The study protocol was approved by the Animal Ethics Committee of the National Institute of Immunology (IAEC#443/17). Experimental procedures followed the guidelines of the Animal Ethics and Biosafety Committee of the National Institute of Immunology (New Delhi, India).

Inbred T-cell-deficient TCRβ^−/−, wild-type C57Bl/6, and congenic CD45.1 mice (6–8 weeks) were maintained in the germ-free condition in the animal experimental facility at the National Institute of Immunology according to the guidelines for laboratory animals.

Mycobacterium tuberculosis (strain H37Rv and H37Rv-GFP) was grown in shaking conditions (120 RPM) at 37°C. 7H9 culture media (Difco™ Middlebrook) was prepared in sterile water following the manufacturer’s instruction. Culture media was supplemented with 0.1% tween-80, 10% Albumin Dextrose Catalase (ADC), and 0.2% glycerol. At OD 0.6, M.tb culture was harvested by centrifugation at 3,000 RPM for 15 min. M.tb infection (200 CFU/mice) was given by aerosol route using Madison inhalation exposure system (Madison Industries, USA) in the BSL-3 facility at Tuberculosis Aerosol Challenge Facility of International Centre for Genetic Engineering and Biotechnology (ICGEB).

Thioglycolate (4%) injection (1.5 to 2 ml) was given intraperitoneally, and after 72 h, the mice were sacrificed and injected with chilled PBS in the peritoneum. The peritoneal exudate was collected and seeded onto 6-, 12-, 24-, and 96-well tissue culture plates (for different experiments) in RPMI media with 10% fetal bovine serum (FBS) for 2–3 h to allow macrophages to adhere to the plates. Subsequently, non-adherent cells were removed by washing twice with RPMI. Macrophages were infected with M.tb at 10× multiplicity of infection (MOI) for 4 h. After infection, the cells were extensively washed twice with PBS to remove non-phagocytosed bacteria and purified CD4+ T cells were added to the culture at 1:2 ratio. Cells were incubated for 24/48 h before further analysis.

Bone marrow from femurs of C57Bl/6 WT and T-cell-deficient TCRβ^−/− mice (6–8 weeks) mice were dispersed and cultured in RPMI medium [containing 100 U/ml of penicillin and supplemented with 10% FBS and 20 ng/ml of mouse macrophage colony stimulating factor (Miltenyi Biotec)] at 37°C and 5% of CO₂. Cells were cultured till 90% of cells were positive for CD11b (a monocyte/macrophage-specific marker) and were harvested after 7 days.

CD4+ T cells were isolated from the spleen of CD45.1 (C57Bl/6) WT mice and enriched by magnetic cell sorting using MACS magnetic beads (Miltenyi Biotec) as per the protocol given. The purity of CD4+ T cells as determined by FACS was found to be 90%–95% ( Figure S1 ). The enriched population of CD4+ T cells was stained with 50 µM carboxy fluorescein succinimidyl ester (CFSE) before adoptive transfer. 2 × 10⁶ enriched CD4+ T cells were then injected intravenously in M.tb-infected C57Bl/6 WT and T-cell-deficient TCRβ^−/− mice.

Flow cytometry assay was done to check the expression of specific cell surface markers (MHCII, CD80, CD86, and CD40) on macrophages. For surface staining, 1 × 10⁶ cells from each group were taken and stained with specific antibodies for cell surface markers (MHCII-APC, CD80-FITC, CD86-PE, F4/80-PECy7, and CD4-FITC) and incubated for 30 min at 4°C. Cells were washed twice in FACS wash buffer after incubation, then resuspended in FACS buffer, and flow cytometric analysis was done (FACS Verse BD Biosciences, USA).

Mice were euthanized by using a Ketamine–Xylazine cocktail (2 mg ketamine + 0.2 mg xylazine/20 g of mice). Lung tissue was collected and chopped with a fine blade in RPMI. The chopped tissues were transferred to digestion enzyme cocktail of hyaluronidase (200 μg/ml), collagenase-A (700 μg/ml), and DNase (30 μg/ml) and incubated at 37°C for 45 min in shaking condition. To get a single-cell suspension, the extracellular matrix was removed by passing the digested lung tissue through a cell strainer (pore size 40 μm). RBCs were lysed using BD Pharm lyse solution. After RBC lysis, cells were allowed to adhere in T75 culture flask for 5 h; non-adherent cells were removed by washing twice with PBS. Culture flasks were maintained on ice throughout washing and tapped firmly in between. After washing, strongly adherent cells (mainly macrophages) were removed by using Zymefree (non-enzymatic cell dissociation solution from Himedia). Purity of these cells were determined by flow cytometry. About 80% of cells were CD11b⁺, CD14⁺, and F4/80⁺ ( Figure S6 ).

