P-glycoprotein targeting: a unique strategy to selectively eliminate immunoreactive T cells

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RoyMartin Guimond, Antonia Balassy, Melanie Barrette, Sylvie Brochu, Claude Perreault and Denis Claude immunoreactive T cellsP-glycoprotein targeting: a unique strategy to selectively eliminate

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P-GLYCOPROTEIN TARGETING: A UNIQUE STRATEGY

TO SELECTIVELY ELIMINATE IMMUNOREACTIVE T CELLS

Martin Guimond,1 Antonia Balassy,1,2 Mélanie Barrette,1,2

Sylvie Brochu,1 Claude Perreault,1 and Denis Claude Roy1

1Division of Hematology-Immunology, Maisonneuve-Rosemont Hospital Research Center Department of Medicine, Université de Montréal, and

2Theratechnologies Inc. Montreal, Canada

Scientific heading: ImmunobiologyRunning title: Pgp-modulation to eliminate immunoreactive T cellsWord count - Total text: 4444 words; abstract: 197 words.

DCR is the recipient of a clinician-scientist award of the F.R.S.Q.Supported by a grant from Theratechnologies-F.R.S.Q. and the Cancer Research Society of Canada.

Correspondence to: Denis Claude Roy, MDDepartment of Hematology-ImmunologyMaisonneuve-Rosemont Hospital Research Center5415 L'Assomption Blvd.Montreal, QC, H1T 2M4, Canada Tel.: (514) 252-3404Fax: (514) 252-3430E-mail: [email protected]

Copyright 2002 American Society of Hematology

Blood First Edition Paper, prepublished online April 17, 2002; DOI 10.1182/blood-2001-12-0353 For personal use only. by guest on June 13, 2013. bloodjournal.hematologylibrary.orgFrom



ABSTRACT

T lymphocytes have been found to harbor P-glycoprotein (Pgp) and to demonstrate modulation

of its ion channel transporter function according to the state of activation of T lymphocytes. We

hypothesized that cytotoxic chemicals that are extruded by Pgp could be used to specifically

eliminate immunoreactive T cell populations. In this study, we evaluated the capacity of 4,5-

dibromorhodamine methyl ester (TH9402), a photosensitizer structurally similar to rhodamine, a

dye transported by Pgp, and which becomes highly cytotoxic upon activation with visible light to

selectively deplete alloreactive T lymphocytes. Stimulation of T cells with mitogens or

allogeneic major histocompatibility complex mismatched cells resulted in the preferential

retention of the TH9402 rhodamine-derivative in activated T cells, both CD4+ and CD8+.

Photodynamic cell therapy of TH9402-exposed T cells lead to the selective elimination of

immunoreactive T cell populations. In addition, this treatment preserved resting T cells and their

capacity to respond to third party cells. Inhibition of Pgp enhanced cellular trapping of the dye in

non-activated T cells and resulted in their depletion after exposure to light. Targeting of Pgp-

deficient cells may therefore represent an appealing strategy for the prevention and treatment of

graft-versus-host disease, and other allo- or auto-immune disorders.

E-mail: [email protected]




INTRODUCTION

Graft-versus-host disease (GVHD) is the principal cause of mortality and the primary limitation

to the early and widespread use of allogeneic stem cell transplantation (SCT), a treatment that

often represents the only curative option for numerous patients with malignant diseases and

hereditary metabolic disorders. Depletion of T cells capable of recognizing and mounting an

immune response toward host cells from stem cell grafts abrogates GVHD.1-4 However, the

elimination of T cells also results in delayed T cell reconstitution, and thus, an increased rate of

infection, particularly with viral agents such as cytomegalovirus, herpes zoster, and Epstein-Barr

virus.5-7 In addition, the eradication of mature T cells is associated with an increased risk of graft

rejection and an increased incidence of relapse of malignant disease.1,5,8-10 Thus, T cells are

required early after allogeneic transplantation and depleting the graft of its T cell content is not

an ideal approach to prevention of post-transplant complications. Although new

immunosuppressive agents offer options to decrease the incidence and severity of GVHD, most

of the time these strategies are only partially effective and may also increase the incidence of

viral and fungal infections, and other adverse effects of profound immunosuppression. To

provide a solution to this conundrum, selective inactivation or elimination of alloreactive donor

T lymphocytes could allow early immune recovery and response toward infectious agents, and

potentially preserve graft-versus-leukemia (GVL) activity.11-13 In addition, a strategy to

selectively eliminate immunoreactive T cells could represent an important advance for the

treatment of a large number of patients with autoimmune disorders.

