Graft-versus-host disease (GvHD) is a limiting toxicity of bone marrow transplantation in humans. Donor T cells are the effectors of GvHD and must first interact with dendritic cells (DC) to become activated. The particular role of recipient DC has been highlighted by several recent murine studies (1, 2). GvHD commonly affects the skin and Langerhans cells (LC) of the epidermis appear to be potent inducers of the allogeneic T-cell response (2, 3). Strategies to reduce the impact of GvHD in humans focus on the attenuation of donor T-cell responses through the use of ciclosporin, methotrexate, and direct T depletion by CD34 selection of stem cells or treatment with antibodies such as alemtuzumab (3). Incorporation of agents to deplete DC during transplant conditioning may enhance GvHD prophylaxis in the acute posttransplant period without compromising long-term antiviral and antitumor T-cell responses to the same extent as T-depletion strategies (4). We have recently shown that a mouse monoclonal IgM, CMRF-44, which binds to a restricted determinant on activated DC (5–7), is able to deplete blood DC and suppress the mixed leukocyte reaction and primary immune responses in vitro (8). Here, we investigate the activity of CMRF-44 against LC, which are directly involved in skin GvHD and serve as a model for all tissue-derived DC. CMRF-44 is compared with alemtuzumab to illustrate the potential of new therapeutic antibodies to enhance existing means of controlling GvHD.
MATERIALS AND METHODS
Migration of LC: RPMI 1640; 2 mM glutamine; 1% penicillin and streptomycin; 10% FCS (Gibco/Invitrogen, Paisley, United Kingdom); 500 U/mL GM-CSF (Peprotech, Reading, United Kingdom). Complement-mediated lysis: RPMI 1640; 20 mM HEPES (Gibco/Invitrogen). Allostimulation assay: RPMI 1640; 2 mM glutamine; 1% penicillin and streptomycin; 10% pooled human AB serum (Gibco/Invitrogen); 500 U/mL GM-CSF.
Complement, Antibodies, and FACS
Lyophilized human complement serum was obtained from Sigma-Aldrich, Dorset, United Kingdom (S1764). CMRF-44 was purified in-house; other antibodies: mouse monoclonal IgM ‘TEPC 183′: Sigma-Aldrich (M3795); PE-conjugated sheep anti-mouse F(Ab’)2: Vector Laboratories, California; alemtuzumab/CAMPATH 1H: Schering Healthcare Ltd, West Sussex, United Kingdom; pooled human immunoglobulin ‘flebogamma’: Grifols, Cambridge, United Kingdom; CAMPATH-1G (rat anti-CD52 IgG2b) and PE-labeled isotype control: Serotec, Oxford, United Kingdom; FITC-conjugated anti-CD1a clone NA 1/34: Dakocytomation, Cambridge, United Kingdom; APC-conjugated anti-HLA-DR clone L324: BD Biosciences, Oxford, United Kingdom; FITC/APC-conjugated mouse isotype control antibodies: BD Biosciences. Stained cells were analyzed on a FACScalibur instrument using Cellquest software (BD Biosciences).
Breast reduction skin was obtained with ethical approval. Three hundred micrometer sheets cut with a Webster skin graft knife (N Stenning, Brisbane, Australia) were incubated with Dispase (Gibco/Invitrogen) 1 mg/mL in RPMI for 60 min at 37°C. Epidermal sheets were peeled from the dermis using forceps and rinsed in PBS. Fresh LC were prepared by mincing the epidermis and further incubation for 10 min at room temperature with trypsin 0.25% (Gibco/Invitrogen). The resulting cell suspension was centrifuged and washed in PBS. Migratory LC were isolated by floating epidermal sheets for 60–72 hr in medium incubated in a 37°C humidified 5% CO2 atmosphere.
This was performed essentially as described (8). 2×105 migratory LC in 150 μL medium were incubated for 30 min on ice with CMRF-44 or TEPC 183 at 20 μg/mL and alemtuzumab or pooled human immunoglobulin at 10 μg/mL. An equal volume of fresh or heat-inactivated complement serum was then added for 60 min at 37°C. Viability was assessed by Trypan blue exclusion.
Allostimulation by Migratory LC
PBL were prepared from peripheral blood mononuclear cells depleted of monocytes by adherence. Allostimulation was performed using 2×105 responder PBL per well in 150 μL medium in a round-bottomed microtitre plate (Nalge Nunc International). Epidermal sheets were prepared as described above, cut into 4-mm discs with a punch biopsy and then into halves with a scalpel. Each well received a half-disc of epidermis, floating on the medium. After 48 hr of culture, CMRF-44 or TEPC 183 was added to 10 μg/mL followed by 50 μL of fresh or heat-inactivated complement serum. Epidermis was removed after 72 hr. Proliferation of the responder PBL was assessed by 3H-thymidine incorporation at day 4 or 6 of culture (0.5 μCi/well for 16–20 hr). Percentage reduction in proliferation by CMRF-44 + C′ was calculated using the formula: 1 − [(CMRF-44 + C′) − (responder) /(mean of controls) − (responder)] × 100%.