The level of TNF-α, IL-12, IL-6, IFN-γ, IL-2, IL-10, IL-1β, IL-4, and IL-17 cytokines in the serum and peritoneal macrophages culture supernatants was quantified by cytokine beads array (BD Biosciences, San Jose, CA, USA) as per the manufacturer’s protocol.

The level of nitric oxide in serum and peritoneal macrophage culture supernatant was determined indirectly by measuring nitrite concentration using Griess reagent [1% sulfanilamide in 5% H₃PO₄ and 0.1% naphthyl ethylene diamine in distilled water] according to the manufacturer’s protocol. One hundred microliters of culture supernatants/serum was transferred into 96-well plates and then 100 μl of Griess reagent was added, and absorbance was measured at 540 nm.

For mRNA isolation, lung macrophages were isolated according to the protocol mentioned in Material and Methods section. mRNA expression of relevant chemokines in lung macrophages from different groups of mice was determined by qRT-PCR. RNA was isolated from lung macrophages using One Step RNA Reagent (RNAiso Plus, Takara) as per the manufacturer’s protocol. The quantification of mRNA was done using a spectrophotometer and agarose gel electrophoresis was used to determine the quality of mRNA. One microgram of mRNA was used to prepare cDNA using cDNA Synthesis Kit (Revert Aid First Strand, Thermo Scientific, USA) according to the protocol. Real-time PCR was set up using the cDNA and primers specific to each gene, using an RT-PCR master-mix (TB Green Premix Ex Taq Takara). The 2^−ΔΔCt method was used to determine the relative quantities of these genes and were normalized with respective genes from uninfected control group.

Peritoneal macrophages were seeded on UV sterilized coverslips in a 12-well plate, and M.tb-GFP infection was given for 4 h. Then, extracellular bacteria were removed by washing (twice with PBS), and culture was incubated in a CO₂ incubator for 24 h at 37°C. After incubation, cells were then fixed overnight at 4°C in 1% paraformaldehyde followed by washing with PBS twice. Lysosomal compartment of the cells was stained by using 100 nM LysoTracker Red for 30 min at 37°C followed by DAPI staining. M.tb-GFP inside the lysosome was observed under a confocal microscope. In each category, a total of 50 fields were analyzed, M.tb-GFP outside lysosomes (green spots) and M.tb-GFP inside the lysosome (yellow spots) were counted.

Whole lungs were isolated from M.tb-infected mice at different time points, suspended in 1 ml of PBS and homogenized using PolytronR PT 1600 homogenizer (Kinematica, Lucerne, Switzerland). One hundred microliters of lung homogenates were diluted serially, plated in triplicates on 7H11 agar plates, and incubated for 3 to 4 weeks at 37°C. After incubation, bacterial colonies were counted. Similarly, bacterial load in the spleen and liver were determined by counting the number of colonies at different time points.

Collection of lung lobes was done aseptically after systemic perfusion with PBS, fixed overnight with 4% w/v paraformaldehyde, embedded in paraffin, and sectioned to 2-μm tissue sections. The tissue sections were stained with hematoxylin–eosin.

Core body temperature was measured using a rectum probe, and for surface body temperature, an infrared camera thermometer (seek thermal camera) was used.

Prism7 (GraphPad, San Diego, CA, USA) was used for statistical analysis. Data were compiled as mean ± SEM/standard deviation (SD). Comparison between the groups was carried out using two-way analysis of variance (ANOVA). Significance was determined using the Holm-Sidak method. Tukey’s correction was used for multiple comparisons. Any p-value less than 0.05 was considered to be statistically significant.

