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The Journal of Immunology, 2008, 180, 6892 -6902
Copyright © 2008 by The American Association of Immunologists, Inc.
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*Substance via MeSH
Medline Plus Health Information
*Bone Marrow Transplantation
*Cytomegalovirus Infections

Host and Donor Immune Responses Contribute to Antiviral Effects of Amotosalen-Treated Donor Lymphocytes following Early Posttransplant Cytomegalovirus Infection1

Mohammad S. Hossain*, John D. Roback{dagger}, Fengrong Wang* and Edmund K. Waller2,*

* Department of Hematology and Oncology, Division of Stem Cell and Bone Marrow Transplantation, Winship Cancer Institute and {dagger} Department of Pathology and Laboratory Medicine, Emory University School of Medicine, Atlanta, GA 30322


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
We have previously shown that amotosalen-treated splenocytes rescued allorecipients from a lethal dose of mouse CMV (MCMV) administered on day 0 in experimental parent C57BL/6->CB6F1 allogeneic bone marrow transplant. In this study, we investigated the mechanism of antiviral activity of amotosalen-treated donor splenocytes when sublethal MCMV infections were administered 7 days posttransplant. Recipients of 3 x 106 untreated splenocytes were used as control. Following MCMV infection, recipients of untreated splenocytes had 40% early mortality due to acute graft-vs-host disease compared with no deaths among recipients of 10 x 106 treated splenocytes. However, recipients of both types of donor splenocytes effectively cleared MCMV from their liver. Like the untreated CD8+ T cells, amotosalen-treated CD8+ T cells equally retained their in vivo CTL activity against MCMV early peptide-pulsed targets and expressed similar levels of granzyme B within 11 days of infection. In contrast to full donor chimerism in recipients of untreated splenocytes, recipients of amotosalen-treated splenocytes showed mixed chimerism with both donor spleen- and host-derived anti-MCMV CD8+ T cells in their blood and lymphoid organs, with significantly higher numbers of host-derived CD4–CD8– (double negative) T cells in the spleens of recipients of treated splenocytes compared with the recipients of untreated splenocytes. Additionally, recipients of amotosalen-treated splenocytes had lower levels of serum IFN-{gamma} and TNF-{alpha} in response to MCMV infection compared with untreated recipients. Thus, adoptive immunotherapy with treated T cells is a novel therapeutic approach that facilitates hematopoietic engraftment and permits antiviral immunity of both donor and host T cells without graft-vs-host disease.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Graft-vs-host disease (GvHD)3 and opportunistic infections are the major causes of morbidity and mortality in cancer patients treated with allogeneic bone marrow transplant (BMT). In allogeneic BMT patients, donor-derived T cells that recognize host alloantigens help eradicate residual cancer but also cause GvHD, a major deleterious effect of allogeneic transplant. Moreover, donor T cells also play a critical role in promoting stem cell engraftment, encouraging rapid recovery of cellular immunity and preventing opportunistic infections (1, 2). Thus, to establish a therapeutically useful strategy of adoptive T cell immunotherapy, separation of the beneficial anti-opportunistic infection and antitumor effects of donor T cells from the deleterious GvHD effects are highly desirable.

Clinically, a number of immunosuppressive drugs such as calcium inhibitors, corticosteroids, methotrexate, and antimetabolites, etc. are generally used to control GvHD as pharmacological prophylaxes. The clinical responses to prophylactic immunosuppression are often incomplete, with breakthrough GvHD, and patients may experience significant drug-related toxicities. Although T cell depletion from the hematopoietic progenitor cell graft prevents the development of GvHD, patients who receive T cell-depleted allografts are at significantly increased risk for relapse and opportunistic infections (3, 4). In the context of the severe immunodeficiency that follows transplantation of a T cell-depleted allograft, drug-resistant chronic viral infections may develop (5). Due to the alloreactivity and GvHD potential of donor lymphocytes, attempts were made to establish alternative approaches of adoptive immunotherapy by expanding virus-specific donor T cell clones in vitro and infusing donor CTL clones as a strategy to prevent opportunistic viral infections without causing GvHD. In this small pilot study, donor CTL persisted for at least 12 wk and cleared existing viremia without causing clinical GvHD (6). However, adoptive immunotherapy with cultured CTL lines has not been broadly adopted in the transplant community due to the lengthy culture methodology, cost, and specialized technical expertise required.

Murine CMV (MCMV), a member of the β-herpes virus group, can cause disseminated and fatal disease in immunodeficient animals (7). It has structural and biological similarities to human CMV (8, 9), which causes similar disease in immunodeficient humans (10). First-line antiviral innate immunity against MCMV infection is mediated by the NK cells (11) and macrophages (12) and also by the CD4–CD8– (double negative (DN)) TCR{alpha}β+CD3+ T cells (13). The second line of antiviral immunity requires adaptive immune responses with the expansion of Ag-specific T cells that eventually clear viral infected cells from the host tissues. We developed a mouse model of allogeneic BMT with superimposed MCMV infection induced in the early posttransplant period to recapitulate the pathophysiology of acute GvHD (aGvHD) and CMV infections that are usually seen in human transplant recipients. Using ex vivo amotosalen-treated (also referred to as "treated" throughout) splenocytes from murine donors previously immunized against MCMV, we have demonstrated that recipients of amotosalen-treated cellular adoptive immunotherapy survived MCMV infection without GvHD (14, 15), but a detailed mechanism of antiviral immune responses was not performed.

In this study, we have investigated whether the prophylactic administration of amotosalen-treated donor T cells could confer protective antiviral immune responses in a murine model of myeloablative conditioning and allogeneic BMT. Amotosalen is a water-soluble synthetic psoralen compound developed by Cerus. It forms DNA crosslinks between the pyrimidine bases in T cells following short exposure to UVA light. Amotosalen/UVA light-induced DNA crosslinks completely abrogated in vitro T cell proliferation following stimulation with plate-bound anti-CD3 Ab without inhibiting the ability of T cells to produce IFN-{gamma} (14). Transplant recipients were infected with sublethal dose of MCMV on 7 days posttransplant to model the early posttransplant CMV reactivation seen in clinical transplantation. We found that adoptive immunotherapy using prophylactic administration of treated donor splenocytes reduced donor T cell-mediated aGvHD without increasing mortality and preserved both donor- and host-derived innate and adaptive immunity. Rapid hematopoietic engraftment and effective immune reconstitution among recipients of treated splenocytes led to successful clearance of virus-infected cells from target organs.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Mice

CB6F1 (C57BL/6 x BALB/c)(H-2b/d, CD45.2/Thy1.2) mice and PepBoy (B6.SJL-PtprcaPep3b/BoyJ)(H-2b, CD45.1/Thy1.2) mice on the C57BL/6 background were obtained from The Jackson Laboratory. Perforin–/– mice were purchased from Taconic Farms. BA mice (H-2b, CD45.2/Thy1.1) on the C57BL/6 background (from Dr. M. Lieberman, Stanford University, Stanford, CA) were bred at Emory University (Atlanta, GA). C57BL/6 CD45.1+CD45.2+ heterozygous mouse were bred by mating male PepBoy (CD45.1+) and female BA (CD45.2+) mouse strains in the Emory University Animal facility. Procedures conformed to guidelines set by the National Research Council, National Academy of Science and were approved by the Emory University Institutional Animal Care and Use Committee.

