Timed Regulation of 3BP2 Induction Is Critical for Sustaining CD8+ T Cell Expansion and Differentiation

Abstract

Successful anti-viral response requires the sustained activation and expansion of CD8⁺ T cells for periods that far exceed the time limit of physical T cell interaction with antigen-presenting cells (APCs). The expanding CD8⁺ T cell pool generates the effector and memory cell populations that provide viral clearance and long-term immunity, respectively. Here, we demonstrate that 3BP2 is recruited in cytoplasmic microclusters and nucleates a signaling complex that facilitates MHC:peptide-independent activation of signaling pathways downstream of the TCR. We show that induction of the adaptor molecule 3BP2 is a sensor of TCR signal strength and is critical for sustaining CD8⁺ T cell proliferation and regulating effector and memory differentiation.

Keywords: 3BP2; CD8(+) T cells; TCR signaling; differentiation; microclusters; proliferation.

Figure 1.. CD8⁺ T Cells, Unlike CD4⁺ T Cells, Require 3BP2 for Optimal In Vitro Activation and Proliferation

(A) Flow cytometry analysis of CD8⁺ T cell activation markers in 3BP2^+/+ and 3BP2^−/− mice after 24 hr of in vitro stimulation with varying concentrations of anti-CD3 alone or with anti-CD3 plus anti-CD28 antibody. Representative plots from at least three independent experiments are shown. (B and C) In vitro proliferation of CD8⁺ T cells from 3BP2^+/+ and 3BP2^−/− mice was measured by either ³H-thymidine incorporation (B) or CFSE dilution (C). In (B), data are shown as mean ± SE from three independent experiments. In (C), a representative flow cytometry histogram is shown at 48 hr upon in vitro stimulation. (D) The proliferation of CD4⁺ T cells from 3BP2^+/+ and 3BP2^−/− mice was measured by flow cytometry analysis at 48 hr upon in vitro stimulation (anti-CD3 + anti-CD28). (E) Western blotanalysisofthe3BP2 protein expression in CD8⁺ T cellsfrom 3BP2^+/+and 3BP2^KI/KI mice ex vivo. Three independent experimentswere performed. (F) Flow cytometry analysis of the ex vivo expression of surface activation markers In CD8⁺T cells from 3BP2^+/+ and 3BP2^kI/kI mice. Representative plots from at least three independent experiments are shown. (G) Representative flow cytometry histograms of CFSE dilution analysis are shown from 3BP2^+/+ and 3BP2^kI/kI CD8⁺ T cells at 48 hr upon in vitro stimulation. (H) The proliferation of CD4⁺ T cells from 3BP2^+/+ and 3BP2^kI/kI mice was measured by flow cytometry analysis at 48 hr upon in vitro stimulation (anti-CD3 + anti-CD28). (I) CFSE dilution analysis at 48 hr upon P14⁺T cell stimulation in vitro with increasing concentrations of theGp33and the L6F antigenic peptides. Representative of more than three experiments is shown. (J) In vitro proliferation of 3BP2^+/+ or 3BP2-deficient P14⁺ T cells was determined by ³H-thymidine incorporation at 48 hr upon antigenic peptide stimulation. (K) IL-2 production by peptide stimulated P14⁺ T cells was determined using an ELISA assay. Gray areas and dotted lines in all histograms indicate unstimulated cells from 3BP2^+/+ and 3BP2^−/− mice or 3BP2^+/+ and 3BP2^ki/ki mice, respectively.

Figure 2.. 3BP2 Is Critical for the Expansion, Memory Generation, and Memory Response of P14⁺ T Cells upon Antigen Stimulation In Vivo

WT (CD45.2⁺) or 3BP2 (CD45.2⁺) deficient P14⁺ T cells were adoptively transferred In WT (CD45.1⁺) mice, and splenocytes were harvested 8 days post-lnfectlon with LCMV virus. (A) Representative fluorescence-activated cell sorting (FACS) plots showing percentage of CD8⁺Gp33⁺ tetramer-stained cells in the spleens of virus infected mice (left) and percentages of endogenous (CD45.1⁺) or transferred (CD45.2⁺) antigen-specific cells (right). (B) Pooled data from three independent experiments (n = 11 mice per group) showing total numbers of endogenous and adoptively transferred Gp33-specific CD8⁺ T cells in the spleens of infected animals. (C) Absolute numbers of IFN-γ-producing CD8⁺ T cells from infected spleens, determined after in vitro stimulation with Gp33 peptide. Pooled data from three independent experiments are shown (n = 11 mice per group). (D) In vivo effector differentiation of CD8⁺ T cells was examined by flow cytometry analysis of expression of effector markers. The gray areas indicate the expression on naive P14⁺ T cells. (E) Splenocytes from LCMV infected mice were harvested 100 days post-primary infection and total numbers of endogenous or transferred memory Gp33-specific CD8⁺ T cells were determined. (F) Spleen lymphocytes were harvested on day 3 post-challenge with vaccinia virus-Gp. Total numbers of endogenous (CD45.1⁺) and adoptively transferred (CD45.2⁺) Gp33-specific CD8⁺ T cells in the spleens of infected animals are shown. Brackets and p values indicate statistically significant differences.

