Monoclonal antibodies (mAb*) directed against T lymphocyte surface molecules are useful in transplantation immunology and cancer immunotherapy (1-6). One of the most extensively studied monoclonal antibodies, OKT3, is an antibody directed against the epsilon (ε) chain of the TcR/CD3 complex on human T lymphocytes (1, 7-10). Previous studies have shown that administration of OKT3 results in transient T lymphocyte activation, followed by an immunomodulatory effect (11-16). In vitro, CD3 is modulated (internalized) from the T-cell surface (17, 18). In patients, T cells are opsonized and removed temporarily from the circulation by hepatic and splenic mononuclear phagocytes (19, 20). When the T cells return to the circulation, they express only a low density of TcR/CD3 molecules as a result of CD3 modulation (17, 19).
We recently developed an improved human peripheral blood lymphocyte-severe combined immunodeficiency (hu-PBL-SCID) mouse model that can be used for the study of human lymphocytes in vivo. SCID mice have a severe combined immune deficiency, lacking mature murine B and T cells due to a dysfunctional V(D)J recombinase system (21), and can be reconstituted with human peripheral blood lymphocytes (PBLs) (22). Pretreatment with anti-asialo-GM1 (α-ASGM1) (to deplete murine natural killer cells and monocytes) and sublethal radiation (3 Gy) results in high level engraftment of SCID mice with human CD45+ lymphocytes, including CD3+ T cells (23). The human immune cells in these mice are functional, as demonstrated by the induction of primary and secondary immune responses and by the generation of an in vitro proliferative response to phytohemagglutinin (23, 24). Thus, pretreatment of SCID mice with radiation and α-ASGM1 provides a potentially useful model for the study of transplantation immunology and cancer immunotherapy.
The purpose of this study was to analyze the physiological responses of human T lymphocytes to OKT3 in the hu-PBL-SCID mouse model.
MATERIALS AND METHODS
SCID mice. An original pair of homozygous C.B-17 scid/scid mice was obtained from Dr. R. Philips, Hospital for Sick Children, Toronto, Ontario. Mice were subsequently bred and maintained at the Research Annex of the Samuel Lunenfeld Research Institute, Mount Sinai Hospital, Toronto, Ontario. Animals were fed autoclaved food and water, and all manipulations were performed under laminar flow. SCID mice were engrafted at 8-10 weeks of age with human PBLs from buffy coats or whole blood.
Pretreatment of SCID mice. To improve human PBL engraftment (23), SCID mice were pretreated with α-ASGM1 (Wako Pure Chemical Industries, Ltd., Osaka, Japan) and sublethal radiation (3 Gy, 137Cs source, Gammacell, Atomic Energy of Canada Ltd. Commercial Products). α-ASGM1, a rabbit polyclonal antibody that depletes murine natural killer cells and macrophages (25), was administered 1 day before human PBL injection and every 5-7 days after reconstitution. Mice were irradiated once, just before PBL injection.
Engraftment of SCID mice with human PBLs. Buffy coats generously provided by the Canadian Red Cross, or whole blood from volunteers, were used in these studies. PBLs were isolated by Ficoll-Hypaque gradient centrifugation (Pharmacia, Piscataway, NJ). Buffy coats or heparinized whole blood were diluted 1:1 with phosphate-buffered saline, layered over a Ficoll-Hypaque column, and centrifuged at 1500 rpm for 30 min. Cells at the interface were collected, washed three times with RPMI 1640 (Life Technologies, Grand Island, NY), counted, then used for intraperitoneal injection into SCID mice. A total of 3.3-5.0×107 human PBLs per mouse were injected under sterile conditions.
Monoclonal antibodies. The following antibodies specific for human cell surface antigens were used for flow cytometric analysis: CD3, CD4, CD8, CD45, CD16, and isotype-specific controls, directly conjugated to either fluorescein isothiocyanate or phycoerythrin (all from Becton Dickinson, Mountain View, CA).