Preparation of lymphocytes for adoptive immunotherapy

Immune splenocytes were harvested from PepBoy donors previously inoculated with 105 PFU of MCMV (Smith strain; American Type Culture Collection) 2–4 mo earlier. Splenocytes were cultured at 107 cells/ml in complete medium (RPMI 1640 plus 10% heat-inactivated FBS, 1 mM sodium pyruvate, 1 mM nonessential amino acids, 5 x 10–5 M 2-ME, 100U/ml penicillin, 100 µg/ml streptomycin, and 29 mg/ml L-glutamine) at 37°C in 5% CO2 for 24 h according to our previously described methods (14). The cultured cells were resuspended at 20 x 106 per ml of PBS with 5% heat inactivated FBS (HyClone) containing 2 nM amotosalen (S-59 psoralen; Cerus). Twenty-five milliliters of total volume was added per 75-cm2 tissue culture flask and the cells were illuminated with UVA light (320–400 nm, 3.0 J/cm2 UVA dose; Cole Parmer) for a total of 5 min divided into 2.5-min fractions separated by a 1- to 2-min period of gentle agitation. Treated cells were washed and live cells were counted again under fluorescence microscopy using ethidium bromide mixed with acridine orange dye (16). The viability was usually >80% after photochemical treatment and an appropriate dosage of viable nucleated cells was used for adoptive immunotherapy. Cells cultured for 24 h without amotosalen treatment were used as controls.

Irradiation, cell transfer, and MCMV infection of BMT recipients

On day –1, CB6F1 mice received a total of 11 Gy of irradiation divided into two doses (5.5 Gy each) 3 h apart (17). The following day, bone marrow (BM) was flushed from the femora and tibia of naive BA donor mice and CD3+ T cells were depleted as described (17). CD3+ T cell-depleted (TCD) BM cells were transplanted alone or with 3 x 106 untreated or 10 x 106 amotosalen-treated donor splenocytes via tail vein injection into irradiated CB6F1 recipient mice. Recipient mice were infected with 2.5 x 104 PFU of MCMV i.p. or vehicle on day 7 posttransplant. The development of clinical GvHD was monitored twice weekly by weight loss and clinical signs of hair loss, ruffled fur, diarrhea, and decreased activity (17). Moribund mice were euthanized and considered to have died on the day following euthanasia for analysis of posttransplant survival.

Lymphocyte isolation

Splenocytes and thymocytes were harvested from BMT recipients by gently crushing tissue between frosted glass slides (Fisher Scientific) (15). Single cell suspensions were prepared by passing the resulting cell suspension through a cell strainer (BD Biosciences). Live lymphocytes were quantitated by fluorescence microscopy.

Determination of liver viral load

Liver viral load was determined as we described previously (18, 19). Briefly, livers were aseptically collected from the MCMV-infected recipients in 2 ml of RPMI 1640 with 2% FBS. The tissue was homogenized by using a Wheaton overhead stirrer (Wheaton Science Products) and centrifuged and different serial diluted supernatants were added over 3T3 confluent monolayers of a 24-well tissue culture plate and incubated for 90 min at 37°C. The wells were overlaid with 1 ml of 2.5% methyl cellulose in complete medium. PFU were directly counted under light microscope after removing the methylcellulose and stained with methylene blue.

Flow cytometry

Flow cytometry was performed as previously described (17). The origin of CD8+ and CD4+ T cells in BMT recipients was determined by staining with mAbs specific for donor BM (Thy1.2–CD45.2+), donor spleen (Thy1.2+CD45.2–), or host T cells (CD45.2+Thy1.2+) in combination with mAbs to CD8 and CD4. Anti-MCMV-specific CD8+ T cells were quantitated using an allophycocyanin-conjugated HGIRNASFI-H-2Db tetramer (NIAID Tetramer Core Facility, Atlanta, GA) (20). For quantitation of the activation status of donor spleen-derived T cells, cells were stained with mAbs for CD44 (FITC) and CD62L (biotin-streptavidin-allophycocyanin) along with mAbs for CD45.1 (PE) and T cell-specific Abs. To measure host-derived CD4–CD8– DN T cells, whole splenocytes were stained with the combined mAbs for CD4 and CD8 (PerCP) and CD3 (allophycocyanin) along with the mAbs CD45.2 (FITC) and Thy1.2 (PE). Granzyme B-producing CD4+ and CD8+ T cells were determined by using antihuman granzyme B (cross-reacts with mouse granzyme B) and their respective isotypes mAbs (purchased from Caltag Laboratories/Invitrogen) through intracellular staining. Stained cells were acquired by FACSAria (BD Biosciences) and analyzed by using FlowJo software. All Abs were purchased from BD Pharmingen.

Measurement of NK cell lytic activity

Splenocytes were harvested from the allogeneic BMT recipients of 10 x 106 treated splenocytes, 3 x 106 untreated splenocytes, and TCD BM alone on days 0 (day 7 after transplant) and 3 after 2.5 x 104-PFU MCMV infection. The harvested splenocytes were cocultured with different E:T ratios of 51Cr-pulsed YAC-1 target cells for 4 h at 37°C. The NK cell cytolytic activity of splenocytes were determined through 51Cr release assay in the culture supernatants as described in detail by Hossain et al. (19).

