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Adoptive transfer of virus-specific T cells can treat infections complicating allogeneic hematopoietic cell transplants. However, autologous antigen-presenting cells (APCs) are often limited in supply. Here, we describe a panel of artificial APCs (AAPCs) consisting of murine 3T3 cells transduced to express human B7.1, ICAM-1 and LFA-3 that each stably express one of a series of 6 common HLA class I alleles. In comparative analyses, T cells sensitized with AAPCs expressing a shared HLA allele or autologous APCs loaded with a pool of 15-mers spanning the sequence of CMVpp65 produced similar yields of HLA-restricted CMVpp65 specific T cells; significantly higher yields could be achieved by sensitization with AAPCs transduced to express the CMVpp65 protein. T cells generated were CD8+, IFNγ+ and exhibited HLA-restricted CMVpp65 specific cytotoxicity. T cells sensitized with either peptide-loaded or transduced AAPCs recognized epitopes presented by each HLA allele known to be immunogenic in man. Sensitization with AAPCs also permitted expansion of IFNγ+ cytotoxic effector cells against subdominant epitopes that were either absent or in low frequencies in T cells sensitized with autologous APCs. This replenishable panel of AAPCs can be used for immediate sensitization and expansion of virus-specific T cells of desired HLA restriction for adoptive immunotherapy. It may be of particular value for recipients of transplants from HLA disparate donors.
Adoptive transfer of in-vitro generated, antigen-specific T cells has recently emerged as a therapeutically effective approach for the prevention and/or treatment of potentially lethal infections caused by cytomegalovirus (CMV) and Epstein-Barr virus (EBV) complicating allogeneic hematopoietic cell (HSCT) or organ transplants (1-6). Clinical trials using donor T cells specific for alloantigen (5) or oncofetal proteins differentially expressed by host tumors are also being explored (7-9). In order to generate sufficient numbers of therapeutically active virus-specific or tumor-selective donor-derived T cells that are adequately depleted of alloreactive T cells capable of initiating graft vs host disease (GVHD) or organ allograft rejection, requires that the T cells be sensitized with antigen presenting cells that present immunogenic epitopes on HLA alleles shared by the donor and diseased host tissues while failing to co-present major or minor alloantigens which might be expressed by the host or an organ allograft. Extended in vitro sensitization with autologous cytokine activated monocytes (CAMs), dendritic cells or EBV transformed B cells (EBV-BLCL) loaded with or transduced to express antigenic epitopes insures such specificity(4, 5). However, because the frequencies of T cells reactive against several viral pathogens and most antigens differentially expressed by tumor cells are low, their expansion in vitro usually necessitates repeated sensitizations with antigen bearing antigen presenting cells(APCs) that are often limited in supply and both time consuming and logistically difficult to produce. Furthermore, for patients receiving allogeneic HSCT transplants from HLA disparate donors, the clinical activity of donor-derived virus-specific T cells sensitized on autologous APCs may be nullified if the immunodominant T cells generated are restricted by HLA alleles not shared by the host (4).
To address these constraints, several groups have proposed the use of different types of artificial antigen presenting cells (AAPCs) using either cell based (immortalized cell lines of Drosophila, mouse or human origin) or acellular systems (polymer beads or liposomes; reviewed in Ref. (10). AAPCs, engineered to express both an HLA allele and important co-stimulatory molecules can present immunogenic viral or tumor antigens on a single expressed HLA allele so as to generate HLA - restricted T cells of desired specificity (11-16). Alternatively, AAPCs expressing co-stimulatory molecules alone have been employed to non-specifically stimulate expansion of unselected or antigen-specific T cells for therapeutic use (17-19). Latouche et al (12) were the first to demonstrate that mouse 3T3 cells sequentially transduced to express the human co-stimulatory molecules ICAM-1, B7.1 and LFA-3 as well as human β2 microglobulin and the HLA- A*0201 heavy chain could be used as an AAPC to sensitize human A*0201+ T cells against co-expressed virus-specific or tumor selective antigenic peptides. Subsequently, Papanicolaou et al (20) demonstrated that CMV-specific T cells could be generated from seropositive HLA- A*0201+ donors at high frequency in vitro by sensitization with the same 3T3-based HLA- A*0201-expressing AAPCs transduced to express either the CMVpp65 peptide NLVPMVATV presented by HLA- A*0201 or the full length CMVpp65 protein. Since then, other studies employing the human K562 leukemic cell line transduced to express HLA- A*0201 or other AAPCs expressing this allele have confirmed the potential of AAPCs to induce antigen-specific HLA- A*0201-restricted T cells (13, 21). However, to date there have been no reports of the construction or function of AAPCs expressing other HLA alleles. While HLA- A*0201 is the most commonly inherited class I allele and AAPCs expressing this allele have provided a useful proof of principle, at least 60% of patients lack this allele (22). Furthermore, we were concerned that the potential of AAPCs expressing this HLA allele to process and present epitopes of CMVpp65 that elicit functional cytotoxic T cells might be overestimated, since HLA- A*0201 restricted human T cells capable of killing CMV infected cells are almost exclusively specific for a single peptide, NLVPMVATV (23).
