Article history:Received 31 January 2013Received in revised form 20 March 2013Accepted 8 April 2013Available online 16 April 2013
In vivo T cell depletion experiments are widely used to establish the role of these cells in avariety of immunological processes. Different clones of monoclonal antibody targeting the CD3molecular complex (mainly 145-2C11 and 17A2) have been successfully used for T celldepletion. In the present work, we assessed the specificity of monoclonal antibody-mediatedCD3 T cell depletion inmouse peripheral blood.We showed that treatment of BALB/Cmicewithmonoclonal antibodies (clones 145-2C11 and 17A2) not only efficiently depletes T cells in vivo,but also leads to a substantial reduction in B cell, granulocyte and platelet counts. In contrast,T cell depletion using a combination of anti-CD4 and anti-CD8 antibodies was efficient andproduced less deleterious effects on other blood cell populations. Therefore, the resultsobtained from T cell depletion experiments using anti-CD3 antibodies must be interpreted withcaution prior to draw definitive conclusions on the role of T cells in a given immunologicalprocess.
In vivo treatment of mice with monoclonal antibodies(mAb) is commonly used for the depletion of various leu-kocyte subsets from peripheral lymphoid organs and blood,and thereby to analyze their role in immune regulation andtreatment of experimental immunological diseases. Morespecifically, treatment of mice with mAb directed againstcell surface antigens expressed on T cells has been used tomodify the course of immune responses in various systems.For example, it has been possible to block humoral responses(Cobbold et al., 1984;Wofsy et al., 1985b), to induce toleranceto specific antigens (Benjamin and Waldmann, 1986;Chatenoud, 2003; Chatenoud and Bluestone, 2007), to delayallograft rejection (Debure et al., 1987; Kreis et al., 1989;Webster et al., 2006) and to retard experimental autoimmune
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diseases (Wofsy et al., 1985a; Ranges et al., 1987; Vanhove,2009) by depleting T cell populations in experimental animals.One of the most commonly used targets for T cell depletionis the CD3 molecular complex. Treatment of mice with anti-CD3 mAb has been shown to induce a rapid and sustainedreduction of T cell numbers (Hiruma et al., 1992) and to inhibitthe acute cellular but not the humoral rejection in the kidneygrafts of monkeys (Armstrong et al., 1998). Finally, anti-CD3mAb have been used to prolong skin allograft survival and toinhibit cytokine secretion and cytotoxic T cell functions inmice(Alegre et al., 1995).
However, not all mAb are suitable to eliminate theirtargets in vivo.Whether amAb eliminates its target or not hasbeen suggested to depend on the structure of the Fc part of theantibody, on the isotype, on the surface antigenic density andthe antigen modulation or internalization of the antigen–antibody complex (Ledbetter and Herzenberg, 1979; Howardet al., 1982; Cobbold et al., 1984; Kaminski et al., 1986;Countouriotis et al., 2002). In addition, the expression ofcomplement regulatory proteins by the target cell is also ofconsiderable importance (Jurianz et al., 1999). In in vivo studiesinvolving T cell depletion, the extent of specific depletion is
39L. Loubaki et al. / Journal of Immunological Methods 393 (2013) 38–44
usually monitored to confirm the reduction in target cellnumbers. In these experiments, it is assumedbut not confirmedexperimentally that, given the specificity of the mAb used fordepletion (e.g. CD3-specific), the cell populations other than Tcells remain untouched. We herein show that two differentclones of anti-CD3 mAb currently used for depletion not effi-ciently only eliminate the CD3-positive targeted cells in vivo,but unexpectedly also affect CD3-negative populations such asB cells, granulocytes and platelets.
2. Materials and methods
2.1. Animals
Wild-type female BALB/c mice (18–22 g) were obtainedfrom Charles River (Montreal, Canada). Mice were kept at theanimal facility at Laval University (Quebec, Canada) and allprocedures were approved by the Laval University Animal CareCommittee.
2.2. T cell depletion
BALB/c mice were injected intraperitoneally (ip) with 1,20 or 100 μg of anti-CD3 clone 145-2C11 (Armenian hamsterIgG), or 1 μg of anti-CD3 clone 17A2 (rat IgG2b kappa). Bothantibodies were purchased from eBioscience, Inc. (San Diego,CA). In a different set of experiments, BALB/c mice wereinjected ip with 30 μg of anti-CD4 (clone RM4-4, rat IgG2bkappa) and 20 μg of anti-CD8 (clone 53–6.7, rat IgG2a kappa)(both from eBioscience, Inc.) in combination or separately,instead of anti-CD3. Blood samples were recovered 24 h afterantibody injection and a cell suspension was prepared forflow cytometry analysis.