In vivo CTL assay

MCMV-immunized splenocytes were collected from the congeneic C57BL/6 (BA, CD45.2, Thy1.1) mice previously vaccinated with Lm-MCMV 14 days before. Lm-MCMV is a CMV vaccine consisting of a killed but metabolically active Listeria monocytogenes (Lm) platform (21) engineered to secrete the MCMV H-2b immunodominant peptide HGIRNASFI (20). This vaccine can induce high levels of anti-HGIRNASFI-specific CD8 T cells around day 7 of vaccination. The numbers of Ag- specific T cells in the blood declined rapidly thereafter, without causing morbidity or lethality in vaccinated recipients (our unpublished data). Half of the splenocytes were treated with 6 nM amotosalen and UVA light, resulting in a >95% reduction in proliferation after stimulation with plate-bound anti-CD3 mAbs in vitro as described previously (14). Amotosalen-treated splenocytes (20 x 106) and amotosalen-treated CD8-depleted splenocytes (20 x 106) were transplanted into irradiated (11 Gy) C57BL/6 recipients that were subsequently infected with a nonlethal dose of MCMV (1 x 104 PFU/mouse i.p.) on day 7 posttransplant. Untreated (20 x 106) splenocytes from vaccinated donors were similarly transplanted in irradiated syngeneic C57BL/6 recipients that were subsequently infected with MCMV and used as positive control for in vivo CTL activity. All BMT recipients received 5 x 106 CD3-depleted BM cells along with the splenocytes. Naive splenocytes (~80 x 106/ml in 3% FBS containing RPMI 1640) were harvested from CD45.1+CD45.2+ heterozygous C57BL/6 mice and pulsed with 3 µM MCMV early peptide (HGIRNASFI) for 90 min at 37°C, and washed thrice with ice cold sterile RPMI 1640 with 3% FBS. MCMV peptide-pulsed target splenocytes (15 x 106) and nonpulsed splenocytes (15 x 106) from CD45.1+ (CD45.2–) PepBoy mice were mixed together and injected i.v. (30 x 106 target cells per mouse) into MCMV-infected transplant recipients of amotosalen-treated donor splenocytes, recipients of amotosalen-treated CD8-depleted donor splenocytes, and untreated donor splenocytes 10 days after MCMV infection. The same numbers of MCMV peptide-pulsed target splenocytes and nonpulsed splenocytes were injected i.v. into nontransplanted MCMV-infected and uninfected naive C57BL/6(CD45.2, Thy1.2) mice as positive and negative controls for in vivo killing, respectively. Sixteen hours following target cell injection, all recipients were sacrificed, their splenocytes were harvested, and the residual MCMV peptide-pulsed CD45.1+CD45.2+ and nonpulsed CD45.1+CD45.2– target splenocytes were quantified by FACS analysis. For graphical presentation, the data from each individual mouse were normalized by dividing the corresponding percentages of residual target cells recovered from their spleens with the mean values of pulsed and nonpulsed targets recovered from the uninfected naive C57BL/6 control recipients. The mean values of these percentages are presented in a bar diagram as a percentage of control values (see Fig. 4B).

Measurement of serum cytokines by ELISA

Sera were collected from the recipients of amotosalen-treated and untreated splenocytes on days 0, 3, 5, 7, 11, and 18 after MCMV infection. Serum levels of IFN-{gamma} and TNF-{alpha} were determined by using ELISA following the directions of the specific ELISA kits purchased from the BD Pharmingen.

Statistical analyses

Mean values were compared using Student’s t test as described in the figure legends. Differences were considered significant when p < 0.05 was obtained.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
Short-term prophylactic adoptive immunotherapy with treated splenocytes protected recipients from MCMV infection without aGvHD

Our previous studies demonstrated that amotosalen-treated donor T cells retained short-term antiviral activity without significant in vivo proliferation or GvHD activity (14, 15). To examine the ability of amotosalen-treated donor T cells to protect against a delayed MCMV infection, we infected recipients of 10 x 106 treated splenocytes plus 5 x 106 TCD BM with a lethal dose of MCMV on day 7 posttransplant. For controls, we infected recipients of 5 x 106 TCD BM alone (no donor immunity) or recipients of 3 x 106 untreated splenocytes plus 5 x 106 TCD BM (positive donor immunity). After MCMV infection, 100% of the recipients of treated splenocytes survived to day 45 posttransplant whereas only 60% recipients of untreated splenocytes survived to day 45 posttransplant. In contrast, recipients of 5 x 106 TCD BM alone all died within the first 21 days posttransplant. In the absence of MCMV infection, all uninfected recipients of TCD BM alone or recipients of TCD BM plus treated splenocytes survived to day 45, while 80% of recipients of untreated splenocytes survived to the termination of this experiment (Fig. 1A). Recipients of treated splenocytes plus TCD BM and recipients of TCD BM alone steadily gained weight without any clinical signs of aGvHD in the absence of MCMV infection. Weight loss occurred among all recipients immediately after MCMV infection, but recipients of treated splenocytes had less weight loss compared with the recipients of untreated splenocytes (Fig. 1B) and developed mild hair loss and dry skin consistent with the development of mild chronic GvHD (data not shown).


Figure 1
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  		FIGURE 1. Prophylactic adoptive immunotherapy with amotosalen-treated splenocytes prevented MCMV mortality without producing aGvHD. Irradiated CB6F1 recipients were transplanted with 5 x 106 TCD BM cells with or without 10 x 106 amotosalen-treated (Treated) or 3 x 106 untreated donor splenocytes. Half of the recipients in each group were infected with MCMV (2.5 x 104 PFU i.p) on day 7 posttransplant. A, Percentage of survival. B, Percentage of weight loss as calculated from the initial weight measured on the day of transplant. The groups of experimental mice presented here are TCD BM ({square}), TCD BM plus MCMV ({blacksquare}), treated splenocytes ({triangleup}), treated plus MCMV ({blacktriangleup}), untreated ({circ}), and untreated plus MCMV (•). The data are representative of one of three similar experiments with five or six mice used per group per experiment.

