Stem Cells, Vol. 15, No. 2, 154-160,
March 1997
© 1997 AlphaMed Press
Hematopoietic Changes Induced by a Single Injection of Anti-CD3 Monoclonal Antibody into Normal Mice
Elke Schneider,
Véronique Salaün,
Asmâa Ben Amor,
Michel Dy
Université René Descartes - Paris V, CNRS URA 1461, Hôpital Necker, Paris, France
Key Words. CFU-C • IL-3 • GM-CSF • Histamine
Dr. Elke Schneider, CNRS URA 1461, Hôpital Necker, 161 rue de Sèvres, 75743 Paris Cedex 15, France.
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Abstract
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The present study evaluates hematopoietic modifications consecutive to in vivo treatment of mice with anti-CD3 monoclonal antibodies (mAb). The hamster mAb 145-2C11, administered in a single i.v. injection of 10 µg, induced the release of both interleukin 3 (IL-3) and GM-CSF into the circulation. IL-3 could be detected in the serum within 1 h, attained maximal levels after 4 h and had disappeared after 24 h. Three days later, treated mice exhibited a two- to threefold rise in blood neutrophil levels and increased spleen cell counts. Concomitantly, the incidence of nucleated erythroid cells in these spleens increased around 10-fold, relative to controls having received hamster Ig. At the same time point, clonogenic progenitor frequencies were 10-fold higher in spleens from treated mice than in those from control mice. Furthermore, the responsiveness of these splenocytes to IL-3, in terms of histamine synthesis, was enhanced. In contrast, bone marrow cell populations were only slightly affected by anti-CD3 injection. All hematopoietic changes required multivalent crosslinking of the mAb for induction, since F(ab')2 fragments lacked this activity. A return to normal occurred 7-10 days after treatment. Two i.v. injections of recombinant murine IL-3 together with recombinant murine GM-CSF on a single day had a less pronounced effect on progenitor cell frequencies in the spleen than treatment with anti-CD3. This difference is probably due to the amplification of growth factor-induced hematopoiesis by the interaction with other cytokines generated in response to anti-CD3.
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Introduction
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The murine monoclonal antibodies (mAb) OKT3 with specificity for the human CD3 complex exerts two seemingly contradictory effects, namely a transient activation of T cells occurring shortly after the stimulation [1] and a persistent immunosuppression which has given rise to a number of clinical applications to prevent organ rejection [2-4].
The hamster mAb 145-2C11 [5], directed against the CD3
portion of the murine T cell receptor for antigen (TCR) complex shares similar features. It induces a monocyte-dependent T cell activation, resulting in a massive release of both T cell- and monocyte-derived cytokines into the circulation [6-8], as well as a long-term immunosuppression in vivo [9]. Among the cytokines generated in response to 145-2C11 mAb, one can distinguish between those mainly involved in the immune response, like tumor necrosis factor-
, interferon-
, interleukin 4 (IL-4) and IL-2, and those with potential effects on hematopoiesis, namely IL-3, GM-CSF and IL-6. So far, most of the studies addressing the mechanisms of anti-CD3-induced tumor rejection and short-term morbidity have focused on the first category of cytokines [10, 11]. The purpose of the present study was to examine hematopoietic modifications resulting from anti-CD3-induced release of growth factors. To this end, we injected the antibody at a dose which induces a potent T cell activation without significant T cell depletion from the spleen [10], as well as a substantial release of GM-CSF and IL-3 into the circulation. We then compared the hematopoietic changes observed to those promoted by the injection of the recombinant forms of IL-3 and GM-CSF.
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Materials and Methods
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Animals
Six- to eight-week-old male or female C57 BL/6 mice were used in all experiments. They were bred in our own facilities under pathogen-free conditions and matched for sex and body weight in each experiment.
Cytokines and Antibodies
Recombinant murine (rm) IL-3 (specific activity: 1-2 x 107 U/mg, as assessed on the NFS-60 cell line) and rmGM-CSF (1-2 x 107 U/mg, as assessed on the DA-3 cell line) were purchased from R & D (Oxon, Great Britain).
