Anti-CD3 Therapy Promotes Tolerance by Selectively Depleting Pathogenic Cells while Preserving Regulatory T Cells

Cristina Penaranda, Qizhi Tang and Jeffrey A. Bluestone
J Immunol August 15, 2011, 187 (4) 2015-2022; DOI: https://doi.org/10.4049/jimmunol.1100713

Abstract

Monoclonal anti-CD3 Abs have been used clinically for two decades to reverse steroid-resistant acute graft rejection. In autoimmune diabetes, short course treatment with FcR-nonbinding (FNB) anti-CD3 mAb in mice with recent onset of diabetes induces long-term disease remission. Induction of tolerogenic regulatory T cells (Tregs) has been implicated to be one of the mechanisms of action by FNB anti-CD3 mAb in these settings. In this study, we examined the effect of FNB anti-CD3 mAb treatment on the homeostasis of naive, effector, and regulatory T cells in vivo. Anti-CD3 treatment induced a transient systemic rise in the percentage but not absolute number of CD4+Foxp3+ Tregs due to selective depletion of CD4+Foxp3− conventional T cells. T cell depletion induced by FNB anti-CD3 mAb was independent of the proapoptotic proteins Fas, caspase-3, and Bim and was not inhibited by overexpression of the anti-apoptotic protein, Bcl-2. Tregs were not preferentially expanded and we found no evidence of conversion of conventional T cells into Tregs, suggesting that the pre-existing Tregs are resistant to anti-CD3–induced cell death. Interestingly, expression of the transcription factor Helios, which is expressed by thymus-derived natural Tregs, was increased in Tregs after FNB anti-CD3 mAb treatment, suggesting that the anti-CD3 treatment can alter, and potentially stabilize, Treg function. Taken together, the results suggest that FNB anti-CD3 therapy promotes tolerance by restoring the balance between pathogenic and regulatory T cells.

Immunotherapy targeting CD3 molecules has shown tremendous promise for the treatment of autoimmune diseases and the prevention of allograft rejection in humans and in murine preclinical models (1). In patients, the anti-CD3 mAb OKT3 was described as an efficient treatment to prevent acute renal allograft rejection >20 y ago (2). However, the development of OKT3 and other anti-CD3 mAb therapeutics was hampered by serious side-effects associated with a “cytokine storm” released as a consequence of generalized T cell activation (3, 4). T cell activation by anti-CD3 mAb depends on immobilization of the mAb on APCs via FcRs and crosslinking of TCR/CD3 complexes at the interface of T cells and APCs (5). Thus, various forms of FcR-nonbinding (FNB) anti-CD3 mAb were developed to avoid cytokine storm elicited by multivalent crosslinking of TCR/CD3. Remarkably, FNB anti-CD3 mAbs retained their immunosuppressive properties in preclinical models in vivo without the toxicity associated with the parental anti-CD3 mAb, and they demonstrated clinical efficacy in the treatment of acute renal allograft rejection (6–9).

Despite the great promises of FNB anti-CD3 mAb treatment in autoimmunity and transplantation, the mechanisms of action of these reagents in vivo are still not clearly defined. Previous data from our laboratory has shown that FNB anti-CD3 mAbs are not passive blockers of TCR/MHC interactions but instead induce partial phosphorylation of the CD3ζ- and CD3ε-chains and inefficient recruitment and phosphorylation of CD3-associated kinase ZAP70 (10). As a consequence, downstream activation of PLCγ, calcium flux and NFATc nuclear translocation, and MAPK phosphorylation were reduced when compared with that induced by multivalent anti-CD3 mAb engagement (10, 11). These in vitro observations correlated well with experiences in human patients treated with FNB anti-CD3. Although both FcR-binding and FNB anti-CD3 mAb induced transient T cell depletion in NOD mice and patients (12), the depletion was more complete with FcR-binding anti-CD3 mAb (13).

CD4+Foxp3+ regulatory T cells (Tregs) can suppress effector T cell (Teff) responses in vitro and in vivo and they are crucial for the maintenance of peripheral tolerance and the prevention of autoimmunity (14, 15). Thus, it is not surprising that recent studies have suggested that rather than altering Teffs, FNB anti-CD3 mAb treatment results in the generation of Tregs (16–18). However, these conclusions have been controversial (19, 20), as potential changes in the relative frequency of Teffs and Tregs following FNB anti-CD3 mAb treatment, as well as the potential for T cell migration out of the blood and secondary lymphoid tissues, have made it difficult to evaluate the direct effect of the therapy on each cell subset.

In this study, we examined the influence of FNB anti-CD3 mAb on conventional versus regulatory T cells. We demonstrate that although FNB anti-CD3 mAb induced an increase in the relative percentage of Tregs, this process was not due to de novo generation or expansion of Tregs. Instead, the increased Treg/Teff ratio was due to preferential depletion of conventional T cells in vivo through Fas- and caspase-3–independent pathways. Furthermore, FNB anti-CD3 mAb treatment led to increased expression of Helios in Tregs, suggesting stabilization of Tregs, which may account for the protracted efficacy of the drug.

Materials and Methods

Mice

BALB/c and C57BL/6 (B6) mice were purchased from Charles River Laboratories (Wilmington, MA), NOD mice were purchased form Taconic (Germantown, NY), and B6.caspase-3–deficient mice were purchased from The Jackson Laboratory (Bar Harbor, ME). DO11.10 TCR transgenic mice, B6.Bcl-2 transgenic mice (a gift from Marisa Alegre), B6.Bim-deficient mice, Fas-deficient BALB/c.lpr/lpr mice, BALB/c.FasL-deficient gld/gld mice, NOD.Foxp3.GFP-Cre, and NOD.Foxp3.GFP-Cre × Rosa26.flox.stop.YFP were bred at our facility. All mice were housed in a specific pathogen-free facility at the University of California at San Francisco. All experiments complied with the Animal Welfare Act and the National Institutes of Health guidelines for the ethical care and use of animals in biomedical research and were approved by the Institutional Animal Care and Use Committee of the University of California at San Francisco.

