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Prepublished online as a Blood First Edition Paper on October 10, 2002; DOI 10.1182/blood-2002-04-1093.
IMMUNOBIOLOGY
From the Laboratory of Cellular Immunobiology,
Department of Medicine; Department of Epidemiology and Biostatistics;
Surgical Pathology Service, Department of Pathology; Allogeneic
Transplantation and Clinical Immunology Services, Division of
Hematologic Oncology, Department of Medicine; Memorial Sloan-Kettering
Cancer Center, New York, NY; and Weill Medical College of Cornell
University, New York, NY.
Alemtuzumab (anti-CD52; Campath 1-H) depletes both host and donor T
cells when used in preparative regimens for allogeneic transplantation.
This promotes engraftment even after nonmyeloablative conditioning and
limits graft-versus-host disease (GVHD) even after unrelated or major
histocompatibility complex (MHC) disparate allografts. We asked whether
anti-CD52 differentially targets antigen-presenting cells (APCs), in
addition to depleting T cells. Monocyte-derived dendritic cells (moDCs)
expressed abundant CD52 as expected. Langerhans cells (LCs) and
dermal-interstitial DCs (DDC-IDCs), however, never expressed CD52.
Immunostaining of skin and gut confirmed the absence of CD52 on these
resident DC populations under both steady-state and inflammatory
conditions. Although anti-CD52 functions primarily by
antibody-dependent cellular cytotoxicity (ADCC) in vivo, assessment of
its activity in vitro included complement-dependent lysis of
CD52+ cells. Anti-CD52 did not impair DC-T-cell adhesion,
diminish DC-stimulated T-cell proliferation, or alter moDC development in vitro. We propose that anti-CD52 abrogates GVHD not only by T-cell
depletion, but also by removing moDCs and their precursors. This would
mitigate moDC phagocytosis and presentation of host-derived antigens to
donor T cells in the inflammatory peritransplantation environment,
thereby limiting GVHD. The sparing of LCs and DDC-IDCs by anti-CD52, as
well as the recovery of donor-derived moDCs in a less inflammatory
environment later after transplantation, may allow all these DCs to
exert formative roles in graft-versus-tumor (GVT) reactions and immune
reconstitution. Whether these results support a separation of
deleterious from beneficial graft-host interactions at the level of
antigen presentation, rather than solely at the level of T cells, will
require further evaluation.
(Blood. 2003;101:1422-1429) CD52 is a small, 12-amino acid,
phosphatidylinositol (GPI)-anchored membrane glycoprotein expressed by
lymphocytes, especially T cells, as well as monocytes, macrophages,
monocyte-derived dendritic cells (moDCs), and the epithelial cells of
the distal epididymis and vas deferens.1-3 All of the
functions of CD52 may not yet be known, but it constitutes at least a
target for complement-mediated cell lysis and antibody-mediated
cellular cytotoxicity.4,5 Complement activation, however,
is neither necessary nor sufficient for monoclonal antibody (MAb)
depletion of CD52+ cells in vivo.6,7
Antibody-dependent cellular cytotoxicity (ADCC) is likely the more
important mechanism for depleting CD52+ cells sensitized in
vivo by approximately 1000-fold less antibody than required for
complement-dependent lysis.8 These amounts of antibody
approximate the levels detected in patients receiving alemtuzumab in vivo.9
Alemtuzumab is a recombinant DNA-derived, humanized MAb directed
against CD52 that is very efficient in mediating lymphocyte depletion
both in vitro and in vivo.10 Treatment with humanized anti-CD52 in vivo mitigates graft-versus-host disease (GVHD) and promotes engraftment, even in adult recipients of unmodified allografts from unrelated or mismatched donors.11,12 This is
especially noteworthy because other regimens that achieve comparable
degrees of T-cell depletion or immune suppression have not always
proven similarly successful in these settings. This led us to ask
whether anti-CD52 targets different types of dendritic cells (DCs) in addition to depleting T cells.
