In the present study, the expression of TLRs among purified CD4+ and CD8+ T cells was demonstrated. Importantly, evidence for a physiological function of TLR on T cells is shown. Treatment of purified CD4+ or CD8+ T cells with poly(I:C) results in changes in the expression of certain TLRs. Additionally, activation with the T cell mitogen PMA also modulated the expression of TLRs. Furthermore, addition of poly(I:C) at the time of antigen priming of CD8+ T cells in vitro increased expression of its cognate receptor TLR3 in these cells. Importantly, treatment of purified naïve OT-1 cells with poly(I:C) induced activation of these cells, and drove them to mount higher expansion in vivo upon their adoptive transfer followed by vaccination. These results demonstrate a direct effect of poly(I:C) on CD8+ T cells and support the notion of direct involvement of TLRs in adaptive immune responses.
Given that addition of certain TLR agonists to different vaccine regimens results in enhanced T cell responses, a better understanding of the functionality of TLRs on CD8+ T cells would improve the rationale application of TLR-based immunotherapy. Until recently, most investigations on TLR have focused on cells of the innate immune system and on the role of TLRs in the initiation of antigen-specific responses following recognition of microbial products by antigen presenting cells. We have recently reported that poly(I:C) is a potent adjuvant for CD8+ T cell responses through stimulation of innate immune responses, including the rapid release of inflammatory cytokines and chemokines in serum, as well as the rapid activation of NK cells, macrophages, and DCs [36
]. The present study further extends the potent adjuvant effects of poly(I:C) to the Ag-specific responses of T cells both in vitro and in vivo. It was found herein, however, that conditioning of DCs in vitro with poly(I:C) is not sufficient to optimize the OT-1 T cell responses in vivo unless the recipient host was conditioned again with poly(I:C) (). Therefore, it could be hypothesized that other cellular components such as NK cells or T cells in the host microenvironment are involved in the adjuvant effects mediated by triggering TLR3 signaling pathway. In our previous studies we explored the roles mediated by NK cells and the cytokines induced by these cells in mediating the adjuvant effects of poly(I:C) [38
]. Therefore, our studies were focused herein to explore whether T cells also respond directly to poly(I:C). We found clearly that both CD4+ and CD8+ T cells express different TLRs. Expression of TLR1, TLR2, TLR6, TLR7 and TLR9 mRNA in murine, feline, and human T cells have also been reported [24
]. Some studies performed on CD4+ cells in BALB/c mice [24
], however, showed no expression of TLR2 in these cells. In contrast, our data showed that expression of TLR2 on CD4+ cells is the highest compared to the expression of other TLRs. This discrepancy in TLR2 expression could be attributed to a possible strain-dependent quantitative and qualitative differences in TLR expression, or because we used real-time RT-PCR analysis which is more accurate and quantitative than the conventional RT-PCR. This led us to compare the expression of TLRs in purified subsets of CD4+ or CD8+ T cells from C57BL/6 or BALB/c mice. Our detailed analysis showed that splenic CD4+ T cells purified from BALB/c and C57BL/6 mice express different levels of TLRs with the magnitude of TLR2 > TLR4 > TLR6 > TLR7 > TLR5 > TLR3 > TLR9 for CD4+ in C57BL/6 mice, and TLR4 > TLR6 > TLR2 > TLR9 > TLR7 > TLR3 > TLR5 for CD4+ in BALB/c mice. Of note, the TLR expression was comparable in CD4+ T cells of BALB/c and C57BL except for TLR2 which was two-fold higher in C57BL/6 mice (). In the case of CD8+ T cells, their TLR expression showed TLR2 > TLR6 > TLR7 > TLR4 > TLR3 > TLR9 > TLR5 in C57BL/6 mice and TLR6 > TLR4 > TLR9 > TLR2 > TLR7 > TLR3 > TLR5 in BALB/c mice. Of note, expression of TLR4, TLR6, and TLR9 was higher in BALB/c mice than in C57BL/6 mice. Overall, the magnitude of TLR expression in CD8+ T cells was higher than in CD4+ T cells. Together, these data suggest a marked difference in TLR expression is dependent on both the animal strain and the cell type. Alteration of TLR expression in T cells after their activation has also been reported in preclinical and clinical studies under different activation settings. For instance, anti-CD3 activated CD4+ T cells showed increases in TLR3, TLR5, and TLR9 and decreases in TLR2 and TLR4 expression levels [24
]. TLR expression showed significant increase in memory (CD44+) CD4 and CD8 T cells after 14 days of burn injury [41
]. Virally-infected T cell lines [40
], as well as CD8+ T cells and B cells [42
] from virally-infected individuals also showed altered TLR expression levels. Thus, altogether, these data suggest that modulation of TLR expression in cells of adaptive immunity upon their activation might be a possible mechanism to regulate the ongoing T cell-mediated immunity.