Recently there have been significant efforts to try to identify and eliminate T cell subsets capable

of mounting an immune response toward host cells and mediating GVHD. Strategies targeting

CD6+ or CD8+ T cells demonstrated convincing potential for the prevention of GVHD in HLA-

matched transplants using related and even unrelated donors.14-16 Interestingly, the elimination of

donor T cell subsets did not translate into a greater incidence of graft rejection.17 Moreover, the

partial loss of GVL activity that occurs with T cell depletion was able to be restored through the




administration of donor lymphocyte infusions at a time when patients are at lower risk for

GVHD.18 Delaying the infusion of donor T cells until the early post-transplant surge in pro-

inflammatory cytokines has abated may contribute to limiting the development of GVHD.19

However, in the context of HLA-mismatched transplantation, antigenic differences between

donor and host cells are particularly immunogenic. In addition, it is extremely difficult to

identify a priori T cell antigens that will be unique markers for cells capable of targeting both

major histocompatibility complex (MHC)-disparate antigens and the numerous distinguishing

peptide sequences expressed by host tissues.20 In contrast, when donor cells are exposed to host

cells ex vivo, cell subsets capable of recognizing host MHC antigens become activated and thus

display the peculiar antigenic and biochemical properties rendering these cells “visible”.

Importantly, a few monoclonal antibody-based strategies have been developed to specifically

eliminate such alloreactive cells.21-25

Rhodamine enters all cells and is extruded from the intracellular milieu through P-glycoprotein

(Pgp) active transport.26 Pgp, the product of the multidrug-resistance-1 (MDR1) gene, is a protein

expressed not only in normal stem cells, but also in T lymphocytes.27-30 Investigators have

proposed that T cell activation may actually lead to the inactivation of Pgp.29 Thus, activated T

cells should fail to extrude rhodamine. While rhodamine is not cytotoxic, 4,5-dibromorhodamine

methyl ester (TH9402), a rhodamine derivative, was found to harbor important photosensitizing

potential.31-33 Its phototoxicity is mediated primarily by singlet oxygen production, with

oxydative damage concentrated to mitochondria by the virtue of drug localization.31,34 The

structural similarity between rhodamine and TH9402 prompted us to evaluate the capacity of the

latter photosensitizing agent to be preferentially retained in Pgp-deficient activated T cells and

thus, lead to their selective elimination after exposure to visible light (514 nm).35

In the present study, we found that photodynamic cell therapy (PDCT) with TH9402 was highly

toxic against CD4+ and CD8+ T cells activated in response to mitogens or MHC-mismatched




antigens. This PDCT selectively preserved resting T lymphocytes and their ability to proliferate

and to demonstrate cytotoxicity toward third-party antigens. PDCT may therefore have clinical

utility for the selection of non-MHC reactive T cells to prevent GVHD and accelerate immune

reconstitution post-transplantation, or for the treatment of immunoreactive disorders. Moreover,

our findings identify targeting of MDR1 inhibition as a unique physiologic approach to

specifically eliminate activated T cells.

MATERIALS AND METHODS

Human cells. Blood samples were obtained with the informed consent of healthy donors under

clinical protocols approved by the Human Subjects Protection Committee of the Maisonneuve-

Rosemont Hospital. Peripheral blood (PB) samples were collected in preservative-free heparin

and mononuclear cells separated by ficoll-hypaque density gradient centrifugation (Ficoll-Paque;

Pharmacia, Piscataway, NJ). The T lymphoblastic cell line CEM and the Pgp-expressing KG1a

cells were obtained from the American Type Culture Collection (ATCC, Rockville, MD).

PHA stimulation. Peripheral blood mononuclear cells (PBMC) were cultured for 72 hours at a

concentration of 3 X 106cells/mL in flasks (Nunclon, Nunc, Denmark) with 2 µg/mL

phytohemagglutinin (PHA; Sigma, St Louis, MO) in X-Vivo 15 medium (Bio-Whittaker,

Walkersville, MD) supplemented with 15% human AB serum (HAB)(Sigma), 2mM L-

glutamine, 1 mM sodium pyruvate, 100 U/mL penicillin, and 100 µg/mL streptomycin (all from

Gibco, Grand Island, NY).

