Th17 cells have been implicated in the response to a number of pathogens and we show here that both CD4 and CD8 IL-17-secreting cells can be detected in the lungs of mice responding to influenza A infection. Somewhat higher levels of IL-17-secreting cells were seen in the secondary response to influenza (data not shown).
We speculate that Tc17 cells represent highly differentiated effector cells that are recruited to the lung during a pulmonary infection.
The injection of blocking Abs to IL-17 enhanced weight loss and diminished survival in response to lethal heterosubtypic challenge, suggesting that IL-17 plays some role in protection. The blocking of IL-17A does not block IL-17F or IL-22 and it may be that these factors can make up for the lack of IL-17A.
IL-17-secreting CD8 effector T cells can be generated in vitro using similar strategies to those used to generate Th17 effectors. In critical experiments, we used cell-sorted naive CD44low populations as our starting cell population, but in other experiments using unselected CD8 there were only modest differences in the resulting population. Tc17 prepared from naive cells had higher levels of IL-17-secreting cells and lower levels of IFN-γ and granzyme B. They were devoid of message for IL-22, which we presume must be derived from effectors derived from memory cells. We used a basic mixture of cytokines modeled after studies with CD4 T cells, including IL-1β, IL-6, and TGF-β with additional IL-2 and Abs to IL-4 and IFN-γ, two cytokines that had been shown to be inhibitory to the development of Th17. In later studies, we omitted IL-2 and added Ab to IL-2 which improved yields. The inclusion of IL-21 appeared to be crucial and enhanced proliferation and the development of the capacity to secrete IL-17 upon restimulation. The addition of IL-23, either in the presence or absence, of IL-21, led to only a modest improvement in yield and differentiation.
RA has been shown to antagonize the effect of TGF-β
in the generation of Th17 effectors (41
). In our studies here, we found that the addition of the RA inhibitor LE540 had relatively little effect (data not shown). Dendritic cells from MedLN (used in the Mucida study (41
)) have been shown to secrete RA (63
) and it seems likely that the lack of effect in our studies is due to the fact that we used LPS-stimulated B cells for Ag presentation, which we presume secrete no RA. IFN-γ
and IL-4 inhibit Th17 generation by directing development toward Th1 or Th2 effectors (12
) and we found that the yield of Tc17 effectors was significantly increased when effectors were generated using CD8 T cells from IFN-γ
-deficient rather than wild-type OT-1 mice when effectors were prepared from unselected CD8 cells. In other experiments (data not shown), we found that the T cells had already differentiated into IL-17-producing cells in the first 2 days of incubation and their messenger RNA expression was almost the same as day 4 Tc17 effector cells. The number of Tc17 effector cells expanded linearly through days 2–5 with a similar ratio fraction of IL-17-secreting cells.
The cell surface phenotype of the Tc17 effector population was very similar to that of Tc1 and Tc2 effectors. CD25, CD44, and CD69 were all up-regulated. CD62L was down-regulated as in Tc1 and Tc2 but expression was unusually low. We have previously shown that only the most highly differentiated, CD62Llow
) or CD8 (65
) effectors migrate to the lung and the finding here supports the idea that this is also true for Tc17 cells.
Several features of effector function of the Tc17 population emerged from the analysis of intracellular cytokine staining and by PCR assays of effector cell RNA. Fifty to 70% of the in vitro-generated effector population secreted IL-17 upon restimulation as judged by intracellular cytokine staining. A large fraction of the IL-17-secreting cells also secreted TNF and a smaller fraction also secreted IL-2. It was striking that there were very few cells in the IL-17-positive or negative effector population that secreted IFN-γ or stained for granzyme B. Only 2–5% of the IL-17-positive cells were also positive for IFN-γ. This appears to be a consequence of the special conditions used for in vitro generation and was not characteristic of IL-17-secreting cells in vivo. The IL-17-negative population also secreted TNF but still only a few cells were positive for IFN-γ. Tc17 effector cells from sorted naive CD44low CD62Lhigh CD8 T cells (nTc17) had fewer IFN-γ and granzyme B- and FoxP3-positive cells than Tc17 effector cells from unselected CD8 T cells (Tc17), and we conclude that these cells maybe come from memory CD8 (CD44high) T cells.
CD8 T cells of this Tc17 phenotype are unique in that they are able to produce IL-17A and IL-17F and lack perforin and granzyme B expression and have no cytolytic activity. The coexpression of TNF is probably part of the same phenotype, but how far TGF-β
and IL-2 secretion are linked or come from cells of a separate subset is not clear. FoxP3 and IL-10 appeared to be made by IL-17-negative cells. Th17 have been shown to secrete IL-22. We found, however, that although Tc17 effectors from unselected CD8 T cells had a significant amount of message for IL-22, Tc17 effector cells made from naive CD8 T cells were negative. This result suggests that IL-22 detected in Tc17 effector populations also comes from effectors derived from CD44high
memory cells. A similar observation has been made for CD4 T cells by Kreymborg et al. (34
) who showed that IL-22 was induced by IL-23 but only in the CD62Llow
One can make a Venn diagram of overlapping circles, one for each cytokine and define a considerable number of cytokine secretion patterns but it is questionable that these should be considered discrete subsets.
