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*
Institute for Human Gene Therapy and Departments of Medicine and of Molecular and Cellular Engineering, University of Pennsylvania, and Wistar Institute, Philadelphia, PA 19104; and
Department of Immunology, SmithKline Beecham Pharmaceuticals, King of Prussia, PA 19406
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Numerous model systems have used various in vivo systems to evaluate the importance of Th cells on Ag-specific immune functions (9, 10). The key role of CD4+ T cell activation was shown in prevention of experimental autoimmune encephalopathy, type II collagen-induced arthritis, allograft tolerance, and virus-induced pathology (11). We have been interested in evaluating adenovirus vector-induced immune responses to determine their role in the persistence of transgene expression and the ability to readminister these vectors for gene therapy. Previous studies have indicated that depletion of CD4+ T cell with anti-CD4 Ab, GK1.5 resulted in persistent transgene expression and efficient ability to readministrate vector in mice (7). However, depletion of CD4+ T cells is not desired in human clinical trials. In this study we have evaluated the efficacy of a nondepleting anti-human CD4 Ab.
The CD4 molecule is a nonpolymorphic membrane glycoprotein of 55 kDa that consists of four extracellular domains (D1–D4). This extracellular domain of murine CD4 molecules shows 55% sequence homology with its human counterpart (12). Residues at positions 19, 89, and 165 on the human CD4 molecule are critical for interaction with MHC class II molecules in both human and mice. Interspecies CD4/MHC class II interactions can mediate functional immune responses (13, 14, 15, 16). It has also been demonstrated that human CD4 can interact with mouse MHC class II molecules and signal through mouse p56lck (17, 18). Killeen et al. (13) have demonstrated that the human CD4 transgene reconstitutes, phenotypically and functionally, the CD4+ T cell compartment in murine CD4-deficient mice (muCD4KO).3 The current study used these HuCD4 mice to evaluate the immune-suppressive effects of a nondepleting mAb to human CD4 in adenoviral vector-mediated immune responses to the lung.
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Materials and Methods |
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Mice deficient for the murine CD4 gene (muCD4KO mice) and muCD4KO mice transgenic for human CD4 (HuCD4) were bred in a specific pathogen-free facility under contract at Charles River (Wilmington, MA). HuCD4 mice have been previously described (13). Briefly, (C57BL/6 x SJL)F1 eggs fertilized by (129/SV x C57BL/6 x DBA2) males were used for microinjection by crossing of male founder to (C57BL/6 x 12/SV) females. These mice were subsequently bred to homozygocity on the H2d locus.
Recombinant adenoviruses
The construction of E1-deleted recombinant adenoviruses expressing Escherichia coli ß-galactosidase (H5.010CBlacZ, henceforth called Ad-lacZ), human alkaline phosphatase (H5.100CBALP, henceforth called Ad-ALP), green fluorescent protein (H5.000CMVGFP, henceforth called Ad-GFP), and luciferase (H5.000CMVLuc, henceforth called Ad-Luc) were amplified on 293 cells and purified on cesium chloride gradients as described previously (19, 20).
Antibodies
Clenoliximab, a Primatized mAb to human CD4, contains the variable domains from an mAb generated in cynomolgus macaque and human IgG4 constant domains. It also contains two single residue mutations, the first in the hinge region and the second within the CH domain, designed to enhance heavy chain dimer formation and reduce Fc receptor binding and (M. Reddy et al., manuscript in preparation). This mAb has no C1q binding or complement-fixing activity and has a dramatically reduced Fc receptor binding activity. The preparation of purified Clenoliximab used in this study was provided by the Department of Pharmaceutical Technologies, SmithKline Beecham Pharmaceuticals (King of Prussia, PA). In one experiment a human IgG1 isotype Ab of irrelevant specificity was used as a control.