 
Recipients of amotosalen-treated T cells developed donor and host-derived antiviral CTLs that effectively cleared MCMV from infected organs

To study antiviral immune response in recipients of amotosalen-treated donor splenocytes, we first measured the kinetics of viral load in the liver after infecting recipients with a sublethal dose (2.5 x 104 PFU) of MCMV. We selected the liver for measuring the viral load because the liver is one of the primary early target organs infected by this virus (22). Recipients of untreated and treated donor splenocytes cleared virus from the liver within day 11 of infection. In the treated group the viral load peaked on day 7 postinfection whereas in recipients of untreated splenocytes the viral load had peaked on day 3 after infection (Fig. 2). To investigate the role of host and donor cellular antiviral immunity, we isolated host- and donor-derived splenocytes from experimental mice at different time points after MCMV infection. Five days after viral infection, very low numbers of splenocytes were recovered from the spleens of recipients of both amotosalen-treated and untreated splenocytes. Among recipients of treated splenocytes the total cell number per spleen dramatically increased, reaching a peak on day 11 postinfection followed by a rapid contraction. In contrast, there was no significant expansion of total splenocyte numbers among recipients of untreated splenocytes (Fig. 3A). Recipients of treated splenocytes had a rapid expansion of host-derived T cells (Fig. 3D) and a slower expansion of donor spleen-derived T cells (Fig. 3B). Recipients of untreated splenocytes had a rapid expansion of donor spleen-derived T cells following MCMV infection with a corresponding decrease in the frequency of host-derived T cells (Fig. 3, B and D). Next, we determined the kinetics of MCMV peptide-specific tetramer+CD8+ T cells by FACS. Both donor spleen- and host-derived antiviral T cell populations gradually increased in the spleens of the recipients of treated splenocytes after viral infection (Fig. 3, C and E). In recipients of untreated splenocytes the donor spleen-derived MCMV peptide-specific tetramer+CD8+ T cell populations peaked on day 3 after MCMV infection followed by a abrupt contraction phase without any expansion of host-derived MCMV tetramer+CD8+ T cells (Fig. 3, C and E). In recipients of treated T cells infected with MCMV, MCMV peptide-specific tetramer+ antiviral CTLs of donor spleen origin peaked on day 7 after viral infection while an expansion of a smaller number of host-derived MCMV peptide-specific tetramer+CD8+ T cells occurred with a peak on day 5 after viral infection (Fig. 3E). Thus, expansion of both donor spleen- and host-derived MCMV peptide-specific tetramer+CD8+ T cells occurred among recipients of amotosalen-treated splenocytes that facilitated effective viral clearance from infected organs.


Figure 2
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  		FIGURE 2. The recipients of treated splenocytes successfully cleared viral load from the liver after MCMV infection. Irradiated CB6F1 recipients of 10 x 106 treated or 3 x 106 untreated donor splenocytes along with 5 x 106 TCD BM were infected with a sublethal dose of MCMV (2.5 x 104 pfu i.p.) 7 days posttransplant and livers were aseptically collected on days 3, 5, 7, 11, and 18 after infection. Liver tissue was homogenized and viral load per liver was determined by quantitative viral culture. Mean viral PFU and SD values obtained from the each infected group of five or six recipient mice are shown.

 

Figure 3
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  		FIGURE 3. Recipients of treated splenocytes had both donor- and host-derived antiviral T cells. Spleens from the irradiated CB6F1 recipients of 10 x 106 treated and 3 x 106 untreated donor splenocytes along with 5 x 106 TCD BM as described in the Fig. 2 legend were collected on days 0, 3, 5, 7, 11, and 18 after infection. Splenocytes were harvested as described in Materials and Methods. A, Kinetics of reconstitution of nucleated cells in the spleen after MCMV infection. B and D, Total donor spleen- and host-derived T cells per spleen as measured by FACS analysis, respectively. C and E, Total donor spleen- and host-derived MCMV peptide-specific tetramer+CD8+ T cells per spleen as measured by FACS analysis, respectively. Data are displayed as mean cell numbers with SD values per group of five or six mice from one of two similar experiments.

 
Adoptive immunotherapy with amotosalen-treated-splenocytes produces in vivo CTL activity and clears MCMV from the infected organs

Next, we assessed the antiviral immunity of amotosalen-treated T cells by measuring in vivo CTL cytolytic activity against syngeneic target cells. BMT recipients of 20 x 106 treated splenocytes, 20 x 106 untreated splenocytes, and 20 x 106 CD8-depleted treated splenocytes were infected with a sublethal dose of MCMV (1 x 104 PFU, i.p) on day 7 posttransplant. MCMV peptide-pulsed CD45.1+CD45.2+ C57BL/6 splenocytes (15 x 106) and nonpulsed CD45.1+ C57BL/6 splenocytes (15 x 106) were injected i.v. into all of the MCMV-infected BMT recipients on day 10 after MCMV infection. Similar numbers of target cells were i.v. transferred into congeneic C57BL/6 mice that were neither transplanted nor infected with MCMV (MCMV-naive; negative control) and into congeneic C57BL/6 mice that had been previously infected i.p. with 5 x 104 PFU of MCMV but were not transplanted (MCMV-immune mice; positive control). Sixteen hours later all of the recipients of congeneic target cells were sacrificed and their spleens were analyzed for the presence of residual target cells by FACS analysis (Fig. 4A). In MCMV-naive mice, equal numbers of peptide-pulsed and nonpulsed targets were recovered from the spleens of recipient mice with a normalized value shown as 100%. The peptide-pulsed targets were efficiently cleared from the spleens of nontransplanted MCMV-immune mice (17% of the number of nonpulsed targets) as well as the BMT recipients of treated (19% of number of nonpulsed targets) and untreated splenocytes (18% of number of nonpulsed targets). In contrast, CD8 depletion of the treated splenocytes abolished the differential clearance of the peptide-pulsed targets vs the nonpulsed targets (88% of number of nonpulsed targets) (Fig. 4B). Although the treated donor T cells had limited proliferation, analysis of tetramer staining on the donor T cell population revealed expansion of the MCMV Ag-specific CD8+ T cell population in both the treated (7.1 ± 3.2% of donor spleen-derived T cells) and untreated (4.1 ± 0.9% of donor spleen-derived T cells) donor cells, consistent with the observed in vivo clearance of the MCMV peptide-pulsed naive splenocytes (data not shown) and the retention of antiviral cytotoxic activity among treated CD8+ donor T cells.