The presence of IL-17-negative cells in the effector population could be taken as evidence that the population was incompletely polarized. Arguing against contamination, at least with Tc1, was the finding that almost none of the cells secreted IFN-γ
or stained for granzyme B. The effector population also lacked any detectable cytolytic activity, also arguing against contamination. An absence of cytolytic activity in IL-17-secreting CD8 T cells was also observed by Liu et al. (66
) who termed them Tnc17 cells (for non-cytolytic). It is of interest that stimulation of naive CD8 T cells under rather different conditions (in the presence of IFN-γ
and absence of IL-6 and TGF-β
) led to the development of strong cytolytic activity (67
The PCR analyses showed that the Tc17 effector population contained message for IL-17A and IL-17F, as expected, but was negative for perforin message, again in contrast with Tc1. The Tc17 effector RNA was negative for IL-4 message, arguing against contamination with Tc2 but showed some message level for IL-10 and TGF-β, raising the possibility of contamination with some sort of CD8 equivalent of regulatory T cells or contaminating CD4 T cells. However, Tc17 effector cells from FACS-sorted naive CD8 T cells still showed some message level for IL-10 and TGF-β, and this did not come from CD4 T cells. This was further supported by the presence of message for FoxP3 in addition to RORγt while the absence of message for T-bet and GATA-3 reenforced that concept that there Tc1 and Tc2 were both absent. Alternatively, it is possible that T cell subsets should be regarded more as a continuum rather than solely as a limited number of discrete subsets and we are inclined to the latter belief.
We have made a preliminary investigation of the role of Tc17 cells in vivo. The fact that they can be seen in the lungs of flu-infected mice is consistent with that they do play some role. In a second model, we transferred Tc17 or Tc1 effectors from OT-1 mice into naive recipient mice that were then challenged with ~2 LD50 of influenza A/PR8ova1. The Tc17 effectors reversed weight loss and increased survival after what would otherwise have been a lethal challenge. Tc17 effectors prepared from perforin-deficient mice were equally as effective as those prepared from wild-type mice, while Tc1 effectors from perforin-/- mice were less effective. Tc17 effectors, however, were somewhat less effective if made from IFN-γ-/- mice while Tc1 effectors were unchanged. Tc17 effectors in vivo are double producers, making IL-17 and IFN at later stages in the response, as noted in the description of Figs. and , and it would appear that this IFN can play some role in the protection afforded by Tc17 effectors. The results indicate that Tc17 and Tc1 effectors protect by different mechanisms. The Tc17 effectors were able to protect the recipient mice from lethal challenge despite the fact that they lacked any cytolytic activity, perforin message, or granzyme B expression in vitro. The Tc17 were as potent as equivalent numbers of Tc1 effectors and it is thus unlikely that a small contaminating subset of other cells was responsible for the protection. Cytolytic destruction of infected epithelial cells by CD8 T cells is generally considered to play a key role in the control of influenza virus and we did not exclude the possibility that the Tc17 effectors developed cytolytic activity after injection, although the protective ability of Tc17 prepared from perforin-deficient mice argues against this. Tc17 effectors generated from sorted naive cells also protected against lethal infection and the protection was Ag specific. Tc17 effector cells from OT-1 mice did not protect against lethal influenza using A/PR8 (without the SIINFEKL insert) and the Tc17 effector cells from P14 TCR-transgenic mice did not protect lethal influenza A/PR8ova1-infected mice (data not shown).
The protection afforded by transfer of Tc17 effectors was accompanied by an increased influx of neutrophils into the lung, suggesting one mechanism of protection.
Tc17 effector and memory cells can make several potent cytokines upon restimulation including IL-17A, IL-17F, IL-21, TNF, and IL-22 and several chemokines including CCL2, CXCL9, CXCL10, CXCL11, and CXCL13. They can also interact with other cells such as dendritic cells, macrophages, and epithelial cells and induce a further cascade of events.
We believe that Tc17 cells may provide protection by recruiting host T cells, as shown by Khader et al. (23
), by recruiting host neutrophils, macrophages, and other cells, as shown by a number of investigators (reviewed in Ref. 17
), by enhancing B cell responses in a number of ways including the secretion of IL-21, stimulating B cell proliferation, and differentiation (reviewed in Ref. 68
) and by the B cell chemoattractant CXCL13, the ligand for CXCR5, which is preferentially expressed by human Th17 cell clones (69
) and finally by IL-17A which has been shown to promote growth of airway epithelial cells (70