In addition to the difference in TLR expression based on the H2 background or T cell type, our data also showed that antigen-dependent activation of T cells induced alteration in the expression profile of TLR with a tendency to increase the expression levels of TLR3. When OT-1 cells were conditioned with OVAp and poly(I:C) in vitro, the cells showed higher activation phenotype (CD62Lhigh)
), higher proliferation, and higher capability of producing large amount of IFN-γ (). These in vitro adjuvant effects of poly(I:C) on OT-1 cells could be attributed to its direct effects on CD8+ T cells, since these cells showed appreciated levels of TLR expression (), hypothesizing that CD8+ T cells respond directly to the TLR3L poly(I:C). By testing this hypothesis directly, it was found that triggering TLR3 signaling pathway in CD8+ T cells instructed these cells to express better Ag-specific functionality upon their adoptive transfer in vivo (). Similar to our results, the TLR1/2L Pam3 was reported to co-stimulate Ag-activated T cells in vitro, which was associated with increases in the cell proliferation, survival and functions [43
], and that ligation of TLR3 in effector CD8+ T cells, but neither naïve nor central memory cells, in vitro increased their IFN-γsecretion [44
]. Furthermore, activation of naïve CD4+ T cells led to increases in the expression levels of TLR3, TLR5, and TLR9 and decreases in the expression of TLR2 and TLR4, explaining why treatment of these activated CD4+ T cells only with the TLR3L poly(I:C) and the TLR9L CpG, but not with the TLR2L peptidoglycan or the TLR4L LPS, directly enhanced their survival in vitro and in vivo [24
]. Of note, this study showed that this TLR3 and TLR9-induced CD4+ T cell activation required NF-αB activation and was associated with Bcl-xL
up-regulation. Recent studies have also shown direct effect of CpG on CD4+ T cells [24
] through a MyD88 and PI3K-dependent mechanism [46
]. The direct effects of TLRLs on T cells discussed above would explain some of their potent adjuvant effects and also explain why T cells stimulated by antigenic peptide in vivo divide well, but fail to accumulate efficiently unless TLR agonists are present [47
]. Taken with our results, it can be suggested that TLRLs can directly target cellular components of adaptive immunity including CD4+ and CD8+ T cells.
Expression of functional TLRs on T cells has been found to be extended to regulatory cells since recent observations also suggest that TLRLs have the capacity to directly regulate T cell responses and modulate the suppressive activity of CD4+CD25+ regulatory T (Treg
) cells. For instance, it has been found that Treg
cells express TLR5 at levels comparable to those on monocytes and DCs [48
], and that the TLR9L CpG synergizes with anti-CD3 to induce partial abrogation in the suppressive activity of Treg
]. By contrast, costimulation of Treg
cells with the TLR5L flagellin potently increased their suppressive capacity and enhanced expression of FOXP3, the surrogate marker for Treg
], by inducing the regulatory molecule SOCS-1 (suppressor of cytokine signalling-1) [51
]. These data indicate that the quality of the T cell response depends on what type of TLR is triggered. Therefore, the nature of TLR/TLRL signaling pathway should be carefully considered during application of TLR-based stimulation of innate or adaptive immunity. Large-scale analysis of immune cell TLR expression in the mouse revealed that cells of the innate immune system express a broader number of TLR than cells of the adaptive immune system [29
]. It remains, however, to perform quantitative analysis at the level of each TLR expression by cells of innate and adaptive immunity, since the former might express much higher message of TLR than the latter. These studies might explore whether the quantity of TLR expression impacts on the quality of the cell responses to TLRLs.
The current concept on the mechanisms underlying the adjuvant effects of TLRLs is the direct targeting and stimulation of innate immune cells. Our data presented in this study support the concept that TLRLs can also directly target the adaptive immune cells. Considering this concept during TLRL-based treatments would improve the application of these adjuvants to active vaccination. Furthermore, this direct responsiveness of T lymphocytes to TLRLs offers new perspectives for the immunotherapeutic manipulation of T cell responses in the adoptive immunotherapy setting. Ultimately, TLRLs can condition T cells during their stimulation with cognate Ag in vitro and can also condition the host microenvironment upon adoptive transfer of in vitro TLRL-conditioned T cells. Such dual conditioning system would lead to robust T cell responses.