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Brief Communications: Immunobiology

In Vitro Depletion of Tissue-Derived Dendritic Cells by CMRF-44 Antibody and Alemtuzumab: Implications for the Control of Graft-Versus-Host Disease

Collin, Matthew P.1,3; Munster, David2; Clark, Georgina2; Wang, Xiao-Nong1; Dickinson, Anne M.1; Hart, Derek N.2

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doi: 10.1097/01.TP.0000149321.86104.C4
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Abstract

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

Media

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).

Human Skin

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.

Complement-Mediated Lysis

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.

F1-19
FIGURE 1.:
A. Expression of CMRF-44 antigen on fresh and migratory LC. Freshly isolated or migratory Langerhans cells (LC) were identified by staining with antibodies to HLA-DR and CD1a as shown in the dot plots. Fresh LC were typically 1%–2% of total cells (50,000 events analyzed) and migratory LC, 20%–40% of total cells (10,000 events analyzed). Gated double positive cells were then tested for reactivity with CMRF-44 as shown in the histograms (data labels indicate mean fluorescence intensity). Triple staining was performed in PBS with 5% FCS on ice with CMRF-44 (10 μg/mL) detected by PE-conjugated sheep antimouse secondary antibody (1/50) followed by normal mouse serum (10%) and finally directly conjugated anti-HLA-DR-APC (2 μL) and anti-CD1a-FITC (5 μL). Artefactual loss of antigen due to enzyme treatment in the preparation of fresh cells was excluded by exposing migratory cells to trypsin, without loss of reactivity to antibodies (not shown). Shaded portions show isotype-matched controls. B. Complement-mediated lysis of LC by CMRF-44. Mean values ± SD of three independent experiments for percent lysis of migratory LC and PBL. LC were easily differentiated from keratinocytes by their characteristic dendritic morphology and refractile appearance. Lysed cells were identified by trypan blue staining and counted directly under a hemocytometer. C, complement; HI, heat-inactivated; isotype control antibody, TEPC 183 antibody.

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).

F2-19
FIGURE 2.:
A. Expression of CD52 on fresh and migratory LC. Freshly isolated and migratory Langerhans cells were identified using the same gating strategy as described in Figure 1. CD52 expression was tested by reactivity to PE-conjugated CAMPATH 1G (rat anti-CD52 IgG2b) as shown in the histograms (data labels indicate mean fluorescence intensity). Anti-CD52-PE was combined with directly conjugated anti-HLA-DR-APC and anti-CD1a-FITC antibodies in RPMI with 5% FCS on ice. Filled shaded portions show isotype-matched controls. B. Complement-mediated lysis of LC by alemtuzumab. Mean values ± SD of three independent experiments for percent lysis of LC and PBL. Lysed cells were identified by trypan blue staining. LC were differentiated from keratinocytes by their characteristic dendritic morphology. C, complement; HI, heat-inactivated; alem, alemtuzumab; isotype control antibody, pooled human immunoglobulin.

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).

F3-19
FIGURE 3.:
Inhibition of alloresponse to LC by CMRF-44 and complement. The response of allogeneic PBL measured by 3H thymidine incorporation on day 4 and day 6. Data from one of three experiments performed show the mean of four wells ± SEM ‘Rescue’ denotes wells treated with CMRF-44 + C to which 5,000 LC were added on day 4. Wells were treated with antibodies and complement serum as described in Methods. C, complement; HI, heat-inactivated; isotype control antibody, TEPC 183.

CONCLUSION

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.

ACKNOWLEDGMENTS

Rohit Sinah for purification of CMRF-44, Ken Field for assistance with flow cytometry, and Noel Williams for laboratory management.

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Keywords:

CMRF-44; Dendritic cell; Graft-versus-host disease

© 2005 Lippincott Williams & Wilkins, Inc.