Allogeneic T cell activation. Activation of responder (A) T lymphocytes against stimulator (B)

cells was conducted in a one-way mixed lymphocyte reaction (MLR).36 A and B individuals

presented three major HLA mismatches. Briefly, responder cells from subject A were cultured

for 4 days at 37°C with the same number of irradiated PBMC from subject B (50 Gy)(137Cs;




Gamma Cell, Atomic Energy of Canada, Ottawa, ON, Canada) in medium supplemented with 50

U/mL rhIL-2 (R&D Systems, Minneapolis, MN).

Photodynamic cell therapy with TH9402. After in vitro activation, cells were harvested, washed,

resuspended at a final concentration of 1 X 106 cells/mL, and incubated at 37°C with 10 µM

TH9402 (Theratechnologies Inc., Montreal, QC, Canada) in X-vivo 15 medium with 2.5% HAB.

After a 40-minute incubation, cells were centrifuged and dye efflux favored by resuspending

cells in TH9402-free medium for 90 minutes. At the end of the latter dye efflux period, cells

were exposed to a fluorescent light-scanning device (PDCT-Xerox Series 4, Theratechnologies

Inc.) delivering 5 joules/cm2 at a wavelength of 514 nm.

T cell proliferation assay. Proliferative activity of responder cells exposed to photodynamic

therapy and non-treated controls was assessed on day 5 of a MLR in a standard 3H-thymidine

labeling assay. The total number of cells (responder (A) and irradiated stimulator (B) cells)

present before PDCT was not adjusted after PDCT. These cells were restimulated with a fixed

number (1 x 105) of irradiated stimulator (B) or third-party (C) cells at different

responder/stimulator cell ratios (2:1, 1:1, 1:2, and 1:4) in 96-well U-bottomed microtiter plates

(Nunc). Cultures in triplicate were labeled with 1 µCi (0.037 MBq) 3H-thymidine (Perkin Elmer,

Woodbridge, Canada) per well for 18 hours, harvested onto glass fibre filter mats, and 3H-

thymidine incorporation was measured using a liquid scintillation counter (Wallac, Gaithersburg,

MD).

CTL-precursor and limiting dilution assays. Limiting dilution analyses (LDA) were used to

calculate the frequencies of responding cytotoxic T lymphocyte precursor (CTLp) cells and

clonogenic CEM and KG1a cells in treated and untreated conditions using previously described

methods.37,38 Briefly, to determine CTLp frequency: 24 replicates of graded numbers of treated

or untreated responder (A) cells (3 X 106 to 450 cells/well) were seeded in 96-well microtiter




plates in the presence of 1 X 105 irradiated (50 Gy) fresh PBMC stimulator cells (B or C).

Control wells consisted of stimulator cells only. After 9 days of culture in medium supplemented

with 50 U/mL of rhIL-2, each well was tested for cytolytic activity against 5 X 103 initial

stimulator (B) and third party (C) cells using a standard 51Cr- release assay. The supernatant (100

µL) was harvested from each well and counted in a gamma counter. Spontaneous release was

less than 15%. Results for individual wells were expressed as a percentage of specific lysis

calculated as follows: % specific lysis = 100 X (experimental release – spontaneous release

[medium only]) / (maximum release [1% Triton X-100] – spontaneous release). To measure

CEM and KG1a, clonogenic cell frequencies, cells were grown in a similar LDA (from 5 X 105

to 0.5 cells per well) in RPMI-1640 medium (Life Technologies, Inc., Gaithersburg, MD)

supplemented with 10% fetal bovine serum, fed every 4 days and scored for growth under an

inverted phase microscope.

Immunophenotypic analysis. Expression of cell surface T cell antigens was evaluated by direct

immunofluorescence using standard techniques.39 MAbs used in this study were anti-CD3-FITC

(UCHT1), anti-CD25-PE (B1.49.9), anti-CD3-APC (UCHT1)(Coulter Immunology, Hialeah,

FL), anti-CD4-APC (RPA-T4), anti-CD8-APC (RPA-T8) (Pharmigen, San Diego, CA).

Nonspecific binding was determined using appropriate isotypic controls. Immunofluorescence

reactivity was determined by automated multi-parameter flow cytometry analyzing at least 104

cells in each sample (FACSCalibur; Becton Dickinson, Mountain View, CA) and processed

using Cell Quest software (Becton Dickinson).