We focused on DCs because these are the most potent
antigen-presenting cells (APCs) and the most critical to initiation of cellular immune responses.13-15 A growing body of data has
also emerged regarding the development of DCs and their hematopoietic relationship to other leukocytes.16-25 From these studies
one can distinguish at least 3 different types of myeloid DCs, for
which investigators increasingly find different specialized functions. These myeloid DCs comprise at least CD34+ hematopoietic
progenitor cell (HPC)-derived Langerhans cells (LCs) and
dermal-interstitial dendritic cells (DDC-IDCs), as well as
CD14+ blood moDCs.16-25 Phenotypically, all 3 mature myeloid DC types are class II major histocompatibility complex
(MHC)bright, CD86++, CD14 CD14+ monocytes express abundant CD52, as do moDCs
identified by a MAb CMRF-56.2 We evaluated expression and
function of the CD52 antigen on all 3 myeloid DC types, however. We
focused especially on the CD52 expression pattern of CD34+
HPC-derived, cytokine-generated LCs and DDC-IDCs, as well as their
resident counterparts in skin and gut mucosa. We compared these with
the well-characterized moDCs generated in vitro, which have all the
properties ascribed to and expected of DCs, but which have proven
difficult to identify specifically in vivo.
Our findings suggest additional mechanisms that may underlie the
efficacy of anti-CD52, which go beyond T-cell depletion. These results
may also have important implications for future studies to determine
whether deleterious graft-host interactions such as GVHD can be
distinguished at the level of antigen presentation from beneficial
processes like graft-versus-tumor (GVT) activity and immune reconstitution.
Media and reagents
Fresh human plasma (50% vol/vol) that was not heat inactivated, or
commercial human complement (no. S1764, Sigma, St Louis, MO) was used
for MAb/complement-dependent lysis. The commercial product, used
strictly according to manufacturer's instructions, proved more stable
and reproducible.
Recombinant human cytokines used for in vitro generation of DCs were
granulocyte-macrophage colony-stimulating factor (GM-CSF; Immunex,
Seattle, WA); interleukin 4 (IL-4), tumor necrosis factor Cell purification and generation of DCs
The moDC precursors were obtained from tissue culture plastic-adherent
peripheral blood mononuclear cells (PBMCs) after standard separation
over Ficoll-Paque PLUS (no. 17-1440-03, Amersham Pharmacia Biotech,
Uppsala, Sweden). Tissue culture plastic-adherent mononuclear cells
were cultured in GM-CSF (1000 IU/mL) and IL-4 (500 IU/mL) as
published.28 Medium and cytokines were replenished every 2 days. At approximately day 6, large forward scatter (FSC),
HLA-DR+ cells expressed intracellular CD83, confirming
commitment to DC differentiation, but very little cell surface CD83
(not shown). An inflammatory cytokine cocktail was added to these
immature moDCs for terminal maturation and activation. This
mixture comprised IL-1 CD34+ HPCs were obtained by positive immunomagnetic
selection from bone marrow or G-CSF-elicited PBMCs separated over
Ficoll-Paque PLUS (Amersham Pharmacia Biotech) according to
manufacturer's instructions (CD34+ isolation kit and
LS separation columns, Miltenyi Biotec, Bergisch Gladbach,
Germany). LCs and DDC-IDCs were separately generated from
CD34+ HPCs in the media described and as previously
published17,18,20,22,23. Specific cytokine supplements
included GM-CSF (1000 IU/mL), TNF- Two amendments were made to these cytokine mixtures. For the specific
generation of LCs, TGF- T cells were obtained from the PBMC fraction that was nonadherent to tissue culture plastic. Nonadherence and elution from nylon wool columns (Polysciences, Warrington, PA) further purified these cells in excess of 95%. Phenotypic analyses by flow cytometry Direct fluorescein isothiocyanate (FITC)-conjugated and phycoerythrin (PE)-conjugated mouse antihuman MAbs included anti-CD3-FITC, anti-CD3-PE, anti-CD14-PE, anti-CD16-PE, anti-CD20-PE, anti-CD11b-PE, anti-CD11c-PE (Pharmingen, Franklin Lakes, NJ); anti-CD4-PE, anti-CD8-PE, anti-CD14-PE, anti-CD14-FITC, anti-CD34-FITC, anti-CD11c-allophycocyanin (Becton Dickinson, Franklin Lakes, NJ); and anti-CD34-PE, anti-CD83-PE (Immunotech, Marseille, France). Isotype controls included IgG1-FITC, IgG1-PE, and IgG2a-FITC (Dako, Carpinteria, CA); and rat IgG2b-FITC (Serotec, Oxford, United Kingdom). Rat antihuman CD52-FITC and its respective rat IgG2b-FITC control (Serotec) were used for phenotypic but not