Th1 polarized CD4 T cell responses are important for containment of Mycobacterium tuberculosis (Mtb) infection, but otherwise the features of host protective CD4 T cell responses during Mtb infection are poorly understood. This lack of robust correlates of protection is a major barrier to developing new vaccine and therapeutic strategies. We previously showed that the pulmonary effector T cell response against Mtb is composed of two major subpopulations that preferentially localize to either the lung parenchyma or lung blood vasculature. Intravascular Mtb-specific CD4 T cells display greater potential for cytokine production as well as active production of interferon-gamma (IFNg) in vivo compared to their tissue parenchymal counterparts. However, CD4 T cells with the ability to migrate into the tissue parenchyma were highly protective against Mtb infection, while CD4 T cells that were high producers of IFNg but poorly migrate into the lung displayed minimal control of the infection. In the above studies, we found that the non-protective CD4 T cells expressed high levels of Tbet, the classic Th1 promoting transcription factor, so we investigated the relative roles of Tbet in the protective and non-protective CD4 T cell responses. We found that Tbet-/- mice mounted robust Mtb-specific CD4 T cell responses following infection, indicating that Tbet was not required for the priming of CD4 T cells in Mtb infection. Moreover, the Tbet-/- CD4 T cells showed profound defects in IFNg production as expected, and dramatic increases in IL-17A+ CD4 T cells. Furthermore, the Tbet -/- mice showed greatly increased frequencies of parenchymal T cells and an almost complete lack of the intravascular T cells. To ensure that these immunological differences were T cell intrinsic, we adoptively transferred bulk CD4 T cells from naive WT or Tbet KO mice into Mtb infected WT recipients and analyzed the response of the donor T cells. We found again, that Tbet-/- CD4 T cells expanded and migrated into the lungs, but did not differentiate into the non-protective intravascular phenotype. Therefore, while Tbet is clearly important for the production of IFNg, it also has a unexpected and potentially detrimental role in that it promotes the generation of CD4 T cells that cannot migrate into the lungs and mediate protection against Mtb. Another important observation discussed above was that the CD4 T cells that mediate the best protection against Mtb produce relatively low amounts of IFNg. There is a great deal of data in humans and experimental animals models to show that complete IFNg deficiency leads to extreme disease in Mtb infection, but there is little quantitative information on the relative contribution of CD4 T cell-derived IFNg to the control of Mtb infection in different tissues. This prompted us to re-evaluate the role of CD4 T cell-derived IFNg in Mtb infection. We first transferred WT or IFNgKO CD4 T cells into RAGKO mice and followed the bacterial replication in the lungs and spleen. We found that the vast majority of the reduction in bacterial loads in the lung mediated by CD4 T cells was independent of their ability to produce IFNg. In contrast to the lungs, in the spleen we found that almost all of the anti-bacterial effects of CD4 T cells was due to IFNg production. We next asked if the relatively low contribution of IFNg to CD4 T cell-mediated protection was due to its low expression by the parenchymal CD4 T cells. To do so, we adoptively transferred increasing ratios of WT:IFNgKO CD4 T cells into T cell deficient recipient mice and measured bacterial growth. We found that IFNg-dependent control of Mtb in the lung plateaued at a ratio of 40% WT to 60% IFNgKO and was no better when 100% WT donor T cells were injected. In contrast, in the spleen the best control of the infection occurred at 100% WT T cell transfer. Collectively, these data indicate that CD4 T cell-derived IFNg is critical for control of infection in the spleen, but contributes very little to their protective capacity in the lung. We next asked if forcing CD4 T cells to produce increased levels of IFNg enhanced control of the infection in the lungs and spleen by adoptively transferring CD4 T cells from mice that overproduce IFNg due to a deletion of a regulatory element in the 3 region of the mRNA (ARE CD4 T cells). We found that adoptive transfer of ARE CD4 T cells enhanced control of the infection in the spleen compared to WT cells, but impaired control of the infection in the lung and resulted in the early death of the mice. Therefore, not only does CD4 T cell-derived IFNg contribute very little to control of the infection in the lungs, increasing the production of IFNg by CD4 T cells (2 fold in these experiments) resulted in fatal immunopathology. We next asked if our previous work showing the early death of mice lacking the inhibitory receptor PD-1 could be due to the over-production of IFNg. To do this, we generated IFNg/PD-1 double knock out mice. We tested the role of IFNg production by PD-1KO CD4 T cells with a series of adoptive transfer experiments. We found that adoptive transfer of a mixture of WT and PD-1KO CD4 T cells leads to the early death of the mice. In contrast, transferring WT + IFNg/PD-1DKO CD4 T cells resulted in normal host survival. Therefore, the PD-1 normally serves to inhibit the production of IFNg by CD4 T cells limiting its host detrimental effects. Currently, we are investigating the mechanisms downstream of IFNg-induced mortality in Mtb infected mice. The hope is that we may identify novel targets for immunotherapeutic intervention that work by limiting the detrimental effects of CD4 T cell-derived IFNg in pulmonary Mtb infection. Collectively these data show that the Tbet/IFNg axis is only a minor component of the host-protective response in the lung and may primarily be involved in control of disseminated extra-pulmonary infection. This led us to search for novel effector molecules that contribute to the control of Mtb infection in the lung. To do so, we examined the gene expression profile of purified populations of host-protective and non-protective CD4 T cells from the lungs of infected mice. We have identified a list of candidate effector molecules that are highly upregulated in the protective subset of CD4 T cells compared to the non-protective subset or naive T cells, and are currently interrogating the role of these pathways in control of Mtb infection. Identification of novel anti-tuberculosis CD4 T cell effector molecules will provide critical insight into the logical development of tuberculosis vaccines and potentially identify target pathways for the host-directed therapies for tuberculosis. We have previously developed experimental mouse model of mycobacterial-associated IRIS where CD4 T cell injection into T cell-deficient mice chronically infected with Mycobacterium avium leads to rapid host mortality, and identified several major pathways that have a role in the immunopathology including IFNg and TNF and IL-6. Here use our murine model to examine myeloid cell responses in IRIS. We show that CD4+ T cell reconstitution leads myeloid cell production of effector molecules (NO, TNF, IL-1a and IL-1b) at levels far exceeding an intact host. Along with the increase in the magnitude of inflammatory mediator production, there is also a significant increase in the poly-functionality of myeloid cells in IRIS compared to WT mice. Importantly, changes in both the function and composition of myeloid cells were dependent on CD4 T cell-derived IFNg. Collectively our data demonstrate that T cell reconstitution of chronically-infected hosts triggers a substantial increase in myeloid cell activation, and may implicate dysregulated myeloid cells in the pathogenesis of IRIS.
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