Study design
Particles of each vector (1 x 1011) were
instilled intratracheally in a volume of 50 µl. For this purpose,
animals were anesthetized, and a skin incision was made on the ventral
surface of the neck. Muscles were separated to expose the trachea into
which 50 µl of vector was instilled using a 27-gauge needle. The skin
incision was then closed using a 4-0 vicryl suture material.
Clenoliximab was administered i.p. in a volume of 100 µl as several
doses, at 2 mg/dose/mouse. Study designs are detailed in Fig. 1
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The CTL assay was performed using H-2d P815 cells as previously described (21).
Neutralizing Ab assays
Neutralizing Ab titers were measured by analyzing the ability of serum or bronchoalveolar lavage (BAL) fluid to inhibit transduction of reporter virus, Ad-GFP, into HeLa cells. Various dilutions of serum/BAL were preincubated with reporter virus for 1 h at 37°C and added to 90% confluent HeLa cell cultures. Cells were incubated for 16 h. Expression of GFP was measured by fluoroimaging (Molecular Dynamics, Sunnyvale, CA). The neutralizing titer of Ab was calculated by the highest dilution of the sera at which 50% of the cells turned green.
Adenovirus-specific Igs
Serum (diluted 1/200) and BAL (diluted 1/20) samples from animals were analyzed for adenovirus-specific isotype specific Igs (IgM, IgG1, IgG2a, and IgA) by ELISA. For the ELISA, 96-well flat-bottom, high binding Immulon-IV plates were coated with 200 µl of Ad-LacZ Ag (5 x 106 particles/ml) in PBS overnight at 4°C, washed four times in PBS containing 0.05%Tween, and blocked in PBS with 1% BSA for 2 h at 4°C. Appropriately diluted samples were added to Ag-coated plates and incubated overnight at 4°C. Plates were washed four times in PBS with 0.5% Tween and incubated with biotin-conjugated rat anti-mouse IgM, IgG1, IgG2a, or IgA (1/2000 dilution; PharMingen, San Diego, CA) for 2 h at 4°C. Plates were washed as described above, and a 1/20,000 dilution of peroxidase-conjugated streptavidin was added. Color development was induced by addition of ASBT substrate (Kirkegaard & Perry, Gaithersburg, MD). OD values were read at 405 nm on a MRX Dynatech Microplate reader (Dynatech, Chantilly, VA).
Lymphoproliferative assays
Splenocytes from mice were obtained at various time points during the study. Mouse splenocytes were disaggregated to a single-cell suspension made on a wire mesh that is passed through a nylon filter. Triplicate cultures of 100 µl of 1 x 106 cells/ml lymphocytes were cultured with a multiplicity of infection of 10 of inactivated Ad-lacZ, 100 ng/ml Staphylococcus enterotoxin B (SEB), or medium alone. Proliferation was measured by a 16-h [3H]thymidine (1 µCi/well) pulse. Results are expressed as stimulation index, which is a ratio of the counts per minute of Ag/SEB-stimulated cultures per counts per minute of cultures in medium alone.
Cytokine release assays
Splenocytes were cultured with or without Ag (i.e., inactivated
Ad-lacZ at a multiplicity of infection of 10) for 48 h
in a 24-well plate. Cell-free supernatants were collected and analyzed
for the presence of IL-2, IL-4, IFN-
, and IL-10 by ELISA as
described previously (21).
FACS analysis
Heparinized blood obtained from individual mice was stained with OKT4A-FITC, OKT4-PE (Ortho Diagnostics, Raritan, NJ), and murine CD8-CyC (PharMingen, San Diego, CA) or with isotype controls for 10 min at room temperature. Red cells were lysed in buffer (FACS lysing solution, Becton Dickinson, San Jose, CA). Excess Ab was washed, and cells were fixed in 1% paraformaldehyde. Fluorescence was analyzed on a Coulter Elite flow cytometer (Coulter, Hialeah, FL). The percentage of positive cells were measured from those above background staining of isotype controls.
