Polyclonal T-cell activation protocol stimulates preferential expansion of EBV-specific T-cell clones in vitro

Abstract

Purpose

Allogeneic bone marrow transplantation (AlloBMT) can be curative for patients with leukemia. The most important anti-leukemic effect may be mediated by the T-cells contained within the graft; however, the allogeneic T-cells may also give rise to graft-vs-host disease (GVHD). One way to control GVHD might be to transduce the donor T-cells with a drug-inducible suicide gene. If a retrovirus vector is to be used for this transduction, activation of the T-cells is required for integration of the transgene to occur. The activation protocol should ensure expansion of a broad repertoire of donor T-cells. Notably, T-cells specific for herpes virus family antigens are important for adoptive immunoprotection.

Methods

To define optimal activation conditions for retrovirus-mediated suicide gene transduction of donor T-cells, we examined the repertoire of CD8+ T-cells in general, and Epstein-Barr virus (EBV) specific T-cells in particular, following two different activation and expansion procedures.

Results

We found that repeated CD3/CD28 stimulation resulted in a high level of activation-induced T-cell death, affecting in vivo expanded clones, some of which were specific for EBV, in particular. In contrast, initial CD3/CD28 activation followed by proliferation in interleukin-2 lead to expansion of EBV-specific clones over and above the expansion observed for CD8+ T-cells in general.

Conclusion

These results should impact on protocols for ex vivo activation of T-cells prior to suicide gene transduction.

Introduction

The only treatment modality providing a possible cure for patients with chronic myeloid leukemia (CML) involves allogeneic bone marrow transplantation (AlloBMT), preferably from a human leukocyte antigen (HLA) identical family donor, combined with high-dose chemotherapy with or without radiotherapy. An increasing body of evidence suggests that the cure, rather than resulting from the chemo/radiotherapy, is mediated by the anti-leukemic effect of donor T-lymphocytes included in the AlloBMT graft [5]. The specificity of this effect, termed graft-vs-leukemia (GVL) effect, is not fully understood [16]. For this reason, polyclonal allogeneic T-cells, rather than antigen-specific T-cell lines or clones, are frequently used to obtain a GVL effect; however, within a polyclonal T-cell population there is normally a relatively high proportion of allospecific cells [17]. In the setting of AlloBMT, allospecific T-cells have been shown to be responsible for graft-vs-host disease (GVHD). While GVHD of low severity is normally well tolerated, and has been shown to correlate with GVL effect, severe GVHD is potentially life threatening and requires treatment urgently.

The best treatment for a T-cell mediated disorder should be to remove the T-cells. Recently, introduction of the herpes simplex virus thymidine kinase (HSV TK) “suicide” gene into donor T-cells has been suggested as a method to achieve this [3]. Following infusion of transduced allogeneic T-cells, HSV TK will normally be inactive; however, if exposed to the non-toxic prodrug ganciclovir (GCV), HSV TK will phosphorylate GCV. The GCV monophosphate is then further phosphorylated by endogenous kinases to GCV triphosphate, which in turn is incorporated into DNA of dividing cells causing chain termination, single-strand breaks and eventually death of the transduced T-cells. The best vectors described so far for transduction of T-cells are all based on murine retroviruses; however, if murine retrovirus vectors are to be used, the T-cells need to be activated for efficient integration of the transduced gene to occur [15].

Adoptively transferred donor T-cells, in addition to their GVL effect, should also provide the patient with sufficient-cellular immunity to survive until a competent repertoire of self T-cells is established; thus, the activation protocol used for the transduction of donor T-cells should ensure expansion of memory cells specific for a wide range of antigens, including those derived from viruses that are resident in most humans, as well as naïve T-cells among which the leukemia-specific cells are most likely to be found. The present article shows how two activation protocols, despite being quite similar, have widely different effects on in vivo expanded, Epstein-Barr virus (EBV)-specific CD8+ T-cell clones.