Abs and other reagents

FNB mouse-specific anti-CD3 mAb, 145-2C11-γ3 (2C11-IgG3), was produced in our laboratory. Another FNB mouse-specific anti-CD3 mAb-producing cell line, 145-2C11-IgG2a-Ala-Ala, was provided as a gift from Centocor/Johnson & Johnson and the Ab was produced in our laboratory. Anti-FcR mAb 2.4G2 (University of California at San Francisco Cell Culture Facility, San Francisco, CA), mitogenic hamster anti-CD3 mAb 145-2C11 (BioLegend), anti-CD4 mAb RM4-5 (eBioscience), anti-CD8 mAb 53-6.7 (SouthernBiotech); anti-CD25 mAb PC61 (eBioscience), anti-Foxp3 mAb FJK-16 (eBioscience), anti-Thy1.1 mAb OX-7 (BioLegend), anti–PD-1 mAb J43 (eBioscience), anti–Neuropilin-1 polyclonal Abs (R&D Systems), and anti-Helios mAb 22F6 (BioLegend) were purchased. CFSE was purchased from Molecular Probes (Eugene, OR). FTY720, provided as a gift by Novartis Pharmaceuticals (St. Louis, MO), was administered daily i.p. at a dose of 20 μg/day. EasySep mouse CD4 T cell enrichment kit was purchased from StemCell Technologies (Vancouver, BC, Canada). Mouse IgG whole molecule from Rockland Immunochemicals (Gilbertsville, PA), a gift from Amplimmune (Rockville, MD), was used as a control.

Flow cytometry and cell sorting

Single-cell suspensions were prepared from the spleen and lymph node (LN) of indicated mice using standard procedures. For flow cytometry, cells were stained for 20–30 min on ice in staining buffer (2% FCS and 0.01% sodium azide in PBS). For cell sorting, cells were stained and washed in medium containing 2% FCS and sorted on a MoFlo cytometer (Beckman Coulter, Miami, FL) to >95% purity. Flow cytometric analyses were performed on a BD LSRII flow cytometer with FACSDiva software (BD Pharmingen, San Jose, CA).

Adoptive transfer experiments and in vivo treatments

Unless otherwise indicated, wild-type (WT) NOD or BALB/c mice between 8 and 20 wk age were treated i.v. with 10 μg FNB anti-CD3 mAb daily for 5 consecutive days, and control mice received whole mouse Ig using the same regimen. For adoptive transfer experiments, 9–10 × 106 sorted CD4+Foxp3−GFP− cells, 12 × 106 CFSE-labeled CD4+ T cells or 0.4–0.5 × 106 sorted PD-1−Nrp1− (Helioslo/−) or PD-1+Nrp1+ (Helioshi) Tregs were transferred into syngeneic recipients via retro-orbital injection on day 0. On days 1–5, recipients were treated with FNB anti-CD3 mAb or control Ig as described above. Eight days after adoptive transfer, T cell proliferation was examined by flow cytometry. For analysis of transferred Helioslo/− or Helioshi Tregs, Thy1.1+ cells were enriched prior to flow cytometric analysis: cells from all LNs and spleen were harvested and incubated with 2 μg/ml Thy1.1-allophycocyanin in 200 μl for 30 min at 4°C. Cells were washed, resuspended in 200 μl, and incubated with 50 μl anti-allophycocyanin magnetic beads (Miltenyi Biotec, Bergisch Gladbach, Germany) for 30 min at 4°C. Cells were washed and cell suspension was passed though a MACS LS separation column (Miltenyi Biotec) to obtain a Thy1.1-enriched fraction, which was used for flow cytometric analysis.

PBL counts were obtained using a Hemavet 950 instrument (Drew Scientific, Dallas, TX). To determine trafficking into peripheral tissues, mice were treated for 5 consecutive days with 20 μg FNB anti-CD3 mAb and harvested 1 d after treatment. Mice were perfused using PBS before harvesting spleen, peripheral LNs, bone marrow, lungs, liver, small intestine, and colon. Bone marrow isolated from femurs and tibias was dissociated using mechanical disruption by passing through 23-gauge needles repeatedly. Lung tissues were digested with a mixture of collagenase and DNase. Livers were initially minced into single-cell suspensions using syringe plungers, and leukocytes were enriched using Percoll density gradient centrifugation. The intestinal tracts were washed extensively with PBS to remove fecal content and mucus coating, and the tissue was then cut into small pieces and digested using a mixture of collagenase and DNase to make single-cell suspensions. The total cell counts from each organ and percentages of CD4+ and CD8+ T cells were then used to calculate the numbers of CD4+ and CD8+ T cells present in that organ.

Cell death assays

DMEM/GlutaMAX medium (University of California at San Francisco Cell Culture Facility) supplemented with 5% heat-inactivated FCS (Summit Biotechnology, Ft. Collins, CO), 100 U/ml penicillin, 100 U/ml streptomycin, nonessential amino acids, 10 mM HEPES (University of California at San Francisco Cell Culture Facility), and 50 μM 2-ME (Bio-Rad, Hercules, CA) was used for cell culture. To prepare Th1 effector T cells, single-cell suspensions were prepared from the spleen and LN of DO11.10 mice. DO11.10 cells were stimulated with 0.1 μg/ml ova peptide in the presence of 400 ng/ml IFN-γ and 25 μg/ml anti–IL-4 for 7–10 d, and medium was supplemented with 20 U/ml recombinant human IL-2 on days 4–5 after the initiation of the culture. The cells were restimulated with 0.1 μg/ml ova peptide without added IFN-γ and anti–IL-4 (clone 11B11). The DO11.10 cells were harvested 1 wk later and adoptively transferred or restimulated with FNB anti–CD3-IgG3 overnight and the numbers of viable cells were determined using flow cytometry after anti–Thy-1 and propidium iodide staining.

For measurement of cell death after in vivo treatment, mice were treated with two 10-μg doses FNB anti-CD3 mAb or control Ig 24 h apart and harvested 38 h after the first injections. Single-cell suspensions from LNs were incubated in vitro in media only, or in the presence of 1 ng/ml rIL-2 (eBioscience, San Diego, CA) or 10 ng/ml rIL-7 (PeproTech, Rock Hill, NJ). Cultures were harvest at 26 or 48 h and cell death was assessed by DAPI (Invitrogen, Carlsbad, CA) inclusion by flow cytometry.

Statistical analysis

The statistical significance of differences between groups in Fig. 1 was determined by the two-tailed Student t test using Excel software. The statistical significance of differences between groups in all other figures was determined by the Mann–Whitney U test using Prism software.

Results

FNB anti-CD3 mAb preferentially induces cell death of CD4+Foxp3− cells in vivo

In agreement with previous reports (13), 10 μg/d × 5 d FNB anti-CD3 mAb treatment induced a transient decrease in the percentage of CD4+ T cells and an increase in the percentage of CD4+Foxp3+ Tregs in secondary lymphoid organs (Fig. 1A, 1B). Although the absolute CD4+ cell numbers decreased (Fig. 1C) mirroring the percentage decrease, the absolute CD4+Foxp3+ cell numbers decreased (Fig. 1D) in contrast to the percentage increase. The decrease in CD4+Foxp3+ Treg numbers was not as pronounced as that observed for the total CD4+ T cell population. These results suggest that the FNB anti-CD3 mAb treatment may not induce the expansion of endogenous Tregs, but that it transiently alters the proportion of Tregs due to a selective loss of CD4+Foxp3− cells in secondary lymphoid organs.