The hamster 145 2C11 mAb (IgG), specific for the murine CD3 chain, as well as its F(ab')2 fragments, prepared by pepsin digestion, were kindly provided by Dr. L. Chatenoud. Hamster IgG from Jackson ImmunoResearch Laboratories (West Grove, PA) were used as specificity control. Polyclonal sheep antisera against IL-3 [12] and GM-CSF [13] were kindly provided by Dr. H. Ziltener.
Injection Schedule
Mice received a single i.v. injection of 10 µg of anti-CD3 mAb, 10 µg of control hamster IgG or 50 µg of the anti-CD3 F(ab')2 fragment diluted in 100 µl saline. rmIL-3 and rmGM-CSF were administered i.v. twice on a single day, at a dose of 1 µg of each factor diluted in 100 µl saline.
WBC Count
Mice were anesthetized with ether, and orbital plexus blood was obtained using micropipettes. WBCs were counted in hemocytometers (Unopette, Becton Dickinson; Rutherford, NJ) and differential counts were performed on blood films stained with May-Grünwald-Giemsa. No mouse was examined more than once to avoid misleading cell counts from previously damaged vessels.
Cell Preparations
Bone marrow cells were removed from femurs and tibias by flushing with Hanks' balanced salt solution (HBSS; GIBCO; Grand Island, NY). Spleen cells were homogenized in HBSS by gently teasing with forceps. The cells were centrifuged, resuspended in minimal essential medium (MEM) supplemented with 10% horse serum at a concentration of 107 cells/ml. These cell suspensions, prepared at different times after treatment, were used for progenitor cell assays and cytocentrifuge preparations examined after May-Grünwald-Giemsa staining.
Spleen and bone marrow cell suspensions were also distributed into 96-well microtiter plates (200 µl/well) at a concentration of 107 and 2.5 x 106 cells per ml respectively. They were incubated for 48 h in MEM supplemented with 10% horse serum with or without 1 ng/ml of rmIL-3. Cell-free supernatants were then prepared and stored at -20°C until histamine and cytokine assay.
Histamine and Cytokine Assays
Histamine was measured by an automated continuous flow fluorometric technique previously described [14], whose lower limit of sensitivity is about 0.5 ng/ml. The specificity of this assay has been verified by a radioimmunoassay (Immunotech; Marseilles, France).
Sera from anti-CD3-injected and control mice were tested for their proliferative effect on the FDCP-2 cell line which in our hands responds exclusively to IL-3. The GM-CSF assay was performed on the FDCP-1 cell line that also responds to IL-3 [15]. For this reason, all samples were tested in the presence of a 1/40 dilution of polyclonal sheep antiserum against IL-3 resulting in 90% inhibition of the proliferation induced by 1 ng of rmIL-3 per ml. Cell proliferation was evaluated after a 48-h incubation by the colorimetric MTT assay. The cytokines were quantified in relation to rmIL-3 and rmGM-CSF at optimal concentrations (1 ng/ml and 5 ng/ml, respectively), 1 unit/ml being defined as the concentration that leads to half-maximal cell proliferation in the assay. The specificity of the IL-3 and GM-CSF assays was assessed in the presence of the neutralizing anti-IL-3 and anti-GM-CSF antisera.
Colony Forming Assays
Nonerythroid colony forming units-culture (CFU-C) were quantified in semisolid culture medium (-modification of Dulbecco's MEM) containing 0.8% methylcellulose (Fluka; Buchs, Switzerland), 10% fetal calf serum (FCS) and 1 ng/ml of rmIL-3. Cells were plated in a final volume of 1 ml at concentrations ranging from 5-50 x 104 cells per culture dish (Falcon 1008). Colonies were scored on day 7. To evaluate BFU-E, and CFU-Mix frequencies, cultures were performed in the same medium, supplemented with 20% FCS, 1% deionized bovine serum albumin, 1 ng/ml of rmIL-3, 2 U/ml of human erythropoietin, 200 ng/ml of rm stem cell factor and 1,000 U/ml of rmIL-6. Colonies were scored on days 7-10 for BFU-E and CFU-Mix.