To investigate the effect of adoptive CD4+ T cell transfer from M.tb-infected CD45.1 (C57Bl/6) mice into TCRβ^−/− mice during chronic mycobacterial infection, TCRβ^−/− and C57Bl/6 WT mice were challenged with M.tb (about 200 CFU/mice) by aerosol route ( Figure 1A ). After 30 days of infection, enriched CFSE-labeled CD4+ T cells (2 × 10⁶) from M.tb-infected CD45.1 mice were injected intravenously into TCRβ^−/− mice and C57Bl/6 WT control mice. Recipient TCRβ^−/− mice rapidly lost 20%–30% of their initial body weight within 15 days of CD4+ T-cell transfer ( Figure 1B ). All the TCRβ^−/− mice in which CD4+ T cells were adoptively transferred succumbed by day 25 ( Figure 1C ). In contrast, infected C57Bl/6 WT recipient mice showed no significant weight loss and mortality. Core body and surface body temperature were also checked at different time intervals post-infection and after CD4+ T cell transfer. After M.tb infection, both groups showed increased body temperature, but after 15 days of CD4+ T cell transfer, TCRβ^−/− mice showed a significant drop in their core and surface body temperature ( Figures 1D, E ). This drop in core and surface body temperature could be because of heightened inflammation as part of the body’s natural defense (17). These results suggest that adoptive transfer of M.tb primed CD4+ T cells into M.tb-infected mice lacking T cells is sufficient to drive TB-IRIS, resulting in rapid wasting, excessive inflammation, and death.

It is reported that when CD4+ T cells are introduced in a T cell-deficient environment, they proliferate spontaneously (18). This observation was confirmed in our mouse model of TB-IRIS. Purified CD4+ T cells [cell purity was assessed by flow cytometry and 90%–95% of cells were found CD4 positive ( Figure S1 )] were labeled with CFSE. In vivo proliferation of CFSE-labeled CD4+ T cells from CD45.1 mice transferred to M.tb-infected KO as well as WT mice was studied. After 72 h of transfer, the CFSE dilution of CD4+ T cells in M.tb-infected KO and WT mice blood was compared. Higher dilution of CFSE was observed in KO mice providing evidence of higher proliferation of transferred T cells in M.tb-infected KO mice as compared to WT mice ( Figure S2 )]. These observations confirm that in this TB-IRIS model, proliferation of donor T cells in T-cell-depleted conditions is significantly high as compared to WT mice.

To investigate how efficiently these TCRβ^−/− mice control the M.tb infection, we compared the bacterial burden in lung, spleen, and liver in TCRβ^−/− and WT mice. In contrast to wild-type mice, TCRβ^−/− mice showed at least one log higher bacillary load in the lung, spleen, and liver at day 30 ( Figure 2A ). Interestingly, the transfer of CD4+ T cells into TCRβ^−/− mice significantly reduced the pulmonary and extrapulmonary bacterial burden while no such reduction was observed in WT mice. After 15 days of CD4+ T cell transfer, the bacterial burden in TCRβ^−/− animals was reduced by one log and it became similar in KO and WT mice ( Figure 2B ). This could be due to the hyper immune response in TCRβ^−/− mice, which killed the bacteria.

Lung histopathology analysis was done in all the groups of TCRβ^−/− and WT mice ( Figure 3 ). In M.tb-infected TCRβ^−/− mice in which CD4+ T cells were transferred, a large number of mononuclear cells can be seen in the lung sections as compared to WT mice (shown by green arrow). At higher magnification (100×), foamy macrophages (blue arrows) can be seen in the lung section of KO mice. No difference in the uninfected and only CD4+ T cell transferred group was observed between KO and WT mice. In the M.tb-infected WT mice, a higher number of mononuclear cells (mainly lymphocytes) were observed as compared to M.tb-infected TCRβ^−/− mice because there are no T cells in this group. These observations corroborate with the flow cytometry studies.