Hematopoietic progenitor cell assay. PB cells from healthy donors mobilized with G-CSF

(Amgen, Thousand Oaks, CA) underwent PDCT and were plated in methylcellulose medium

(MethoCult H4434; StemCell Technologies Inc, Vancouver, BC, Canada) on 35-mm plastic

culture dishes, according to the manufacturer's instructions. Colony-forming units-granulocyte-




macrophage (CFU-GM) and colony-forming units-mix (CFU-mix) were scored after 14 days of

culture at 37ºC in a fully humidified 5% CO2 atmosphere.38

Functional evaluation of Pgp-170. Pgp substrate efflux modulation by cyclosporin-A and

verapamil was determined in an accumulation assay using TH9402. Cells were stained with 10

µM TH9402 for 40 minutes, washed and resuspended in either medium alone, with 1 µg/mL

cyclosporin A (Novartis Pharma, Dorval, QC, Canada) or 5 µg/mL verapamil (Sabex,

Boucherville, QC, Canada). Cellular retention of the dye was assessed by flow cytometry

(FACSCalibur, Becton Dickinson).40,41 Calibration beads were used in all experiments to ensure

stable energy delivery (Calibrate3 and -APC, Becton-Dickinson). Positive controls for functional

Pgp-expression consisted of KG1a cells.

Statistical analysis. To determine CTLp frequency, experimental wells were scored positive if

the percent specific lysis of a well exceeded the mean + 3 SD of the wells where only stimulator

cells were present. Cytotoxicity and clonogenicity at each serial cell concentration were assessed

in an "all-or-nothing" (positive or negative) fashion, and frequency within the test population

was estimated by use of chi-square minimization.38,42

RESULTS

Photodynamic elimination of T cells. In order to assess the sensitivity of proliferating T cells to

TH9402 PDCT, the lymphoblastic T cell line CEM was used as target. Cells were incubated for

40 minutes with 10 µM TH9402, washed and incubated with TH9402-free medium for 90

minutes and then exposed to light (5 joules/cm2). These parameters were previously found to

eliminate more than 99.9% of malignant K562 cells, while preserving more than 50% of

hematopoietic progenitor cells.32 TH9402 PDCT when compared to untreated controls depleted

more than 99.97% of CEM T cells measured by LDA (Figure 1A).




Figure 1.




The potential of this TH9402-based PDCT method to eliminate activated T lymphocytes was

evaluated by comparing proliferative responses of treated (PDCT) versus untreated PHA-

activated normal PBMCs toward MHC-mismatched stimulator cells. In untreated controls, PHA-

stimulated cells were able to proliferate when subsequently exposed to MHC-disparate

stimulatory cells in an MLR (Figure 1B). In contrast, TH9402 PDCT completely abrogated the

response of PHA-stimulated cells to MHC-mismatched cells. The specificity of PDCT for

activated cells was evaluated by treating resting PBMC, incubated in IL-2 containing medium

only, and then measuring proliferative response in allogeneic mismatch MLR (Figure 1C).

Interestingly, PDCT did not affect the response of these resting cells, a finding that indicates a

higher level of sensitivity to PDCT for primed versus resting T cells.

Depletion of alloreactive T lymphocyte subsets. The clinical application of PDCT in the context

of allogeneic transplantation must rely on both specific elimination of T cells that are reactive

toward host cells and preservation of T cells capable of subsequent response to infectious or

other foreign antigens. To clarify this issue, PBMCs (individual A) were first exposed for 4 days

to allogeneic stimulator (individual B) cells mismatched at 3 MHC loci (A, B and DR). After this

activation process, cells were exposed to PDCT and then presented with either the same

stimulator (B) cells for a second time, or with third-party (C) cells in a conventional 3H-

thymidine incorporation assay (Figure 2). Increasing concentrations of TH9402 and light

intensity induced gradually decreasing proliferation toward stimulator B cells. In contrast, the

capacity of residual cells to proliferate when exposed to third party C cells was preserved except

at the highest treatment intensity. Moreover, to discriminate between the effect of TH9402, light

and PDCT, A cells were treated with either TH9402 alone, light alone or TH9402 PDCT.

Proliferative responses were preserved after exposure to either TH9402 without light or to light

alone (data not shown) (p = NS). The highest TH9402 concentration (10 µΜ) and light intensity

(5 J/cm2) were selected for all subsequent experiments because these achieved maximum

elimination of specific alloreactivity and only slightly affected response to third party C cells.




Figure 2.




Figure 3.




