Selective Activation of Antigen-Experienced T Cells by Anti-CD3 Constrained on Nanoparticles

Ying-Chun Lo, Michael A. Edidin and Jonathan D. Powell
J Immunol November 15, 2013, 191 (10) 5107-5114; DOI: https://doi.org/10.4049/jimmunol.1301433

Abstract

Activation of T cells through the TCR is mediated by the TCR-CD3 signaling complex. Cross linking of this complex with Abs directed against CD3 leads to potent activation of T cells. However, such activation is not Ag-specific. We exploited the observation that the TCR-CD3 complex is clustered on T cells that have been activated by Ag by using anti-CD3 nanoparticles to selectively activate Ag-experienced mouse T cells. We find that constraining anti-CD3 on the surface of a nanoparticle markedly and selectively enhances proliferation and cytokine production of Ag-experienced T cells but does not activate naive T cells. This effect was recapitulated in heterogeneous cultures containing mixtures of Ag-specific CD4+ or CD8+ T cells and bystander T cells. Furthermore, in vivo anti-CD3–coated nanoparticles increased the expansion of Ag-specific T cells following vaccination. Overall, these findings indicate that anti-CD3–coated nanoparticles could be use to enhance the efficacy of vaccines and immunotherapy. The results also suggest constraining a ligand on the surface of a nanoparticle might as general strategy for selectively targeting clustered receptors.

Introduction

Specificity and memory are key features of the adaptive immune system (1, 2). An adaptive immune response amplifies a small population of Ag-specific B and T lymphocytes to promote the clearance of an infection. Although B cell receptors (antibodies) can recognize soluble intact Ag, T cells recognize cognate peptides presented in the context of MHC molecules on the surface of APCs (3). On naive T cells, the Ag-specific TCR is distributed across the surface of the cell in nanoclusters; these nanoclusters oligomerize into microclusters after T cells are activated by Ag (4–6). Clustering promotes the transmission of intracellular signals via the CD3 signaling complex, leading to T cell activation (7–10). It is also believed to increase the sensitivity for low concentrations of Ag (11) and to generate maximal local signals by providing continuous engagement of TCR/MHC (12). TCR microclusters are observed in both effector and memory cells; their presence correlates with increased sensitivity of Ag-experienced T cells (13).

It has been estimated that the number of TCRs within a nanocluster, prior to activation, ranges from a single receptor to a cluster of ≥20 (11). Binding experiments indicate that these clusters are 1–3 nm in size (5). In contrast, microclusters, which are formed upon T cell activation, have been estimated to be hundreds of nanometers in diameter (14, 15) and contain ∼100 TCR complexes as determined by total internal reflection fluorescence microscopy (16). Furthermore, by employing photoactivated localization microscopy, density domains inside microclusters have been estimated to be 35–70 nm in diameter and contain 7–20 TCRs (17). Based on such data, it is reasonable to assume that the distance between two TCR complexes in the microcluster of activated T cells is ∼20 nm.

We hypothesized that the difference in TCR clustering between naive and recently activated T cells could be exploited to selectively boost Ag-specific responses. To test our hypothesis, we used mAb to CD3, a general T cell activator, bound to quantum dots (QD) (14, 18–20). Anti-CD3–coated Qdots 605 (anti-CD3 QD; Invitrogen) are ∼18 nm in diameter and coupled to multiple anti-CD3 Abs, which are potent T cell agonists. In this report, we demonstrate that anti-CD3 constrained on the surface of a nanoparticle selectively activates only T cells that are Ag-experienced and, in contrast to soluble anti-CD3, does not activate naive T cells.

Materials and Methods

Microscopy

Cells were fixed by 2% formaldehyde and stained with rabbit anti-mouse CD3-γ (Santa Cruz Biotechnology) overnight and goat anti-rabbit DyLight 488 (Jackson ImmunoResearch Laboratories) for 2 h. Cells were then mounted with Prolong Gold Anti-fade reagent (Invitrogen) and imaged with an upright fluorescence microscope with 710NLO-Meta confocal module (AxioExaminer; Zeiss) with a 63×/1.2W C-Apo objective. Microclusters were identified using the “Find objects using intensity (>21044)” and “Separate touching objects (object size guide 0.08 μm2)” functions of Volocity imaging analysis software (PerkinElmer). Data were acquired with Zen imaging software (Zeiss) and analyzed with Volocity analysis software (PerkinElmer).

Mice

Mice were kept in accordance with guidelines of the Johns Hopkins University Institutional Animal Care and Use Committee. 5C.C7 TCR-transgenic RAG2−/− mice and DO11.10 TCR-transgenic RAG2−/− mice (Thy1.2+, Kd; hemagglutinin [HA]-specific) were from Taconic Farms. 6.5 TCR-transgenic (Thy1.1+, Kd; HA-specific) mice, B10.D2 (Thy1.1+, Kd) mice, clone 4 TCR-transgenic (Thy1.1+, Kd; HA-specific) mice, OT-1 TCR transgenic RAG2−/− (Thy1.1+, Kb; HA-specific) mice, and B10.D2 (Thy1.2+, Kd) mice were a gift from Charles Drake (Departments of Oncology, Immunology and Urology, School of Medicine, Johns Hopkins University, Baltimore, MD). C57BL/6 (Thy1.2+, Kb) mice were obtained from The Jackson Laboratory.