Previous studies (19, 20) have shown that hyperactivated CD4+ T cells are responsible for pathogenesis of TB-IRIS, but how these CD4+ T cells become hyperactivated is not completely known. CD4+ T cells require help from antigen-presenting cells for their activation, and according to our hypothesis, these macrophages are somehow differently activated in the TCRβ^−/− mice. Hence, to examine the role of macrophages in the development of TB IRIS, their activation status was analyzed and compared between WT and TCRβ^−/− mice by measuring the levels of secretory cytokines in the culture supernatant of peritoneal macrophages. There were six groups in this experiment, i.e., only macrophages, macrophages infected with M.tb, macrophages co-cultured with CD4+ T cells, M.tb-infected macrophages co-cultured with CD4+ T cells, macrophages with IFN-γ alone, and M.tb-infected macrophages with IFN-γ. Higher levels of pro-inflammatory cytokines IL-6, TNF-α, IL-12, and IL-1β were detected in the culture supernatant of macrophages from TCRβ^−/− mice in which M.tb infection was given as compared to wild type. The levels of these cytokines further increased to a significant level after the addition of CD4+ T cells ( Figure 4A ). Another interesting observation was that in TCRβ^−/− mice macrophages, recombinant IFN-γ along with M.tb infection also increased the secretion of pro-inflammatory cytokines in culture supernatant, which is higher than M.tb alone but less than the M.tb+CD4+ T cell group ( Figure 4A ). Since CD4+ T cells were co-cultured with macrophages, we further checked the T-cell-secreted cytokines and found that level of Th1 (IFN-γ) and Th17 (IL-17) cytokines was high in the KO group while the level of Th2 (IL-4 and IL-10) cytokines was very low in both KO and WT groups ( Figure 4B ). Level of nitric oxide (NO), which plays a key role in the elimination of intracellular M.tb from infected macrophages, was also analyzed. Interestingly, significantly higher NO production by M.tb-infected TCRβ^−/− mouse macrophages was observed as compared to WT macrophages, which further increased after the addition of CD4+ T cells ( Figure 4C ). Previous studies (21) have shown that NO production depends on IFN-γ stimulation, but interestingly, here we found that TCRβ^−/− mouse macrophages produced copious amounts of NO even in the absence of IFN-γ.

To assess the ex vivo activation status of peritoneal macrophages, expression of MHCII, CD80, CD86, and CD40 was analyzed on the macrophages from TCRβ^−/−and wild-type mice. There were four groups in this experiment, i.e., only macrophages, macrophages infected with M.tb, macrophages co-cultured with CD4+ T cells, and M.tb-infected macrophages co-cultured with CD4+ T cells ( Figure 5A ). After M.tb infection, the TCRβ^−/− mice macrophages showed significantly higher expression of MHCII and CD86 while expression of CD80 and CD40 was found to be similar in infected KO and WT mice macrophages, but interestingly, after the addition of CD4+ T cells, significantly higher expression of MHCII, CD80, CD86, and CD40 was observed in M.tb-infected KO mice macrophages as compared to WT macrophages. Co-stimulatory molecules (CD80/86) expressed by macrophages interact with CD28 on CD4+ T cells, which provide a second signal to CD4+ T cells for their activation and proliferation (22, 23). Significantly higher expression of co-stimulatory markers and MHCII provides evidence that macrophages from mice model of TB-IRIS are in more activated state as compared to their WT counterpart.

Phago-lysosomal trafficking is an important innate defense pathway that clears microbes by delivering them to lysosomes (24). Antigen presentation by MHCII requires processing of M.tb antigens in the lysosome. To examine the phago-lysosomal fusion status, co-localization of green fluorescent protein (GFP) expressing M.tb in lysosomes was checked in macrophages by using lysotracker red ( Figure 5B ). Lysotracker red is a specific dye that stains acidic compartments in the cells, so the GFP expressing bacteria that is inside the lysosome appears yellow. The twofold higher number of GFP expressing M.tb were found in the lysosomal compartment of TCRβ^−/− mice macrophages as compared to WT, showing that there is high phago-lysosomal fusion activity in TCRβ^−/− mice macrophages. These results suggested robust antigen presentation efficacy of TCRβ^−/− mice macrophages compared to wild-type macrophages. Furthermore, we got curious whether this higher inflammation affects the survival of macrophages. To investigate this, we examined the survival of KO and WT mice peritoneal macrophages after M.tb infection. For this, culture supernatants from M.tb-infected KO and WT mice, post 96 h of infection, were checked for lactate dehydrogenase secretion in ex vivo culture (LDH, which leaks out from dead cells). A low level of LDH activity in supernatants from M.tb-infected KO mice macrophages demonstrated a lower level of M.tb-induced cell death than WT macrophages ( Figure S3 ). This higher viability of KO mice macrophages suggests that these macrophages can interact with CD4+ T cells for an extended period, potentially resulting in prolonged inflammation.

