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[Cancer Research 59, 3107-3111, July 1, 1999]
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[Cancer Research 59, 3107-3111, July 1, 1999]
© 1999 American Association for Cancer Research

Experimental Therapeutics

Fas-FasL-mediated CD4+ T-Cell Apoptosis following Stem Cell Transplantation1

Rakesh K. Singh, Michelle L. Varney, Suleyman Buyukberber, Kazuhiko Ino, Ana G. Ageitos, Elizabeth Reed, Stefano Tarantolo and James E. Talmadge2

Departments of Pathology and Microbiology [R. K. S., M. L. V., S. B., K. I., A. G. A., J. E. T.] and Internal Medicine-Oncology/Hematology [E. R., S. T.], University of Nebraska Medical Center, Omaha, Nebraska 68198


   ABSTRACT
Top
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
We report the preferential expression of Fas on CD4+ T cells and Fas ligand (FasL) on monocytes and their potential role in the selective loss of CD4+ T cells in breast cancer patients undergoing high-dose chemotherapy and peripheral blood stem cell transplantation (PSCT). A high frequency of apoptotic CD4+ T cells (28–51%) is observed during the first 100 days after PSCT concomitant with a significant increase in monocyte frequency and FasL expression (11.6–23%) on monocytes. The preferential expression of Fas on CD4+ T cells (73–92%) in the peripheral blood (PB) of these patients is associated with a significantly higher frequency of CD4+ T-cell apoptosis compared with CD8+ T cells (28–47%) and CD4+ T cells (46 ± 5.7%) in normal PB. These data suggest that "primed" Fas+ CD4+ lymphocytes interact with activated monocytes that express FasL, resulting in apoptosis, leading to deletion of CD4+ T cells, an inversion in the CD4:CD8 T-cell ratio, and immune dysfunction. The prevention of CD4+ T-cell apoptosis and improved immune reconstitution by the manipulation of PB stem cell products, blockade of Fas-FasL interactions, or cytokine support after transplantation may be important adjuvant immunotherapeutic strategies in patients undergoing high-dose chemotherapy and PSCT.


   INTRODUCTION
Top
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Immune dysfunction occurs after HDT3 and autologous PSCT despite restoration of T cell numbers and may contribute to disease relapse (1, 2, 3, 4, 5, 6, 7) . This dysfunction includes an inversion in the CD4:CD8 T-cell ratio and a depression in T-cell function (1 , 2) . Studies from our laboratory and others have demonstrated a cell-mediated suppression of T-cell function in the PB of cancer patients (1 , 2 , 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18) . In addition, high levels of type 2-associated cytokines are found in the infused T cells and monocytes (16 , 19 , 20) .

Apoptosis provides one mechanism for the regulation of peripheral CD4+ T-cell homeostasis. This form of cell death is highly regulated and dependent on the expression of a family of ligands, including FasL, tumor necrosis factor-related apoptosis-inducing ligand, and their receptors (21 , 22, 23, 24, 25, 26, 27, 28) . Enhanced monocyte-dependent apoptosis of uninfected CD4+ T cells is postulated to contribute to CD4+ T-cell depletion in HIV-infected individuals and to leads to an inverted CD4:CD8 T-cell ratio (25 , 29 , 30) . Recent data from our laboratory suggest that cancer patients undergoing HDT and PSCT, like HIV-infected individuals, have a prolonged immunodeficiency, an inverted CD4:CD8 ratio, and peripheral tolerance (1 , 2 , 15) . However, the cellular and molecular mechanism(s) underlying this immune dysfunction remains unclear. This study demonstrates that monocyte-associated FasL expression and the selective depletion of CD4+ T cells expressing Fas are important mechanisms in the inversion of the CD4:CD8 T-cell ratio and immune dysfunction in breast cancer patients after HDT and PSCT.


   MATERIALS AND METHODS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Patients.
High-risk breast cancer patients who were candidates for HDT and PSCT (n = 12) at the University of Nebraska Medical Center were entered into this study. Written informed consent for stem cell collection and autologous transplantation was obtained from each patient. All of the patients received an average of four cycles of chemotherapy before HDT and stem cell transplantation, and all received Cytoxan, thiotepa, and Hydrea as the HDT regimen. PSCT patients were mobilized with granulocyte colony-stimulating factor (31) , and a target dose of 6.5 x 108 mononuclear cells/kg body weight was collected and cryopreserved. The samples were obtained using protocols approved by the Institutional Review Board of the University of Nebraska Medical Center.