Morphological gene transfer analyses
X-Galactosidase and alkaline phosphatase histochemistry was analyzed in frozen sections (6 µm) as described previously (1). Green fluorescent protein (GFP) expression in lung tissue was analyzed in paraffin sections by fluorescent microscopy. Luciferase activity was measured in lung tissue, frozen immediately upon harvesting, and subsequently ground in a prechilled mortar and pestle. The tissue was transferred to a 1.5-ml microfuge tube and resuspended in 500 µl of 1x Reporter Lysis Buffer (Promega, Madison, WI). The tubes were vortex mixed, and the samples were freeze/thawed three times. Lysates were cleared by centrifugation. Luciferase assays were performed using the manufacturer’s protocol, and results were read on a luminometer. The protein concentration of each lysate was assayed by bicinchoninic acid protein assay (Pierce, Rockford, IL), and luciferase activities were expressed as luciferase activity (arbitrary light units) per milligram of protein.
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Results |
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), but not Th2-type responses (no secretion of IL-4
or IL-10), in response to in vitro adenovirus Ag stimulation (Fig. 2). Consistent with their ability to
secrete IFN-, the muCD4KO mice generated a modest
lymphoproliferative (data not shown) and CTL response (Fig. 3A). Low levels of
neutralizing Abs were found in serum, but not in BAL (Fig. 4). Further, muCD4KO generated antiviral
IgM and IgG2a responses, but failed to generate IgG1 in serum or IgA in
BAL (Fig. 4). These observations indicate that muCD4KO mice are able to
mount modest Th1-type, but not Th2-type responses. Consequently,
antivector CTLs are diminished, and neutralizing Abs are absent.
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) and Th2-type (IL-4 and IL-10)
responses were observed in HuCD4 mice that received Ad-lacZ
into the airway (Fig. 2
). Robust CTL activity to viral Ags was also
observed (Fig. 3B). High levels of adenovirus-specific
neutralizing Abs were detected in serum and BAL (Fig. 4). The full
spectrum of antiviral IgM, IgG2a, IgG1, and IgA isotypes were measured
(Fig. 4). IgG isotypes were not detectable in BAL on day 28 (data not
shown). These observations confirm that the HuCD4 transgene in HuCD4
mice confers complete immune responsiveness to adenoviral vectors.
Having established the role of HuCD4 in T and B cell responses to
vector, we evaluated the potential of CD4+ T cell
inhibition in blunting these undesirable effector functions. A
nondepleting Primatized Ab to CD4, called Clenoliximab, was evaluated.
This Ab is in advanced clinical development for the treatment of
rheumatoid arthritis and psoriasis. Our initial experiments studied the
impact of CD4+ T cell inhibition in the HuCD4
mice on a primary response to vector instilled into the lung.
Clenoliximab treatment markedly suppressed Th2-type activation (Fig. 2), whereas Th1-type responses (Fig. 2) and CTL function (Fig. 3C) were markedly diminished below those in the muCD4KO
animals. Neutralizing Ab was not formed in serum or BAL (Fig. 4).
Isotype fractionation was consistent with an inhibition of Th2-type
responses with no IgG1 (serum) or IgA (BAL) to adenovirus, but normal
levels of IgM and IgG2a in serum (Fig. 4). IgA levels in the serum were
minimal; however, these were completely inhibited in the anti-CD4
mAb-treated animals (data not shown).
Experimental protocols were established to evaluate the potential of
Clenoliximab as an adjunct to vector in the long term treatment of
cystic fibrosis (CF). A summary of the study groups is presented in
Fig. 1. Group 5 received chronic doses of Clenoliximab during a regimen
of four sequential vector administrations spaced 1 mo apart. All
vectors were E1-deleted adenoviruses, although each expressed a
different reporter gene, to easily distinguish them from one another
(vector 1, lacZ; vector 2, ALP; vector 3, GFP; vector 4,
Luc). Groups 1–4 represent treatment with each vector of naive animals
without Clenoliximab and treatment with the next vector 30 days later;
animals were euthanized, and tissues were harvested for transgene
expression 4 days after each vector administration. Group 6 was
designed to evaluate the durability of transient Clenoliximab treatment
by following the animals long after the initial Ab effect diminished
(i.e., study readministration with GFP vector 90 days after treatment
with lacZ vector and short course of Clenoliximab).