FIGURE 1.
FIGURE 1.

FNB anti-CD3 mAb increases the percentage but not the absolute number of CD4+Foxp3+ T cells. Mice were treated with FNB anti-CD3 mAb (▪) or control Ig (○) and peripheral LNs were harvested at the indicated times. Total cell numbers were determined and T cells subsets were analyzed by flow cytometry. Data shown are normalized to the Ig-treated age-matched mice that were harvested on the same day to control for changes in cell number due to age. A, Normalized percentage of CD4+ cells within lymphocyte gate. B, Normalized percentage of Foxp3+ cells within the CD4+ gate. C, Normalized total CD4+ cell number. D, Normalized total CD4+Foxp3+ cell number. Each datum point represents an individual mouse. Data are representative of two independent experiments.

Previous reports have demonstrated that FNB anti-CD3 treatment is particularly effective at controlling autoreactive T cells at the time of diabetes onset in NOD mice (13, 18). In that setting, many autoreactive T cells are activated Teffs. To determine the relative sensitivity of recently activated effector versus naive T cells to FNB anti-CD3 mAb-induced depletion in vivo, we compared the rate of depletion of endogenous naive T cells and adoptively transferred activated Teffs after FNB anti-CD3 mAb treatment. The Th1 Teffs were generated in vitro by activating T cells from DO11.10 TCR transgenic mice with their cognate peptide under Th1 skewing conditions. FNB anti-CD3 treatment induced 12–37% reduction in the total number of endogenous splenic T cells (Table I). In the same animals, depletion of transferred activated Th1 Teffs was consistently greater (40–66%) than that of the endogenous naive cells (Table I). Thus, taking all of our data together, T cells showed a hierarchy in their sensitivity to FNB anti-CD3 mAb-induced depletion: recently activated Teffs are the most sensitive, followed by naive T cells, whereas Tregs are the most resistant.

View this table:
Table I. Rate of FNB anti-CD3 mAb depletion of naive and Th1 effector CD4 cells in vivo

The loss of CD4+ cells after FNB anti-CD3 mAb treatment could result from preferential migration out of LNs or depletion of these cells. First, analysis of T cell counts in other tissues such as bone marrow, intestine, liver, and lung in mice treated with FNB anti-CD3 mAb revealed no significant increase in CD4+ or CD8+ T cells that could account for the loss of these cells from the secondary lymphoid organs (data not shown). To examine the possibility of T cell migration more directly, mice were treated with FNB anti-CD3 mAb under the cover of FTY720 to block exit of T cells from LNs (21) and the number of CD4+ T cells and percentage of Tregs were examined in LNs. FTY720 treatment led to a significant drop in the lymphocyte count in the peripheral blood, demonstrating that LN exit was blocked (data not shown). FNB anti-CD3 mAb induced a similar drop in the CD4+ T cell count and an increase in the percentage of Tregs in the FTY720- and control-treated mice (Fig. 2A and data not shown). Thus, the selective loss of CD4+Foxp3− cells after FNB anti-CD3 mAb treatment was not due to their preferential trafficking out of LNs, suggesting that the relative increase of CD4+Foxp3+ compared with CD4+Foxp3− was due to preferential depletion of CD4+Foxp3− cells.

FIGURE 2.
FIGURE 2.

Depletion of T cells induced by FNB anti-CD3 mAb is due to cell death in vivo. A, Mice were treated with FTY720 daily on days 1–8 to block lymphocytes from exiting LNs and with FNB anti-CD3 mAb on days 2–6. Percentage of CD4+ T cells in LN of saline- and FTY720-treated NOD mice were analyzed on day 9. Individual mice from two independent experiments are shown. B–E, Mice were treated twice with FNB anti-CD3 mAb 24 h apart. Thirty-eight hours after the first injection, cells from LNs were harvested and cultured in vitro for the times specified. B, Representative plots gated on CD4+ cells showing viability measured by DAPI uptake at the indicated times after incubation in media only. C, Viability of cells cultured in media only, or media supplemented with (D) 1 ng/ml IL-2 or (E) 10 ng/ml IL-7. Data shown represent average of five to six mice pooled from at least two independent experiments. Bars represent SD between samples.

Mechanism of FNB anti-CD3 mAb-mediated cell death

To measure cell death caused by in vivo treatment with FNB anti-CD3 mAb, cells harvested from mice that had been treated with FNB anti-CD3 mAb in vivo were incubated in vitro. Cells harvested from FNB anti-CD3 mAb-treated mice showed increased cell death, as measured by DAPI uptake, compared with mice treated with control Ab (Fig. 2B, 2C). Cell death was not prevented by addition of IL-2 or IL-7 (Fig. 2D, 2E), suggesting that FNB anti-CD3 mAb-induced cell death was not a consequence of cytokine deprivation in vitro and resulted from an apoptotic program that was initiated by FNB anti-CD3 mAb in vivo.

Typically, T cell death can be induced by either cell surface death receptors such as Fas and TNF-αRI or mitochondrial cell death effector molecules such as Bim (22, 23). T cells deficient in Fas ligand (gld) or Fas (lpr) were resistant to FNB anti-CD3 mAb-induced cell death in vitro (Fig. 3A and data not shown). However, lpr mice treated with FNB anti-CD3 mAb showed a similar level of T cell depletion as in WT mice (Fig. 3B), suggesting that the in vivo mechanism of cell death was distinct from that seen in vitro.

FIGURE 3.
FIGURE 3.

FNB anti-CD3 mAb induces T cell death via distinct mechanisms in vitro and in vivo. A, Th1 cell lines generated from WT or Fas ligand-deficient gld DO11.10 TCR transgenic mice were exposed to a titrated dose of FNB anti-CD3 mAb in vitro and the number of viable cells, after overnight culture, was determined by flow cytometry by counting CD4+annexin−propidium iodide− cells. B–D, WT, lpr, Bim-deficient, or Bcl-2 transgenic mice were treated with FNB anti-CD3 mAb or PBS as control and the relative number or percentage of CD4+ cells in the LNs was determined 1 wk after the initiation of the treatment. E, Caspase-3 deficient mice were treated with control Ig or FNB anti-CD3 and the percentage of CD4+ cells in LNs was determined by flow cytometry on day 7. Data shown are a summary of at least two independent experiments.

