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Research ArticleAIDS/HIVImmunologyInfectious disease
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10.1172/jci.insight.199314
1Department of Pediatrics, Children’s Hospital of Pittsburgh of the University of Pittsburgh Medical Center, Pittsburgh, Pennsylvania, USA.
2Center for Vaccine Research,
3Department of Microbiology and Molecular Genetics, and
4Division of Laboratory Animal Research, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania, USA.
5Department of Immunology and Infectious Diseases, Harvard T. H. Chan School of Public Health, Boston, Massachusetts, USA.
Address correspondence to: Philana Ling Lin, UPMC Children’s Hospital of Pittsburgh 2310 AOB, 4401 Penn Ave., Pittsburgh, Pennsylvania, 15224, USA. Phone: 412.383.2178; Email: linpl@chp.edu.
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1Department of Pediatrics, Children’s Hospital of Pittsburgh of the University of Pittsburgh Medical Center, Pittsburgh, Pennsylvania, USA.
2Center for Vaccine Research,
3Department of Microbiology and Molecular Genetics, and
4Division of Laboratory Animal Research, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania, USA.
5Department of Immunology and Infectious Diseases, Harvard T. H. Chan School of Public Health, Boston, Massachusetts, USA.
Address correspondence to: Philana Ling Lin, UPMC Children’s Hospital of Pittsburgh 2310 AOB, 4401 Penn Ave., Pittsburgh, Pennsylvania, 15224, USA. Phone: 412.383.2178; Email: linpl@chp.edu.
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1Department of Pediatrics, Children’s Hospital of Pittsburgh of the University of Pittsburgh Medical Center, Pittsburgh, Pennsylvania, USA.
2Center for Vaccine Research,
3Department of Microbiology and Molecular Genetics, and
4Division of Laboratory Animal Research, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania, USA.
5Department of Immunology and Infectious Diseases, Harvard T. H. Chan School of Public Health, Boston, Massachusetts, USA.
Address correspondence to: Philana Ling Lin, UPMC Children’s Hospital of Pittsburgh 2310 AOB, 4401 Penn Ave., Pittsburgh, Pennsylvania, 15224, USA. Phone: 412.383.2178; Email: linpl@chp.edu.
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1Department of Pediatrics, Children’s Hospital of Pittsburgh of the University of Pittsburgh Medical Center, Pittsburgh, Pennsylvania, USA.
2Center for Vaccine Research,
3Department of Microbiology and Molecular Genetics, and
4Division of Laboratory Animal Research, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania, USA.
5Department of Immunology and Infectious Diseases, Harvard T. H. Chan School of Public Health, Boston, Massachusetts, USA.
Address correspondence to: Philana Ling Lin, UPMC Children’s Hospital of Pittsburgh 2310 AOB, 4401 Penn Ave., Pittsburgh, Pennsylvania, 15224, USA. Phone: 412.383.2178; Email: linpl@chp.edu.
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1Department of Pediatrics, Children’s Hospital of Pittsburgh of the University of Pittsburgh Medical Center, Pittsburgh, Pennsylvania, USA.
2Center for Vaccine Research,
3Department of Microbiology and Molecular Genetics, and
4Division of Laboratory Animal Research, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania, USA.
5Department of Immunology and Infectious Diseases, Harvard T. H. Chan School of Public Health, Boston, Massachusetts, USA.
Address correspondence to: Philana Ling Lin, UPMC Children’s Hospital of Pittsburgh 2310 AOB, 4401 Penn Ave., Pittsburgh, Pennsylvania, 15224, USA. Phone: 412.383.2178; Email: linpl@chp.edu.
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1Department of Pediatrics, Children’s Hospital of Pittsburgh of the University of Pittsburgh Medical Center, Pittsburgh, Pennsylvania, USA.
2Center for Vaccine Research,
3Department of Microbiology and Molecular Genetics, and
4Division of Laboratory Animal Research, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania, USA.
5Department of Immunology and Infectious Diseases, Harvard T. H. Chan School of Public Health, Boston, Massachusetts, USA.
Address correspondence to: Philana Ling Lin, UPMC Children’s Hospital of Pittsburgh 2310 AOB, 4401 Penn Ave., Pittsburgh, Pennsylvania, 15224, USA. Phone: 412.383.2178; Email: linpl@chp.edu.
