean reduction 65%). To exclude the possibility that a decreased lymphoproliferative response to stimulation through the TCR/CD3 complex simply reflects a reduced number of proliferating T cells contained in PBMCs after TCD therapy, the [3H]thymidine incorporation upon stimulation with immobilized anti-CD3 of 1 × 105 PBMCs was compared with that of a cell population in which 7.5 × 104 purified T cells were recombined with 2.5 × 104 monocytes. A similarly reduced response of patient samples in both cell preparations (Figure 1) indicated that in fact the responsiveness of T cells to stimulation after TCD therapy was reduced.
To attempt to identify, on a molecular level, a correlate to the
defective T-cell proliferative response, we examined by flow cytometry
the induction of the cell-surface markers CD69, CD25, and HLA-DR on T
cells, all of which are essentially absent on normal, unstimulated T
cells but are readily induced upon T-cell activation in a
time-dependent fashion.35 In these studies, immobilized
anti-CD3 or PHA were selected as stimulants as they most strongly
induced the expression of the above cell-surface molecules and gave
similar results. The monitoring of the expression of the respective
cell-surface markers by T-helper cells for 72 hours of culture (the
time period for determination of the proliferative response measured by
[3H]thymidine incorporation) showed marked differences
between patient and control samples (5 experiments; patients 3, 10, 18, 19, and 20). First, patient samples (Figure
2, left column) contained higher
proportions of CD4+CD25+ cells (9% median
[range, 8%-14%] versus 3% median [range, 2%-5%], patient
versus control) and of CD4+HLA-DR+ cells (24%
median [range, 21%-25%] versus 7% median [range, 3%-8%], patient versus control) but not of CD4+CD69+
cells (2% median [range, 3%-8%] versus 6% median [range,
1%-11%], patient versus control), a finding compatible with an
increased proportion of activated T cells in patients having received
high-dose chemotherapy, as previously described.28 In
cultures stimulated with anti-CD3, however, the increase of expression
of the above activation markers, given as the mpc on an arbitrary log
scale in histogram plots
Inability of exogenous IL-2 to overcome the impaired up-regulation of cellular activation markers induced by in vitro stimulation One possible explanation for a defective T-cell activation is a lack of T-cell stimulating cytokines, particularly of IL-2, the production of which has been consistently observed to be reduced in patients having received TCD therapy.36,37 To examine a role of IL-2 on the impaired induction of cell-surface activation molecules, we explored the potential of exogenous IL-2 to restore the capability of T-helper cells to up-regulate CD69 and CD25 upon polyclonal stimulation. In an initial control experiment, we showed that, in line with previous experimental evidence,38 IL-2 induced CD69 on natural killer (NK) cells (CD3 CD56+) in a dose-dependent manner, the
percent of CD69+ cells rising from 2% to 50% in the
presence of 25 U/mL to 200 U/mL IL-2 at 24 hours of culture. However,
essentially no change in CD69 cell-surface expression by
CD4+ T cells was observed (Figure
3A) comparing cultures stimulated with
immobilized anti-CD3 only to stimulated cultures supplemented with 25 U/mL exogenous IL-2 (the increase of the mpc being 1.2-fold, 1.1-fold,
and 0.9-fold at 4, 24, and 48 hours of culture, respectively; results
obtained from patients 18-21). IL-2 (25 U/mL) slightly enhanced the
density of expression of CD25 (Figure 3B) as indicated by a 1.6-fold
(range, 1.2-fold to 1.6-fold) and 1.5 fold (range, 1.0-fold to 1.9 fold) increase of the mpc at 24 hours and 48 hours of culture,
respectively.36 Increasing the dose of IL-2 from 25 U/mL to 100 U/mL did not further enhance CD25 expression nor affect
CD69 expression by stimulated or unstimulated CD4+ T cells
(not shown). Further, similar to a previous experience,37 IL-2 did not alter the proliferative responses to immobilized anti-CD3
(the change of cpm ranging from 0.9-fold to 1.2-fold in both controls
and patients, not shown).