Groups 5 (chronic treatment) and 6 (short course) were evaluated for
the effect of Clenoliximab on peripheral CD4+ and
CD8+ T cells as detected by flow cytometry with
mAbs specific to CD4 (OKT4A-FITC and OKT4-PE) and CD8 (CD4-CyC; see
Fig. 5). OKT4A binds to an epitope within
domain 1 (D1) of CD4 that overlaps with the binding site of
Clenoliximab, whereas OKT4 binds to a nonoverlapping epitope within
D3D4. Chronic treatment with Clenoliximab did not result in a decrease
in OKT4 or CD8 binding below the range in naive animals, indicating it
did not deplete these cells. OKT4A binding was inhibited initially
following the repeated administration of Clenoliximab at the time of
vector 1. OKT4A binding returned to normal levels during periods when
there was no treatment with Chlenoliximab for >7 days. The OKT4A
epitope was masked again when blood samples were drawn from the mice
within a few days of Chlenoliximab dosing. These data are consistent
with other observations, which show the presence of circulating Ab for
3–7 days at the 2-mg dose used in this study (data not shown).
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A) and SEB (Fig. 6B). Serum (Fig. 6C) and BAL (Fig. 6D)
were also harvested for neutralizing Ab to adenovirus. Fig. 7 shows the outcome of gene transfer.
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A). Similarly, stimulation to SEB was persistently
diminished (Fig. 6B). Neutralizing Ab in serum and BAL were
also suppressed (Fig. 6, C and D). As predicted,
this allowed for efficient administration of vector 2 (ALP, Fig. 7G), vector 3 (GFP, Fig. 7H), and vector 4
(luciferase), which did not occur when animals were not treated with
Clenoliximab (2) (Fig. 7, A–E).
Study group 6 was designed to evaluate the effect of Clenoliximab after
the activity of the Ab had waned. Vector 1 was administered with a
short course (spanning days -3 to +28) of Clenoliximab, and vector 2
was administered on day 90. Binding of OKT4A to PBMC returned to normal
by day 43 (Fig. 5A), as did the responsiveness of these
cells to SEB by day 60 (Fig. 6B). Importantly, Ag-specific
responses to adenovirus (Fig. 6A) and neutralizing Ab in
serum (Fig. 6C) and BAL (Fig. 6D) all remained
suppressed beyond the time Clenoliximab was no longer active. Ad-GFP
was effectively readministered on day 90 (Fig. 7J).
Transient inhibition of CD4+ T cells also
markedly prolonged the expression of lacZ from the first
vector administration, consistent with its suppression of CTL activity
(Fig. 8).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The critical role of CD4 molecules in regulation of T cell functional responses is clearly established. CD4-MHC class II interactions have been shown to be involved in both enhanced cell adhesion and transducing signals to T cells, which contribute to the strength of the TCR/CD3-mediated signal (15, 22, 23). The nature of the signals transduced through the CD4 molecule that contribute to TCR/CD3 signals have been extensively studied (24). The in vivo role of CD4 molecules in differentiation of Th cells was elucidated in early studies in muCD4KO mice, which suggested that the CD4 molecule was not required for some Th1 cell functions (13, 25). Two recent studies using Nippostrongylus brasiliensis and Leishmania major (22, 26) have demonstrated that the CD4 molecule is critical for Th2 cell differentiation. In our study, administration of Ad-lacZ to muCD4KO mice generated a detectable, but significantly decreased, Th1-type response with the absence of a Th2-type response. Reconstitution of immune function through the human CD4 transgene in the HuCD4 mouse resulted in humoral and cellular immune responses to vector that were equivalent to those observed in MHC-matched BALB/c mice (data not shown). Similar effects have been reported in clinical trials with anti-CD4 Abs in humans (27). These observations are consistent with the finding that CD4 molecules contribute to differentiation of the immune response to a Th2 phenotype (22, 26). Thus, HuCD4 mice generate normal immune responses and, therefore, are a good model for evaluating therapeutics that interfere with human CD4 function.