Reagents and Abs

Hamster anti-mouse CD3 (145-2C11) Qdot 605 and Qdot 655 streptavidin (SA) conjugate were purchased from Invitrogen. Abs against the following proteins were purchased from BD Biosciences: CD4 (GK1.5), CD8a (53-6.7), Thy1.1 (OX-7), Thy1.2 (53-2.1), Vβ8.1/8.2 (MR5-2), IFN-γ (XMG1.2), and IL-4 (11B11). Biotin-labeled Abs against 6.5 TCR and stimulatory anti-CD3 (145-2C11) Abs as well as neutralizing anti–IL-4 (11B11) and anti–IFN-γ (XMG1.2) Abs were purified from hybridoma supernatants prepared in-house. Neutralizing anti–IL-12p40 (C17.8) Abs were from eBioscience. Other regents used: CFSE cell proliferation kit (Invitrogen), eFluor 670 cell proliferation dye (eBioscience), fluorophore-conjugated SA (BD Biosciences), IL-2, IL-7, IFN-γ, IL-12 p40, and IL-4 cytokines (all from PeproTech), PCC protein and OVA protein (Sigma-Aldrich), HA class II peptide (SFERFEIFPKE), HA class I peptide (IYSTVASSL) (both from Johns Hopkins Synthesis and Sequencing Facility), OVA class II peptide (ISQAVHAAHAEINEAGR), OVA class I peptide (SIINFEKL) (both from AnaSpec), and Imject Freund's Complete Adjuvant (Thermo Scientific).

Flow cytometry and intracellular staining

All experiments were performed on a BD FASCalibur (BD Biosciences) and analyzed using FlowJo analysis software (Tree Star). Brefeldin A (GolgiPlug; BD Biosciences) or monensin (GolgiStop; BD Biosciences) was used for cytokine staining. Cells were surface stained and underwent fixation/permeabilization followed by staining for intracellular proteins in Perm/Wash Buffer (reagents all from BD Biosciences). Gates were set appropriately with unstimulated and controls. Voltages were determined from unstained controls.

Proliferation and ELISA

Proliferation was measured by dilution of CFSE or eFluor 670 cell proliferation dyes. Cells were labeled according to the manufacturer’s protocol. The cytokines IFN-γ and IL-4 were measured in supernatants by ELISA as described by the manufacturer (eBioscience).

Cell culture

Unless otherwise stated, splenocytes were cultured in 50% RPMI 1640/50% EHAA media supplemented with 10% heat-inactivated low-LPS FBS, 1% penicillin/streptomycin, and 1% glutamine. APCs were from the non-CD4+, column-bound fraction of CD4+ T cell isolation. For Th1 and Th2 cultures, splenocytes were stimulated in media supplemented with 5 μM PCC and different skewing cytokines and Abs for 48 h. Skewing conditions were as follows: Th1: IL-12 (5 ng/ml), IFN-γ (100 ng/ml), and anti–IL-4 (100 μg/ml); and Th2: IL-4 (1 ng/ml), anti–IL-12 (100 μg/ml), and anti–IFN-γ (100 μg/ml).

Adoptive transfer

For adoptive transfer experiments, 6.5 TCR-transgenic mice were sacrificed via CO2 asphyxiation. Spleens and lymph nodes were collected and homogenized, and RBCs were lysed. CD4+ T cells were purified using Miltenyi magnetically labeled beads (Miltenyi Biotec) according to the manufacturer’s protocol. Cells were then washed and resuspended with PBS for i.v. injections. Typically, 1–5 × 106 cells were injected per mouse in 0.2 ml PBS by retro-orbital i.v. injection.

Results

Ag recognition leads to TCR clustering

Emerging studies reveal that after Ag recognition, there is clustering of TCRs not only at the T cell–APC interface but also on the whole surface of the T cell (12). We employed confocal microscopy to image the distribution of TCRs on T cells before and after Ag exposure. We imaged 1 μm below the top of the cell, trading off sharpness for the largest area of membrane in a single image. It has been reported that membrane molecules remain mobile after fixation (21), and the observed clustering could be amplified by the Ab used for staining. However, the observed clustering differences between naive and primed cells are consistent with those on live cells.

Naive RAG2−/− CD4+ 5C.C7 TCR-transgenic T cells, stained for the TCR signaling complex using purified anti-CD3 and Alexa 488–labeled secondary Ab, showed few clusters and a relatively uniform distribution of TCR on the surface (Fig. 1A). After 24 h of stimulation in vitro with 0.05 μM PCC protein, we observed the formation of TCR microclusters over the surface of the activated T cells (Fig. 1B). To determine whether these microclusters persisted, 5C.C7 cells stimulated in vitro with 0.05 μM PCC protein were then rested in media supplemented with 100 ng/ml IL-2 and 10 ng/ml IL-7 without Ag for another 6 d. At this time point, 6 d after the encounter with Ag, the TCR microclusters were still present (Fig. 1C).

FIGURE 1.
FIGURE 1.

Ag stimulation induces TCR clustering. Fresh naive 5C.C7 splenocytes (A), 5C.C7 splenocytes stimulated with 50 nM PCC for 24 h (B), and 5C.C7 splenocytes stimulated with 50 nM PCC for 24 h, washed with PBS, and rested in 1 μg/ml IL-2 and IL-7 without PCC for another 6 d (C) were fixed by 2% formaldehyde, stained for CD3, and imaged by confocal microscopy. (D–F) Microclusters were classified by size and size classes color-coded using Volocity imaging analysis software. Objects with area <0.01 μm2 were excluded. Scale bars, 1 μm. (G) The average number of microclusters identified on each cell (n ≥ 25). (H) The average area of each microcluster. (I) The mean intensity of each cell (n ≥ 25). Data are representative of three independent experiments. **p < 0.01, ***p < 0.001 (two-tailed t test).

To further quantify the numbers of microclusters on T cells, images of at least 25 cells for each condition were collected and microclusters were identified using the Volocity software (PerkinElmer) (Fig. 1D–F). Fresh, naive 5C.C7 cells had an average of 15 microclusters on a single imaging slice. In contrast, there was a marked increase in the number of microclusters, to an average of 50 per cell, after 24 h of stimulation. Similarly, after resting for 6 d, we still observed an average of 35 microclusters on the surface of the cells (Fig. 1G). The average area of the microclusters on the activated cells was 0.1255 μm2. The size of the microclusters decreased to 0.0844 μm2 after 6 d of rest; however, their size remained larger than those observed on the naive T cells, which was 0.0667 μm2 (Fig. 1H). The total amount of TCRs expressed on the surface of the naive and 6-d activated T cells, as determined by mean fluorescence intensity, was similar (Fig. 1I). Thus, although the overall number of TCRs on the surface of naive and recently activated T cells was similar, the size and intensity of TCR microclusters differentiated Ag-experienced 5C.C7 T cells from naive cells.

