The present study analyses the influence of high-dose chemotherapy (HD) and autologous stem cell transplantation on natural and vaccine-induced specific immunity in breast cancer patients. Peripheral blood was collected from five breast cancer patients at serial time points in connection with treatment and in a follow-up period of 1 year. The frequencies of CD8+ and CD4+ T cells responsive to cytomegalovirus (CMV), varicella zoster virus (VZV), and tetanus in antigen-activated whole blood were determined by flow cytometric analysis of CD69, TNFα, IFNγ and IL-4 expression. Mononuclear cells were labelled with PKH26 dye and the CMV, VZV, and tetanus toxoid-specific proliferation of T cell subpopulations was analysed by flow cytometry. In none of the patients did the treatment result in loss of overall T cell reactivity for any of the antigens. Prior to chemotherapy 5/5 patients possessed TNFα expressing T cells specific for CMV, 4/5 for VZV, and 3/5 for tetanus. One year after stem cell transplantation all patients possessed TNFα expressing T cells specific for CMV, VZV and tetanus. The highest percentages of cytokine-responding T cells were seen after stimulation with CMV antigen. In general, the lowest reactivity (close to zero) was measured in G-CSF-mobilised blood at the time of leukapheresis. In spite of a continuously reduced CD4 to CD8 ratio after transplantation, recovery of CD4+ T cells usually occurred prior to CD8+ recovery and often to a higher level. The study demonstrates that natural as well as vaccine-induced specific immunity established prior to HD can be regained after stem cell transplantation. These data indicate that introduction of a preventive cancer vaccination in combination with intensive chemotherapy may be a realistic treatment option.
High-dose chemotherapy (HD) has been used as an adjuvant treatment for breast cancer patients with a high risk of disease recurrence. The dose-intensive regimen profoundly impairs the immune function, especially cell mediated functions, as indicated by several reports describing both qualitative and quantitative T cell imbalances, as well as defects in the proliferative response of T cells to common activators.1,2,3,4,5,6,7 The aim of this study was to determine the influence of HD and autologous peripheral blood stem cell transplantation (PBSCT) on natural and vaccine-induced memory T cell immunity. For this purpose a prospective study of breast cancer patients treated with HD was carried out. Qualitative and quantitative evaluation of the cellular immune system were performed in connection with treatment and in a follow-up period of 1 year. The frequencies of cytomegalovirus (CMV), varicella zoster virus (VZV) and tetanus-responsive CD8+ and CD4+ subpopulations of T lymphocytes were analysed in antigen (Ag)-activated whole blood and determined by flow cytometric analysis.8,9,10,11,12,13
Analyses of the specific immune reactivity of cancer patients undergoing treatment with HD impart knowledge about the impact of intensive chemotherapy on low frequency subpopulations of Ag-specific T lymphocytes. T lymphocytes are able to perform killing of tumour cells3,14,15 and are therefore a potential target for therapeutic immune modulation in combination with stem cell transplantation. The analyses carried out may help to develop strategies for adding a preventive vaccination program.
Materials and methods
Patients and treatment
Five women with breast cancer who had undergone primary tumour excision and axillary lymph node dissection showing ⩾6 tumour positive lymph nodes were included in the study. The patients received adjuvant HD and PBSCT. The treatment was initiated with three cycles of standard-dose induction chemotherapy (FEC: 5-fluorouracil, epirubicin, cyclophosphamide) with 3 weeks interval followed by peripheral blood stem cell harvest. PBSC were mobilised using recombinant human G-CSF. After PBSC harvest, patients received HD with cyclophosphamide, thiotepa and carboplatin given daily for 4 consecutive days.16 Stem cells were reinfused 48 h after completion of chemotherapy. Samples of heparinised peripheral blood were obtained from patients at serial time points in connection with treatment and with follow-up the first year. All patients were sero-positive for VZV IgG antibodies and 4/5 were sero-positive for CMV IgG antibodies before initiation of treatment. Furthermore, all patients had received vaccination against tetanus within 10 years.
Antigens and antibodies
Tetanus toxoid (purified, 3 mg/ml) and tetanus vaccine preparation (Cat. No. 468801) was provided from Statens Seruminstitut, Copenhagen, Denmark and used at an optimal stimulatory concentration of 10 μl (1:50) toxoid for cell culturing and 15 μg/ml for full blood activation.
