Reactivation of CMV can cause severe disease after allogeneic hemopoietic stem cell transplantation. Adoptive T cell therapy was successfully used for patients who had received transplants from CMV-positive donors. However, patients with transplants from CMV-negative donors are at highest risk, and an adoptive therapy is missing because CMV-specific T cells are not available from such donors. To address this problem, we used retroviral transfer of CMV-specific TCR genes. We generated CMV-specific T cell clones of several HLA restrictions recognizing the endogenously processed Ag pp65. The genes of four TCRs were cloned and transferred to primary T cells from CMV-negative donors. These CMV-TCR-transgenic T cells displayed a broad spectrum of important effector functions (secretion of IFN-γ and IL-2, cytotoxicity, proliferation) in response to endogenously processed pp65 and could be enriched and expanded by strictly Ag-specific stimulation. Expansion of engineered T cells was accompanied by an increase in specific effector functions, indicating that the transferred specificity is stable and fully functional. Hence, we expect these CMV-TCR-transgenic T cells to be effective in controlling acute CMV disease and establishing an antiviral memory.

Reactivation of CMV is an important cause of disease and death after allogeneic hemopoietic stem cell transplantation (allo-HSCT).3 Antiviral medication is accompanied by severe side effects and is often ineffective due to the development of resistant virus strains. Furthermore, antiviral drugs often only delay CMV-associated disease (1). The development of CMV-related disease is strongly correlated with absent or delayed reconstitution of CMV-specific T cells (1, 2, 3, 4, 5). Thus, current therapeutic concepts focus on the reconstitution of the CMV-specific cellular immune response. Adoptive transfer of CMV-specific cytotoxic T cell clones (6) and T cell lines (7, 8, 9) has been very effective in treating and preventing CMV-related disease (10). In these studies, the transferred T cells had been isolated from CMV-positive HSCT donors. With CMV-seronegative donors, isolation and enrichment of CMV-specific T cells are difficult, if not impossible, because naive CMV-specific T cells are too rare to be detected or directly accessed (11, 12). As a consequence, CMV-seropositive recipients of stem cell grafts from CMV-negative donors are at particular risk to develop severe CMV-related disease because CMV-specific T cell reconstitution is highly deficient (5, 13). To prepare T cells with specificities absent from the donor’s repertoire, TCR gene transfer is a promising option (14, 15, 16, 17). Recently, clinical application of TCR-transgenic T cells in malignant melanoma patients resulted in tumor remissions and was well tolerated (18). In contrast, TCR-transgenic T cells for antiviral therapy have not yet been used in the clinic. Adoptive transfer of CMV-TCR-transgenic T cells should be especially suitable for clinical application, because CMV-specific TCRs, which were selected in vivo for antiviral function and maintenance, are readily accessible from the T cell memory of CMV-seropositive donors. Healthy individuals with persistent CMV infection carry large proportions of CMV-specific CD8+ and CD4+ memory T cells (19, 20). Many epitopes derived from the immunodominant CMV Ags pp65, IE-1, and others, which bind to various HLA molecules, have been characterized (19, 21, 22). Several CMV-specific TCR chain sequences, some of which are shared by various donors (public TCRs), are already known (23, 24, 25, 26, 27, 28, 29, 30, 31).

Previously, we showed that Ag-specific T cell lines and clones can be efficiently generated, expanded, and tested using B cell-based Ag presentation systems (22, 32, 33). In this study, we used B cells to establish CMV-specific CD8+ T cell clones of various HLA restrictions which recognize endogenously processed CMV Ag. Their TCRs were cloned and transferred to primary T cells from CMV-negative donors, and the resulting TCR-transgenic T cells were analyzed for various effector functions. We tested their capacity to selectively expand in response to endogenously processed Ag, and investigated their reactivity spectrum after repeated Ag contact to ensure the stability of their Ag-specific function. Taken together, we intend to show that CMV-TCR-transgenic T cells are promising tools for adoptive T cell therapy.

The standard cell culture medium was RPMI 1640 (Invitrogen) supplemented with 10% FCS (PAA Laboratories), 100 U/ml penicillin/100 μg/ml streptomycin (Invitrogen), and 100 nM sodium selenite (ICN Biochemicals). 293T cells were cultivated in DMEM (Invitrogen) with the same supplements.

PBMCs from healthy donors (Table I) were collected with donors’ informed consent following the requirements of the local ethical board and the principles expressed in the Helsinki Declaration. PBMCs were obtained by centrifugation on Ficoll/Hypaque (Biochrom). HLA typing was performed by PCR-based methods (IMGM). Stably pp65-expressing mini-lymphoblastoid cell lines (mLCL) and control (pp65-negative) mLCLs were generated by infection of PBMCs with B cell-transforming mini-EBV vectors (32). CD40-activated B-blast (BBL) cultures were established as described (33) and maintained by weekly replating PBMCs on irradiated (140 Gy) murine fibroblasts, stably expressing the human CD40L, in the presence of 2 ng/ml rIL-4 (R&D Systems). The TCRαβ-deficient T cell lines Jurkat76 (J76) (34) and J76 stably expressing the human CD8α chain (J76CD8; kindly provided by W. Uckert, Max-Delbrück-Center, Berlin, Germany) were used as recipient cells for TCR transfer studies. 293T cells were used for packaging of the retroviral vectors.

Table I.

HLA types and virus carrier state of donors

DonorHLA-AHLA-BHLA-CCMV SerostatusEBV Serostatus
*0201, *2502 *1503, *51 *1203, *1402 Nega Pos 
*0201, – *44, *51 *02, *05 Neg Pos 
*0101, *2601 *3501, *5701 *0401, *0602 Neg Neg 
*03, *24 *3501, *4002 *02, *04 Neg Pos 
*01, *0201 *1501, *37 *03, *06 Neg Pos 
*0201, – *3503, *5701 *04, *06 Pos Pos 
*01, *11 *08, *1501 *03, *07 Pos Pos 
*02, *03 *35, *4001 *03, *15 Pos Pos 
*02, *26 *07, *38 *07, *12 N.D. N.D. 
10 *02, *34 *14, *44 *04, *08 N.D. N.D. 
DonorHLA-AHLA-BHLA-CCMV SerostatusEBV Serostatus
*0201, *2502 *1503, *51 *1203, *1402 Nega Pos 
*0201, – *44, *51 *02, *05 Neg Pos 
*0101, *2601 *3501, *5701 *0401, *0602 Neg Neg 
*03, *24 *3501, *4002 *02, *04 Neg Pos 
*01, *0201 *1501, *37 *03, *06 Neg Pos 
*0201, – *3503, *5701 *04, *06 Pos Pos 
*01, *11 *08, *1501 *03, *07 Pos Pos 
*02, *03 *35, *4001 *03, *15 Pos Pos 
*02, *26 *07, *38 *07, *12 N.D. N.D. 
10 *02, *34 *14, *44 *04, *08 N.D. N.D. 

Neg, Negative; Pos, positive; N.D., not determined.

We generated T cells against the following HLA class I-restricted epitopes from pp65: NLVPMVATV, aa 495–503, HLA-A*0201-restricted (abbreviated NLV); IPSINVHHY, aa 123–131, HLA-B*3501-restricted (IPS); YSEHPTFTSQY, aa 363–373, HLA-A*0101-restricted (YSE).

