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The Journal of Immunology, 1998, 160: 4481-4489.
Copyright © 1998 by The American Association of Immunologists

Thrombopoietin (TPO) Knockout Phenotype Induced by Cross-Reactive Antibodies Against TPO Following Injection of Mice with Recombinant Adenovirus Encoding Human TPO1

Mohammed-Amine Abina2,*, Micheline Tulliez{ddagger}, Marie-Thérèse Duffour{dagger}, Najet Debili*, Catherine Lacout*, Jean-Luc Villeval*, Françoise Wendling*, William Vainchenker* and Hedi Haddada{dagger}

* Institut National de la Santé et de la Recherche Médicale, Unité 362, and {dagger} Centre National de la Recherche Scientifique Unité de Recherche Associée 1301, Institut Gustave Roussy, Villejuif, France; and {ddagger} Service d’anatomo-pathologie, Hôpital Cochin, Paris, France


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Adenovirus vectors have emerged as potent agents for gene transfer. Immune response against the vector and the encoded protein is one of the major factors in the transient expression following in vivo gene transfer. A single injection of an adenovirus encoding human thrombopoietin (TPO) into mice induced transient thrombocytosis, followed by a chronic immune thrombocytopenia. Thrombocytopenic mice had anti-human TPO Abs of the IgG2a and IgG1 isotypes. Thrombocytopenic mice sera neutralized more efficiently human than murine TPO, and exhibited no detectable anti-murine TPO Abs. Despite their low affinity for murine TPO, anti-TPO Abs induced a TPO knockout-like phenotype, i.e., low number of marrow megakaryocytes and of all kinds of hemopoietic progenitors. Hybridomas derived from a thrombocytopenic mouse revealed cross-reactivity of all of the secreted anti-TPO Ab isotypes. Mice subjected to myelosuppression after virus injection showed that anti-human TPO of IgG1 and IgG2a isotypes disappeared. Thus, sustained human TPO production was responsible for platelet elevation for at least 5 mo. Compelling results showed that elevated IgG2a/IgG2b ratios are always associated with thrombocytopenia, whereas low ratios are associated with tolerance or normal platelet counts. Finally, we hypothesize that in humans some chronic thrombocytopenia associated with a low TPO plasma level are due to anti-TPO Abs.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Recombinant adenoviruses are DNA viruses capable of efficiently transducing a wide range of cell types in vitro and in vivo (1). However, gene expression is often transient, due to extrachromosomal delivery of the genetic material and to immunologic clearance of viral transduced cells (2). Cellular immunity has been described as the major mechanism limiting the duration of transduced gene expression and is mediated initially by CD8+ cytotoxic T cells (3, 4). Ab-mediated humoral immunity is the major effector of the secondary response (5, 6). Overall, the response to a systemic adenoviral infection involves Th1 and Th2 T cell subsets with production of IFN-{gamma}, IL-2 (Th1), IL-4, and IL-10 (Th2) (7). The antiviral Ab response shows a predominance of IgG2a, IgG1, and IgM isotypes (8, 9, 10). However, the immune response may also be directed against the transgene-encoded protein, as shown by Ab production against various foreign proteins such as canine factor IX (5) or human {alpha}1-antitrypsin in mice (9). The presence of cross-reactive Abs with neutralizing activity against human and murine erythropoietin (Epo)3 has also been described in the mouse after injection of an adenovirus encoding human Epo (11). The injection of an adenovirus encoding the murine Epo cDNA produced a sustained biologic effect for up to 112 days, suggesting that the immune response against the transgene-encoded protein is a major factor in transient gene expression following adenoviral gene transfer (11).

Thrombopoietin (TPO) is a recently isolated growth factor that regulates platelet production (12) and has about 50% homology with Epo. We demonstrate in this study that a single injection of a recombinant adenovirus containing the huTPO cDNA to DBA/2J mice induces a transient platelet elevation within the first 3 wk, followed by a stable chronic immune thrombocytopenia due to the generation of cross-reactive anti-TPO Abs. The thrombocytopenic mice showed anti-TPO Abs of IgG2a, IgG1 isotypes. Moreover, these Abs were capable of completely clearing endogenous TPO, leading to a phenotype similar to that observed in TPO knockout mice. In contrast, when DBA2/J mice were subjected to a myeloablative regimen 7 days after adenovirus injection, prolonged expression of the transgene was observed and correlated with inhibition of anti-TPO Abs of IgG2a and IgG1 isotypes.

An immune response against Epo was implicated in a type of immunologically induced anemia in humans (13, 14); we hypothesize a similar mechanism for some cases of immune thrombocytopenia, especially those related to a low TPO production.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Construction of recombinant E1-deleted adenovirus vector

The huTPO cDNA was inserted in the EcoRV restriction site of the adenovirus (Ad) Rous sarcoma virus (RSV) ß-galactosidase (ßgal) plasmid after excision of the ßgal gene by SalI. The huTPO cDNA under control of the RSV viral promoter is followed by a fragment of Ad5 (mu 9.4-17; BglII-HindIII) to permit homologous recombination for the generation of the recombinant adenovirus AdRSVhuTPO. The resulting plasmid was cotransfected into the human embryonic 293 cell line with ClaI-digested Ad5dl324 DNA using precipitation by calcium phosphate, as previously described (15). AdRSVßgal carrying the nuclear localization site Escherichia coli lacZ marker gene under the control of the same viral promoter was used as a control and has been previously described (15). Viral stocks were prepared by infection of the 293 cell line, purified and concentrated by a double cesium chloride gradient, dialyzed, aliquoted, and stored in 10% glycerol at -80°C. Titers of the viral stocks were determined by limiting dilution on plaque assays using 293 cells and expressed as PFU.

Animal procedures

DBA/2J-specific pathogen-free mice were obtained from Janvier (Orleans, France). All animals were bred in negative pressure isolators for adenovirus injection experiments in the animal facilities of Institut Gustave Roussy (Villejuif, France). Female mice (6–8 wk old) were injected with recombinant adenoviruses via the retroorbital sinus. DBA/2J mice were injected with 3 to 6 x 109 PFU of AdRSVhuTPO, while control mice were injected with the same doses of AdRSVßgal or with PBS.

TPO concentrations

Serum TPO concentrations were measured using a microwell assay (16). Assays were performed in duplicate by adding 200 cells from the human c-mpl-transfected Ba/F3 cell line (12) in a 10-µl vol of DMEM plus 10% FCS to serial twofold dilutions of the serum. TPO concentrations were calculated by assigning 1 U/ml to the concentration, resulting in 50% cell survival after 2 to 3 days of incubation at 37°C in a humidified atmosphere of 10% CO2 in air. In a dose-response analysis using the full-length rhuTPO, 1 U is approximately the equivalent of 100 pg of the molecule.