Constraining anti-CD3 Abs on nanoparticles selectively activates Ag-experienced CD4+ T cells

Soluble anti-CD3 activates all T cells regardless of their Ag-specificity by cross linking the TCR-CD3 signaling machinery (22, 23). We postulated that the different degrees of TCR clustering on the surface of Ag-experienced versus naive T cells could be exploited to selectively activate only the T cells that had previously engaged Ag (Fig. 2A, 2B). We reasoned that in general, the cross linking ability of anti-CD3 when constrained to a nanoparticle would be limited when the TCRs were scattered across the surface of the cell, as is the case for naive T cells, but that the anti-CD3 QD could engage the TCR in the microclusters that develop after T cell activation.

FIGURE 2.
FIGURE 2.

Anti-CD3 QD activate Ag-experienced cells. (A) Receptors are mostly dispersed on the surface of a naive T cell. Upon activation, receptors cluster. (B) The dispersed receptors are not readily cross linked by binding of a ligand-coated nanoparticle, and this interaction delivers a minimal signal. However, ligand-coated nanoparticles can bind multiple receptors in a cluster; this induces strong signaling. (C) eFluor 670 dilution in naive 5C.C7 splenocytes stimulated for 3 d with 0, 17, or 50 nM PCC in combination with various concentration of anti-CD3 QD. (D) Naive purified CD4+ 5C.C7 cells and Ag-experienced CD4+ 5C.C7 cells as shown in Fig. 1C were labeled with CFSE and incubated with irradiated APCs (in the absence of PCC) in the presence or absence of 1 nM anti-CD3 QD for 3 d. CFSE dilution measures cell proliferation. Data are representative of at least three independent experiments.

To this end, naive 5C.C7 splenocytes were incubated in vitro with media or PCC protein together with anti-CD3 QD for 72 h and evaluated for proliferation. T cells that were not previously activated by Ag failed to proliferate in response to the anti-CD3 QD even at the highest concentration, but responded perfectly well to soluble anti-CD3 (data not shown for 5C.C7, but see below for another example). T cells incubated with low- dose peptide proliferated modestly, whereas the addition of anti-CD3 QD resulted in markedly enhanced proliferation. This selective enhancement also increased with increasing amounts of anti-CD3 QD (Fig. 2C). In addition, the ability of the anti-CD3 QD to enhance proliferation was maintained even if we removed the Ag from the culture (Supplemental Fig. 1).

We hypothesize that TCR clustering on the surface of previously activated T cells enables them to be stimulated by the anti-CD3–coated QD. To test this hypothesis, 5C.C7 cells were stimulated with PCC protein for 2 d, rested in IL-2 and IL-7 for 7 d, and then were CFSE labeled and restimulated with anti-CD3 QD and fresh APCs. T cells that had seen Ag 6 d earlier proliferated vigorously in response to 1 nM of anti-CD3 QD but did not respond to fresh APCs alone. Also, naive 5C.C7 cells responded minimally to the same concentration of anti-CD3 QD (Fig. 2D, Supplemental Fig. 2A). Thus, Ag-experienced T cells, for which TCR are clustered, are responsive to anti-CD3 QD, whereas T cells that have yet to see their Ag (and have most TCR diffusely distributed) are unresponsive.

The ability of anti-CD3 QD to selectively enhance the proliferation of Ag-experienced T cells was Ag-specific. PCC-specific 5C.C7 T cells were incubated with specific Ag, PCC, or with control OVA and then assayed for their ability to respond to the anti-CD3 QD. After 72 h, naive 5C.C7 cells responded minimally to anti-CD3 QD without the presence of peptide (Fig. 3, Supplemental Fig. 2B). 5C.C7 T cells coincubated with OVA Ag failed to respond to 18.5 pM anti-CD3 QD (Fig. 3). In addition, stimulation of Ag-experienced T cells was not a property of unmodified QD, because SA-coated QD lacking anti-CD3 failed to stimulate proliferation of the Ag-experienced 5C.C7 T cells (Fig. 3).

FIGURE 3.
FIGURE 3.

CD4+ T cell activation by anti-CD3 QD is Ag-specific and does not occur with empty QD. CFSE dilution in naive 5C.C7 splenocytes stimulated with no Ag, 50 nM PCC, or 50 nM OVA with or without 18.5 pM anti-CD3 QD or 18.5 pM SA-QD for 3 d. Data are representative of three independent experiments.

Constraining anti-CD3 Abs on nanoparticles selectively activates both CD4+ and CD8+ T cells

First, we repeated our experiments with splenocytes from RAG+/+ 6.5 TCR-transgenic mice. The 6.5-transgenic TCR is specific for class II HA peptide. However, because these mice are wild-type (WT) for the RAG gene, only 10–20% of the CD4+ T cells express the transgenic TCR. The remaining cells, which are negative for the transgenic TCR (6.5−), express TCR with other specificities. This allows us to simultaneously evaluate the specificity of anti-CD3 QD for Ag-specific and endogenous T cells in a single culture. We stimulated the splenocytes with HA II peptide for 48 h followed by the addition of anti-CD3 QD or soluble anti-CD3. Proliferation was analyzed 3 d after addition of anti-CD3 QD. Both 6.5+ and 6.5− T cells proliferated in response to 23 nM soluble anti-CD3 regardless of whether the cells were preincubated with HA peptide (Fig. 4A). In contrast none of the 6.5+ or 6.5− T cells proliferated in response to 8.7 nM anti-CD3 QD alone without HA peptide. Low-dose HA II peptide, as expected, only stimulated the 6.5+ T cells. The addition of anti-CD3 QD to such cultures led to a marked enhancement of proliferation of the 6.5+ T cells but not of the 6.5− T cells (Fig. 4A). There were similar concentrations of anti-CD3 in both culture conditions: 23 nM in soluble Ab and ∼45 nM in anti-CD3 QD, assuming there are 5–10 Abs on a QD (15). Because the amounts of anti-CD3 Abs are similar, the enhancement of activation by anti-CD3 QD does not reflect a large difference in Ab available for cross-linking TCR, but rather the difference between the cross-linking capacity of soluble Ab molecules and that of Abs constrained onto nanoparticles.