Figure 4
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  		FIGURE 4. Donor spleen-derived T cells showed in vivo antiviral CTL activity against MCMV infection. A, MCMV-immunized splenocytes were collected from the congeneic C57BL/6 (BA, CD45.2, Thy1.1) mice previously vaccinated with Lm-MCMV 14 days before. Amotosalen-treated splenocytes (20 x 106) and amotosalen-treated CD8-depleted splenocytes (20 x 106) were transplanted into irradiated (11 Gy) C57BL/6 recipients that were subsequently infected with a nonlethal dose of MCMV (1 x 104 PFU/mouse i.p.) on day 7 posttransplant. Untreated splenocytes (20 x 106) from vaccinated donors were similarly transplanted into irradiated syngeneic C57BL/6 recipients that were subsequently infected with MCMV and used as BMT control for in vivo CTL activity as described in Materials and Methods. Sixteen hours following target cell injection, the residual MCMV peptide-pulsed CD45.1+CD45.2+ and CD45.1+ nonpulsed target cells in the spleens were quantified by FACS analysis. The FACS data are representative of splenocytes from one mouse of 6–10 mice per group. B, The data from each individual mouse (6 to 10 mice per group) were normalized as described in Materials and Methods. The mean values of these percentages are presented in a bar diagram as a percentage of control values. The average percentages of no peptide target splenocytes (open bars) and CD45.1+CD45.2+ splenocytes with MCMV peptide-pulsed target splenocytes (closed bars) are given + SE. *, p < 0.0001; Student’s t test. C, Represents FACS data for granzyme B-producing CD4 and CD8+ T cells on day 11 after MCMV infection in an independent syngeneic BMT experiment similar to those in A and B. Appropriate isotype control Abs were used. D, Congeneic C57BL/6 and perforin–/– mice were vaccinated with 1 x 106 Lm-MCMV CFU i.p. On day 14 after vaccination, splenocytes were harvested and 30 x 106 Lm-MCMV-vaccinated, amotosalen-treated splenocytes or 9 x 106 untreated splenocytes were transplanted into irradiated C57BL/6 mice. Perforin–/– splenocytes (9 x 106) were similarly transplanted into irradiated C57BL/6 mice. After 7 days, recipients were infected with MCMV i.p. Livers and spleens were harvested on day 3 and 7 after infection and viral load per organ was determined. The data presented here is from one experiment using five mice per group at each time point.

 
Because antiviral T cell function is related to the production of granzyme B and perforin (23), we next tested granzyme B expression by the CD4+ and CD8+ T cells on day 11 after MCMV infection in the splenocytes harvested from the recipients of treated and untreated splenocytes. The granzyme B-producing CD4+ and CD8+ T cells (as measured by intracellular staining) were found to be similar in the recipients of both amotosalen-treated and untreated splenocytes (Fig. 4C). We next compared the ability of the treated splenocytes to clear MCMV infection from infected organs with splenocytes from perforin–/– donors using a similar syngeneic BMT model. We harvested donor splenocytes from Lm-MCMV-vaccinated congeneic C57BL/6 and perforin–/– mice on day 14 postvaccination. Syngeneic BMT recipients of 30 x 106 treated splenocytes, 9 x 106 untreated splenocytes, and 9 x 106 perforin–/– splenocytes were infected with 2.5 x 104 PFU of MCMV i.p. and their spleen and liver viral loads were determined on days 3 and 7 after infection. Significantly higher viral loads (p < 0.005) were observed in the spleens of recipients of perforin–/– splenocytes as compared with recipients of treated C57BL/6 splenocytes on day 3 after infection. In the livers, viral loads were similar between treatment groups. All animals had cleared MCMV to essentially very low but detectable levels by day 7 after infection (Fig. 4D). Thus, treated spleen-derived T cells show antiviral immunity that appears to depend on the secretion of granzyme B and perforin, like untreated T cells.

BMT recipients of amotosalen-treated splenocytes showed no NK lytic activity

NK cells play an important role in the early management of MCMV infection in immunocompetent normal mice (11). To explore the effect of NK cells against MCMV infection in our BMT model, we next investigated the splenic NK cell response against MCMV infection in recipients of amotosalen-treated and untreated splenocytes as well as TCD BM alone on days 0 and 3 after infection (days 7 and 10 after transplant, respectively). The spleens of BMT recipients contained mostly donor BM-derived CD3–NK1.1+ NK cells on day 7 after transplant (before infection), and their number decreased by day 10 (3 days after MCMV infection; Fig. 5A). Donor spleen- and host-derived CD3-NK1.1+ cells were negligible in number and are not shown here. Although the splenocytes harvested from the recipients of treated, untreated, and TCD BM alone had higher numbers of CD3–NK1.1+ NK cells in uninfected condition, they did not kill NK-sensitive Yac-1 target cells (Fig. 5B). Splenocytes harvested from the recipients of untreated donor splenocytes 3 days after MCMV infection produced a modest level (20%) of target cell lysis at a 100:1 E/T ratio. Splenocytes recovered from recipients of treated splenocytes and recipients of TCD BM alone showed almost no NK cell cytolytic activities (Fig. 5B). Therefore, the recipients of TCD BM alone ultimately died due to cumulative replication viral loads in infected organs such as the liver, supporting the observation that host- and donor-derived NK cells have limited antiviral activity in this model system (Fig. 5C).


Figure 5
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  		FIGURE 5. Recipients of amotosalen-treated splenocytes showed no NK cytolytic activity against MCMV infection. CB6F1 recipients of treated splenocytes, untreated splenocytes, and TCD BM alone were infected with 2.5 x 104 PFU i.p. on day 7 after transplant. Normal C57BL/6 mice were infected with 5 x 104 PFU i.p. A, The presence of donor BM-derived NK cells (CD3-NK1.1+) was measured by FACS analysis. B, Splenocytes were collected on days 0 and 3 after infection and NK lytic activity was measured by using YAC-1 target cells pulsed with 51Cr. C, Kinetics of viral load per liver was measured in recipients of TCD BM alone after viral infection. The data represents one of two similar experiments using four or five mice per group at each time point.

 
Adoptive immunotherapy with amotosalen-treated T cells results reduced levels of inflammatory cytokines in response to MCMV infection

In recipients of allogeneic BMT, aGvHD causes injury to host tissue due to in part to dysregulated production of inflammatory cytokines by the donor T cells (24). To investigate the effects of inflammatory cytokines in our model of early CMV infection in allogeneic BMT, we next determined the kinetics of serum IFN-{gamma} and TNF-{alpha} levels after infecting BMT recipients with a sublethal dose of MCMV 7 days posttransplant. Recipients of amotosalen-treated and untreated splenocytes had similar levels of IFN-{gamma} in their serum on the day of MCMV infection, and very significantly higher levels of IFN-{gamma} were measured in the serum of recipients of untreated splenocytes on day 3 of MCMV infection compared with the recipients of treated splenocytes (Fig. 6, upper panel). TNF-{alpha} levels were also found to be similar on the day of MCMV infection and for the following 5 days among recipients of either treated or untreated splenocytes. A transient increase in TNF-{alpha} levels was seen on day 11 after infection only in recipients of untreated splenocytes (p > 0.05; Fig. 6, lower panel).