Peripheral blood hematologic measurements

Blood samples were obtained from ether-anesthetized animals by puncture of the retroorbital sinus. After RBC lysis in Unopette vials (Becton Dickinson, Franklin Lakes, NJ), platelets and white cells were counted by microscopy and microhematocrits were determined following blood centrifugation.

Analysis of clonogenic committed progenitor cells

Femoral marrow (8 x 104) and spleen cells (1 x 106) of DBA/2J mice, harvested at various times following injection of AdRSVhuTPO, were cultured in 1 ml of 0.8% methylcellulose in Iscove’s medium supplemented with 20% FCS supplemented with rmuIL-3 (100 U/ml; Immunex, Seattle, WA) and rhuEpo (1 U/ml; Cilag, Paris, France) to determine the number of granulocyte-macrophage CFU (CFU-GM) and erythroid burst-forming cells (BFU-E). Megakaryocyte CFU (CFU-MK) were grown in 0.3% agar supplemented with rmuTPO (10 ng/ml; ZymoGenetics, Seattle, WA), rmuIL-3, and recombinant murine stem cell factor (50 ng/ml; Immunex), as previously described, using 1 x 105 marrow cells and 5 x 105 spleen cells/500 µl agar medium (12). For each determination, cultures for one noninjected and one AdRSVhuTPO-injected mouse were performed in duplicate at 37°C/5% CO2 in air for 5 days.

TPO-neutralizing activity in the sera of thrombocytopenic mice

To determine the anti-TPO activity in the sera of thrombocytopenic mice, microwell assays were performed by adding 200 cells from the human or murine c-mpl-transfected Ba/F3 cell line to serial dilutions of the serum previously incubated for 1 h at 37°C with 2 U/ml (200 pg/ml) of rhuTPO or rmuTPO, respectively. rhuTPO was added at a high concentration (5 µg/ml) to serial dilutions of the serum to reverse the neutralization. To exclude nonspecific toxicity of the mouse serum, Ba/F3-mpl-transfected cells were also stimulated with 50 U/ml of rmuIL-3 added to the serial dilutions of the sera to be tested. All dilutions were tested in duplicate.

Detection of anti-human and anti-murine TPO Abs

Ninety-six-well Nunc Maxisorb plates were coated with 1 µg/ml of huTPO (Genzyme, Cambridge, MA) or muTPO (ZymoGenetics, Seattle, WA) in PBS/0.1% BSA overnight at 4°C. PBS/2% FCS was used to block nonspecific binding. Plates were washed (PBS/0.1% Tween-20), and serial dilutions of sera from AdRSVhuTPO- and AdRSVßgal-injected mice were incubated in the coated wells for 90 min at 37°C. The plates were washed five times with PBS/0.1% Tween-20 and then incubated with 100 µl of a 1/5000 dilution of peroxidase-conjugated goat anti-mouse IgG + IgM or goat anti-mouse IgM (Jackson ImmunoResearch Laboratories, West Grove, PA) for 1 h at 37°C. For determination of anti-huTPO Ab isotypes, the following peroxidase-conjugated Abs were used: goat anti-mouse IgG2a, goat anti-mouse IgG2b, and goat anti-mouse IgG1 (Southern Biotechnology, Birmingham, AL). All Abs were used at a dilution of 1/5000. Following washing, the wells were incubated with 100 µl of substrate (o-phenylenediamine-dihydrochloride; Sigma, St. Louis, MO). The reaction was stopped after 5 to 10 min by adding 50 µl of 12% H2SO4. The OD was measured with a spectrophotometer at 492 nm. Wells were considered as positive when the OD was approximatively twofold that of the OD observed with 5-wk serum from an AdRSVßgal-injected mouse. The IgG2a/IgG2b ratio was calculated by dividing the inverse of the last positive dilution of IgG2a anti-huTPO Ab by the inverse of the last positive dilution of IgG2b anti-huTPO Ab. For each mouse, the first dilution assayed was 1/40; if no positivity was found at this dilution, the titer was arbitrarily considered to be 1/10 for purposes of calculation.

Detection of anti-viral Abs

Microtiter plates as described above were coated for 18 h at 4°C with 100 µl/well of PBS containing 1 µg/ml of heat-inactivated AdRSVßgal particles treated with SDS (0.01%). Plates were washed (PBS/0.1% Tween-20), and serial dilutions of sera from AdRSVhuTPO- and PBS-injected mice were incubated in the coated wells for 90 min at 37°C. The plates were washed five times with PBS/0.1% Tween-20 and then incubated with 100 µl of a 1/5000 dilution of peroxidase-conjugated goat anti-mouse IgG + IgM for 1 h at 37°C. For determination of anti-adenoviral Ab isotypes, the same Abs used for determination of anti-TPO isotypes were used at the same dilutions.

Histology

Organs (spleen, femur, tibia, kidney, liver, and lung) of mice sacrificed at different times after the injection of the recombinant adenovirus vectors were fixed in Bouin’s solution or buffered formaldehyde and embedded in paraffin. Thin sections (3–5 µm) were stained by hematoxylin/eosin (HE), May-Grünwald-Giemsa, or periodic acid-Schiff (PAS) stains.

Myeloablative regimen

Seven days after the injection of PBS, AdRSVßgal, or AdRSVhuTPO, mice were subjected to a myeloablative regimen consisting of the combination of 5 Gy irradiation and a single i.p. injection of 1.2 mg of carboplatin (Paraplatin; Bristol-Myers-Squibb, Princeton, NJ).


   Results
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 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Kinetics of platelet production in DBA2/J after i.v. injection of AdRSVhuTPO

AdRSVhuTPO (3–6 x 109 PFU/mouse) was administered via the retroorbital sinus to a total of 14 DBA/2J mice in two independent experiments. A 1.6-fold increase in the platelet count after the first week (n = 13) and a 2.8-fold increase after the second week (n = 13) following injection were observed (Fig. 1Go). After the third week, 6 of the 14 mice were thrombocytopenic (the mean platelet count of the thrombocytopenic mice was 78.5 x 104/µl ± 0.7), while 4 mice had high platelet counts. At week 5, all animals except two (8 of 10) were thrombocytopenic (mean platelets count of the thrombocytopenic mice was 47.5 x 104/µl ± 0.7). The thrombocytopenia worsened over the following weeks and remained stable (13–36% of normal platelet count) by week 9. One thrombocytopenic mouse was analyzed 10 mo after the injection of the AdRSVhuTPO and still had a low platelet count (14% of normal). Another mouse did not exhibit thrombocytopenia over 8 mo of observation, but became thrombocytopenic at 10 mo, with 44% of the normal platelet count. No platelet variation was observed during the follow-up in the PBS- or the AdRSVßgal-injected mice. The platelet count was 144.2 x 104/µl ± 12.3 in the PBS-injected mice.