OKT3 (generously provided by Stella Tymkiewicz, Ortho Pharamaceutical Corp., Toronto, Canada) is a murine IgG2a mAb directed against the ε-chain of the CD3 antigen of human T lymphocytes. OKT3 was injected at a dose of 5, 0.5, or 0.05 μg (all in a volume of 100 μl) intravenously into the tail vein of reconstituted hu-PBL-SCID mice. 145-2C11, a hamster monoclonal antibody against the murine CD3 surface antigen, was generously provided by Dr. Bluestone. This mAb was injected intravenously into hu-PBL-SCID mice at a dose of 5 μg per mouse.
Assessment of human PBL engraftment and OKT3 effect in hu-PBL-SCID mice. The effect of OKT3 was evaluated 1-18 hr and 1-3 weeks after reconstitution with human lymphocytes. Mice were killed at different time points after the administration of OKT3 (1 hr, 4 hr, 12-18 hr, and 6-18 days). The phenotype of human lymphocytes before and after injection of OKT3 was evaluated by flow cytometry. Reconstituted hu-PBL-SCID mice treated with OKT3 were compared with mice treated with saline or with 145-2C11.
For two-color flow cytometric analysis, splenocytes (1×105 cells/sample) were incubated with antibodies for 45 min on ice, then washed three times with phosphate-buffered saline containing 1% fetal bovine serum (Life Technologies). At least 5×103 cells were counted on an EPICS C flow cytometer (Coulter Electronics, Miami Lakes, FL). Splenocytes from unreconstituted SCID mice were tested and were negative when stained with any of the anti-human lymphocyte markers.
Histopathology. The spleen, liver, and lungs of hu-PBL-SCID mice were removed 7-20 days after OKT3 or saline injection. The organs were fixed immediately in 10% formalin and then embedded in paraffin. Sections (4-6 μm) were cut and stained with hematoxylin and eosin and examined microscopically.
Detection of human tumor necrosis factor-α (TNF-α) serum levels in hu-PBL-SCID mice. Serum was obtained from clotted whole blood after cardiac puncture of the hu-PBL-SCID mice. Mice were killed for serum collection before OKT3 injection and 1, 4, or 24 hr after injection; samples were stored at -70°C until used for testing. Serum levels of human TNF-α were measured by species-specific enzyme-linked immunosorbent assay using a fluorescence detection system (26). Briefly, 96-well flat-bottom plates were coated with goat anti-mouse (IgG Fc portion-specific) antibody and incubated overnight. Nonspecific binding sites were blocked with 6% bovine serum albumin solution. Anti-human TNF mAbs (BioSource International, Camarillo, CA) and either sample serum or standard recombinant TNF-α (Genzyme, Cambridge, MA) were added and incubated on a shaker. This was followed by incubation with polyclonal rabbit anti-human TNF-α-neutralizing antiserum. After a wash, goat anti-rabbit antiserum conjugated to alkaline phosphatase (Jackson Immunoresearch Labs, BioCan, Mississauga, Canada) was added and incubated for 1 hr. After being washed, the plates were incubated with substrate solution, 5-fluorosalicyl phosphate, and the developing reagent, terbium EDTA. The fluorescence was measured with a time-resolved fluorometer, and the calibration curve and data reduction were performed by an automatic immunoanalyzer (Cyberfluor 615).
Statistical analysis. Means between groups were compared by Student's t test.