Previous studies by our group and others showed that in vivo depletion of naive T cells by the classical anti-CD3 mAb (clone 145-2C11) or by superantigen is mediated by the proapoptotic protein Bim and blocked by overexpression of the anti-apoptotic proteins Bcl-2 or Bcl-xL (24, 25). We investigated whether depletion of T cells in vivo by FNB anti-CD3 mAb was mediated by a similar mechanism. CD4+ T cells were depleted to a similar extent in Bim-deficient and WT mice (Fig. 3C). This depletion resulted in a similar rise in the percentages of Tregs as observed in earlier experiments (data not shown). It was possible that other Bim-related mitochondrial proapoptotic proteins, such as Bad and Bax, mediated FNB anti-CD3 mAb-induced T cell death. Cell death mediated by these proteins is blocked by overexpression of the anti-apoptotic protein Bcl-2 (26). To determine potential roles of other mitochondrial proapoptotic factors in FNB anti-CD3 mAb-induced T cell depletion, T cell numbers in WT and Bcl-2 transgenic mice were compared after FNB anti-CD3 mAb treatment. The results showed that CD4+ T cell depletion was similar in WT and Bcl-2 transgenic mice (Fig. 3D). Finally, we analyzed the role of caspase-3, which is the downstream caspase activated by both intrinsic and extrinsic apoptosis pathways and shown to be required for anti-CD3 mAb-induced death of T cells in vitro (27). Treatment of caspase-3–deficient mice with FNB anti-CD3 mAb resulted in depletion of CD4+ T cells in the LNs similar to that seen in WT mice (Fig. 3E).

FNB anti-CD3 mAb does not promote the conversion or expansion of Tregs

Our results show that one of the effects of FNB anti-CD3 treatment is T cell depletion. We next examined the effects of FNB anti-CD3 mAb treatment on the homeostasis of Tregs. To first determine whether FNB anti-CD3 treatment promoted the conversion of naive Foxp3− T cells into Foxp3+ Tregs, CD4+Foxp3− cells from Foxp3-GFP reporter mice were sorted and transferred to lymph-replete recipient mice that were then treated with FNB anti-CD3 mAb. Analysis after 7 d showed that the percentage of Foxp3+ cells within the transferred population was not statistically different between control and FNB anti-CD3 mAb-treated mice (Fig. 4A), suggesting that stable Foxp3 expression was not induced by FNB anti-CD3 mAb. To confirm the lack of transient Foxp3 induction following FNB anti-CD3 treatment, NOD.Foxp3.GFP-Cre × Rosa26.flox.stop.YFP mice (28) were treated and the proportions of YFP+GFP+ “bona fide” Tregs and YFP+GFP− “transient” Foxp3-expressing cells were analyzed on day 14 after treatment (Fig. 4B). Although the percentage of CD4+ cells that expressed Foxp3 at any time (total YFP+) was increased in FNB anit-CD3 treated mice, which can be attributed to a loss of Foxp3− cells (Fig. 4C), the percentage of bona fide Tregs (YFP+GFP+) did not change (Fig. 4D). Thus, there was no evidence that FNB anti-CD3 mAb promoted generation of adaptive Tregs or transient expression of Foxp3 in vivo.

FIGURE 4.
FIGURE 4.

FNB anti-CD3 mAb does not promote the conversion or expansion of Tregs. A, CD4+GFP− cells, genetically marked by the Thy 1 congenic marker, were sorted from Foxp3-GFP reporter mice and transferred to WT recipients that were treated with FNB anti-CD3 mAb. On day 7, LNs were harvested and expression of Foxp3 protein was analyzed on the transferred cells by flow cytometry. Representative dot plots and summary of two independent experiments are shown. B, Foxp3.GFP-Cre × Rosa26.flox.stop.YFP mice were treated with FNB anti-CD3 mAb and LNs were analyzed on day 14 by flow cytometry. Representative FACS plots gated on CD4+ T cells are shown. C, Percentage of YFP+ cells within the CD4+ population (sum of upper and lower right quadrants of plots shown in B). D, Percentage of GFP+ cells within the YFP+ population (upper right quadrant divided by sum of upper and lower right quadrants of plots shown in B). Summary of two independent experiments is shown.

The previous experiments clearly demonstrated that FNB anti-CD3 mAb treatment does not induce Treg conversion. However, these data could not distinguish whether the increased frequency of Tregs was due solely to preferential loss of Foxp3− cells or involved the concomitant expansion of Foxp3+ Tregs (16–18). To determine the effects of FNB anti-CD3 mAb on the proliferation of Foxp3− versus Foxp3+ CD4+ T cells, total CD4+ cells, marked congenically, were CFSE labeled and transferred to recipient mice that were either treated with control Ab or FNB anti-CD3 mAb (Fig. 5A). As expected, the percentage of Foxp3+ cells within the transferred population increased as a result of preferential depletion of Foxp3− cells (Fig. 5A). The therapy resulted in a decrease in the ratio of Foxp3− to Foxp3+ cells in FNB anti-CD3 mAb-treated mice (Fig. 5B). Interestingly, FNB anti-CD3 mAb treatment led to a decrease in the percentage of undivided Foxp3− cells (Fig. 5C). Importantly, there was no difference in the percentage of Foxp3+ cells that had not divided (Fig. 5D), demonstrating that FNB anti-CD3 mAb does not induce proliferation of existing Foxp3+ Tregs.

FIGURE 5.
FIGURE 5.

FNB anti-CD3 mAb preferentially depletes CD4+Foxp3− T cells. Purified CD4+ T cells, marked by the Thy1 congenic marker, were labeled with CFSE and transferred to WT recipient mice that were treated with FNB anti-CD3. LNs were harvested on day 7 analyzed by flow cytometry. A, Representative FACS plots and pooled data showing CFSE dilution and Foxp3 expression gated on transferred CD4+ T cells. B, Ratio of Foxp3− to Foxp3+ cells. C, Representative histograms and pooled data showing the percentage of Foxp3− cells that remained undivided. D, Percentage of Foxp3+ cells that remained undivided. Each dot represents an individual mouse and data shown are pooled from two independent experiments.