Mononuclear Cell Isolation.
The fresh uncultured PSC products from patients were diluted 1:2 in HBSS (Life Technologies, Inc., Grand Island, NY), layered on Ficoll-Hypaque (Organon Teknika, Durham, NC) and centrifuged for 20 min at 400 x g using a Beckman TJ6R centrifuge with swinging bucket rotor. Mononuclear cells from normal donors were used as controls. The cells were washed twice with HBSS and adjusted to 4 x 106 cells/ml in RPMI 1640 containing 10% fetal bovine serum, 10 mM HEPES, gentamicin, and L-glutamine.

Cell Isolation and Separation.
Mononuclear cells isolated from PB were used to isolate CD4+ and CD8+ T cells and CD14+ monocytes using discontinuous Percoll gradient and immunomagnetic isolation technique, as described previously (15) . The morphology of each fraction was assessed using Wright-Giemsa staining of cytospin preparations and flow cytometry.

Flow Cytometric Analysis.
For phenotypic analysis, mononuclear cells isolated from PSC product, and PB were counted and adjusted to 1 x 106 cells in PBS (Life Technologies, Inc.) containing 2.5% fetal bovine serum to maintain cell viability. Nonspecific binding was blocked with human IgG, and an aliquot of 1 x 105 cells was stained for 30 min at 4°C with each mixture of monoclonal antibodies (at saturating concentrations) to various human leukocyte antigens. The antibodies used in this study were purchased from Becton Dickinson (San Jose, CA) and Coulter Corporation (Hialeah, FL). All three-color data were acquired on a Becton Dickinson FACStarPlus (San Jose, CA). Detailed three-color data analysis was performed using the Attractors and CellQuest software from Becton Dickinson, as described previously (15) . Each subpopulation was gated using forward and side scatter and fluorescence intensity for the specific markers CD14, CD4, CD8, CD95 (Fas), and CD95L (FasL). With the use of this technique, CD14+ monocytes were defined as cells with intermediate side scatter and expressing high levels of CD14, which allowed us to distinguish them from lymphocytes, polymorphonuclear neutrophils, and granulocytes. The lymphocyte population was distinguished based on lower side scatter and high expression of CD4 and CD8.

Assay for Apoptosis.
A multicolor flow cytometric analysis, including a TUNEL assay for apoptosis, was used to determine the frequency of apoptotic T cells in the PB cells of the patients undergoing HDT and PSCT, as described previously (15) . Briefly, cell suspensions were stained with either phycoerythrin or biotin-adenomatous polyposis coli, washed twice in PBS, resuspended in ice-cold PBS/1% BSA (100 µl), and fixed with 10% of buffered formalin. The cells were permeabilized using 0.1% Triton X-100/0.1% sodium citrate solution by incubating at 0°C for 2 min. After washing, the cells were resuspended in 50 µl of TUNEL reaction mixture (0.03 {gamma} mol FITC-UTP, 3 µmol dATP, 2 µl of 25 mM CaCl2, and 25 U TdT; Boehringer Mannheim, Indianapolis, IN) and incubated for 60 min at 37°C. The cells were washed and analyzed by flow cytometry. A forward scatter by side scatter plot was used to gate all of the cells while excluding cell debris and aggregated cells. CD4+ and CD8+ cells were then individually backgated to determine the frequency of cells undergoing apoptosis.

Analysis of FasL mRNA Expression.
Total cellular RNA was isolated from PSC products, PB lymphocyte, and purified CD4+-, CD8+-, and CD14+-treated or untreated cells using Trizol reagent (Life Technologies, Inc., Gaithersburg, MD) reverse transcription-PCR was performed as described earlier (19 , 20) . First-strand cDNA was synthesized using total RNA (2 µg), oligo (dT)18 primer, and superscript RT (Life Technologies, Inc., Gaithersburg, MD). First-strand cDNA (2 µl; 1:10 dilution) was amplified using a PCR primer set table and a DNA thermal cycler (Perkin-Elmer, Foster City, CA) for different cycles. Each cycle set the denaturing temperature at 94°C for 60 s, annealing temperatures (55°C for ß-actin and FasL; Table 1Citation ) for 90 s, and extension at 72°C for 90 s for a total of 20 cycles for ß-actin and 40 cycles for FasL. PCR fragments were separated on 2% ethidium bromide containing agarose gel, visualized, and photographed using UV trans-illuminators (Kodak, Rochester, NY). For quantitative studies and to confirm the specificity of the amplified sequences, gels were blotted on GeneScreen membrane (DuPont) and processed for Southern blot analysis, as described earlier (19 , 20) . The membranes were hybridized with 32P-labeled specific oligonucleotide or cDNA probes for 4 h, washed under stringent conditions, and analyzed by digital autoradiography (Phosphor-Imager; Molecular Dynamics, Sunnyvale, CA). Relative amounts of radioactivity between samples blotted on each membrane were examined using an ImageQuant analysis system on Phosphor-Imager (digital autoradiography). Relative mRNA transcript levels were obtained by using an equal number of cells with simultaneous amplification within the linear range, blotting and probing the samples to be compared. The level of mRNA expression for FasL is expressed as the ratio (expression ratio) obtained by dividing FasL (expressed as density units from the phosphor imager) by the signal from the housekeeping gene ß-actin.