Effects of OKT3 or 145-2C11 injection on the surface phenotype of engrafted human lymphocytes. We evaluated human T lymphocyte responses to intravenous OKT3 treatment in hu-PBL-SCID mice by characterizing the phenotype of splenic human lymphocytes before OKT3 treatment and at various time points after OKT3 treatment. Flow cytometric analysis of human lymphocytes in the spleen of hu-PBL-SCID mice 12-18 days after engraftment, immediately before OKT3 treatment, revealed 16.6±10% human CD3+ and 37.2±13% CD45+ T cells. Four to 12 hr after the injection of 5 μg of OKT3, most human lymphocytes were CD3-/CD4+ and CD3-/CD8+, which suggests that human CD3+ lymphocytes undergo antigenic modulation immediately after the binding of the anti-CD3 mAb. Although no CD3+ lymphocytes were detected by flow cytometry during the first 12-14 hr after OKT3 injection, the cells were still present in the spleens of engrafted SCID mice, as demonstrated by the presence of CD4+ and CD8+ cells (Table 1). CD3+ human lymphocytes reappeared in small numbers 4-6 days after the initial antigenic modulation, with high variability in the degree of CD3 expression (3.4±6.8% CD3+ T cells). This antigenic modulation was followed by depletion of human CD3+ lymphocytes, as no CD4+ or CD8+ human lymphocytes were found at later time points. However, cells positive for the pan-leukocyte marker, CD45, were still detectable. The engraftment level and phenotype of human cells in the spleens of OKT3-treated hu-PBL-SCID mice at different time points after OKT3 injection are shown in Table 2.
Human T-cell depletion was an OKT3 dose-dependent effect. The magnitude of T lymphocyte depletion was analyzed 5 days after the injection of three different doses of OKT3. The percentage of human CD3+ lymphocytes in the spleen after injection of 5, 0.5, and 0.05 μg of OKT3 were 0.5±0.4%, 1.7±0.5%, and 9.1±9.6%, respectively (Fig. 1). Control mice treated with saline alone had 13.6±7.2% CD3+ lymphocytes in this experiment.
The depletion of human lymphocytes was OKT3-specific, since levels of CD3+ human T lymphocytes did not fall when mice were treated with 5 μg of 145-2C11, a hamster antimouse CD3 antibody (data not shown).
Release of TNF-α after the injection of OKT3. OKT3 (5 μg/mouse) was administered 12 days after SCID mice were engrafted with human PBLs. Serum samples were collected 4 hr after the intravenous OKT3 injection. Mice treated with OKT3 had increased levels of human TNF-α in their serum, with a mean of 3140 pg/ml (n=3), compared with 1980 pg/ml, in mice treated with saline (n=5; P<0.1, Student's t test).
Graft-versus-host disease (GVHD). In our previous study (23), we demonstrated that a substantial number of engrafted hu-PBL-SCID mice develop fatal acute GVHD 3-4 weeks after PBL injection, manifested by altered behavior, weight loss, ruffled fur, anemia, and dehydration. We therefore followed a group of reconstituted animals for signs of GVHD after intravenous injection of 5 μg of OKT3 (OKT3 given 12 days after PBL injection). Control hu-PBL-SCID mice were injected with saline. The animals were examined for manifestations of the disease every 24 hr; all animals were killed on day 18 after PBL infection. In the control group, six of eight hu-PBL-SCID mice developed acute GVHD, confirmed on histological examination. In contrast, none of the OKT3-injected hu-PBL-SCID mice developed signs of GVHD.
An additional group of animals was given a single dose of OKT3 after manifesting early symptoms of GVHD. As a result of OKT3 treatment, all injected hu-PBL-SCID mice showed complete recovery from GVHD within 2-3 days of the OKT3 injection, demonstrated by a return to normal behavior and weight gain.
Histopathology. Histologic examination of sections from the liver and lung of hu-PBL-SCID mice showed similar findings. In the liver, there were patchy infiltrates of mononuclear cells (Fig. 2A). These were composed mostly of small lymphocytes and were distributed predominantly in the portal triads. There was minimal hepatocyte necrosis and insignificant numbers of cells within the sinusoids. After treatment with OKT3, there was a marked reduction in the cellular infiltrate (Fig. 2B). Sections of lung from untreated hu-PBL-SCID mice also showed scattered aggregates of small lymphocytes. These were distributed mainly around small arteries and venules (Fig. 2C). There was no evidence of vascular thrombosis or acute inflammation. After treatment with OKT3, the volume of cellular infiltrates was substantially reduced (Fig. 2D).