CMV Ag and VZV Ag were used as partially purified virally infected cell lysates (Biowhittaker, Walkersville, MD, USA). Matched partially purified CMV or VZV control Ag prepared from non-infected cell lysates (Biowhittaker) was utilised in the assays to detect non-specific activation. Optimal stimulatory concentration of the viral Ags was 60 μl/ml blood or cell culture. Lipopolysaccharide (LPS) 1 μg/ml blood was used for monocyte-specific stimulation (Sigma, Cat. No. L2654; St Louis, MO, USA).
Monoclonal antibodies (mAbs) used for surface staining were CD3 APC, CD8 PerCP (5 μl/test), CD4 FITC (5 μl/test), CD69 PE (5 μl/test), CD69 FITC (10 μl/test), CD33 PE (5 μl/test). Mabs used for intracellular staining were anti-TNFα FITC, anti-IFNγ FITC (10 μl/test), anti-IL-4 PE. MAbs used for activation were CD28, CD2/CD2R. All mAbs were purchased from BD Bioscience (San José, CA, USA). The antibodies were used at concentrations recommended by the manufacturer or as indicated in brackets.
Intracellular cytokine detection (ICC) in Ag-activated T lymphocytes
Cell preparation and antigenic stimulation:
This was carried out according to the method described by Suni et al8 slightly modified. Sodium heparinised venous blood was aliquoted into 4 ml minisorp-tubes (Cat. No. 466982; Life Technologies, Taastrup, Denmark) at 500 μl per tube. For Ag-specific T cell activation the costimulatory mAb CD28 was added to the samples at 3 μg/ml together with Ag or control Ag. For polyclonal T cell activation mAb CD28 was used in combination with mAb CD2/CD2R. Brefaldin A (BFA, Sigma Cat. No. B7651) was added at a concentration of 10 μg/ml blood after 1 h. After another 5 h of incubation 50 μl of 20 mM EDTA were added for 15 min for detachment of adhering cells.
Activated blood samples were aliquoted into staining tubes, 100–125 μl/tube. Samples were lysed and fixed in 4 ml 1 × FACS lysing solution (BDIS) and permeabilized with 500 μl of 1 × FACS permeabilizing solution. The cells were washed with cold wash buffer (PBS, 0.5% BSA, 0.1% NaN3) and four-colour analyses of the T cell cytokine responses were carried out using a staining antibody cocktail consisting of CD3 APC, CD8 PerCP, CD69 PE or FITC and anti-TNFα FITC, anti-IFNγ FITC or anti-IL-4 PE. After staining, samples were washed and fixed in 1% paraformaldehyde in PBS.
Flow cytometric analysis:
Four-colour flow cytometric analysis was performed on a FACSCalibur flow cytometer (BD Bioscience). Day-to-day consistency of measurements was checked by Calibrite Calibration Beads (Cat. No.340486 and 340487; BD Bioscience). Data were acquired using CellQuest software (BD Bioscience). Thirty thousand gated CD3+ events were collected using FL4 as a fluorescent trigger. Forward scatter vs side scatter gating of lymphocytes were employed in data analysis and CD4 helper T cells were defined as CD3+, CD8− gated lymphocytes. CD69 staining was included to enhance the identification of specific Ag-responsive T cells,9 which were defined as double-positive (cytokine+ and CD69+) cell subsets.
Measurement of Ag-specific proliferation of T lymphocytes
Peripheral blood mononuclear cells (PBMC) were isolated from heparinised blood over Lymphoprep (Nycomed, Oslo, Norway) using standard procedures.
Labelling was carried out according to the method described by Allsopp et al13 PBMC (1 × 107) were pelleted in polypropylene tubes (Cat. No. 352097; Falcon (BD Bioscience)) resuspended in 1 ml diluent C and incubated for 2 min with 1 ml of 3 μM PKH26 dye (Cat. No. PKH26-GL; Sigma). Staining reaction was stopped by addition of 2 ml of heat inactivated 100% FCS. Labelled cells were washed and cell number and viability were assessed using Trypan blue.