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  		Table 1 Primers for reverse transcription-PCR analysis

 
Statistical Analysis.
SPSS for Windows (SPSS Inc., Chicago, IL) was used for the independent sample t test (two-tailed) to compare means. The data presented are a composite of all of the patients analyzed in each group.


   RESULTS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Frequency of Apoptotic T Lymphocytes in the PB of Patients Undergoing HDT and PSCT.
The frequency of apoptotic CD4+ T cells was significantly higher on days 10, 14, and 26, but not day 100, in the PB of breast cancer patients after HDT and PSCT as compared with normal individuals and on days 10 and 14, relative to pretransplant levels (Fig. 1)Citation . A representative dot plot of CD4 and TUNEL-positive cells from the PB of normal and day 14 posttransplant donors demonstrating the differences in the CD4+ T-cell apoptosis is shown in Fig. 2Citation . In general, higher levels of apoptotic cells in the PB of patients were observed compared with normal donors (Fig. 2)Citation . The frequency of CD4+ and CD8+ T cells (12.85 + 2.08 and 9.79 + 2.7, respectively) in the PB of patients on day 14 after transplantation was not significantly different as compared with normal donors (11.68 + 1.3 and 6.67 + 1.06, respectively). In addition, the frequency of apoptotic CD4+ T cells in the PB of patients on day 14 after transplant (50.3 + 8.0) was significantly higher as compared with normal donors (10.1 + 2.3; Figs. 1Citation and 2Citation ). Furthermore, the patients had no significant difference in the frequency of apoptotic CD4+ T cells before transplantation as compared with normal donors, suggesting that prior chemotherapy had no role in this apoptosis. Similarly, the frequency of apoptotic CD8+ T cells was significantly higher on days 14 and 26 as compared with pretransplant levels. However, on days 10 and 14, the frequency of apoptotic CD4+ T cells was significantly higher than that of CD8+ T cells. Interestingly, the frequency of apoptotic CD8+ T cells before transplantation was significantly lower than that observed in normal PB. The presence of increased numbers of apoptotic cells in the PB of patients undergoing HDT and PSCT was confirmed by DNA fragmentation analysis (data not shown).



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  		Fig. 1. The frequency of apoptotic CD4+ or CD8+ T cells in a total cell gate of CD4+ or CD8+ T cells was determined using three-color flow cytometry. Results are reported as the mean ± SE. *, significantly different from pretransplant levels (P <= 0.05); #, significantly different from normal PB (P <= 0.05); $, significantly different from the frequency of apoptotic CD8+ T cells at corresponding time points (P <= 0.05).

 


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  		Fig. 2. Representative dot plots that demonstrate the presence of apoptotic CD4+ T cells in the PB of normal donors (A) and the day 14 posttransplant PB of a breast cancer patient (B).

 
Abnormal CD4:CD8 T-Cell Ratio in Patients Undergoing HDT and PSCT.
The phenotypic analysis of CD4+ and CD8+ T cells revealed an inverted CD4:CD8 T-cell ratio in patients after HDT and PSCT. However, there was no significant difference in the ratios before and 10 days after HDT and PSCT (Fig. 3)Citation . In contrast, from day 14 onward, the CD4:CD8 T-cell ratio was significantly lower than pretransplant levels and that observed in normal donors (Fig. 3)Citation .



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  		Fig. 3. The ratio between CD4+ and CD8+ cells. The values are the mean of CD4:CD8 ratios ± SE for each group. *, significantly different from normal PB; #, significantly different from pretransplant levels.

 
Increased Fas (CD95/Apo1) Expression on CD4+ and CD8+ T Cells.
A significantly higher frequency of CD4+Fas+ T cells was observed in the PB of patients as compared with normal donors. However, there was no difference in the frequency of CD4+Fas+ T cells after HDT and PSCT as compared with pretransplant levels. The data suggest that the expression of Fas+ is independent of HDT and PSCT and that the increased CD4+ T-cell apoptosis was associated with FasL expression on monocytes. In contrast, there was no significant difference in the frequency of CD8+Fas+ T cells before transplant or at different time points after transplant compared with normal donors (Fig. 4)Citation . Furthermore, 70–80% of the CD4+ T cells were Fas+, a percentage significantly higher at all time points examined than the 20–45% of CD8+ cells that were Fas+ (Fig. 4)Citation .