We further analyzed the infiltration of macrophages in lungs of M.tb-infected KO mice and found that it was increased by about 15% as compared to uninfected mice. After adoptive transfer of CD4+ T cells, this was further increased by 50%–60% in infected KO mice while no such increase was observed in WT mice ( Figure S4 ). When macrophage population was compared between infected KO vs. infected WT mice, 1.5-fold higher level of macrophages was observed in M.tb-infected KO lungs as compared to infected WT lungs. In addition, infiltration of neutrophils was analyzed. Neutrophil infiltration was found to be significantly high in M.tb-infected TCR β KO mice (40%–50% increase as compared to uninfected controls), which further increased by 50%–55% after transfer of CD4+ T cells. So, neutrophil infiltration was much higher than macrophages.

Since we obtained interesting results in the ex vivo culture of peritoneal macrophages and found higher activation status of TCRβ^−/− mice macrophages, we further analyzed the activation status of lung and systemic macrophages in in vivo conditions. For this, mice were challenged with M.tb (about 200 CFU/mice) by aerosol route, and after 30 days of infection, CD4+ T cells were transferred from M.tb-infected WT mice to KO mice. There were four groups, i.e., uninfected control group, M.tb-infected group, only CD4+ T cells transferred group, and M.tb-infected group in which CD4+ T cells were transferred. After 15 days of CD4+ T-cell transfer, lungs from each group were collected and single-cell suspension was prepared and analyzed for the expression of macrophages activation markers. Similar to the ex vivo study, after M.tb infection, expression of MHCII and CD86 was higher in TCRβ^−/− mice ( Figure 6 ). Lung macrophages from M.tb-infected TCRβ^−/− mice in which CD4+ T cells were adoptively transferred showed significantly higher expression of MHCII, CD80, CD86, and CD40 compared to WT. Since TB-IRIS is a systemic disease, expression of these activation markers was also checked and compared between blood monocytes of TCRβ^−/− and WT mice. Only M.tb infection resulted in the increased expression of MHCII and CD80 in TCRβ^−/− mice blood monocytes while similar to lung macrophages, adoptive transfer of CD4+ T cells in M.tb-infected TCRβ^−/− mice resulted in significantly higher expression of MHCII, CD80, CD86, and CD40 on blood monocytes compared to WT ( Figure 7 ), which would make them potent cells to modulate the adaptive immune response. These results suggest that macrophages from TCR-β ^−/−animals are robust and abnormally activated, which supports our hypothesis.

Furthermore, to study the systemic response, serum cytokines in M.tb-infected TCRβ^−/− and WT mice were checked. After M.tb infection, a higher level of IL-6, TNF-α, IL-12, and IL-1β was observed in TCRβ^−/− mice, as compared to WT mice, which further increased significantly when CD4+ T cells were transferred ( Figure 8A ). When compared to WT, M.tb-infected TCRβ^−/− mouse serum showed substantially more NO level, which increased further when CD4+ T cells were introduced in M.tb-infected TCRβ^−/− mice ( Figure 8C ). We also checked the level of anti-inflammatory cytokines in serum; a low level of IL-4 and IL-10 was observed in both TCRβ^−/− and WT mice serum ( Figure 8B ). Pro-inflammatory cytokines are in charge of launching a strong response against exogenous pathogens while anti-inflammatory cytokines are important for reducing the inflammatory response and preserving homeostasis for proper organ function. The excessive output of these mediators could be playing crucial role in the development of IRIS syndrome.

As a critical component of innate immunity, macrophages serve as the first-line defense of M.tb infection (25). To further explore the effect of CD4+ T cell deficiency and M.tb infection on macrophages, lung macrophages were isolated from different groups of M.tb-infected and uninfected, TCRβ^−/−and WT animals post 15 days of CD4+ T cell transfer. mRNA expression of various inflammation-associated markers like chemokines, matrix metalloproteases, M.tb-associated pattern recognition molecules, and other inflammation regulatory molecules was analyzed.