Figure 6
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  		FIGURE 6. Recipients of amotosalen-treated splenocytes induced significantly lower levels of inflammatory cytokines following MCMV infection. Sera were collected from the recipients of treated and untreated splenocytes on days 0, 3, 5, 7, 11, and 18 after MCMV infection. Serum levels of IFN-{gamma} and TNF-{alpha} were determined by following the directions of the specific ELISA kits purchased from BD Pharmingen as described in Materials and Methods. The data represents the mean and SE of each group and each time point of four or five mice used per group. *, p < 0.005; Student’s t test.

 
Adoptive immunotherapy with amotosalen-treated splenocytes allowed the generation of host-derived CD4–CD8– DN T cells during the early phase of MCMV infection

The significantly lower levels of virus in the liver among recipients of treated splenocytes compared with the recipients of untreated splenocytes on day 3 after infection (Fig. 2) suggested an antiviral effect of innate immunity among the former group. To further characterize early antiviral innate immune responses, we next measured the different cellular elements associated with innate immunity. Although NK cells represent the major part of the early antiviral innate immunity in normal immunocompetent mice, we could not measure detectable levels of NK cell cytolytic activity in splenocytes from the MCMV-infected BMT recipients (Fig. 5). Next, we determined the kinetics of host-derived CD4–CD8– (DN) T cells, another component of antiviral innate immunity that might respond during the early phase of MCMV infection (13). Surprisingly, we found very significantly (p = 0.005) increased levels of host-derived CD4–CD8– (DN) T cells on day 3 after MCMV infection in the spleens of recipients of treated splenocytes vs minimal numbers of host T cells in the spleens of recipients of untreated splenocytes (Fig. 7A). All of the CD4–CD8– (DN) T cells expressed CD44high without CD62L, a phenotype of activated T cells (Fig. 7B).


Figure 7
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  		FIGURE 7. Adoptive immunotherapy with amotosalen-treated splenocytes allowed the expansion of host-derived CD4–CD8– DN T cells following MCMV infection. Recipients of treated and untreated splenocytes sacrificed on days 0, 3, 5, and 7 after MCMV infection and the kinetics of the expansion of host-derived (Thy1.2+CD45.2+) CD4–CD8– (DN) T cells per host spleen were analyzed from their splenocytes by FACS analysis. A, Kinetics of host-derived CD4–CD8– (DN) T cells per spleen. B, Expression of CD62L and CD44 (solid lines; dashed lines are for isotype controls) by the CD4–CD8– (DN) T cells on days 0 and 3 after MCMV infection. *, p < 0.005; Student’s t test.

 
Recipients of amotosalen-treated splenocytes had persistent mixed T cell chimerism in the presence of viral infection

To characterize the activation status of T cells following viral infection, we determined the kinetics of T cell expansion, donor-host T cell chimerism, and T cell activation status in the spleens of MCMV-infected transplant recipients. The total number of splenocytes harvested per spleen from uninfected BMT recipients of amotosalen-treated and untreated splenocytes was very low (treated spleen: (3.5 ± 1.1) x 106; untreated spleen: (4.7 ± 1.8) x 106), consistent with the effects of myeloablative conditioning and the limited expansion of T cells within 7 days posttransplant (Fig. 3A). In the absence of viral infection, host-derived T cells represent 40–50% of splenocytes at day 7 posttransplant (Fig. 8A). Following viral infection, the donor spleen-derived T cells expanded modestly in the spleens of recipients of treated splenocytes and expanded mostly in the recipients of untreated splenocytes, concomitant with a marked depletion of host T cells resulting in almost 100% donor chimerism within 5 days after infection (12 days posttransplant) (Fig. 8A). The donor spleen-derived CD4+ T cells in the recipients of untreated splenocytes were significantly higher (p < 0.005) compared with the recipients of treated splenocytes on day 7 posttransplant. In response to viral infection, donor spleen-, BM-, and host-derived CD8+ T cells expanded significantly in recipients of treated splenocytes compared with the recipients of untreated splenocytes on day 11 after infection (Table I). After day 18 of infection, recipients of treated splenocytes showed considerably higher levels of CD4+ and CD8+ T cells derived from donor spleens, BM, and host compared with the recipients of untreated splenocytes (Table I). We next characterized the activation status of the donor spleen-derived T cells harvested from the spleens of recipients of both treated and untreated splenocytes on the day of MCMV infection (7 days posttransplant) and 11 days after MCMV infection. As the subsets of effector, memory, and naive activity of T cells are mostly defined by the expression of CD62L (25) and CD44 (26) surface Ags, we next determined the relative proportions of these subsets in the CD4 and CD8 compartments. Before transplant, most of the CD4 and CD8 T cells of both treated and untreated splenocytes had a naive phenotype with high levels of CD62L expression and low to intermediate (int) levels of CD44 expression (CD44low or CD44int). At day 7 posttransplant (i.e.; day 0 after MCMV infection) and day 11 after MCMV infection, donor spleen-derived CD4+ and CD8+ T cells harvested from the spleens of recipients of both treated and untreated splenocytes had lost CD62L expression and become CD44high, consistent with either their transition to an effector or memory phenotype and/or a greater relative expansion of memory T cells vs naive T cells (Fig. 8B). Thus, the loss of CD62L expression and the gain of CD44 expression following MCMV infection were consistent with T cell activation and conversion from a predominantly naive phenotype to an effector/memory phenotype (26).


Figure 8
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  		FIGURE 8. Donor spleen-derived, amotosalen-treated T cells expand in response to MCMV infection. Recipients of treated or untreated splenocytes were sacrificed at serial time points after MCMV infection to assess changes in T cell populations derived from the BMT recipient, the BMT donor, and the splenocyte donor. A, Kinetics of T cells derived from donor spleens (Thy1.2+CD45.2–), host (Thy1.2+CD45.2+), and donor BM (Thy1.2–CD45.2+) along with host-derived non-T cells). The FACS data shown here represent a single mouse at each time point and are representative of the four or five mice tested per time point. B, Expression of CD62L and CD44 (solid lines; dashed lines are for isotype controls) by the donor spleen-derived CD8 and CD4 T cells before transplant on days 0 and 11 after MCMV infection. These data are representative of the four or five mice used per group.