RESULTS AND DISCUSSION
Expression of CMRF-44 Antigen and Lysis with Complement In Vitro
LC isolated freshly by enzymatic digestion and LC migrating from epidermis over 60–72 hr were tested for reactivity with CMRF-44. Fresh LC had no detectable expression of CMRF-44 antigen but migration increased expression 43- to 60-fold (range of three experiments; Fig. 1A). These findings confirm histological studies showing that other tissue DC rarely express CMRF-44 antigen in situ but that rapid up-regulation follows differentiation (5, 7, 9), in this case induced by migration of LC (10–12). Although CMRF-44 binding is restricted to activated cells, the sparing of resting DC, which may be promoting tolerance, is potentially advantageous.
CMRF-44 and complement caused 97.2%±2.1% lysis of migratory LC (Fig. 1B). This result extends our previous observation that blood DC is sensitive to lysis with CMRF-44 and complement (8). CMRF-44 removes 89% of DC from bulk PBMC and 95% of purified CD11c myeloid DC. Thus LC and myeloid blood DC appear to be equally sensitive to complement-mediated lysis with CMRF-44.
Comparison with Alemtuzumab
Alemtuzumab is a humanized antibody used to control GvHD in bone marrow transplantation (13, 14) by depleting T cells, which express high levels of CD52 (15, 16). Blood DC analyzed by FACS express CD52 but LC and tissue DC appear negative by immunohistochemistry (17, 18). The effect of alemtuzumab on LC and tissue DC has not been directly tested in vivo.
Using flow cytometry, a more sensitive method than immunohistochemistry, we found low expression of CD52 on fresh and migratory LC (Fig. 2A). Peripheral blood lymphocytes and monocytes labeled with approximately two logs greater mean fluorescence (not shown). In keeping with their low CD52 expression, only 4.7%±1.4% of LC were lysed with alemtuzumab and complement (Fig. 2B). Primary LC have not been tested previously but this result is in keeping with a report showing that the susceptibility of various in vitro-derived DC subsets is proportional to their CD52 expression (17). The resistance of LC and presumably other tissue DC to alemtuzumab is in contrast to the rapid lysis in vitro (17, 18) or clearance in vivo of blood DC (17, 19).
Complement-mediated lysis is an imperfect model for the in vivo action of these antibodies for several reasons. First, it is difficult to make a strict comparison of a murine IgM with a humanized IgG antibody, although the results indicate an in vitro activity of CMRF-44, which is not matched by alemtuzumab. Second, multiple additional mechanisms of antibody-mediated depletion may operate in vivo. Some of these, such as antibody-dependent cell cytotoxicity (ADCC) are well described for alemtuzumab (15) and although the expression of CD52 by LC may be insufficient for ADCC, other indirect effects such as depletion of monocytoid LC precursors (18) or mobilization of LC by cytokine activation (20), may lead to a degree of tissue DC depletion by alemtuzumab. A full comparison of the two antibodies will be possible when a humanized form of CMRF-44 has been prepared, through work currently in progress.
Inhibition of allostimulation by LC Using CMRF-44 and Complement
The previous results suggest that CMRF-44 has the potential to control alloresponses by depleting migratory tissue DC. To confirm this, we tested CMRF-44 in a novel system of allostimulation using a fragment of epidermis floating over responder PBL. This allows LC to migrate directly into contact with allogeneic lymphocytes. CMRF-44 and complement reduced proliferation of allogeneic PBL by 69%–95% (range of three experiments) compared with controls (Fig. 3). The proliferative response could be restored to wells treated with CMRF-44 and complement by addition of further LC on day 4 (‘Rescue’; Fig. 3), confirming that the responder PBL had remained intact during CMRF-44 treatment. Absolute values for thymidine incorporation were lower than frequently seen in standard mixed leukocyte reactions due to a relatively low stimulator to responder ratio (approximately 1:1,000; data not shown) and relative immaturity of migratory LC, which require additional signals to achieve full antigen-presenting potency (10, 12).
New strategies are required to control GvHD that do not compromise immunity to infections and graft-versus-tumor effects. Temporary depletion of DC by antibodies may be one way to achieve this. Therapeutic antibodies such as alemtuzumab have demonstrated the power of depleting specific cellular targets in preventing GvHD but so far none, including alemtuzumab, has shown activity against tissue DC. This report demonstrates the activity of CMRF-44 against LC, a tissue-derived DC and key player in skin GvHD. Humanization of this antibody is in progress to allow CMRF-44 to be evaluated as a clinical reagent, either as an adjunct or alternative to alemtuzumab in conditioning for stem cell transplantation. As for alemtuzumab, originally a rat monoclonal IgM, concomitant modification to IgG1 isotype will be necessary to allow CMRF-44 access to extravascular tissue compartments and to facilitate both complement-mediated killing and ADCC.
Rohit Sinah for purification of CMRF-44, Ken Field for assistance with flow cytometry, and Noel Williams for laboratory management.
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