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1Department of Pediatrics, Children’s Hospital of Pittsburgh of the University of Pittsburgh Medical Center, Pittsburgh, Pennsylvania, USA.
2Center for Vaccine Research,
3Department of Microbiology and Molecular Genetics, and
4Division of Laboratory Animal Research, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania, USA.
5Department of Immunology and Infectious Diseases, Harvard T. H. Chan School of Public Health, Boston, Massachusetts, USA.
Address correspondence to: Philana Ling Lin, UPMC Children’s Hospital of Pittsburgh 2310 AOB, 4401 Penn Ave., Pittsburgh, Pennsylvania, 15224, USA. Phone: 412.383.2178; Email: linpl@chp.edu.
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1Department of Pediatrics, Children’s Hospital of Pittsburgh of the University of Pittsburgh Medical Center, Pittsburgh, Pennsylvania, USA.
2Center for Vaccine Research,
3Department of Microbiology and Molecular Genetics, and
4Division of Laboratory Animal Research, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania, USA.
5Department of Immunology and Infectious Diseases, Harvard T. H. Chan School of Public Health, Boston, Massachusetts, USA.
Address correspondence to: Philana Ling Lin, UPMC Children’s Hospital of Pittsburgh 2310 AOB, 4401 Penn Ave., Pittsburgh, Pennsylvania, 15224, USA. Phone: 412.383.2178; Email: linpl@chp.edu.
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1Department of Pediatrics, Children’s Hospital of Pittsburgh of the University of Pittsburgh Medical Center, Pittsburgh, Pennsylvania, USA.
2Center for Vaccine Research,
3Department of Microbiology and Molecular Genetics, and
4Division of Laboratory Animal Research, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania, USA.
5Department of Immunology and Infectious Diseases, Harvard T. H. Chan School of Public Health, Boston, Massachusetts, USA.
Address correspondence to: Philana Ling Lin, UPMC Children’s Hospital of Pittsburgh 2310 AOB, 4401 Penn Ave., Pittsburgh, Pennsylvania, 15224, USA. Phone: 412.383.2178; Email: linpl@chp.edu.
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1Department of Pediatrics, Children’s Hospital of Pittsburgh of the University of Pittsburgh Medical Center, Pittsburgh, Pennsylvania, USA.
2Center for Vaccine Research,
3Department of Microbiology and Molecular Genetics, and
4Division of Laboratory Animal Research, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania, USA.
5Department of Immunology and Infectious Diseases, Harvard T. H. Chan School of Public Health, Boston, Massachusetts, USA.
Address correspondence to: Philana Ling Lin, UPMC Children’s Hospital of Pittsburgh 2310 AOB, 4401 Penn Ave., Pittsburgh, Pennsylvania, 15224, USA. Phone: 412.383.2178; Email: linpl@chp.edu.
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1Department of Pediatrics, Children’s Hospital of Pittsburgh of the University of Pittsburgh Medical Center, Pittsburgh, Pennsylvania, USA.
2Center for Vaccine Research,
3Department of Microbiology and Molecular Genetics, and
4Division of Laboratory Animal Research, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania, USA.
5Department of Immunology and Infectious Diseases, Harvard T. H. Chan School of Public Health, Boston, Massachusetts, USA.
Address correspondence to: Philana Ling Lin, UPMC Children’s Hospital of Pittsburgh 2310 AOB, 4401 Penn Ave., Pittsburgh, Pennsylvania, 15224, USA. Phone: 412.383.2178; Email: linpl@chp.edu.
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1Department of Pediatrics, Children’s Hospital of Pittsburgh of the University of Pittsburgh Medical Center, Pittsburgh, Pennsylvania, USA.
2Center for Vaccine Research,
3Department of Microbiology and Molecular Genetics, and
4Division of Laboratory Animal Research, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania, USA.
5Department of Immunology and Infectious Diseases, Harvard T. H. Chan School of Public Health, Boston, Massachusetts, USA.
Address correspondence to: Philana Ling Lin, UPMC Children’s Hospital of Pittsburgh 2310 AOB, 4401 Penn Ave., Pittsburgh, Pennsylvania, 15224, USA. Phone: 412.383.2178; Email: linpl@chp.edu.
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1Department of Pediatrics, Children’s Hospital of Pittsburgh of the University of Pittsburgh Medical Center, Pittsburgh, Pennsylvania, USA.