Immunophenotypic analysis. In order to evaluate the specificity of PDCT for activated T

lymphocytes, the expression of the inducible α chain of the IL-2 receptor (IL-2R: CD25) was

measured on CD4+ and CD8+ T cell populations from treated samples and untreated controls

(Figure 3). At the end of 4-day MLR, cells were exposed to TH9402 PDCT or medium, and

immunophenotypic analysis performed after 3-day culture in IL-2 containing medium. At least

98% of CD8+CD25+ cells and 96% of CD4+CD25+ cells were eliminated by PDCT when

measured by flow cytometry (Table 1). In contrast, most unactivated (CD25-) T cells were

spared; their increased proportion after PDCT confirms the selectivity of PDCT for activated

lymphocytes. In addition, CD25- T cells also stained negatively for propidium iodide, a finding

that indicates preservation of T cell integrity (data not shown).

CTL-precursor frequency after photodynamic therapy. To confirm the specificity of PDCT for

anti-host T cells, cytotoxic T lymphocyte precursors (CTLp) were enumerated following

treatment with TH9402 or medium using limiting dilution analysis (LDA). The number of CTL-

p active against B and C cells was determined after TH9402 PDCT of (AXB) MLR primed cells

(Figure 4A). In untreated samples, more anti-B than anti-C CTL-p were detected, a finding

probably reflecting the primary nature of the immune response against C cells versus the

secondary immune reaction against B cells. TH9402 PDCT eliminated anti-B cell CTL-p by a

1000-fold, but anti-C cell CTLp frequency was decreased by only 30-fold, confirming the

preferential targeting of previously activated CTLp.




Figure 4.




Progenitor recovery after photodynamic therapy. The effect of PDCT on mobilized PB cells

from normal individuals (n = 4) was utilized to assess the toxicity of the procedure for other

spontaneously proliferating cells. These cells were exposed to the same PDCT conditions as for

T cell depletion. Survival of hematopoietic progenitor cells was evaluated using a semi-solid

culture assay (Figure 4B). These conditions, which induced 100- to 1000-fold decreases in

activated T cells, did not cause a significant decrease in the growth of CFU-GM (p = 0.09) nor

CFU-mix colonies (p = 0.1).

Kinetics of dye retention. The specificity of TH9402-mediated killing for activated T cells could

be due to a differential accumulation or retention of the dye in resting versus activated T cells.

To test this hypothesis, TH9402 influx/efflux kinetics were evaluated in PHA-stimulated and

resting lymphocytes. At the end of the incubation period, retention of the dye was higher in

PHA-stimulated CD3+ cells than in resting lymphocytes (Figure 5A). In addition, activated

lymphocytes continued to sequester more TH9402 over time than resting lymphocytes, even

after reaching the plateau phase (p<.05). Moreover, after a MLR, CD25 expressing T

lymphocytes, whether CD4+ or CD8+, retained more TH9402 compared to CD25- T cells

(p<.05)(Figure 5B, C). These data indicate that both TH9402 accumulation and TH9402

retention are increased in the proliferating and activated T cells.




Figure 5.




Interestingly, resting T cells not only incorporated lower levels of TH9402 than PHA activated

cells, but also a large proportion of the former cells demonstrated a second peak of lower

fluorescence intensity (Figure 6A). This bimodal distribution indicates the existence among

resting cells of two populations with a different propensity to eliminate the dye. Similar results

obtained using MLR-activated T cells (data not shown) indicate that both TH9402 accumulation

and TH9402 retention are increased in the proliferating and activated T cells.




Figure 6




PgP involvement in TH9402 efflux. In order to study mechanisms of retention of the TH9402

rhodamine derivative, we focussed on Pgp, which has been previously described as the main

channel involved in rhodamine efflux.43,44 In resting T lymphocytes, inactivation of Pgp by

cyclosporin-A lead to a disappearance of the peak of lower fluorescence intensity (M2: MFI =

32), and gave rise to a single peak of TH9402 fluorescence (M1) demonstrating slightly higher

retention of the dye (MFI = 600) than the M1 peak of the CSA-unexposed sample (MFI = 400)

(Figure 6A). In contrast, inactivation of the MDR1 channel had no major impact on retention of

the dye in PHA-stimulated lymphocytes. Moreover, the effect of cyclosporin-A on TH9402

efflux was durable and prevented the appearance of CD3+ T cells with low concentrations of dye

for more than 2 hours (Figure 6B).

