Pre-existing SIV infection increases expression of T cell markers associated with activation during early Mycobacterium tuberculosis co-infection and impairs TNF responses in granulomas

Tuberculosis (TB) is the leading infectious cause of death among people living with HIV (PLHIV). PLHIV are more susceptible to contracting Mycobacterium tuberculosis (Mtb) infection and often have worsened TB disease. Understanding the immunologic defects caused by HIV and the consequences it has on Mtb co-infection is critical in combating this global health epidemic. We previously established a model of simian immunodeficiency virus (SIV) and Mtb co-infection in Mauritian cynomolgus macaques (MCM), and showed that SIV/Mtb co-infected MCM had rapidly progressive TB. We hypothesized that pre-existing SIV infection impairs early T cell responses to Mtb infection. To test our hypothesis, we infected MCM with SIVmac239 intrarectally followed by co-infection with a low dose of Mtb Erdman 6 months later. SIV-naïve MCM were infected with Mtb alone as controls. Six weeks after Mtb infection, animals were necropsied and immune responses were measured by multiparameter flow cytometry. While the two groups exhibited similar TB progression at time of necropsy (Nx), longitudinal sampling of the blood (PBMC) and airways (BAL) revealed a significant reduction in circulating CD4+ T cells and an influx of CD8+ T cells in airways following Mtb co-infection of SIV+ animals. Differences in the activation markers CD69, PD-1, and TIGIT were observed. At sites of Mtb infection (i.e. granulomas), SIV/Mtb co-infected animals had a higher proportion of CD4+ and CD8+ T cells expressing PD-1 and TIGIT. In addition, there were fewer TNF-producing CD4+ and CD8+ T cells in granulomas and airways of SIV/Mtb co-infected animals. Taken together, we show that concurrent SIV infection alters T cell phenotypes in granulomas during the early stages of TB disease. As it is critical to establish control of Mtb replication soon after infection, these phenotypic changes may distinguish the immune dysfunction that arises from pre-existing SIV infection which promotes TB progression. Author Summary People living with HIV are incredibly susceptible to TB and, when co-infected with Mtb, often develop serious TB disease. We do not yet understand precisely how HIV infection impairs the early stages of the adaptive immune response against Mtb bacilli. We employed a non-human primate model of HIV, using SIV as a surrogate for HIV, followed by Mtb co-infection to investigate the immunologic defects associated with pre-existing SIV infection over the first six weeks of Mtb co-infection. Our study focused on CD4+ and CD8+ T cells as these cells are known to play an important role in Mtb control. We found more CD8+ T cells in granulomas, the sites of Mtb infection, from SIV/Mtb co-infected animals, with little difference in CD4+ T cells. SIV/Mtb co-infected animals and animals infected with SIV alone had a higher proportion of both CD4+ and CD8+ T cells expressing activation markers compared to SIV-naïve animals, consistent with SIV-dependent immune activation. Notably, we observed a lower proportion of TNF-producing T cells, a cytokine critical for Mtb control, in granulomas and airways of SIV/Mtb co-infected animals. Taken together, these data show that pre-existing SIV alters T cell phenotypes and reduces TNF responses early in Mtb infection.


Introduction 39
Tuberculosis (TB) is a major global health concern, especially among people living with HIV 40 (PLHIV). TB, caused by the bacterium Mycobacterium tuberculosis (Mtb), is the leading cause of death 41 worldwide among PLHIV, accounting for one-third of AIDS deaths [1]. PLHIV are incredibly susceptible 42 to Mtb and have a 20-fold greater risk of developing TB than HIV-naïve individuals [1]. PLHIV have a 43 greater risk of both developing active TB disease or reactivating a latent TB infection [2,3]. The risk for 44 contracting bacterial infections, including Mtb, is higher in PLHIV even before circulating CD4+ T cell 45 counts fall [4][5][6][7], although susceptibility rapidly increases as CD4+ T cell counts decline [8]. Furthermore, 46 the TB risk remains elevated even after individual are put on antiretroviral therapy [9]. Thus, there is a critical need to identify more precisely the underlying mechanisms by which pre-existing HIV infection 48 impairs the immune response to Mtb.

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Knowledge gaps remain in our understanding of early immune events following Mtb infection [10].

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TIGIT in lung tissue and granulomas. 187 While BAL can be used to sample airway cells longitudinally, it cannot access cells within the lung 188 parenchyma or within granulomas. We conducted comprehensive, PET/CT-guided necropsies 6 weeks after

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Mtb infection to harvest TB granulomas as well as random lung tissue samples to determine whether there 190 were phenotypic and/or functional differences in these tissues between SIV+ and SIV-naïve animals.