2Center for Vaccine Research,
3Department of Microbiology and Molecular Genetics, and
4Division of Laboratory Animal Research, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania, USA.
5Department of Immunology and Infectious Diseases, Harvard T. H. Chan School of Public Health, Boston, Massachusetts, USA.
Address correspondence to: Philana Ling Lin, UPMC Children’s Hospital of Pittsburgh 2310 AOB, 4401 Penn Ave., Pittsburgh, Pennsylvania, 15224, USA. Phone: 412.383.2178; Email: linpl@chp.edu.
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1Department of Pediatrics, Children’s Hospital of Pittsburgh of the University of Pittsburgh Medical Center, Pittsburgh, Pennsylvania, USA.
2Center for Vaccine Research,
3Department of Microbiology and Molecular Genetics, and
4Division of Laboratory Animal Research, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania, USA.
5Department of Immunology and Infectious Diseases, Harvard T. H. Chan School of Public Health, Boston, Massachusetts, USA.
Address correspondence to: Philana Ling Lin, UPMC Children’s Hospital of Pittsburgh 2310 AOB, 4401 Penn Ave., Pittsburgh, Pennsylvania, 15224, USA. Phone: 412.383.2178; Email: linpl@chp.edu.
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1Department of Pediatrics, Children’s Hospital of Pittsburgh of the University of Pittsburgh Medical Center, Pittsburgh, Pennsylvania, USA.
2Center for Vaccine Research,
3Department of Microbiology and Molecular Genetics, and
4Division of Laboratory Animal Research, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania, USA.
5Department of Immunology and Infectious Diseases, Harvard T. H. Chan School of Public Health, Boston, Massachusetts, USA.
Address correspondence to: Philana Ling Lin, UPMC Children’s Hospital of Pittsburgh 2310 AOB, 4401 Penn Ave., Pittsburgh, Pennsylvania, 15224, USA. Phone: 412.383.2178; Email: linpl@chp.edu.
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1Department of Pediatrics, Children’s Hospital of Pittsburgh of the University of Pittsburgh Medical Center, Pittsburgh, Pennsylvania, USA.
2Center for Vaccine Research,
3Department of Microbiology and Molecular Genetics, and
4Division of Laboratory Animal Research, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania, USA.
5Department of Immunology and Infectious Diseases, Harvard T. H. Chan School of Public Health, Boston, Massachusetts, USA.
Address correspondence to: Philana Ling Lin, UPMC Children’s Hospital of Pittsburgh 2310 AOB, 4401 Penn Ave., Pittsburgh, Pennsylvania, 15224, USA. Phone: 412.383.2178; Email: linpl@chp.edu.
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1Department of Pediatrics, Children’s Hospital of Pittsburgh of the University of Pittsburgh Medical Center, Pittsburgh, Pennsylvania, USA.
2Center for Vaccine Research,
3Department of Microbiology and Molecular Genetics, and
4Division of Laboratory Animal Research, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania, USA.
5Department of Immunology and Infectious Diseases, Harvard T. H. Chan School of Public Health, Boston, Massachusetts, USA.
Address correspondence to: Philana Ling Lin, UPMC Children’s Hospital of Pittsburgh 2310 AOB, 4401 Penn Ave., Pittsburgh, Pennsylvania, 15224, USA. Phone: 412.383.2178; Email: linpl@chp.edu.
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1Department of Pediatrics, Children’s Hospital of Pittsburgh of the University of Pittsburgh Medical Center, Pittsburgh, Pennsylvania, USA.
2Center for Vaccine Research,
3Department of Microbiology and Molecular Genetics, and
4Division of Laboratory Animal Research, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania, USA.
5Department of Immunology and Infectious Diseases, Harvard T. H. Chan School of Public Health, Boston, Massachusetts, USA.
Address correspondence to: Philana Ling Lin, UPMC Children’s Hospital of Pittsburgh 2310 AOB, 4401 Penn Ave., Pittsburgh, Pennsylvania, 15224, USA. Phone: 412.383.2178; Email: linpl@chp.edu.
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1Department of Pediatrics, Children’s Hospital of Pittsburgh of the University of Pittsburgh Medical Center, Pittsburgh, Pennsylvania, USA.