FIGURE 4.
FIGURE 4.

Anti-CD3 QD promote selective, Ag-specific, activation of both CD4+ and CD8+ T cells. (A) Splenocytes from 6.5 TCR-transgenic mice were stimulated with or without HA II peptide for 2 d followed by addition of media, soluble anti-CD3 Ab, or anti-CD3 QD and cultured for another 2 d. CFSE dilutions in CD4+6.5− gated T cells and CD4+6.5+ gated T cells from the same cultures are shown. (B) Splenocytes from DO11.10 RAG2−/− (Thy1.2) and WT B10.D2 (Thy1.1) mice were mixed and stimulated with or without OVA II peptide for 1 d followed by addition of media or anti-CD3 QD for another 2 d culture. eFluor 670 dilutions in WT, CD4+Thy1.1+ gated, and DO11.10, CD4+Thy1.2+ gated, cells from the same cultures are shown. (C) Splenocytes from clone 4 TCR-transgenic mice were stimulated with or without HA I peptide for 1 d followed by addition of media or anti-CD3 QD and cultured for another 2 d. CFSE dilutions versus CD8+Vβ8.1.2+ population are shown. (D) Splenocytes from OT-1 RAG2−/− (Thy1.1) and WT C57BL/6 (Thy1.2) mice were mixed and stimulated with or without OVA I peptide for 1 d followed by addition of media or anti-CD3 QD and cultured for another 2 d. eFluor 670 dilutions in WT (CD8+Thy1.2+ gated) and OT-1 (CD4+Thy1.1+ gated) cells from the same cultures are shown. Data are representative of three independent experiments.

We next tested a second experimental system for the selectivity of anti-CD3 QD in activating Ag-specific CD4+ T cells. Splenocytes from class II OVA–specific DO11.10 RAG2−/− (Thy1.2) mice and WT B10.D2 (Thy1.1) mice, which share the same H-2d background, were cocultured with or without class II OVA peptide. Anti-CD3 QD or media was added to the culture 1 d later for an additional 2 d. Without the addition of OVA peptide, neither B10.D2 nor DO11.10 CD4+ cells proliferated (Fig. 4B). The addition of class II OVA peptide led to the proliferation of only the Ag-specific DO11.10 cells. The addition of anti-CD3 QD to the OVA-treated cultures led to the enhanced proliferation of the DO11.10 T cells but did not stimulate the B10.D2 CD4+ T cells (Fig. 4B).

Clustering of TCRs upon Ag-induced activation has also been shown for CD8+ T cells (16). We found that anti-CD3–coated QD also selectively enhance the activation of CD8+ T cells. RAG+/+ CD8+ clone 4 TCR-transgenic T cells, specific for class I HA peptide, were stimulated with 10 nM of HA I for 24 h. Anti-CD3 QD were then added into the culture to a final concentration of 10 nM 2 d before we harvested the cells. As was observed with CD4+ T cells, anti-CD3 QD alone did not induce proliferation in the absence of Ag. However, coculture of HA class I peptide with the anti-CD3–coated QD led to a marked enhancement of the Ag-specific (Vβ8.2+) CD8+ T cells (Fig. 4C). Thus, anti-CD3 QD could also selectively enhance the activation of Ag-specific CD8+ T cells.

We next tested the ability of anti-CD3–coated QD to selectively enhance CD8+ T cell activation in cultures containing T cells of mixed specificity. T cells from OVA-specific RAG2−/− CD8+ OT-1 (Thy1.1+) TCR-transgenic mice were mixed with T cells from WT C57BL/6 (Thy1.2) mice. The cells were cultured with or without class I OVA peptide in the presence and absence of anti-CD3 QD. Anti-CD3 QD alone or media failed to induce T cell proliferation in any of the CD8+ T cells (Fig. 4D). In the presence of class I OVA peptide, only the Ag-specific OT-1 cells proliferated. The addition of anti-CD3 QD to such cultures led to the enhanced activation of the OT-1 CD8+ T cells but not the other CD8+ T cells (Fig. 4D). Thus, anti-CD3 QD have the ability to selectively enhance the activation of Ag-specific CD8+ T cells as well as CD4+ T cells.

Anti-CD3 QD enhance effector generation and function

Thus far, we have demonstrated the ability of anti-CD3 QD to enhance the proliferation of CD4+ and CD8+ T cells. We next tested whether the anti-CD3 QD could promote the generation of specific CD4+ effector cells. Splenocytes from CD4+ 6.5 TCR-transgenic mice were stimulated in vitro with class II HA peptide for 48 h. Anti-CD3 QD were then added to the culture and cells incubated for another 4 d. In these long-term cultures, the addition of anti-CD3 QD led to an increase in the number of IFN-γ–producing 6.5+ T cells (Fig. 5A). This increase in IFN-γ–secreting cells was also reflected in the amount of IFN-γ secreted into the supernatant as measured by ELISA (Fig. 5B).

FIGURE 5.
FIGURE 5.

Anti-CD3 QD enhance cytokine production. (A) IFN-γ production in 6.5 splenocytes stimulated with or without HA II peptide for 2 d, followed by another 4-d culture with or without the presence of anti-CD3 QD. CD4+ gated cells were shown. (B) IFN-γ production by 6.5 splenocytes. Data are representative of three independent experiments. (C) Schematic representation of experiment setup. Naive 5C.C7 splenocytes were CFSE labeled and stimulated with PCC in Th1- or Th2-skewing conditions for 2 d followed by boosting with various concentration of anti-CD3 QD for 1 more d. (D) CFSE dilution and cytokine production of 5C.C7 CD4+ T cells labeled, skewed, and boosted as in (C). Data are representative of three independent experiments. ***p < 0.001 (two-tailed t test).