YSE-specific T cells from donor 7 (Table I) were reactivated within PBMCs by stimulation with the irradiated (50 Gy) autologous pp65-expressing mLCLs (32). Per well of a 12-well plate, 6 × 106 PBMCs and 1.5 × 105 pp65-expressing mLCLs were cocultivated in 3 ml of medium. On day 10 and then every 7 days, cells were pooled, counted using trypan blue staining, and replated at 3 × 106 cells/3 ml of medium per well, adding freshly irradiated (50 Gy) pp65 mLCL as stimulators at an effector-stimulator ratio of 3:1 and 100 U/ml rIL-2 (Proleukin S; Novartis). At day 50 of culture, T cell clones were generated by limiting dilution.

For single-cell cloning, 0.5 or 3 T cells/well were seeded into 96-well round-bottom plates. 2 × 104/well irradiated (50 Gy) autologous pp65-expressing mLCL, 3 × 105/well of a mixture of irradiated (50 Gy) allogeneic PBMCs from three different donors, and 1000 U/ml IL-2 were added. Restimulations of T cell clones were performed every 2 wk, and outgrowing clones were expanded in 96-well round-bottom plates under the same conditions.

For reactivation of NLV- or IPS-specific T cells from donor 6 or 8 (Table I), PBMCs were pulsed with 5 μg/ml of the relevant CMV peptide (JPT), and plated at 8 × 106 cells/4 ml of medium per well in a 12-well plate. After 24 h at 37°C and 5% CO2, the cells were washed three times with PBS, and replated in the 12-well plate, and supplemented with 10 U/ml IL-2. On day 8, the rIL-2 concentration was raised to 50 U/ml. The T cell culture was expanded according to cell proliferation. At day 14, T cell clones were generated as described above.

TCR-PCR analysis of CMV-specific T cell clones was performed as previously described (35). Total RNA from T cell clones was extracted using the RNeasy Mini Kit (Qiagen). cDNA was synthesized using murine leukemia virus (MLV)-reverse transcriptase and oligodeoxythymidylate primer (MBI-Fermentas). Subfamily-specific PCR of the TCR α-chain was performed with 34 different Vα primers (36). The TCR β-chain was analyzed by PCR with degenerated Vβ primers as previously described (37). Specifically amplified gene products were analyzed by DNA sequencing (Sequiserve). TCR nomenclature according to IMGT was used (38).

TCR α- and β-chain genes were separately inserted into the myeloproliferative sarcoma virus-derived vector MP71Gpre (39) as previously described (40). Briefly, specific TCR α- and β-chain genes were amplified from cDNA isolated from CMV-specific T cell clones by specific primers containing 5′-NotI and 3′-EcoRI restriction sites, and cloned as single TCR chain genes into the vector MP71Gpre (MP71-TCRα and MP71-TCRβ) replacing the GFP gene. The GFP-encoding MP71Gpre vector was used as control to evaluate infection efficiencies. All TCR cassettes used in this study were verified by DNA sequencing (Sequiserve).

To produce amphotropic MLV-pseudotyped retroviruses, 293T cells were cotransfected by calcium phosphate precipitation with expression plasmids encoding the Moloney MLV gag/pol genes (pcDNA3.1MLVg/p) and the MLV-10A1 env gene (pALF-10A1) together 1) with the respective TCR-encoding retroviral vector plasmids MP71-TCRα and MP71-TCRβ, or 2) with the GFP-encoding plasmid MP71Gpre (transfection and transduction control), or 3) without retroviral vector plasmids (mock control) (41). Forty-eight hours after transfection, the retroviral supernatant was harvested, filtered (0.45 μm pore size), and used directly for infection of PBMCs, J76 cells, and J76CD8 cells. PBMCs (106 in 1 ml), which had been activated with 50 U/ml rIL-2 and 50 ng/ml anti-CD3 Ab (OKT-3, kindly provided by E. Kremmer, Helmholtz Zentrum, Munich, Germany) 2 days earlier, were transduced in 24-well plates precoated with 5 μg/well RetroNectin (Takara) in the presence of 4 μg/ml protamine sulfate (MP Biomedicals) and 100 U/ml rIL-2. After addition of 1 ml of retrovirus-containing supernatant, the plates were spinoculated for 2 h at 800 × g and 32°C. J76 and J76CD8 cells (105 in 1 ml) were transduced as described above but without adding rIL-2. Medium was replaced after 24 h.

Weekly restimulation of transduced PBMCs was started 6 days after TCR transfer. For Ag-specific stimulation, transduced T cells (1.5 × 106/1.5 ml/well) were cocultivated with irradiated (50 Gy) autologous pp65-expressing mLCL at a ratio of 4:1 with 100 U/ml rIL-2 in 24-well plates. For nonspecific stimulation, transduced T cells (1.5 × 106/1.5 ml/well) were cocultivated with 1.5 × 106 per well of a mixture of irradiated (50 Gy) allogeneic PBMCs from three different donors and 1.5 × 105/well irradiated (50 Gy) autologous pp65-negative mLCL with 100 U/ml rIL-2 and 30 ng/ml OKT-3 in 24-well plates.

Multimer staining was performed by incubating the T cells for 10 min at room temperature with PE-labeled HLA/peptide tetramer or unlabeled HLA/peptide pentamer. The cells were counterstained on ice for 15 min with anti-CD4-FITC, anti-CD3-PE-Cy5, anti-CD8-APC Abs (all BD Pharmingen), and, in the case of unlabeled pentamers, with Pro5 Fluorotag R-PE (Proimmune). Directly after staining, the cells were fixed by 1.6% formaldehyde (Carl Roth). As control, T cells were stained as explained above but without adding the respective multimer. PE-labeled NLV/A*0201 tetramer was purchased from Beckman Coulter; unlabeled IPS/B*3501 and YSE/A*0101 pentamers were purchased from Proimmune. Cells were analyzed on a BD Biosciences FACSCalibur flow cytometer. Data analysis was performed using FlowJo 8.8.4 software (Tree Star). For analysis, viable lymphocytes were gated in a forward-sideward scatter dot plot. All shown dot plots span, on both coordinates, a range from 1 to 10,000 arbitrary units of fluorescence intensity in a logarithmic scale.

CMV-specific T cell clones and TCR-transduced PBMCs were analyzed for cytokine secretion by ELISA. Effector cells (104) were cocultivated overnight with target cells (2 × 104) in 200 μl/well of a 96 V-well plate at 37°C and 5% CO2. Then supernatants were harvested, and IFN-γ and IL-2 ELISAs were performed according to the manufacturer’s recommendations (Mabtech).

Cytotoxicity of TCR-transduced PBMCs was analyzed by calcein release assay as described previously (33).

We generated CMV-specific CD8+ T cell clones against the epitopes YSE (HLA-A*0101), NLV (HLA-A*0201), and IPS (HLA-B*3501) derived from pp65. CMV-specific T cells were enriched by Ag-specific stimulation of primary T cells from three different CMV-seropositive donors. NLV- and IPS-specific T cells were obtained by stimulating PBMCs with the epitope peptide, as shown for NLV-specific T cells in Fig. 1,A. YSE-specific T cells were enriched by repeated stimulation with the autologous pp65-expressing mLCL (Fig. 1 B). Subsequently, T cell clones were generated by limiting dilution.

FIGURE 1.