To determine if the higher retention of the dye caused by Pgp inhibition translated into higher

cytotoxicity, we incubated resting T cells with verapamil and exposed them to PDCT. This

MDR1 inhibitor significantly enhanced the PDCT elimination of resting T cells (p<0.01) (Figure

7A). To investigate the extent of MDR1 involvement in TH9402-mediated effects, we repeated

the same experiment using the KG1a cell line, which demonstrates high levels of constitutive

expression of Pgp.45 Phototherapy with TH9402 was not cytotoxic to KG1a cells, but the

addition of verapamil led not only to increased retention of the dye (data not shown), but also to

depletion of 99.99% of clonogenic cells (Figure 7B). When used without PDCT, verapamil did

not deplete T or KG1a cells. These findings clearly identify Pgp as the principal modulator of

TH9402 cellular concentration and photodynamic cytotoxicity.




Figure 7.




DISCUSSION

Selective elimination of donor T cell subsets recognizing host histocompatibility antigens

represents an appealing strategy to eradicate GVHD. However, in order to limit complications

with viral and fungal infections, graft rejection and relapse, which occur after physical or

functional T cell depletion, T cells capable of generating an immune response toward foreign

antigens must be preserved.5,11,46 Nevertheless, the specific elimination of such host-reactive T

cells represents a difficult task. In our present study, we uncovered a unique cytotoxic pathway

that takes advantage of the intrinsic modulation of the Pgp channel transporter to eradicate

immunoreactive T cells. Activated T lymphocytes demonstrated preferential accumulation and

retention of the TH9402 rhodamine derivative over resting T cells. Indeed, we found that resting

T lymphocytes, which express MDR1,29 extruded TH9402 through this channel transporter,

while the cellular activation process lead to an impairment in MDR1-mediated TH9402 efflux.

These kinetics of accumulation of TH9402 have resulted in the photodynamic eradication of

responder cells immunized ex vivo against stimulator cells, in conditions simulating MHC-

mismatched transplantation. In addition, this PDCT achieved drastic elimination of IL-2 receptor

expressing CD4+ and CD8+ cell populations. Notably, these findings translated into highly

efficient depletion of host-reactive cytotoxic T lymphocyte precursors. Moreover, the efficiency

of Pgp spared resting T cells and preserved their ability to generate proliferative and cytotoxic

responses against antigens other than host MHC.

Immunological tolerance may result from a variety of mechanisms, including deletion, anergy,

ignorance, and suppression.47- 51 PDCT using the TH9402 photosensitizer abrogated anti-host




reactivity of T lymphocytes activated either with mitogens or with an allogeneic MLR. Flow-

cytometric evaluations demonstrated that most of the effect of TH9402 treatment was

attributable to depletion of alloreactive T cells expressing CD25, the inducible high affinity IL-2

receptor. Both activated CD4+ and CD8+ cells were sensitive to the PDCT shown by the

detection of less than 1% of the total pool of lymphocytes expressing CD25 after treatment.

While the scant number of CD25+ cells detected could represent activated T cells escaping

photodynamic eradication, it is also possible that they correspond to cells bound to die from

lethal damage of PDCT-mediated oxidative damage. Alternatively, these lymphocytes could

represent non-activated T lymphocytes, such as regulatory T cells, which have been found to

constitutively express CD25.52-54 The preservation of a regulatory T cell population would be

particularly useful since it has been shown to play an important role in induction of tolerance to

alloantigen via costimulatory blockade.55 The latter scenarios would explain why the evaluation

of the impact of PDCT on CTLp demonstrated greater elimination of anti-host clonogenic

cytotoxic precursors (Figure 4A) than of CD25+ T cell populations (Figure 3). Moreover, the 3

logs of depletion of CTLs observed with the LDA is of the same order as the threshold of 2 to 3

logs of T cell depletion thought to be required for the prevention of GVHD.56

Because T cell receptor diversity post-transplant is decreased according to the number of T cells

present in the graft, it is crucial that as many T cells as possible be spared.57 Our results

demonstrate that while phototherapy using TH9402 is highly toxic for activated T cells, it

remains selective and preserves a large proportion of the CD4+ and CD8+ cells that do not

express the IL-2 high affinity receptor and other cell lineages such as myeloid and erythroid

progenitors. Interestingly, the administration of such T cells has the potential to restore T cell




receptor diversity through expansion in response to homeostatic signals of the host and to