FNB anti-CD3 mAb treatment alters the balance of Helios-expressing Tregs

Although the preferential depletion of conventional T cells following anti-CD3 mAb therapy in NOD mice could account for short-term remission of diabetes, it remained possible, if not likely, that qualitative changes in Tregs (other than expansion or induction) may play a role in long-term stable remission. It was recently reported that the transcription factor Helios is expressed by thymus-derived natural Tregs but is reduced or absent in adaptive Tregs induced in vitro by TGF-β (29). Furthermore, Helioslo/− Tregs produce more IFN-γ and IL-2 than do Helioshi Tregs (29), suggesting that the Helioslo/− population may be more “plastic” or unstable. We hypothesized that FNB anti-CD3 mAb may alter the relative proportion of Helioshi to Helioslo/− Tregs, thereby increasing the proportion of stable/suppressive Tregs. The treatment of mice with FNB anti-CD3 mAb led to an increase in the percentage and absolute number of Helioshi Tregs (Fig. 6A, 6B). This increase could be due to proliferation or preferential survival of Helioshi Tregs or induced expression of Helios in Helioslo/− cells. As shown in Fig. 5 above, FNB anti-CD3 mAb did not induce proliferation of Tregs. Therefore, it is unlikely that proliferation of Helioshi cells was responsible for the increased percentage of this Treg. To obtain Helioshi and Helioslo/− Tregs, we used the surface markers PD-1 and Neuropilin-1, which are preferentially expressed by Helioshi Tregs (M. Yadav, S. Bailey-Bucktrout, D. Davini, C. Louvet, R. Head, D. Kuster, P. Ruminski, D. Von Schack, and J.A. Bluestone, submitted for publication). Sorted CD25+PD-1−Nrp1− cells, of which 44–55% were Helioslo/−, and CD25+PD-1+Nrp1+ cells, of which 69–70% were Helioshi, were transferred to lymphoreplete recipients that were then treated with FNB anti-CD3 mAb. An average of 24% of transferred Helioslo/− Tregs expressed Helios after control Ig treatment, whereas 68% of Helioshi Tregs expressed high levels of Helios, consistent with a stable phenotype in vivo (Fig. 6C). FNB anti-CD3 mAb treatment resulted in an increased percentage of Helios-expressing Tregs in both the sorted Helioslo/− (68%) and Helioshi (82%) populations. Our transfer experiments could not rule out preferential survival of Helioshi Tregs, but nonetheless our results show that the treatment induces Helios expression in Tregs and causes previously unrecognized qualitative changes in this cell population.

FIGURE 6.
FIGURE 6.

FNB anti-CD3 mAb alters the balance of Helios-expressing Tregs. A and B, NOD mice were treated with FNB anti-CD3 mAb. On day 7 all LNs and spleen were harvested and the percentage and total number of CD4+Foxp3+Helioshi were calculated. Data shown are a summary of three independent experiments. C, Thy1.1+CD25+PD-1−Nrp1− (Helioslo/−) and CD25+PD-1+Nrp+ (Helioshi) Tregs were sorted and transferred to Thy1.2+ mice. Recipients were treated with FNB anti-CD3 mAb. All LNs and spleen were harvested and Thy1.1 cells were enriched as described in Materials and Methods. Helios expression on transferred Foxp3+ cells was analyzed by flow cytometry. Representative histograms are shown on top and pooled data from two independent experiments are shown on bottom.

Discussion

FNB anti-CD3 mAbs effectively control autoimmunity and transplant rejection in preclinical murine models and clinical trials in patients (1). The mechanisms underlying the tolerogenic outcome of FNB anti-CD3 mAb therapy are still ill-defined. In particular, effects of FNB anti-CD3 mAbs on conventional T cells versus Tregs are controversial. In this study, we addressed this issue directly by comparing the impact of FNB anti-CD3 mAbs on conventional CD4+Foxp3− T cells and CD4+Foxp3+ Tregs. Our data showed that although both T cell subsets underwent partial depletion following FNB anti-CD3 mAb treatment, there was a relative enrichment of Tregs. Although killing of T cells induced by FNB anti-CD3 mAb was dependent on the Fas pathway in vitro, it occurred independently of Fas, Bim, and caspase-3 in vivo. Importantly, the increase in the frequency of Tregs was not associated with induction or expansion of adaptive Tregs. Additionally, FNB anti-CD3 mAb treatment increased the proportion of Helios-expressing Tregs. Thus, our study suggests that FNB anti-CD3 mAb therapy promotes tolerance and prevents autoimmunity by restoring the balance between pathogenic autoreactive Teffs and suppressive Tregs and potentially increases the stability of the Treg population.

Current theories on the underlying mechanisms of the preclinical and clinical efficacy of FNB anti-CD3 mAb therapy often involve the generation of Tregs in addition to its effects on Teffs. The notion of FNB anti-CD3 induction of Tregs was supported by increases of CD4+CD25+ Treg frequencies observed after FNB anti-CD3 mAb treatment in several murine models, but absolute numbers of Tregs were not reported in these studies (16–18). Our study demonstrates that the relative enrichment of Tregs following FNB anti-CD3 mAb treatment was due to differential sensitivity of conventional and regulatory T cells to cell death, such that activated Teffs are most sensitive and Tregs are least sensitive. We found no evidence of Treg induction or expansion after FNB anti-CD3 treatment. Similar to our results, other studies have not observed increased Treg percentages after FNB anti-CD3 mAb treatment in vivo (19, 20, 30). Considering the therapeutic applications of FNB anti-CD3 mAbs, this finding has important implications and clarifies the mode of action of these reagents. For instance, the preferential effects of the FNB anti-CD3 mAbs on Teffs help to explain why anti-CD3 treatment is most effective at the time of peak disease activity (13, 18).

The mechanism responsible for cell death after FNB anti-CD3 mAb treatment remains to be elucidated. Single genetic deficiency of Fas, Bim, and caspase-3 or overexpression of Bcl-2 did not prevent cell depletion. Thus, these data suggest FNB anti-CD3 mAb may activate multiple apoptotic pathways in vivo such that absence of one does not prevent cell death or that one main pathway is activated, but in its absence, there is compensation by the other pathways. Alternatively, it is possible that FNB anti-CD3 mAb activates cell death pathways that are independent of caspases such as necrosis or autophagy. Additionally, it remains unclear why conventional T cells are more susceptible to FNB anti-CD3–induced death than are Tregs. A possible hypothesis is that TCR signaling is altered in Tregs compared with non-Tregs such that different downstream signals are activated in each cell type. Indeed, it was shown that overexpression of Foxp3 results in decreased PLCγ1, ERK1/2, and S6 phosphorylation after TCR engagement (31). Alternatively, there may be differential expression of pro- or anti-apoptotic factors in Tregs versus conventional T cells that are responsible for the preferential death of conventional T cells. Conversely, if expression of these proteins is similar, posttranslational modifications that cause their sequestration and affect their activity may be different (32). Interestingly, Tregs have also been shown to be less susceptible to cell death caused by low levels of whole body irradiation (33), suggesting that this mechanism may also be relevant in protecting Tregs from other signals that induce cell death.