Cell culture and Ag stimulation:
Cells were plated out at 2 × 105 cells/well in a 96 U-bottomed plate (Nunclon; Gibco, Taastrup, Denmark) in RPMI1640 (Cat. No. 72400; Gibco) with 2 mM glutamax, 100 U/ml penicillin, 100 μg/ml streptomycin, 25 mM Hepes, and containing 10% heat inactivated AB serum. The cells were cultured in a final total volume of 200 μl and each condition was set out in triplicate. Tetanus toxoid, VZV and CMV Ags were added in optimal concentrations. Phytohaemagglutinin (PHA-L) (Cat. No. 153322, ICN Biomedicals, Aurora, OH, USA) was used at a final concentration of 1 μg/ml. To detect non-specific proliferation viral control Ag was used and for negative control no Ag was added to the cells. Plates were incubated for 7 days at 37°C, 5% CO2.
Preparation of cells for flow cytometry:
The cells were harvested into staining tubes, washed with wash buffer, and stained for 30 min. Four-colour analyses of the T cell proliferation responses were carried out using a staining antibody cocktail consisting of CD3 APC, CD8 PerCP, and CD4 FITC as PKH26 emission was detected in the FL2 channel. After staining, samples were washed and fixed in 1% paraformaldehyde in PBS.
Flow cytometric analysis:
Four-colour flow cytometric analysis was performed on a FACSCalibur flow cytometer. Data were acquired using CellQuest software. The flow cytometer was set to acquire events for a given length of time (240 s). Forward scatter vs side scatter gating of viable lymphocytes was employed in data analysis. Cell division was determined as reduced fluorescence intensity of the PKH26 dye (the fluorescence intensity of membrane staining halves with each cell division).
Descriptive statistics for each immunological measure were generated separately by time of assessment. When applicable results are expressed as mean values and standard deviation (s.d.). No statistical tests were performed due to low number of observations.
Reconstitution of lymphocyte subsets
The recovery of T lymphocytes of the individual breast cancer patients after HD is illustrated in Figure 1. Mean total lymphocyte concentration in the blood before initiation of chemotherapy was 1580 cells/μl compared to 1180 cells/μl 12 months after HD (Figure 1a). The blood concentration of CD3+, CD8+ T cells increased rapidly after HD (Figure 1c) and was almost equivalent before and 12 months after HD: 382 and 408 cells/μl, respectively. In contrast, a significant decrease in the mean blood content of CD3+ CD4+ T cells from 802 cells/μl to 280 cells/μl was observed even 12 months after HD (Figure 1b). Consistent with these findings the CD4 to CD8 ratio was continuously reduced from 2.13 to 0.76 after HD (Figure 1d).
Ag-specific T cell cytokine response during HD
CMV Ag, VZV Ag and tetanus-specific activation of T cells, as measured by CD69 and cytokine expression was evaluated during the HD treatment. Three different cytokines, TNFα, IFNγ and IL-4, were assessed simultaneously. In all patients a close correlation between TNFα and IFNγ expression was observed, with the fraction of activated T cells expressing TNFα being either slightly higher or equivalent to the fraction of T cells expressing IFNγ. Expression of IL-4 of the activated T cells, on the other hand, was either minimal or undetectable.
A representative course is depicted in Figure 2. A gradual decrease in the fraction of cytokine-producing T cells was seen during induction chemotherapy, interrupted by temporary spikes indicating an increase in Ag-specific T cell reactivity induced by FEC. In general, the fraction of Ag-reactive, cytokine-producing T cells was close to zero at the time of leukapheresis followed by a gradual increase after HD. The recovery time and level was highly variable, however, in the majority of patients the recovery of Ag-specific, cytokine-producing CD4+ T cells occurred prior to CD8+ T cell recovery and to a higher level than previous.
TNF α response to CMV Ag:
The highest percentages of cytokine-responding T cells were seen after stimulation with CMV Ag, and CMV-specific CD4 and CD8 cytokine-responding T cells were demonstrated in all patients prior to chemotherapy, as well as 4–7 months and 12 months after HD (Table 1). The mean percentage of CMV-responding CD8 T cells was 0.89 before chemotherapy and 0.67% 12 months after HD (Table 2). An even higher mean level of responding CD4 T cells was seen; 2.89% before chemotherapy and increasing to 3.53% 12 months after HD.