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  		FIGURE 1. Early onset of thrombocytopenia in DBA/2J mice injected i.v. with AdRSVhuTPO. Thrombocytosis was observed at weeks 1 and 2 following injection of AdRSVhuTPO. Thrombocytopenia appeared at about 3 to 4 wk and progressively increased to reach 13 to 36% of the normal platelet counts at about 8 wk. Individual mice are presented; bar represents mean of platelet counts of normal mice.

 
In addition, serum levels of huTPO were measured by a biologic assay after the injection of 3 to 6 x 109 PFU of AdRSVhuTPO. huTPO production was transient. Elevated levels were detected during the first week, rapidly declined during the second and the third week after injection, and returned to undetectable levels after 4 wk. Thereafter, no TPO bioactivity (either human or murine) was measurable (data not shown).

Histologic analysis

At week 2 following administration, AdRSVhuTPO-injected mice had mononuclear and polymorphonuclear cell granulomas in the lobular area of the liver associated with focal necrosis (Fig. 2GoA) and megakaryocytic hyperplasia. The spleen and bone marrow also revealed marked megakaryocytic hyperplasia. At wk 4, the inflammatory granulomas were absent in the liver sections. Later (>=9 wk), the histologic sections of the liver of markedly thrombocytopenic mice showed plasma cell infiltration in the periportal area (Fig. 2GoB). These mice also displayed a significant decrease in megakaryocyte numbers both in marrow and spleen. In the marrow, megakaryocytes were estimated to be 10% of normal (Fig. 2Go, C and D), and hyperplasia of the splenic white pulp, especially in the marginal area (B cell area) was observed (Fig. 2GoE). Spleen of a normal mouse was used as control (Fig. 2GoF).



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  		FIGURE 2. Histopathologic analysis of mice injected with AdRSVhuTPO. A, Liver sections show polymorphonuclear cells infiltrating the periportal and the intralobular area of the liver at wk 2 (original magnification, x40). B, Liver sections of a thrombocytopenic mouse at 9 wk show plasma cells (arrows) infiltrating the periportal area (original magnification, x40). C, Bone marrow sections show rare megakaryocytes (arrows) compared with D, normal bone marrow (original magnification, x10). E, Spleen sections show hyperplasia of the marginal area compared with F, normal spleen from control mice (original magnification, x4). The arrows indicate the granuloma in A and the marginal area in E and F. v, portal blood vessel.

 
Analysis of committed progenitors in thrombocytopenic mice

The number of CFU-GM- and BFU-E-derived colonies was analyzed in four severely thrombocytopenic mice at weeks 9, 12, and 13, and 10 mo after the AdRSVhuTPO injection. In the marrow (n = 4), CFU-GM and BFU-E numbers were markedly decreased (56 and 33% of control). In the spleen, a decrease in these progenitors was also observed (37 and 50% of controls for CFU-GM and BFU-E, respectively) (n = 3; Table IGo). When CFU-MK were analyzed, similar changes were noted (51 and 19% of control values in the marrow and spleen, respectively, at week 12).


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  		Table I. Low levels of erythroid and myeloid progenitors in thrombocytopenic mice following injection of AdRSVhuTPOa

 
To obviate difficulties of interpretation of variation of progenitor cell numbers among experiments, progenitors from normal animals were used as internal controls for each experiment. Analysis of progenitors in AdRSVßgal-injected mice showed no differences compared with noninjected mice (data not shown).

Presence of a TPO-neutralizing activity in the sera of thrombocytopenic mice

The presence of thrombocytopenia associated with a decrease in marrow megakaryocytes and an undetectable level of TPO strongly suggests the possibility of anti-huTPO Abs cross-reactive with muTPO. In a first set of experiments, anti-TPO-neutralizing activity in the sera of thrombocytopenic mice was investigated. Sera of markedly thrombocytopenic mice were taken at weeks 7, 9, and 13 after the initial injection of the AdRSVhuTPO, when the platelet counts were 21, 14, and 12%, respectively, of normal, and were analyzed for their capacity to inhibit the proliferation of Ba/F3-hu-c-mpl and Ba/F3-mu-c-mpl cells in the presence of rhuTPO or rmuTPO.

One milliliter of the 7-wk serum inhibited the equivalent of 256 U of the muTPO and 8000 U of the huTPO, while 1 ml of the 9-wk serum inhibited the equivalent of 8000 U of the muTPO and 32,000 U of the huTPO (Fig. 3Go). For the 13-wk serum, 1 ml of serum inhibited the equivalent of 32,000 U of the huTPO; this inhibition was neutralized by an excess of huTPO.



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  		FIGURE 3. Presence of neutralizing activity against human and murine TPO in the sera of thrombocytopenic mice. Neutralizing activity was calculated as follows: inverse of the maximal dilution giving neutralization x concentration of TPO used for the neutralization assay on the human or murine c-mpl-transfected Ba/F3 cell line. Higher neutralizing activity was observed against huTPO than against muTPO.

 
Detection of anti-TPO Abs in the sera of thrombocytopenic and nonthrombocytopenic mice

Since TPO-neutralizing activity was detected in the sera of thrombocytopenic mice, the presence of Abs directed against human and murine TPO was directly assayed in seven thrombocytopenic mice. Total Ig and different isotypes of the Abs against human and murine TPO were analyzed. All of the thrombocytopenic mice had high titers of anti-huTPO Abs of the IgG1 (Fig. 4GoA) and IgG2a (Fig. 4GoB) isotypes, whereas the IgG2b (Fig. 4GoC) isotype was observed at low titers and in only some thrombocytopenic mice early after adenovirus injection (at 5 wk and earlier). Only one mouse had high titers of anti-huTPO of the IgM isotype (Fig. 4GoD). The mouse that remained nonthrombocytopenic for 8 mo following injection was analyzed at 8 mo and had IgG1, but not IgG2a, anti-huTPO Abs; IgG2b Abs were also detectable, but at low titer. The thrombocytopenic mouse that had anti-huTPO Abs of the IgM isotype also displayed IgM anti-muTPO Abs at exactly the same titer and the same OD (Fig. 4GoD). IgG2a, IgG2b, and IgG1 anti-muTPO were undetectable in mice sera. However, when anti-TPO mAbs were produced from splenocytes of a thrombocytopenic mouse (IgG2a, IgG2b, and IgM isotypes), cross-reactivity was observed for all of them (2 clones of IgG2a, 1 clone of IgG2b, and 2 clones of IgM).