In the present study, we demonstrated that a single injection of OKT3 into hu-PBL-SCID mice results in a physiologic response of engrafted human lymphocytes. The response of the human T cells in hu-PBL-SCID mice resembles the events that take place in humans after OKT3 injection and in mice after injection of 145-2C11 (11-15, 27). Moreover, OKT3 given to engrafted mice with early signs of GVHD results in the abrogation of the symptoms of GVHD, followed by full recovery. It has been demonstrated repeatedly both in vitro and in vivo that OKT3 leads to the activation of human lymphocytes (11-15), as demonstrated by T-cell proliferation, increased serum levels of TNF-α, γ-interferon, and interleukin (IL)-2, and induction of the expression of activation markers such as IL-2R and Leu-23 (16, 27-31). Alegre et al. (32) have shown that OKT3 induces up-regulation of CD69 and the release of human IL-2 in the hu-SPL-SCID mouse model (using splenocytes from cadaveric organ donors). Our results also indicate early in vivo T-cell activation after OKT3 injection. Interestingly, significant levels of human TNF-α were detected in the serum of hu-PBL-SCID mice before OKT3 injection. These levels of human TNF-α in the hu-PBL-SCID model are associated with the activation of human T lymphocytes due to ongoing GVHD (33). However, an additional peak of TNF-α was also noted 1-4 hr after OKT3 injection, suggesting further T-cell activation. The exact source of TNF-α in our model is difficult to determine; the two major sources of serum TNF-α are macrophages and lymphocytes. It is possible that direct activation of engrafted lymphocytes after OKT3 binding to CD3 causes TNF-α release. Data from animal studies indicate that anti-CD3 antibody-activated murine T lymphocytes are indeed the major source of TNF-α secretion (15). However, the other possibility is macrophage-mediated secretion of TNF-α via binding of OKT3 to FcγIII receptors. In fact, we have demonstrated that human macrophages are present in the spleen of engrafted hu-PBL-SCID mice pretreated with α-ASGM1 and sublethal radiation (23).
In humans, OKT3 injection results in a cytokine-release syndrome. Clinically, the first dose causes headaches, hypothermia, diarrhea, and hypotension during the first few hours after OKT3 injection (28, 34). A similar syndrome of first dose response to an anti-mouse CD3 mAb (145-2C11) injection has been observed in BALB/c mice, with large doses of 145-2C11 being lethal (15, 35). In doses similar to those used in the present study, the cytokine-release syndrome was manifested by hypomotility, hypothermia, diarrhea, and piloerection (15, 35). In our hu-PBL-SCID mouse model, however, no similar response was noticed during the first few hours after OKT3 injection. Perhaps there are too few human lymphocytes in the mouse model to elicit a sufficiently strong systemic response. Alegre et al. noted similar findings in hu-SPL-SCID mice when the percentage of human cell engraftment was moderate (32), but animals engrafted with a higher percentage of human T cells became lethargic and more likely to die after OKT3 therapy. Whether these animals died of GVHD or of a cytokine-release syndrome is not clear (32).
One of the main events in early lymphocyte-OKT3 interaction includes modulation of the CD3 epitope (12, 13, 36, 37), suggesting that antibody-mediated modulation represents an important mechanism of OKT3-mediated immunosuppressive activity. In our hu-PBL-SCID model, as in patients, administration of OKT3 results in the early transient emergence of CD3-/CD4+ and CD3-/CD8+ lymphocytes. These findings suggest that the CD3 antigen is probably coated by the OKT3 antibody, although the cells are still present in the spleens of engrafted mice. Our data are in keeping with previously reported observations in humans, showing antigenic modulation of lymphocytes as a result of interaction of OKT3 and CD3/TcR complex expressed on human T cells (12). Gebel et al. used flow cytometry to show that OKT3 can be detected bound to the lymphocytes of patients soon after OKT3 injection, and the failure to detect CD3 antigen is due to the sequestering of the specific epitope (12). Magnussen et al. reported that OKT3-mediated cross-linking of δ-chains of the CD3 complex causes shedding of CD3 ε-chains, and probably the CD3 complex as a whole (36). Other T-cell surface molecules are unaffected by OKT3, so that the usual array of surface molecules (CD2, CD4, CD8, etc.) are expressed on all T cells that repopulate the peripheral blood compartment during OKT3 treatment (37).