The recent report of Helios expression as a marker for thymus-derived natural Tregs (29) led us to ask whether FNB anti-CD3 mAb had preferential effects on natural versus adaptive Tregs. Our data show increased expression of Helios after FNB anti-CD3 mAb treatment, suggesting that Helios expression can be induced on a previously Helioslo/− population, although we cannot rule out the possibility that Helioslo/− cells are preferentially depleted by the treatment. Helios expression does not seem to directly affect Treg function (29); however, data from our laboratory suggests that Helioslo/− adaptive Tregs are not able to control autoimmune responses in vivo (M. Yadav, J.A. Bluestone, et al., submitted for publication). Our laboratory also showed that at the time of disease onset, the Tregs in the pancreas of NOD mice express lower levels of Foxp3 and CD25, likely making them unstable and dysfunctional, and that treatment with IL-2/anti–IL-2 complexes can prevent disease by augmenting the percentage of Tregs in the pancreas (34). Finally, a greater proportion of Helioslo/− Tregs produces cytokines (35); thus, we suggest that Helios expression is a marker of stable and functional Tregs. Therefore, we hypothesize that in addition to restoring the balance between Teffs and Tregs, FNB anti-CD3 mAb also affects the balance between stable/functional and unstable/nonfunctional Tregs.

Finally, whether these results can be extended to the human setting is still controversial. Interestingly, a population of CD8+Foxp3+ cells arises in human patients treated with OKT3-γ1(Ala-Ala) (36), which we do not observe in mice treated with FNB anti-CD3 mAb. Unlike murine T cells, human T cells transiently express Foxp3 protein upon activation (37); however, Foxp3 expression does necessarily not confer suppressive function. Additionally, in humans, Foxp3 expression may not be a reliable marker of Tregs as it is in mice. Furthermore, the effect of FNB anti-CD3 mAb on human cells has been mostly studied in vitro; however, it is possible that in vitro effects differ from in vivo effects, as we show that in vitro T cell death is Fas mediated but genetic depletion of Fas has no effect on T cell depletion in vivo.

In conclusion, our results suggest that FNB anti-CD3 mAb treatment reverses autoimmunity by deleting autoreactive pathogenic T cells and restoring the balance of pathogenic versus regulatory T cells toward increased regulation. This outcome is achieved by the selective sensitivity of Teffs and naive T cells versus Tregs to FNB anti-CD3 mAb-induced cell death but not by de novo induction of adaptive Tregs or expansion of natural Tregs. Our results have important implications for the design of FNB anti-CD3 mAb-based immunotherapies in autoimmune diseases and suggest the pertinence of developing combination therapies that would strengthen FNB anti-CD3 mAb modes of action. For instance, although counterintuitive, anti-CD3 immunotherapy might be best when coupled with Ag immunization that drives the differentiation of conventional cells into an activated state, which will be more sensitive to anti-CD3 mAb-induced cell death.

Disclosures

J.A.B. is a paid consultant for Macrogenics and has a financial interest in the commercialization of anti-CD3 mAb. The other authors have no financial conflicts of interest.

Acknowledgments

We thank Shuwei Jiang for cell sorting, Nicolas Martinier and Dorothy Fuentes for animal husbandry, Jackie Fu for technical assistance, and Greg Szot and Jiena Lang for assistance in the purification of the Ab. We thank members of the Bluestone, Anderson, and Abbas Laboratories, as well as Dr. Scott Oakes for helpful discussions. We thank Drs. Helene Bour-Jordan, Abul Abbas, and Hans Dooms for critical reading of the manuscript.

Footnotes

  • This work was supported by National Institutes of Health Grants R37 AI46643 and P30 DK63720 (for core support), and C.P. was supported by National Institute of General Medical Sciences Grant 1 R25 GM56847.

  • Abbreviations used in this article:

    B6
    C57BL/6
    FNB
    FcR-nonbinding
    LN
    lymph node
    Teff
    effector T cell
    Treg
    regulatory T cell
    WT
    wild-type.

  • Received March 11, 2011.
  • Accepted June 8, 2011.