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  		FIGURE 4. Presence of IgG2a and IgG1 anti-huTPO Abs in the sera of thrombocytopenic mice following AdRSVhuTPO injection. Presence of anti-huTPO Abs of IgG1 (A), IgG2a (B), IgG2b (C), and IgM (D) isotypes in the sera of AdRSVhuTPO-injected mice was determined, as described in Materials and Methods by an ELISA assay on huTPO-coated plates. Revelation following incubation of mice sera was done with the corresponding peroxidase-conjugated antiisotype. Two mice (Ma and Mb) representative of seven tested mice in two independent experiments are presented; two other mice subjected to irradiation and carboplatin (IC) 1 wk following the AdRSVhuTPO injection (M1 IC and M2 IC) are also presented at week 5 (wk5) and week 11 (wk11) after virus injection. One mouse (Mb) had IgM anti-huTPO Abs in its serum, and also IgM anti-muTPO with the same titer (D).

 
Impairment of anti-TPO Ab production with a myeloablative regimen

A protocol combining irradiation and carboplatin injection was assessed for its capacity to modify the production of anti-TPO Abs and prevent secondary thrombocytopenia.

Kinetics of platelets and huTPO production after a myeloablative regimen

Control mice and animals injected 1 wk earlier with 6 x 109 PFU of AdRSVhuTPO or AdRSVßgal were treated with 5 Gy total body irradiation and carboplatin (1.2 mg i.p.). The platelet nadir was identical in all groups. The control mice died due to the consequences of marrow aplasia, whereas two of three AdRSVhuTPO-treated mice survived the myeloablative regimen. Platelets of the two surviving mice progressively increased to normal levels at approximately week 7 and continued to increase up to 2.1- to 2.5-fold the normal level at week 10. These values were constant for at least 5 mo (Fig. 5Go).



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  		FIGURE 5. Sustained platelet production after myelosuppression following AdRSVhuTPO injection. Myeloablative regimen (5 Gy irradiation and i.p. injection of 1.2 mg carboplatin) was given 1 wk after the AdRSVhuTPO injection. Data (3 mice per group) are shown as mean ± SD. {blacksquare}, AdRSVhuTPO-injected mice; {diamond} AdRSVßgal-injected mice; {circ} noninjected control mice (NC).

 
Analysis of huTPO production 4 mo following virus injection showed the presence of 20 U/ml for mouse 1 and 200 U/ml for mouse 2, demonstrating that transgene expression was still efficient.

Analysis of anti-TPO Abs after the myeloablative regimen

When analyzed at 10 wk following the myeloablative regimen, the two AdRSVhuTPO-injected mice had low levels of anti-huTPO Abs of the IgG2b isotype (Fig. 4GoC). The absence of anti-TPO Abs of the IgG1 (Fig. 4GoA) and IgG2a (Fig. 4GoB) isotypes correlates with the prolonged expression of huTPO in these mice. To determine whether the absence of IgG1 and IgG2a Abs was due to inhibition of production of these isotypes secondary to aplasia or to absence of efficient immunization during the week before the myeloablative regimen, we analyzed the sera of these mice at 5 wk. The two mice had low but detectable levels of IgG1 (Fig. 4GoA); one had a high titer of IgG2a (Fig. 4GoB) and IgG2b (Fig. 4GoC), while the other had detectable IgG2b (Fig. 4GoC), but not IgG2a (Fig. 4GoB).

Analysis of anti-adenovirus Abs

Since anti-TPO Abs of mixed Th1 (IgG2a) and Th2 (IgG1) subsets disappeared after the myeloablative regimen, we wanted to know whether a similar inhibition could also concern anti-adenovirus Abs. Abs against adenovirus appeared as early as 1 wk after injection and remained at high titer for at least 10 mo. Isotype analysis also showed mixed Th1 and Th2 subsets, with high titers of IgG1, IgG2a, and IgG2b (Fig. 6Go, A, B, and C, respectively). However, no alteration in the anti-adenovirus Ab isotypes was observed after the myeloablative regimen in the two surviving mice treated with AdRSVhuTPO, with all of the isotypes observed at week 11 postadenoviral injection (Fig. 6Go, A, B, and C, respectively).



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  		FIGURE 6. Anti-adenovirus Abs in the sera of AdRSVhuTPO-injected mice. Presence of anti-adenovirus Abs of IgG1 (A), IgG2a (B), and IgG2b (C) isotypes in the sera of AdRSVhuTPO-injected mice was determined, as described in Materials and Methods, by an ELISA assay on adenovirus-coated plates. Revelation, following incubation of mice sera, was done with the corresponding peroxidase-conjugated antiisotype. Two representative mice (Ma and Mb) are presented; two other mice subjected to irradiation and carboplatin 1 wk after AdRSVhuTPO injection (M1 IC and M2 IC) are also presented at week 4 (wk4) and week 10 (wk10) following virus injection.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
To investigate the potential use of a recombinant adenovirus encoding huTPO in gene transfer protocols, we have injected this vector in mice and followed its biologic effects. Our results show that i.v. injection of this vector into immunocompetent adult mice led to a transient thrombocytosis, followed by a chronic thrombocytopenia in the majority of animals. The elevation of platelets in immunocompetent mice injected with the AdRSVhuTPO in our study was about twofold lower to the values reached by mice injected with an adenovirus in which the huTPO cDNA is under the control of the CMV promoter (17). Previous studies in mice models showed that production of secreted proteins by adenovirus vectors was higher when the transgene is under the control of the CMV promoter when compared with the RSV promoter (18). Thrombocytopenia appeared about 3 to 4 wk following virus injection and progressively worsened to a chronic stable condition at about 8 wk. One mouse analyzed 10 mo after the injection remained thrombocytopenic. The early onset of the disease appeared in all injected mice, except one. Mice had 13 to 36% of the normal platelet count when the nadir was reached. Analysis of the sera from thrombocytopenic mice on the Ba/F3 cell line transfected with either the human or the murine TPO receptor (hu-c-mpl and mu-c-mpl) revealed neutralizing activities against both human and murine TPO, although huTPO was more efficiently neutralized than muTPO. Further analysis of the sera from thrombocytopenic mice revealed high titers of Abs against huTPO. The phenotype of the anti-huTPO Abs indicated mixed Th1 and Th2 subset activation, while analysis of the humoral response against the adenovirus vector showed the same mixed phenotype. All thrombocytopenic mice presented simultaneously anti-huTPO Abs of the IgG2a and IgG1 isotypes, and one mouse also had the IgM isotype. In contrast, a mouse that developed thrombocytopenia 10 mo following injection had no anti-huTPO Abs of the IgG2a isotype, low titers of IgG2b, and higher titers of the IgG1 isotype. These results suggest that polyclonal B cell activation seems to be necessary for the early onset of the thrombocytopenia. The presence of IgG2a seems important for neutralization. In all of the sera from thrombocytopenic mice tested, Abs against muTPO of the IgG1, IgG2a, and IgG2b isotypes were not detectable, except for the mouse with anti-huTPO Abs of the IgM isotype that also displayed anti-muTPO Abs of the IgM isotype. However, when mAbs against huTPO were derived from the spleen of one thrombocytopenic mouse, we observed that all of the Abs, irrespective of their isotype, cross-reacted with both human and murine TPO. These data indicate that, in contrast to IgM anti-TPO Abs, all of the IgG Abs have a low affinity for muTPO.