2Center for Vaccine Research,
3Department of Microbiology and Molecular Genetics, and
4Division of Laboratory Animal Research, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania, USA.
5Department of Immunology and Infectious Diseases, Harvard T. H. Chan School of Public Health, Boston, Massachusetts, USA.
Address correspondence to: Philana Ling Lin, UPMC Children’s Hospital of Pittsburgh 2310 AOB, 4401 Penn Ave., Pittsburgh, Pennsylvania, 15224, USA. Phone: 412.383.2178; Email: linpl@chp.edu.
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1Department of Pediatrics, Children’s Hospital of Pittsburgh of the University of Pittsburgh Medical Center, Pittsburgh, Pennsylvania, USA.
2Center for Vaccine Research,
3Department of Microbiology and Molecular Genetics, and
4Division of Laboratory Animal Research, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania, USA.
5Department of Immunology and Infectious Diseases, Harvard T. H. Chan School of Public Health, Boston, Massachusetts, USA.
Address correspondence to: Philana Ling Lin, UPMC Children’s Hospital of Pittsburgh 2310 AOB, 4401 Penn Ave., Pittsburgh, Pennsylvania, 15224, USA. Phone: 412.383.2178; Email: linpl@chp.edu.
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1Department of Pediatrics, Children’s Hospital of Pittsburgh of the University of Pittsburgh Medical Center, Pittsburgh, Pennsylvania, USA.
2Center for Vaccine Research,
3Department of Microbiology and Molecular Genetics, and
4Division of Laboratory Animal Research, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania, USA.
5Department of Immunology and Infectious Diseases, Harvard T. H. Chan School of Public Health, Boston, Massachusetts, USA.
Address correspondence to: Philana Ling Lin, UPMC Children’s Hospital of Pittsburgh 2310 AOB, 4401 Penn Ave., Pittsburgh, Pennsylvania, 15224, USA. Phone: 412.383.2178; Email: linpl@chp.edu.
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Published February 23, 2026 - More info
Published in Volume 11, Issue 4 on February 23, 2026
AbstractCoinfection with both HIV and M. tuberculosis (Mtb) results in disseminated tuberculosis (TB) and accelerated HIV progression. Despite greater access to antiretroviral treatment (ART), it remains unclear whether suppression of HIV replication protects against severe Mtb infection. Here, using a macaque model of SIV/Mtb coinfection, we investigated whether treatment of SIV infection with ART influenced control of a subsequent Mtb challenge compared with SIV-infected macaques that were not treated with ART. Macaques were first infected with SIVB670, SIVB670 with ART, or saline followed by a low-dose Mtb inoculation with serial clinical and PET-CT imaging assessments. At necropsy, gross pathology, viremia, bacterial burden, and immunologic parameters were compared. SIV-TB animals had greater gross pathology and total bacterial burden than TB-only and SIV/ART/TB groups. However, despite normal blood CD4 counts and undetectable SIV RNA, SIV/ART/TB macaques showed similar clinical parameters and extrapulmonary involvement as SIV/TB animals. Analysis of barcoded-Mtb suggests that ART control of SIV replication did not prevent Mtb extrapulmonary dissemination. These data indicate that people living with HIV on ART remain at high risk of bacterial dissemination and extrapulmonary TB disease. Understanding the mechanisms of extrapulmonary spread and disease severity during HIV/TB coinfection remains an important issue.
IntroductionTuberculosis (TB), the disease caused by Mycobacterium tuberculosis (Mtb), is currently the most common infectious cause of death, leading to 1.23 million deaths in 2024 with 10.7 million new active TB cases (1). HIV is the greatest risk factor for TB, even when CD4 T cell counts are in the normal range, and HIV/Mtb coinfection accelerates progression of both TB and AIDS (2). Clinical manifestations of TB among people living with HIV (PLWHIV) often vary by degree of immune suppression. While pulmonary disease remains the most common clinical manifestation of TB, atypical presentations can occur and are often associated with low CD4 counts. Symptoms are often more subtle, chest radiographs can have atypical findings with negative sputum smears, and the rates of extrapulmonary disease (defined as TB pathology at anatomical sites outside of the thoracic cavity) are higher than HIV-naive individuals (3–5). The presence of extrapulmonary TB is more likely to be diagnosed in HIV/Mtb coinfected people and is associated with increased mortality (6).