The statistical significance of all data was established by Student's t-test.
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Results
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Induction of IL-3 and GM-CSF by Anti-CD3 Injection
As shown in Figure 1, 
a single injection of 10 µg of anti-CD3 mAb promoted rapid release of IL-3 into the circulation, as assessed by the proliferative effect of sera from treated mice on the FDCP-2 cell line. This cytokine was detected as soon as 1 h after anti-CD3 injection, maximal levels being attained after 4 h and maintained up to at least 8 h. The growth factor had disappeared from the circulation 24 h post-treatment. After neutralization of IL-3 by anti-IL-3 Abs, the sera conserved about 50% of their proliferative effect on the FDCP-1 cell line. This activity was abolished in the presence of anti-GM-CSF Abs, proving that both IL-3 and GM-CSF were present in the circulation. Sera collected 4 h after anti-CD3 injection contained 54.5 ± 6.9 U/ml of GM-CSF (mean ± SE from three different samples), as quantified after neutralization of IL-3 in relation to optimal concentrations of rmGM-CSF. In the same experimental conditions, GM-CSF was not detected in sera from IgG-injected control mice.
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Figure 1. Detection of IL-3 in sera from C57BL/6 mice injected with 10 µg anti-CD3 mAb. IL-3 was quantified by its proliferative effect on FDCP-2 cells, in relation to optimal concentrations of rmIL-3. Data are means ± SE from three different samples assayed in triplicate. IL-3 was not detected in sera from mice injected with hamster IgG used as an isotype control.
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Changes in the Blood of Anti-CD3-Injected Mice
As shown in Figure 2, anti-CD3 injection within 24 h resulted in a dramatic fall of lymphocyte levels in peripheral blood, compared to controls. Lymphocyte counts were then progressively restored, with a significant overshoot on day 3 and day 4 (p < 0.001). Anti-CD3 treatment increased neutrophil levels on day 1, followed by a return to normal on day 2 and a secondary rise between day 3 and day 4. We detected only a few eosinophils, whether mice were treated or not. Monocytes were also not modified by the injection of anti-CD3, and the hematocrit remained normal.
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Figure 2. Changes in lymphocyte and neutrophil levels in peripheral blood from C57BL/6 mice injected with 10 µg anti-CD3 mAb. Each point represents the mean value ± SE from three mice. The shadowed area comprises control values ± SE averaged from three normal and three IgG-injected mice.
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Effect of Anti-CD3 Injection on Spleen and Bone Marrow Cell Populations
A single injection of the anti-CD3 mAb 145-2C11 promoted striking changes in spleen cell populations (Table 1). On day 1, the proportion of granulocytes increased significantly, matching the rise of this population in peripheral blood. The proportion of CD4+ and CD8+ T cells (16.0 ± 0.9% and 8.6 ± 1.1%, respectively) was not significantly modified relative to IgG-injected controls (18.0 ± 0.9% and 11.0 ± 0.8%, respectively), as assessed by cytofluorometry analysis of spleen cells from 14 anti-CD3-injected and 13 control mice. The number of spleen cells remained normal at this time point but increased thereafter, reaching a maximum on day 4 post- injection. Nucleated RBCs were then about 10 times more frequent than in normal spleens and the proportion of immature myeloid cells was significantly augmented. From day 4 on, the different spleen cell populations returned progressively to baseline, except mature granulocytes which increased slightly and remained so until day 10, when all other populations were back to normal (3.6 ± 1.3% in control spleens versus 6.8 ± 2.2% in spleens from anti-CD3-injected mice; means ± SE from three mice, p < 0.05).
The cytological changes in the spleen resulting from anti-CD3 injection were paralleled by modifications in progenitor cell frequencies, as illustrated in Figure 3A. The incidence of CFU-C was maximal on day 4, and back to normal on day 9. BFU-E and CFU-Mix were similarly expanded on day 4 post-injection (62.3 ± 15.9 BFU-E and 15.5 ± 5.1 CFU-Mix per 106 spleen cells from anti-CD3-injected mice, versus 7.6 ± 2.5 BFU-E and 3.1 ± 1.5 CFU-Mix per 106 spleen cells from Ig-injected controls; means ± SE from three separate experiments).