In vivo antigenic modulation of human lymphocytes has been studied in transplant patients treated with OKT3 in the immediate posttransplantation period or after diagnosis of acute rejection of a renal graft (13). Examination of graft-infiltrating mononuclear cells before OKT3 treatment reveals a predominance of CD3+/CD4+ and CD3+/CD8+ cells. OKT3 treatment results in the appearance of infiltrating lymphocytes that show a typical antigenic modulation pattern manifested by CD3-/CD4+ and CD3-/CD8+ lymphocytes (13). This antigenic modulation is followed by removal of CD3+ lymphocytes from the peripheral circulation. As in these human studies, we have demonstrated that, within 14 hr of OKT3 administration, human lymphocytes in the spleen of hu-PBL-SCID mice are modulated to the CD3-/CD4+ and CD3-/CD8+ phenotype. Although some CD3+ cells are still present in the spleen 4-6 days after OKT3 injection, no CD3+, CD4+, or CD8+ human lymphocytes are detectable by day 9-12. The deletion of human T cells is also accompanied by the recovery of the mice from GVHD.
We have further demonstrated that this phenomenon occurs in a dose-dependent fashion, as lower doses result in only partial depletion of CD3+ human T lymphocytes. The precise mechanisms of human T-cell depletion in hu-PBL-SCID mice treated with OKT3 are not known. In humans, several mechanisms may be responsible for rapid T-cell depletion, including sequestration of the lymphocytes in lymphatic organs, antibody-dependent cellular cytotoxicity, and complement-mediated lysis (13). Additional studies are needed to explore the mechanisms of T-cell depletion in the hu-PBL-SCID mouse model.
Our previous study showed that pretreatment with sublethal radiation and α-ASGM1 results in high-level engraftment of SCID mice with human immune cells, but a substantial number of engrafted mice develop fatal GVHD (23). We have demonstrated in this study that injection of OKT3 results in the resolution of this severe GVHD. Deletion of T lymphocytes after injection of OKT3 results in complete clearance of lymphocytic infiltration from various organs and prolonged survival of the mice. Alegre studied a humanized mAb (Ala-Ala-IgG4, formerly named gOKT3-7(γ4-a/a)) derived from OKT3 and bearing mutations of two amino acids in the Fc portion to impede its binding to Fcγ receptors (32). They reported a reduction in the symptoms of GVHD (and a possible prevention of death from GVHD) in hu-SPL-SCID mice treated with Ala-Ala-IgG4, but their numbers were too small to generate statistical differences (32).
In conclusion, our data provide evidence for transient activation of engrafted human T lymphocytes, followed by antigenic modulation and subsequent deletion of the T cells after OKT3 injection in hu-PBL-SCID mice. These data suggest a physiologic human T-cell response to OKT3 administration similar to that observed in both human studies and murine models (after injection of an anti-murine CD3 antibody). Further experimentation with the hu-PBL-SCID mouse should be useful in transplantation immunology. The success of the hu-PBL-SCID model should also allow the possibility of using this system to study the response of PBLs from patients who will themselves undergo (or have undergone) transplantation in the clinical setting.
This study was supported by a research fellowship from the National Cancer Institute of Canada (with funds provided by the Canadian Cancer Society) (S.D.), the Lederle Fellowship in Surgical Oncology (S.D.), and the Lederman Research Fund (B.S.), an Israel Physician Fellowship (B.S.), and a Milvizky Award (B.S.).
Abbreviations: α-ASGM1, anti-asialo-GM1; GVHD, graft versus host disease; hu-PBL-SCID, human PBL-SCID; IL, interleukin; mAb, monoclonal antibody; PBL, peripheral blood lymphocyte; SCID, severe combined immune deficiency; SPL, splenocytes; TNF, tumor necrosis factor.
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