Anti-CD3 QD selectively enhanced effector cell generation under specific Th skewing conditions (Fig. 5C). Naive 5C.C7 splenocytes were incubated with PCC peptide under conditions that would promote the generation of either Th1 (IFN-γ plus IL-12 plus anti–IL-4 Ab) or Th2 (IL-4 plus anti–IFN-γ Ab plus anti–IL-12 Ab) effector cells for 2 d. On the third day, the cells were incubated with different concentrations of anti-CD3 QD and then assayed for both proliferation and cytokine production. As expected, anti-CD3 QD enhanced the proliferation of Ag-activated T cells in both Th1 and Th2 conditions (Fig. 5D). Furthermore, the addition of the anti-CD3 QD enhanced the production of IFN-γ in the cells activated under Th1 conditions and production of IL-4 in the cells activated under Th2 conditions. Thus, the anti-CD3 QD enhanced both proliferation and cytokine production in Ag-experienced CD4+ effector cells.

Anti-CD3 QD boosts responses to vaccines in vivo

Next, we wanted to determine if activation of T cells in vivo rendered them susceptible to activation by anti-CD3 QD. We transferred 6.5+Thy1.1+CD4+ T cells into WT B10.D2 Thy1.2+ mice, which were then injected with class II HA peptide mixed with CFA. Six days later, draining lymph nodes were harvested. The harvested cells were CFSE labeled and then cultured in media only or with anti-CD3 QD. After 3 d of in vitro restimulation with anti-CD3 QD, we observed that only the 6.5+CD4+ T cells responded. That is, the anti-CD3 QD only stimulated the lymph node T cells that had previously encountered Ag in vivo (Fig. 6A). Both 6.5+ and 6.5− T cells proliferated in positive control cultures, with soluble anti-CD3 (Fig. 6B) and abrogated specificity. Thus, in vivo–activated Ag-specific T cells were as responsive to anti-CD3 QD to cells that were activated in vitro.

FIGURE 6.
FIGURE 6.

Anti-CD3 QD boost Ag-specific responses of T cells stimulated in vivo. (A) CD4+ 6.5 splenocytes were transferred to recipient B10.D2 mice. The recipients were immunized s.c. with HA II peptide and CFA. Single-cell suspensions of draining lymph nodes obtained 6 d after vaccination were CFSE labeled and rechallenged with media, soluble anti-CD3, or anti-CD3 QD for another 2 d. Recovery of Ag-specific CD4+6.5+ cells is shown. (B) CFSE dilutions in CD4+6.5− gated cells and CD4+6.5+ gated cells from the same cultures were shown. Note, the decreased percentage of 6.5+ T cells in the cultures treated with soluble anti-CD3 is due to the relative increase in 6.5− T cells in response to this nonspecific stimulus. (C) Proportion of CD4+Vβ8.1.2+ cells in single-cell suspensions of draining lymph nodes from B10.D2 (Thy1.2) mice given adoptive transfer of CD4+ 6.5 splenocytes (Thy1.1) and immunized as in (A) or with PBS 2 d prior to s.c. boost with media or anti-CD3 QD; single-cell suspensions of draining lymph nodes were obtained 3 d after boost. (D) B10.D2 mice were given adoptive transfer and immunized as in (A) 1 d prior to s.c. boost with media or anti-CD3 QD. Mice were rechallenged with vaccinia-HA at day 11. Recovery of adoptively transferred cells at day 14 is shown as Thy1.1+ cells. IL-2 (E) and IFN-γ (F) production by splenocytes and cells from draining lymph nodes from (D) stimulated with HA-II peptide for overnight. Data are representative of three (A–C) or two (D–F) independent experiments. *p < 0.05 (two-tailed t test). dLN, Draining lymph node; O/N, overnight.

Finally, we wanted to determine if anti-CD3 QD could enhance the response to a vaccine in vivo. Again, 6.5+Thy1.1+CD4+ T cells were adoptively transferred into WT B10.D2 Thy1.2+ mice, which were then vaccinated s.c. with PBS or class II HA peptide with CFA to generate newly activated 6.5+ T cells. Two days later, mice were injected with PBS or anti-CD3 QD at the same site. Three days later, draining lymph nodes were harvested, and the frequency of Ag-specific CD4+Vβ8.1.2+ cells was evaluated. Treatment with anti-CD3 QD led to an increase in the frequency of vaccine-induced Ag-specific CD4+Vβ8.1.2+ T cells in the draining lymph nodes compared with the frequency in nodes of animals given PBS (Fig. 6C), but did not lead to widespread activation of endogenous memory T cells (Supplemental Fig. 2C–E). Thus, the sequenced administration of Ag and anti-CD3 QD enhanced the initial vaccine-induced expansion of Ag-specific T cells in vivo. To expand these findings, we performed a similar experiment to determine if the addition of anti-CD3 QD could enhance the generation of memory cells. Mice were vaccinated with peptide and then injected with PBS or anti-CD3 QD 1 d later. After an additional 11 d, the mice were infected with vaccinia virus that expresses HA peptide. We observed that mice which received the peptide (day 0) followed by the anti-CD3 QD (day 1) had an increased recall response when challenged with vaccinia virus that expresses HA peptide administered on day 12 (Fig. 6D). In addition, Ag-specific T cells from the anti-CD3 QD-treated mice had higher IFN-γ and IL-2 production than controls (Fig. 6E, 6F). These results are consistent with the hypothesis that the sequenced administration of Ag and anti-CD3 QD enhance the generation of functional memory cells. These data also argue against the possibility that enhanced proliferation of T cells induced by anti-CD3 QD results in cell death after the initial burst of proliferation.