Despite their low affinity, these circulating Abs are able to completely neutralize endogenous TPO production, since mice analyzed at late stages (9, 12, and 13 wk, and at 10 mo), when the thrombocytopenia was stably established, showed a phenotype comparable with that described for TPO- or c-mpl-knockout mice (19, 20, 21, 22). Indeed, the decrease in platelet numbers (13–36% of the baseline levels) was similar, and the absolute number of hemopoietic progenitor cells was also reduced by 50%.

Histologic analysis showed the development of systemic autoimmunity rather than organ-specific autoimmunity since no visible destructive T cell infiltrate was observed in the examined tissues (bone marrow, liver, lung, and kidney) that were analyzed when the thrombocytopenia was clearly established. It is of interest that histologic aspects of the spleens were comparable with that seen in human immune thrombocytopenia (23) with a hyperactive white pulp, especially in the marginal area (B cell zone). In contrast with the cellular infiltrates observed in liver sections (comprising mainly mononuclear and polymorphonuclear cell granuloma in the lobular area around regions of focal necrosis) 2 wk postinjection, plasma cell infiltrates in the periportal area of the liver were the striking feature seen 9 wk postinfection. The absence of similar infiltrates in the kidney (one of the major organs producing TPO) (24) suggests that B cells specifically trigger Ag in the liver, where they are probably activated.

In the second part of this work, we investigated whether anti-TPO Ab production could be modified by a myeloablative regimen. Mice were injected at day 0 with either AdRSVhuTPO or a control virus (AdRSVßgal) or PBS, and all mice were subjected to myeloablation 7 days later. In the three groups, the same nadir in platelet counts was reached at week 2 posttreatment. Thereafter, one of the three AdRSVTPO-injected mice died, most probably from bleeding, and all of the control mice died from bacterial septicemia (PBS- and AdRSVßgal-injected mice) (49). The two surviving mice injected with AdRSVhuTPO developed chronic thrombocytosis that persisted for at least 5 mo. In contrast with the transient elevation of TPO observed in normal mice, a high level of TPO in the serum of myeloablated mice was still detectable 4 mo posttreatment. Analysis of Abs in the serum of these two mice showed the presence of anti-huTPO of IgG1, IgG2a, and IgG2b isotypes for mouse 1, and IgG1 and IgG2b for mouse 2 at week 5, whereas at week 11 only the presence of lower titers of IgG2b was detected in the serum from mouse 1, while no anti-TPO Abs were detectable in mouse 2.

The data indicate that the myeloablative regimen resulted in an inhibition of the production of IgG2a and IgG1 anti-TPO Abs. These anti-TPO Ab isotypes are probably responsible for the TPO neutralization since platelet counts return to high levels when they completely disappear. The IgG2a anti-TPO isotype seems particularly important for neutralization. In contrast, the presence of IgG2b correlates with the delayed thrombocytopenia and also with tolerance following myeloablation. The elevated IgG2a/IgG2b (>4) ratio is always correlated with thrombocytopenia, whereas a low ratio (<=1) correlates with normal or high platelet counts (Fig. 7Go). At week 5 after AdRSVhuTPO injection, the presence of all isotypes demonstrates that an efficient immunization occurs during the first week following virus injection before myeloablation.



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  		FIGURE 7. Correlation between platelet counts and IgG2a/IgG2b ratio of anti-huTPO Abs. IgG2a/IgG2b ratio was calculated as follows: inverse of the last positive dilution of IgG2a anti-huTPO Ab/inverse of the last positive dilution of IgG2b anti-huTPO Ab from the ELISA assay (described in Materials and Methods). One mouse was not thrombocytopenic at wk 5 and had no detectable IgG2a, whereas high titer of IgG2b was observed ({diamond}). This mouse became thrombocytopenic at wk 11, when no detectable IgG2b isotype was found, but high titer of IgG2a was observed ({diamondsuit}). The other groups presented are: thrombocytopenic mice at week 11 (n = 3) and one thrombocytopenic mouse at 8 mo (n = 1) (•), mouse nonthrombocytopenic at 8 mo ({triangleup}), and two mice with high platelet counts following a myeloablative regimen ({circ}). Vertical bar represents the mean of platelet counts of normal mice. Horizontal bar represents a limit between low and high ratios.

 
Does adenovirus act as an adjuvant in the locally induced immunization? Viruses are suspected in participating in many autoimmune disorders such as diabetes type 1, subacute thyroiditis, and multiple sclerosis. Local tissue destruction associated with local secretion of inflammatory cytokines and the molecular mimicry between viral and self Ags probably plays an important role (25, 26, 27). Adenovirus induces IFN-{gamma} secretion (7, 28) and IL-12 gene expression after infection of mice (29). Because local expression of IFN-{gamma} and IL-12 participates in some instances in the development of autoimmunity (30, 31, 32, 33), we speculate that such a mechanism could initiate an efficient immune response against huTPO (huTPO shares 76.5% identity at the amino acid level with the muTPO (34)) produced following adenovirus gene transfer, and thereafter induce a loss of tolerance for the endogenous muTPO. The persistence of the thrombocytopenia over time suggests that muTPO must be responsible for the continuous stimulation of the generated autoreactive B cell clones via Th subsets. Histopathologic analysis revealed the presence of plasma cells infiltrating the liver, but not the kidney, which is one of the major organs producing TPO. This is probably related to the fact that i.v. injection of recombinant adenovirus leads to highly efficient transduction of liver cells (4), suggesting an important role of adenovirus infection in the development of this immune response. In addition, previous uses of rhuTPO in imunocompetent mice have not induced such an immune thrombocytopenia (35, 36, 37). However, recent data showed that injection of rhuTPO in dogs could also lead to chronic immune thrombocytopenia (38).