Antiretroviral treatment (ART) to control HIV replication has dramatically improved HIV and HIV/Mtb coinfection care, especially as it is now available in up to 77% of PLWHIV (1). Delays in ART and low CD4 T cell counts correlate with increased TB incidence over time (1, 7, 8), and ART has been shown to reduce mortality from TB and the overall incidence of TB (9–11). Among HIV/Mtb coinfected individuals, ART can prevent or remedy the loss of CD4 T cells (7, 12, 13), improve Mtb-specific CD4 and CD8 T cell responses (14), and reduce macrophage dysfunction (15). However, ART only partially ameliorates the HIV-induced increase in susceptibility to TB (16), as PLWHIV on ART are still more susceptible to TB than HIV-uninfected individuals (17–19). The mechanisms by which HIV increases susceptibility to TB and why ART only partially reduces that risk are not well understood. It was initially presumed that a low CD4 count was the primary cause of the increase in TB susceptibility in PLWHIV (12), yet we and others have shown that there are CD4-independent risks associated with SIV-induced TB pathogenesis (20–24). Human cohort studies are challenging, as the order in which the infections occur (i.e., primary versus secondary HIV infection) and the duration of each infection are often unknown. For example, acute Mtb infection after chronic HIV infection may lead to worse severity than longstanding asymptomatic Mtb infection (latent) and subsequent acute HIV infection. To dissect these factors, highly controlled animal models to interrogate the phenomena and mechanisms of pathogenesis are beneficial.
Nonhuman primate (NHP) models of TB recapitulate many key features of human Mtb infection (25, 26), and we and others have shown that they can be productively infected with SIV to better understand HIV-Mtb coinfection (20–23, 27, 28). While many studies in NHP have addressed the role of SIV infection induced reactivation of latent Mtb infection (22, 23, 28, 29), to the best of our knowledge, this is the first study to address the influence of SIV infection aggressively controlled with ART and subsequent Mtb infection. Here, cynomolgus macaques were infected with SIV with or without ART and subsequently infected with Mtb to determine the influence of SIV replication or ART suppression of SIV on susceptibility to acute Mtb infection, using a barcoded strain of virulent Mtb. Our data confirm that SIV increases TB pathology and influences immunological functions. Although ART reduced pulmonary TB pathology, it did not prevent extrapulmonary spread of TB.
ResultsSIV/ART/TB animals developed similar signs of TB disease as SIV/TB with immune activation. To address the effect of ART controlled viral suppression on SIV/Mtb coinfection, NHP were randomized into 4 groups: TB-only, SIV/TB, SIV/ART/TB, and SIV-only. SIV infection occurred for 16 weeks prior to Mtb infection with a subset of animals initiated on ART at day 3 of SIV infection, based on prior studies in which viral replication was suppressed despite established viral tissue reservoirs (30) (Figure 1A). Subsequent Mtb infection was planned for 12 weeks in all relevant groups. Serial positron emission tomography using 18-flourodeoxyglucose (FDG) probe and computed tomography (PET-CT) imaging and clinical, immunologic, and bacterial outcomes were compared at necropsy. We focused our comparisons between SIV/TB and SIV/ART/TB groups with secondary analyses including TB- and SIV-only groups.
Figure 1Study design and markers of disease. (A) Adult cynomolgus macaques were randomized to receive either SIV infection alone (n = 3), SIV infection for 16 weeks and then M. tuberculosis (Mtb) challenge (n = 9), SIV with antiretroviral treatment (ART) for 16 weeks and then Mtb challenge (n = 10), or Mtb infection alone (n = 9). ART was continued throughout the course of SIV and Mtb infection. Mtb infection (low dose Erdman) progressed for 12 weeks with serial blood, airway and lymph node sampling and PET-CT performed. (B) Serial SIV RNA plasma levels before and after Mtb infection and serial frequencies of CD4 T cells in the peripheral blood are shown. Median with IQR error bars shown. (C) Clinical scores within each treatment group are shown over the course of Mtb infection. (D and E) Interval change in immune activation markers, sCD14 and sCD163, are shown by group. Limit of detection for sCD14 is 125 pcg/mL and sCD163 is 0.469 ng/mL. (F) Erythrocyte sedimentation rates (ESR) during the course of infection by experimental group are shown. Each light-colored line represents a single animal over time; dark lines represent median at each time point. Normal range is 0–2 mm. (B) Statistical analysis was restricted to compare only SIV/ART/TB and SIV/TB groups. Mann-Whitney U test run at each time point and adjusted for multiple comparisons by Holm-Šídák method (0.05 < +P < 0.10, 0.01 < *P < 0.05, 0.001 < **P < 0.01, ***P < 0.001). Black, TB-only; Blue, SIV/TB; Red, SIV/ART/TB; Gray, SIV-only. (D and E) Wilcoxon matched-pairs signed rank test used for analysis in B and C. TB-only (n = 9), SIV/ART/TB (n = 10), SIV/TB (n = 8–9).
