Adoptive immunotherapy after HLA mismatched stem cell transplantation Oosten, L.E.M.

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Adoptive immunotherapy after HLA mismatched stem cell

transplantation

Oosten, L.E.M.

Citation

Oosten, L. E. M. (2007, November 21). Adoptive immunotherapy after HLA

mismatched stem cell transplantation. Retrieved from

https://hdl.handle.net/1887/12446

Version: Corrected Publisher’s Version

License: Licence agreement concerning inclusion of doctoral

thesis in the Institutional Repository of the University

of Leiden

Downloaded from: https://hdl.handle.net/1887/12446

Note: To cite this publication please use the final published version (if

applicable).

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35

Artiﬁ cial antigen-presenting

constructs efﬁ ciently stimulate minor

histocompatibility antigen-speciﬁ c

cytotoxic T lymphocytes

Liesbeth E.M. Oosten

¹

, Els Blokland

¹

, Astrid G.S. van Halteren

¹

,

Julie Curtsinger

²

, Matthew F. Mescher

²

, J.H. Frederik

Falkenburg

³

, Tuna Mutis

¹

, Els Goulmy

¹

Chapter 2:

Blood 2004;104(1):224-226

1Department of Immunohematology and Blood Transfusion, and ³Department of Hematology, Leiden University Medical Center, Leiden, The Netherlands; ²Center of Immunology, Department of Laboratory Medicine and Pathology, University of Minnesota, Minneapolis, USA

This work was funded in part by grants from the Dutch Cancer Society (KWF Kankerbestrijding), the Leukemia & Lymphoma Society, and Foundation ‘De Drie Lichten’.

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Chapter 2

37 ABSTRACT

Cytotoxic T lymphocytes (CTLs) specific for hematopoietic system-restricted minor histocompatibility antigens (mHags) are important reagents for adoptive immunotherapy of relapsed leukemia after allogeneic stem cell transplantation.

However, expansion of these CTLs to therapeutic numbers is often hampered by the limited supply of antigen-presenting cells (APCs). Therefore, we evaluated whether cell- sized latex beads coated with human leukocyte antigen (HLA)/mHag complexes HLA- A2/HA-1 or HLA-A2/HA-2, and recombinant CD80 and CD54 molecules can replace professional APCs. The artifi cial antigen-presenting constructs (aAPCs) effectively stimulated HA-1- and HA-2-specifi c CTL clones as shown by ligand-specifi c expansion, cytokine production, and maintenance of cytotoxic activity, without alteration of CTL phenotype. Furthermore, HA-1-specifi c polyclonal CTL lines were enriched as effi ciently by aAPCs as by autologous HA-1 peptide-pulsed dendritic cells. Thus, aAPCs coated with HLA/mHag complexes, CD80 and CD54 may serve as tools for in vitro enrichment of immunotherapeutical mHag-specifi c CTL lines.

INTRODUCTION

The successful application of donor lymphocyte infusions for the treatment of relapsed leukemia after allogeneic stem cell transplantation illustrates the feasibility of adoptive immunotherapy of hematological malignancies¹. To minimize graft- versus-host disease, we earlier proposed the use of mHags HA-1 and HA-2 as immunotherapeutical reagents². HA-1 and HA-2 display hematopoietic system- restricted tissue distribution and relevant expression on leukemic cells and their progenitors^3-6. Moreover, CTLs directed against these mHags do not cause graft- versus-host disease in an ex vivo skin explant model⁷, and coincide with complete remission of relapsed leukemia and multiple myeloma after HLA-matched HA-1- or HA-2-mismatched donor lymphocyte infusions⁸.

HA-1- and HA-2-specifi c CTLs can be generated in vitro using peptide-pulsed or mHag- transduced autologous dendritic cells (DCs) as antigen-presenting cells (APCs)^9,10. However, expansion of CTLs is diffi cult due to the limited availability of donor derived DCs. To date, several reports indicate effective stimulation of T cells by HLA/peptide ligands expressed on aAPCs such as liposomes^11,12 or microbeads^13-16. We developed aAPCs that can be manufactured under good manufacturing practice conditions and used to expand mHag-specifi c CTLs for adoptive immunotherapy. Hereto, cell-sized latex microbeads coated with HLA-A2/HA-1 or HLA-A2/HA-2 complexes, CD80, and CD54 were investigated for their potential to effi ciently stimulate HA-1- and HA-2-specifi c CTL clones and lines while keeping their antigen-specifi c cytolytic properties.