Previous studies using depleting Abs to murine CD4 in adenovirus vector-treated immune (28)-competent mice have demonstrated that the helper functions of CD4+ T cells are critical for induction of CD8+ CTL and secretion of neutralizing Ab by B cells. Clenoliximab is being developed for the treatment of rheumatoid arthritis, an autoimmune disease, and it is a potential candidate as an adjunct to in vivo gene therapy in humans with viral vectors. Administration of Clenoliximab caused coating of but did not lead to depletion of CD4+ T cells. The change in CD4+ and CD8+ T cells is a dynamic process, since adenovirus vector instillation resulted in expansion of T cells, which was affected by Clenoliximab treatment. T cell responsiveness was markedly inhibited in Clenoliximab-treated animals, as shown by loss of both adenovirus and SEB responses on days 11–28. Interestingly both short course and chronic regimens of Clenoliximab resulted in persistent loss of Ag-specific responsiveness following an exposure to vector. The restoration of SEB responses following a short course of Clenoliximab correlated directly with the recovery of the OKT4A epitope in these mice, which corresponds to the loss from circulation of any remaining Clenoliximab. The prolonged suppression of Ag-specific immune responses (including generation of neutralizing Abs) in animals receiving a short course of Clenoliximab was confirmed by the ability to readminister the Ad-GFP on day 90 (see group 6). These results demonstrate that a 4-wk regimen of CD4 Clenoliximab leads to long term abrogation of Ag-specific immune responses to the coadministered vector. This appears to be due to the complete suppression of the initial response, allowing the mice to behave as in first time exposure to Ag and is not tolerance because a secondary exposure to vector without coadministration of Clenoliximab will stimulate normal responses to vector (data not shown).
We next asked whether adenoviral vectors could be repeatedly administered under conditions of CD4+ T cell suppression, which would better simulate clinical applications. An experimental protocol was developed in which virus expressing different transgenes was instilled into the lung at 4-wk intervals along with i.p. injections of Clenoliximab. This study demonstrates that Clenoliximab is very effective in inhibiting the generation of neutralizing Ab responses after multiple injections of vector. The lack of functional humoral responses was confirmed by the ability to readminister adenoviral vectors with four sequential treatments. These findings indicate that Clenoliximab is an effective immunosuppressive agent to block generation of humoral and cellular immune responses during repeated adenovirus vector administrations to the lung.
In conclusion, the ability to readminister vectors will be critical for the successful application of gene therapy for chronic diseases. Inhibition of CD4+ T cell function with a nondepleting Primatized anti-human CD4 Ab effectively blocks the T cell-dependent B cell response to adenoviral vectors in the lung. Application of this strategy for gene therapy to lung, such as CF, will require a more careful assessment of safety.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. James M. Wilson, Wistar Institute, 3601 Spruce Street, Philadelphia, PA 19104. E-mail address:
3 Abbreviations used in this paper: muCD4KO, murine CD4 knockout; HuCD4, human CD4; GFP, green fluorescent protein; BAL, bronchoalveolar lavage; SEB, Staphylococcus enterotoxin B; CF, cystic fibrosis; D1, domain 1.
Received for publication December 31, 1998. Accepted for publication April 22, 1999.
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) or stimulated with Ad-lacZ
(
). These analyses were performed with muCD4KO, HuCD4, or HuCD4
treated with Clenoliximab (CD4 mAb). *, p <
0.001, using Student’s t test.
) P815 target cells in a
51Cr release assay. The percent specific lysis is expressed
as a function of different E:T cell ratios. The results are the mean
and SEM of two separate experiments in the muCD4KO mice and three
separate experiments in the HuCD4 mice.
25% of
the epithelial cells.