Materials and methods

Cell cultures and tetramer staining

The CD8+ T-cells were obtained from peripheral blood mononuclear cells (PBMC) from healthy blood-bank donors and healthy HLA A*0201+ (EBV) seropositive laboratory personnel by positive selection using immunomagnetic beads (Dynal, Oslo, Norway) and Detachabead (Dynal). Memory cells were obtained by removal of CD45RA+ cells by cell sorting (FACS Vantage, Becton Dickinson Immunocytometry Systems, San Jose, Calif.). Cells were cultured in RPMI 1640, supplemented with 10% fetal calf serum and antibiotics (culture medium, CM). For initial activation of all cell cultures, anti-CD3 monoclonal antibody (MAb; UCHT-1, Diatec, Oslo, Norway) was coated onto the bottom of 48-well plates. All cell cultures were initiated by establishing cells in anti-CD3 coated wells in CM supplemented with anti-CD28 MAb (clone CD28.2, Pharmingen, San Diego, Calif.) at 1 μg/ml and interleukin 2 (IL-2; R&D Systems Europe, Abingdon, UK) at 10 U/ml. At the time of transfer to a larger culture well, on days 3 or 4, each culture was split; one-half was transferred to a well coated with anti-CD3 MAb. These cells were maintained in CM supplemented with anti-CD28 MAb and IL-2, and further transferred to flasks coated with anti-CD3 MAb. Most cultures managed in this way were restimulated by new anti-CD3 every 3–4 days and maintained in medium containing anti-CD28 MAb and IL-2 (termed repeatedly stimulated cells). The other half of the initial culture was transferred to an uncoated well, and the CM was supplemented with IL-2 only (termed IL-2-expanded cells); thus, these cells were activated with anti-CD3/anti-CD28 only at the start of the culture. Counting and viability testing of cells in culture was performed using acridine orange/ethidium bromide staining and fluorescence microscopy. Quantification and sorting of cells specific for the EBV lytic cycle peptide GLCTLVAML among CD8+ T-cells before and after culture was performed using a PE-conjugated tetramer of HLA A*0201 coupled to the antigenic peptide (A*0201/GLC; ProImmune, Oxford, UK).

Spectra-type analysis of CDR3 length

Cells were suspended in PBS without protein, pelleted and frozen at 1–3×10⁶ cells per pellet. Analysis of tetramer+ cells was performed on cells sorted using the FACSVantage. Here, the number of cells pelleted for analysis was considerably less (11–18×10³ from day 0 cells, 28–410×10³ from day 14 cells). Spectra-type analysis of the distribution of complementarity determining region 3 (CDR3) lengths within T-cell receptor (TCR) BV families was performed as described previously [14]. Briefly, cell pellets were thawed and lysed in lysis and binding buffer, mixed with magnetic oligo-dT beads for isolation of mRNA (Dynal) and the eluate incubated at 37°C for 1 h with a reverse transcriptase mix for cDNA synthesis. Following reverse transcription, amplification of TCR genes was performed with BV-specific primers from the T-Cell Receptor BV Typing Amplimer Kit (Clontech Laboratories, Palo Alto, Calif.) and a fluorochrome-labeled BC primer. The PCR products were run on a 4.25% polyacrylamide gel and analyzed using ABI Prism 377 DNA Sequencer and Genotyper software (Perkin Elmer, Foster City, Calif.). CDR3 lengths were quantified by peak height and considered to be expanded if they exceeded the 5% upper confidence limit for the same CDR3 length within that BV family, as given by a reference panel [14].

Results

In vivo expanded CD8+ T-cell clones are particularly susceptible to activation-induced cell death in vitro.

We have recently examined the effect of the two different activation protocols on T-cell expansion, activation-induced cell death, and changes in T-cell phenotype (I.A. Hedfors and J.E. Brinchmann, Scand J Immunol, in press). In that study, following the initial anti-CD3/anti-CD28 activation signal, a considerable proportion of CD8+ T-cells were found to be dead on day 2 (naïve CD8 median 22% dead cells, memory CD8 median 38% dead cells). The percentage of dead cells gradually decreased in the IL-2-expanded cultures, to reach <10% from day 8. From day 14, subset expansion ceased in the cultures maintained in IL-2 only. For the next 2 weeks, cell numbers were maintained (CD8+ T-cells) or slightly reduced (CD4+ T-cells). If the IL-2-expanded cells were restimulated on day 14, cell numbers were increased only slightly (CD4+ T-cells) or maintained (CD8+ T-cells). In contrast, in the cultures which were repeatedly stimulated during the first 2 weeks, the percentage of dead cells rebounced to reach 45% (median) on day 8. As a result, the numbers of viable cells were always greater in the IL-2-expanded cultures (I.A. Hedfors and J.E. Brinchmann, Scand J Immunol, in press). Cell counts from the present study confirmed these observations (Table 1).

