Immune Netw.  2014 Aug;14(4):207-218. 10.4110/in.2014.14.4.207.

Enhancing T Cell Immune Responses by B Cell-based Therapeutic Vaccine Against Chronic Virus Infection

• Kim MK
• Lee A
• Hwang YK
• Kang CY
• Ha SJ

• KMID: 2150809
• DOI: http://doi.org/10.4110/in.2014.14.4.207

Abstract

Chronic virus infection leads to the functional impairment of dendritic cells (DCs) as well as T cells, limiting the clinical usefulness of DC-based therapeutic vaccine against chronic virus infection. Meanwhile, B cells have been known to maintain the ability to differentiate plasma cells producing antibodies even during chronic virus infection. Previously, alpha-galactosylceramide (alphaGC) and cognate peptide-loaded B cells were comparable to DCs in priming peptide-specific CD8+ T cells as antigen presenting cells (APCs). Here, we investigated whether B cells activated by alphaGC can improve virus-specific T cell immune responses instead of DCs during chronic virus infection. We found that comparable to B cells isolated from naive mice, chronic B cells isolated from chronically infected mice with lymphocytic choriomeningitis virus (LCMV) clone 13 (CL13) after alphaGC-loading could activate CD1d-restricted invariant natural killer T (iNKT) cells to produce effector cytokines and upregulate co-stimulatory molecules in both naive and chronically infected mice. Similar to naive B cells, chronic B cells efficiently primed LCMV glycoprotein (GP) 33-41-specific P14 CD8+ T cells in vivo, thereby allowing the proliferation of functional CD8+ T cells. Importantly, when alphaGC and cognate epitope-loaded chronic B cells were transferred into chronically infected mice, the mice showed a significant increase in the population of epitope-specific CD8+ T cells and the accelerated control of viremia. Therefore, our studies demonstrate that reciprocal activation between alphaGC-loaded chronic B cells and iNKT cells can strengthen virus-specific T cell immune responses, providing an effective regimen of autologous B cell-based therapeutic vaccine to treat chronic virus infection.

Keyword

Chronic virus infection; B-cell based therapeutic vaccine; alpha-galactosylceramide; T cell immune responses

MeSH Terms

Animals
Antibodies
Antigen-Presenting Cells
B-Lymphocytes
Clone Cells
Cytokines
Dendritic Cells
Glycoproteins
Lymphocytic choriomeningitis virus
Mice
Natural Killer T-Cells
Plasma Cells
T-Lymphocytes
Viremia
Antibodies
Cytokines
Glycoproteins

Figure

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  Figure 1 Comparison of αGC-loaded naïve or chronic B cells for bidirectional activation of iNKT and B cells in naïve mice. naïve and chronic B cells were isolated from splenocytes of naïve mice and chronically infected mice that were initially depleted of CD4+ T cells and subsequently infected with LCMV CL13 (over 90 d p.i.), respectively. (A) Schedule for αGC-loading onto B cells and generation of in vivo activated iNKT cells and B cells. Naive and chronic B cells isolated from Ly5.1+ naïve and chronically infected mice were cultured with vehicle (veh) or αGC in vitro for 18~20 h. 2×106 cells of αGC-loaded B cells were adoptively transferred into Ly5.2+ congenic naive mice. The recipient mice were sacrificed 6 and 24 h after adoptive transfer of B cells for analysis of the activation of iNKT cells and donor B cells, respectively. (B) In vivo activation of iNKT cells by αGC-loaded naïve and chronic B cells in naïve mice. Frequency of iNKT cells among CD4+ T cells was examined by staining with CD1d tetramer (tet) and TCR-β (CD1d tet+ TCR-βinter) and their function was evaluated by intracellular staining of IFN-γ and TNF-α. The number in the plot indicates the percent of corresponding population. (C) In vivo activation of donor αGC-loaded B cells by activated iNKT cells in naïve mice. Ly5.1+ CD19+ donor B cells were gated and analyzed for the expression of surface molecules. The number in histogram plot represents mean fluorescence intensity (MFI) of the expressed protein. The vertical grey line in histogram plot indicate a geometric mean level of the protein expressed on naïve B cells loaded with vehicle. Results are representative of at least three independent experiments.

• 

  Figure 2 Comparison of αGC-loaded naïve or chronic B cells for bidirectional activation of iNKT and B cells in chronically infected mice. Naïve and chronic B cells were isolated from splenocytes of naïve mice and chronically infected mice that were initially depleted of CD4+ T cells and subsequently infected with LCMV CL13 (over 90 d p.i.), respectively. (A) Schedule for αGC-loading onto B cells and generation of in vivo activated iNKT cells and B cells. Naive and chronic B cells isolated from Ly5.1+ naïve and chronically infected mice were cultured with veh or αGC in vitro for 18~20 h. 2×106 cells of αGC-loaded B cells were adoptively transferred into Ly5.2+ congenic mice that were already infected with LCMV CL13 (over 90 d p.i.). The recipient mice were sacrificed 6 and 24 h after adoptive transfer of B cells for analysis of the activation of iNKT cells and donor B cells, respectively. (B) In vivo activation of iNKT cells by αGC-loaded naïve and chronic B cells in chronically infected mice. Frequency of iNKT cells among CD4+ T cells was examined by staining with CD1d tet and TCR-β (CD1d tet+ TCR-βinter) and their function was evaluated by intracellular staining of IFN-γ and TNF-α. The number in the plot indicates the percent of corresponding population. (C) In vivo activation of donor αGC-loaded B cells by activated iNKT cells in chronically infected mice. Ly5.1+ CD19+ donor B cells were gated and analyzed for the expression of surface molecules. The number in histogram plot represents mean fluorescence intensity (MFI) of the expressed protein. The vertical grey line in histogram plot indicate a geometric mean level of the protein expressed on naïve B cells loaded with vehicle. Results are representative of at least three independent experiments.

