We previously showed that triggering TLR7/8 causes changes to the lymphoid microenvironment that are unfavorable for HIV replication 
. Here we sought to identify the individual contributions of TLR7 and 8 to the overall anti-HIV activity and to gain insights into the mechanisms of how TLR trigger anti-viral defenses. We found that (i) the TLR7- or 8-mediated anti-HIV activity depended on the origin of the lymphoid tissue, (ii) dendritic cells are indispensable for the anti-HIV effect, (iii) the main effector cells for the anti-HIV activity were NK-cells and CD8+ T-cells, and iv) soluble factors mediated the anti-HIV effects and were not reversed when the antiviral active cytokines IFN-α, -γ, or TNF-α were neutralized.
The TLR7/8 agonist R-848 shows strong anti-HIV activity in PBMC and lymphoid suspension cells from tonsils 
. In this study, we used two agonists which selectively trigger either TLR7 or 8 
. Their potency depended strongly on the origin of lymphoid tissue. Triggering TLR7 was primarily responsible for the anti-HIV activity in tonsillar lymphoid suspension cells, but it had only limited and inconsistent effects in PBMC. In contrast, triggering TLR8 had strong anti-HIV activity in PBMC; it had also consistent but somewhat less anti-HIV activity in tonsillar lymphoid suspension cells. Because of limited availability of tonsillar tissue, we focused in most experiments on PBMC for elucidating the anti-HIV mechanism(s) of TLR7 and/or 8 triggering.
Based on the sentinel function and the expression of TLR8, we speculated that MDC initiate the complex changes in PBMC that are eventually unfavorable for HIV replication. Indeed, when we depleted DC and DC precursors from PBMC, the anti-HIV effect from TLR7/8 triggering was mostly lost. Not surprisingly, depletion of these cell subsets from untreated cultures resulted in modest increases of HIV replication, pointing to HIV's ability to trigger TLR directly 
and thus to initiate an anti-viral defense program, although less potently than the TLR agonists examined. The potent antiviral defense program initiated by TLR8 or 7/8 was also reflected when exposing MoMDC to the TLR agonists: they showed cell clumping, up-regulation of the cell surface marker CD80, CD83 and CD86 as well as pronounced secretion of a number of cytokines. Triggering solely TLR7 had no visible effect on MoMDC.
Previously, we reported that depletion of B cells partially reversed the anti-HIV activity of R-848 
. Strikingly, when we depleted NK and CD8+ T-cells prior to adding the TLR8 or 7/8 agonists to PBMC, we observed a rather complete reversal of the anti-HIV effects; depleting either NK or CD8+ T-cells showed only partial inhibition, suggesting that there is an additive effect when stimulating NK and CD8+ T-cells or there is an eminent cross-talk between these cell-populations necessary for this striking anti-HIV effect. Triggering TLR7 in PBMC depleted from NK and CD8+ T-cells showed inconsistent effects which reflect its overall inconsistent anti-HIV effects. Since NK and CD8+ T-cells are the key effector cells for the TLR8-mediated HIV inhibitory activity, their low or even absent numbers in tonsillar lymphoid suspension cultures may explain the lower TLR8 agonist's anti-HIV effects in that tissue as compared to PBMC. The robust HIV inhibitory activity in tonsillar lymphoid tissue and the rather poor one in PBMC in response to TLR7 triggering speak in favor of additional tissue type specific mechanism(s) beyond NK and CD8+ T-cells which may explain the differences between TLR7 and TLR8 agonists. Notably, while we have not assessed functional differences, the percentages of DC are similar in the lymphoid tissue studied.
The potent cell activation subsequent to triggering TLR8 or TLR7/8 is reflected by an up-regulation of intracellular IFN-γ and TNF-α and increased cytolytic activity of NK and CD8+ T-cells. Surprisingly, levels of expression of inhibitory (CD94, KIR2DL2, KIR3DL1, ILT2) and activating receptors (NKp30, NKp46, NKp44, DNAM-1) on NK cells were similar between the controls and PBMC treated with TLR agonists (data not shown).
