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Microfluidic squeezing for intracellular antigen loading in polyclonal B-cells as cellular vaccines.

Lee Szeto G, Van Egeren D, Worku H, Sharei A, Alejandro B, Park C, Frew K, Brefo M, Mao S, Heimann M, Langer R, Jensen K, Irvine DJ - Sci Rep (2015)

Bottom Line: However to date, a significant barrier to utilizing B-cells as APCs is their low capacity for non-specific antigen uptake compared to "professional" APCs such as dendritic cells.Squeezed B-cells primed and expanded large numbers of effector CD8(+)T-cells in vitro that produced effector cytokines critical to cytolytic function, including granzyme B and interferon-γ.Altogether, these data demonstrate crucial proof-of-concept for mechano-poration as an enabling technology for B-cell antigen loading, priming of antigen-specific CD8(+)T-cells, and decoupling of antigen uptake from B-cell activation.

View Article: PubMed Central - PubMed

Affiliation: 1] Department of Materials Science &Engineering, MIT [2] Department of Biological Engineering, MIT [3] David. H. Koch Institute for Integrative Cancer Research, MIT [4] The Ragon Institute of Harvard, MIT, and MGH.

ABSTRACT
B-cells are promising candidate autologous antigen-presenting cells (APCs) to prime antigen-specific T-cells both in vitro and in vivo. However to date, a significant barrier to utilizing B-cells as APCs is their low capacity for non-specific antigen uptake compared to "professional" APCs such as dendritic cells. Here we utilize a microfluidic device that employs many parallel channels to pass single cells through narrow constrictions in high throughput. This microscale "cell squeezing" process creates transient pores in the plasma membrane, enabling intracellular delivery of whole proteins from the surrounding medium into B-cells via mechano-poration. We demonstrate that both resting and activated B-cells process and present antigens delivered via mechano-poration exclusively to antigen-specific CD8(+)T-cells, and not CD4(+)T-cells. Squeezed B-cells primed and expanded large numbers of effector CD8(+)T-cells in vitro that produced effector cytokines critical to cytolytic function, including granzyme B and interferon-γ. Finally, antigen-loaded B-cells were also able to prime antigen-specific CD8(+)T-cells in vivo when adoptively transferred into mice. Altogether, these data demonstrate crucial proof-of-concept for mechano-poration as an enabling technology for B-cell antigen loading, priming of antigen-specific CD8(+)T-cells, and decoupling of antigen uptake from B-cell activation.

No MeSH data available.


Related in: MedlinePlus

Squeeze-delivered B-cells prime T-cells to produce effector CTL cytokines, while B-cell stimuli program distinct cytokine milieus.A) Secretion of major effector cytokines and molecules (granzyme B, IFN-γ, TNF-α, IL-2, CD137) were shown for co-cultures indicating T-cell activation state. B) Differential IL-6/IL-10 milieus induced in co-cultures by activating with different B-cell stimuli (n = 3 independent experiments for A & B). All data were represented as means±standard deviation. Pairs of conditions were tested for statistically significant pairwise differences with repeated measures 1-way ANOVA followed by Holm Sidak multiple comparisons test; multiplicity adjusted p-values < 0.05 were considered significant, and exact p-values were shown where significant.
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f4: Squeeze-delivered B-cells prime T-cells to produce effector CTL cytokines, while B-cell stimuli program distinct cytokine milieus.A) Secretion of major effector cytokines and molecules (granzyme B, IFN-γ, TNF-α, IL-2, CD137) were shown for co-cultures indicating T-cell activation state. B) Differential IL-6/IL-10 milieus induced in co-cultures by activating with different B-cell stimuli (n = 3 independent experiments for A & B). All data were represented as means±standard deviation. Pairs of conditions were tested for statistically significant pairwise differences with repeated measures 1-way ANOVA followed by Holm Sidak multiple comparisons test; multiplicity adjusted p-values < 0.05 were considered significant, and exact p-values were shown where significant.

Mentions: The functionality of B-cell-primed CD8+ T-cell populations was assayed by measuring secretion of effector molecules at days 2 and 4 of co-cultures. B-cells loaded with antigen by squeezing, whether resting or activated, primed T-cells to secrete substantial quantities of granzyme B, IFN-γ, and TNF-α, while B-cells loaded with antigen by endocytosis produced basal levels of cytokines (Fig. 4A). CpG-activated, squeezed B-cells primed CD8+T-cells that produced less granzyme B, but more IFN-γ and TNF-α when compared to resting squeezed B-cells, though these differences did not reach statistical significance. IL-2 and activation-induced release of soluble CD137 were also detected40, consistent with T-cell activation by both resting and activated mechano-porated B-cells (Fig. 4A). Effector cytokine secretion levels were similar between squeezed B-cells and positive controls--beads or peptide. In addition to T-cell-derived cytokines, we also examined potential cytokines in the co-culture milieu that could direct CTL priming and output. Different B-cell stimuli produced distinct cytokine secretion patterns as expected: CpG- and LPS-stimulated cells generated divergent ratios of IL-6 and IL-10 secretion, while mCD40L induced low levels of both (Fig. 4B). Thus, B-cells loaded with antigen by mechano-poration primed functional effector T-cells and produced an IL-6/IL-10 milieu that is known to be important in programming different priming, expansion, and differentiation potentials for effector41424344 and memory CD8+T-cells4546. Consistent with previous studies, combined IL-6 and IL-10 expression correlated with increased proliferation indices in innate-stimulated (CpG B, CpG C, LPS) co-cultures, while resting (SQZ) co-cultures had relatively lower proliferation and produced negligible IL-6/IL-10 (Supplementary Fig. S4).


