B cell, CD8 T cell and gamma delta T cell lymphocytic alveolitis alters alveolar immune cell homeostasis in HIV-infected Malawian adults [version 1; referees: 1 approved with reservations]

: HIV infection is associated with increased risk to lower respiratory tract Background infections (LRTI). However, the impact of HIV infection on immune cell populations in the lung is not well defined. We sought to comprehensively characterise the impact of HIV infection on immune cell populations in the lung. : Twenty HIV-uninfected controls and 17 HIV-1 infected ART-naïve adults Methods were recruited from Queen Elizabeth Central Hospital, Malawi. Immunophenotyping of lymphocyte and myeloid cell populations was done on bronchoalveolar lavage fluid and peripheral blood cells. : We found that the numbers of CD8 T cells, B cells and gamma delta T cells Results were higher in BAL fluid of HIV-infected adults compared to HIV-uninfected controls (all p<0.05). In contrast, there was no difference in the numbers of alveolar CD4 T cells in HIV-infected adults compared to HIV-uninfected controls (p=0.7065). Intermediate monocytes were the predominant monocyte subset in BAL fluid (HIV-, 63%; HIV+ 81%), while the numbers of classical monocytes was lower in HIV-infected individuals compared to HIV-uninfected adults (p=0.0006). The proportions of alveolar macrophages and myeloid dendritic cells was lower in HIV-infected adults compared to HIV-uninfected controls (all p<0.05). : Chronic HIV infection is associated with broad alteration of immune Conclusions cell populations in the lung, but does not lead to massive depletion of alveolar CD4 T cells. Disruption of alveolar immune cell homeostasis likely explains in part the susceptibility for LRTIs in HIV-infected adults. +


Introduction
HIV-infected individuals have increased susceptibility to lower respiratory tract infections (LRTIs) 1,2 , which account for 75-98% of lung complications in antiretroviral therapy (ART)-naïve HIVinfected adults worldwide 3,4 . Predisposition to LRTIs is largely attributed to HIV-induced impairment of lung immunity, including reduced frequency of respiratory antigen-specific alveolar CD4 + T cells 5-7 as well as impaired alveolar macrophage function 5,8 . HIV infection is also associated with CD8 + T cell alveolitis, a condition characterized by the influx of HIV-specific CD8 + T cells into the lung 9,10 . While these immune cell perturbations partly underlie propensity for LRTIs in HIV-infected individuals, the impact of HIV infection on the composition and functions of other immune cell populations in the lung is not well defined.
Several studies have reported alterations in the proportions and functions of different immune cell populations in peripheral blood in HIV-infected individuals 11-14 . While peripheral blood CD4 + T cell depletion and an increase in CD8 + T cells are hallmarks of progressive untreated chronic HIV infection 15 , depletion of B cells 11 and aberrant NK cell function and redistribution from CD56 dim towards CD56 neg subsets has been observed during early and chronic HIV infection 12 . Two major human γδ T cells subsets (designated Vδ1 or Vδ2) are also altered in HIV-infected individuals, with an increase in the Vδ1 subset and a decrease in the Vδ2 subset 13 . Furthermore, increased proportions of non-classical and intermediate monocytes and depleted myeloid and plasmacytoid dendritic cell subsets have been reported in individuals with high plasma HIV viral load 14,16,17 .
We, therefore, undertook a comprehensive characterisation of the impact of HIV infection on immune cell populations in the lung. We obtained paired bronchoalveolar lavage (BAL) fluid and peripheral blood from HIV-uninfected and asymptomatic HIV-infected, antiretroviral therapy (ART)-naïve Malawian adults. We analysed and compared the proportions and numbers of CD4 + and CD8 + T cells, B cells, NK cell subsets, γδ T cells, monocytes, dendritic cell subsets, neutrophils and alveolar macrophages in samples from HIV-infected and uninfected individuals.

