Mycobacterial containment relies on the ability of granulomas to spatially and temporally co-localize critical cellular components from the innate and adaptive arms of the immune system to generate anti-bacterial effector responses. We previously demonstrated that these lesions in infected mice consist of a largely static macrophage network that supports the rapid recruitment and constrained localization of a highly motile effector T cell population (Egen et al., 2008
). Communication between these two cell types is critical for the anti-mycobacterial response and consists in part of antigen presentation to T cells by infected macrophages and possibly DCs and the subsequent local activation and production of macrophage-stimulating cytokines by the T cells (Flynn and Chan, 2001
Using a well-established liver infection model, we demonstrated that few potential effector T cells were stimulated sufficiently by antigen at any one time within a mycobacterial granuloma to elicit inflammatory cytokine production, and that this low frequency correlated with the paucity of antigen-specific T cells showing migration arrest under several conditions. Yet nearly all the T cells were capable of showing migration arrest and responding with a much higher amount of per cell cytokine production if confronted with artificially raised amounts of antigen. From these data we conclude that, despite being sufficient for constraining the infection, the display of antigenic complexes by the myeloid compartment within a granuloma is functionally limited, such that at any moment, only a small number of T cells can be triggered to arrest migration and produce relatively modest levels of cytokines.
We speculate that these data using a hepatic infection model are highly relevant to and predictive of events occurring under more physiological pulmonary infections with Mtb. Indeed, systemic infection models have been used extensively to examine the cellular and molecular pathways involved in host responses to Mtb. However, similar experiments in low-dose aerosol Mtb infection models will be an important step in determining the extent to which our findings relate to the pathogenesis of human tuberculosis. In addition, our studies primarily examined the effector response to a single mycobacterial antigen using a TCR transgenic adoptive transfer model. While we demonstrate that the bulk population of endogenous CD4+ T cells behaves similarly to our transferred population in terms of producing a muted, sub-maximal effector cytokine response, future work aimed at examining T cell recognition of additional mycobacterial antigens is needed to determine the extent to which our findings can be generalized.
Although often speculated upon, the correlation between T cell motility and effector cytokine production has not been well established. Our analysis of local cytokine production within the liver without additional in vitro
restimulation revealed that, despite highly efficient Th1 cell polarization during priming and recruitment of mycobacterial antigen-specific effector T cells into granulomas, only small numbers of these cells actively produce IFN-γ and/or TNF-α at any one time. Thus, cytokine production appears linked with motility and similar TCR signaling thresholds may be in place for regulating both migration arrest and upregulation of effector function. The rare T cells that do receive sufficient signals to cause arrest synthesize cytokine that, at least in the case of IFN-γ, is secreted in a spatially polarized fashion, presumably resulting in highly localized delivery of the effector protein to limited numbers of target APCs. These data are consistent with previous findings demonstrating polarized secretion of IFN-γ by CD8+
T cells towards virally infected targets in the brain (Barcia et al., 2008
). Interestingly, we also observed rare cases of clustered cytokine-producing T cells, suggesting the existence of extremely localized regions of more robust antigen presentation within some granulomas. These data are reminiscent of previous findings showing heterogeneity and local variation in T cell responses to Leishmania major-infected
cells in the skin (Filipe-Santos et al., 2009
Numerous studies have identified mycobacterial pathways capable of limiting antigen presentation by infected cells, especially via the MHC class II pathway (Harding and Boom, 2010
). In addition, down-regulation of costimulatory molecules from the surface of APCs and upregulation of inhibitory receptors capable of locally suppressing the T cell response could also result in limited effector function within the granuloma (Schreiber et al., 2010
). It is likely that these immune evasion mechanisms are at least partly responsible for the low effector CD4+
T cell arrest and cytokine production observed in this study. However, other factors may also influence the frequency of arrested and cytokine-producing T cells detected in granulomas. During the initial response to sub-optimal TCR stimuli in the LN, naïve T cells are thought to integrate signals derived from multiple encounters with APCs until a threshold is reached for arrest (Henrickson et al., 2008
; Mempel et al., 2004
). We did observe a high frequency of T cells showing signs of activation shortly after homing to the infected liver but were unable to determine if those T cells receiving low levels of stimulatory signals became more prone to arrest at later time points. Examination of in vitro
-generated effector T cell motility 12–24 hours post-transfer did not reveal greater frequencies of arrested T cells compared to early time points (not shown), but these experiments are difficult to interpret as we were unable to prevent continual T cell homing into the granuloma, thus leading to a temporally asynchronous population at later time points. An additional possibility is that rare T cells encountering high amounts of cognate antigen could eventually desensitize TCR signaling pathways leading to an increased threshold for arrest. Consistent with this idea, in vivo
-primed p25-EGFP T cells that had homed to the granuloma over the course of days to weeks did not show the difference in motility between BCG- and rBCG30-infected animals that was apparent in experiments using acutely transferred effector T cells examined within 4 hours of their entry into these structures. Thus, an interesting possibility is that effector T cells at sites of chronic inflammation can adjust their stimulation threshold in order to strike a balance between protection and immune-mediated tissue destruction.
Recently, several publications have concluded that effector T cell arrest is rare at sites of chronic infection (Beattie et al., 2010
; Filipe-Santos et al., 2009
; Schaeffer et al., 2009
; Wilson et al., 2009
). While these studies did not examine the relationship between motility and cytokine production, together with our study they suggest that limited long-term T cell-APC interactions and correspondingly low levels of effector responses may be a general feature of persistent infections and emphasize the importace of determining whether bulk population behavior or the dynamic properties of only a minor fraction of imaged cells is the relevant parameter for measurement in such cases. Comparing these data to future studies aimed at examining effector T cell motility and effector cytokine production in acutely resolving infection models should be informative, as we speculate that rapidly resolving infections may induce more robust effector T cell responses capable of effectively clearing the invading pathogen.
The data presented here reveal that, relative to their potential, effector T cells migrating within mycobacterial granulomas produce an extremely muted response as a result of limited local antigen presentation and/or recognition. An implication of these findings is that strategies aimed solely at expanding the pool of antigen-specific effector T cells in individuals infected with some persistent pathogens, such as Mtb, may meet with limited success, as there may be insufficient antigen present at sites of infection to support additional effector responses. Rather, immunotherapeutic approaches designed to both increase levels of local antigen presentation and maintain a high frequency of effector T cells within infected tissues will likely have the best chance at successfully reversing the course of the disease.