Table 1.

Cell counts in cultures of repeatedly stimulated and IL-2-expanded cells

Donor	Cell subset	Day 0	Day 14
			Restimulated	IL-2 expanded
1	Memory CD8	0.5	22.8 (45.6)	153.4 (306.8)
2	Memory CD8	0.5	5.2 (10.4)	59.6 (119.2)
3	Total CD8	1.0	13.8 (13.8)	107 (107)
4	Total CD8	0.5	17.6 (35.2)	136 (272)
5	Total CD8	1.0	35.6 (35.6)	281.6 (281.6)

Memory or total CD8+ T-cells were cultured for 14 days according to repeatedly stimulated or IL-2-expanded protocols. Values are number of cells (×10⁶). Values in parentheses are number of cells per input cell after 2 weeks in culture. The cells from donors 1 and 2 have been phenotypically characterized as part of a previous study (I.A. Hedfors and J.E. Brinchmann, Scand J Immunol, in press)

Using spectra-type analysis of the distribution of lengths within 22 TCR BV families, we wanted to determine the possible differential effect of the two activation protocols on the TCR repertoire of the expanded cells. Previous studies have shown that spectra-type analysis of CD8+ T-cells from healthy adults almost invariably reveal some TCR BV families with expanded peaks, representing TCR of clones expanded in vivo. Such clonally expanded cells may persist through many years of follow-up [13]. Many of the persistently expanded CD8+ T-cell clones have been shown to be specific for resident viruses of the herpes virus family [19]. It was no surprise to us, therefore, to find expansions such as that shown in Fig. 1A in many of the BV families in uncultured CD8+ T-cells from different donors (Fig. 1E).

Fig. 1A–E.

The CD8+ T-cell clones expanded in vivo are lost following repeated in vitro stimulation. Spectra-type profile of A TCR BV 14 from donor 2 unstimulated memory CD8+ T-cells, B restimulated memory CD8+ T-cells, and C IL-2-expanded cells. D Spectra-type profile of BV14 of phytohemagglutinin blasts from a different donor run at the same time and included as a control. E Results for donors 1–4 are summarized. The numbers for day 0 designate the total number of BV families containing expanded peaks representing in vivo expanded clones. Day 14 values are the number of expanded peaks of the same complementarity-determining region-3 length within the same BV families after 14 days in restimulated or IL-2-expanded cell culture

Spectra-type analysis at the end of the culture period revealed marked differences between the two activation protocols. For the restimulated cells, expanded peaks had disappeared or were dramatically reduced in all cultures (Fig. 1B, E). In contrast, most of the expansions were maintained in the IL-2-expanded cells (Fig. 1C,E). Combined with our observations showing excessive cell death in the restimulated cell cultures compared with the IL-2-expanded cells, this shows that the clones represented by the expanded spectra-type peaks were more susceptible to activation-induced cell death than other CD8+ T-cells.

In vivo expanded EBV-specific CD8+ T-cell clones may be enlarged or diminished in culture depending on the T-cell activation procedure.

Spectra-type analysis shows the presence of expanded clones but does not determine their specificity. To determine if EBV-specific clones were among those particularly susceptible to activation-induced cell death, CD8+ T-cells were stained with A*0201/GLC before and after culture. Figure 2 shows that, while A*0201/GLC specific T-cells were reduced or absent in the restimulated cultures of cells from the three donors studied, they were actually increased in the IL-2-expanded cultures. To determine if the same clones were present before and after expansion in the IL-2-only culture, tetramer staining cells were sorted and subjected to spectra-type analysis. Table 2 shows that, of the 32 A*0201/GLC specific clones from three donors identified on day 0, 19 were still present in the A*0201/GLC specific population of IL-2-expanded cells. Sorting of tetramer staining cells could not be performed on restimulated cells, as the numbers of tetramer+ cells in these cultures were too low.

Fig. 2A–D.