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Adoptive Immunotherapy after HLA-mismatched Stem Cell Transplantation

38

MATERIALS AND METHODS

MHAG-SPECIFIC CTL CLONES AND POLYCLONAL CTL LINES, CD4⁺ T HELPER CELLS AND DCS

In vivo and in vitro generation of mHag-speciﬁ c CTL clones, polyclonal CTL lines, and DCs is documented in detail elsewhere^9,17. CD4⁺ T helper cells (CD4⁺ Th cells) were generated by culturing peripheral blood mononuclear cells (PBMCs) for 14 days in Iscove’s Modiﬁ ed Dulbecco’s medium (IMDM) containing 10% pooled human serum and 0.5% standard Dutch diphteria/pertussis/tetanus/polio vaccine (National Institute of Public Health and the Environment), and 20 U/ml interleukin-2 (IL-2) (Cetus) from day 6 onwards.

FUNCTIONAL ASSAYS

All functional assays were performed in IMDM +10% human serum. Stimulator cells were irradiated (30 Gy) before use. Results of proliferation, interferon-γ (IFN-γ) production, and cytotoxicity assays are expressed as the mean of duplicate samples.

Proliferation and IFN-γ production were determined by co-culturing 2.5x10⁴ responders with 5x10⁴stimulators. After 48 hours, supernatant was harvested for IFN-γ enzyme-linked immunosorbent assay (Sanquin), detection limit: 2 pg/ml. The remaining cultures were labeled with 1.0 μCi ³H-thymidine for 16 hours, after which incorporation was measured using liquid scintillation counting.

Cytotoxicity was evaluated by incubating 2500 ⁵¹Cr labeled target cells with serial dilutions of effector CTLs for 4 hours; supernatants were harvested for gamma counting. % speciﬁ c lysis = (experimental release-spontaneous release)/(maximal release-spontaneous release) x 100%

Cytokine proﬁ les were tested using the Human Th1/Th2 Cytokine Cytometric Bead Assay (BD Biosciences), detection limits: IFN-γ 7.1 pg/ml, tumor necrosis factor-α (TNF-α) 2.8 pg/ml, IL-10 2.8 pg/ml, IL-5 2.4 pg/ml, IL-4 2.6 pg/ml, IL-2 2.6 pg/ml.

SYNTHETIC PEPTIDES AND HLA-A2/MHAG PEPTIDE COMPLEXES

HA-1 and HA-2 peptides were synthesized according to the reported sequences^18,19. The biotinylated recombinant HLA-A2/HA-1 and HLA-A2/HA-2 complexes were generated as described and used as monomers for aAPC coating or as tetramers for analysis of HA-1- or HA-2-speciﬁ c CTLs²⁰.

LIGAND IMMOBILIZATION ON LATEX BEADS

5.3 μm polystyrene sulfate latex beads (Interfacial Dynamics) were incubated sequentially with streptavidin-allophycocyanin (1 μg per 10⁷ beads) (Molecular Probes, Leiden), with recombinant human CD80/Fc-chimera and CD54 (0.5 μg and 1.5 μg per 10⁷ beads respectively) (R&D Systems), and with 1% human albumin (Sanquin) for 20 minutes in 0.5 ml phosphate-buffered saline (PBS) per 10⁷ beads at 4°C. The beads were then incubated with biotinylated HLA-A2/mHag peptide complexes (0.5 μg per 10⁷ beads) for 40 minutes in 1 ml PBS per 10⁷ beads at 4°C. After each incubation step, the beads were washed with 1 ml PBS.

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Chapter 2

39 RESULTS AND DISCUSSION

OPTIMIZATION OF AAPCS FOR THE STIMULATION OF CTL CLONES AND LINES

Prior to the use of aAPCs in stimulation and expansion studies, the aAPCs’ various components were analyzed for optimal stimulation of CTL clones and lines. To ensure appropriate presentation of the HLA/peptide complex to the T cell receptor, the aAPCs were ﬁ rst coated with streptavidin and then with biotinylated HLA-A2/mHag complexes.

Any remaining non-speciﬁ c binding sites were blocked by coating with human albumin.

Optimal HLA-A2/mHag ligand density was determined by stimulating four CTL clones with aAPCs coated with different amounts of HLA-A2/mHag complexes and surplus streptavidin, and established at 1 μg/ml (Figure 1a,b; data shown for HA-1-specifi c CTL clone 2.12 and HA-2-specifi c CTL clone 1.7). Optimal densities for CD80 and CD54 were determined by stimulating four CTL clones with aAPCs coated with different amounts of CD80 or CD54 and 1 μg/ml HLA-A2/mHag ligand, and established at 0.5 μg/ml for CD80 and 1.5 μg/ml for CD54 (Figure 1c,d; data shown for HA-1-specifi c CTL clone

Proliferation (A) and IFN-γ production (B) of HA-1-specifi c CTL clone 2.12 and HA-2-specifi c CTL clone 1.7 stimulated with aAPCs that were coated with various concentrations of specifi c ligand (x axis) but without the costimulatory molecules CD80 and CD54. Proliferation (C) and IFN-γ production (D) of CTL clones 2.12 and 1.7 stimulated with aAPCs coated with the specifi c ligand in the absence of costimulatory molecules, with aAPCs additionally coated with CD80 or CD54, or with fully coated aAPCs (HLA-A2/mHag complexes, CD80, CD54).