Analysis of anti-adenovirus Abs revealed, as was observed for the anti-huTPO Abs, the same mixed Th1- and Th2-dependent Ab isotypes. However, myelosuppression does not lead to the disappearance of IgG1 and IgG2a anti-adenovirus Abs, as was observed for the anti-huTPO Abs, all of the anti-adenovirus Ab isotypes detected in the normal mice are still observed 11 wk following myeloablation. These results suggest that the humoral response to adenovirus is more potent, perhaps due to the complex viral structure, as has been described for other viruses such as vesicular stomatitis virus (39). However, persistence of transgene expression for more than 5 mo suggests that transduced cells are not rejected by the CTL response directed against viral Ag-expressing cells. Recent data showed that the presence of CTL specific for adenoviral Ags is not sufficient to eliminate the transduced cells, and suggest that the immune response against the transgene is the major mechanism in the transient gene expression following adenovirus gene transfer (40, 41). Another possible hypothesis is that, as shown following total lymphoid irradiation treatment (42), the myeloablative regimen used inhibited the IL-2 pathway in activated CD4 cells, and thus the proliferation of specific CTL clones. Our results confirm previous data showing that transient immunosuppression beginning before or during immunization, using either immunosuppressive drugs or anti-CD4 Abs, permits prolongation of transgene expression following adenoviral gene transfer (43, 44, 45, 46).

In humans, chronic immune thrombocytopenia is an autoimmune disorder due to destructive Abs directed against platelet-associated Ags. The platelet glycoprotein complex IIb/IIIa is the principal autoantigen, although other platelet membrane components have been implicated, such as Ib/IX complex Ag and rarely glycoprotein IIIa alone (47). The sensitized platelets are removed by the macrophages in the spleen and the liver. However, in some autoimmune diseases such as systemic lupus erythematosus, thrombocytopenia has been related to a defect in TPO production (48). Our data show that it is conceivable that Abs against TPO may induce immune thrombocytopenia. We hypothesize that some of these immune thrombocytopenias immune related to a defect in TPO production could be in fact due to anti-TPO Ab; thus, immune complexes may block ELISA or biologic TPO detection assays.

Natural neutralizing Abs against Epo were described in one patient with pure red cell aplasia (13), and an Ab response was shown in patients with renal failure injected with recombinant Epo (14). TPO has about 50% homology with Epo, and thus the thrombocytopenia described in this work presents many similarities with the immune response against Epo.

The immune thrombocytopenia described hereby provides a novel model for the understanding of such pathology. The myeloablative protocol used confirms that transient immunosuppression can induce long-term transgene expression, and shows that prolonged expression can also be achieved even if initial immunization has occurred.

Finally, we hypothesize that some cases of immune thrombocytopenia in humans, especially those with decreased megakaryocytes, may be due to Abs directed against TPO or its receptor (c-mpl), even if they have low affinity and are undetectable by classical ELISA assays.


   Acknowledgments
 
We thank Rafick-Pierre Sékaly, Sam Burstein, Ali Turhan, and Antonio Coutinho for helpful discussions and critical reading of the manuscript; D. Cosman (Immunex) for the gift of rmuIL-3 and recombinant murine stem cell factor; and D. Foster (ZymoGenetics) for rmuTPO. We are grateful to the staff of Institut Gustave Roussy animal facilities for its valuable help and to Catherine Marcilhac for her assistance in preparing the manuscript.


   Footnotes
 
1 This work was supported by grants from Institut National de la Santé et de la Recherche Médicale, Institut Gustave Roussy, Centre National de la Recherche Scientifique, and Ligue Nationale Contre le Cancer. Back

2 Address correspondence and reprint requests to Dr. M. A. Abina at the current address, Laboratoire d’immunologie, IRCM, 110 avenue des Pins Ouest, Montreal, Quebec, H2W 1R7, Canada. E-mail address: Back

3 Abbreviations used in this paper: Epo, erythropoietin; Ad, adenovirus; ßgal, ß-galactosidase; BFU-E, erythroid burst-forming unit; CFU-GM, granulocyte-macrophage colony-forming unit; CFU-Mk, megakaryocyte colony-forming unit; hu, human; mu, murine; PFU, plaque-forming unit; RSV, Rous sarcoma virus; TPO, thrombopoietin. Back