References

  1. ↵
    1. Chatenoud L.,
    2. J. A. Bluestone
    . 2007. CD3-specific antibodies: a portal to the treatment of autoimmunity. Nat. Rev. Immunol. 7: 622–632.
    OpenUrlCrossRefPubMed
  2. ↵
    1. Cosimi A. B.,
    2. R. B. Colvin,
    3. R. C. Burton,
    4. H. J. Winn,
    5. R. Rubin,
    6. G. Goldstein,
    7. P. C. Kung,
    8. R. A. Hoffman,
    9. W. P. Hansen,
    10. P. S. Russell
    . 1981. Immunologic monitoring with monoclonal antibodies to human T-cell subsets. Transplant. Proc. 13: 1589–1593.
    OpenUrlPubMed
  3. ↵
    1. Abramowicz D.,
    2. L. Schandene,
    3. M. Goldman,
    4. A. Crusiaux,
    5. P. Vereerstraeten,
    6. L. De Pauw,
    7. J. Wybran,
    8. P. Kinnaert,
    9. E. Dupont,
    10. C. Toussaint
    . 1989. Release of tumor necrosis factor, interleukin-2, and γ-interferon in serum after injection of OKT3 monoclonal antibody in kidney transplant recipients. Transplantation 47: 606–608.
    OpenUrlCrossRefPubMed
  4. ↵
    1. Chatenoud L.,
    2. C. Ferran,
    3. A. Reuter,
    4. C. Legendre,
    5. Y. Gevaert,
    6. H. Kreis,
    7. P. Franchimont,
    8. J. F. Bach
    . 1989. Systemic reaction to the anti-T-cell monoclonal antibody OKT3 in relation to serum levels of tumor necrosis factor and interferon-γ. [corrected] N. Engl. J. Med. 320: 1420–1421.
    OpenUrlCrossRefPubMed
  5. ↵
    1. Alegre M. L.,
    2. J. Y. Tso,
    3. H. A. Sattar,
    4. J. Smith,
    5. F. Desalle,
    6. M. Cole,
    7. J. A. Bluestone
    . 1995. An anti-murine CD3 monoclonal antibody with a low affinity for Fcγ receptors suppresses transplantation responses while minimizing acute toxicity and immunogenicity. J. Immunol. 155: 1544–1555.
    OpenUrlAbstract
  6. ↵
    1. Alegre M. L.,
    2. A. M. Collins,
    3. V. L. Pulito,
    4. R. A. Brosius,
    5. W. C. Olson,
    6. R. A. Zivin,
    7. R. Knowles,
    8. J. R. Thistlethwaite,
    9. L. K. Jolliffe,
    10. J. A. Bluestone
    . 1992. Effect of a single amino acid mutation on the activating and immunosuppressive properties of a “humanized” OKT3 monoclonal antibody. J. Immunol. 148: 3461–3468.
    OpenUrlAbstract
    1. Friend P. J.,
    2. G. Hale,
    3. L. Chatenoud,
    4. P. Rebello,
    5. J. Bradley,
    6. S. Thiru,
    7. J. M. Phillips,
    8. H. Waldmann
    . 1999. Phase I study of an engineered aglycosylated humanized CD3 antibody in renal transplant rejection. Transplantation 68: 1632–1637.
    OpenUrlCrossRefPubMed
    1. Woodle E. S.,
    2. J. R. Thistlethwaite,
    3. I. A. Ghobrial,
    4. L. K. Jolliffe,
    5. F. P. Stuart,
    6. J. A. Bluestone
    . 1991. OKT3 F(ab′)2 fragments: retention of the immunosuppressive properties of whole antibody with marked reduction in T cell activation and lymphokine release. Transplantation 52: 354–360.
    OpenUrlCrossRefPubMed
  9. ↵
    1. Woodle E. S.,
    2. D. Xu,
    3. R. A. Zivin,
    4. J. Auger,
    5. J. Charette,
    6. R. O’Laughlin,
    7. D. Peace,
    8. L. K. Jollife,
    9. T. Haverty,
    10. J. A. Bluestone,
    11. J. R. Thistlethwaite Jr..
    1999. Phase I trial of a humanized, Fc receptor nonbinding OKT3 antibody, huOKT3γ1(Ala-Ala) in the treatment of acute renal allograft rejection. Transplantation 68: 608–616.
    OpenUrlCrossRefPubMed
  10. ↵
    1. Smith J. A.,
    2. Q. Tang,
    3. J. A. Bluestone
    . 1998. Partial TCR signals delivered by FcR-nonbinding anti-CD3 monoclonal antibodies differentially regulate individual Th subsets. J. Immunol. 160: 4841–4849.
    OpenUrlAbstract/FREE Full Text
  11. ↵
    1. Smith J. A.,
    2. J. Y. Tso,
    3. M. R. Clark,
    4. M. S. Cole,
    5. J. A. Bluestone
    . 1997. Nonmitogenic anti-CD3 monoclonal antibodies deliver a partial T cell receptor signal and induce clonal anergy. J. Exp. Med. 185: 1413–1422.
    OpenUrlAbstract/FREE Full Text
  12. ↵
    1. Herold K. C.,
    2. J. B. Burton,
    3. F. Francois,
    4. E. Poumian-Ruiz,
    5. M. Glandt,
    6. J. A. Bluestone
    . 2003. Activation of human T cells by FcR nonbinding anti-CD3 mAb, hOKT3γ1(Ala-Ala). J. Clin. Invest. 111: 409–418.
    OpenUrlCrossRefPubMed
  13. ↵
    1. Chatenoud L.,
    2. J. Primo,
    3. J. F. Bach
    . 1997. CD3 antibody-induced dominant self tolerance in overtly diabetic NOD mice. J. Immunol. 158: 2947–2954.
    OpenUrlAbstract
  14. ↵
    1. Brusko T. M.,
    2. A. L. Putnam,
    3. J. A. Bluestone
    . 2008. Human regulatory T cells: role in autoimmune disease and therapeutic opportunities. Immunol. Rev. 223: 371–390.
    OpenUrlCrossRefPubMed
  15. ↵
    1. Tang Q.,
    2. J. A. Bluestone
    . 2008. The Foxp3+ regulatory T cell: a jack of all trades, master of regulation. Nat. Immunol. 9: 239–244.
    OpenUrlCrossRefPubMed
  16. ↵
    1. Belghith M.,
    2. J. A. Bluestone,
    3. S. Barriot,
    4. J. Mégret,
    5. J. F. Bach,
    6. L. Chatenoud
    . 2003. TGF-β-dependent mechanisms mediate restoration of self-tolerance induced by antibodies to CD3 in overt autoimmune diabetes. Nat. Med. 9: 1202–1208.
    OpenUrlCrossRefPubMed
    1. Bresson D.,
    2. L. Togher,
    3. E. Rodrigo,
    4. Y. Chen,
    5. J. A. Bluestone,
    6. K. C. Herold,
    7. M. von Herrath
    . 2006. Anti-CD3 and nasal proinsulin combination therapy enhances remission from recent-onset autoimmune diabetes by inducing Tregs. J. Clin. Invest. 116: 1371–1381.
    OpenUrlCrossRefPubMed
  18. ↵
    1. Kohm A. P.,
    2. J. S. Williams,
    3. A. L. Bickford,
    4. J. S. McMahon,
    5. L. Chatenoud,
    6. J. F. Bach,
    7. J. A. Bluestone,
    8. S. D. Miller
    . 2005. Treatment with nonmitogenic anti-CD3 monoclonal antibody induces CD4+ T cell unresponsiveness and functional reversal of established experimental autoimmune encephalomyelitis. J. Immunol. 174: 4525–4534.
    OpenUrlAbstract/FREE Full Text
  19. ↵
    1. Chen G.,
    2. G. Han,
    3. J. Wang,