Animals randomized to SIV infection without ART showed substantial reductions of CD4 T cells in blood, airways, and tissues (Figure 1B and Supplemental Figure 1; supplemental material available online with this article; https://doi.org/10.1172/jci.insight.199314DS1). Plasma viremia peaked at 1 week after infection and reached a setpoint by approximately 4 weeks (Figure 1B). The level of plasma SIV RNA among SIV/TB NHP was similar to the SIV-only animals. Sustained suppression of SIV replication occurred by 3 weeks of ART in the SIV/ART/TB NHP. This effectively prevented the loss of CD4 T cells within multiple compartments, resulting in significantly higher CD4 T cells counts in the blood, airway, and peripheral lymph nodes compared with SIV/TB NHP across all time points before and after Mtb infection (Figure 1B and Supplemental Figure 1). SIV-only and SIV/TB NHP also maintained similar CD4 T cell frequencies across all time points both before and during Mtb infection in blood, airway, and peripheral lymph nodes. Reductions in CD4 T cells in the SIV only and SIV/TB NHPs were associated with higher CD8 T cell counts (Supplemental Figures 1 and 2).
After Mtb challenge, both SIV/TB and SIV/ART/TB macaques displayed more clinical and immunologic signs of early Mtb infection than animals with Mtb infection alone (Figure 1C). To quantify and compare clinical signs of disease, we modified our previously published clinical scoring system (31) to numerically quantify signs of TB disease (see methods) (Figure 1C and Supplemental Figure 3). Peaking at 4–6 weeks after Mtb infection, 40% of the TB-only NHPs developed at least 1 sign of TB similar to previously published studies (25). However, 60%–90% of animals in the SIV/ART/TB and SIV/TB groups had at least 1 sign of disease at 4–6 weeks that continued throughout infection, often with both microbiologic markers and clinical signs of disease (Figure 1C and Supplemental Figure 3). We measured markers of immune activation, soluble CD14 (sCD14) (macrophage activation marker) and sCD163 (monocyte/macrophage scavenger receptor), often observed in humans with HIV/Mtb coinfection (32). Both the SIV/ART/TB and SIV/TB NHP had significant elevations in sCD14 after Mtb infection but only the SIV/TB group had increased levels of sCD163 (Figure 1, D and E). Erythrocyte sedimentation rate (ESR), a marker of systemic inflammation, can be increased transiently during acute Mtb or SIV infection and remain elevated with worsening TB disease (25). In the TB-only group, the median ESRs were normal throughout Mtb infection, although there were increases in some animals (26). Conversely, SIV/TB NHP maintained high ESRs starting at 4–6 weeks after Mtb infection (median = 10 mm IQR25–75, 3.25–45.75, n = 9), peaking at 6–8 weeks (47.5 mm, 3.125–55, n = 8) and many NHPs had elevated ESR thereafter (Figure 1F). SIV/ART/TB animals experienced high ESRs at 4–6 weeks after Mtb infection (19.5 mm, 0.875–44.13, n = 10) and remained slightly above normal levels thereafter. Interestingly, SIV/TB animals had higher plasma levels of Th1 cytokines (e.g., IL-12, IL-2) and IP-10 (CXCL10) before and during early Mtb infection compared with TB-only groups and/or SIV/ART/TB (Supplemental Figure 4). None of the TB-only and SIV/ART/TB animals reached humane endpoints prior to their predetermined time of necropsy after Mtb challenge (12 weeks). In contrast, the SIV/TB animals median time to necropsy was 8.9 weeks (Supplemental Figure 5).