In the present study, we established a panel of AAPCs each expressing a single common HLA class I allele (i.e. HLA- A*0201, A*0301, A*2402, B*0702, B*0801 or C*0401) which could be used to sensitize T cells from up to 80% of our healthy hematopoietic progenitor cell transplant donors against virus specific or tumor selective antigens. We then evaluated each AAPC for its capacity to sensitize and stimulate the expansion of HLA-restricted T cell populations specific for peptides of CMVpp65 known to be expressed on CMV infected human cells. We chose to evaluate responses to the CMVpp65 protein because it is the immunodominant antigen that is most frequently targeted by CD8+ cytotoxic T cells in CMV seropositive donors (24, 25) and because a large series of epitopes of CMVpp65 have been identified that can be presented by the HLA alleles in the AAPC panel and can elicit virus-specific cytotoxic T cell responses (25-27). Our results demonstrate that this panel can be used to generate CMVpp65-specific IFNγ+ and cytotoxic CD8+ T cells of desired HLA restriction, including T cells specific for subdominant epitopes that may be represented at low or undetectable frequencies in T cells sensitized with peptide-pool loaded autologous cytokine-activated monocytes (CAMs) or dendritic cells.
Blood samples were obtained from 13 healthy CMV- seropositive consenting donors according to protocols approved by the Institutional Review Board of Memorial Sloan-Kettering Cancer Center (New York, NY); 5 leukocyte units from healthy CMV seropositive volunteer donors were purchased from the New York Blood Center. Among these donors, 8 expressed HLA- A*0201 (1 co-expressed A*0301, 1 co-expressed HLA B*0801), 7 expressed HLA- A*2402 (2 co-expressed A*0201), 5 expressed HLA- B*0702 (3 co-expressed A*0201), 4 expressed C*0401 (2 co-expressed A*2402, and 1 co-expressed A*0301), 2 expressed HLA- B*0801 and 1 donor expressed HLA- A*0301. HLA typing was performed by the Histocompatibility Testing Laboratory at Memorial Sloan Kettering Cancer Center, by analysis of HLA allele-specific nucleotide sequences using standard high-resolution typing techniques. Donor CMV serostatus was determined by standard serologic techniques in the clinical microbiology laboratory at Memorial Sloan-Kettering Cancer Center.
The panel of AAPCs was constructed according to methods previously described(12, 20). Briefly, NIH 3T3 fibroblast cell lines (ATCC, Manassas, VA), were first transduced sequentially with 4 replication incompetent SFG retroviral vectors encoding human B 7.1 (CD80), ICAM-1(CD54) and LFA-3 (CD58) and human β2-microglobulin. After each transduction, the cells were sterilely sorted by fluorescence-activated cell sorting (FACS) (Moflo, Beckman-Coulter, CA) to select populations expressing high levels of each vector encoded human protein in the sequence. The resultant cell line was called 3T3-4. The cDNA sequences for HLA- A*0201, A*2402, A*0301, B*0702, B*0801 and C*0401 were obtained from the International Histocompatibility Working Group (IHWG) Workshop Cell and Gene Bank (Fred Hutchinson Cancer Center, Seattle WA, USA). Each HLA cDNA sequence was then amplified and cloned into an SFG retroviral vector as previously described (12, 25). Separate aliquots of the 3T3-4 line were then transduced with a vector encoding a single HLA heavy chain. The transduced cells were then sorted and cloned to isolate AAPCs with the highest level of HLA and co-stimulatory molecule expression. These cells are referred to as AAPC class-I. The expression of all the transduced co-stimulatory molecules as well as HLA alleles was verified by flow cytometry using FITC labeled anti-CD80, PE-Cy5 labeled anti-CD58, APC labeled anti-CD54, FITC-labeled anti- β2-microglobulin, and PE or FITC labeled anti-human HLA class-I antibodies (Beckton Dickinson, CA). AAPCs were maintained in Dulbecco's Modified Eagle Medium (DMEM; Invitrogen Inc., Carlsbad, CA) supplemented with 10% heat inactivated defined calf serum (DCS; Hyclone, Logan, UT)
To enhance and stabilize expression of certain HLA alleles, specifically HLA- A*2402 and B*0801, the SFG vectors encoding the HLA sequences were modified by inserting the Kozak sequence GCCGCCACC immediately prior to the AUG initiator codon of the HLA gene.(28)
The cDNA sequence of CMV pp65 was kindly provided by Dr. N. Cereb (Histogenetics Inc. and the Center for Genetic Polymorphism, Hawthorne, NY.). This gene, linked via an internal ribosomal entry site (IRES) to puromycin N-acetyl transferase, was then cloned into an SFG vector and then used to transduce aliquots of AAPCclass I expressing each HLA allele. Cells co-expressing CMVpp65 were then selected by propagation in media containing puromycin dihydrochloride (10 μg/ml) (Sigma-Aldrich, Inc). These cells will be referred to as AAPCclass-Ipp65.
PBMCs were isolated from whole blood using Ficoll hypaque gradient separation (Accurate Chemical & Scientific, Westbury, NY.), and suspended in IMDM with 10% human AB serum at a concentration of 10 × 106/ml. Aliquots of 2 ml per well were then plated in a 6-well tissue culture plate (Corning Inc, Corning, NY) to facilitate adherence of monocytes. After 1-2 hours, non-adherent mononuclear cells were removed, and adherent monocytes cultured with 2ml serum free IMDM per well containing 2000 IU(50μl) of GM-CSF (Immunex, Seattle, WA) and 700 U (25μl) of IL-4 ( R&D systems Inc, Minneapolis, MN USA). On days 2 and 4, 2000U GM-CSF (50μl) and 700 U of IL-4(25 μl) were again added to each 2ml culture. On day 5, tumor necrosis factor-α (SIGMA, St. Louis) was added to achieve a final concentration of 5ng/ml, Interleukin-1β to 5ng/ml, interleukin-6 (R&D systems, Inc, Minneapolis, MN USA) to 150 ng/ml and prostaglandin-E2 (Calbiochem, La Jolla, CA USA) to 1μg/ml to induce final maturation of the CAMS. On day 7 the mature CAMS were harvested, characterized as to the expression of HLA Class II, CD14 and co-stimulatory molecules by FACS immunocytofluorometry, counted, aliquoted, and used for sensitization of T cell lines as detailed below.