Establishment of CMV pp65-specific polyclonal T cell lines and CD8+ T cell clones. A, The frequency of CMV-specific CD8+ T cells recognizing the HLA-A*0201-restricted epitope NLV was assessed by staining with NLV tetramer and anti-CD8 Ab at days (d) 0, 6, and 14 after initial stimulation of PBMCs from donor 6 with NLV peptide. As a control, the staining procedure was performed without adding NLV tetramer. B, The frequency of CMV-specific CD8+ T cells recognizing the HLA-A*0101-restricted epitope YSE was assessed by staining with YSE-pentamer and anti-CD8 Ab at days 10, 22, and 29 during repeated stimulation of PBMCs from donor 7 using autologous pp65-expressing mLCL. As a control, the staining procedure was performed without adding YSE pentamer. C, Multimer binding of the CMV-specific CD8+ T cell clones JG-24, JG-33, BF-33, and MD-19 was analyzed by staining with the respective HLA-peptide multimer (abbreviated NLV, IPS, YSE) and anti-CD8 Ab. As controls, the staining procedure was performed without adding the respective multimer. D, Release of IFN-γ by the CMV-specific CD8+ T cell clones JG-24 (NLV2-TCR) and JG-33 (NLV3-TCR) after recognition of endogenously presented pp65 was analyzed by ELISA. JG-24 T cells (▪) or JG-33 T cells () were tested against autologous, allogeneic HLA-A2-positive, or allogeneic HLA-A2-negative target cells (donors are indicated in brackets). Mean values and ranges of duplicates are shown. E, Release of IFN-γ by the CMV-specific CD8+ T cell clone BF-33 (IPS-TCR) after recognition of endogenously presented pp65 was analyzed by ELISA. BF-33 T cells were tested against autologous, allogeneic HLA-B*3501-positive, or allogeneic HLA-B*3501-negative target cells (donors are indicated in brackets). Values are mean values and ranges of duplicates.

FIGURE 1.

Establishment of CMV pp65-specific polyclonal T cell lines and CD8+ T cell clones. A, The frequency of CMV-specific CD8+ T cells recognizing the HLA-A*0201-restricted epitope NLV was assessed by staining with NLV tetramer and anti-CD8 Ab at days (d) 0, 6, and 14 after initial stimulation of PBMCs from donor 6 with NLV peptide. As a control, the staining procedure was performed without adding NLV tetramer. B, The frequency of CMV-specific CD8+ T cells recognizing the HLA-A*0101-restricted epitope YSE was assessed by staining with YSE-pentamer and anti-CD8 Ab at days 10, 22, and 29 during repeated stimulation of PBMCs from donor 7 using autologous pp65-expressing mLCL. As a control, the staining procedure was performed without adding YSE pentamer. C, Multimer binding of the CMV-specific CD8+ T cell clones JG-24, JG-33, BF-33, and MD-19 was analyzed by staining with the respective HLA-peptide multimer (abbreviated NLV, IPS, YSE) and anti-CD8 Ab. As controls, the staining procedure was performed without adding the respective multimer. D, Release of IFN-γ by the CMV-specific CD8+ T cell clones JG-24 (NLV2-TCR) and JG-33 (NLV3-TCR) after recognition of endogenously presented pp65 was analyzed by ELISA. JG-24 T cells (▪) or JG-33 T cells () were tested against autologous, allogeneic HLA-A2-positive, or allogeneic HLA-A2-negative target cells (donors are indicated in brackets). Mean values and ranges of duplicates are shown. E, Release of IFN-γ by the CMV-specific CD8+ T cell clone BF-33 (IPS-TCR) after recognition of endogenously presented pp65 was analyzed by ELISA. BF-33 T cells were tested against autologous, allogeneic HLA-B*3501-positive, or allogeneic HLA-B*3501-negative target cells (donors are indicated in brackets). Values are mean values and ranges of duplicates.

Close modal

To test whether the T cell clones recognize pp65 Ag when intracellularly expressed, processed and presented, we analyzed their reactivity to pp65-expressing mLCLs, control (pp65-negative) mLCLs, and CD40-activated BBLs from various donors. For characterization of their TCR, we selected T cell clones which strongly and exclusively recognized autologous or HLA-matched pp65-expressing mLCLs, as shown for NLV- and IPS-specific clones (Fig. 1, D and E). All T cell clones with this reaction pattern showed a clear staining with the corresponding HLA/peptide multimer (Fig. 1 C).

CMV-specific TCRs of these T cell clones were characterized by TCR subfamily-specific PCR (Table II). The NLV-specific T cell clones derived from donor 6 used different TRAV and TRBV genes. The TCR NLV2 from T cell clones JG-9 and JG-24 is a public TCR and was also found in NLV-specific T cell clones of another donor not included in this study. In addition, other groups described NLV-specific T cells from various donors which used the same TCR α- or β-chain or closely related variants (25, 26, 30, 31). In contrast, the TCRs NLV3, YSE, and IPS have not been described thus far. We found the CDR3α region of the NLV3-TCR to be extremely short; this TCR might recognize Ag by adopting an unusual structure, meriting further investigation.

Table II.

TCR α- and β-chain sequences derived from pp65-specific CD8+ T cell clones

DonorT Cell CloneEpitopeHLA Restrictionα-Chainβ-ChainTCR Name
AVCDR3αAJBVCDR3βBJ
JG-9/JG-24 NLV A*0201 35*02 CAG PMKTSYDKV IFG 50*01 12-4*01 CAS SSANYGY TFG 1–2*01 NLV2 
JG-33 NLV A*0201 3*01 CAV FFG 35*01 27*01 CAS SPTGGSPSPL HFG 1–6*01 NLV3 
MD-19 YSE A*0101 12-1*01 CVA WGGYSSASKI IFG 3*01 9*01 CAS SVVGDEQ YFG 2–7*01 YSE 
BF-33/BF-38 IPS B*3501 5*01 CAE RGWDNDM RFG 43*01 11-2*01 CAS SADSNGEL FFG 2–2*01 IPS 
DonorT Cell CloneEpitopeHLA Restrictionα-Chainβ-ChainTCR Name
AVCDR3αAJBVCDR3βBJ
JG-9/JG-24 NLV A*0201 35*02 CAG PMKTSYDKV IFG 50*01 12-4*01 CAS SSANYGY TFG 1–2*01 NLV2 
JG-33 NLV A*0201 3*01 CAV FFG 35*01 27*01 CAS SPTGGSPSPL HFG 1–6*01 NLV3 
MD-19 YSE A*0101 12-1*01 CVA WGGYSSASKI IFG 3*01 9*01 CAS SVVGDEQ YFG 2–7*01 YSE 
BF-33/BF-38 IPS B*3501 5*01 CAE RGWDNDM RFG 43*01 11-2*01 CAS SADSNGEL FFG 2–2*01 IPS 

The TCR α- and β-chain genes derived from these four CMV-specific T cell clones were individually inserted into the retroviral vector plasmid pMP71Gpre, and infectious retroviral supernatants were produced. To investigate transgenic TCR expression in the absence of an endogenous TCR, we transferred the CMV-specific TCRs to TCRαβ-deficient J76 cells and to J76CD8 cells stably expressing the human CD8α chain (Fig. 2 A). All TCRs were expressed on J76CD8 cells and were strongly stained with the respective HLA/peptide multimer. Staining levels on J76 cells without CD8α were reduced, but three of the four TCRs could be detected on these cells, except for the IPS-TCR. We conclude that the TCRs NLV2, NLV3, and YSE did not strictly require CD8 to bind their target HLA/peptide complex.

FIGURE 2.