TNFα response to VZV Ag:
Prior to chemotherapy, T cells with cytokine response to VZV Ag were measurable in 4/5 patients. When evaluated 4–7 months after HD only 2/5 patients showed VZV-specific T cell response, however, after 12 months all patients possessed cytokine-responding T cells specific for VZV (Table 1). CD8+ T cell response (0.54%) was only detected in one patient prior to chemotherapy, whereas, 4/5 patients possessed VZV-specific CD8+ T cells 12 months after HD with a mean percentage of 0.11% (Table 2). CD4+ response was almost equivalent before and 12 months after HD.
TNFα reponse to tetanus Ag:
Prior to chemotherapy, T cells with cytokine response to tetanus Ag were measurable in 3/5 patients. When evaluated 4–7 months and 12 months after HD the remaining two patients had gained reactivity, thus 5/5 patients possessed cytokine-responding T cells specific for tetanus (Table 1). However, CD4 response was prevalent as only two patients had CD8 response. An increase in the mean percentage of tetanus-responding CD8 T cells as well as CD4 T cells succeeded the HD treatment (Table 2).
In none of the patients did the treatment result in loss of overall T cell reactivity for any of the Ags. In some patients, however, a loss of Ag-specific CD8 T cell reactivity (n = 2) or the emergence of Ag-specific CD4 and CD8 T cell reactivity (n = 2) succeeded the treatment.
CD2-induced T cell cytokine response during HD
The cytokine response after polyclonal activation of the T cells was assessed by CD2 stimulation. In parallel with the Ag-specific response, the polyclonal cytokine response gradually reduced during FEC, although interrupted by temporary spikes. However, the lowest level of reactive T cells was measured 2–4 weeks after PBSCT and reduction of the polyclonal cytokine response in association with leukapheresis was only observed in one patient. An increase in the mean percentage of CD2-activated CD8+ T cells from 3.8 to 23.6 succeeded treatment, while the fraction of CD2-activated CD4+ T cells were close to constant: 10.8% before and 12.6% after.
Monocyte cytokine response during HD
For evaluation of monocyte functionality during HD treatment, Ag-induced activation of monocytes as measured by TNFα expression was analysed by stimulation with VZV-Ag (Figure 3). Monocytes were identified among other cell populations by forward side scatter signals and CD33 expression. Prior to chemotherapy the percentage of TNF α-responding monocytes of the five patients was highly individual with a mean of 37.9 % (range, 6.1–80.7). In general, the fraction of responding monocytes was markedly reduced at the time of leukapheresis and 6 months after transplantation: 6.3% (range, 1.0–13.2) and 8.6% (range, 1.9–21.4), respectively. Finally, 12 months after transplantation the mean percentage of TNFα-responding monocytes was 17.4 (range, 2.9–27.6). One patient with a low starting value (6.1%) had gained some reactivity 12 months after end of treatment (18.2%), whereas in one patient, only marginal monocyte response was found (2.9%) even after 12 months. In this patient a low TNFα response of the monocytes after activation with LPS was also observed.
T cell proliferation in response to specific Ags
Four patients were available for evaluation of T cell proliferation in response to CMV Ag, VZV Ag and tetanus toxoid as assessed by PKH26 labelling of the cells prior to Ag stimulation. To compute the frequency of viable T cells which had proliferated during the 7 days of activation, viable T cell subsets were positively identified among other cell populations by light scatter signals and CD3, CD4/8 expression.
Proliferation in response to CMV Ag:
CMV-specific proliferation of CD4 and CD8 T cells was demonstrated in all patients prior to chemotherapy, as well as 4–7 months and 12 months after HD (Table 1). Only one patient lacked CD8 proliferative response to CMV prior to chemotherapy. The highest percentages of proliferating T cells were seen after stimulation with the CMV Ag. For both CD4+ and CD8+ T cells, a marked increase in the CMV-specific proliferation was found after transplantation (Table 3). A proliferative response to CMV was coincident with a positive cytokine response.