T cells were sensitized using APCs loaded with a pool of 138 overlapping pentadecapeptides spanning the sequence of CMVpp65 as described previously (31). Subpools of these peptides, or single peptides, were used to identify epitopes of CMV pp65 eliciting T cell responses observed using an epitope mapping strategy previously described (31, 32). Each of the 138 pentadecapeptides was synthesized according to specifications of validated sequence, purity, sterility and absence of endotoxin (Natural and Medical Sciences Institute, Tuebingen, Germany).
Aliquots of 0.5 × 106 AAPC class-I were allowed to adhere in 6 well plates for 6-8 hours in 1 ml serum free DMEM per well and pulsed with the pool of synthetic overlapping CMVpp65 pentacedapeptides as described previously (31). 2.5 μg of each nonamer peptide (Research Genetics, Huntsville, AL) or single pentadecapeptide or a total of 24 μg pooled pentadecapeptides (2 μg of each pentadecapeptide in the subpools and 0.18 μg of each pentadecapeptide in the complete pool) was added to 1 × 106 APCs in 1 ml serum free IMDM for 3 hours at room temperature. An additional 2 ml IMDM was then added and the entire peptide containing supernatant removed. The CMVpp65 peptide pool-loaded (PL) AAPC class-I were then cultured in 2ml of AIM V medium (Invitrogen Inc, Carlsbad, CA) with 5% DCS and irradiated to 1500cGy prior to addition of T cells for sensitization.
Aliquots of 1 × 106 autologous CAMs were suspended in 1ml serum free IMDM in 15 ml tubes and pulsed with CMV pp65 peptide pool (referred to as PL CAMs) using the same approach as described above.
Following separation of PBMC from whole blood (ficoll hypaque), T cells were enriched by depletion of CD19+, CD14+ and CD56+ cells, RBC, granulocytes and DCs using monoclonal antibody coated immunomagnetic beads (Pan T-Cell Isolation Kit II, Miltenyi Biotec Inc, Auburn, CA USA) as previously described (29-31).
Aliquots of these T cells were then sensitized by co-culture with one of the following set of antigen presenting cells: (1) AAPCs Class-Ipp65, (2) CMVpp65 peptide pool loaded AAPC Class-I (PL AAPC Class-I), (3) CMVpp65 peptide pool loaded CAMs (PL CAMs), (4) AAPC Class-I alone or (5) autologous CAMs alone.
0.5 × 106 cell aliquots of each type of AAPC were co-cultured with 5 ×106 T cells per well in 6 well plates in AIM V medium with 5 % DCS. T cells were maintained at a concentration of 1× 106/ml and were re-stimulated with PL AAPC class I or AAPC class-I pp65 every 10 days at an effector to stimulator ratio of 10:1. T cell cultures were supplemented with IL-2 (20U/ml) starting on day 12 and then 3 times per week.
T cells were sensitized using autologous CAMs and PL CAMs as previously described (29, 31). Briefly, 0.5 × 105/ml irradiated CAM (3000 rads) were cultured with 1× 106/ ml T cells in 20 ml volume of IMDM medium supplemented with 10% heat inactivated human AB serum (Gemini, CA) in 25 cm2 flasks for 7 days. T cells were re-stimulated weekly with autologous PL CAMs, at an effector to stimulator ratio of 20:1. Interleukin-2 (IL-2) (20U/ml) was added to T cell cultures on day 10, and 2-3 times per week thereafter.
Tetramer analysis was performed on days 0, 7, 14 and 21 for all T cell lines using commercially available CMVpp65 MHC-peptide tetramers for HLA- A*0201, A*2402 and B*0702 bearing peptide sequences NLVPMVATV, QYDPVAALF and TPRVTGGGAM respectively (Beckman Coulter, Inc Fullerton, CA). Analyses performed on day 21 were used for comparisons between T cells sensitized using the different APCs. T cells were incubated with CD3 FITC, CD8 PE , CD4 PerCP (BD Bioscience, San Jose, CA) and APC conjugated tetrameric complex for 20 minutes on ice. The stained cells were washed and subsequently analyzed by FACS using FACSCalibur flow cytometer with dual laser for 4-color capability. Data were analyzed using Flowjo software (Tree Star Inc, Ashland, OR). T cells were gated on CD3 and CD8 positive cells to determine the percentage of tetramer positive CD8 T lymphocytes.
T cell responses to specific peptides or subpools of CMV pp65 were quantitated by measuring the number of IFNγ positive T cells generated upon secondary stimulation with autologous APCs loaded with the peptides or peptide pool (PL) of interest, according to the technique of Waldrop et al (33) as modified by Koehne et al (29). Peptide-loaded autologous PBMC or autologous BLCL were used as APC to stimulate the responding T cells.
All T cells lines were assessed for their capacity to lyse CMVpp65 loaded targets using a standard 51chromium release assay as previously described (29, 30). Targets used in all experiments consisted of a panel of EBV-BLCL, each sharing with T cells of a given donor a single HLA allele. These cells were pulsed, as specified for a given experiment, with the complete pool of CMVpp65 peptides, or specific sub-pools thereof, single pentadecapeptides, or a CMV pp65 nonamer known to be presented by that allele (e.g. NLVPMVATV for HLA- A*0201, QYDPVAALF for HLA A2402 and TPRVTGGGAM and RPHERNGFTV for HLA B0702) (31). Targets pulsed with other CMV pp65 peptides not presented by the shared HLA allele were used as controls. Absence of EBV reactivity was ascertained by lack of cytotoxicity against BLCL lines without peptide. HLA restriction was identified by reactivity against targets pulsed with an identified peptide epitope presented on a specific shared HLA allele, and absence of reactivity against peptide loaded on either EBV BLCL bearing other shared alleles or fully mismatched EBV BLCL.