FIGURE 1. OPTIMIZATION OF AAPC COATING

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40

Proliferation (A) and IFN-γ production (B) of various HA-1-speciﬁ c CTL clones (2.12, 3HA15, 5W38) and HA-2-speciﬁ c CTL clones (1.7, 1.9) incubated for 48 hours with medium only, with aAPCs coated with the costimulatory molecules CD80 and CD54, with aAPCs coated with costimulatory molecules and either HLA-A2/HA-1 or HLA-A2/HA-2 complexes, or with HA-1⁺/HA-2⁺ EBV-LCLs.

Cytotoxic activity (C) of two CTL clones (2.12, 1.7) after incubation for 7 days with medium only, with HA-1⁺/HA-2⁺ EBV-LCLs, or with aAPCs coated with speciﬁ c ligand. Cytotoxic activity is shown for an effector:target ratio of 8:1. Data are presented as the mean percentage of lysis ± SD.

FIGURE 2. AAPC-MEDIATED STIMULATION OF MHAG-SPECIFIC CTL CLONES

2.12 and HA-2-speciﬁ c CTL clone 1.7). Addition to aAPCs of CD80 and CD54 at these concentrations resulted in an enhancement of proliferation and IFN-γ production of 105%

to 245% at the optimal HLA-A2/mHag ligand density, and up to 390% at suboptimal HLA- A2 ligand densities (data not shown). Subsequently, aAPCs coated with 1 μg/ml HLA-A2/

mHag ligand, 0.5 μg/ml CD80 and 1.5 μg/ml CD54 were used throughout this study.

AAPC-MEDIATED STIMULATION OF MHAG-SPECIFIC CTL CLONES

The capacity of aAPCs to stimulate mHag-specific CTLs in an efficient and ligand- specific manner was analysed and compared with that of professional APCs. Three HA-1-speciﬁ c CTL clones (2.12, 3HA15, 5W38) and two HA-2-speciﬁ c CTL clones (1.7, 1.9) were stimulated with a) aAPCs coated with the costimulatory molecules CD80 and

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Chapter 2

41 CD54 only, b) aAPCs coated with costimulatory molecules and either HLA-A2/HA-1 or

HLA-A2/HA-2 complexes, or c) HLA-A2⁺ Epstein-Barr virus-transformed lymphoblastoid cell lines naturally expressing HA-1 and HA-2 (HA-1⁺/HA-2⁺ EBV-LCLs) (Figure 2a,b).

All mHag-speciﬁ c CTL clones showed signiﬁ cant proliferation and IFN-γ production in response to aAPCs coated with the relevant HLA-A2/mHag complexes and costimulatory molecules, comparable to the responses to HA-1⁺/HA-2⁺ EBV-LCLs. No proliferation or IFN-γ production was induced by aAPCs coated with irrelevant HLA-A2/mHag complexes or with costimulatory molecules only.

Improper or partial T cell receptor signaling by altered peptide ligands or T cell receptor engagement in the absence of adequate costimulatory signals can lead to changes in cytokine secretion proﬁ les or anergy of T cells^21-23. Similarly, aAPCs may alter the phenotype and function of CTL clones. Therefore, mHag-speciﬁ c CTL clones (2.12 and 1.7) were stimulated for 7 days with aAPCs or with HA-1⁺/HA-2⁺ EBV-LCLs. Subsequently, cytotoxic activity and secretion of IFN-γ, TNF-α, IL-10, IL-5, IL-4 and IL-2 were measured.

The speciﬁ c cytotoxicity (Figure 2c) and the cytokine proﬁ les (data not shown) were comparable for either mode of stimulation.