2.3. Flow cytometry analysis
Leukocytes were labeled using the following anti-mouseantibodies: anti-CD45-PE, anti-CD3-Alexa 647 (BDPharmingen,Mississauga, Canada), anti-Gr-1-Alexa 488, anti-B220-APC-efluor 780, anti-CD8-efluor 450 and anti-CD4-PECy7(eBioscience, Inc.). For leukocyte enumeration, 5 μl of bloodwas first incubated with the above antibodies followed byfixation and red cell lysis (CyLyse Erythrocyte Lysing kit, PartecNorth America, Inc., Swedesboro, NJ) in a final volume of800 μl. A volume of 200 μl of each sample was analyzed on aCyFlow ML cytometer (Partec North America, Inc.) to establishthe concentration of each cell population. For platelet and redcell enumeration, 5 μl of each blood sample was first labeledwith anti-CD61-PE (eBioscience, Inc.) followed by dilution(1/20,000) prior to analysis (30 μl) by flow cytometry.Platelets were gated according to PE fluorescence while redcells were gated based on the FSC/SSC profile.
2.4. Statistical analyses
All statistical analyses were performed using the GraphPadInStat software (GraphPad Software, La Jolla, CA). Values ofP b 0.05 were considered to indicate statistical significance.
3. Results
3.1. The anti-CD3 clone 145-2C11 induces the depletion ofB cells and granulocytes
To deplete T cells in mice, we first tested the anti-CD3 clone145-2C11 using protocols described by several groups ofinvestigators (Mysliwietz and Thierfelder, 1992; Alegre et al.,1995; Alvarez et al., 2004; van der Fits et al., 2009). Before andtwenty-four hours after antibody injection, blood samples weretaken and analyzed by flow cytometry to confirm T cell deple-tion. Cells were first labeled with CD45 to gate on leukocytes.The side scatter (SSC, granularity)/CD45 profile obtained fromthe same mouse, before and after injection of 100 μg of145-2C11 mAb is shown on Fig. 1A. The reduction in lymphoidcell populations was evident, but curiously, cells with highgranularity (mostly corresponding to granulocytes) were alsoapparently decreased (Fig. 1A). This prompted us to analyze themain blood cell populations (T, B cells and granulocytes) usingCD3 (clone 17A2), CD45R (B220) and Ly6G (Gr-1)-specificmAbs respectively, before and after injection of different dosesof 145-2C11 mAb. The results showed that clone 145-2C11efficiently depleted T cells from the bloodof BALB/cmice (Fig. 1Band C), even in animals injected with the very low dose of 1 μg.The 20 μg and 100 μg doses of 145-2C11 led to an even moreextensive T cell depletion (Fig. 1C). The analysis of the CD4 andCD8 subsets using CD4- and CD8-specific antibodies (clonesRM4-4 and 53–6.7 respectively) confirmed the depletion of bothsubsets of T cells (data not shown). However, the analysis ofB220+ cells revealed a significant reduction in the B cell pop-ulation in the same animals (Fig. 1B and C). Indeed, B cell countswere decreased by the same magnitude as that of T cells, atthe three doses of 145-2C11 tested. Similarly, the granulocytepopulation (Gr-1+) was decreased following 145-2C11 injec-tion (Fig. 1B and C), albeit to a lesser extent.
3.2. The anti-CD3 clone 17A2 also induces a pan-leukocytedepletion
To determine whether the bystander effect observed on Bcell populations during T cell depletion was restricted to theanti-CD3 clone 145-2C11, we performed depletion experi-ments using another commonly used clone of anti-CD3 (clone17A2) (Mysliwietz and Thierfelder, 1992; ten Hagen et al.,1998). We first determined the minimal dose necessary toinduce more than 90% T cell depletion (1 μg), to minimizepotential bystander effects on other blood cell populations.Micewere thus injectedwith 1 μg of 17A2 and the extent of celldepletionwas evaluated in blood samples 24 h later.We foundthat, similarly to 145-2C11, the anti-CD3 17A2 led to an exten-sive T cell depletion that was associated with a significant re-duction in the B cell population (Fig. 2). In addition, a statisticallysignificant two-fold decrease in granulocyte counts was ob-served (Fig. 2). Altogether, our results show that two commonlyused clones of anti-CD3 not only efficiently deplete T cells, butalso induce the depletion of other leukocyte populations.