Transfer of four different CMV-specific TCRs into T cell lines and PBMCs. A, The CMV pp65-specific TCRs NLV2, NLV3, IPS, and YSE were transferred into J76 cells (top) and J76CD8 cells (bottom). Expression of the introduced TCR was analyzed by staining with the respective HLA/peptide-multimer (thick line) at day 4 after transfer. As control, mock-transduced cells were analyzed by staining with the same multimer (thin line). Numbers indicate the percentage of multimer-positive cells. B, PBMCs from CMV-seronegative donors were transduced with the CMV pp65-specific TCRs NLV2 (donor 1), NLV3 (donor 1), IPS (donor 3), and YSE (donor 3; bottom), or mock-transduced (top). Expression of the introduced TCR was analyzed by staining with the respective HLA-peptide multimer at day 4 after transfer. C, The TCRs NLV2, NLV3, IPS, and YSE were transferred into PBMCs from different CMV-seronegative donors, and the frequency of multimer-positive cells within different T cell subsets, CD3+ cells (black bars), CD8+CD4 cells (dark gray bars), and CD8CD4+ cells (light gray bars), was determined by costaining with the respective HLA/peptide-multimer and Abs against CD3, CD4, and CD8 at day 4 after TCR transfer. D and E, CMV-specific reactivity of TCR-transgenic T cells against cells endogenously presenting the target Ag pp65 was analyzed by IFN-γ ELISA (D) and IL-2 ELISA (E) at day 6 after TCR transfer. NLV2 (black bars), NLV3 (dark gray bars), or mock-transduced (light gray bars) PBMCs from donor 1 were tested against autologous, allogeneic HLA-A2-positive, or allogeneic HLA-A2-negative target cells (donors are indicated in brackets). Mean values and ranges of duplicates are shown. The percentage of multimer-positive cells at day 4 after transfer is indicated.

FIGURE 2.

Transfer of four different CMV-specific TCRs into T cell lines and PBMCs. A, The CMV pp65-specific TCRs NLV2, NLV3, IPS, and YSE were transferred into J76 cells (top) and J76CD8 cells (bottom). Expression of the introduced TCR was analyzed by staining with the respective HLA/peptide-multimer (thick line) at day 4 after transfer. As control, mock-transduced cells were analyzed by staining with the same multimer (thin line). Numbers indicate the percentage of multimer-positive cells. B, PBMCs from CMV-seronegative donors were transduced with the CMV pp65-specific TCRs NLV2 (donor 1), NLV3 (donor 1), IPS (donor 3), and YSE (donor 3; bottom), or mock-transduced (top). Expression of the introduced TCR was analyzed by staining with the respective HLA-peptide multimer at day 4 after transfer. C, The TCRs NLV2, NLV3, IPS, and YSE were transferred into PBMCs from different CMV-seronegative donors, and the frequency of multimer-positive cells within different T cell subsets, CD3+ cells (black bars), CD8+CD4 cells (dark gray bars), and CD8CD4+ cells (light gray bars), was determined by costaining with the respective HLA/peptide-multimer and Abs against CD3, CD4, and CD8 at day 4 after TCR transfer. D and E, CMV-specific reactivity of TCR-transgenic T cells against cells endogenously presenting the target Ag pp65 was analyzed by IFN-γ ELISA (D) and IL-2 ELISA (E) at day 6 after TCR transfer. NLV2 (black bars), NLV3 (dark gray bars), or mock-transduced (light gray bars) PBMCs from donor 1 were tested against autologous, allogeneic HLA-A2-positive, or allogeneic HLA-A2-negative target cells (donors are indicated in brackets). Mean values and ranges of duplicates are shown. The percentage of multimer-positive cells at day 4 after transfer is indicated.

Close modal

To confer CMV-specific reactivity on primary T cells from CMV-negative donors, we performed retroviral transfer of our CMV-specific TCRs. Four days after transfer, all TCRs were expressed on PBMCs and could be stained with peptide/HLA multimers, whereas mock-transduced cells were multimer negative (Fig. 2, B and C). Multimer-staining levels of the individual TCRs were comparable between different donors with different HLA backgrounds (Table I), but the levels varied among the four different TCRs (Fig. 2,C). Proportions of multimer-positive cells were similar within different T cell subsets (CD3+, CD8+, CD4+) except for the IPS-TCR. This TCR was not detectable on CD4+CD8 T cells, suggesting CD8 dependency, which is consistent with our previous observation that this TCR could not be detected on J76 cells (Fig. 2,A). The general transduction efficiency, as determined with GFP-expressing retrovirus, was comparable for all donors (45–60%). To check whether the TCR-transgenic T cells had acquired reactivity against pp65-expressing cells already at this early stage, we cocultivated the transduced PBMCs with autologous, allogeneic HLA-matched and HLA-mismatched target cells, namely pp65-expressing and pp65-negative mLCLs and BBLs, at day 6 after TCR transfer, and determined the amount of released IFN-γ and IL-2. NLV2-TCR-transduced as well as NLV3-TCR-transduced PBMCs specifically produced considerable amounts of IFN-γ (Fig. 2,D) and some IL-2 (Fig. 2,E) after coculture with HLA-A2-positive pp65-expressing mLCLs. Cocultivation with BBLs (pp65 and EBV negative) did not induce IFN-γ, but low amounts of IFN-γ were released when the TCR-transduced cells were tested against pp65-negative mLCLs, indicating the presence of some EBV-specific memory T cells, which was expected because the donor (donor 1) was EBV positive (Table I).

These results show that CMV-specific T cells recognizing endogenously processed Ag can be rapidly produced by TCR transfer in a simple 8-day procedure from PBMCs from seronegative donors.

For successful T cell therapy, TCR-transgenic T cells must be able to recognize the endogenously processed Ag and proliferate in an Ag-specific manner in vivo to achieve sufficient function and maintenance after therapeutic T cell transfer. Therefore, we investigated whether CMV-TCR-transduced T cells could be expanded and enriched by Ag-specific stimulation. As Ag-specific stimulators, we used the autologous pp65-expressing mLCL, which presents endogenously processed pp65 epitopes on class I and II HLA molecules (22, 32). For comparison, we performed nonspecific stimulation with anti-CD3 Ab, a mix of allogeneic PBMCs derived from three unrelated donors, and the autologous mLCL without pp65 expression. The stimulation protocol is schematically shown in Fig. 3 A.

FIGURE 3.

Enrichment of CMV-specific TCR-transgenic T cells by Ag-specific stimulation. A, Schematic overview over the transduction and stimulation procedure. B, PBMCs from donor 1 were transduced with the NLV2-TCR or mock-transduced, followed by Ag-specific compared with nonspecific stimulation. The frequency of NLV-specific T cells was analyzed at days 4, 13, 20, and 28 after TCR transfer by staining with NLV tetramer and anti-CD8 Ab. C, Intensity of multimer staining over time in PBMCs from donors 1 and 2, transduced with the NLV2-TCR. The geometric mean fluorescence intensity (MFI) of gated CD8+ multimer+ cells after specific or nonspecific stimulation is presented.

FIGURE 3.

Enrichment of CMV-specific TCR-transgenic T cells by Ag-specific stimulation. A, Schematic overview over the transduction and stimulation procedure. B, PBMCs from donor 1 were transduced with the NLV2-TCR or mock-transduced, followed by Ag-specific compared with nonspecific stimulation. The frequency of NLV-specific T cells was analyzed at days 4, 13, 20, and 28 after TCR transfer by staining with NLV tetramer and anti-CD8 Ab. C, Intensity of multimer staining over time in PBMCs from donors 1 and 2, transduced with the NLV2-TCR. The geometric mean fluorescence intensity (MFI) of gated CD8+ multimer+ cells after specific or nonspecific stimulation is presented.