functional assessments of CD52. Annexin-V (Early Apoptosis Detection Kit, Kamiya Biomedical, Seattle, WA) and propidium iodide staining, respectively, distinguished apoptotic and necrotic cells. Cytofluorographic evaluation used a FACScan (Becton Dickinson, Immunocytometry Systems, San Jose, CA), gating for live events. For analysis of specific epitope expression by DCs, candidate cells were first gated for large FSC, HLA-DRbright cells, after which 10 000 events were collected for analysis.MAb and complement-dependent cell lysis We used MAb/complement-dependent lysis in vitro as a surrogate measurement of anti-CD52 function in vivo. Assessment by ADCC in vitro, which is the more likely mechanism through which anti-CD52 exerts its effects in vivo,6 proved not to be logistically feasible.Cells were cultured in complete RPMI supplemented with 50% fresh human plasma without heat inactivation or with commercial complement equivalent to 50% plasma. As negative controls for complement, heat-inactivated (56°C for 30 minutes) normal human plasma or serum was used. Humanized anti-CD52 (alemtuzumab) and nonreactive control humanized anti-CD20 (rituximab) were added at the concentrations indicated for each experiment. Cells were opsonized with MAb for 30 to 40 minutes on ice, thoroughly washed, and exposed to complement or plasma (50% vol/vol) for 1 hour. When certain DCs proved resistant to MAb/complement-dependent lysis, owing to their lack of CD52 expression, complement exposure of MAb-opsonized cells was extended overnight to exclude the possibility that low-level CD52 expression might mediate some degree of cell targeting and lysis by alemtuzumab. After washing, the remaining viable cells were counted directly by trypan blue exclusion on a hemacytometer. Immunohistochemistry Immunohistochemical studies were performed on formalin-fixed and paraffin-embedded tissues. The antibodies used included anti-S-100 protein (1:50 000; Biogenix, San Ramon, CA) and anti-CD52 (1:40; Serotec). The tissue sections were exposed to the antibodies in citrate buffer solution at pH 6.0. Detection of the primary antibody was performed with a biotinylated secondary antibody (1:100; Vector, Burlingame, CA) for 30 minutes followed by an avidin-biotin complex system (Vector), using diaminobenzidine tetrahydrochloride (DAB; Biogenix) as chromogen. The slides were counterstained with Mayer hematoxylin (Sigma).Allogenic mixed leukocyte reactions DCs were cocultured with 105 purified allogeneic T cells (alloMLRs) in triplicate round-bottomed microwells at either a constant ratio of 30:1 for T/DCs and variable concentrations of MAbs, or at variable T/DC ratios of 30:1 to 1000:1 in the presence of a constant MAb concentration. Medium for allogeneic MLRs consisted of complete RPMI supplemented with 10% single-donor serum or plasma as described in "Media and reagents," but with no exogenous cytokines. DCs were extensively washed to remove cytokines before adding to T cells.Because only moDCs expressed CD52 in appreciable amounts (see "Results"), only moDCs were evaluated in alloMLRs after treatment with anti-CD52. The alloMLRs were cultured in the continuous presence of alemtuzumab, which targeted both moDC stimulators and T-cell responders. Alternatively, the moDCs were pretreated with humanized anti-CD52 (alemtuzumab) or control humanized anti-CD20 (rituximab) and complement, thoroughly washed, and then added to allogeneic stimulators in doses based on the DC yield in the control condition. Proliferating T cells incorporated
[methyl-3H]-thymidine (3HTdR; 1 µCi/well [0.037 MBq]; PerkinElmer Life Sciences, Boston, MA) during the last 12 hours of a 4- to 5-day culture. The amount of
3HTdR incorporated was measured in a
CD52 is differentially expressed on DC subsets and their precursors CD52 expression was determined by flow cytometry using a FITC-conjugated rat antihuman CD52 compared with a rat IgG2b-FITC control (Figure 1; Table 1). Purified T cells were included as positive controls given their known expression of CD52. Fresh PBMCs were also stained and gated for candidate circulating, immature DCs, as CD11c+, lineage-negative cells (CD3 ,
CD14, CD16, CD20), from which
we determined that all expressed CD52. Monocytes also expressed
abundant amounts of CD52, as expected, and more than 95% of the
differentiated immature and mature moDCs expressed CD52 as well. The
mean fluorescent intensity (MFI) decreased somewhat with maturation,
indicating a decrease in CD52 density on the cell surface.