Discussion

Nanoparticles have been explored for various applications not only because of their miniature size but also because of their relatively large surface area, which allows immobilization of multiple ligands (24–26). Owing to these unique physicochemical and functional properties, nanoparticles have been used as carriers for Ag delivery, with successful examples including enhancing the efficacy of dendritic cell–based cancer immunotherapy (24, 27–30). Colloidal semiconductor nanocrystals, often referred to as QD, are a new generation of fluorescent dyes with advantage of brightness, photobleaching resistance, good chemical stability, and tunable spectral properties compared with traditional organic fluorophores and fluorescent-tagging molecules (15, 16). Ab-conjugated QD were developed for high-resolution labeling and used to show the coclustering of TCR and CD4 or CD8 coreceptor in microclusters in recently activated T cells (6, 31). In this report, we are able to exploit the unique properties of Ab-coated QD, not for imaging, but rather to selectively activate Ag-specific T cells.

Although soluble anti-CD3 acts as a potent T cell mitogen, activating T cells indiscriminate of TCR specificity (23), we find that anti-CD3 constrained on the surface of a nanoparticle specifically activates Ag-experienced T cells. Thus, by constraining anti-CD3 on the surface of nanoparticles, we have imparted selectivity. We hypothesize that this selectivity is due to the fact that, unlike naive T cells, Ag-activated T cells display microclusters of the TCR on their surface (5, 6, 16). TCRs in these clusters are more sensitive to cross linking by the anti-CD3–coated nanoparticles than are TCR dispersed in smaller clusters across the surface of naive cells. In support of this hypothesis is our finding that T cells that have been rested for 6 d after Ag stimulation, and still display TCR microclusters, remain sensitive to activation by anti-CD3–coated nanoparticles. In addition, preliminary studies employing (larger) 100-nm CD3-gold particles activated both naive and Ag-experienced T cells (data not shown). Future studies employing a series of anti-CD3–coated gold particles of varying sizes will better define the relationship between the cluster size and the selective responsiveness to anti-CD3–constrained agonists. Alternatively, it is possible that the ability of anti-CD3–coated nanoparticles to selectively activate Ag-specific T cells is not due to the differences in microclusters exhibited by activated and resting T cells. It may be that the signaling machinery of previously activated T cells is more sensitive to limited exposure of anti-CD3 than resting T cells. Indeed, the kinetic segregation model suggests that these differences in response to Ag are due to the local changes in the balance of kinases and phosphatases associated with the TCR (32). Thus, it is possible that our results are not secondary to clustering per se, but rather due to changes in the topography of kinases and phosphatases in the Ag-experienced cells that make them more sensitive to activation when anti-CD3–coated nanoparticles engage TCR.

Our in vivo data suggest that anti-CD3–coated nanoparticles could be employed clinically to enhance the magnitude of the response to vaccines for both infectious diseases as well as tumors. In vivo, the administration of the anti-CD3–coated nanoparticles enhanced the frequency of the Ag-specific T cells without any evidence of nonspecific T cell activation. Such findings suggest that the coadministration of Ag and anti-CD3–coated nanoparticles might lead to the enhancement of the T cell responses. Furthermore, we find that the administration of anti-CD3–coated nanoparticles during the initial encounter with Ag can result in increased generation of memory cells. Indeed, the mice treated with the anti-CD3–coated nanoparticles demonstrated increased recall responses upon rechallenge 10 d later. Such a finding suggests that this strategy might be employed to enhance the efficacy of preventative vaccines by boosting the generation of memory cells.

We believe that our findings have broad implications for promoting specificity even outside of the immune system. The selectivity of receptor–ligand interactions imparts signaling specificity. The specific expression of receptors on different cell types enables biologic selectivity. However, broad distribution of a receptor can present a hurdle to developing pharmacologic agents. The benefits of the specific biochemical pathways blocked or induced by ligand specificity may be mitigated by the fact that receptor is expressed on a diversity of cells. One such example of this is seen in the use of the anti-lymphoma agent anti-CD20 (rituximab) (33). CD20 is expressed on lymphoma cells (34), and indeed, rituximab is efficient at destroying tumor cells. In contrast, the expression of CD20 on all B cells means that a consequence of treatment is the depletion of nonmalignant B cells. If CD20 is clustered on lymphoma cells when compared with normal B cells, it is possible that constraining anti-CD20 on the surface of a nanoparticle might demonstrate greater selectivity for lymphoma cells. That is, constraining a particular ligand on the surface of a nanoparticle may promote the targeted activation of its receptor selectively on the desired cell type.

Disclosures

The authors have no financial conflicts of interest.

Acknowledgments

We thank the members of the Powell and Drake Laboratories for helpful suggestions and reagent contributions. We also thank Dr. Scot Kuo and others at the Johns Hopkins University School of Medicine Microscope Facility for advice on confocal microscopy.

Footnotes

  • This work was supported by National Institutes of Health Grants R56AI099276 and P01AI072677.

  • The online version of this article contains supplemental material.

  • Abbreviations used in this article:

    HA
    hemagglutinin
    QD
    quantum dot
    SA
    streptavidin
    WT
    wild-type.

  • Received May 31, 2013.
  • Accepted September 12, 2013.