Since NK and CD8+ T-cells do not respond directly to TLR7/8 agonists 
, their activation has to occur either by cell-cell contact with a TLR7/8-responsive cell or by soluble factors. The most likely candidates are IL-12, -15, and -18, which are released from DC upon their activation. However, blocking the individual cytokines or all of them with neutralizing antibodies did not reverse the anti-HIV activity and neither prevented the cellular activation of NK and CD8+ T-cells that is consistent with a recently published study 
. Thus, we assume that cell-cell contact and/or a variety of soluble redundant factors will ultimately result in the activation of these effector cells. Indeed, the significance of bi-directional interactions between DC and NK cells has been convincingly demonstrated for an efficient immune defense 
So how do NK and CD8+ T-cells block HIV infection? To answer this question, we used trans-well experiments, in which HIV-infected CD4+ T-cells were separated from PBMC devoid of CD4+ T-cells. We found that separating the effector from the target cells did not abrogate the anti-HIV effects, thus implicating soluble factors in the process. Indeed, triggering TLR7 and/or 8 resulted in prominent changes in the cytokine profile. However, we were not able to assign the anti-HIV effect to one distinct cytokine even though we neutralized a large number of soluble factors either alone or in combination. The lack of any reversal of the antiviral activity subsequent to triggering TLR8 or TLR7/8 when IFN-α was neutralized is most puzzling and speaks in favor of very potent unknown factor(s) inhibiting HIV. In contrast, the inhibitory activity in response to TLR7 triggering was partially reversed when IFN-α was neutralized: this indicates that these above postulated factor(s) are not secreted or not to the same amount when exposing lymphoid tissue to TLR7 agonists. While not statistically significant, it appeared that blocking IL-12, -15 and -18 resulted also in some reversal of the anti-HIV effects in the case of triggering TLR7. We assume that TLR7 triggering activates DC which in turn releases IL-12, -15 and -18 critical for activating downstream effector cells such as NK or CD8+ T-cells.
While the data with the trans-wells were unambiguous, cell-cell contact may substantiate the anti-HIV effects. However, NK cells showed only minor changes of activating and/or inhibiting receptors (see above) and thus, we do not assume that they play a major role. In contrast, TRAIL showed a prominent up-regulation on NK cells. This increased TRAIL expression is reminiscent of a recent report describing the TLR7 triggering dependent generation of killer PDC expressing TRAIL able to induce apoptosis of HIV-infected SupT1 cells 
. The potential effects of TRAIL may be masked by the potent inhibitory anti-HIV effects of soluble factors in our experimental set-up that may explain the lack of any reversal of anti-HIV activity when neutralizing TRAIL (data not shown). The potent HIV-inhibition by soluble factors is reminiscent of the long-searched CD8+ T-cell antiviral factor, CAF 
Triggering TLR7/8 appears to inhibit HIV before its integration into the host-genome. Downregulation of CCR5 after release of β-chemokines may contribute to the anti-HIV activity when challenging the lymphoid tissue with CCR5-tropic strains. However, other anti-HIV mechanism(s) must be involved. Down-regulation of CCR5 was not complete, and more importantly, X4-tropic strains were also inhibited. We excluded an unspecific effect on the fusion process of the TLR agonists as examined by a cell-cell based fusion assay. Furthermore, we used replication-incompetent viruses pseudotyped with VSV envelope, which enter cells by an entirely different mechanism(s) than HIV. We observed a similar inhibition of the reporter gene in tissue exposed to TLR agonists, also suggesting that these agonists act after entry but before integration/transcription. To substantiate this finding, we investigated whether the TLR agonists induced changes influence HIV replication at the level of transcription: for that purpose CD4+ T-cells were infected overnight and spreading infection was inhibited by adding the fusion inhibitor enfurvitide. The infected CD4+ T-cells were subsequently co-cultured with previously TLR7/8 agonist stimulated autologous PBMC devoid of CD4+ T-cells. Thus, resulting HIV output from this co-culture reflects exclusively HIV transcription from HIV-infected CD4+ T-cells. In this setup, the TLR agonists had no inhibitory activity as compared to the control excluding any inhibitory action at the transcriptional level.
In summary, we propose a new model, in which triggering TLR7 and/or 8 of DC activates NK cells and CD8+ T-cells. These cells, in turn, induce the release of soluble factors that result in a microenvironment unfavorable to HIV infection. Since B cells express TLR7, they may be activated directly or indirectly by the corresponding trigger, but in any case, they have less anti-HIV activity than NK and CD8+ T-cells. The inability to identify a specific soluble factor is characteristic of the vast array of changes induced by triggering pattern recognition receptors and points to the poorly understood redundancy of the innate immune responses. As outlined in the introduction, we would like to reiterate the fact that compounds triggering TLR7 and/or 8 also strengthen the adaptive immune responses. Thus, these compounds may combine by acting on diverse arms of the immune response optimal properties as immunomodulatory drugs in HIV infection.