TLR3L and TLR9L enhance proliferation and cytokine production of T cells during antigen stimulation in vitro
Given our recent studies in which we reported the beneficial effects of IL-12 to enhance CD8+ T cell responses in vitro 24, we wanted first to test if conditioning unfractionated Pmel-1 splenocytes with TLRLs can induce greater cell expansion and cytokine production in vitro as compared to IL-12. To this end, un-fractionated Pmel-1 splenocytes were cultured with either IL-12, the TLR3L poly(I:C), or the TLR9L CpG in the presence of peptide stimulation for 72 hrs. we found that both Poly(I:C) and CpG enhanced the proliferation of Pmel-1 cells (Fig. 1A) as well as their function measured by IFN-γ (Fig. 1B), TNF-α (Fig. 1C), and IL-2 (Fig. 1D). Of note, the cells conditioned with CpG elicited higher effects than those of poly(I:C) and IL-12. This supports the role of TLRLs in general and TLR9 in particular in augmenting cytokine production. Together, these data indicate that conditioning T cells with TLRLs during antigen presentation can result in comparable if not better production of inflammatory cytokines as compared to IL-12.
Treatment of splenocytes in vitro with TLR3 or TLR9 agonists induced proliferation of non-CD8+ cells with a CD62Llow phenotype
Because we used unfractionated splenocytes in Figure 1 above, we next determined whether the fraction of non-Pmel-1 T cells (i.e. antigen non-specific CD8+ cells) that exists in the culture contributed to the enhanced overall proliferation shown in Figure 1. To this end, unfractionated splenocytes from Pmel-1 mice were labeled with CFSE and cultured in media alone, poly(I:C), or CpG in the presence or absence of antigen.Then, the cell proliferation was measured after 72 hrs by flow cytometry after gating on CD8+ T cells and non-CD8+ T cells (i.e. other cells in the culture such as CD4+ T cells, B cells, macrophages and dendritic cells). Under this setting, most of the CD8+ T cells are reactive to peptide.
We found that in absence of peptide stimulation, TLR3L and TLR9L induced proliferation of only non-CD8+ T cells (Fig. 2A, upper panel). As expected, antigen stimulation alone resulted in greater proliferation of Pmel-1 CD8+ T-cells (antigen-reactive) than co-stimulation with either TLRL (Fig. 1A, lower panel). However, concomitant stimulation with peptide and each of TLRL increased the proliferation of both non-CD8+ and CD8+ T cell levels (Fig. 1A, lower panel). Down regulation of the homing molecule CD62L on immune cells indicates to their activation. Indeed, treatment with either of TLRL alone induced a CD62Llow phenotype on non-CD8+ T-cells, with CpG inducing a vastly larger population than poly(I:C) (Fig. 2B upper panel), indicating that non-CD8+ T cells also expressed the activation phenotype. These cells were still increased in number in the presence of peptide stimulation (Fig. 2B, lower panel), suggesting that conditioning cells with TLRLs can induce activation and proliferation of non-CD8+ T cells even in the absence of antigen-stimulation.
TLR3L and TLR9L induced activation and proliferation of B cells
Noting the effects of TLR3L and TLR9L on non-CD8 cells above, we sought to identify the non-CD8+ T cell population responsible for this marked proliferation. To this end, we cultured CFSE-labelled Pmel-1 cells as above with poly(I:C) or CpG. Cells were then harvested and stained for specific immune cells, including dendritic cells (CD11c+), CD4+ T cells, NK cells (NK1.1), and B cells. Interestingly, both TLR3L and TLR9L induced expansion of B cells in absence of antigen stimulation (Fig. 3A). Notably, the TLR9L CpG induced higher proliferation (> 90%) of B cells as compared to poly(I:C) as well as the TLR7L imiquimode, which we usd here as another control for TLR9L.
Given this unique higher effect of CpG, we repaeated the same experiments but with only CpG in the presence or absence of antigen stimulation. Interestingly, CpG also induced B cell proliferation (Fig. 3B lower panel) but with less effects than in the absence of antien stimulation (Fig. 3B, upper panel), demonstrating that triggering of TLR9 signaling pathway during antigen-specific activation of CD8+ T cells can yield concomitant expansion of B ((B220+) cells and CD8+ T cells in vitro. Accordingly, these data indicate that the TLR-induced enhancement in cell proliferation in Fig. 1A is mediated in part by the increased non-specific proliferation of B cells.
CpG-induced proliferation of B cells associates with their activation
We tested whether the TLR9L-induced proliferation of B220+ cells paralleled the reported up-regulation of co-stimulatory molecules. To this end, we cultured unfractionated splenocytes from pmel-1 mice in vitro with or without peptide stimulation and treated these cells with media alone or with CpG for 24 hours. We found that both in the presence (Fig. 4A) and in the absence (Fig. 4B) of peptide stimulation, B220+ cells showed expression of CD25, CD69, CD40, CD80, and CD86. CD80 and MHC-I were only marginally affected (data not shown). Treatment of pmel-1 cells with with CpGin the presence of antigen stimulation increased the percentage of B cells (B220+) expressing CD25, CD69 and CD40 as compared to with no CpG (Fig. 4A, upper panel). Interestingly, treatment of pmel-1 cells with CpG in the absence of antigen stimulation also increased the pmel-1 cells expressing these CD25, CD69 and CD40 molecules as well as CD86 as compared to cells treated with media alone (Fig. 4B, lower panel). Of note, the effects of CpG on the activation of B cells was higher in the abence of antigen stimulation as compared with the presence of antigen.