3.3. Anti-CD3 injection induces a reduction in platelet counts
To gain more insight into the non-specific effect ofanti-CD3 treatment on blood cell populations, we performed
Fig. 1. Effect of 145-2C11 mAb treatment on blood cell populations of BALB/c mice. (A) Representative flow cytometry profile of blood leukocytes (CD45+ cells)from BALB/c mice before (left panel) or after (right panel) treatment with 100 μg of 145-2C11 mAb. Cells were labeled with anti-CD45-PE prior to analysis.(B) Analysis of the different leukocyte populations in the blood of BALB/c mice before (left panel) or after (right panel) treatment with 100 μg of 145-2C11 mAb.Cells were labeled with anti-CD3-Alexa 647 and anti-B220-APC-efluor 780 prior to analysis by flow cytometry. The major cell populations are shown (T, T cells;B, B cells, Gran, granulocytes). (C) Leukocyte counts in mice prior to (NT) or 24 h after the injection of different doses (1, 20 or 100 μg) of 145-2C11 mAb. T andB cell counts were established by analysis of 200 μl of a cell suspension labeled and gated as in (B); granulocytes counts were determined using cells labeled withanti-Gr-1-Alexa 488. ns, not significant; *P b 0.05; **P b 0.01; ***P b 0.001, ANOVA with Bonferroni post-test, n = 3–5 mice per group.
40 L. Loubaki et al. / Journal of Immunological Methods 393 (2013) 38–44
Fig. 2. Effect of 17A2 mAb treatment on blood leukocytes. Leukocyte countsin mice before or 24 h after treatment with 1 μg of 17A2 mAb. Counts wereestablished as described in Fig. 1C. **P b 0.01; ***P b 0.001, Mann–Whitney,n = 3–5 mice per group.
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platelet and red cell counts on blood samples recoveredbefore and after anti-CD3 injection. The results showed that,in addition to its effect on B cells and granulocytes, anti-CD3also reduced the platelet numbers in mice injected with145-2C11 (Fig. 3) or 17A2 (data not shown). The reductionwas apparent, although not statistically significant at the 1 μgdose, and reached about 50% with either the 20 μg or 100 μgdose. In contrast, the red cell counts before and after anti-CD3treatment remained constant at all doses, in all animals(10.0 ± 1.3 vs 9.9 ± 0.9 × 106/μl before and after 145-2C11injection respectively, or 10.1 ± 0.1 106/μl after 17A2 injection).
Fig. 3. Effect of 145-2C11mAb treatment on blood platelets. Platelet enumer-ation in the blood of BALB/c mice was done prior to (NT) or 24 h after theinjection of different doses of 145-2C11 mAb (1, 20 or 100 μg). Plateletcounts were established following analysis of a volume of 30 μl of a 1/20,000dilution of blood samples labeledwith anti-CD61-PE, by flow cytomet. Plateletswere gated according to PE fluorescence. ns, not significant; *P b 0.05;**P b 0.01, Kruskal–Wallis with Dunn post-test, n = 3–5 mice per group.
3.4. Monoclonal anti-CD4 and anti-CD8 induce a more specificT cell depletion
In an attempt to achieve T cell depletion without affectingother cell populations, we tested mAb directed towards theCD4 and CD8 cell surface molecules. BALB/c mice were in-jected with anti-CD4, anti-CD8 or a combination of both. Thedose of each antibody used was optimized in preliminaryexperiments and was set to 30 μg for anti-CD4 and 20 μg foranti-CD8. The different blood cell populations (T, B cells,granulocytes, platelets and red cells) were monitored by flowcytometry 24 h after injection, as before. The results ob-tained showed the specific reduction in CD4+ or CD8+ Tcells, in animals injectedwith anti-CD4 (Fig. 4A) and anti-CD8(Fig. 4B) respectively, although a small reduction in CD4+ Tcell numbers was observed in anti-CD8-treated mice (P =0.0447, paired T test). As expected, a corresponding reductionin CD3+T cell numberswas observed following anti-CD4 andanti-CD8 treatments. The other blood cell populations (B cells,granulocytes, platelets and red cells) were not significantlydecreased (Fig. 4A and B). However, there was a tendencytowards reduction in B cell and granulocyte counts, especiallyin mice injected with the anti-CD8 mAb (Fig. 4B). When micewere injected with a combination of both anti-CD4 and anti-CD8, both T cell subsets as well as the CD3+ T cell populationwere significantly depleted (Fig. 4C). There was also a sig-nificant reduction in B cell and granulocyte counts whereasthe platelet population remained untouched.