reconstitute the peripheral T cell pool.58,59 Indeed, non-activated T cells remain

immunocompetent and able to proliferate in response to new antigenic stimuli, whether

previously cultured in IL-2 only, mitogens, or stimulated with allogeneic cells. This is

corroborated by the ability of TH9402 exposed cells to generate CTLp against third-party

antigens, a finding that also confirms the selectivity of PDCT. Future studies will challenge us to

determine if the small decrease in reactivity toward third party cells observed after PDCT could

reflect the elimination of T cell clones with dual specificity and demonstrating the capacity to

react toward both host and third-party cells.60,61

Since only T cells recognizing an antigen expressed by stimulator cells will be eliminated, PDCT

should spare T cells recognizing tumor antigens (developmentally regulated antigens or

leukemia-specific antigens) provided care is taken to exclude neoplastic cells from the stimulator

cell population.62,63 NK cells also express high levels of Pgp and should be protected from PDCT

toxic effects.28,64 Though we demonstrate here that PDCT is effective at eliminating T cells

reactive to stimulator cell MHC antigens, the effect of PDCT on minor histocompatibility

antigen (MiHA) stimulated T cells has yet to be addressed.36,65 In addition, the observation that

TH9402 treated T cells respond to third party MHC antigens indicates preservation of T cell

signaling and effector pathways that should translate into elimination of viral and fungal

invaders.22 Moreover, the addition of donor T cells, although non-reactive toward host MHC

antigens, could help lower the incidence of graft rejection associated with T cell depletion.3,5,8,20

Indeed, T cells present after PDCT have the potential to act as veto cells to block anti-donor

reactivity of host T cells without requiring recognition of host alloantigens.66,67 In future studies,




it will be important to delineate the nature of the various T and NK cell populations that escape

elimination by TH9402 PDCT and their contribution to the prevention of immunologic and

infectious post-transplant complications.

Our findings indicate that TH9402 PDCT does not exhibit a broad antiproliferative effect, but

rather acts specifically against activated T cells according to intrinsic physiologic properties of

target cells. Modulation of Pgp activity, which results in differential retention and cytotoxicity

from TH9402, could reflect biomechanical modifications of such channel transporters with the

activation process.68,69 Interestingly, Pgp could also be inactivated by PDCT itself,70 an

inhibitory mechanism that would augment retention of the photosensitizer in those activated T

cells with partial inhibition of MDR1 function, without affecting resting T cells that have already

extruded most of the dye at the time of light application. The increased retention of TH9402, and

potentially of its photoproducts, in activated cells could enhance the efficacy and specificity of

the treatment. While we cannot exclude a contribution of metabolic changes induced by T cell

activation to altered mitochondrial targeting by this rhodamine-derivative,71 our findings clearly

indicate that Pgp plays an important role in the intracellular handling of TH9402, and identify a

novel approach that takes advantage of the functional inhibition of this pathway of resistance to

selectively eradicate activated T cells.

The current photodynamic approach could be applied directly for the ex vivo treatment of stem

cell grafts or donor lymphocyte infusions in order to prevent GVHD in the context of MHC-

mismatched allogeneic transplantation.72,73 Moreover, recent identification and sequencing of

several MiHAs and improvements in immunization strategies using dendritic cells should




facilitate the activation of effector cells directed at host MiHAs.74 This may broaden the

applicability of this PDCT to allogeneic HLA-matched transplant strategies. Finally, the

selectivity of TH9402 PDCT for activated T lymphocytes could be exploited for the targeted

elimination of both alloreactive T cell clones that develop after solid organ transplants and

autoreactive clones responsible for diseases such as lupus erythematosus, rheumatoid arthritis

and systemic sclerosis.75-78

ACKNOWLEDGEMENTS

The authors thank Drs B. Leonard, N. Beauger and G. Krosl for insightful scientific advice; Dr

M.A. Caligiuri for critical review of the manuscript; and C. LeHouillier and the members of the

Cell Therapy Laboratory and Apheresis Unit for their excellent technical assistance. We thank all

scientists at Theratechnologies Inc. for their close collaboration and technical support.