ART did not prevent extrapulmonary dissemination of TB among SIV/ART/TB macaques. At necropsy, inflammation in the lungs and thoracic lymph nodes appeared greater in SIV/TB animals (Figure 2A). PET-CT–identified lung granulomas and other sites of disease progression were tracked during the course of Mtb infection. Greater total lung inflammation (FDG activity) is noted in the SIV/TB groups at 8 and 12 weeks after Mtb infection (Figure 2B). At necropsy, SIV/TB NHP had greater overall TB pathology, especially in the lungs and extrapulmonary sites, compared with TB-only NHP (Figure 3A). However, SIV/ART/TB NHP had greater extrapulmonary involvement than the TB-only group (Figure 3B). A higher proportion of SIV/TB NHP developed TB pneumonia (indicating severe disease) compared with the TB-only and SIV/ART/TB NHP groups (78%, 20%, and 30%, respectively; P = 0.03) (Supplemental Figure 6A). Total bacterial burden (including lung and thoracic lymph node) among SIV/TB animals was higher than the TB-only and SIV/ART/TB groups (Figure 3C). Individual Mtb bacterial growth per lung granuloma varied across groups (Supplemental Figure 6, B and C). SIV/TB animals had higher CFU per lung granuloma (and total live and killed Mtb measured by chromosomal equivalents [CEQ]) indicating increased growth and/or reduced killing than the SIV/ART/TB and TB-only groups (Figure 4). Higher Mtb growth was also present in thoracic lymph nodes (with and without granulomas being present) in SIV/TB NHP compared with SIV/ART/TB and TB-only NHP (Figure 4). While it is not surprising that SIV infection increases overall pathology and Mtb growth within NHP, early control of SIV replication with ART appeared to prevent disease progression in the lungs and lymph node but did not ameliorate the dissemination of Mtb to extrapulmonary sites.
Figure 2PET-CT images of tuberculosis-involved lung and thoracic LN at necropsy. (A) Top row, TB-only; middle row, SIV/ART/TB; bottom row, SIV/TB. Images are arranged in each group by total CFU (lowest CFU of the group on the left and highest on the right). (B) Total lung FDG activity is presented at 4, 8, and 12 weeks after Mtb infection for each cohort. Each dot represents an animal at each time point, and lines represent means. A mixed-effects analysis was utilized with Tukey’s multiple-comparison test P values are shown.
Figure 3Gross pathology and Mtb burden at necropsy. (A) Tuberculosis-associated gross pathology is estimated using a quantitative score at necropsy (necropsy score) that includes disease specific to the lung, lymph node, and extrapulmonary sites. (B) Tuberculosis-associated extrapulmonary gross pathology and Mtb growth from these sites is used to estimate extrapulmonary score for each animal at necropsy. (C) Total bacterial burden, lung bacterial burden, and thoracic LN burden are shown across experimental groups. Lines represent means; each dot represents an animal. Kruskal-Wallis test with Dunn’s multiple-comparison–adjusted P values reported in A and B. One-way ANOVA with Tukey’s multiple-comparison adjusted P values reported in C. TB-only (n = 9), SIV/ART/TB (n = 10), SIV/TB (n = 9).
Figure 4Comparison of viable and total (viable and killed) Mtb per granuloma by group. (A) Viable Mtb growth (CFU) by experimental group in lung granulomas and thoracic lymph nodes (with and without granuloma) are shown. (B) Total Mtb burden includes both viable and killed Mtb that is measured by chromosomal equivalents (CEQ) in lung granulomas and thoracic lymph nodes. Mixed effect model (animal as a random effect and treatment group as a fixed effect) was used; Tukey HSD adjusted P values (for P < 0.10) reported. Each dot represents an individual sample (granuloma or lymph node), and each symbol represents a different animal; lines represent medians.