CMV peptide-HLA tetramer+ T cells were analyzed for TCRVβ repertoire via flow cytometry using commercially available kit containing antibodies to 24 subfamilies of the Vβ region of the human TCR (IO Test® Beta Mark, Beckman Coulter, Inc, France) according to procedures provided by the manufacturer (34).
Sequential transduction and sorting of the NIH 3T3 cells with SFG vectors directing the constitutive expression of the human co-stimulatory molecules B7.1, 1CAM-1 and LFA-3 as well as β2 microglobulin permitted the selection of a transduced line, termed 3T3-4, which exhibits stable, high expression of each of the introduced co-stimulatory molecules as well as β2 microglobulin (supplemental Fig 1A). The level of expression of these transduced genes has been sustained over more than two years of re-culturing.
Transduction of aliquots of 3T3-4 cells with SFG vectors encoding specific HLA class-I heavy chains also permitted selection and cloning of AAPCs expressing each of the single HLA alleles introduced. For AAPCs selectively expressing HLA- A*0201, A*0301, B*0702 or C*0401, the level of HLA allele detected on the surface of the AAPCs, as assessed by FACS analysis with a FITC-labeled HLA class-I specific monoclonal antibody, has remained stable for periods of culture exceeding 5 months (supplemental Fig 1B). However, even when the AAPCs transduced with vectors encoding HLA- A*2402 or B*0801 were cloned for high expression of HLA, sequential analyses of these AAPCs over an additional 4 weeks of culture demonstrated progressive reductions in the proportion of cells expressing these HLA alleles and in the level of each HLA expressed. In contrast, expression of β2 -microglobulin by these AAPCs was sustained.
In order to enhance and potentially stabilize expression of HLA- A*2402 and B*0801 on transduced AAPCs, we modified the SFG vectors by inserting an initiation sequence GCCGCCACC described by Kozak (28, 35) into the 5′ end of the leader sequence of the gene encoding each of these HLA alleles. These modified vectors were then transduced into 3T3-4 cells. Thereafter, cells expressing each allele were isolated by FACS sorting, and compared with 3T3-4 cells transduced with the unmodified vector for expression of the transduced HLA allele over extended periods of culture. AAPCs transduced with vectors encoding HLA- A*2402 or B*0801 lacking the Kozak sequence exhibited progressive reductions in both the number of cells expressing the vector encoded HLA allele and the level of HLA expressed by the transduced cells (supplemental Fig. 2 A and B). AAPCs transduced with vectors including the Kozak sequence exhibited higher initial expression of the transduced HLA allele. In the AAPCs transduced to express HLA- B*0801, this high expression was sustained through the 4 weeks of observation (supplemental Fig. 2B). In the HLA- A*2402 transduced AAPCs, expression of the allele decreased slightly in the first week post transduction, but was sustained thereafter (supplemental Fig. 2A).
We compared the level of HLA and co-stimulatory molecules expressed on the AAPCs with that expressed by T cell donor derived autologous CAMs and EBV BLCLs. Following maturation in-vitro, the CAMs used in our studies strongly expressed both HLA class-I and Class-II, CD40, CD86, LFA-3 and ICAM-1. They were also CD14+, but CD83 dim. As such, they exhibited the phenotype of monocyte derived dendritic cells. As shown in a representative sample in Figure 1, the AAPCs exhibited higher expression of B7.1, LFA-3 and ICAM-1 than CAMs, and comparable expression of HLA class-I.
Following establishment of this panel of AAPC class I, aliquots of cells expressing each HLA allele were further transduced with an SFG bicistronic vector encoding the full length sequence of CMV pp65 and puromycin-N-acetyltransferase. After selection in medium containing puromycin, expression of CMV pp65 by AAPC class I-pp65 was found in >90% of the cells by indirect immunofluorescence staining (supplemental Fig. 3 ).
We next comparatively assessed each AAPC for its capacity to sensitize human T lymphocytes either as AAPCs Class-I pulsed with CMV pp65 peptide pool (PL AAPC Class-I) or as AAPC class I-pp65. Accordingly, T cells from groups of up to 8 CMV seropositive donors, sharing an HLA allele expressed by a given AAPC, were sensitized with either PL AAPCclass-I or AAPCclass-I pp65 for 21 days in vitro, and then assessed for the number of CMV peptide specific T cells generated.
For T cells sensitized with AAPCs expressing HLA A0201, A2402, and B0702, we initially measured the number and proportion of T cells binding HLA-peptide tetramers. One representative example of the increases in tetramer+ T cells is presented for each of these alleles in Fig. 2A.
Quantitations of tetramer+ T cells generated after sensitization with PL CAMs, PL AAPCsclass I sharing single HLA alleles with the donor T cell, as well as responses to AAPCclass I pp65 bearing the same HLA allele are shown in Fig. 2, B and C. Cultures sensitized with PL AAPCclass I exhibited 50- to200-fold increases in T cells binding tetramers containing known immunogenic epitopes presented by the HLA alleles expressed by the sensitizing AAPC. These responses were similar to those elicited in response to the same epitopes when the T cells were sensitized with autologous PL CAMs. Strikingly, when the same T cells were sensitized with AAPC class I-pp65, they generated a 550- to 1000-fold increase in T cells binding the same tetramers. As shown in Fig. 2C, the absolute numbers of peptide-specific T cells generated in response to AAPC class I-pp65 were higher for each allele tested. For the group of HLA- A*0201+ donors, this increase was statistically significant (p<0.03).