References

  1. ↵
    1. Bonilla F. A.,
    2. H. C. Oettgen
    . 2010. Adaptive immunity. J. Allergy Clin. Immunol. 125(Suppl 2): S33–S40.
    OpenUrlCrossRefPubMed
  2. ↵
    1. Litman G. W.,
    2. J. P. Rast,
    3. S. D. Fugmann
    . 2010. The origins of vertebrate adaptive immunity. Nat. Rev. Immunol. 10: 543–553.
    OpenUrlCrossRefPubMed
  3. ↵
    1. Davis M. M.,
    2. P. J. Bjorkman
    . 1988. T-cell antigen receptor genes and T-cell recognition. Nature 334: 395–402.
    OpenUrlCrossRefPubMed
  4. ↵
    1. Dustin M. L.,
    2. J. T. Groves
    . 2012. Receptor signaling clusters in the immune synapse. Annu Rev Biophys 41: 543–556.
    OpenUrlCrossRefPubMed
  5. ↵
    1. Fahmy T. M.,
    2. J. G. Bieler,
    3. M. Edidin,
    4. J. P. Schneck
    . 2001. Increased TCR avidity after T cell activation: a mechanism for sensing low-density antigen. Immunity 14: 135–143.
    OpenUrlPubMed
  6. ↵
    1. Zhong L.,
    2. G. Zeng,
    3. X. Lu,
    4. R. C. Wang,
    5. G. Gong,
    6. L. Yan,
    7. D. Huang,
    8. Z. W. Chen
    . 2009. NSOM/QD-based direct visualization of CD3-induced and CD28-enhanced nanospatial coclustering of TCR and coreceptor in nanodomains in T cell activation. PLoS ONE 4: e5945.
    OpenUrlCrossRefPubMed
  7. ↵
    1. Anikeeva N.,
    2. D. Gakamsky,
    3. J. Schøller,
    4. Y. Sykulev
    . 2012. Evidence that the density of self peptide-MHC ligands regulates T-cell receptor signaling. PLoS ONE 7: e41466.
    OpenUrlCrossRefPubMed
    1. Fernández-Miguel G.,
    2. B. Alarcón,
    3. A. Iglesias,
    4. H. Bluethmann,
    5. M. Alvarez-Mon,
    6. E. Sanz,
    7. A. de la Hera
    . 1999. Multivalent structure of an alphabetaT cell receptor. Proc. Natl. Acad. Sci. USA 96: 1547–1552.
    OpenUrlAbstract/FREE Full Text
    1. Grakoui A.,
    2. S. K. Bromley,
    3. C. Sumen,
    4. M. M. Davis,
    5. A. S. Shaw,
    6. P. M. Allen,
    7. M. L. Dustin
    . 1999. The immunological synapse: a molecular machine controlling T cell activation. Science 285: 221–227.
    OpenUrlAbstract/FREE Full Text
  10. ↵
    1. Minguet S.,
    2. M. Swamy,
    3. B. Alarcón,
    4. I. F. Luescher,
    5. W. W. Schamel
    . 2007. Full activation of the T cell receptor requires both clustering and conformational changes at CD3. Immunity 26: 43–54.
    OpenUrlCrossRefPubMed
  11. ↵
    1. Schamel W. W.,
    2. I. Arechaga,
    3. R. M. Risueño,
    4. H. M. van Santen,
    5. P. Cabezas,
    6. C. Risco,
    7. J. M. Valpuesta,
    8. B. Alarcón
    . 2005. Coexistence of multivalent and monovalent TCRs explains high sensitivity and wide range of response. J. Exp. Med. 202: 493–503.
    OpenUrlAbstract/FREE Full Text
  12. ↵
    1. Huppa J. B.,
    2. M. Axmann,
    3. M. A. Mörtelmaier,
    4. B. F. Lillemeier,
    5. E. W. Newell,
    6. M. Brameshuber,
    7. L. O. Klein,
    8. G. J. Schütz,
    9. M. M. Davis
    . 2010. TCR-peptide-MHC interactions in situ show accelerated kinetics and increased affinity. Nature 463: 963–967.
    OpenUrlCrossRefPubMed
  13. ↵
    1. Kumar R.,
    2. M. Ferez,
    3. M. Swamy,
    4. I. Arechaga,
    5. M. T. Rejas,
    6. J. M. Valpuesta,
    7. W. W. Schamel,
    8. B. Alarcon,
    9. H. M. van Santen
    . 2011. Increased sensitivity of antigen-experienced T cells through the enrichment of oligomeric T cell receptor complexes. Immunity 35: 375–387.
    OpenUrlCrossRefPubMed
  14. ↵
    1. Alivisatos P.
    2004. The use of nanocrystals in biological detection. Nat. Biotechnol. 22: 47–52.
    OpenUrlCrossRefPubMed
  15. ↵
    1. Mattoussi H.,
    2. J. M. Mauro,
    3. E. R. Goldman,
    4. G. P. Anderson,
    5. V. C. Sundar,
    6. F. V. Mikulec,
    7. M. G. Bawendi
    . 2000. Self-assembly of CdSe-ZnS quantum dot bioconjugates using an engineered recombinant protein. J. Am. Chem. Soc. 122: 12142–12150.
    OpenUrlCrossRef
  16. ↵
    1. Boyle S.,
    2. D. L. Kolin,
    3. J. G. Bieler,
    4. J. P. Schneck,
    5. P. W. Wiseman,
    6. M. Edidin
    . 2011. Quantum dot fluorescence characterizes the nanoscale organization of T cell receptors for antigen. Biophys. J. 101: L57–L59.
    OpenUrlCrossRefPubMed
  17. ↵
    1. Lillemeier B. F.,
    2. M. A. Mörtelmaier,
    3. M. B. Forstner,
    4. J. B. Huppa,
    5. J. T. Groves,
    6. M. M. Davis
    . 2010. TCR and Lat are expressed on separate protein islands on T cell membranes and concatenate during activation. Nat. Immunol. 11: 90–96.
    OpenUrlCrossRefPubMed
  18. ↵
    1. Bruchez M., Jr..,
    2. M. Moronne,
    3. P. Gin,
    4. S. Weiss,
    5. A. P. Alivisatos
    . 1998. Semiconductor nanocrystals as fluorescent biological labels. Science 281: 2013–2016.