Epstein-Barr-virus-specific cells are lost following repeated in vitro stimulation. Histogram representation of A A*0201/GLC tetramer staining of total CD8+ T-cells from donor 5 on day 0, B day-14 restimulated cells, and C IL-2-expanded cells. D The percentage of tetramer+ cells for donors 3–5 are summarized

Table 2.

Recovery of tetramer+ CD8+ T-cell clones after 14 days in IL-2-only cell cultures. HLA human leukocyte antigen

Donor	No. of clones in tetramer+ cells, day 0	Recovery in IL-2-expanded tetramer+ cells, day 14	Clones in tetramer+ cells, day 14, not observed day 0
3	11	6	5
4	8	5	2
5	13	8	4
Total	32	19	11

HLA A*0201/GLC tetramer+ cells were sorted before and after 14 days of IL-2-expanded cell cultures. Spectra-type analysis was subsequently performed on the sorted cells. The numbers for day 0 represent all significantly expanded peaks within all 22 blood volume (BV) families among the sorted, tetramer+ cells on that day. The numbers for recovery in IL-2-expanded cells are the number of expanded peaks on day 14 with counterparts within the same length and BV family on day 0. The column to the right is the number of expanded clones in the tetramer+ cells on day 14 without an expanded counterpart within tetramer+ cells day 0. In both cell populations, there were some BV families with multiple, normally distributed peaks (data not shown)

Discussion

Treatment with allogeneic, suicide gene transduced T-cells frequently involves patients with iatrogenic T-cell immunodeficiency. These patients are dependent on the donor T-cells for immunoprotection until a new repertoire of “self” T-cells develops based on the allogeneic stem cells in the transplant. For this reason the adoptively transferred T-cells should include cells specific for environmental antigens in addition to cells which may exert a GVL effect. For patients made immunodeficient in association with AlloBMT, diseases caused by members of the herpes virus family are common and frequently serious [20]; thus, if suicide gene transduced donor T-cells are transferred in order to provide immunoprotection for AlloBMT patients, one should make sure that the T-cell activation protocol used for the transduction does not lead to deletion of clones specific for herpes viruses. We show here that this may easily occur if the wrong T-cell activation protocol is chosen.

Following activation, T-cells cluster into two major groups dependent on their response to activation: those who proliferate, and those who die by activation-induced cell death (AICD). Major determinants of post-activation T-cell fate seem to be costimulation through CD28, binding of IL-2 to its receptor on T-cells, and the state of activation and subset affiliation of the T-cell [1, 4, 7, 9, 10, 21]. However, the exact roles played by these factors have not yet been determined; thus, CD28 co-stimulation normally reduces AICD, but if given alone, CD28 signaling may induce apoptosis [4, 10]. While IL-2 has been associated with rescue from apoptosis [21], IL-2 has also been shown to induce apoptosis in activated T-cells [11, 12]. Generally, primed or memory T-cells have been found to be more resistant to AICD than naïve cells [7, 9]. Specifically, EBV specific memory CD8+ T-cells that have re-expressed CD45RA have been found to be resistant to apoptosis, and to retain replicative potential [6]. Against this background, we decided to examine the effect of two different T-cell activation programs (repeated CD3 restimulation in the presence of anti-CD28 and IL-2 vs initial CD3/CD28 stimulation followed by expansion in IL-2 only) on two populations of T-cells (greatly expanded CD8+ T-cell clones vs the whole CD8+ T-cell population).

Using CDR3 length analysis to characterize the TCR repertoire of T-cell subsets, greatly expanded CD8+ T-cell clones have been shown to persist over many years [8, 13]. Recently, the specificity of many of these persistently expanded clones has been shown to be for resident viruses of the herpes virus family [19]. We have shown that such clones frequently express CD45RA [13] and as such might be expected to be resistant to AICD [6]. As these clones may represent an important part of the memory CD8+ T-cell repertoire, we chose to examine whether the two activation protocols would expand such clones in parallel with the whole CD8+ T-cell population. The results suggest that such clones proliferate to a greater extent than the general CD8+ T-cell population on one initial activation signal mediated by the CD3 and CD28 cell-surface molecules followed by expansion in an IL-2 containing medium. In contrast, repeated anti-CD3/anti-CD28 stimulation seemed to kill cells within the in vivo expanded clones to a greater extent than the CD8+ T-cell population in general. As these cells were maintained in IL-2 containing medium, the presence of IL-2 was clearly insufficient to suppress the AICD observed in these cultures. One explanation for these observations may be that most of these persistently expanded clones are indeed specific for resident (viral) antigens. If so, these cells may be repeatedly and specifically stimulated via their TCR in vivo. In contrast, other memory cells for which no antigenic stimulation remains in the body, or naïve cells that have not yet encountered antigen, probably exist in a completely resting state in vivo. In this scenario, the state of activation of the T-cell in vivo may be a determinant of the differential response to the two activation procedures in vitro. Our results are, in fact, completely in accordance with observations made by Dunne et al. [6] using an assay measuring survival in unstimulated culture. Here, EBV-specific CD8+ T-cells showed much lower survival than the total CD8+ T-cell population; however, within the excessively dying EBV-specific T-cells, those expressing CD45RA were more resistant to cell death.