Close modal

For all four CMV-TCRs, CMV-TCR-transgenic CD3+ T cells were specifically enriched by stimulation with the endogenously processed CMV Ag pp65 (Fig. 4,C). The same was true if only CMV-TCR-transgenic CD8+ T cells were evaluated (Fig. 4,D). Examples of multimer stainings are shown in Fig. 3,B. As before, for a given TCR proportions of TCR-transgenic T cells were similar among different PBMC donors (Fig. 4, C and D). Ag-specific stimulation led to an expansion of each TCR-transduced T cell culture in terms of absolute cell numbers (Fig. 4,A). For the two NLV-TCRs, specific and nonspecific stimulation were comparable in terms of total cell expansion (Fig. 4,A). For the IPS- and YSE-TCRs, nonspecific stimulation led to the strongest overall cell expansion (Fig. 4,A), but only specific stimulation raised the proportion of TCR-transgenic T cells (Fig. 4, C and D). Both stimulation protocols preferentially expanded CD8+ T cells (Fig. 4 B).

FIGURE 4.

Expansion and composition of CMV-TCR-transduced PBMC cultures during stimulation. CMV-specific TCRs NLV2, NLV3, IPS, and YSE were transferred into PBMCs from different CMV-seronegative donors followed by Ag-specific or nonspecific stimulation. A, The total cell number of the cultures was determined by counting viable cells after trypan blue staining at days (d) 6, 13, and 20 after TCR transfer. The cell count at day 6 was set equal to 1. For the IPS-TCR, a different scale (range, 0–150) was used than for the other TCRs (range, 0–100). B, Enrichment of CD8+ T cells within the PBMC culture was determined by staining with anti-CD8 Ab and flow cytometric analysis at days 4, 13, and 20 after TCR transfer. CE, Proportion of CMV multimer-positive cells within different T cell subsets (CD3+, CD8+CD4, and CD8CD4+) during the stimulation period was assessed by staining with the respective HLA-peptide multimer and Abs against CD3, CD4, and CD8 at days 4, 13, and 20 after TCR transfer. The proportion of multimer-positive cells was <0.5% for mock-transduced and for control-stained TCR-transduced cells. For each of the four TCRs, a different scale was used in C–E.

FIGURE 4.

Expansion and composition of CMV-TCR-transduced PBMC cultures during stimulation. CMV-specific TCRs NLV2, NLV3, IPS, and YSE were transferred into PBMCs from different CMV-seronegative donors followed by Ag-specific or nonspecific stimulation. A, The total cell number of the cultures was determined by counting viable cells after trypan blue staining at days (d) 6, 13, and 20 after TCR transfer. The cell count at day 6 was set equal to 1. For the IPS-TCR, a different scale (range, 0–150) was used than for the other TCRs (range, 0–100). B, Enrichment of CD8+ T cells within the PBMC culture was determined by staining with anti-CD8 Ab and flow cytometric analysis at days 4, 13, and 20 after TCR transfer. CE, Proportion of CMV multimer-positive cells within different T cell subsets (CD3+, CD8+CD4, and CD8CD4+) during the stimulation period was assessed by staining with the respective HLA-peptide multimer and Abs against CD3, CD4, and CD8 at days 4, 13, and 20 after TCR transfer. The proportion of multimer-positive cells was <0.5% for mock-transduced and for control-stained TCR-transduced cells. For each of the four TCRs, a different scale was used in C–E.

Close modal

From these data, we determined the increase in absolute numbers of TCR-transgenic CD8+ T cells (expansion) as well as the increase in the proportion of TCR-transgenic CD8+ T cells (enrichment), after both Ag-specific and nonspecific stimulation (Table III). For each TCR, both stimulation conditions led to an impressive expansion of CD8+ T cells expressing the transgenic TCR (36- to 672-fold; Table III). With the exception of the IPS-TCR-transgenic T cells, Ag-specific stimulation resulted in a superior expansion compared with nonspecific stimulation. For all four TCRs, enrichment of TCR-transgenic CD8+ T cells was stronger after specific stimulation than after nonspecific stimulation (Table III). Together, these data show that endogenously processed Ag stimulates a robust and specific expansion and enrichment of T cells carrying transgenic CMV-specific TCRs.

Table III.

Total expansion and enrichment of CD8+ T cells expressing transgenic TCRs by Ag-specific or nonspecific stimulation

CD8+Multimer+ CellsNLV2NLV3IPSYSE Donor 3
Donor 1Donor 2Donor 1Donor 2Donor 3Donor 4
Expansiona        
 Ag-specific stimulation 148 60 141 224 228 88 672 
 Nonspecific stimulation 36 40 47 40 640 183 301 
Enrichmentb        
 Ag-specific stimulation 1.74 1.02 1.44 1.64 11.15 4.78 31.6 
 Nonspecific stimulation 0.37 0.23 0.23 0.27 1.13 0.79 0.79 
CD8+Multimer+ CellsNLV2NLV3IPSYSE Donor 3
Donor 1Donor 2Donor 1Donor 2Donor 3Donor 4
Expansiona        
 Ag-specific stimulation 148 60 141 224 228 88 672 
 Nonspecific stimulation 36 40 47 40 640 183 301 
Enrichmentb        
 Ag-specific stimulation 1.74 1.02 1.44 1.64 11.15 4.78 31.6 
 Nonspecific stimulation 0.37 0.23 0.23 0.27 1.13 0.79 0.79 
a

Expansion of CMV-TCR-transgenic CD8+ T cells was calculated by dividing the absolute number of multimer+CD8+ T cells on day 20 by the absolute number of multimer+CD8+ T cells on day 4 (see also Fig. 4). Ag-specific or nonspecific stimulation was performed on days 6 and 13 (see Fig. 3 A).

b

Enrichment of CMV-TCR-transgenic CD8+ T cells was calculated by dividing the proportions of multimer+CD8+ T cells within total cells on day 20 by the proportions of multimer+CD8+ T cells within total cells on day 4 (see Fig. 4 D).

CD4+ T cells expressing these HLA class-I-restricted TCRs were maintained (NLV2, NLV3) or even expanded (YSE) by specific stimulation, except for the IPS-TCR (Fig. 4,E), which was the only TCR that could not be detected on J76 cells lacking CD8 (Fig. 2 A).

We observed no great variations over time in the intensity of multimer staining of gated multimer-positive populations during specific or nonspecific stimulation, as shown for NLV2-TCR-transduced cells from two donors in Fig. 3 C, suggesting that TCR expression levels were relatively stable.

To check whether CMV-TCR-transgenic T cells had maintained their specific function after Ag-specific expansion, we reassessed their pp65-specific cytokine release after three rounds of stimulation. Ag-specifically or nonspecifically stimulated TCR-transduced cultures were cocultivated with a panel of target cells endogenously expressing pp65 and HLA-mismatched or pp65-negative controls (Fig. 5). Compared with the situation 6 days after TCR transfer (Fig. 2, D and E), we found strongly increased pp65-specific release of IFN-γ for each CMV-TCR-transduced T cell line after Ag-specific expansion (Fig. 5, A and B). After nonspecific expansion, secretion was much lower (Fig. 5, A and B), consistent with lower numbers of multimer-positive cells (Fig. 4 C). HLA-mismatched and pp65-negative controls were only weakly recognized. As expected, mock-transduced T cell cultures showed no pp65-specific reactivity.