4. Discussion
In this report, we show that T cell depletion induced byCD3-specific mAb is associated with a nonspecific but signifi-cant depletion of B cells, granulocytes and platelets. We alsoshow that the T cell depletion induced by anti-CD4 oranti-CD8 antibodies alone is relatively specific for their re-spective T cell subset. The combination of anti-CD4 andanti-CD8 permits to deplete CD3+ T cells as efficiently asanti-CD3, with less dramatic effects on other cell populations.These observations were made in blood samples taken 24 hafter antibody injection, but similar observations were alsomade in blood samples taken 48 h after injection (data notshown).
The nonspecific depletion of cell populations other than Tcells observed in the present work is unexpected, since themAb used are very specific for their targets, as confirmed byflow cytometry. Depletion of cells other than T cells must thusoccur as a secondary event in response to non-specific cyto-toxic effects induced during T cell depletion rather than by adirect interaction with the injected mAb. Whether thedecrease in non-T cell populations in the blood of treatedmice is the result of true depletion or cell sequestration inlymphoid or other organs is currently unknown. Examinationof splenic cells recovered at sacrifice (48 h after antibodyinjection) showed normal B cell and granulocyte counts (datanot shown), in contrast to their significantly reduced numbersin blood. This suggests that true depletion occurs in the bloodas a bystander cytotoxic effect of T cell depletion rather thansequestration in the spleen. However additional experimentsare required to definitively conclude on that matter.
Fig. 4. Effect of anti-CD4 and anti-CD8 treatment on blood cell populations of BALB/c mice. (A) Leukocyte and platelet counts in BALB/c mice, before (black bars)and after (white bars) injection of 30 μg of anti-CD4. Cell counts were determined as described in Figs. 1C and 3. (B) Leukocyte and platelet counts in BALB/c mice,before (black bars) and after (white bars) injection of 20 μg of anti-CD8. (C) Leukocyte and platelet counts in BALB/c mice, before (black bars) and after (whitebars) injection of a combination of 30 μg of anti-CD4 and 20 μg of anti-CD8. ns, not significant; *P b 0.05; **P b 0.01; ***P b 0.001, paired T test, n = 3 mice pergroup. Data are representative of 2 separate experiments.
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Actually, several mechanisms have been proposed to ex-plain mAb-mediated cell depletion. A number of studies haveshown that therapeutic mAb are able to induce complement-dependent cytotoxicity (CDC) and antibody-dependent cell-
mediated cytotoxicity (ADCC) (Raasveld et al., 1993; Iannelloand Ahmad, 2005; Weiner, 2007). Therapeutic mAb can alsoinduce apoptosis through an activation induced cell death(AICD) pathway, cell-cycle arrest, or a combination of these
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mechanisms to deplete target cells have also been reported(Janssen et al., 1992; Chatenoud, 2003; Villamor et al., 2003;Olszewski and Grossbard, 2004; Lowenstein et al., 2006).Considering these mechanisms, we tried to explain the non-specific deleterious effects of anti-CD3 on other blood cellpopulations. For example, administration of anti-CD3 mAb inboth humans (OKT3) and mice (145-2C11) have been asso-ciated with a massive systemic release of cytokines includingIFN-γ, IL-2, IL-4, IL-6, CSF and TNF-α and a concomitantgeneration of complement system components such as C4d,C3a, C5a and SC5b-9 (Alegre et al., 1991; Raasveld et al., 1993;Alegre et al., 1995; Vallhonrat et al., 1999). Cytokines such asTNF-α and INF-γ, and complement C5a have been shown toincrease the surface expression of leukocyte's adhesionmole-cules such as VCAM-1 or integrin β2 family member, thusincreasing the adhesiveness of leucocytes to the endothelialcells and then favoring their trapping in endothelial lining(Thornhill et al., 1991; Kinnaert et al., 1996). In addition,Raasvelt et al. reported a peripheral blood granulocytopeniawhich was associated with a pulmonary sequestration ofactivated granulocytes in the lungs, immediately followingthe first dose of OKT3 (Raasveld et al., 1993). Therefore, thedecrease in B cells and granulocytes observed may be due tothe trapping of these cells in the blood vessels or lungs fol-lowing the increase in cell surface adhesion molecules.