To confirm that T-cell clones specific for resident viruses were among those depleted by repeated stimulation, we tested for the concentration of T-cells specific for the immunodominant EBV peptide GLCTLVAML presented by HLA A*0201 by tetramer staining before and after expansion according to the two different activation protocols. We found that tetramer+ cells were greatly reduced or absent in repeatedly stimulated cells; however, detection of tetramer+ cells is dependent on the expression of the TCR/CD3 complex on the cell surface, and repeated stimulation could lead to persistent down-regulation of cell surface TCR/CD3. We have examined this, and found that CD3/TCR was indeed practically removed from the cell surface immediately following the activation procedure employed in these experiments; however, TCR/CD3 returned to the cell surface, and even in repeatedly stimulated cells the concentration of the TCR/CD3 complex was similar to that observed in day-0 unstimulated cells from day 8 onwards (I.A. Hedfors and J.E. Brinchmann, Scand J Immunol, in press); thus, the absence of tetramer+ cells in the repeatedly stimulated cells was not due to the failure of these cells to express TCR.

It was interesting to observe that, even when small numbers of cells were sorted, the same clones could be detected among tetramer+ cells before and after IL-2 only T-cell expansion. This suggests that, even though a surprisingly large number of peaks were actually detected within the tetramer sorted cells, a limited number of clones dominate the A*0201/GLC specific T-cell response.

The experiments presented here were performed using a murine retroviral vector. Recently, gene transduction experiments have been performed in several laboratories using lentiviral vectors. Lentiviruses are complex retroviruses with accessory genes which have important regulatory functions during viral life cycle and viral pathogenesis. Lentivirus vectors may infect nondividing, terminally differentiated mammalian cells, including lymphocytes [15]. Thus, if lentiviral vectors could be used for suicide gene transduction, activation and expansion of the T-cells might not be necessary; however, most of the lentiviral vectors used for gene therapy approaches are based on the human immunodeficiency virus type 1 (HIV-1). Clearly, a large number of safety and efficacy issues have to be addressed before HIV-1-based vectors can be used in clinical settings. Murine retroviral vectors, on the other hand, have been used for transduction of mature lymphocytes for more than a decade, probably involving manipulation of more than 10¹² cells, with no evidence of insertional mutagenesis or other transgene mediated threats to patient health [2]. As murine retroviral vectors are dependent on T-cell activation for integration of the transgene to occur, detailed information about the effect of activation and expansion on T-cell phenotype (I.A. Hedfors and J.E. Brinchmann, Scand J Immunol, in press) and T-cell receptor repertoire (this paper) will continue to be important for protocols involving suicide gene transduction of T-lymphocytes.

Our results show that if T-cells expanded in vitro are to maintain their content of clones specific for resident antigens, the activation procedure must be carefully chosen. Recently, it was shown that EBV-specific T-cells were lost following suicide gene modification and certain activation procedures [18]. Our data are consistent with those results; however, we extend these observations by showing that the repertoire of EBV-specific cells may be maintained, and their number greatly increased following in vitro culture using a different activation and expansion procedure. Our results show that T-cells specific for the EBV/GLC antigen are more prone to activation induced cell death but also easier to expand given the right cell culture conditions, compared with CD8+ T-cells in general. Further studies are required to identify the molecular mechanisms responsible for this differential effect. These results should impact on protocols for ex vivo activation and transduction of T-cells to be used for AlloBMT.