Chemokines are chemical messengers secreted by immune and non-immune cells that play a critical role in recruiting other immune cells to the infection site (26). In lung macrophages, mRNA expression of chemokines CCL2, CCL3, CCL5, CXCL1, CXCL2, CXCL8, CXCL9, and CXCL11 and chemokine receptors CCR1 and CCR2 was compared between different groups of TCRβ^−/− and wild-type mice by qRT-PCR. In only M.tb-infected group, mRNA expression of CXCL8, CXCL11, and CCL5 was approximately threefold high in TCRβ^−/− and twofold high in wild type as compared to uninfected control mice ( Figure 9A ). Interestingly, after adoptive transfer of CD4+ T cells in M.tb-infected groups, mRNA expression of CXCL11 and CCL5 increased about 8-fold, and CXCL8 increased about 10-fold in TCRβ^−/− mice, while no change was observed in wild-type mice ( Figure 9A ). Similarly, mRNA expression of CXCL9 increased about 5-fold in only M.tb-infected TCRβ^−/− mice group and about 15-fold in M.tb-infected TCRβ^−/− mice group in which CD4+ T cells were transplanted ( Figure 9A ). In contrast, about twofold rise was seen in the wild-type group in both M.tb-infected and M.tb+ CD4+ T-cell-transferred groups. After M.tb infection, mRNA expression of CCR2 increased about 5-fold in M.tb-infected TCRβ^−/− mice compared to 1.5-fold increase in WT mice whereas CD4+ T cell transfer resulted in an approximately 8-fold rise in KO mice, but no such increase was found in WT. No difference in the mRNA expression of CCR1 was observed between different groups of KO and WT mice ( Figure S5 ). mRNA expression of other chemokines, CCL2, CCL3, CXCL1, and CXCL2, was also analyzed but no significant change was detected (data not shown).

Matrix metalloproteinases (MMPs) are a group of proteases that destroy extracellular matrix components and are believed to play a role in tuberculosis-related lung injury (27, 28). During TB infection, MMPs may facilitate various stages of lung remodeling (29). MMP1, MMP3, MMP8, MMP9, and MMP11 mRNA expression in lung macrophages have been examined before and after M.tb infection and CD4+ T cell transfer. mRNA expression of MMP1 was about 6-fold higher in M.tb-infected TCRβ^−/− mice lung macrophages, which increased to about 10-fold upon CD4+ T-cell transfer, compared to the 2-fold increase in M.tb + CD4 T-cell-transferred group of WT mice ( Figure 9B ). No change was observed in mRNA expression of MMP9 in TCRβ^−/− and wild-type mice after M.tb infection, but after CD4+ T-cell transfer, mRNA expression of MMP9 increased to about 1.5 times in M.tb-infected KO mice. These results show higher expression of CXCL8, CXCL9, CXCL11, CCL5, MMP1, and MMP9 in the mouse model of TB-IRIS, which could be possibly involved in the pathogenesis of IRIS disease.

Pattern recognition molecules like TLRs and feedback inhibition regulating molecules like Suppressor of Cytokines Signaling (SOCS) were also analyzed and compared between TCRβ^−/− and wild-type mice lung macrophages. Toll-like receptors are very crucial pattern recognition receptors in the recognition of M.tb. Recognition of microbial products by these receptors trigger functional maturation and initiation of antigen-specific adaptive immune responses (30). So, we further analyzed whether these are differentially expressed in TCRβ^−/− vs. WT mice. mRNA expression of TLR2, TLR4, and TLR9 were found to be significantly increased in lung macrophages of M.tb-infected TCRβ^−/− mice as compared to WT ( Figure 10A ).

The SOCS proteins function as feedback inhibitors of the various signaling pathways and can terminate innate and adaptive immune responses and thus control inflammation in a normal situation (31). So, the expression of SOCS1 and SOCS3 in lung macrophages was analyzed. Interestingly, mRNA expression of SOCS1 and SOCS3 was found to be low in infected lung macrophages of TCRβ^−/− mice as compared to WT lung macrophages ( Figure 10B ).

Tumor necrosis factor receptor-associated factor 6 (TRAF6), an E3 ubiquitin ligase, is a signaling protein of the interleukin-1 receptor (IL-1R)/Toll-like receptor (TLR) family and the TNFR superfamily (32). In this study, we also examined the mRNA expression of TRAF-6 in lung macrophages. Following M.tb infection, it was observed that TRAF-6 mRNA expression in TCRβ^−/− and WT mice lung macrophages increased by 2.5-fold when compared to uninfected control, and this further increased to 5-fold after CD4+ T cell transfer in TCRβ^−/− mice ( Figure 10C ). After M.tb infection and CD4+ T-cell transfer, no statistically significant change in the mRNA expression of TRAF-6 was detected in wild-type lung macrophages.