Approximately 80% of the starting CD34+ HPCs expressed CD52, although at a lower surface density than PBMCs and moDCs based on the MFI. Most importantly, however, neither immature nor mature LCs or DDC-IDCs derived from these CD34+ HPCs in vitro ever expressed CD52 at any subsequent stage of differentiation or maturation. This was confirmed by serial phenotyping from approximately day 3 until the end of the 2-week culture. CD52 has no proliferative or adhesive function in DC/T-lymphocyte interactions To evaluate whether CD52 alone influences DC function, allogeneic T cells and DCs were cocultured in the presence of humanized anti-CD52 (alemtuzumab) compared with the nonreactive humanized anti-CD20 (rituximab) control. To avoid the introduction of complement, 10% (vol/vol) heat-inactivated plasma or serum was used. The more potent immunostimulatory, mature CD83+ moDCs were combined in a fixed DC/T-cell ratio with allogeneic T cells (1 DC/30 T cells), and the dose of MAb added to the cultures was varied from 1 µg/mL to 1 mg/mL final. Humanized anti-CD52 did not inhibit the formation of DC/T-cell clusters, from which reactive T-cell blasts emerge, as assessed by direct inspection using inverse phase microscopy (not shown). T-cell proliferation also remained comparable to control conditions at all MAb doses (Figure 2). Hence, we conclude that CD52 plays no role in adhesion or proliferation in allogeneic DC/T-cell interactions.
Alemtuzumab (anti-CD52) does not impair development and maturation of moDCs MAbs have several mechanisms by which they may exert their cellular effects. In addition to complement-mediated cytotoxicity and ADCC, these mechanisms may also include the induction of apoptosis and the inhibition of metabolically active proteins such as cytokines and growth factors.10,32 To evaluate any role of anti-CD52 in this respect, humanized anti-CD52 (alemtuzumab) was added in excess at 1 mg/mL during the generation of moDCs from CD14+ monocytes in 1% heat-inactivated autologous plasma with cytokines. Cell counts as well as flow cytometric analysis for apoptosis and maturation did not reveal any differences between anti-CD52-supplemented cultures compared with controls (Table 2). Anti-CD52 therefore causes neither apoptosis nor inhibition of DC development and maturation from CD52+ CD14+ class II MHC+ monocyte precursors.
Humanized anti-CD52 induces complement-mediated lysis in proportion to the level of CD52 expression We used MAb/complement-dependent lysis in vitro as a surrogate measurement of anti-CD52 function in vivo, which is likely more dependent on ADCC.6 Cells were treated with anti-CD52/complement or control anti-CD20/complement, and percent lysis was calculated based on the recovery of viable cells detected by trypan blue exclusion on a direct hemacytometer count. Humanized anti-CD52 lysed CD52+ cells in the presence of complement and in proportion to the level of surface CD52 expression, with the exception of CD34+ HPCs (Figure 3).