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Figure 3. Effect of anti-CD3 injection on the incidence of CFU-C in the spleen (A) and on the capacity of splenocytes to generate histamine in response to a 48-h stimulation with 1 ng/ml of rmIL-3 (B). Data represent means ± SE from three separate experiments.
– – – • – – – control IgG-injected mice
— O — anti-CD3-injected mice
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The rise in clonogenic progenitor frequencies in the spleen coincided with enhanced responsiveness to IL-3, in terms of histamine production (Fig. 3B
) that was not associated with increased mast cell or basophil levels. Furthermore, histamine production was abrogated by -fluoromethylhistidine, a specific inhibitor of histamine synthesis, thus excluding degranulation. It is also noteworthy that spleen cells from anti-CD3-injected mice displayed a transient incapacity to produce histamine in response to IL-3 the day following the injection.
In the same experimental conditions, we detected only minor alterations in the bone marrow. Progenitor frequencies, as well as the capacity to respond to IL-3 in terms of histamine synthesis increase, remained the same after anti-CD-3 injection as in untreated mice (data not shown). Cytological analysis (Table 2) revealed a slight rise in the frequency of immature myeloid cells on day 1 post-injection and a more marked diminution of lymphocyte counts on day 4. The granulocyte compartment was also slightly increased at this time point, while other subsets were not modified by the treatment with anti-CD3.
Injection of F(ab')2 fragments instead of the entire anti-CD3 mAb did not induce any of the hematopoietic modifications described above, even though they were injected at a higher dose (50 µg per mouse). It could therefore be concluded that multivalent crosslinking of the antibody was requisite for these biological effects.
Hematopoietic Changes Induced by i.v. Injection of IL-3 and GM-CSF
To evaluate the part of anti-CD3-induced IL-3 and GM-CSF in the hematopoietic changes observed, we tried to block the activity of these cytokines by injecting their corresponding antisera prior to anti-CD3 treatment. The antibodies used in this study were only effective at high concentrations in vitro (1 mg/ml). They did not neutralize IL-3 and GM-CSF in vivo. For this reason, we injected IL-3 and GM-CSF and compared the hematopoietic changes induced to those resulting from anti-CD3 mAb treatment. On account of the transient growth factor release promoted by the mAb, we limited the treatment to two injections performed on a single day at an interval of 6 h.
We observed a number of qualitative and quantitative differences between the effect of anti-CD3 and that of the two hematopoietic growth factors. In contrast with anti-CD3 mAb, IL-3 and GM-CSF did not modify spleen cell populations on the first day after injection (see uninjected controls in Table 1). On day 4, we observed a significant increase in the proportion of immature myeloid cells and nucleated RBCs, with a preferential expansion of the former population (Table 3). Unlike anti-CD3 mAb, IL-3 and GM-CSF did not increase the number of spleen cells. Growth factor injection did, however, augment the incidence of nonerythroid clonogenic progenitors on day 4, though to a lesser extent than anti-CD3 (Table 4). As shown in the same table, these cells also displayed an enhanced response to IL-3, in terms of histamine synthesis. Injection of IL-3 and GM-CSF had no consistent effect on the number and distribution of bone marrow cells, except for a small increase in myeloid precursors on day 4 (data not shown). The proportion of clonogenic progenitors in this organ also remained unchanged.
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Table 4. Effect of i.v. injection of IL-3 and GM-CSF on histamine production and colony formation by spleen cells
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Discussion
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Antibodies directed against the CD3 complex of the TCR promote, both in vitro and in vivo, a striking T cell activation resulting in the induction of a variety of cytokines which are massively released into the circulation [6-8, 11]. These cytokines include IL-3, thus providing one of the rare experimental situations where substantial amounts of this cytokine become available in serum. Given the potential clinical applications of anti-CD3, this observation led us to examine the hematopoietic changes caused by this treatment [16].