As also shown in Fig. 2C, the absolute yields of tetramer positive T cells generated in response to AAPCclass I and AAPCclass I-pp65 expressing HLA- A*0201 tended to be higher than those generated against AAPCs expressing either HLA- A*2402 or B*0702.
We also analyzed the phenotype of the tetramer+ T cells at sequential times in their generation. As shown in one representative culture presented in Fig. 3A, tetramer positive T cells were CD8+ and predominantly of a central memory phenotype at day 7 of culture. By day 10, almost half of the cells exhibited an effector memory phenotype. At days 17 and 24, the tetramer+ cells were almost exclusively of an effector memory phenotype.
We also compared the T cell receptor repertoires of T cells generated against epitopes presented by PL CAMs versus AAPCclass I-pp65 by examining the Vβ of tetramer+ T cells. Fig. 3B presents results from one representative donor and demonstrates that the Vβ chains represented in the TCRs of T cells sensitized with HLA- A*0201 AAPC class I-pp65 were the same as those detected among T cells sensitized with autologous PL CAMs.
When analyzed for T cells generating intracellular 1FNγ in response to secondary stimulation with PL autologous donor PBMC, the PL AAPCclass I and AAPCclass I-pp65 expressing HLA- A*0201, A*2402, B*0702, B*0801 and C*0401 each elicited significant numbers of IFNγ+ CMV pp65-specific T cells (Fig. 4A). Again, absolute yields of CMVpp65-specific IFNγ+ T cells from cultures sensitized with the AAPCclass I-pp65 were consistently greater than those generated from T cell cultures sensitized with PL AAPCclass I . The absolute numbers of IFNγ+ T cells generated in response to any one of these AAPCclassI-pp65 expressing an HLA allele shared by the donor were similar to the total numbers of IFNγ+ T cells generated after sensitization with PL autologous CAMs.
T cells sensitized with each of these PL AAPCclassI and AAPCclass I-pp65 also exhibited significant cytolytic activity against PL EBV-BLCL but did not lyse the same EBV-BLCL without peptide.
We also compared T cell cytotoxic activity against PL EBV-BLCL matched at the HLA allele shared by the donor and sensitizing AAPC. In these comparative assays, as shown in Fig. 4B, T cells sensitized with PL AAPCclass I and AAPC class I-pp65 exhibited equivalent cytotoxic activity against these targets, but did not lyse unloaded HLA-sharing EBV-BLCL or peptide loaded targets lacking the restricting HLA allele. When T cells from the same donor were sensitized with PL CAMs and tested against PL EBV-BLCL sharing single HLA alleles, cytotoxic responses were detected against PL EBV-BLCL sharing A*0201, A*2402 and B*0701 but not PL EBV-BLCL sharing C*0401 or B*0801, reflecting the fact that responses restricted by these alleles were subdominant in each of the donors studied.
We then examined to what degree the proportion of CMVpp65 specific IFN γ + CD8+ T cells generated using PL CAMs or PL AAPC class I was correlated with the level of in-vitro cytotoxicity exhibited by the sensitized T cells against peptide pool loaded autologous BLCL. As shown in Figure 4C, the % cytotoxicity was significantly correlated with the proportion of IFNγ+CD8+ T cells for T cells sensitized with either PL AAPC class-I (r=0.53) or AAPC class-Ipp65. However the same tight correlation was not found for T cells sensitized using PL CAMs (r=0.44). Among the T cells sensitized with PL CAMs, we usually observed T cells reactive against more than 1 epitope of CMVpp65. While such epitopes might elicit IFNγ+ responses, their relative capacity to lyse peptide loaded targets may differ (23) potentially reflecting competition between cytotoxic T cells todifferent epitopes presented on the cell surface. In contrast, T cells sensitized with PL or transduced AAPC are directed against specific targets. As a result, correlation between IFNγ+ T cells and cytotoxicity in circumstances wherein there is target excess would be expected to be high.
T cells from 3 HLA- A*0301+ donors, when sensitized with either PL HLA- A*0301 AAPCclass I or HLA- A*0301+ AAPCclassI-pp65, did not generate CMVpp65- specific T cells. When T cells from these donors were sensitized with PL autologous CAMs, they each generated CMVpp65-specific 1FNγ+ and cytotoxic CD8+ T cells. However, no HLA- A*0301 restricted CMVpp65 specific T cells were detected (data not shown).
Our studies of T cells generated in response to AAPCclass I-pp65 expressing HLA- A*0201, B*0701 or A*2402 suggested that these transduced AAPCs elicited responses to immunogenic epitopes known to be presented by human APCs expressing these alleles as reflected by the generation of high numbers of T cells binding HLA-peptide tetramers bearing such epitopes. We wished to ascertain whether these transduced AAPCs might also process and present other CMV pp65 epitopes not normally presented by human APCs. Accordingly, we mapped the epitopes recognized by T cells sensitized with PL autologous CAM or AAPCclass I and compared them to those elicited in response to AAPCclassI-pp65. Results for AAPC expressing A*2402, B*0702, C*0401 and B*0801 are presented in Fig. 5. As can be seen, the pattern of responses against the matrix of subpools of CMVpp65 15-mers for T cells sensitized with AAPCclass I-pp65 and PL AAPCclass I were largely identical. Epitope mapping demonstrated that the T cells sensitized with AAPCclassIpp65 expressing HLA- A*2402 and C*0401 recognized peptides 85 and 86 containing the nonamer QYDPVAALF shared in subpools 1,2 and 20 (Fig. 5, A and C). Responses to this specific nonamer were also confirmed. T cells sensitized with HLA B*0702+ AAPCclass I pp65 responded to pools 9 and 21 containing the HLA- B*0702 epitope TPRVTGGGAM while those sensitized with PL HLA- B*0702+ AAPCclass I also responded to the peptide RPHERNGFTV present in subpools 7 and 18 (Fig. 5B). Each of these peptides is a known immunogenic epitope of CMVpp65 capable of eliciting T cell responses restricted by HLA- B*0702 (26, 27).