As expected, T cells were lysed very efficiently, almost up to 100%. CD14+ monocytes, immature moDCs, and mature moDCs also proved sensitive to complement-mediated lysis by humanized anti-CD52. LCs and DDC-IDCs, however, proved resistant regardless of their maturation state, in keeping with their lack of CD52 expression. The only CD52+ cells that were resistant to MAb/complement-dependent lysis were CD34+ HPCs, which had a lower surface density of CD52 based on MFI. Although moDCs expressed abundant CD52, independent of the maturation
state, the MFI decreased somewhat with maturation. Mature moDCs proved
less sensitive to anti-CD52/complement-mediated lysis than immature
moDCs and with much greater variation at the highest dose of
complement. Reduced concentrations of complement to 12.5% resulted in
diminished lysis and increased survival of approximately 40% of mature
moDCs (Figure 4). In contrast,
MAb/complement-dependent lysis of immature moDCs and T cells remained
complete at all concentrations of complement (Figure 4). MAb was used
at 1 mg/mL in the experiments depicted to confirm that resistant cells
were insensitive even to anti-CD52 used in excess.
Humanized anti-CD52/complement alters moDC stimulation of allogeneic T-cell proliferation in a dose-dependent manner We evaluated the functional consequences of anti-CD52 targeting of CD52+ cells in the context of a fully allogeneic MLR. LCs and DDC/IDCs were not evaluated in these experiments, owing to their lack of CD52 expression and their resistance to anti-CD52/complement-dependent lysis.Cells were exposed continuously throughout the alloMLR culture period
to humanized anti-CD52 (alemtuzumab)/complement versus control
humanized anti-CD20 (rituximab)/complement. Mature moDCs were used in
these experiments as the optimal stimulators, even though they were
more resistant to MAb/complement lysis (Figure 3) than their less
immunostimulatory, day 5 to 6 immature moDC precursors. The dominant
effect, as shown in Figure 5A, may
therefore have been directed at the T-cell responders. By holding
constant the amount of complement, there was a wide range of inferred
lytic activity, especially at MAb doses below 30 µg/mL, which
correspond to the levels recoverable from patients treated in
vivo.9
Alternatively, day 5 to 6 immature moDCs were opsonized with anti-CD52, washed, and exposed to complement before subsequent cytokine-induced maturation and addition to allogeneic T cells in the MLR. We used immature moDCs that were matured after MAb/complement exposure, because already matured moDCs do not exhibit consistent susceptibility to humanized anti-CD52 (alemtuzumab)/complement. Pretreatment of the moDCs also spared T cells from the effects of the MAb and instead specifically targeted the moDCs. As shown in Figure 5B, the percent inhibition relative to control humanized anti-CD20 (rituximab) was most pronounced at the lower stimulator doses, likely due to the strength of an allogeneic stimulus when higher moDC doses were used. In any case these lower stimulator doses would approximate T/DC ratios expected in vivo, even under inflammatory conditions. In lieu of exposing MAb-treated cells to complement for the experiments
depicted in Figure 5B, we opsonized immature moDCs with humanized
anti-CD52 (alemtuzumab) or control humanized anti-CD20 (rituximab),
then cultured these cells with nonadherent autologous PBMCs dosed to
provide a ratio of natural killer (NK) cell
(CD3 Resident populations of LCs and DDC-IDCs do not express CD52, similar to the progeny generated in vitro under cytokine-supported conditions One of the hazards of drawing conclusions based on cells generated in vitro under the aegis of nonphysiologic doses of recombinant cytokines is that the culture itself may have artifactually skewed the results. We therefore examined CD52 protein expression immunohistochemically on archival human tissue (Figure 6). The specimens included 2 samples each of normal skin and intestinal tissue, skin biopsies from patients with drug hypersensitivity reactions, and skin biopsies from patients with GVHD. None of the biopsies showed coexpression of S-100 protein-positive cells with CD52. Thus, resident epidermal or mucosal LCs were immunonegative for CD52. Likewise, coexpression of CD52 and S-100 protein-positive inflammatory cells of the dermis never occurred. Hence, DDC-IDCs were also immunonegative for CD52. These results argue definitively against the possibility that exogenous cytokines in vitro could have modulated CD52 surface expression compared with that found on resident populations of DCs in vivo.