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  		Fig. 4. The frequency of Fas+, CD4+, or Fas+ CD8+ T cells in a total cell gate of CD4+ or CD8+ T cells was determined by three-color flow cytometry. Results are reported as the mean ± SE. *, significantly different from pretransplant levels (P <= 0.05); #, significantly different from normal PB (P <= 0.05); $, significantly different from frequency of Fas+CD8+ T cells at corresponding time points (P <= 0.05).

 
Increased FasL (CD95L) Expression on CD14+ Monocytes But Not T Cells.
A significantly increased frequency of FasL expression was observed on CD14+ monocytes on days 10, 14, 26, and 100 after HDT and PSCT as compared with pretransplant levels and those observed on monocytes from normal PB (Fig. 5A)Citation . FasL+CD4+ and FasL+CD8+ cells were rarely observed in the PB of these patients (Fig. 5A)Citation . Furthermore, at all of the time points analyzed, the frequency of FasL+CD14+ cells was significantly higher than FasL+CD4+ or FasL+CD8+ cells (Fig. 5)Citation . Besides an increase in FasL+ monocytes, there was a significant increase in the frequency and absolute number of monocytes in the PB of patients after HDT and PSCT (Fig. 5B)Citation . In contrast, there was no significant difference in the frequency or absolute number of monocytes before HDT and PSCT as compared with normal donors (Fig. 5B)Citation .



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  		Fig. 5. A, flow cytometry analysis of FasL expression on CD4+ T cells, CD8+ T cells, and CD14+ monocytes in the PB of breast cancer patients after HDT and PSCT. The results are shown as the frequency of FasL+ cells in their respective total cell gate. *, significantly different from pretransplant levels (P <= 0.05). Results are reported as the mean ± SE. #, significantly different from normal PB (P <= 0.05); $, significantly different from the frequency of FasL+CD8+ or FasL+CD4+ T cells at corresponding time points (P <= 0.05). B, the frequency and absolute number of CD14+ monocytes in the PB of patients undergoing HDT and PSCT was determined by three-color flow cytometry. Results are reported as the mean ± SE. *, significantly different from pretransplant levels (P <= 0.05); #, significantly different from normal PB (P <= 0.05).

 
As shown in Fig. 6Citation , FasL mRNA in mononuclear cells from the PB of patients before transplantation was similar to that of normal donors. In contrast, the expression of FasL mRNA in the PB of transplant patients was significantly higher at all time points examined after HDT and PSCT as compared with pretransplant levels and normal PB (Fig. 6)Citation .



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  		Fig. 6. Levels of FasL mRNA transcripts in the PB of patients after HDT and PSC and normal donors. Expression indexes for FasL mRNA were calculated by comparing with housekeeping gene ß-actin. Results are reported as the mean ± SE. *, significantly different from pretransplant levels (P <= 0.05); #, significantly different from normal PB (P <= 0.05).

 

   DISCUSSION
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
The present studies demonstrate that the selective depletion of CD4+ T cells is one mechanism for the peripheral tolerance observed in breast cancer patients after HDT and PSCT. We observed a preferential apoptosis of CD4+ T cells, which is associated with the frequency of monocytes and monocytes expressing FasL in the PB. In addition, the CD4+ T cells in the PB of these patients preferentially express Fas as compared with CD8+ T cells. We suggest that the high frequency of monocytes expressing FasL in the PB of these patients results in the preferential deletion of the Fas+CD4+ T cells. These studies provide a mechanism for the immune dysfunction peripheral tolerance and depressed CD4:CD8 T-cell ratio that are observed after HDT and PSC (1 , 2) . Furthermore, we hypothesize that the high frequency of FasL expression on monocytes is associated with the secretion of high levels of monocyte-activating cytokines by the T cells after transplantation (20) .

Recently, we reported that monocytes in mobilized PSC products, as well as PB lymphocytes, after transplantation inhibit T-cell function (1 , 2 , 14 , 15) by inducing T-cell apoptosis (14) . Our present data demonstrate a higher frequency of apoptotic CD4+ T cells in the PB of patients after HDT and PSCT, which might contribute to the abnormal immune reconstitution. Similar reports by Donnenberg et al. (32 , 33) suggest that T-cell apoptosis parallels lymphopoiesis in patients who have had bone marrow transplants. An accelerated spontaneous and activation-induced T-cell apoptosis is also observed in the PB of individuals infected with HIV, which may occur by a similar mechanism (34, 35, 36, 37, 38) .