• 

  Figure 3 In vivo priming of antigen-specific CD8+ T cells by αGC and epitope-loaded naïve and chronic B cells. Naïve and chronic B cells were isolated from splenocytes of naïve mice and chronically infected mice that were initially depleted of CD4+ T cells and subsequently infected with LCMV CL13 (over 90 d p.i.), respectively. Naïve P14 CD8+ T cells were purified from splenocytes of naïve P14 mice. (A) Schedule for analysis for in vivo activity of αGC and peptide-loaded naïve and chronic B cells to prime antigen-specific CD8+ T cells. 5×106 of CellTrace™ Violet (CTV)-labeled P14 Thy1.1+ CD8+ T cells were adoptively transferred into Thy1.2+ congenic naïve mice. After 24 h, the mice were given with 1×106 of naïve or chronic B cells loaded with veh, αGC, veh plus GP33-41 peptide (GP33), or αGC plus GP33. The recipient mice were sacrificed 48 h after adoptive transfer of B cells for analysis of donor P14 cells. (B and C) Proliferation (1st row) and cytokine production (2nd to 4th row) of P14 cells primed with αGC plus GP33-loaded naïve (B) and chronic B cells (C). Donor Thy1.1+ CD8+ T cells in the spleen were gated and examined for CTV dilution along with cytokine production. Division time and frequency of dividing cell population are indicated in histogram plot. The number as shown in each quadrant of the plot represents percentage of the corresponding cell population. Results are representative of at least three independent experiments.

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  Figure 4 Effect of therapeutic vaccination with αGC and epitope-loaded naïve B cells on T cell responses and virus control during chronic virus infection. LCMV CL13-infected mice were vaccinated with 1×107 of veh, αGC, veh plus GP33, or αGC plus GP33-loaded naïve B cells at 25 d p.i. (A) Frequency of DbGP33-41 tet-positive cells among CD8+ T cells in the chronically infected mice after therapeutic vaccination with B cells. At 0, 7, and 14 d post-vaccination (25, 32, and 39 d p.i.), CD8+ T cells in the blood were gated and evaluated for the expansion of DbGP33-41 tet+ cells. (B) Fold-increase of DbGP33-41 tetramer specific CD8+ T cells in PBMCs between 0 and 7 d post-vaccination. (C) Change of virus titer in the blood post-vaccination. Serum virus titer was determined from individual mice before (17 d p.i.) and after therapeutic vaccination with B cells (39 d p.i.). Vertical line in the graph indicates time point when therapeutic vaccination was performed. (D) Reduction of serum virus titer after B cell therapeutic vaccination. Fold-decrease of serum virus titer was calculated by serum virus titer before therapeutic vaccination (17 d p.i.) divided by that after therapeutic vaccination with B cells (39 d p.i.). Results are representative of two independent experiments. n=4 mice per group in each experiment. ns, not significant; *p<0.05; **p<0.01.

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  Figure 5 Expansion of epitope-specific CD8+ T cells and their effector function after therapeutic vaccination with αGC and epitope-loaded naïve B cells. LCMV CL13-infected mice were vaccinated with 1×107 of veh, αGC, veh plus GP33, or αGC plus GP33-loaded naïve B cells at 25 d p.i. and sacrificed at 39 d p.i. (14 d post-vaccination). (A) Frequency of DbGP33-41 tet+ cells among CD8+ T cells in the spleen. (B) Total number of DbGP33-41 tet+ cells in the spleen and lung. (C) IFN-γ and CD107a expression in CD8+ T cells in the spleen after in vitro stimulation with GP33-41 or GP276-286 peptide. (D) Proportion of CD8+ T cells producing IFN-γ and CD107a in the spleen after peptide stimulation. Number in the plot indicates percentage of the corresponding cells. Results are representative of two independent experiments. n=3 mice per group in each experiment. ns, not significant; *p<0.05; ***p<0.001.

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  Figure 6 Therapeutic efficacy of αGC and epitope-loaded chronic B cells during chronic virus infection. Four different groups of B cells were purified from LCMV CL13-infected mice (25 d p.i.) and treated with veh plus GP33 or αGC plus GP33. LCMV CL13-infected mice were vaccinated at 25 d p.i. with 1×107 of each group of B cells as therapeutic vaccine and sacrificed at 39 d p.i. (14 d post-vaccination). (A) Fold-increase of DbGP33-41 tetramer specific CD8+ T cells in PBMCs between 0 and 7 d post-vaccination (between 25 and 32 d p.i.),. (B) Reduction of serum virus titer after B cell therapeutic vaccination. Fold-decrease of serum virus titer was calculated by serum virus titer before therapeutic vaccination (17 d p.i.) divided by that after therapeutic vaccination with B cells (39 d p.i.). (C) Frequency of DbGP33-41 tet+ cells among CD8+ T cells in the spleen. (D) Total number of DbGP33-41 tet+ cells in the spleen. (E) IFN-γ and CD107a expression in CD8+ T cells in the spleen after in vitro stimulation with GP33-41 or GP276-286 peptide. (F) Proportion of CD8+ T cells producing IFN-γ and CD107a in the spleen after peptide stimulation. Number in the plot indicates percentage of the corresponding cells. Results are representative of two independent experiments. n=3 mice per group in each experiment. ns, not significant.

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