LEGENDS TO FIGURES

Figure 1. TH9402 eliminates proliferating T cells. A) CEM T cells were incubated with

10 µM TH 9402 for 40 minutes, washed and exposed to 5 J/cm2 of light after a dye efflux period

of 90 minutes. Survival of clonogenic cells for treated (white) and untreated (black) cells was

evaluated using a limiting dilution assay. B) PBMC exposed to PHA or C) medium only for 72

hours underwent PDCT. Proliferation to MHC-incompatible cells was evaluated in an MLR at

different responder:stimulator (R:S) ratios. Results expressed as mean ± SEM of experiments

performed in triplicate.

Figure 2. Effect of TH9402 concentration and light intensity on the depletion of host- and

third party-reactive T cells. Donor cells were first primed against host cells in a one-way, 4-

day MLR, and then treated with increasing concentrations of TH9402 and light intensity. A)

After treatment, cells were co-cultured with irradiated stimulator cells from the same host or B)

third party cells for 5 days and proliferation was measured after addition of 3H-thymidine.

(*p<.05; **p<.01) Results expressed as mean ± SEM of the percentage of proliferation of the

untreated control at a R:S ratio of 2:1; experiments performed in triplicate.

Figure 3. TH9402 PDCT eliminates activated CD4+ and CD8+ cells. MLR-activated cells

underwent TH9402 PDCT and after 72 hours, T cell populations were assessed for CD25

expression using flow cytometry. Numbers indicate the percentage of cells, and dot plots are

representative of 3 experiments.

Figure 4. PDCT eliminates anti-stimulator and preserves anti-third party CTLp, and

normal hematopoietic progenitor cells. A) Effect of TH9402 and medium treatment of MLR-

activated cells on the frequency of CTLp directed against stimulator B and third party C cells.

Results are expressed as mean ± SEM of 3 experiments. B) Mobilized peripheral blood




progenitors from 5 normal donors were treated with TH9402 or medium. Results are expressed

as mean ± SEM of CFU-GM and CFU-Mix colonies.

Figure 5: Kinetics of incorporation of TH9402 in resting and activated lymphocytes. A)

TH9402 dye retention was analyzed in the CD3+ cells from samples incubated (diamonds) or not

(circles) with PHA. B) Dye retention was also measured in activated (CD25+) and non-activated

(CD25-) CD4+ and C) CD8+ cells within the same MLR-activated sample. MFI ± SEM of 3 to 6

experiments and p < 0.05 for all evaluations.

Figure 6. Impact of PgP inhibition on TH9402 content in resting and activated

lymphocytes. A) PHA-stimulated and resting lymphocytes were stained with TH9402 for 40

minutes and resuspended in medium alone or with cyclosporin-A. Flow cytometric assessment of

TH9402 content in CD3+ cells was performed 90 minutes after the end of the incubation period.

Numbers in parentheses indicate the MFI of corresponding cell populations. B) The impact of

cyclosporin-A exposure on the proportion of PHA-stimulated lymphocytes capable of

eliminating TH9402 (MFI of less than 100 units) was measured over time, starting after

completion of the 40-minute incubation period. The results are representative of 3 experiments.

Figure 7. Down-modulation of MDR1 function enhances TH9402-mediated cytotoxicity. A)

Resting PBMC were exposed to TH9402 in medium supplemented or not with verapamil.

Elimination of CD4+ and CD8+ cell populations was measured 3 days after PDCT using flow

cytometry, and compared with untreated controls. Inhibition of MDR1 function increased the

photodynamic elimination of T cells. B) Cytotoxicity of PDCT on KG1a cells, an MDR1

expressing cell line, was measured using a limiting dilution assay. Verapamil alone or PDCT

with TH9402 had no effect on KG1a cells but combining the inhibition of MDR1 with verapamil

to TH9402 PDCT resulted in the elimination of more than 3 logs of clonogenic cells. Results are

expressed as mean ± SEM of at least 2 experiments.




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Table 1. Impact of TH9402 PDCT on activated and non-activated CD4+ and CD8+ cells

evaluated using flow-cytometry.

CD4+ cells

(x106 cells)

CD8+ cells

(x106 cells)

Treatment† CD25+ CD25- CD25+ CD25-

Medium 1.68±0.61 0.96±0.69 1.11±0.40 0.50±0.34

TH9402 0.060±0.011 0.21±0.09 0.026±0.012 0.26±0.09†After exposure to TH9402 PDCT or medium, AXB* cells were cultured for 3 days in IL-2 supplemented medium. Absolute cell numbers are expressed in million cells (mean ± SEM of 3 experiments).




P-glycoprotein targeting: a unique strategy to selectively eliminate immunoreactive T cells

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