Proliferation in response to VZV Ag:
Only one patient possessed a CD8+ T cell proliferative response to VZV Ag prior to treatment while 3/4 patients had a CD4 response (Table 1). After transplantation VZV-specific T cell proliferation was induced in one patient (see below). The mean level of proliferative response before and 12 months after treatment was unchanged for the CD8+ T cell subset, whereas the CD4+ response was enhanced (Table 3). A proliferative response to VZV was coincident with a positive cytokine response. However, cytokine responses were observed without measurable proliferation.
Proliferation in reponse to tetanus toxoid:
T cells with proliferative response to tetanus Ag were measurable in 3/4 patients prior to chemotherapy and 4–7 months after PBSCT but only in two patients after 12 months (Table 1). Thus, tetanus-specific proliferation of T cells was totally lost in one patient. CD8 proliferation was lost and CD4 proliferation became marginal in one patient, whereas maginal CD8 proliferation emerged in a previously negative patient. A decline in CD4+ as well as CD8+ T cell proliferation was seen 4–7 months after transplantation. However, after 12 months the level of CD8 proliferation was similar to pre-treatment (Table 3). A proliferative response to tetanus was not necessarily coincident with a positive cytokine response and vice versa.
Treatment-induced clinical herpes zoster eruption
Due to the severe immune suppression induced by the HD patients are at risk of reactivation of varicella zoster virus. About 25% of breast cancer patients treated with HD suffer from localised herpes zoster eruptions during treatment or within the first year after treatment. Figure 4 illustrates the VZV reactivity of CD4+ and CD8+ T cells from a patient during HD. This patient had cutaneous herpes zoster approximately 5 months after transplantation. Accordingly, a marked increase in VZV reactivity was demonstrated 7 and 10 months after transplantation.
This study aims to characterise the persistence of natural and vaccine-induced memory T cells through HD and autologous stem cell transplantation in breast cancer patients. The results are preliminary and data should be interpreted with the limitations of the low number of observations, which precluded statistical analyses. Overall, however, the analyses indicate that HD induces considerable quantitative alterations in the Ag-specific T lymphocyte reactivity, the level and duration of immune suppression varying between patients. The lowest T cell reactivity was measured in the leukapheresis samples after G-CSF mobilisation of stem cells. In the cytokine assay employed, processing and presentation of Ag to the T cells is necessary for induction of the Ag-specific response. Blood monocytes are assumed to perform this Ag presentation and could therefore be critical for T cell activation.17,18 In accordance with this, monocyte functionality was found to be reduced at the time of leukapheresis, whilst increasing T cell activation was measured in several patients 4–7 months after transplantation when monocyte activation was still low. The reason for this is not evident and has not been clarified by this study. However, several studies have addressed the correlation between monocytes and T cells in mobilised blood; they found that G-CSF mobilisation increases the blood content of monocytes suppressing polyclonal T cell responsiveness, presumably due to interleukin-10-mediated interference with CD28 signal transduction in the T cells.19,20,21
Several groups have successfully used leukapheresis products to generate dendritic cells in vitro.22,23,24,25 In contrast, in vitro expansion of Ag-specific T cells from leukapheresis products has been found to be extremely difficult (G Gaudernack, personal communication). These observations could be of significance for the future use of harvested mobilised stem cells for immune modulating therapy and indicate that further investigations on this subject are necessary.
In defiance of the intensive chemotherapy, in general, a regain of the Ag-specific reactivity was observed after treatment. However, recovery time and level were Ag-dependent and varied between patients. Quantitative T cell reconstitution is known to occur first in the CD8+ T cell population and with delay in the CD4+ T cell population causing an inverted CD4/CD8 ratio for months.2,4,7 Accordingly, we found a reduced CD4/CD8 ratio even 12 months after transplantation. In contrast to these data, no delay of CD4+ T cell recovery of Ag-specific, cytokine production compared to CD8+ T cells was demonstrated. This could be explained by the fact that mainly CD45RA+, naive T cells are reduced after transplantation, while response in vitro to recall Ags is performed by the CD45RO+ memory T cells.7,26,27
CMV is a member of the herpes virus family, which persists in cells of monocyte progenitor lineage after primary infection. Reinfection with CMV may occur during the period of immunosuppression connected with HD and stem cell transplantation, particularly in sensitised patients.28 CD4+ and CD8+ T cells reactive to CMV were measurable by ICC in all patients prior to treatment initiation and were found to be much more frequent and with a higher proliferative response than VZV and tetanus-reactive T cells. In healthy adults a similarly high level has been explained by a requirement of a more active immune surveillance to restrict replication of persistent CMV in cells of the monocyte/macrophage lineage.29 After transplantation CMV-specific T cell recovered within months to the same or an even higher level without any of the patients having clinical symptoms or signs compatible with CMV disease. A similar association has been observed after bone marrow transplantation.30
Monocytes and macrophages are highly effective presenters of MHC class I as well as MHC class II-associated Ags. Consequently, even limited replication of CMV in these cells due to HD-induced immune suppression is very likely to cause an efficient re-exposure of CD8/CD4 restricted immunogenic CMV epitopes, which might explain the observed increase in immune reactivity.