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Elevated immune activation in the lungs has been suggested previously to cause more severe TB

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A higher frequency of CD8+ T cells expressing PD-1 was present in the granulomas of SIV/Mtb 204 co-infected MCM (red squares; Fig 8F) compared to those infected with Mtb alone (blue circles).

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Conversely, there were no differences in the frequency of CD8+ T cells expressing PD-1 in lung tissue 206 between animals infected with Mtb only (blue circles), SIV/Mtb (red squares), and SIV only animals (black 207 triangles; Fig 8E). There was a trending increased frequency of CD8+ T cells expressing TIGIT in lung 208 tissue of SIV/Mtb animals ( Fig 8G, H).

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Granulomas from SIV+ animals contain lower frequencies of T cells producing TNF.

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The frequency of T cells producing the cytokines IFN- and TNF were measured in lung tissue and 212 granulomas collected at necropsy 6 weeks after Mtb infection (Fig 9). IFN- production by CD4+ and CD8+

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T cells differed little between SIV+ (red squares) and SIV-naïve (blue circles) animals in both lung tissue 214 and granulomas (Fig 9A-D

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T cells and some studies suggest that a reduction in the ratio of CD4:CD8 T cells is associated with poor 246 TB disease outcome [38]. We found CD4:CD8 T cell ratios were lower across all tissue compartments 247 measured in SIV/Mtb co-infected animals (Fig 2). A subset of animals chronically infected with SIV alone 248 was necropsied to provide insight into the immunologic environment in tissues prior to Mtb infection. These 249 animals also exhibited lower CD4:CD8 ratios in the blood and lung tissue, indicating that the decrease observed in SIV/Mtb animals was a consequence of their pre-existing SIV infection. In humans, HIV 251 infection lowers CD4:CD8 T cell ratios in blood by a continuous loss of CD4+ T cells [48]. We observed 252 a similar decline of circulating CD4+ T cells, without significant changes to CD8+ T cells, in SIV-infected 253 animals, both before and after Mtb infection (Fig 3). Interestingly, in the airways, where immune cells first 254 encounter inhaled Mtb, SIV/Mtb co-infected animals showed significant accumulation of CD8+ T cells (Fig   255  4). This is likely a consequence of SIV infection as SIV-specific CD8+ T cells have been shown to 256 accumulate in airways following SIV infection [41].

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The CD4:CD8 T cell ratios in both lung tissue and granulomas were lower in SIV/Mtb co-infected 258 animals and was also noted in the lung tissue of SIV+ MCM, indicating that SIV infection alone drove the 259 decline in the CD4:CD8 ratio (Fig 2). Looking more closely at the T cell populations in lung granulomas, co-infected MCM and assess SIV-specific CD8+ T cell responses to test this hypothesis.

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We did not observe a difference in the frequencies of IFN--producing CD4+ or CD8+ T cells  activation signature were elevated in blood and tissues when compared to animals infected with Mtb alone.

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In the blood from SIV+ MCM, we observed a higher frequency of both CD4+ and CD8+ T cells expressing 293 markers associated with T cell activation (Fig 6). Interestingly, while peripheral CD4+ T cells were 294 activated only after Mtb infection, CD8+ T cells were activated prior to Mtb infection in an SIV-dependent 295 manner (Fig 6). In lung tissue as well as granulomas, there was a higher frequency of CD4+ T cells    thoracic lymph nodes, regardless of whether pathology was grossly apparent, and plating as described 414 above. The CFU from each sample were summed to yield total thoracic lymph node bacterial load.

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Summing the total lung and total thoracic lymph node CFU provided the total thoracic bacterial burden To assess conventional CD4+ and CD8+ T cells, cells from PBMC, BAL, lung tissue, lymph nodes,

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and granulomas were stained as previously described [84]. Cell markers and antibodies are listed in Table  1. To assess frequencies of circulating T cell subsets, cryopreserved PBMC were used for staining.

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Corresponding whole blood samples were sent to the clinical hematology laboratory at the University of 455 Pittsburgh Medical Center for complete blood counts (CBC). We used the flow cytometry data to convert 456 the total lymphocyte numbers from the CBC to total CD4+ and CD8+ T cells/microliter of blood.

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For analysis of BAL samples, freshly isolated cells were stained. Cells were resuspended in media

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(RPMI 1640 supplemented with 10% human albumin, 1% L-glutamine, and 1% HEPES) and divided for a 459 phenotype flow panel and a stimulation flow panel ( Table 1). The staining procedure for both panels were 460 identical, except for the antibody cocktails used (

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To stain tissues obtained at necropsy, freshly isolated tissue homogenates were used.

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Cells were resuspended in media as described above and were either left unstimulated or stimulated with 477 phorbol 12,13-dibutyrate (PDBu) and ionomycin for 3 hours at 37C, 5% CO 2 . The stimulators described above and brefeldin A were added simultaneously. Cells were then resuspended in 500 nM dasatinib and