FIGURE 5.

CMV Ag-specific release of effector cytokines by CMV-TCR-transduced PBMCs after three rounds of stimulation. PBMCs derived from CMV-seronegative donors (A, donor 2; B and C, donor 3) were retrovirally transduced with different CMV-specific TCRs, or mock-transduced as indicated. Three rounds of Ag-specific (▪) compared with nonspecific () stimulation were performed, and CMV-specific reactivity of TCR-transgenic T cells against cells endogenously presenting the target Ag pp65 was analyzed by IFN-γ ELISA (A and B) and IL-2 ELISA (C) at day 25 after TCR transfer. As target cells, autologous and allogeneic (matched or mismatched to the HLA restriction of the transgenic TCRs) pp65-expressing mLCLs, control mLCL, or CD40-activated BBLs were used. w/o, Without. Mean values and range of duplicates are shown.

FIGURE 5.

CMV Ag-specific release of effector cytokines by CMV-TCR-transduced PBMCs after three rounds of stimulation. PBMCs derived from CMV-seronegative donors (A, donor 2; B and C, donor 3) were retrovirally transduced with different CMV-specific TCRs, or mock-transduced as indicated. Three rounds of Ag-specific (▪) compared with nonspecific () stimulation were performed, and CMV-specific reactivity of TCR-transgenic T cells against cells endogenously presenting the target Ag pp65 was analyzed by IFN-γ ELISA (A and B) and IL-2 ELISA (C) at day 25 after TCR transfer. As target cells, autologous and allogeneic (matched or mismatched to the HLA restriction of the transgenic TCRs) pp65-expressing mLCLs, control mLCL, or CD40-activated BBLs were used. w/o, Without. Mean values and range of duplicates are shown.

Close modal

Additionally, TCR-transduced T cell cultures specifically secreted considerable amounts of IL-2 in response to endogenously processed pp65 after Ag-specific stimulation (Fig. 5 C). In contrast, there was no CMV-specific release of IL-2 by nonspecifically stimulated cultures.

These results show that Ag-specific expansion significantly enhanced the specific function of the TCR-transduced T cells (Fig. 5), consistent with the increase in CMV multimer-positive T cells (Fig. 4). To more precisely quantify the stability of Ag-specific function of TCR-transgenic T cells, we determined the amount of IFN-γ secreted by one multimer-positive T cell before and after three rounds of stimulation, under the assumption that only multimer-positive T cells will specifically secrete IFN-γ upon challenge with CMV APCs. As shown for NLV2-transduced cells in Fig. 6, the amount of cytokine secreted per multimer-positive cell in response to the CMV mLCL was 12-fold higher after Ag-specific stimulation than it was before stimulation. Nonspecific stimulation led to a 2.6-fold increase in cytokine release per cell under the above assumption. Thus, Ag-specific function appears to be a durable property of TCR-transgenic T cells; moreover, their functional capacity can even be intensified, especially by repeated contact with endogenously processed Ag.

FIGURE 6.

CMV Ag-specific cytokine release per multimer-positive cell before and after stimulation of CMV-TCR-transduced PBMC. PBMCs from CMV-seronegative donor 1 were transduced with the NLV2-TCR, and IFN-γ secretion in reaction to target cells was analyzed before (day 6) and after (day 25) three rounds of Ag-specific (left) or nonspecific (right) stimulation. Target cells included autologous and HLA-mismatched pp65-expressing mLCLs and control mLCLs. Cytokine release per multimer-positive cell was calculated by dividing the total amount of cytokine detected in the reaction supernatant by the number of total T cells per reaction multiplied by the proportion of multimer-positive cells assessed at the nearest available time point: 4.23% on day 4 (before stimulation); 11.2% on day 28 (after specific stimulation); 0.79% on day 28 (after nonspecific stimulation).

FIGURE 6.

CMV Ag-specific cytokine release per multimer-positive cell before and after stimulation of CMV-TCR-transduced PBMC. PBMCs from CMV-seronegative donor 1 were transduced with the NLV2-TCR, and IFN-γ secretion in reaction to target cells was analyzed before (day 6) and after (day 25) three rounds of Ag-specific (left) or nonspecific (right) stimulation. Target cells included autologous and HLA-mismatched pp65-expressing mLCLs and control mLCLs. Cytokine release per multimer-positive cell was calculated by dividing the total amount of cytokine detected in the reaction supernatant by the number of total T cells per reaction multiplied by the proportion of multimer-positive cells assessed at the nearest available time point: 4.23% on day 4 (before stimulation); 11.2% on day 28 (after specific stimulation); 0.79% on day 28 (after nonspecific stimulation).

Close modal

Ag-specific cytotoxicity of CD8+ T cells is essential for the control of viral infection. After three rounds of stimulation, we investigated Ag-specific killing by expanded CMV-TCR-transgenic T cells in cocultures with pp65-positive and pp65-negative target cells (Fig. 7). After Ag-specific stimulation, NLV2-TCR-transduced T cells from CMV-negative donors efficiently lysed the autologous pp65-expressing mLCL even at very low E:T ratios. The HLA-mismatched pp65-expressing mLCL, pp65-negative mLCLs and BBLs were not or only very weakly recognized and killed by the T cells (Fig. 7,A). After nonspecific stimulation, NLV2-TCR-transduced T cells were less efficient in killing the pp65-positive cells and showed an increased reactivity against the autologous as well as the HLA-mismatched control mLCLs. Mock-transduced PBMCs showed an EBV-specific or nonspecific killing pattern (Fig. 7,A). Similar to the NLV2-TCR, transfer of the three other CMV-specific TCRs to primary T cells followed by stimulation resulted in efficient CMV-specific lysis of target cells (Fig. 7 B). In each case, the specificity or intensity of lysis or both were superior after Ag-specific stimulation.

FIGURE 7.

CMV Ag-specific cytotoxic reactivity of CMV-TCR-transduced PBMCs after three rounds of stimulation. PBMCs derived from different CMV-seronegative donors were retrovirally transduced with different CMV-specific TCRs or mock-transduced as indicated. Three rounds of Ag-specific or nonspecific stimulation were performed, and cytotoxic reactivity of TCR-transgenic T cells against cells endogenously presenting the target Ag pp65 was assessed in a calcein release assay at day 25 or 26 after TCR transfer. A, NLV2-transduced (top) and mock-transduced (bottom) cells derived from donor 1 were tested against autologous and HLA-A2-mismatched mLCLs, either expressing pp65 or not, as well as CD40-activated BBLs, at different E:T ratios as indicated. Values are means and SDs of triplicates. B, NLV3-, IPS-, and YSE-transduced cells derived from different donors were tested against autologous or HLA-matched mLCLs, either expressing pp65 or not, as well as CD40-activated BBLs, at different E:T ratios as indicated. Values are means and SDs of triplicates.

FIGURE 7.