Anti-CD3 mAb injection results in rapid and pronounced hematopoietic modifications in peripheral blood and spleen, while the bone marrow remains relatively unchanged. The dose of anti-CD3 used throughout this study has been previously shown to promote a potent T cell activation but no significant T cell depletion from the spleen [10, 17]. Yet, even in these conditions, we observed a striking fall of blood lymphocyte levels within 24 h after injection. The disappearance of these cells might be explained either by destruction or sequestration following TCR coating [9].
The day after anti-CD3 injection, the percentages of CD4+ and CD8+ subsets in the spleen vary little from those found in control mice. Among the early changes promoted by anti-CD3 injection, a significant increase in neutrophil levels both in peripheral blood and in the spleen occurs on day 1. This phenomenon, like the decrease of circulating lymphocytes, was not mimicked by IL-3 and GM-CSF injection.
Since in vivo treatment of mice with antisera against IL-3 and GM-CSF did not neutralize the corresponding activities in the circulation of anti-CD3-injected mice, we injected the recombinant growth factors to evaluate to what extent they were implicated in the hematopoietic changes observed. The proportion of immature myeloid cells and nucleated RBCs is substantially increased by either treatment, though growth factors expand preferentially myeloid rather than erythroid cells, conversely to anti-CD3. Clonogenic progenitor frequencies are also augmented in both situations. Yet, anti-CD3 is much more effective in this respect since it increases the incidence of CFU-C around 10-fold and nearly doubles the number of spleen cells. At the doses injected, IL-3 and GM-CSF do not change the total number of spleen cells, and clonogenic progenitor frequencies are not increased more than two to three times. The expansion of the immature spleen compartment is accompanied by enhanced responsiveness to IL-3 in terms of histamine synthesis, though this effect is less marked in response to the growth factor combination. This is in accordance with previous results, associating this biological activity with relatively undifferentiated myeloid cells [18].
It is also noteworthy that neither anti-CD3 nor growth factor injection consistently modifies bone marrow cell populations, as previously reported for in vivo experiments with IL-3 [19, 20]. The diminution of lymphocytes in the bone marrow of mice having received anti-CD3 four days before does not occur after injection of IL-3 and GM-CSF, and also has not been documented by other in vivo studies on these factors [20, 21].
One of the characteristic features of IL-3 is its capacity to trigger bone marrow CFU-spleen (CFU-S) into the cell cycle [22, 23]. The higher percentage of dividing stem cells is associated in vivo with elevated numbers of circulating CFU-S [24] and increased frequencies of these early progenitors in spleen and liver. These cells probably achieve their full maturation in extramedullary sites. They may therefore account for the increase of CFU-C, BFU-E, CFU-Mix and morphologically recognizable precursor cells occurring four days after injection of anti-CD3 in the spleen, rather than locally differentiated stem cells.
Even though injection of IL-3 and GM-CSF reproduces most of the hematopoietic changes induced by anti-CD3, the latter is probably directly responsible for the rapid loss of lymphocytes from peripheral blood. The early increase in granulocyte levels in these compartments is also not promoted by growth factor injection, but may be due to other mediators generated in response to anti-CD3. This notion is also consistent with the less striking hematopoietic modifications seen in response to IL-3 and GM-CSF, suggesting that other cytokines released in response to anti-CD3 [7] might amplify the effect of the growth factors.
Given the variety of clinical settings in which anti-CD3 mAb could be of potential use, the striking hematopoietic modifications induced by this treatment deserve consideration. Our observations could be of particular interest in the context of allogeneic bone marrow transplantation, where a beneficial effect of endogenous colony-stimulating factors on engraftment has been documented [25]. In this situation, the protection against allograft rejection by colony-stimulating factors might be a consequence of their capacity to accelerate hematopoietic recovery [26].
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Acknowledgments
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The technical assistance of A. Arnould and F. Machavoine is gratefully acknowledged. This work was supported in part by Grant ARC 6365 from the "Association pour la Recherche contre le Cancer."
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References
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accepted for publication December 6, 1996.
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Abstract
Introduction