T cells reactive against the same peptides as those presented by PL AAPCclass I and AAPCclass I pp65 were also detected among T cells sensitized with PL CAM with three exceptions: 1) T cells from donor C, sharing HLA- C*0401, when sensitized with PL CAMs, selectively responded to a peptide, VYALPLKML, presented by HLA- A*6801, and did not respond to the QYDPVAALF elicited by PL HLA- C*0401+ AAPCclassI and HLA- C*0401+ AAPCclassI pp65 (Fig. 5C). 2) T cells from donor B, sharing HLA- B*0702 responded to RPHERNGFTV when sensitized with either PL CAMs or HLA- B*0702+ AAPC class I , but only responded to TPRVTGGGAM when sensitized with HLA- B*0702+ AAPCclass I pp65 (Fig. 5B). 3) T cells from another donor (Fig. 5D), whose genotype includes HLA- B*0801 as well as HLA- A*1101, responded exclusively to pools 3, 4 and 13 that contain pentadecapeptides 3 and 4 which share the GPISGHVLK peptide known to be immunogenic when presented by the HLA- A*1101 expressed by her own CAMs while the same donor's T cells sensitized with PL HLA- B*0801+ AAPCclassI or HLA- B*0801+ AAPCclass I pp65 responded to two peptides LTMTRNPQPF and LARNLVPMV contained in peptides 63/64 and peptide 123 respectively. Both of these peptides have high predicted affinity for HLA- B*0801 (36, 37).
To further ascertain whether the murine AAPCclass-Ipp65 presented epitopes on their expressed HLA alleles that were also processed and presented by human antigen presenting cells, we epitope mapped the responses of T cells from a series of donors sensitized with these AAPCclass-Ipp65 to determine whether they responded to epitopes of CMVpp65 known to be presented by the expressed HLA allele. The results are summarized in Table.1. As can be seen, all HLA- A*0201+ seropositive donors sensitized with HLA- A*0201+ AAPC class-Ipp65 generated T cells specific for the NLV peptide presented by this allele. Similarly, all HLA- B*0702+ donors responded to the TPR peptide. Among the 7 HLA- A*2402+ donors tested, 5 responded to the QYD nonamer presented by this allele; two others responded to newly identified epitopes presented by HLA- A*2402 that were also recongnized by their T cells when sensitized with PL CAMs. Similarly, 2 of 3 HLA- C*0401+ donors responded to the QYD nonamer known to be presented by this allele; T cells from the third C*0401+ donor, when sensitized with either AAPC class-Ipp65 or autologous PL CAMs responded to another epitope presented by this allele, KDVALRHVV. Taken together, these data provide evidence that the murine 3T3 derived AAPCclass-Ipp65 are able to process and present epitopes of CMVpp65 on their expressed HLA alleles that are known to be presented by the same alleles on human APCs.
To ascertain if we could generate antigen-specific T cell responses to sub-dominant epitopes of CMVpp65 using this panel of AAPC, we compared T cell responses from selected donors after sensitization with PL CAMs to those of T cells sensitized with PL AAPCclass-I expressing different class I alleles expressed by the same donor.
As shown in Fig. 6A, when T cells from a donor co-expressing HLA- B*0702 and HLA- A*0201 were sensitized with PL CAMs, a large population of tetramer+ T cells specific for the CMV pp65 epitope TPRVTGGGAM presented by HLA- B*0702 were generated, as well as a significant (14%) population of T cells binding HLA- A*0201 tetramers containing the nonamer NLVPMVATV. Functional analysis and epitope mapping revealed that these T cells selectively generated IFNγ in response to two epitopes presented by HLA- B*0702, the TPRVTGGGAM peptide noted above and RPHERNGFTV; only a small number of IFNγ+ T cells were generated in response to the NLVPMVATV presented by HLA- A*0201 (Fig. 6B). Furthermore, these T cells lysed HLA- B*0702+ EBVBLCL loaded with these epitopes but failed to lyse HLA- A*0201+ EBVBLCL loaded with the NLV peptide (Fig. 6C). In contrast, when the same donor's T cells were sensitized with HLA- A*0201+ AAPCclassIpp65, T cells restricted by HLA- A*0201 and specific for the NLVPMVATV peptide were generated, as demonstrated by tetramer analysis (Fig. 6A), generation of IFNγ+ T cells in response to specific peptide containing subpools and targeted nonamers (Fig. 6B), and the capacity of these sensitized T cells to lyse HLA- A*0201+ human targets loaded with this peptide (Fig. 6C).
Table II summarizes a comparison of the responses of T cells from 7 donors sensitized with PL CAMs with those of T cells sensitized with PL AAPCclassI and AAPCclassI-pp65 from the same donor, each expressing a different HLA allele expressed by the donor. In all donors tested, sensitization with PL CAMs selectively induced T cells specific for 1-2 immunodominant CMV pp65 epitopes. While sensitization with peptide loaded or transduced AAPCs expressing the dominant presenting HLA alleles regularly elicited responses to the same dominant epitopes, we could also generate comparable cytotoxic T cell responses to subdominant epitopes which were either not produced or only present at low frequencies in T cells sensitized with PL CAMs (Table II).
