Dataset 5: IL-15 increases cross-reactive antibody responses to H3N2 viruses

(A) B cells from healthy donors were co-stimulated with CpG ODN 2006 inactivated A/Vic11 viruses and interleukin 15. Strain-specific and HA stalk-reactive IgG in supernatants of activated B cells were detected after 6 days. (B) Fold change in cross-reactive antibodies. (B) Correlation between influenza specific antibodies and HA antigenic sequence. No correlation between cross-reactive antibodies and HA sequence was detected

Dataset 6: Stalk-reactive antibody responses to H3 viruses enhanced by Il-15

Purified B cells were costimulated with CpG₂₀₀₆, A/Victoria/361/2011 viruses and IL-15. Stalk-reactive IgG in supernatants was detected at day 6. Chimeric molecules cH5/3, cH4/7 and cH5/1 were used for assessing antibodies against H3 stalk, H7 stalk and H1 stalk, respectively. Each symbol and line represents an individual donor. (A) H3 stalk-reactive antibodies. (B) H7 Stalk-reactive antibodies. (C) H1 stalk-reactive antibodies

Dataset 3: Influenza viruses induce cross-reactive antibody responses in vitro

B cells were obtained by negative selection, and then stimulated with CpG ODN 2006 or together with A/Vic11 for 6 days. Cross-reactive antibodies binding to H1, H2, H5, H6, H7 and B influenza subtypes in B cell culture were measured by mPlex-Flu assay. (A) Fold change in cross-reactive antibodies. All values of IgG levels (MFI) were subtracted those of medium before calculating fold change. Only those values of IgG induced by CpG plus A/Vic11 viruses, which were greater than 100, were selected to calculate fold change. Each symbol represents the median of fold change in levels of IgG induced by CpG plus H3 to IgG stimulated by CpG 2006 ODN alone. (B) Correlation between influenza specific antibodies and HA antigenic sequence

Dataset 4 - Induction of HA stalk-reactive antibodies by H3 viruses

B cells from healthy donors were stimulated with CpG 2006 ODN alone or together with inactivated A/Vic11 viruses, A/SH13 (H7N9), and A/Hong Kong/33982/2009 (A/HK09) (H9N2) and pandemic H1N1 viruses. The levels of IgG against H3 HA, H5 head and chimeric molecules cH5/3 are shown for individual honors. (A) Nine of 13 donors displayed increases in stalk-reactive IgG after A/Vic11 stimulation. (B, C) A correlation model assuming different coefficients for different H3N2 strains-specific antibodies was fitted to evaluate the relationship between HA stalk-reactive and strain-specific IgG

Mesenchymal Stromal Cells Support the Viability and Differentiation of Follicular Lymphoma-Infiltrating Follicular Helper T-Cells

<div><p>The biology of follicular lymphoma (FL) is largely dictated by the immune-effector and stromal cells that comprise its tumor microenvironment. FL-infiltrating T-cell populations that are thought to be fundamental to FL biology are follicular helper T-cells (TFH), follicular regulatory T-cells (TFR), a recently described population that regulates TFH activity, and regulatory T-cells (Treg). These T-cell populations have dynamic interactions with mesenchymal stromal cells (MSCs) in the tumor microenvironment. Whereas MSCs have been shown to support FL B-cell and Treg viability, their effects on FL-infiltrating TFH and TFR cells have not been described. Herein we show that MSCs support the viability of FL-infiltrating TFH and TFR, as well as Tregs, in part through an IL-6-dependent mechanism. We further demonstrate that MSCs mediate TFH to TFR conversion by inducing the expression of FoxP3 in TFH cells, demonstrating for the first time that human TFR can be derived from TFH cells. Given that the balance of TFH and TFR populations likely dictate, in part, the biology of this disease, our data support the potential for targeting MSCs as a therapeutic strategy.</p></div

Characterization of FL-infiltrating T-cells by flow cytometry.

<p>Shown is an example of the gating strategy used to define TFH, Treg and TFR subsets. Viable lymphocytes were gated according to size, granularity and expression of CD3 and CD4. T-cell subsets were classified as: TFH (CXCR5⁺PD-1⁺CD25⁻Bcl-6⁺FoxP3⁻), Treg (FoxP3⁺CD25⁺) and TFR (CXCR5⁺PD-1⁺CD25⁺Bcl-6⁺FoxP3⁺).</p

Cell contact is not required for MSC support of T-cell populations.

<p>(<b>A</b>) Flow cytometry plot depicting increases in the proportion of FL-infiltrating T-cells with phenotypes consistent with TFH, Treg and TFR populations in transwell cultures of FL-SCS and FL-SCS/TN-MSC. (<b>B</b>) Bar graph representing average fold change of FL-infiltrating T-cell populations cultured in transwell plates with TN-MSCs compared to T-cell populations cultured in transwell plates without TN-MSCs after 48 hrs (n = 7, <b>*</b>p<0.05, <b>**</b>p<0.01).</p

MSCs support FL-infiltrating TFH, Treg and TFR subsets.

<p>The proportion of T-cells with phenotypes indicating TFH, Treg and TFR were analyzed by intracellular flow cytometry after culture for 48 hrs in media alone or with TN-MSCs (<b>A</b>). Flow cytometry histograms showing reduced active caspase-3 in TFH, Treg and TFR cells after culture with TN-MSCs (<b>B</b>). The proportion of T-cells with phenotypes indicating TFH, Treg and TFR were analyzed after 48 hrs of culture with FL BM-derived MSCs n = 2 (<b>C</b>). Average fold change from T-cells cultured without MSCs is indicated by solid line, <b>*</b>p<0.05, <b>**</b>p<0.01.</p

MSCs support FL-infiltrating T-cell subsets in the absence of B-cells.

<p>(<b>A</b>) CD19⁺ pan B-cell Dynabeads® were used to deplete FL-SCS of B-cells, the remaining cells were cultured alone or with TN-MSCs. Non-depleted FL-SCS cultured in parallel with B-cell depleted experiments served as controls. (<b>B</b>) CD3⁺CD4⁺ FACS isolated FL T-cells were cultured alone or with TN-MSCs, Non-depleted FL-SCS cultured with or without TN-MSC served as controls TN-MSCs supported TFH, Treg and TFR populations in purified T-cell cultures.</p

MSCs induce FoxP3 expression in TFH subsets.

<p>(<b>A</b>) Flow cytometry gating scheme used to isolate TFH cells and post-sort analysis showing Bcl-6 and FoxP3 expression in the isolated cells. (<b>B</b>) The percentage of isolated TFH cells expressing CD25 and FoxP3 (TFR) immediately after sorting, at 48 hrs after culture alone and at 48 hrs after co-culture with TN-MSCs. (<b>C</b>) Flow cytometry histogram showing FoxP3 expression in TFH after 48 hrs in culture alone and after 48 hrs in culture with TN-MSCs.</p