The successful application of anti-CD52 in clinical transplantation derives in large part from its depletion of alloreactive T cells. This mechanism supports recent reports that recombinant DNA-derived, humanized anti-CD52 (alemtuzumab), administered in vivo, reduces the incidence and severity of GVHD after allogeneic HSCT while preserving the GVT benefit of the allograft, although follow-up is admittedly still limited.11,12 Investigators have also now shown that anti-CD52 MAb eliminates host moDCs, compared with standard chemotherapy and radiation-based preparative regimens, but does not impair recovery of donor-derived moDCs.3 We asked whether, in addition to T lymphocytes, distinct types of DCs differentially expressed CD52 for targeting by alemtuzumab. Using 3 carefully defined populations of myeloid DCs, we found that moDCs expressed CD52 to the exclusion of other myeloid DC populations. The most surprising and novel finding was that LCs and DDC-IDCs were, in fact, always negative for the CD52 epitope, either as resident populations in normal or inflamed skin or gut or as cytokine-generated progeny of CD34+ HPCs in vitro. Although the operative mechanism by which humanized anti-CD52 (alemtuzumab) exerts its effects in vivo is likely by ADCC,6 MAb/complement-dependent lysis provided a useful approximation of anti-CD52 function for our studies in vitro. Alemtuzumab, or anti-CD52, caused complement-dependent lysis in proportion to the amount of surface CD52 expression and was therefore lytic only for moDCs but not for either immature or mature LCs or DDC-IDCs. CD34+ HPCs also proved resistant, perhaps reflecting their lower surface density of CD52 expression. This resistance, however, is also consistent with the well-documented preservation of transplantable stem cells after anti-CD52 purging in vitro.33,34 It may also account for the paucity of actual cases of aplasia from treatment with alemtuzumab in vivo. CD52 exerted no adhesive or proliferative functions between DCs and T cells, because anti-CD52 alone in the absence of complement inhibited neither DC/T-cell aggregation nor alloantigen-specific T-cell proliferative responses. CD52 also had no effect on moDC development because binding of CD52 by the MAb did not alter cytokine-driven differentiation in vitro. The fact that anti-CD52 MAb therapy with alemtuzumab does not result in increased GVHD, given the persistence of LCs and DDC-IDCs, suggests 2 possible explanations. One is that the dominant effect of alemtuzumab is to cause such profound T-cell depletion that survival of LCs and DDC-IDCs is irrelevant. Our data suggest an additional possibility, which is that elimination of moDCs from the inflammatory environment early after transplantation removes a highly phagocytic and potent APC that could otherwise present antigen from dying host cells to surviving or newly generated donor T cells. We offer several lines of evidence in support of this reasoning. First of all, despite the absence of randomized trials, comparable levels of T-cell depletion by other transplantation regimens have not regularly achieved success similar to anti-CD52-containing regimens in terms of immune reconstitution and reduced GVHD, especially in older adults receiving unrelated or mismatched allografts. This is true even when G-CSF-elicited, T cell-depleted PBSCs containing disproportionate numbers of Th2-inducing DCs35 have been used as the source of the allograft. Alemtuzumab may therefore mediate either qualitative differences in T-cell depletion or differentially affect antigen presentation in contrast to other T-cell depletion approaches. Secondly, infections by viruses like cytomegalovirus (CMV) seem not to be exerting negative effects on patient survival after alemtuzumab-facilitated transplantations, to the same extent as comparable CMV reactivation rates after other methods of T-cell depletion,11 suggesting more effective immune reconstitution. Although follow-up is still limited, relapse is so far not a greater cause of treatment failure after preparative regimens using anti-CD52.11,12 We would therefore propose that preservation of LCs and DDC-IDCs after anti-CD52 treatment allows these myeloid DCs to play formative roles in the redevelopment of acquired and beneficial immunity. Several lines of evidence support the concept of moDCs as prime candidates for eliciting GVHD reactions, at least acutely. Monocytes and moDCs are intensely phagocytic,36 especially compared with other myeloid DCs, such as LCs and DDC/IDCs. In the inflammatory environment of an allogeneic transplantation, circulating monocyte precursors and immature moDCs would be ideally suited for uptake of dying host cells. Differentiation into mature moDCs and presentation of these host