 

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  		Table I. CD4+ and CD8+ T cells per spleen of recipients with treated and untreated splenocytesa

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
We have previously shown that transplant grafts containing 10 x 106 amotosalen-treated splenocytes conferred protection from lethal doses of MCMV without causing GvHD (14, 15). Because ex vivo photochemical treatment of T cells significantly inhibits their replicative capacity, the antiviral activity of treated T cells could be due to their short-term cytotoxic activity in vivo (in the absence of T cell proliferation) or the ability of an antigenic stimulus to rescue antiviral T cells from apoptosis, which usually follows the amotosalen-induced double-strand DNA crosslinking. The mechanism of early antiviral immune response of treated splenocytes and the question why the treated splenocytes that apparently regained proliferative capacity did not induce aGvHD were not addressed in our earlier reports. In this study, we have developed a more clinically relevant model of MCMV infection with a sublethal dose of virus administered on day 7 posttransplant to simulate the early MCMV reactivation that occurs in CMV seropositive recipients undergoing allogeneic BMT (4). Our present data confirm that treated donor T cells retained their antiviral activity without causing aGvHD, even when the viral challenge was delayed by 1 wk after transplant and the infusion of treated donor splenocytes. Although the T cells that originated from the treated splenocytes had limited proliferation in vivo following acute MCMV infection (consistent with the antiproliferative effects of DNA crosslinking), recipients of treated splenocytes successfully cleared MCMV from the infected target organs and had 100% survival compared with 0% survival in recipients of TCD BM alone and 60% survival among mice transplanted with a low nonlethal dose of untreated splenocytes (Fig. 1). Moreover, recipients of treated splenocytes had better immune reconstitution, better antiviral immune responses in lymphoid and nonlymphoid organs, and lower levels of inflammatory cytokines than recipients of a lower dose of untreated splenocytes. The procedure of using ex vivo treated donor lymphocytes to adoptively transfer immunity in allogeneic transplantation has been developed to facilitate posttransplant immune reconstitution without GvHD (16, 27, 28).

One striking finding in our present study is that the recipients of treated splenocytes had mixed chimerism with both donor- and host-derived immune cells and expansion of viral Ag-specific T cells of both host and donor origin. Allogeneic BMT with mixed chimerism has several advantages over the full donor chimerism (29). In mixed chimeric recipients, hematopoietic progenitor cells from both the host and the donor are allowed to populate in the thymus and undergo negative selection deleting both host- and donor-reactive T cells, resulting in a pool of peripheral T cells with a broad repertoire that are tolerant toward donor and host allo-Ags (30, 31). Furthermore, T cell mixed chimerism has been correlated with a decreased risk of moderate to severe aGvHD and death by GvHD (32). Of note, the clearing of the MCMV infection in recipients of amotosalen-treated splenocytes was temporally associated with the initial proliferation of host-type immune cells followed later by a slower, limited proliferation of donor spleen-derived T cells and the absence of high levels of inflammatory cytokines. Thus, early mixed chimerism in recipients of treated splenocytes likely helped generate an antiviral immune response following an early posttransplant viral infection. Furthermore, because viral infection produces long-lived memory T cells that are restricted to both host type and donor type MHC (33), early mixed chimerism may result in protective, synergistic, long-term antiviral immune responses.

We investigated the mechanism by which treated donor splenocytes with limited proliferative capacity in vitro confer antiviral activity in vivo. Because anti-MCMV-immune response is controlled by both innate and adoptive immunity in normal mice, macrophages, NK cells, and antiviral cytokines such as type 1 IFN, IFN-{gamma}, etc. may play an important role in controlling MCMV infection within the first 5 days postinfection, with a primary role for CD8+ T cells in clearing virus-infected cells from the organs of transplant recipients at later time points (11, 12, 34, 35, 36). Although treated donor T cells had lost >90% of their proliferative capacity after in vitro stimulation with plate-bound anti-CD3 mAb and had limited in vivo expansion of MCMV peptide-specific tetramer+CD8+ T cells in the presence of MCMV infection, the recipients of treated CD8+ T cells had showed similar levels of vivo CTL activity compared with recipients of untreated splenocytes using a syngeneic BMT model and MCMV peptide-pulsed target cells as described in Fig. 4, A and B. Comparing recipients of treated and untreated splenocytes, we saw highly effective clearing of the peptide-pulsed targets compared with the nonpulsed targets in both groups. Similar levels of granzyme B expression were also seen in both treated and untreated T cells (Fig. 4C), further supporting their antiviral function against MCMV in vivo. Of note, the numbers of donor nonpulsed and MCMV peptide-pulsed target cells recovered from the spleens of both groups of BMT recipients were approximately half the number of target cells recovered from nonirradiated, nontransplanted normal C57BL/6 mice. The likely explanation for this observation is that lethal irradiation may affect the migration of donor target cells in vivo.

We studied the role of donor and host NK cells in clearing virus in the transplant model systems because amotosalen-resistant donor NK cells could potentially be responsible for the observed antiviral activity of the treated splenocytes. A large number of donor BM-derived CD3–NK1.1+ cells were detected in the spleens of BMT recipients within 7 days after transplant (Fig. 5A), but they had limited NK cytolytic activity against Yac-1 targets when tested 3 days postinfection (Fig. 5B). The lack of NK cell-mediated antiviral immunity may be due to a lack of the intercellular immunoregulatory communications required to activate or mature cytolytic NK cells or to an altered NK cell repertoire during the first weeks posttransplant with a predominance of NK cells expressing NK cell receptor (34, 35, 36, 37). As a result, BMT recipients that received only TCD BM alone died due to MCMV infection (Fig. 5C). Therefore, antiviral protection is dependent upon donor spleen-derived MCMV memory CD8+ T cells in this experimental BMT model. Interestingly, although the amotosalen-crosslinked CD8+ T cells showed limited proliferation and did not cause aGvHD, they played a major role in clearing virus from infected tissues. Donor-treated spleen-derived MCMV peptide-specific tetramer+CD8+ T cells expanded following MCMV infection and expressed granzyme B (an important effector molecule for CTL immunity; Ref. 23), with similar kinetics as those of donor-derived T cells among recipients of untreated splenocytes (Fig. 4). Recipients of 9 x 106 perforin–/– splenocytes from MCMV-immune donors had significantly higher viral loads in the spleen compared with recipients of 30 x 106 treated splenocytes from MCMV immunized donors (p < 0.005), suggesting that effective viral clearance requires perforin expression on donor T cells in this tissue, but there were no differences in the liver viral load between these groups. A reduced dose of perforin–/– donor splenocytes (9 x 106) was chosen to permit observations of antiviral immune responses without early lethality due to GvHD, because perforin–/– T cells retain significant GvHD potential (38). The similar levels of virus in the liver on days 3 and 7 postinfection indicates that MCMV in the liver is not cleared by perforin-mediated immunity as has been previously described by Tay and Welsh (39) although the exact mechanism of antiviral immunity of perforin–/– donor splenocytes has not been defined in our experimental system.

