{'fraction_non_alphanumeric': 0.03510971786833856, 'fraction_numerical': 0.02962382445141066, 'mean_word_length': 4.785131459655485, 'pattern_counts': {'":': 0, '<': 0, '<?xml version=': 0, '>': 1, 'https://': 0, 'lorem ipsum': 0, 'www.': 0, 'xml': 0}, 'pii_count': 0, 'substrings_counts': 0, 'word_list_counts': {'cursed_substrings.json': 0, 'profanity_word_list.json': 0, 'sexual_word_list.json': 0, 'zh_pornsignals.json': 0}}
CD4CD8 Double Positive T cell responses during Mycobacterium tuberculosis infection in cynomolgus macaques

Abstract Background Tuberculosis (TB) kills millions of people every year. CD4 and CD8 T cells are critical in the immune response against TB. T cells expressing both CD4 and CD8 (CD4CD8 T cells) are functionally active and have not been examined in the context of TB. Methods We examine peripheral blood mononuclear cells (PBMC) and bronchoalveolar lavage cells (BAL) and lung granulomas from 28 cynomolgus macaques during Mycobacterium tuberculosis (Mtb) infection. Results CD4CD8 T cells increase in frequency during early Mtb infection in PBMC and BAL from pre‐infection. Peripheral, airway, and lung granuloma CD4CD8 T cells have distinct patterns and greater cytokine production than CD4 or CD8 T cells. Conclusion Our data suggest that CD4CD8 T cells transient the blood and airways early during infection to reach the granulomas where they are involved directly in the host response to Mtb.

functionally respond to HIV, 17,21,22 SHIV, 23 Mycobacterium leprae, 24 and various cancers. 19,20,30,31 CD4CD8 T cells from adults with rheumatoid arthritis produce more IFNγ than peripheral CD4 and CD8 T cells stimulated with mitogens. 32 Taken together, CD4CD8 T cells express both pro-inflammatory and anti-inflammatory cytokines production and are able to exhibit cytolytic function. However, they have not been examined in the context of TB.
Here, we sought to characterize these CD4CD8 T cells during the course of Mtb infection in the blood, airways, and lung granulomas using a cynomolgus macaque model of TB. We show that CD4CD8 T cells increase in frequency in both blood and the airways during early Mtb infection and within lung granulomas with Mtb-specific responses that are similar but distinct in pattern from conventional single-expressing CD4 and CD8 T cells.

Adult (>4 years of age) cynomolgus macaques (Macaca fascicularis)
were screened for other co-morbidities (eg parasites, SIV) before infection (Valley Biosystems, Sacramento, CA). Animals were infected with low-dose (~25 CFU per monkey) Mtb (Erdman strain) via bronchoscopic instillation to the lower lung lobe and housed in Biosafety Level 3 (BSL-3) primate facility. 2,33,34 Infection was confirmed as previously described. 34 Animals were declared as having active disease (ie repeated presence of Mtb growth in bronchoalveolar lavage [BAL] or gastric aspirate, elevated sedimentation rate [ESR], and/or clinical signs of active TB after infection) or latent infection (ie no clinical signs of active TB, normal ESR, and lack of persistent Mtb growth by 6 months post-infection) based on previously published criteria. 34 Twenty-four monkeys were included in the PBMC intracellular cytokine (ICS) analysis, 28 monkeys were included in the BAL profile, and eight monkeys were included in the lung granuloma profile. Due to restrictions on animal procedures or low cell counts, some time points use fewer monkeys. Immune data for eight of these animals were previously published 2,35 but without CD4CD8 T cell analysis. All animal protocols and procedures were approved by the University of Pittsburgh's Institutional Animal Care and Use Committee that adheres to the national guidelines established in the Animal Welfare Act and Guide for the Care and Use of Laboratory Animals as mandated by the U. S. Public Health Service Policy.

| Procedures
Cells from the BAL and PBMCs were obtained for analysis prior to Mtb infection, 4 weeks, 12 weeks post-infection, and at time of clinical diagnosis (latent or active TB) as previously described. 36,37 Isolated cells were stimulated with ESAT6-CFP10 peptide pools (10 μg/mL of every peptide) or incubated with only media (RPMI+10%hAB).
PBMC incubated with or without the peptide pools for 1 hour in media, followed by the addition of Brefeldin A (Golgiplug: BD biosciences) for 5 hours at 37°C with 5% CO 2 . ESAT6-CFP10 responses within PBMC T cells are presented after subtracting responses from PBMC incubated with media. BAL and granuloma cells were isolated and stimulated for 4 hours with peptide pools in the presence of Brefeldin A. When used, CD107a antibody was added prior to incubation. A minimum of 5 × 10 5 BAL or PBMC cells were incubated with each stimulation condition and acquired.
A total of 29 granulomas were used for analysis from eight macaques (2-5 granulomas per animal) using the same ICS protocol as above for BAL. The range of cells analyzed from granulomas included CD4 (median = 116, IQR 25%-75% = 80-347), CD8 (median = 135, IQR 25%-75% = 78-304), and CD4CD8 (median = 111, IQR 25%-75% = 74-260). T cells were in the estimated range of a previously published threshold. 2 Granuloma-specific T cell responses were only available from clinically latent animals as animals with active TB were dedicated for other studies and immune data were not available.
Granuloma cells were stained for CD3, CD4, CD8, IFNγ, TNF, IL-2, IL-10, IL-17, and granzyme B. Data acquisition was performed using an LSR II (BD) and analyzed using FlowJo Software v.9.7 (Treestar Inc, Ashland, OR). Gating strategies used for analysis are presented using ESAT6-CFP10-stimulated PBMC ( Figure S1). To eliminate the possibility that CD4CD8 T cell population is a result of fluorophore spillover, the population was confirmed using combinations of fluorophores that are excited by different lasers ( Figure S1).   (Table 1) but not CD8 T cells. In contrast, a higher proportion of perpiheral CD4CD8 T cells producing Mtb-specific IFN-γ and TNF were observed at 4 weeks after infection compared to CD8 T cells (Table 1). Taken together, peripheral CD4CD8 T cells appear to have higher cytolytic potential during acute Mtb infection than CD4 T cells but more Mtb-specific cytokine producing properties than CD8 T cells.

| RE SULTS
Similar to blood, the frequency of CD4CD8 T cells in the airway was significantly lower compared to CD4 and CD8 T cells. In contrast to blood, airway CD4CD8 T cells expressed significantly greater IFNγ, IL-10, IL-17, IL-2, and TNF than CD4 and CD8 T cells prior to Mtb infection suggesting that these cells may play a non-specific role but could contribute to the immune response in the airways. At

CO N FLI C T S O F I NTE R E S T
The authors do not have any conflicts of interest associated with this manuscript.