SIV-induced T cell changes are not completely ameliorated by ART. Given that peripheral blood responses do not accurately reflect those in the lung, we focused on the immunological responses in lung granulomas (33). Reduced frequency and total CD4 T cells were noted within lung granulomas of SIV/TB NHP but were preserved in SIV/ART/TB NHP (Figure 5, A and B). Similarly, the absolute number of HLA-DR expressing CD4 T cells was lower in the SIV/TB NHP compared with the other cohorts (Figure 5, C and D). Given the reduction in CD4 T cells in SIV/TB granulomas, it is not surprising that higher frequencies of CD8 T cells were observed in this group compared with others. The frequencies of CD8 T cells (though not total numbers) producing any Th1 cytokine (TNF, IFN-γ, or IL-2) were higher in the SIV/TB and SIV/ART/TB granulomas compared with TB-only NHP, possibly due to increased bacterial burden and stimulation by SIV or Mtb antigens (Figure 5, E and F) (though not among SIV/ART/TB groups). A higher percentage (but not total cells) of CD8 T cells producing IL-10 was also observed in SIV/ART/TB and SIV/TB NHP compared with TB-only NHP (Figure 5, G and H). Greater CD4 T cell expression of CD38 (activation marker and T cell regulator) and reduced IL-17 was observed among the SIV/TB NHP compared with TB-only controls (Supplemental Figure 7), which has been observed in blood of HIV/Mtb coinfected humans (34). We also utilized t-SNE to visualize more complex cell populations (Supplemental Figure 8) on a subset of samples that were available. Subtle shifts in phenotypic character among the CD4 and CD8 populations between the TB-only and the SIV (± ART) groups were noted, though they were not statistically significant, given the limited sample size.
Figure 5Immunophenotyping of granulomas by experimental group. (A and B) Frequency and absolute numbers of CD4 and CD8 T cells per granuloma are shown. (C and D) Frequency and absolute number of HLA-DR expressing CD4 and CD8 T cells within granulomas are shown. (E and F) Frequency and absolute numbers of the CD4 and CD8 T cells expressing any Th1 cytokine (at least one of the following: IFN-γ, TNF, or IL-2) within granulomas is shown. (G and H) Frequency and absolute numbers of IL-10 expressing CD4 and CD8 T cells is shown. Each circle represents a granuloma; lines are medians. Mixed effect model (animal as a random effect and treatment group as a fixed effect) was used; Tukey HSD adjusted P values (for P < 0.10) reported.
Thoracic lymph nodes play an important role in T cell priming that is critical to the adaptive immune response and can harbor both Mtb and SIV (35, 36). While the frequency of CD4 T cell among SIV/TB lymph nodes was significantly lower than other groups (and the frequency of CD8 T cells consequently higher), the SIV/ART/TB animals had higher CD4 T cells than the other groups (Supplemental Figure 9). CD4 and CD8 T cells expressing CD38 in thoracic lymph nodes were significantly higher in SIV/TB NHP compared with TB-only and SIV/ART/TB NHP. Lastly, greater CD4 T cell expression of the degranulating protein CD107a was observed in the SIV/TB group compared the other groups. Based on t-SNE visualization of a subset of samples, subtle changes in surface marker and functional phenotypes were noted in both CD4 and CD8 populations in the lymph nodes (Supplemental Figure 10). Overall, thoracic lymph node T cell responses among SIV/ART/TB groups were more similar to the TB-only groups.
ART did not prevent Mtb-barcode dissemination to extrapulmonary sites. While it was clear that there was greater dissemination in the SIV/TB animals, we were surprised to see increased extrapulmonary dissemination in the SIV/ART/TB animals (Figure 6), prompting further analysis. We tracked the Mtb barcodes with PET-CT images across time points to compare bacterial establishment and dissemination within and across experimental groups (37). There was no difference in the number of unique barcodes established between each cohort (Figure 7A). There was no difference in the number of unique barcodes observed in early lung granulomas (granulomas established at 4 weeks after Mtb challenge by PET-CT) among the different cohorts (Figure 7B). Nor were there differences in unique barcode counts in thoracic lymph nodes across all groups (Figure 7C). However, both SIV/ART/TB and SIV/TB animals had greater numbers of barcodes shared between extrapulmonary and thoracic lymph node sites compared with the TB-only NHP (Figure 7D). Given that there are multiple thoracic lymph nodes (e.g., bilateral hilar and carinal lymph nodes) and extrapulmonary sites (e.g., liver, spleen, kidney), we examined the proportion of tissues in each compartment that shared barcodes. Again, both the SIV/ART/TB and SIV/TB NHPs had greater proportions of tissues sharing barcodes than the TB-only animals (Figure 7, E and F). No differences were observed in the overall diversity of barcodes within each animal based on treatment group (Supplemental Figure 11). In short, unexpectedly SIV did not influence the establishment of Mtb infection; we expected a greater number of unique barcodes in SIV/TB NHPs compared with other groups. Thus, while initial Mtb infection in the lungs and thoracic lymph nodes was unc
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