Received for publication September 18, 1997. Accepted for publication January 7, 1998.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Ali, M., N. R. Lemoine, C. J. A. Ring. 1994. The use of DNA viruses as vectors for gene therapy. Gene Ther. 1:367.[Medline]
  2. Horwitz, M. S.. 1996. Adenoviridae and their replication. B. N. Fields, and D. M. Knipe, and P. M. Howley, eds. Virology 3rd Ed.2149.-2171. Raven Press, New York.
  3. Yang, Y., F. A. Nunes, K. Berencsi, E. E. Furth, E. Gonczol, J. M. Wilson. 1994. Cellular immunity to viral antigens limits E1-deleted adenoviruses for gene therapy. Proc. Natl. Acad. Sci. USA 91:4407.[Abstract/Free Full Text]
  4. Yang, Y., H. C. J. Ertl, J. M. Wilson. 1994. MHC class I-restricted cytotoxic T lymphocytes to viral antigens destroy hepatocytes in mice infected with E1-deleted recombinant adenoviruses. Immunity 1:433.[Medline]
  5. Dai, Y., E. Schwarz, D. Gu, W.-W. Zhang, N. Sarvetnick, I. M. Verma. 1995. Cellular and humoral immune responses to adenoviral vectors containing factor IX gene: tolerization of factor IX and vector antigens allows for long-term expression. Proc. Natl. Acad. Sci. USA 92:1401.[Abstract/Free Full Text]
  6. Yang, Y., L. Qin, H. C. J. Ertl, J. M. Wilson. 1995. Cellular and humoral immune responses to viral antigens create barriers to lung-directed gene therapy with recombinant adenoviruses. J. Virol. 69:2004.[Abstract]
  7. Yang, Y., J. Wilson. 1996. CD40 ligand-dependent T cell activation: requirement of B7-CD28 signaling through CD40. Science 273:1862.[Abstract/Free Full Text]
  8. Juillard, V., P. Villefroy, D. Godfrin, A. Pavirani, A. Venet, J.-G. Guillet. 1995. Long-term humoral and cellular immunity induced by a single immunization with replication-defective adenovirus recombinant vector. Eur. J. Immunol. 25:3467.[Medline]
  9. Kay, M. A., A.-X. Holterman, L. Meuse, A. Gown, H. D. Ochs, P. S. Linsley, C. B. Wilson. 1995. Long-term hepatic adenovirus-mediated gene expression in mice following CTLA4Ig administration. Nat. Genet. 11:191.[Medline]
  10. Gahery-Segard, H., V. Juillard, J. Gaston, R. Lengagne, A. Pavirani, P. Boulanger, J.-G. Guillet. 1997. Humoral immune response to the capsid components of recombinant adenoviruses: routes of immunization modulate virus-induced Ig subclass shifts. Eur. J. Immunol. 27:653.[Medline]
  11. Tripathy, S., H. B. Black, E. Goldwasser, J. M. Leiden. 1996. Immune response to transgene-encoded proteins limits the stability of gene expression after injection of replication-defective adenovirus vectors. Nat. Med. 2:545.[Medline]
  12. Wendling, F., E. Maraskovsky, N. Debili, C. Florindo, M. Teepe, M. Titeux, N. Methia, J. Breton-Gorius, D. Cosman, W. Vainchenker. 1994. c-MPL ligand is a humoral regulator of megakaryocytopoiesis. Nature 369:571.[Medline]
  13. Casadevall, N., E. Dupuy, P. Molho-Sabatier, G. Tobelem, B. Varet, P. Mayeux. 1996. Autoantibodies against erythropoietin in a patient with pure red-cell aplasia. N. Engl. J. Med. 334:630.[Free Full Text]
  14. Peces, R., M. De La Torre, R. Alcazar, J. M. Urra. 1996. Antibodies against recombinant human erythropoietin in a patient with erythropoietin resistant anemia. N. Engl. J. Med. 335:523.[Free Full Text]
  15. Stratford-Perricaudet, L. D., M. Levrero, J.-F. Chasse, M. Perricaudet, P. Briand. 1990. Evaluation of the transfer and expression in mice of an enzyme encoding gene using a human adenovirus vector. Hum. Gene Ther. 1:241.[Medline]
  16. Gough, N. M., D. Metcalf, J. Gough, D. Grail, A. R. Dunn. 1985. Structure and expression of the mRNA for murine granulocyte-macrophage colony stimulating factor. EMBO J. 4:645.[Medline]
  17. Ohwada, A., S. Rafii, M. A. S. Moore, R. G. Crystal. 1996. In vivo adenovirus vector-mediated transfer of the human thrombopoietin cDNA maintains platelet levels during radiation- and chemotherapy-induced bone marrow suppression. Blood 88:778.[Abstract/Free Full Text]
  18. Guo, Z. S., L.-H. Wang, R. C. Eisensmith, S. L. C. Woo. 1996. Evaluation of promoter strength for hepatic gene expression in vivo following adenovirus-mediated gene transfer. Gene Ther. 3:802.[Medline]
  19. Gurney, A. L., K. Carver-Moore, F. de Sauvage, M. W. Moore. 1994. Thrombocytopenia in c-mpl-deficient mice. Science 265:1445.[Abstract/Free Full Text]
  20. Alexander, W., A. Roberts, N. A. Nicola, R. Li, D. Metcalf. 1996. Deficiencies in progenitor cells of multiple hematopoietic lineages and defective megakaryocytopoiesis in mice lacking the thrombopoietin receptor c-mpl. Blood 87:2162.[Abstract/Free Full Text]
  21. Carver-Moore, K., H. E. Broxmeyer, S. M. Luoh, S. Cooper, J. Peng, S. A. Burstein, M. W. Moore, F. J. de Sauvage. 1996. Low levels of erythroid and myeloid progenitors in thrombopoietin- and c-mpl-deficient mice. Blood 88:803.[Abstract/Free Full Text]
  22. de Sauvage, F. J., K. Carver-Moore, S.-M. Luoh, A. Ryan, M. Dowd, D. L. Eaton, M. W. Moore. 1996. Physiological regulation of early and late stages of megakaryocytopoiesis by thrombopoietin. J. Exp. Med. 183:651.[Abstract/Free Full Text]
  23. Hassan, N. M. R., R. S. Neiman. 1985. The pathogeny of the spleen in steroid treated immune thrombocytopenic purpura. Am. J. Clin. Pathol. 84:433.[Medline]
  24. Sungaran, R., B. Markovic, B. H. Chong. 1997. Localization and regulation of thrombopoietin mRNA expression in human kidney, liver, bone marrow, and spleen using in situ hybridization. Blood 1:101.
  25. Theofilopoulos, A. N.. 1995. The basis of autoimmunity: part I mechanisms of aberrant sel-recognition. Immunol. Today 16:90.[Medline]
  26. Gianani, R., N. Sarvetnick. 1996. Viruses, cytokines, antigens, and autoimmunity. Proc. Natl. Acad. Sci. USA 93:2257.[Abstract/Free Full Text]
  27. Hemmer, B., B. T. Fleckenstein, M. Vergelli, G. Jung, H. McFarland, R. Martin, K. H. Wiesmuller. 1997. Identification of high potency microbial and self ligands for a human autoreactive class II-restricted T cell clone. J. Exp. Med. 185:1651.[Abstract/Free Full Text]