We here describe a panel of murine 3T3 cell-based AAPCs, each expressing human ICAM-1, B7.1 and LFA-3 as well as β2 microglobin and a single HLA class- I heavy chain: HLA- A*0201, A*0301, A*2402, B*0702, B*0801 or C*0401. The potential utility of this panel is suggested by the fact that, 78% of the 168 patients at our center received an HLA non -identical HSCT between 2001 and 2005 because we could not identify an HLA matched unrelated donor from the National Marrow Donor Program (NMDP) registry who inherited one or more of these HLA alleles. Planned expansion of this panel to include AAPCs expressing HLA- A*0101, A*1101 and B*4402 will cover >90% of patients for whom virus-specific T cells restricted by an HLA allele expressed by one of the AAPCs in the panel can be generated.
We chose to construct this panel of AAPCs from a murine 3T3 cell line rather than a human cell line such as K562 that has been proposed by other groups (17, 21) primarily because of concerns that K 562 and other human cells deficient in their expression of HLA could process and present minor allo-antigens in the context of a transduced HLA allele that might stimulate donor T cells capable of inducing graft-versus-host disease following adoptive transfer. We had additional concerns regarding the use of the K 562 cells because they expresses the human MHC Class-I chain related genes (MICA and MICB) which, on the one hand, can be a significant allo-antigen (38-40) and on the other, can release soluble MICA and MICB, which, by downregulating surface expression of NKG2D on CD8+ T cells can interfere with their effector functions (41). In contrast, while 3T3 cells might elicit a xeno-antigenic response, our studies have shown that T cells generated against either peptide-pulsed or CMV pp65-transduced AAPCs generate IFN-γ+ T cells and lyse targets upon secondary stimulation only against human targets bearing CMV pp65 epitopes and the T cells' restricting the HLA allele, no alloresponses are recorded. A similar level of alloreactivity of T cells sensitized with the AAPCs coexpressing HLA- A*0201 and telomerase has been reported (42). Similarly, 3T3 cells genetically modified to express CD 40L have also been safely employed to provide costimulation in trans to T cells sensitized in-vitro against autologous melanoma cells for adoptive therapy (43). Furthermore, clinical trials using human epithelial cells cultured on a 3T3 cell matrix for skin transplants (44-46)or corneal repair (47) have provided evidence indicating that their capacity to elicit alloantigenic responses is low.
The NIH 3T3 based AAPCs in this panel stably express each of the transduced human co-stimulatory molecules as well as β2 microglobulin. Similarly, the expression of HLA- A*0201, A*0301 and B*0702 has remained constant in cultures for more than 5 months. The basis for the loss of expression of HLA- A*2402 and B*0801 in AAPC transduced with the same vector is unclear. It is unlikely that preferential outgrowth of untransduced AAPCs was the cause, since even clones selected for high expression of these alleles exhibited this fall off. Gene silencing from selective insertion of SFG vectors at susceptible sites was also thought to be unlikely since the same instability of HLA expression was exhibited in 2-3 different aliquots of transduced 3T3-4s. Based on the possibility that the initiation sequences of these alleles are susceptible to inhibition of translation, the vectors were modified to include the KOZAK sequence (28, 35) immediately upstream of the initiator codon of the gene encoding these HLA H chains. This sequence has been shown to optimize mRNA translation when inserted proximal to AUG start codons of genes in eukaryotic cells (28, 48, 49). This permitted generation of AAPCs with sustained high expression of each of these alleles
Our results demonstrate that AAPCs individually expressing HLA- A*0201, A*2402, B*0702, B*0801 or C*0401, can each stimulate the generation of large populations of CMVpp65 specific HLA-restricted T cells. From a starting population of 10 × 106 unselected T cells containing approximately 104 CMV pp65 peptide reactive T cells, sensitization with PL AAPCclass I or AAPCclass-I pp65 can generate as many as 5 × 106 to 1 × 108 epitope specific T cells over 3 weeks of culture. Such numbers can provide doses of CMVpp65 specific T cells well within the range of numbers used in ongoing trials of adoptive cell therapy for the prevention and or treatment of CMV infections (2, 3, 50). The yields of tetramer+ CMV pp65 epitope specific T cells generated using AAPCclass I-pp65 were consistently higher than those generated in response to PL CAMs or AAPCclass I. This may reflect the potential of living APCs like AAPCclass I pp65 to provide more effective sensitization of T cells and superior yields of Ag-specific T cells by continuously processing and presenting immunogenic epitopes throughout in- vitro culture. Indeed, greater or equal number of peptide specific T cells have also been generated using dendritic cells transduced to express an immunogenic CMVpp65 peptide presented by HLA- A*0201(51).
Although T cell sensitization with AAPCclass I pp65 regularly induced higher yields of CMVpp65-specific T cells, the utility of AAPC class-Ipp65 would be limited if the epitopes of CMVpp65 presented by these mouse 3T3 derived AAPCs differ from those recognized by T cells sensitized with PL autologous CAMs or AAPCclass I. To be presented by AAPCclassI-pp65, the CMV pp65 protein must be processed by the murine ubiquitin proteosome pathway, and then loaded on the expressed HLA heavy chain/β2microglobulin complex by murine TAP proteins (52-55). Earlier studies suggested that the antigenic peptides presented would differ significantly due to differences between the murine and human TAP proteins. (56) However, in mice transgenic for HLA- A*0201, A*1101 or B*0702, immunization with peptide epitopes of either viruses or human tumor antigens with high binding affinity for these HLA alleles, and known immunogenicity in man, elicited murine T cell responses specific for the same peptides when presented by the transgenic human HLA alleles (57-62). These studies thus provided evidence that mouse cells could process proteins and present on the transgenic HLA allele the same epitopes as naturally presented on human APCs. Reports from our own group also demonstrate that 3T3-based HLA- A*0201 AAPCs transduced to express either CMVpp65 or telomerase can elicit human T cell responses specific for epitopes normally presented by HLA- A*0201 on human cells(20, 42).