    4. R. Wang,
    5. R. Xu,
    6. B. Shen,
    7. J. Qian,
    8. Y. Li
    . 2008. Essential roles of TGF-β in anti-CD3 antibody therapy: reversal of diabetes in nonobese diabetic mice independent of Foxp3+CD4+ regulatory T cells. J. Leukoc. Biol. 83: 280–287.
    OpenUrlAbstract/FREE Full Text
  20. ↵
    1. Yang W.,
    2. S. Hussain,
    3. Q. S. Mi,
    4. P. Santamaria,
    5. T. L. Delovitch
    . 2004. Perturbed homeostasis of peripheral T cells elicits decreased susceptibility to anti-CD3-induced apoptosis in prediabetic nonobese diabetic mice. J. Immunol. 173: 4407–4416.
    OpenUrlAbstract/FREE Full Text
  21. ↵
    1. Mandala S.,
    2. R. Hajdu,
    3. J. Bergstrom,
    4. E. Quackenbush,
    5. J. Xie,
    6. J. Milligan,
    7. R. Thornton,
    8. G. J. Shei,
    9. D. Card,
    10. C. Keohane,
    11. et al
    . 2002. Alteration of lymphocyte trafficking by sphingosine-1-phosphate receptor agonists. Science 296: 346–349.
    OpenUrlAbstract/FREE Full Text
  22. ↵
    1. Green D. R.
    2003. Overview: apoptotic signaling pathways in the immune system. Immunol. Rev. 193: 5–9.
    OpenUrlCrossRefPubMed
  23. ↵
    1. Hildeman D.,
    2. T. Jorgensen,
    3. J. Kappler,
    4. P. Marrack
    . 2007. Apoptosis and the homeostatic control of immune responses. Curr. Opin. Immunol. 19: 516–521.
    OpenUrlCrossRefPubMed
  24. ↵
    1. Hildeman D. A.,
    2. Y. Zhu,
    3. T. C. Mitchell,
    4. P. Bouillet,
    5. A. Strasser,
    6. J. Kappler,
    7. P. Marrack
    . 2002. Activated T cell death in vivo mediated by proapoptotic bcl-2 family member bim. Immunity 16: 759–767.
    OpenUrlCrossRefPubMed
  25. ↵
    1. Tang Q.,
    2. J. A. Smith,
    3. G. L. Szot,
    4. P. Zhou,
    5. M. L. Alegre,
    6. K. J. Henriksen,
    7. C. B. Thompson,
    8. J. A. Bluestone
    . 2003. CD28/B7 regulation of anti-CD3-mediated immunosuppression in vivo. J. Immunol. 170: 1510–1516.
    OpenUrlAbstract/FREE Full Text
  26. ↵
    1. Núñez G.,
    2. R. Merino,
    3. D. Grillot,
    4. M. González-García
    . 1994. Bcl-2 and Bcl-x: regulatory switches for lymphoid death and survival. Immunol. Today 15: 582–588.
    OpenUrlCrossRefPubMed
  27. ↵
    1. Woo M.,
    2. R. Hakem,
    3. M. S. Soengas,
    4. G. S. Duncan,
    5. A. Shahinian,
    6. D. Kägi,
    7. A. Hakem,
    8. M. McCurrach,
    9. W. Khoo,
    10. S. A. Kaufman,
    11. et al
    . 1998. Essential contribution of caspase 3/CPP32 to apoptosis and its associated nuclear changes. Genes Dev. 12: 806–819.
    OpenUrlAbstract/FREE Full Text
  28. ↵
    1. Zhou X.,
    2. S. L. Bailey-Bucktrout,
    3. L. T. Jeker,
    4. C. Penaranda,
    5. M. Martínez-Llordella,
    6. M. Ashby,
    7. M. Nakayama,
    8. W. Rosenthal,
    9. J. A. Bluestone
    . 2009. Instability of the transcription factor Foxp3 leads to the generation of pathogenic memory T cells in vivo. Nat. Immunol. 10: 1000–1007.
    OpenUrlCrossRefPubMed
  29. ↵
    1. Thornton A. M.,
    2. P. E. Korty,
    3. D. Q. Tran,
    4. E. A. Wohlfert,
    5. P. E. Murray,
    6. Y. Belkaid,
    7. E. M. Shevach
    . 2010. Expression of Helios, an Ikaros transcription factor family member, differentiates thymic-derived from peripherally induced Foxp3+ T regulatory cells. J. Immunol. 184: 3433–3441.
    OpenUrlAbstract/FREE Full Text
  30. ↵
    1. von Herrath M. G.,
    2. B. Coon,
    3. T. Wolfe,
    4. L. Chatenoud
    . 2002. Nonmitogenic CD3 antibody reverses virally induced (rat insulin promoter-lymphocytic choriomeningitis virus) autoimmune diabetes without impeding viral clearance. J. Immunol. 168: 933–941.
    OpenUrlAbstract/FREE Full Text
  31. ↵
    1. Carson B. D.,
    2. S. F. Ziegler
    . 2007. Impaired T cell receptor signaling in Foxp3+ CD4 T cells. Ann. N. Y. Acad. Sci. 1103: 167–178.
    OpenUrlCrossRefPubMed
  32. ↵
    1. Puthalakath H.,
    2. A. Strasser
    . 2002. Keeping killers on a tight leash: transcriptional and post-translational control of the pro-apoptotic activity of BH3-only proteins. Cell Death Differ. 9: 505–512.
    OpenUrlCrossRefPubMed
  33. ↵
    1. Qu Y.,
    2. B. Zhang,
    3. S. Liu,
    4. A. Zhang,
    5. T. Wu,
    6. Y. Zhao
    . 2010. 2-Gy whole-body irradiation significantly alters the balance of CD4+CD25− T effector cells and CD4+CD25+Foxp3+ T regulatory cells in mice. Cell. Mol. Immunol. 7: 419–427.
    OpenUrlCrossRefPubMed
  34. ↵
    1. Tang Q.,
    2. J. Y. Adams,
    3. C. Penaranda,
    4. K. Melli,
    5. E. Piaggio,
    6. E. Sgouroudis,
    7. C. A. Piccirillo,
    8. B. L. Salomon,
    9. J. A. Bluestone
    . 2008. Central role of defective interleukin-2 production in the triggering of islet autoimmune destruction. Immunity 28: 687–697.
    OpenUrlCrossRefPubMed
  35. ↵
    1. McClymont S. A.,
    2. A. L. Putnam,
    3. M. R. Lee,
    4. J. H. Esensten,
    5. W. Liu,
    6. M. A. Hulme,
    7. U. Hoffmüller,
    8. U. Baron,
    9. S. Olek,
    10. J. A. Bluestone,
    11. T. M. Brusko
    . 2011. Plasticity of human regulatory T cells in healthy subjects and patients with type 1 diabetes. J. Immunol. 186: 3918–3926.
    OpenUrlAbstract/FREE Full Text
  36. ↵
    1. Bisikirska B.,
    2. J. Colgan,
    3. J. Luban,
    4. J. A. Bluestone,
    5. K. C. Herold
    . 2005. TCR stimulation with modified anti-CD3 mAb expands CD8+ T cell population and induces CD8+CD25+ Tregs. J. Clin. Invest. 115: 2904–2913.
    OpenUrlCrossRefPubMed
  37. ↵
    1. Walker M. R.,
    2. D. J. Kasprowicz,
    3. V. H. Gersuk,
    4. A. Benard,
    5. M. Van Landeghen,
    6. J. H. Buckner,
    7. S. F. Ziegler
    . 2003. Induction of FoxP3 and acquisition of T regulatory activity by stimulated human CD4+CD25− T cells. J. Clin. Invest. 112: 1437–1443.
    OpenUrlCrossRefPubMed
Back to top

In this issue

Print
Download PDF
Article Alerts
Email Article
Citation Tools

Jump to section