In recipients of untreated splenocytes, donor spleen-derived CD4+ T cells expanded to significantly higher levels (p < 0.005) compared with recipients of amotosalen-treated splenocytes on day 7 posttransplant (Table I), which may be a manifestation of acute GvHD in the former group because CD4+ T cells are the primary agents responsible for acute GvHD in this strain combination (40). In presence of CMV infection the intensity of acute GvHD is exacerbated, which further inhibits splenic immune function in allogeneic transplant recipients (41). However, in response to viral infection, CD8+ T cells expanded in the spleens of recipients of treated splenocytes and their immune functions remained intact, which further supports the absence of acute GvHD in these mice. The mechanism that allogeneic donor CD8+ T cells recover from the anti-proliferative effects of amotosalen/UVA light treatment is hypothesized to be a differential effect of ex vivo amotosalen exposure on T cells with a naive phenotype, because this donor cell population contains the majority of alloreactivity in the transplant graft (Ref. 16 and our unpublished data).

We noted an extensive expansion of host-derived CD4–CD8– (DN) T cells during the early phase following MCMV infection in recipients of treated splenocytes (Fig. 7). These host DN T cells might be responsible for a number of important antiviral therapeutic functions. First, DN T cells are a component of innate immunity and are able to produce a number of immunoregulatory cytokines and chemokines such as IFN-{gamma}, TNF-{alpha}, MCP-1 and Eta-1 (early T cell activation-1) that initiate adaptive immune responses (42, 43) (13). Thus, the significantly lower liver viral load on day 3 following MCMV infection in recipients of treated splenocytes compared with the recipients of untreated splenocytes might be the result of the antiviral activity associated with innate immune responses initiated by host-derived antiviral DN T cells (Fig. 7). Second, the DN T cells might help in the generation of both host- and donor-derived Ag-specific cytotoxic CD8+ T cells that effectively clear virus-infected cells during the late phase of MCMV infection (13). Third, the extensive expansion of DN T cells might help to ameliorate the potential for donor T cells to cause aGvHD (44, 45, 46).

One of the significant differences in transplant outcomes that we observed between recipients of treated vs untreated splenocytes was the absence of high levels of TNF-{alpha} and IFN-{gamma} in the former group. High levels of inflammatory cytokines in the early posttransplant period are part of the pathogenesis of aGvHD (47). TNF-{alpha} is reported to be the major inflammatory cytokine that is responsible for much of the cytokine-mediated damage to host tissues in patients with aGvHD (48). In the present model system, by using prophylactic administration of amotosalen-treated lymphocytes 1 wk before MCMV infection we found that serum levels of TNF-{alpha} and also IFN-{gamma} in recipients of treated splenocytes were low and were not increased following MCMV infection, whereas the administration of a low dose of untreated splenocytes was associated with marked transient increases of IFN-{gamma} (Fig. 6). The low level of serum TNF-{alpha} observed in recipients of 3 x 106 untreated splenocytes might be due to the presence of mild aGvHD or to the lower doses of total body irradiation used in these experiments with less tissue injury than that used in other model systems (49). The use of higher numbers of untreated splenocytes as a better positive control for aGvHD was not feasible because of the higher mortality among recipients of larger numbers of untreated splenocytes (14). The high serum level of IFN-{gamma} on day 3 postinfection seen in recipients of untreated splenocytes (Fig. 6) was associated with the extensive expansion of MCMV-immunized donor T cells and likely contributed to their antiviral activity (50, 51) by enhancing MCMV peptide processing (52). Thus, the abrupt decrease in viral load per liver within 5 days of infection in recipients of treated splenocytes and the slower clearance of virus in recipients of treated splenocytes (Fig. 2) might be due to higher levels of IFN-{gamma} during the early phase of MCMV infection in the former group (Fig. 6).

In summary, we have studied the mechanisms by which prophylactic administration of treated splenocytes protect allogeneic BMT recipients against a lethal dose of MCMV administered on day 7 posttransplant. Recipients of treated splenocytes had expansion of both host- and donor-derived MCMV peptide specific tetramer+CD8+ T cells and lower levels of the inflammatory cytokines that are associated with the pathogenesis of acute GvHD. The in vivo killing of MCMV-pulsed naive target cells by treated donor T cells in a syngeneic BMT model also demonstrated that treated T cells retained antiviral activity in a model in which aGvHD is absent. These results suggest that prophylactic administration of treated donor lymphocytes may be an ideal method of enhancing posttransplant immunity in recipients of allogeneic BMT: a method of adoptive cellular immunotherapy that avoids the thymic damage associated with GVHD while producing a protective antiviral effect. Photo-activated amotosalen-DNA crosslinked treatment of T cells is an established treatment process that has already been used in human to treat cutaneous T cell lymphoma, GvHD, and autoimmune disease (53, 54, 55). The present data demonstrating the antiviral activity and lack of toxicity following prophylactic adoptive cellular immunotherapy using amotosalen-treated donor lymphocytes suggests that clinical trials of this approach in patients undergoing allogeneic BMT may be warranted.


   Acknowledgments
 
We thank Cerus Corporation for providing amotosalen (S-59 psoralen).


   Disclosures
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
The authors have no financial conflict of interest.


   Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 This research was supported by National Institutes of Health Grants HL70997 (to J.D.R.) and CA-74364 (to E.K.W.). Back

2 Address correspondence and reprint requests to Dr. Edmund K. Waller, Department of Hematology and Oncology, Winship Cancer Institute, Emory University, 1701 Upper Gate Drive, WCI Building, 4th Floor, Atlanta, GA 30322. E-mail address: ewaller{at}emory.edu Back

3 Abbreviations used in this paper: GvHD, graft-vs-host disease; aGvHD, acute GvHD; BM, bone marrow; BMT, bone marrow transplant; DN, double negative; Lm, Listeria monocytogenes; MCMV, murine CMV; TCD, CD3+ T cell depleted. Back

Received for publication August 15, 2007. Accepted for publication March 9, 2008.


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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
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*Bone Marrow Transplantation
*Cytomegalovirus Infections

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