Recent studies from our laboratory and others suggest that T-cell apoptosis is associated with T-cell activation by either CD3+ cross-linking, phorbol myristate acetate, or phytohemagglutinin (1 , 2 , 9 , 14 , 15 , 17 , 39) . This occurs through a MHC nonrestricted monocyte-dependent mechanism (15) . The requirement for T-cell activation seems to be a common feature of monocyte-dependent apoptosis mediated by Fas-FasL interaction (25 , 30 , 34) . Our previous results suggest that the T cells and monocytes in the PB of the HDT and PSCT patients are highly activated based on the expression of immunoregulatory cytokines (19 , 20) . These results are similar to the finding that circulating T lymphocytes from HIV-infected individuals are activated (36) , have an increased expression of Fas on their membranes (37 , 38) , and are more susceptible to FasL-mediated killing (38) . Our data also suggest that the CD4+ T cells in the PB of HDT and PSCT patients undergo apoptosis after encountering monocytes expressing FasL. However, whether this is predominantly a PB or lymph node phenomenon is unknown. Thus, the circumstance whereby a susceptible CD4+ T cell encounters an apoptosis-inducing ligand remains unknown.

These data suggest that monocyte expression of FasL and preferential expression of Fas on CD4+ cells have a mechanistic role in the inverted CD4:CD8 T-cell ratios observed after HDT and PSCT. A higher frequency of CD14+ monocytes and CD14+ FasL+ monocytes are found in the PB at times when a significantly higher frequency of apoptotic CD4+ cells are observed. However, we did not observe a significant increase in the frequency of apoptotic CD8+ cells, which suggests a preferential apoptosis of CD4+ cells. Similar to these findings, recent studies suggest that the death of uninfected CD4+ cells in AIDS patients involves a monocyte-dependent induction of T-cell apoptosis (25 , 29 , 40) . The inability of HIV to infect chimpanzee macrophages has been suggested as an explanation for the lack of T-cell apoptosis and development of AIDS in HIV-infected chimpanzees (41) . These studies suggest that accessory cells can send a death signal to neighboring primed and uninfected CD4+ T cells, which mediates the depletion of T cells. Recent studies also suggest that FasL expression is not restricted to cells of lymphoid origin (29 , 42) observed an up-regulation of FasL expression by the human AIDS virus in macrophages, which mediated apoptosis of T cells expressing Fas, leading to immunodeficiency. We report herein that most of the CD4+ T cells in the PB of patients undergoing HDT and PSCT express Fas, which, combined with the monocyte expression of FasL, mediates the apoptosis of CD4+ T cells.

Previous in vitro studies suggest a requirement for cell-to-cell interactions in monocyte-dependent T-cell apoptosis (15) and a role for a membrane-associated form of FasL, but did not rule out a role for soluble FasL. A recent study demonstrates that human monocytic cells contain high levels of intracellular FasL, which is rapidly released after cellular activation (43) , suggesting an additional FasL mechanism. Furthermore, preclinical studies (44 , 45) and clinical studies (46 , 47) have suggested that cytokine mobilization can result in a type 2 cytokine storm that also contributes to immune deficiency.

In summary, our data suggest that "primed" Fas+CD4+ lymphocytes interact with activated monocytes that express FasL, resulting in apoptosis that leads to preferential deletion of CD4+ T cells, an inversion in the CD4:CD8 T-cell ratio, and immune dysfunction. We suggest that this is one mechanism in the peripheral tolerance observed in cancer patients after HDT and PSCT. Furthermore, the prevention of apoptosis in CD4+ T cells and effective immune reconstitution by the manipulation of PSC products or cytokine/antibody support after transplantation may be an important adjuvant therapeutic strategy in patients undergoing HDT and PSCT.


   FOOTNOTES
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 Supported in part by NIH Grants R01-CA61593 and R29-CA72781 and Grant 97-71 from the Nebraska Department of Health and Human Services. Back

2 To whom requests for reprints should be addressed, at Department of Pathology and Microbiology, University of Nebraska Medical Center, 985660 Nebraska Medical Center, Omaha, NE 68198-5660. Phone: (402) 559-5639; Fax: (402) 559-4990; E-mail: jtalmadg{at}unmc.edu Back

3 The abbreviations used are: HDT, high-dose chemotherapy; PSC, peripheral stem cell; PSCT, PSC transplantation; PB, peripheral blood; TUNEL, terminal deoxynucleotidyl transferase-mediated nick end labeling; FasL, fas ligand. Back

Received 1/ 8/99. Accepted 5/ 3/99.


   REFERENCES
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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