VZV causes varicella in childhood whereupon it remains dormant in the dorsal root ganglia. Prior to treatment, VZV-specific T cell response was mainly detected in the CD4+ T cell subpopulation as only one patient had a CD8+ T cell response. A similarly low frequency of VZV-specific CD8 reactivity has been observed in healthy adults,29 indicating that VZV-induced CD4+ memory T cell response is prevalent and more persistent than the CD8 memory response.
Reactivation of VZV may occur during the period of immunosuppression connected with HD and stem cell transplantation. Thus, frequencies of herpes zoster ranging from 25 to 50% of patients undergoing autologous bone marrow transplantation have been described, with the majority of cases occurring within 6 months after transplantation.31,32,33 Clinical signs of herpes zoster were only reported for one patient, followed by a marked increase in VZV-specific T cell reactivity. However, all patients regained VZV-specific T cell reactivity within 1 year after treatment with a marked increase in two patients. Subclinical reactivation of the latent virus is most likely, as it has been demonstrated that recovery of VZV-specific T cell function after bone marrow transplantation is correlated to re-exposure to the VZV Ag.34 Compared to the regain of CMV reactivity, the recovery of VZV-specific T cell immunity was delayed and at a lower level. A later reactivation of the virus and a lower accessibility of immunogenic viral proteins could explain this.
In contrast to the viral Ags, tetanus-specific immunity is normally attained by vaccination. All patients were immunised with tetanus within the last 10 years and none of the patients received tetanus vaccination during treatment and follow-up. Nevertheless, several patients re-established tetanus immunity after transplantation. Furthermore, some patients gained measurable tetanus-specific reactivity. These findings are important as they verify that vaccine-induced immunity is preserved through HD and autologous stem cell transplantation. Furthermore, even without Ag re-stimulation, expansion of mature specific T cells is taking place, particularly in the CD4+ subset. In accordance with these findings, clonal T cell expansion and regeneration of T cell receptor repertoire have been demonstrated after HD and stem cell transplantation by use of T cell receptor analyses.35
In conclusion, this preliminary study demonstrates that specific immunity induced prior to HD can resist the treatment and reappear after transplantation. A larger series of patients is, however, necessary to draw definitive conclusions.
New anticancer treatment modalities such as cancer vaccination therapy are making alternating therapy a potential research field in the future. The present data indicate that, introduction of a preventive cancer vaccination in combination with intensive chemotherapy could be a realistic treatment option. However, choosing the right combination strategy might be of crucial importance.
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This research was supported by grants from Michaelsen Fonden, Hœrslev-fonden and Dansk Kraeftforsknings Fond. We are also grateful for the financial support from Moltums Fond, Petrus Andersen Fond, Direktr JA Srensen og hustru EI Srensens Mindefond, P & A Simonsens Fond, G & A Haensch's Fond and Direktr J Madsen & hustru O Madsens Fond.
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Svane, I., Nikolajsen, K., Hansen, S. et al. Impact of high-dose chemotherapy on antigen-specific T cell immunity in breast cancer patients. Application of new flow cytometric method. Bone Marrow Transplant 29, 659–666 (2002). https://doi.org/10.1038/sj.bmt.1703521
- stem cell transplantation
- breast cancer
- cellular immune reconstitution
- antigen-specific lymphocytes
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