Microfluidic squeezing for intracellular antigen loading in polyclonal B-cells as cellular vaccines.

Lee Szeto G, Van Egeren D, Worku H, Sharei A, Alejandro B, Park C, Frew K, Brefo M, Mao S, Heimann M, Langer R, Jensen K, Irvine DJ - Sci Rep (2015)

Squeeze-delivered B-cells prime T-cells to produce effector CTL cytokines, while B-cell stimuli program distinct cytokine milieus.A) Secretion of major effector cytokines and molecules (granzyme B, IFN-γ, TNF-α, IL-2, CD137) were shown for co-cultures indicating T-cell activation state. B) Differential IL-6/IL-10 milieus induced in co-cultures by activating with different B-cell stimuli (n = 3 independent experiments for A & B). All data were represented as means±standard deviation. Pairs of conditions were tested for statistically significant pairwise differences with repeated measures 1-way ANOVA followed by Holm Sidak multiple comparisons test; multiplicity adjusted p-values < 0.05 were considered significant, and exact p-values were shown where significant.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4441198&req=5

f4: Squeeze-delivered B-cells prime T-cells to produce effector CTL cytokines, while B-cell stimuli program distinct cytokine milieus.A) Secretion of major effector cytokines and molecules (granzyme B, IFN-γ, TNF-α, IL-2, CD137) were shown for co-cultures indicating T-cell activation state. B) Differential IL-6/IL-10 milieus induced in co-cultures by activating with different B-cell stimuli (n = 3 independent experiments for A & B). All data were represented as means±standard deviation. Pairs of conditions were tested for statistically significant pairwise differences with repeated measures 1-way ANOVA followed by Holm Sidak multiple comparisons test; multiplicity adjusted p-values < 0.05 were considered significant, and exact p-values were shown where significant.
Mentions: The functionality of B-cell-primed CD8+ T-cell populations was assayed by measuring secretion of effector molecules at days 2 and 4 of co-cultures. B-cells loaded with antigen by squeezing, whether resting or activated, primed T-cells to secrete substantial quantities of granzyme B, IFN-γ, and TNF-α, while B-cells loaded with antigen by endocytosis produced basal levels of cytokines (Fig. 4A). CpG-activated, squeezed B-cells primed CD8+T-cells that produced less granzyme B, but more IFN-γ and TNF-α when compared to resting squeezed B-cells, though these differences did not reach statistical significance. IL-2 and activation-induced release of soluble CD137 were also detected40, consistent with T-cell activation by both resting and activated mechano-porated B-cells (Fig. 4A). Effector cytokine secretion levels were similar between squeezed B-cells and positive controls--beads or peptide. In addition to T-cell-derived cytokines, we also examined potential cytokines in the co-culture milieu that could direct CTL priming and output. Different B-cell stimuli produced distinct cytokine secretion patterns as expected: CpG- and LPS-stimulated cells generated divergent ratios of IL-6 and IL-10 secretion, while mCD40L induced low levels of both (Fig. 4B). Thus, B-cells loaded with antigen by mechano-poration primed functional effector T-cells and produced an IL-6/IL-10 milieu that is known to be important in programming different priming, expansion, and differentiation potentials for effector41424344 and memory CD8+T-cells4546. Consistent with previous studies, combined IL-6 and IL-10 expression correlated with increased proliferation indices in innate-stimulated (CpG B, CpG C, LPS) co-cultures, while resting (SQZ) co-cultures had relatively lower proliferation and produced negligible IL-6/IL-10 (Supplementary Fig. S4).

Bottom Line: However to date, a significant barrier to utilizing B-cells as APCs is their low capacity for non-specific antigen uptake compared to "professional" APCs such as dendritic cells.Squeezed B-cells primed and expanded large numbers of effector CD8(+)T-cells in vitro that produced effector cytokines critical to cytolytic function, including granzyme B and interferon-γ.Altogether, these data demonstrate crucial proof-of-concept for mechano-poration as an enabling technology for B-cell antigen loading, priming of antigen-specific CD8(+)T-cells, and decoupling of antigen uptake from B-cell activation.

View Article: PubMed Central - PubMed

Affiliation: 1] Department of Materials Science &Engineering, MIT [2] Department of Biological Engineering, MIT [3] David. H. Koch Institute for Integrative Cancer Research, MIT [4] The Ragon Institute of Harvard, MIT, and MGH.

ABSTRACT
B-cells are promising candidate autologous antigen-presenting cells (APCs) to prime antigen-specific T-cells both in vitro and in vivo. However to date, a significant barrier to utilizing B-cells as APCs is their low capacity for non-specific antigen uptake compared to "professional" APCs such as dendritic cells. Here we utilize a microfluidic device that employs many parallel channels to pass single cells through narrow constrictions in high throughput. This microscale "cell squeezing" process creates transient pores in the plasma membrane, enabling intracellular delivery of whole proteins from the surrounding medium into B-cells via mechano-poration. We demonstrate that both resting and activated B-cells process and present antigens delivered via mechano-poration exclusively to antigen-specific CD8(+)T-cells, and not CD4(+)T-cells. Squeezed B-cells primed and expanded large numbers of effector CD8(+)T-cells in vitro that produced effector cytokines critical to cytolytic function, including granzyme B and interferon-γ. Finally, antigen-loaded B-cells were also able to prime antigen-specific CD8(+)T-cells in vivo when adoptively transferred into mice. Altogether, these data demonstrate crucial proof-of-concept for mechano-poration as an enabling technology for B-cell antigen loading, priming of antigen-specific CD8(+)T-cells, and decoupling of antigen uptake from B-cell activation.

No MeSH data available.


Related in: MedlinePlus