CMV Ag-specific cytotoxic reactivity of CMV-TCR-transduced PBMCs after three rounds of stimulation. PBMCs derived from different CMV-seronegative donors were retrovirally transduced with different CMV-specific TCRs or mock-transduced as indicated. Three rounds of Ag-specific or nonspecific stimulation were performed, and cytotoxic reactivity of TCR-transgenic T cells against cells endogenously presenting the target Ag pp65 was assessed in a calcein release assay at day 25 or 26 after TCR transfer. A, NLV2-transduced (top) and mock-transduced (bottom) cells derived from donor 1 were tested against autologous and HLA-A2-mismatched mLCLs, either expressing pp65 or not, as well as CD40-activated BBLs, at different E:T ratios as indicated. Values are means and SDs of triplicates. B, NLV3-, IPS-, and YSE-transduced cells derived from different donors were tested against autologous or HLA-matched mLCLs, either expressing pp65 or not, as well as CD40-activated BBLs, at different E:T ratios as indicated. Values are means and SDs of triplicates.

Close modal

CCR7 and L-selectin (CD62L) mediate homing of Ag-specific T cells to the lymph nodes, and their expression characterizes central memory T cells (42). We investigated the expression of these markers after TCR transfer (day 6) and Ag-specific or nonspecific stimulation (day 13, day 20). Expression of CCR7 and CD62L by NLV-TCR-expressing cells closely mirrored expression by the complete cell cultures (Fig. 8). The proportion of CCR7+ and CD62L+ cells decreased over time, but they accounted for >15% of CMV-TCR-positive or total T cells after specific expansion even at day 20.

FIGURE 8.

Central memory markers on CMV-TCR-transgenic T cells during stimulation. PBMCs derived from CMV-seronegative donor 1 were retrovirally transduced with the CMV-specific NLV2-TCR or NLV3-TCR followed by Ag-specific or nonspecific stimulation. A, The frequency of CCR7-positive T cells was assessed at days 6, 13, and 20 after TCR transfer by staining with CCR7-specific Ab and NLV tetramer and subsequent flow cytometric analysis. The proportion of CCR7-positive cells within the NLV tetramer-positive population (▪) as well as the proportion of CCR7-positive cells within the total T cell population () is shown. B, Similarly, the frequency of CD62L+ T cells was assessed at days 6, 13, and 20 after TCR transfer by staining with CD62L-specific Ab and NLV tetramer followed by flow cytometric analysis. The proportion of CD62L+ cells within the NLV tetramer-positive population (▪) as well as the proportion of CD62L+ cells within total lymphocytes () is shown.

FIGURE 8.

Central memory markers on CMV-TCR-transgenic T cells during stimulation. PBMCs derived from CMV-seronegative donor 1 were retrovirally transduced with the CMV-specific NLV2-TCR or NLV3-TCR followed by Ag-specific or nonspecific stimulation. A, The frequency of CCR7-positive T cells was assessed at days 6, 13, and 20 after TCR transfer by staining with CCR7-specific Ab and NLV tetramer and subsequent flow cytometric analysis. The proportion of CCR7-positive cells within the NLV tetramer-positive population (▪) as well as the proportion of CCR7-positive cells within the total T cell population () is shown. B, Similarly, the frequency of CD62L+ T cells was assessed at days 6, 13, and 20 after TCR transfer by staining with CD62L-specific Ab and NLV tetramer followed by flow cytometric analysis. The proportion of CD62L+ cells within the NLV tetramer-positive population (▪) as well as the proportion of CD62L+ cells within total lymphocytes () is shown.

Close modal

These findings indicate that a proportion of CMV-TCR-transgenic T cells maintain their central memory phenotype after repeated Ag contact, which might contribute to the establishment of a TCR-transgenic CMV-specific T cell memory.

In this study, we characterized CMV-specific T cells generated from CMV-seronegative donors by TCR transfer. Our intention was to investigate whether such T cells would be suitable for adoptive therapy of patients who suffer from CMV-related disease after allo-HSCT. Transfer of CMV-specific T cells might be ideal to prevent and cure CMV disease, but for the combination CMV-negative donor/CMV-positive recipient (D−/R+) such cells are usually not available. However, for these patients, the risk of CMV disease is highest. D+/R+ is usually the most frequent situation in allo-HSCT (5), but with decreasing CMV prevalence in the population, D−/R+ HSCTs will occur with increasing frequency.

The hypothesis underlying this study was that suitable CMV-specific TCRs are readily available within the memory T cell repertoire of CMV-seropositive donors. It can be expected that the TCR repertoire of CMV-specific memory T cells is shaped, at least in part, by the necessity to control CMV infection and to protect from disease. To select appropriate candidate T cell clones to derive TCRs for transfer, we used a screening procedure that selected T cells specifically reacting to endogenously processed CMV Ag. Furthermore, we assumed that CMV Ag-specific T cells can be engineered by CMV-TCR transfer independent of the HLA restriction of the TCRs, although up to now mainly transfer of HLA-A*0201-restricted tumor-specific TCRs has been studied (17). To verify these assumptions, we isolated, characterized, and cloned several CMV-specific TCRs that recognize different epitopes from pp65 restricted through various HLA allotypes. One of these TCRs, NLV2, was present in both HLA-A*0201-positive donors investigated, confirming the earlier description of this TCR or its β-chain as public (25, 26, 30, 31). A second TCR, NLV3, recognizing the same epitope, has not been described previously and was identified in only one of the donors. Because this TCR has an unusually short CDR3α region (Table II), it might be interesting to investigate the structure of the corresponding TCR-peptide-MHC complex. Additionally, we characterized two previously undescribed TCRs with different HLA restrictions, one recognizing the HLA-A*0101-restricted epitope YSE and one specific for the HLA-B*3501-restricted epitope IPS. We found that retroviral transfer of all these CMV-specific TCRs resulted in TCR-transgenic T cells which exhibited a wide spectrum of desirable functions in response to endogenously processed CMV Ag. By stimulation with endogenously processed Ag, TCR-transgenic T cells could be specifically expanded and enriched in relative and absolute terms (Fig. 4 and Table III), indicating a strong proliferative potential of these cells after Ag contact. This feature will be important for controlling CMV infection or reactivation in vivo. The increase in CMV-TCR-expressing T cells was accompanied by an increase in Ag-specific effector functions (cytotoxicity and cytokine secretion) of similar magnitude (Figs. 5 and 7), showing that the functionality of these TCR-transgenic T cells is fully maintained after Ag contact and Ag-specific proliferation in vitro. In line with these observations, staining intensity of HLA-peptide multimer-positive populations was stable (Fig. 3,C), and the functional capacity of multimer-positive populations even appeared to increase over time (Fig. 6). Furthermore, no stimulation of TCR-transduced T cells, cell sorting or depletion was required to obtain T cells displaying Ag-specific function at very convincing levels as early as 6 days after TCR transfer (Fig. 2).

These results prompt us to suggest that a simple protocol consisting of TCR transfer to PBMCs, without further expansion or selection steps, will produce CMV-TCR-transgenic T cell populations suitable for immediate use in T cell transfer therapy. Care should be taken to keep the total number of allogeneic T cells transferred to a patient low enough to minimize the probability of graft-vs-host disease, for example, <106 cells/kg for HLA-matched donor-recipient pairs (43), because alloreactive or originally tolerant self-reactive T cells might be present in the TCR-transduced T cell preparation. Such potentially harmful T cells might be activated by the anti-CD3 treatment preceding transduction, or by means of a transgenic CMV-specific TCR coexpressed on the same cell and recognizing its target Ag in the patient. However, as long as T cell therapy is performed early enough in a pre-emptive situation, low numbers of CMV Ag-specific T cells may be sufficient to avert disease (9). We consider it likely that Ag-driven T cell expansion in the CMV-infected HSCT patient, possibly favored by lymphopenia (1), will be at least as effective in producing sufficient numbers of specific effector T cells than any further expansion in vitro. The considerable proportions of CMV-TCR-transgenic T cells positive for the central memory markers CCR7 and CD62L (Fig. 8), which were largely retained after Ag contact in vitro, further suggest that TCR-transgenic cells will be able to form an effective CMV-specific memory in vivo.