On the other hand, complement alternative pathway com-ponents such as Bb and SC5b-9 membrane attack complexhave been shown to be increased in patients treatedwith OKT3(Vallhonrat et al., 1999). It is thus possible that administrationof 17A2 and 145-2C11 anti-CD3 in mice induces CDC whichcould lead to the nonspecific reduction of both B cells andgranulocyte counts through their destruction by the comple-ment alternative pathway. Another potential mechanism for Bcell and granulocyte depletion by anti-CD3 could be related tothe release of granzyme and lysozyme during an exceedinglyvigorous ADCC induced following interaction of the Fc part ofthe anti-CD3 with FcγR, as observed by Weng et al. duringrituximab-induced neutropenia (Weng et al., 2010).
The platelet numbers in anti-CD3-treated mice were alsosignificantly reduced. Such reduction may be related to theinduction of cytokine release and complement system acti-vation following anti-CD3 administration, asmentioned above.These two phenomena have been shown to affect the coagu-lation process (Alegre et al., 1991; Raasveld et al., 1993). Indeed,it has been reported that plasma thrombin/antithrombin-IIIcomplex, tissue-type plasminogen-activator and plasmin-alpha2/antiplasmin complex levels were increased as compared topre-treatment levels in OKT3-treated patients, suggesting theactivation of coagulation and fibrinolysis by OKT3 (Raasveldet al., 1992). Abramovicz et al. showed that the OKT3 procoag-ulant effect could also be secondary to TNF-α activation, sinceTNF-α stimulates the synthesis by monocytes and endothelialcells of platelet-activating factor (PAF), themost potent inducerof platelet aggregation (Tijburg et al., 1991; Abramowicz et al.,1992; Okajima, 2001). Furthermore, TNF-α and activated com-plement system components can upregulate the expression ofthe highly thrombogenic tissue factor that plays a central role inthe extrinsic coagulation pathway (Saadi et al., 1995; Taylor etal., 1996; Okajima, 2001).
In contrast to the depletion induced by CD3-specific mAb,T cell depletion using anti-CD4 and anti-CD8 alone or in
combination led to a significant depletion of the targeted Tcell population with fewer side-effects on other blood cellpopulations. This difference may be ascribed to the mainmode of action of these mAbs, which is likely to differ fromthat of anti-CD3. Indeed, cell coating is the main mode actionof anti-CD4 mAb (Bank and Chess, 1985). T cell coating notonly prevents cell-to-cell interactions, but also elicits negativesignals for which the molecular basis remains to be fullyelucidated (Bank and Chess, 1985; Jabado et al., 1997). Moreprecisely, it has been shown that anti-CD4 mAb can transmitnegative signals to T cells and induce their depletion throughan AICD-dependent mechanism (Newell et al., 1990). Al-though the main mode of action of anti-CD8 mAb is not welldescribed, it probably differs from that of anti-CD4, given thatB cells are more affected in anti-CD8 treated animals. It hasbeen reported that anti-CD8 mAb can induce T cell apoptosisby increasing the expression and phosphorylation of LAT(linker for activation of T cells), the predominant target oftyrosine phosphorylation during T cell activation required forapoptosis induction (Grebe et al., 2004; Clarke et al., 2009).However, is it likely that different combinations of several ofthe mechanisms described above are responsible for thenonspecific depletion of blood cell populations other than Tcells, following treatment with T cell-specific mAb. Interest-ingly, splenic T cells of mice sacrificed 48 h after antibodyinjection were more extensively depleted with the blend ofanti-CD4/anti-CD8 (>80% depletion) compared to anti-CD3(145-2C11 at 100 μg, about 50% depletion) (data not shown).This suggests that the anti-CD4/anti-CD8 cocktail is morepotent than anti-CD3 to deplete both blood and splenic T cells.
Our study raises serious concerns about the interpretationof results obtained from experiments involving T cell deple-tion in mice. For example, anti-mouse CD3 treatment (17A2,doses ranging from 25 μg to 400 μg per mouse) has beenshown to suppress chronic graft-versus-host disease (GVHD)in a mouse model, revealing a critical role for T cells in thisprocess (Mysliwietz and Thierfelder, 1992). However, in thelight of the results presented herein, it is not possible to ruleout the possibility that B cell depletion could have also playeda prominent role in GVHD attenuation, since B cells weremostlikely totally depleted from the anti-CD3-treated animals.Indeed, treatment with rituximab, a chimeric anti-CD20-specific mAb which specifically depletes human B cells, hasbeen associated with a reduced incidence and severity ofacute GVHD in treated patients (Shimabukuro-Vornhagenet al., 2009). In conclusion, studies involving T cell depletionwith T cell-specific mAb should include a careful evaluation ofother blood cell populations prior to draw conclusions on therole of T cells in any given biological process.
Acknowledgments
The authors thank Pascal Rouleau for the excellent technicalassistance.
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