  28. Tiensiwakul, P., N. Khoobbyarian. 1983. Adenovirus fiber protein produces synthesis of interferon in mouse spleen and macrophage culture. Intervirology 20:52.[Medline]
  29. Coutelier, J.-P., J. Van Broeck, S. F. Wolf. 1996. Interleukin-12 gene expression after viral infection in the mouse. J. Virol. 69:1955.[Abstract]
  30. Savertnick, N., J. Shizuru, D. Liggitt, L. Martin, B. McIntyre, A. Gregory, T. Parslow, T. Stewart. 1990. Loss of pancreatic islet tolerance induced by beta-cell expression of interferon-gamma. Nature 346:844.[Medline]
  31. Trembleau, S., T. Germann, K. Gately, L. Adorini. 1995. The role of IL-12 in the induction of organ-specific autoimmune diseases. Immunol. Today 16:383.[Medline]
  32. Leonard, J. P., K. E. Wadburg, S. J. Goldman. 1995. Prevention of experimental autoimmune encephalomyelitis by antibodies against interleukin 12. J. Exp. Med. 181:381.[Abstract/Free Full Text]
  33. Von Herrath, M. G., M. B. A. Oldstone. 1997. Interferon-{gamma} is essential for destruction of ß cells and development of insulin-dependent diabetes mellitus. J. Exp. Med. 185:531.[Abstract/Free Full Text]
  34. Bartley, T. D., J. Bogenberger, P. Hunt, Y.-S. Li, H. S. Lu, F. Martin, M.-S. Chang, B. Samal, J. L. Nichol, S. Swift, M. J. Johnson, R.-Y. Hsu, V. P. Parker, S. Suggs, J. D. Skrine, L. A. Merewether, C. Clogston, E. Hsu, M. M. Hokom, A. Hornkohl, E. Choi, M. Pangelinan, Y. Sun, V. Mar, J. McNinch, L. Simonet, F. Jacobsen, C. Xie, J. Shutter, H. Chute, R. Basu, L. Selander, D. Trollinger, L. Sieu, D. Padilla, G. Trail, G. Elliott, R. Izumi, T. Covey, J. Crouse, A. Garcia, W. Xu, J. Del Castillo, J. Biron, S. Cole, M. C.-T. Hu, R. Pacifici, I. Ponting, C. Saris, D. Wen, Y. P. Yung, H. Lin, R. A. Bosselman. 1994. Identification and cloning of a megakaryocyte growth and development factor that is a ligand for the cytokine receptor Mpl. Cell 77:1117.[Medline]
  35. de Sauvage, F. J., P. E. Hass, S. D. Spencer, B. E. Malloy, A. L. Gurney, S. A. Spencer, W. C. Darbonne, W. J. Henzel, S. C. Wong, W.-J. Kuang, K. J. Oles, B. Hultgren, Jr L. A. Solberg, D. V. Goeddel, D. L. Eaton. 1994. Stimulation of megakaryocytopoiesis and thrombopoiesis by the c-Mpl ligand. Nature 369:533.[Medline]
  36. Ulich, T. R., J. del Castillo, G. Senaldi, O. Kinstler, S. Yin, S. Kaufman, J. Tarpley, E. Choi, T. Kirley, P. Hunt, W. P. Sheridan. 1996. Systemic hematologic effects of PEG-rHuMGDF-induced megacaryocyte hyperplasia in mice. Blood 87:5006.[Abstract/Free Full Text]
  37. Hokom, M. M., D. Lancey, O. B. Kinstler, E. Choi, S. Kaufman, J. Faust, C. Rowan, E. Dwyer, J. L. Nichol, T. Grasel, J. Wilson, R. Steinbrink, R. Hecht, D. Winters, T. Boone, P. Hunt. 1995. Pegylated megakaryocyte growth and development factor abrogates the lethal thrombopenia associated with carboplatin and irradiation in mice. Blood 86:4486.[Abstract/Free Full Text]
  38. Dale, D. C., J. L. Nichol, D. A. Rich, D. M. Best, S. J. Slichter, W. Sheridan, P. Hunt. 1997. Chronic thrombocytopenia is induced in dogs by development of cross-reacting antibodies to the Mpl ligand. Blood 90:3456.[Abstract/Free Full Text]
  39. Bachmann, M. F., R. M. Zinkernagel. 1996. The influence of virus structure on antibody responses and virus serotype formation. Immunol. Today 17:553.[Medline]
  40. Michou, A. I., L. Santoro, M. Christ, A. Pavirani, M. Mehtali. 1997. Adenovirus-mediated gene transfer: influence of transgene, mouse strain and type of immune response on persistence of transgene expression. Gene Ther. 4:473.[Medline]
  41. Wadsworth, S. C., H. Zhou, A. E. Smith, J. M. Kaplan. 1997. Adenovirus vector-infected cells can escape adenovirus antigen-specific cytotoxic T-lymphocyte killing in vivo. J. Virol. 71:5189.[Abstract]
  42. Field, E. H., Z. X. Ye, T. M. Rouse. 1996. Mechanisms of tolerance induction, splenocytes from total lymphoid irradiated mice inhibit the IL-2 pathway in TCR-activated CD4 cells. J. Immunol. 157:3796.[Abstract]
  43. Jooss, K., Y. Yang, J. M. Wilson. 1996. Cyclophosphamide diminishes inflammation and prolongs transgene expression following delivery of adenoviral vectors to mouse liver and lung. Hum. Gene Ther. 7:1555.[Medline]
  44. Lochmuller, H., B. J. Petrof, G. Pari, N. Larochelle, V. Dodelet, Q. Wang, C. Allen, S. Prescott, B. Massie, J. Nalbantoglu, G. Karpati. 1996. Transient immunosuppression by FK506 permits a sustained high-level dystrophin expression after adenovirus-mediated dystrophin minigene transfer to skeletal muscles of adult dystrophic (mdx) mice. Gene Ther. 3:706.[Medline]
  45. Smith, T. A., B. D. White, J. M. Gardner, M. Kaleko, A. McClelland. 1996. Transient immunosuppression permits successful repetitive intravenous administration of an adenovirus vector. Gene Ther. 3:496.[Medline]
  46. Kolls, J. K., D. Lei, G. Odom, S. Nelson, W. R. Summer, M. A. Gerber, J. E. Shellito. 1996. Use of transient CD4 lymphocyte depletion to prolong transgene expression of E1-deleted adenoviral vectors. Hum. Gene Ther. 7:489.[Medline]
  47. Kiefel, V., S. Santoso, C. Mueller-Eckhardt. 1992. Serological, biochemical, and molecular aspects of platelet autoantigens. Semin. Hematol. 29:26.
  48. Nichol, J. L.. 1997. Serum levels of thrombopoietin in health and disease. D. J. Kuter, and P. Hunt, and W. Sheridan, and D. S. Zucker-Franklin, eds. Thrombopoiesis and Thrombopoietins 359.-375. Humana Press, Totowa.
  49. Abina, M. A., M. Tulliez, C. Lacout, N. Debili, J.-L. Villeval, F. Pflumio, F. Wendling, W. Vainchenker, and H. Haddada. Major effects of TPO delivered by a single injection of a recombinant adenovirus on prevention of septicemia and anemia associated with myelosuppression in mice: risk of sustained expression inducing myelofibrosis due to immunosuppression. Gene Ther. In press.



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