Triggering of TLR3 and TLR9 in naïve CD8+ T cells in vitro enhanced their homeostatic expansion expansion in vivo.
The data shown above conclude that conditioning of pmel-1 CD8+ T cells with TLRLs, namely CpG, elicited cytokine production comparable to IL-12 as well as superior expansion and activation of a mature population of B cells with up-regulation of co-stimulatory molecules. Given these data, we hypothesized that upon injection of cells conditioned with TLRLs during antigen stimulation in vitro, these cells could affect the number of antigen-specific CD8+ T cells post adoptive transfer. To address this hypothesis, we conditioned unfractionated splenocytes from Pmel-1 mice with IL-12, TLR3L, or TLR9L in the presence of peptide and then adoptively transferred cells into naïve mice on day 0. The percentage of transgenic pmel-1 CD8+ T cells was analyzed in the peripheral blood on day 5 after adoptive transfer to measure the homeostatic-driven proliferation of transgenic CD8+ T cells. Overall, we found that the percentage of these cells was low (<1%) in the blood of all groups (Fig. 5A). However the cells that were conditioned in vitro with CpG showed relatively higher numbers (0.6%) as compared to cells conditioned with PPS (<0.1%), IL-12 (<0.3%) and TLR3L (<0.1%) (Fig. 5A).
Triggering TLR3 and TLR9 in CD8+ T cells during antigen presentation in vitro enhanced antigen-specific CD8+ T cell expansion in vivo.
In addition, our group established poly(I:C) as a potent adjuvant in vaccination regimens (Salem, 2005; Salem, 2006; Salem, 2009; Salem, 2007; Salem et al., 2020). We then conducted the same experiment except this time administering vaccination with or without poly(I:C) on day 7 after adoptive cell transfer and analyzed the cell numbers in the peripheral blood on day 12. Adoptive transfer of T cells was preceded 24 hour with preconditioning of the host with CTX. Vaccination with peptide alone enhanced the expansion of CD8+ cells by about 10-fold (Fig. 5B). The expansion of the cells was further increased by about 10-fold when the adjuvant poly(I:C) was mixed with the peptide during vaccination (Fig. 5C). Comparable to the in vitro experiments (Fig. 1A), conditioning with CpG elicited superior expansion compared to poly(I:C) and IL-12 (Fig. 5C). These data indicate that the enhanced proliferation and activation of B cells during conditioning un-fractionated pmel-1 cells in vitro with TLR9L may explain the enhanced survival and antigen-specific expansion of the pmel-1 cells in vivo. Furthermore, these data indicate that condition of CD8+ T cells in vitro with CpG enhances their homeostatic-driven expansion in vivo.
Brief conditioning of CD8+ T cells in vitro with TLR9L enhances the responses of these cells to vaccination and TLR3L in vivo
The data described above showed that conditioning of unfractionated cells with TLR9L during peptide presentation in vitro can enhance CD8+ T cell expansion in vivo after vaccination with peptide and poly(I:C). To understand whether this beneficial effect of TLR9L to CD8+ T cells depends on the antigen presentation microenvironment in vitro, we conditioned unfractionated Pmel-1 splenocytes with TLR3L or TLR9L for 72 hours and then adoptively transferred them on day 0 into recipient mice pretreated 24 hours before with CTX. Mice transferred with TLR3L-conditioned or TLR9L-conditioned T cells were then vaccinated with peptide mixed with either the TLR3L poly(I:C) or the TLR9L CpG. Under this setting, TLR-conditioned cells were challenged in vivo with the same TLRLs used in vitro or with a different TLRL. The mice were then re-challenged after 30 days, the time point when all cells are contracted and become resting memory cells. As shown in Figure 6, the cells conditioned either in the presence of TLRL showed similar expansion to vaccination in vivo regardless whether the vaccination was mixed with relevant or irrelevant TLRL. Of note, the CpG-conditioned cells showed the highest expansion when measured on day 5 post adoptive transfer and peptide vaccination. When the cells were contracted by day 30 and then challenged with peptide mixed with relevant or irrelevant TLRL, the in vitro TLR9-conditioned T cells showed the highest expansion in vivo but only when TLR3L was added to the vaccination. These data are consistent with those in Figure 5, showing high expansion of cells that was conditioned in vitro with TLR9L and in vivo with peptide + poly(I:C). Of note, when the expansion levels of the adoptively transferred T cells in Figures 5 and 6 are compared, it appears that cells conditioned in vitro with TLR9L in absence of peptide stimulation expand in vivo with a higher rate than those conditioned in vitro in the presence of antigen presentation. Taken the results in Figures 5 and 6 together, it can be suggested that just brief conditioning of T cells in vitro with TLR9L alone can enhance the responses of these cells to vaccination and poly(I:C) in vivo upon adoptive transfer.