The present report confirms and significantly extends prior studies with 3T3-based AAPCs expressing HLA- A*0201 (12, 20, 42), providing evidence that AAPCs expressing several HLA class I alleles can process and present the same epitopes of CMVpp65 that would normally be presented by human APCs. Sensitization with AAPCclassI-pp65 expressing HLA- A*0201, A*2402 and B*0702 elicited tetramer+ T cell responses against the same CMV pp65 epitopes that were recognized by T cells presented by these HLA alleles on autologous CAMs. The TCRs represented in the tetramer+ T cells responding to AAPCclassI-pp65 were also closely matched with those generated in response to PL CAMs. Furthermore, as shown in Figures Figures55 and and66 and Table 2, epitope mapping of T cell responses sensitized with AAPC class-Ipp65 expressing HLA- A*0201, A*2402, B*0702 and C*0401 demonstrated a high frequency of responses against penta-decapeptides and specific nonamers within those known to be immunogenic peptides of CMVpp65 presented by each HLA allele. In those instances in which sensitization with AAPCclass-Ipp65 led to generation of HLA-restricted T cells specific for previously unreported epitopes, T cells responsive to the same epitopes were also consistently detected in cultures sensitized with autologous PL CAMs. Nevertheless, the possibility that processing of proteins such as CMVpp65 by AAPCs of murine origin might yield certain HLA-binding peptide epitopes with enhanced immunogenicity remains open.
Sensitization of T cells with PL CAMs regularly stimulated the propagation of T cells specific for only 1-2 epitopes of CMV pp65 and restricted by no more than 1-2 of the donor's HLA alleles.(31). Such preferential expansion of T cells specific for 1-2 immunodominant epitopes has also been documented in other studies of responses to CMVpp65 and to IE-1 and immunogenic proteins of other viruses (23, 31, 32, 63-65). The basis for such immunodominance is complex and poorly understood. The in vivo selection of dominant responses is likely influenced by the characteristics of the causative viral pathogen and the stage of infection (66, 67), the host cells that process viral proteins and present potentially immunogenic peptides on specific HLA alleles, the quantity and HLA-binding affinity of the antigenic peptides presented, and the types and affinities of the T cell responses elicited (61, 63-71). Studies by Bihl et al for EBV and HIV (70)and Lacey et al for CMVpp65 (72) demonstrate that viral epitopes presented by HLA B alleles elicit stronger responses irrespective of their HLA-binding affinity.
Such immunodominant responses pose a special challenge to the development of effective T cell immunotherapies for the increasing proportion of patients now receiving transplants from HLA disparate donors. Virus-specific T cells that are generated from transplant donors by sensitization with autologous APCs presenting viral antigens, will be therapeutically active only if the immunodominant T cells that preferentially expand in- vitro are restricted by HLA alleles shared by the transplant recipient, and not by HLA alleles unique to the donor. While selective sensitization of donor T cells with peptide epitopes known to be shared by donor and host can circumvent this limitation (26, 27), for most viruses only a limited number of consistently immunogenic peptides have been identified.
Accordingly, we examined the potential of our panel of AAPCs to induce populations of functional T cells specific for subdominant epitopes of CMVpp65 presented by a desired HLA allele which could not be elicited when T cells were sensitized with PL autologous CAMs. As shown in Figure 6 and Table 2, we were able to use this panel of AAPCs to consistently generate CMVpp65 IFNγ+ CD8+ T cells that lysed human cell targets presenting subdominant epitopes of CMVpp65 presented by the restricting HLA alleles expressed by the AAPC from each of the donors tested. This included donors whose T cells, when sensitized with PL autologous CAMs, failed to respond to the epitopes presented by the AAPCs.
Our findings suggest that this panel of AAPCs provides a standardizable, renewable and immediately accessible “off the shelf” source of cellular reagents to generate CMVpp65 peptide specific IFNγ+ and cytotoxic CD8+ T cells of desired HLA restriction in numbers required for adoptive cell therapy. These AAPCs may be particularly useful for generating donor T cells restricted by HLA alleles shared by donor and host for use in HLA disparate transplant recipients whose risk of mortality due to CMV infection remains as high as 15% despite the use of antiviral drugs (73). They may also permit early selection and expansion of HLA-restricted long-lived central memory T cells specific for a given epitope which may confer more sustained T cell mediated resistance after adoptive transfer (74), and. facilitate rapid identification of natural and heteroclitic HLA binding epitopes that elicit viricidal T cell responses and the cloning of T cells of desired epitope specificity and HLA restriction.
We thank Lorna Barnett and Anam Khan for technical assistance, and both Dr. Ekaterina Doubrovina and Dr. Gloria Koo for technical advice and critical review.
1This work was supported through NIH grants CA23766, CA59350, the Burton Abrams Charitable Trust, the Ryan E. McGeough Charitable Fund, the Aubrey Fund for Pediatric Cancer Research, The Larry H. Smead Foundation, the Commonwealth Foundation for Cancer Research, The Dutch Cancer Society and The Claire L. Tow Chair in Pediatric Oncology Research
The authors have no financial conflict of interest.