TABLE I. ENRICHMENT OF MHAG-SPECIFIC POLYCLONAL CTL LINES BY AAPC IN THE ABSENCE OR PRESENCE OF ADDITIONAL AUTOLOGOUS FEEDER CELLS OR BY DCS

Day 0 Day 7¹ CTL lines

aAPCs aAPCs+

PBMCs

aAPCs+

CD4⁺Th DC

#1

HA-1^A2 tetramer*

lysis: HA-1⁺/ HA-1-^†

9.3 68 / 26

11.1 37 / 20

18.2 59 / 25

15.5 38 / 11

16.3 48 / 22

#2

HA-1^A2 tetramer lysis: HA-1⁺/ HA-1-

9 51 / -1

11 29 / 4

19.6 25 / -3

22.2 36 / 3

13.9 21 / 1

#3

5.3 43 / 5

8.7 19 / -1

8.6 24 / 5

16.2 32 / 0

13.8 52 / 1

#4

3.9 40 / 5

2.3 13 / 9

7.5 45 / 19

10.6 47 / 9

9.7 53 / 7

1 mHag-speciﬁ c polyclonal CTL lines were expanded for 7 days in the presence of 20 units/ml IL-2 and 10 ng/ml IL-7. Responder:stimulator ratios were as follows: CTLs:aAPCs 1:1, CTLs:

aAPCs:PBMCs 1:1:1; CTLs: aAPCs:CD4⁺ Th 5:5:1; CTLs:DCs 10:1.

* HA-1^A2 tetramer⁺ cells indicates the % viable CD8⁺ HA-1^A2 tetramer⁺ lymphocytes in the total population of viable CD8⁺ lymphocytes.

† Lysis indicates the % lysis of HLA-A2⁺/HA-1⁺ or HLA-A2⁺/HA-1^- EBV-LCLs; results are shown for an effector:target ratio of 10:1.

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42

Polyclonal CTL lines #4 and #5 were stimulated with aAPCs and either autologous CD4⁺ Th cells (CTL line #4) or autologous PBMCs (CTL line #5) in the presence of 20 units/ml IL-2 and 10 ng/ml IL-7. Responder:stimulator ratios were as follows: CTLs:aAPCs:PBMCs 1:1:1; CTLs:aAPCs:CD4⁺ Th cells 2:2:1. Prior to each restimulation, HA-1^A2 tetramer staining of viable CD8⁺ T lymphocytes and cytotoxicity assays were performed. Cytotoxicity results are shown for an effector:target ratio of 10:1.

The percentages in the scatterplot subpanels represent the percentage of viable CD8⁺ HA-1^A2 tetramer- binding lymphocytes in the total population of viable CD8⁺ lymphocytes.

FIGURE 3. AAPC-MEDIATED EXPANSION OF MHAG-SPECIFIC POLYCLONAL CTL LINES

AAPC-MEDIATED ENRICHMENT OF MHAG-SPECIFIC POLYCLONAL CTL LINES

The main goal of this study was to investigate whether aAPCs can replace autologous DCs for the enrichment of polyclonal mHag-specifi c cultures. Hereto, fi ve different polyclonal HA-1-specifi c CTL lines (#1, #2, #3, #4, #5), originally induced and restimulated twice with HA-1 peptide-pulsed autologous DCs, were stimulated as follows. CTL lines #1, #2,

#3, and #4 were restimulated for 7 days with a) aAPCs only, b) aAPCs and autologous PBMCs, c) aAPCs and autologous CD4⁺ Th cells, or d) HA-1 peptide-pulsed autologous DCs. HA-1Â2 tetramer staining and cytotoxicity assays were performed at day 0 and at day 7 (Table I). For all CTL lines, similar or improved enrichment for HA-1-specifi c CTLs was observed after restimulation with aAPCs and autologous feeder cells when compared to DC mediated enrichment. The feeder cells could either be total PBMCs or activated CD4⁺ Th cells. These responses were driven by antigen presentation via aAPCs as none of the CTL lines showed enrichment after 7 days of incubation with PBMCs or CD4⁺ Th cells only (data not shown). In a separate experiment, CTL lines #4 and #5 were restimulated repeatedly with aAPCs and autologous feeder cells. Both lines showed a 5-fold increase in absolute numbers of HA-1Â2 tetramer-binding cells without loss of HA-1-specifi c cytotoxic activity after 14 days (Figure 3).

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Chapter 2

43 In summary, aAPCs coated with HLA-A2/mHag complexes, CD80 and CD54 can be used

to selectively enrich mHag-speciﬁ c CTLs for adoptive immunotherapy. In combination with autologous feeder cells, aAPCs stimulate CTLs as efﬁ ciently as peptide-pulsed DCs.

The aAPCs can be generated in a non-laborious way and can be easily removed from the cell culture by a density gradient. All aAPC-constituents are obtainable clinical grade.

Future studies will address the question whether aAPCs can be used for the primary in vitro induction of mHag-speciﬁ c CTLs.

ACKNOWLEDGEMENTS

We wish to thank Prof. F.H.J. Claas and Dr. M.J.B. van Stipdonk for critical reading and comments.

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