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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

Antigen-specific CD8+T-cell proliferation is primed in vivo by squeeze-delivered B-cells.A) Representative gating shown on 2D pseudocolor plot (CD8 vs. Thy1.1) to identify adoptively transferred OT-I T-cells, followed by representative histogram overlays of CFSE dilution and gating to identify number of divisions. B, C) Quantitative analysis shown for % divided OT-I T-cells in spleens (B) and inguinal lymph nodes (C). All data are represented as means±standard deviation (n = 3 animals, representative of 3 independent experiments). Pairs of conditions were tested for statistically significant differences with ordinary 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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f5: Antigen-specific CD8+T-cell proliferation is primed in vivo by squeeze-delivered B-cells.A) Representative gating shown on 2D pseudocolor plot (CD8 vs. Thy1.1) to identify adoptively transferred OT-I T-cells, followed by representative histogram overlays of CFSE dilution and gating to identify number of divisions. B, C) Quantitative analysis shown for % divided OT-I T-cells in spleens (B) and inguinal lymph nodes (C). All data are represented as means±standard deviation (n = 3 animals, representative of 3 independent experiments). Pairs of conditions were tested for statistically significant differences with ordinary 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: In addition to having potential as an in vitro antigen-specific T-cell expansion platform, B-cells are an alternative to dendritic cells for use as cellular vaccines. As proof-of-principle for this application, we adoptively transferred CFSE-labelled OT-I CD8+T-cells expressing Thy1.1 as a congenic marker into recipient mice as reporters of antigen presentation. One day later, we injected either resting B-cells immediately after mechano-poration-mediated antigen loading, or mechano-poration-loaded B-cells that were subsequently activated for 24 h with CpG in vitro. Resting B-cells loaded with antigen by endocytosis were used as controls, either immediately or after 24 h of activation with CpG. Four days after B-cell transfer, mice were sacrificed and spleens and inguinal lymph nodes were analysed for OT-I proliferation by flow cytometry. Representative results and flow cytometry gating are shown in Fig. 5A and Supplementary Fig. S5. Consistent with our in vitro results, we found that mechano-porated B-cells were able to elicit division of adoptively transferred OT-I T-cells while endocytosis controls showed only basal division. Both CpG-activated and resting squeezed B-cells caused OT-I proliferation in spleens (Fig. 5B, ~45% and ~35% divided of injected OT-I T-cells with p < 0.001 and p = 0.001 comparing SQZ vs. endocytosis, respectively). CpG-activated and resting squeezed B-cells also elicited similarly enhanced OT-I proliferation in lymph nodes compared to endocytosis B-cells (Fig. 5C, ~40% and ~35% divided of injected OT-I T-cells by CpG B and resting squeezed B-cells, respectively; p < 0.001 comparing SQZ vs. endocytosis for both resting and CpG). Endocytosis controls showed ~4% baseline division in lymph nodes. These promising results suggested that B-cells loaded by microfluidic mechano-poration merit further investigation as an in vivo platform for cellular vaccines.


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)

Antigen-specific CD8+T-cell proliferation is primed in vivo by squeeze-delivered B-cells.A) Representative gating shown on 2D pseudocolor plot (CD8 vs. Thy1.1) to identify adoptively transferred OT-I T-cells, followed by representative histogram overlays of CFSE dilution and gating to identify number of divisions. B, C) Quantitative analysis shown for % divided OT-I T-cells in spleens (B) and inguinal lymph nodes (C). All data are represented as means±standard deviation (n = 3 animals, representative of 3 independent experiments). Pairs of conditions were tested for statistically significant differences with ordinary 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

f5: Antigen-specific CD8+T-cell proliferation is primed in vivo by squeeze-delivered B-cells.A) Representative gating shown on 2D pseudocolor plot (CD8 vs. Thy1.1) to identify adoptively transferred OT-I T-cells, followed by representative histogram overlays of CFSE dilution and gating to identify number of divisions. B, C) Quantitative analysis shown for % divided OT-I T-cells in spleens (B) and inguinal lymph nodes (C). All data are represented as means±standard deviation (n = 3 animals, representative of 3 independent experiments). Pairs of conditions were tested for statistically significant differences with ordinary 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: In addition to having potential as an in vitro antigen-specific T-cell expansion platform, B-cells are an alternative to dendritic cells for use as cellular vaccines. As proof-of-principle for this application, we adoptively transferred CFSE-labelled OT-I CD8+T-cells expressing Thy1.1 as a congenic marker into recipient mice as reporters of antigen presentation. One day later, we injected either resting B-cells immediately after mechano-poration-mediated antigen loading, or mechano-poration-loaded B-cells that were subsequently activated for 24 h with CpG in vitro. Resting B-cells loaded with antigen by endocytosis were used as controls, either immediately or after 24 h of activation with CpG. Four days after B-cell transfer, mice were sacrificed and spleens and inguinal lymph nodes were analysed for OT-I proliferation by flow cytometry. Representative results and flow cytometry gating are shown in Fig. 5A and Supplementary Fig. S5. Consistent with our in vitro results, we found that mechano-porated B-cells were able to elicit division of adoptively transferred OT-I T-cells while endocytosis controls showed only basal division. Both CpG-activated and resting squeezed B-cells caused OT-I proliferation in spleens (Fig. 5B, ~45% and ~35% divided of injected OT-I T-cells with p < 0.001 and p = 0.001 comparing SQZ vs. endocytosis, respectively). CpG-activated and resting squeezed B-cells also elicited similarly enhanced OT-I proliferation in lymph nodes compared to endocytosis B-cells (Fig. 5C, ~40% and ~35% divided of injected OT-I T-cells by CpG B and resting squeezed B-cells, respectively; p < 0.001 comparing SQZ vs. endocytosis for both resting and CpG). Endocytosis controls showed ~4% baseline division in lymph nodes. These promising results suggested that B-cells loaded by microfluidic mechano-poration merit further investigation as an in vivo platform for cellular vaccines.

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