Defective induction of CD69 in naive and memory T-helper cells To determine whether the impaired induction of CD69 in T-helper cells differently affects the naive and memory T-helper cell subpopulation, CD4+CD45RA+ and CD4+CD45RA populations were separately
analyzed for their capability to up-regulate CD69. As the expression of
the CD45RA and CD45RO epitope is mutually exclusive with the exception
of a small double-positive population,18
CD4+CD45RA cells were classified to largely
contain the CD4+CD45RO+ (memory) cell
population. Initial studies confirmed that a stimulation with
immobilized anti-CD3 or PHA for 24 hours did not change the proportions
of CD4+CD45RA+ cells (not shown). As expected,
in the patient samples tested within 3 months after TCD therapy,
the proportions of CD4+CD45RA+ cells contained
in CD4+ T cells were significantly smaller than in controls
(20% median versus 68% median, patients versus controls;
P < .0001, median 43/µL). Notably, however, the
experiments contained samples from patients having undergone a total
leukocyte depletion (WBC < 0.1/µL) as part of their recent
treatment in whom the absolute count of CD4+CD45RA+ cells was more than 90/µL
(patient 4, 109/µL; patient 14, 154/µL; patient 15, 126/µL;
patient 16, 91/µL; patient 22, 138/µL), which indicates ongoing
thymopoiesis.39
The striking finding of these analyses was that a defective induction
of CD69 detectable in total CD4+ cells affected both the
CD4+CD45RA+ and the
CD4+CD45RA
Increased binding of annexin V by naive and memory T-helper cells A recently proposed mechanism of the impaired immune function after chemotherapy is an increased susceptibility to apoptosis of T cells preferentially detectable in cells with an activated/memory phenotype.28,40,41 To test whether such increased susceptibility to apoptosis similar to an impaired induction of cellular activation also affects T-helper cells with a naive phenotype, a series of 12 experiments (patients 9-17) examined the binding of annexin V by flow cytometry to CD4+CD45RA+ and to CD4+CD45RA cell populations after TCD
therapy, in addition to the above-described assessment of (1)
[3H]thymidine incorporation (reduced by 71% median) and
(2) CD69 up-regulation by CD4+ T cells (reduced by 88%
median) upon anti-CD3 stimulation. The proportion of CD4+ T
cells was reduced (22% median versus 51% median, patients versus controls).
The proportion of annexin V binding cells in resting control
CD4+ T cells at 24 hours of culture was 6% median (range,
4%-10%), and in patient cells it was 25% median (range, 6%-56%;
P < .0001). A similar difference was found in
anti-CD3-stimulated CD4+ T cells (P < .001).
To determine annexin V binding of the naive T-helper cell population,
CD4+ T cells were separated into a CD45RA+ and
a CD45RA
Improved stimulatory response of highly enriched T-cell populations Based on the above findings it was tempting to speculate that the suppressed stimulatory response affecting naive and memory T-cell populations examined in bulk PBMCs might be mediated by T-cell extrinsic factors. To address this question, we compared the stimulatory responses to immobilized anti-CD3 of PBMCs to those of highly enriched T cells. Indeed, whereas the in vitro response to anti-CD3 examined in total PBMCs obtained from patients after TCD therapy was reduced (patients 17, 20, and 22), highly enriched T cells showed a markedly improved response evident by both an improved proliferative response and an improved up-regulation of CD69 of total CD4+ T cells including CD4+CD45RA+ cells (Figure 7).
The present findings of an impaired induction of CD69 upon in
vitro stimulation and an increased binding of annexin V affecting T-helper cells, including the naive
(CD4+CD45RA+) subset after TCD therapy, bear
several implications relevant to the as-yet-incomplete understanding of
an impaired T-cell function after cytotoxic anticancer therapy. A
reduced up-regulation of CD69 was detectable as early as 4 hours of
culture and was more pronounced as the concomitantly observed reduced
up-regulation of CD2543 or of HLA-DR. The extent of CD69
induction, similar to observations in healthy
controls35,44 and individuals with HIV
infection,45,46 also in our study correlated with
lymphoproliferation, which has been routinely used to measure T-cell
function after TCD therapy.34 In normal human T cells
stimulated via the TCR/CD3 complex, the expression of CD69 is rapidly
induced; transcripts of CD69 can be identified even 30 minutes after
stimulation.47 Activated Ras plays a central role for
cell-surface expression,48 which reaches a maximum at 18 to 24 hours after stimulation.49,50 Although the function
of CD69 has not been ultimately defined, cross-linking studies have
demonstrated a role for CD69 in regulating the production of T-cell
activating cytokines such as IL-251 as well as the IL-2
receptor (CD25),52 interferon Among other alterations of the cytokine network observed after
TCD therapy,3,54 a decrease of IL-2
production36,37,55 as well as of transcription examined on
a single-cell basis56 may be of particular relevance to
the disturbed T-cell function. In the present study, however, exogenous
IL-2 when added to anti-CD3-stimulated cultures (1) failed to improve
the reduced proliferative response similar to a previous
observation,37 and (2) failed to restore the impaired
capability of post-TCD therapy CD4+ T cells to generate
normal levels of CD69 cell-surface expression. This finding is in
agreement with recent experimental evidence showing that even high
doses (1000 U/mL) of exogenous IL-2 were not able to induce CD69 in
naive human T cells.57 Thus, since after TCD therapy the