Figure 3.. 3BP2 Is Induced in CD8⁺ T Cells upon TCR and CD28 Activation and Nucleates an LCK, ZAP-70, SLP-76, and VAV Signaling Module

(A) Spleen CD8⁺ T cells were isolated and activated in vitro. Samples were collected at 4,15, and 24 hr post-activation and 3BP2 mRNA levels were quantified by RT-PCR relative to GADPH levels. (B and C) Samples of CD8⁺ T cells activated with either plate-bound antibodies (B) or soluble anti-genic peptides (C) were collected and 3BP2 protein levels were visualized by western blot. B-actin levels were used as protein loading control. The numbers represent the fold increase of protein levels over the untreated control. (D and E) Purified spleen CD8⁺ T cells were antibody (D) or peptide (E) activated, and 3BP2 protein was immunoprecipitated from precleared soluble fractions. 3BP2-coimmunoprecipitated complexes were separated by SDS-PAGE and identified by western blot.

Figure 4.. The Adaptor Protein 3BP2 Is Enriched in Microcluster Signaling Complexes and Translocates to the Nucleus upon TCR Stimulation

(A) Live-cell imaging of Jurkat E6.1 cells transfected with WT 3BP2 GFP and plated on anti-CD3 coated coverslips. Stills taken from videos are presented as maximum Z projections. (B) Jurkat E6.1 cells transfected with WT 3BP2 GFP were stimulated for 3 min on anti-CD45 coated coverslips as negative control, fixed in 2.4% para-formaldehyde, and stained for the localization of 3BP2, phospho-LAT, phospho-SRC, and phosphotyrosine residues (pY). (C–E) Jurkat E56.1 cells transfected with WT 3BP2 GFP were stimulated for 3 min on anti-CD3 coated coverslips, fixed in 2.4% paraformaldehyde, and stained for the localization of 3BP2, phospho-ZAP-70 (C), pospho-SLP-76, phospho-LAT (E), phospho-SRC (D), and phosphotyrosine residues (pY) (E).

Figure 5.. 3BP2 Is Not Required for Priming and Early Activation Signaling in CD8⁺ T Cells

(A) Indo-1-loaded splenic CD8⁺ T cells were analyzed for ~120 s before stimulation with soluble anti-CD3 at 10 μg/mL. Changes in [Ca²⁺]_i following anti-CD3 ligation are shown, with arrows indicating the addition of the stimulus. 3BP2^+/+ cells are represented by black lines, and 3BP2^−/− cells are represented by gray lines. The data shown are representative of three independent experiments. (B) The phosphorylation profile of short-term-activated CD8⁺ T cells was detected by western blot using specific anti-phosphotyrosine (pY) antibodies. (C) Splenic CD8⁺ T cells from 3BP2^+/+ and 3BP2^−/− mice were activated in vitro for 5, 15, 30, and 60 min with plate-bound antibodies. Cells were lysed and protein extracts were separated by SDS-PAGE and immunoblotted for phosphory-lated and total LCK. (D) Induction of ZAP-70 phosphorylation was measured in CD8⁺ T cells by flow cytometry. Cells were activated by plate-bound antibodies (average of mean fluorescence intensity from three experiments is shown) or soluble peptides (representative histograms from three independent experiment) for 5, 15, and 30 min. (E) CD8⁺ T cells from 3BP2^+/+ and 3BP2^−/− mice were briefly stimulated in vitro for 2 and 6 hr and transferred in new wells, and their proliferation was measured by CFSE dilution at 48 hr after transfer. Gray areas and dotted lines in (D) and (E) indicate unstimulated cells from 3BP2^+/+ and 3BP2^−/− mice, respectively.

Figure 6.. 3BP2 Modulates Phosphotyrosine Signaling Downstream of the TCR

The phosphorylation profile of activated CD8⁺ T cells from 3BP2^+/+, 3BP2^−/−, and 3BP2^KI/KI mice was determined by western blotting at 24 hr upon in vitro stimulation with either plate-bound antibodies (A and C) or soluble antigenic peptides (B). B-actin levels were used as protein loading control. All lysates were subjected to SDS-PAGE and immunoblotted concurrently. The order of the samples was subsequently rearranged to conform with the text (boxes define WT and 3BP2 KO samples, respectively).

Figure 7.. The Adaptor Protein 3BP2 Is Required for Optimal ERK and NFAT Activation

CD8⁺ T cells from 3BP2^+/+ and 3BP2^−/− mice were activated in vitro for 24 hr. Cells were lysed, and protein extracts were separated by SDS-PAGE and im-munoblotted for phosphorylated and total proteins. (A and C) Plate-bound antibodies stimulation. (B and D) Antigenic peptide stimulation. (E) NFAT nuclear translocation was monitored in CD8⁺ T cells from 3BP2^+/+ and 3BP2^−/− mice activated in vitro with plate-bound antibodies. The numbers in all panels represent average fold increase in the protein phosphorylation levels compared with the untreated or the AV-treated control, respectively. Significance levels are shown in Figure S5. All lysates were subjected to SDS-PAGE and immunoblotted concurrently. The order of the samples was subsequently rearranged to conform with the text (boxes define WT and 3BP2 KO samples, respectively).