Different strategies to obtain CMV-specific T cells for adoptive therapy after D−/R+ HSCT might be taken into consideration. CMV-specific T cells could be obtained directly from a third unrelated CMV-seropositive donor. In this context, third-party-derived EBV-specific T cells had very good therapeutic effects in solid organ transplant recipients (44). However, allogeneic T cells might be rapidly rejected, hampering long-term antiviral protection. Therefore, T cells derived from the HSCT donor are to be preferred for adoptive therapy.

Another alternative to CMV-TCR transfer would be in vitro priming and expansion of rare naive CMV-specific T cells from the donor. Remarkably, with a small proportion of CMV-seronegative donors it has been possible to generate CMV-specific T cells by in vitro priming (45, 46, 47). However, we expect it to be very difficult to translate these observations into feasible clinical procedures, because the precursor frequency of naive CMV-specific CD8+ T cells in CMV-seronegative individuals, estimated from general considerations on TCR diversity (48), is unlikely to be above 1 in 106 naive T cells. Therefore, transfer of CMV-specific TCRs to primary T cells will be the easiest and most efficient method for adoptive therapy of CMV-related complications after HSCT with CMV-seronegative donors.

By using CMV-specific TCRs of various HLA restrictions, population coverage can be optimized. With the selected HLA-A*0101-, HLA-A*0201-, and HLA-B*3501-restricted CMV-specific TCRs, at least 70% of Europeans can be covered, HLA-A*0201 being the most frequent and HLA-A*0101 the second most frequent HLA allotype. Obviously, further extension of this TCR repertoire to cover additional HLA allotypes is highly desirable.

CMV-specific TCR genes have previously been cloned and transferred to T cells (27, 49, 50). These studies established that it is possible to transfer HLA-B7-restricted (49) or HLA-A2-restricted (27, 50) pp65-specific TCRs to γδ T cells (27, 49), αβ T cells (27), or in vitro-differentiated hemopoietic precursors (50), and showed that pp65 peptide-loaded targets (27, 49, 50) and LCLs transduced with pp65 (49, 50) were recognized.

In these studies, CMV served as a model specificity to establish principles of TCR transfer. Aiming at the development of a CMV-specific T cell therapy in an HSCT context, our study extends previous findings with respect to the following points: we identified and transferred CMV-specific TCRs restricted through the frequent HLA alleles A*0101 and B*3501; we used a facilitated method of TCR transfer that does not require cell sorting and the expression of heterologous marker genes in the transduced T cells; we performed CMV-specific TCR transfer with an extended panel of donors and analyzed T cell reactivity against target cells of various HLA types; in accordance with our intended therapeutic goal, we used donors who were CMV-negative, excluding the possibility that endogenous CMV-specific T cells might contribute to the observed effects; we showed that CMV-TCR-transgenic T cells specifically and continuously expand in response to repeated challenge with endogenously presented Ag, while fully maintaining their CMV-specific effector functions.

It was previously described that different CMV-specific TCRs recognizing the HLA-A*0201-restricted NLV epitope significantly differ in their efficiency in conferring CMV-specific function to PBMC cultures (27). This observation suggests that only some CMV-specific TCRs may be suitable for clinical application. Similarly, we observed different surface expression levels for different TCRs (Fig. 2, AC). TCR expression levels appeared to depend on their HLA restriction: the two HLA-A*0201-restricted TCRs, recognizing the NLV epitope, were expressed at higher levels than the HLA-A*0101- and HLA-B*3501-restricted TCRs. This effect was observed both in primary T cells and TCR-deficient J76 cells and therefore appears to be related to “intrinsic properties” (27) of the TCRs. Additionally, a difference in the expression levels among the two HLA-A*0201-restricted TCRs was observed only in primary T cells, but not in J76 cells, which implies that interference by endogenous TCRs might play a role here. Despite these differences early after transduction, surface expression as well as specific function of the different TCRs became rather similar after repeated Ag-specific stimulation (Figs. 4,C, F55, and F66). Because we observed that the lower initial transduction rates of the IPS- and YSE-TCRs were compensated by a stronger Ag-driven enrichment, we speculate that all four TCRs investigated in this study will be similarly efficient in adoptive therapy.

In our study, we performed retroviral transfer of unmodified TCR chain genes. Modifications of the TCR for increased surface expression or reduced TCR chain cross-pairing have been successfully used, e.g., murinization of TCR constant regions, fusion to CD3ζ, or insertion of cysteines to form additional cystine bridges between the transduced α- and β-chains (51). However, these modifications have potential disadvantages, for example altered TCR signaling or introduction of immunogenic protein sequences. Such modifications have often been used for TCRs recognizing tumor-/autoantigens (51); these TCRs may require optimization because a preformed functional T cell memory against such Ags may not exist or be inaccessible. In contrast, for CMV-specific TCRs, a functional T cell memory and therefore TCRs recognizing their viral target Ag with high avidity are readily available. There remains the risk of harmful cross-reactivity of TCR-transgenic T cells due to an accidentally coexpressed allo- or autoreactive endogenous TCR, or due to the formation of a novel specificity against an allo- or autoantigen by cross-pairing of introduced and endogenous TCR chains. To reduce the probability of cross-pairing, careful molecular modification of TCRs might be considered. Before their use in therapy, unmodified TCRs should be extensively tested for cross-reactivity and cross-pairing tendency after TCR transfer into primary T cells. For better characterization of cross-pairing potential, instead of total PBMCs, specific T cells with a defined endogenous TCR repertoire could be used as hosts for TCR transfer (27, 52, 53). For example, EBV-specific T cells transduced with CMV-TCRs would have the added benefit of simultaneous protection against both these HSCT-relevant pathogens (10). Additional safety mechanisms could be introduced to be able to eliminate TCR-transgenic T cells in the patient if adverse effects are observed (54).

In summary, we isolated CMV-specific TCRs of different HLA class I restrictions from memory-derived T cell clones characterized by efficient recognition of endogenously processed viral Ag. We demonstrated that these TCRs can be used to rapidly prepare TCR-transgenic T cells equipped with an arsenal of specific antiviral functions. We believe that such TCR-transgenic T cells qualify as candidates for a successful clinical application in HSCT patients with a CMV-negative donor, a situation of considerable and potentially increasing clinical importance.

We thank Drs. Angela M. Krackhardt and Josef Mautner for stimulating discussions and technical advice. We thank Dr. Wolfgang Uckert for generously providing J76CD8 cells and the myeloproliferative sarcoma virus retroviral gene transfer technology.

The authors have no financial conflict of interest.

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

This work was supported by Deutsche Forschungsgemeinschaft Grants SFB-Transregio 36 and SFB 455 and by the Helmholtz Alliance on Immunotherapy of Cancer funded by the Initiative and Networking Fund of the Helmholtz Association.

3

Abbreviations used in this paper: allo-HSCT, allogeneic hemopoietic stem cell transplantation; mLCL, mini-lymphoblastoid cell line; BBL, CD40-activated B blast; J76, Jurkat 76; J76CD8, Jurkat 76 stably expressing human CD8α; MLV, murine leukemia virus; D, donor; R, recipient; CD62L, L-selectin.

1
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