    OpenUrlAbstract/FREE Full Text
    1. Hotz C. Z.
    2005. Applications of quantum dots in biology: an overview. Methods Mol. Biol. 303: 1–17.
    OpenUrlPubMed
  20. ↵
    1. Michalet X.,
    2. F. F. Pinaud,
    3. L. A. Bentolila,
    4. J. M. Tsay,
    5. S. Doose,
    6. J. J. Li,
    7. G. Sundaresan,
    8. A. M. Wu,
    9. S. S. Gambhir,
    10. S. Weiss
    . 2005. Quantum dots for live cells, in vivo imaging, and diagnostics. Science 307: 538–544.
    OpenUrlAbstract/FREE Full Text
  21. ↵
    1. Tanaka K. A.,
    2. K. G. Suzuki,
    3. Y. M. Shirai,
    4. S. T. Shibutani,
    5. M. S. Miyahara,
    6. H. Tsuboi,
    7. M. Yahara,
    8. A. Yoshimura,
    9. S. Mayor,
    10. T. K. Fujiwara,
    11. A. Kusumi
    . 2010. Membrane molecules mobile even after chemical fixation. Nat. Methods 7: 865–866.
    OpenUrlCrossRefPubMed
  22. ↵
    1. Meuer S. C.,
    2. R. E. Hussey,
    3. D. A. Cantrell,
    4. J. C. Hodgdon,
    5. S. F. Schlossman,
    6. K. A. Smith,
    7. E. L. Reinherz
    . 1984. Triggering of the T3-Ti antigen-receptor complex results in clonal T-cell proliferation through an interleukin 2-dependent autocrine pathway. Proc. Natl. Acad. Sci. USA 81: 1509–1513.
    OpenUrlAbstract/FREE Full Text
  23. ↵
    1. Van Wauwe J. P.,
    2. J. R. De Mey,
    3. J. G. Goossens
    . 1980. OKT3: a monoclonal anti-human T lymphocyte antibody with potent mitogenic properties. J. Immunol. 124: 2708–2713.
    OpenUrlAbstract
  24. ↵
    1. Cho N. H.,
    2. T. C. Cheong,
    3. J. H. Min,
    4. J. H. Wu,
    5. S. J. Lee,
    6. D. Kim,
    7. J. S. Yang,
    8. S. Kim,
    9. Y. K. Kim,
    10. S. Y. Seong
    . 2011. A multifunctional core-shell nanoparticle for dendritic cell-based cancer immunotherapy. Nat. Nanotechnol. 6: 675–682.
    OpenUrlCrossRefPubMed
    1. Sun C.,
    2. J. S. Lee,
    3. M. Zhang
    . 2008. Magnetic nanoparticles in MR imaging and drug delivery. Adv. Drug Deliv. Rev. 60: 1252–1265.
    OpenUrlCrossRefPubMed
  26. ↵
    1. Klippstein R.,
    2. D. Pozo
    . 2010. Nanotechnology-based manipulation of dendritic cells for enhanced immunotherapy strategies. Nanomedicine 6: 523–529.
    OpenUrlCrossRefPubMed
  27. ↵
    1. Hamdy S.,
    2. A. Haddadi,
    3. A. Shayeganpour,
    4. J. Samuel,
    5. A. Lavasanifar
    . 2011. Activation of antigen-specific T cell-responses by mannan-decorated PLGA nanoparticles. Pharm. Res. 28: 2288–2301.
    OpenUrlCrossRefPubMed
    1. Hirosue S.,
    2. I. C. Kourtis,
    3. A. J. van der Vlies,
    4. J. A. Hubbell,
    5. M. A. Swartz
    . 2010. Antigen delivery to dendritic cells by poly(propylene sulfide) nanoparticles with disulfide conjugated peptides: Cross-presentation and T cell activation. Vaccine 28: 7897–7906.
    OpenUrlCrossRefPubMed
    1. Jewell C. M.,
    2. S. C. López,
    3. D. J. Irvine
    . 2011. In situ engineering of the lymph node microenvironment via intranodal injection of adjuvant-releasing polymer particles. Proc. Natl. Acad. Sci. USA 108: 15745–15750.
    OpenUrlAbstract/FREE Full Text
  30. ↵
    1. Nembrini C.,
    2. A. Stano,
    3. K. Y. Dane,
    4. M. Ballester,
    5. A. J. van der Vlies,
    6. B. J. Marsland,
    7. M. A. Swartz,
    8. J. A. Hubbell
    . 2011. Nanoparticle conjugation of antigen enhances cytotoxic T-cell responses in pulmonary vaccination. Proc. Natl. Acad. Sci. USA 108: E989–E997.
    OpenUrlAbstract/FREE Full Text
  31. ↵
    1. Pathak S.,
    2. M. C. Davidson,
    3. G. A. Silva
    . 2007. Characterization of the functional binding properties of antibody conjugated quantum dots. Nano Lett. 7: 1839–1845.
    OpenUrlCrossRefPubMed
  32. ↵
    1. Davis S. J.,
    2. P. A. van der Merwe
    . 2006. The kinetic-segregation model: TCR triggering and beyond. Nat. Immunol. 7: 803–809.
    OpenUrlCrossRefPubMed
  33. ↵
    1. Maloney D. G.,
    2. A. J. Grillo-López,
    3. C. A. White,
    4. D. Bodkin,
    5. R. J. Schilder,
    6. J. A. Neidhart,
    7. N. Janakiraman,
    8. K. A. Foon,
    9. T. M. Liles,
    10. B. K. Dallaire,
    11. et al
    . 1997. IDEC-C2B8 (Rituximab) anti-CD20 monoclonal antibody therapy in patients with relapsed low-grade non-Hodgkin’s lymphoma. Blood 90: 2188–2195.
    OpenUrlAbstract/FREE Full Text
  34. ↵
    1. Anderson K. C.,
    2. M. P. Bates,
    3. B. L. Slaughenhoupt,
    4. G. S. Pinkus,
    5. S. F. Schlossman,
    6. L. M. Nadler
    . 1984. Expression of human B cell-associated antigens on leukemias and lymphomas: a model of human B cell differentiation. Blood 63: 1424–1433.
    OpenUrlAbstract/FREE Full Text
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