induction of CD69 expression Of note, immobilized anti-CD3, which was preferentially used as a stimulant, activates T-helper cells independently of their antigenic determination. Thus, a reduced responsiveness of T cells reflects a functional deficit independent of the repertoire, which has been shown to be frequently limited in T-helper cells in the post-TCD therapy period.25-27 Further, because of the heterogeneity of the patients (Table 1), it appears unlikely that the observed effects are related to the underlying disease or the treatment. Likewise, a recent or concurrent treatment with cyclosporine A or granulocyte colony-stimulating factor (G-CSF), both drugs being used in patients undergoing transplantation (Table 1) and known to be correlated with an altered T-cell function,59-62 apparently was not the cause of the observed T-cell defect, per se, as the defect was similar in patients having received high-dose chemotherapy alone. Most striking was the finding that the T-cell dysfunction of the early post-TCD therapy period did not spare T cells bearing a naive phenotype. A recent exposure to the toxic effects of anticancer chemotherapy as an explanation would implicate that naive T cells had expanded by peripheral pathways of regeneration. Yet, as to the accumulated evidence, such thymus-independent expansion of CD4+CD45RA+ T cells after high-dose chemotherapy or BMT is extremely limited.22,39 Whereas such a thymus-independent regeneration of naive T cells cannot be definitively excluded in patients in whom at the time point of examination the number of circulating CD4+CD45RA+ cells was low, a cell count of more than 90/µL of circulating CD4+CD45RA+ cells, as was found in 5 patients in the present study, conceivably indicated an expansion by use of a thymopoietic pathway.39 As these patients also had an impaired in vitro T-cell immune function, the observed effects extended to the thymus-dependently regenerated T-helper cell population. Thus, while recent evidence suggests that an effective reconstitution of naive T cells coincided with (1) a normalization of the apoptotic decline,41,63 (2) the reconstitution of immune reactivity and responses to revaccinations,64 and (3) the restoration of a diverse T-cell repertoire,65,66 the present study provides evidence that an impaired T-cell function after TCD therapy is not simply explained by a lack or a delay of thymus-dependent T-cell reconstitution. This view is in line with the findings that (1) a decreased bcl-2 level, which is supposed to reflect an increased susceptibility to apoptosis of post-TCD therapy T cells, was demonstrated even in CD4+CD45RA+ cells,40 and (2) a sufficient immune function can be restored even in case of an extremely limited regeneration of naive T-helper cells,22 possibly by transfer of long-lived antigen-experienced cells.67 Rather, our findings suggest that the immunocompetence of regenerated T cells is impaired independently of the source of recruitment. Support for this conclusion comes from the finding that highly enriched T cells showed a markedly improved response of total CD4+ as well as of CD4+CD45RA+ cells to anti-CD3-induced stimulation as compared with bulk PBMCs. This finding warrants future research to explore potential suppressor effects of cell populations contained in the non-T-cell fraction of PBMCs. To this point, recent studies demonstrated a suppressor capacity of monocytes derived from G-CSF-stimulated leukapheresis products68-70 that might involve Fas-Fas ligand-mediated apoptosis.71 Similarly, initial experiments of our own continuing research has shown that a coculture of highly enriched T cells with autologous monocytes reduced T-cell proliferative responses to immobilized anti-CD3 in a dose-dependent fashion in patient samples while having no effect in control cultures (study in progress). Regardless of the final mechanism, the finding that a T-cell dysfunction in the post-TCD therapy period may be mediated by T-cell extrinsic factors, thus eventually involving multiple T-cell populations, might have implications in the setting of adoptive cell therapy to treat viral infections after BMT72 or to eradicate minimal residual disease in cancer patients73 as the efficacy of transferred T cells may be inhibited by the same suppressive factor(s) that inhibit the function of T cells generated by the host. Such a phenomenon has been described in HIV disease in which transferred T cells became susceptible to apoptosis, similar to host T cells.74 Alternatively, however, a modulation of immunosuppression generated by the host may be useful in the management of graft-versus-host disease and tolerance induction in transplant settings.68 Together, future efforts to overcome or manipulate the functional impairment of T cells after TCD therapy will have to not only attempt to accelerate thymopoiesis (eg, by interleukin-7),75,76 but to identify the factor(s) that affect T-helper cells even when presumably being regenerated through a thymus-dependent pathway.
The authors wish to thank Hannelore Kern and Doris Vergeiner for excellent technical assistance.
Submitted July 16, 2001; accepted January 28, 2002.
Supported by a research grant of the Children's Cancer Research Institute, St Anna Children's Hospital, Vienna, Austria (A.H.).
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: Andreas Heitger, St Anna Children's Hospital, Kinderspitalgasse 6, A-1090 Vienna, Austria; e-mail: andreas.heitger{at}uibk.ac.at.
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Top
Abstract
Introduction
thus estimating the density of
expression
) and
patient (
) CD4+ T cells from 4 to 48 hours of culture.
Patient cells were obtained from a 12-year-old girl with ALL (patient
18) 3 months after MUD-BMT.
) and
in CD4+CD45RA
) at 24 hours of
culture. The mpc of CD69 expression in histogram plots is expressed as
percent of control at 1 to 3 months (time point 1) and at 6 to 9 months
(time point 2) after completion of TCD therapy. Patient 1: 8-year-old
girl with stage IV neuroblastoma (patient 1), high-dose chemotherapy
with autologous stem cell rescue; patient 2: 5-year-old boy with stage
IV Ewing sarcoma (patient 15), high-dose chemotherapy with autologous
stem cell rescue; patient 3: 12-year-old girl with Ewing sarcoma
(patient 10), conventional high-dose chemotherapy; patient 4:
15-year-old girl with germ-cell tumor (patient 16), conventional
high-dose chemotherapy.
, and tumor necrosis
factor-
.