antigens to reactive clones of T cells circulating through secondary lymphoid organs would follow. Recent data also indicate that lipopolysaccharide (LPS) and CD14 are critical to the induction of experimental acute GVHD,37,38 further implicating a specific role for CD14+ moDCs in the sensitization arm of this process. In the case of hematopoietic stem cell allografts, moDCs of either host39 or donor origin could phagocytose dying host cellular antigens for presentation to and sensitization of engrafting donor-derived T cells. These would in turn cause GVHD in the periphery, especially in those sites that most abundantly express the same MHC antigens, for example, skin, gut, liver, and lymphoid organs. In the case of other nonmyeloablative preparative regimens that allow persistence of host T cells, donor moDCs could even sensitize host T cells by the direct pathway, leading to host resistance and nonengraftment or rejection. Similar logic applies to solid organ allografts, where MHC disparities are the rule rather than the exception and where chronic rejection and long-term graft survival remain problematic. Donor moDCs transferred among the so-called passenger leukocytes in an allograft could directly sensitize host T cells. Alternatively, host moDCs could phagocytose and present donor-derived cellular antigens from dying cells in the inflammatory environment of the transplanted allograft. Humanized anti-CD52 therapy should alter or eliminate both processes. These results have important implications for the activity of alemtuzumab in allogeneic transplantation. Like other studies in this area,40-42 our findings support greater attention to the afferent arm of the immune response, rather than focusing on alloreactive T-cell responders/effectors to the potential exclusion of evaluating antigen presentation. This leads us to hypothesize that the undesirable complications of GVHD or rejection (host-versus-graft) may be distinguishable from the beneficial graft-versus-leukemia or GVT effects exerted by hematopoietic allografts, based on differences in afferent sensitization of an immune response by different types of myeloid DCs. If true, this would predict that the selective elimination of moDCs by anti-CD52 in the inflammatory environment early after transplantation may promote the long-term tolerance and graft survival that has been so difficult to achieve with only T cell-targeted immune suppression in mismatched or unrelated host-donor pairings. Accordingly, preservation of resident LCs and DDC-IDCs may be as important to generating GVT and reconstituting normal cellular immunity as is depletion of highly phagocytic moDCs to the reduction of GVHD in the early and highly inflammatory posttransplantation environment. This does not exclude a role, however, for moDCs in the generation and maintenance of peripheral tolerance or GVT at later time points when the inflammatory cytokine milieu is substantially diminished. It also does not exclude a more dominant effect of anti-CD52 on T-cell responder populations, regardless of the DC populations that may be left intact or not. These concepts merit further specific testing in preclinical animal models and clinical trials.
We thank Eileen Walsh, RN, Frieda Toomasi, RN, the nurses and attending physicians of the Allogeneic Bone Marrow Transplantation Service, and the Allogeneic Transplant and Cytotherapy Laboratory staff, especially Nancy H. Collins, PhD, Sharon Bleau, and Zankar Desai, all for assistance with sample procurement and processing. We additionally acknowledge the technical assistance and expertise of Kristin Iversen in performing the immunohistochemical studies. We also appreciate the assistance of Scott Freeswick, RPh, in providing us with alemtuzumab and rituximab for these studies.
Submitted April 10, 2002; accepted September 6, 2002.
Prepublished online as Blood First Edition Paper, October 10, 2002; DOI 10.1182/blood-2002-04-1093.
Supported by grants P01 CA 23766 (J.W.Y.), R01 CA 83070 (J.W.Y.), and P01 CA 59350 (J.W.Y.) from the National Cancer Institute, National Institutes of Health; and LLS 6124-99 (J.W.Y.) from the Leukemia and Lymphoma Society of America.
Correspondence: James W. Young, Memorial Sloan-Kettering Cancer Center, 1275 York Ave, New York, NY 10021; e-mail: youngjw{at}mskcc.org.
The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked "advertisement" in accordance with 18 U.S.C. section 1734.
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Top
Abstract
Introduction
(TNF-
(TGF-
(mean cpm of T cells stimulated by
anti-CD52-treated moDCs/mean cpm of T cells stimulated by anti-CD20
treated moDCs) × 100. Shown are the results of 3 independent
experiments and the SD of the mean percent inhibition at each T/DC
dose.