Study participants
The study was conducted at the Queen Elizabeth Central Hospital, a large teaching hospital in Blantyre, Malawi. Participants were recruited from the hospital's voluntary counselling and testing (VCT) and ART clinics. They were adults aged ≥18yrs comprising healthy HIV-1-uninfected and asymptomatic HIV-1-infected volunteers with no clinical evidence of active disease and willing to undergo bronchoscopy and BAL for research purposes 18 . HIV testing was performed on whole blood using two commercial pointof-care rapid HIV test kits, Determine HIV 1/2 kit (Abbott Diagnostic Division) and Unigold HIV 1/2 kit (Trinity Biotech Inc.). A participant was considered HIV-uninfected if the test was negative by both kits or HIV-infected if the test was positive by both kits. If Determine and Unigold results were discordant, a third rapid test using Bioline HIV 1/2 kit (Standard Diagnostics Inc.) was performed to resolve the discordance. None of the participants were on ART at the time of recruitment to the study, but all initiated ART after sample collection according to the 'test and treat' Malawi national treatment guidelines. Exclusion criteria for the study were: current or history of smoking, use of immunosuppressive drugs, severe anaemia (Hb<8g/dl) and known or suspected pregnancy. The research ethics committee of Malawi College of Medicine approved the study under approval number P.03/16/1907 and all participants provided written informed consent.

Sample collection and experimental procedures
Bronchoscopy and BAL were performed on all participants as previously described 5,6,8 . The fluid was filtered using sterile gauze and centrifuged at 500g for 10min. The supernatant was removed, the cell pellet was resuspended and washed with PBS by spinning in a centrifuge at 500g for 10min. The supernatant was removed and discarded while the cell pellet was resuspended in complete media. Peripheral blood was also obtained from study participants for full blood count (FBC) and peripheral blood mononuclear cell (PBMC) isolation using density gradient centrifugation. Cell counts in BAL cells and PBMCs isolated from each sample were performed using a haemocytometer.

Statistical analysis
Statistical analyses and graphical presentation were performed using GraphPad Prism 5 (GraphPad Software, USA). We used FlowJo v10 software (Treestar, USA) to analyse flow cytometry data. The numbers of cell subsets in BAL fluid were estimated by calculating the proportion of a particular subset relative to the total number (1 × 10 6 cells) of stained cells. In PBMCs, the absolute numbers were obtained by calculating the proportion of a particular subset relative to the full blood count (FBC) data. Data were analysed using Mann Whitney U test. Results are given as median and interquartile range (IQR). Differences were considered statistically significant when p<0.05.

CD8 + T cells, B cells and γδ T cells contribute to HIVassociated lymphocytic alveolitis
We investigated the impact of HIV infection on the proportion and numbers of lymphocyte populations using flow cytometry. The gating strategy is illustrated in Figure 1. We found that the proportions and numbers of lymphocytes in BAL fluid were higher in HIV-infected adults compared to HIV-uninfected (median 20.8% vs. 8.5%, p=0.0004 and median 1 × 10 7 vs. 2.7 × 10 6 cells/100ml of BAL fluid, p=0.0005, respectively) ( Figure 2A and 2B). We next determined the cell subsets that were responsible for the increased frequency of lymphocytes in the alveoli. We found that the proportions and numbers of CD8 + T cells (median, 68% vs. 32%, p<0.0001 and median 7 × 10 6 vs. 7 × 10 5 /100ml of BAL fluid, p<0.0001, respectively) and B cells (median 1.8% vs. 0.8%, p=0.0014 and median 7 × 10 4 vs. 1 × 10 4 /100ml of BAL fluid, p<0.0001, respectively) in BAL fluid were higher in HIV-infected adults compared to HIV-uninfected controls ( Figure 2C, 2D, 2E and 2F). The proportion and numbers of γδ T cells were also higher in BAL fluid from HIV-infected adults compared HIV-uninfected controls (median 1.4% vs. 0.8%, p=0.036 and median 1 × 10 5 vs. 2 × 10 4 /100ml of BAL fluid, p=0.0002, respectively) ( Figure 3A and 3B).

Differential impact of HIV infection on lymphocyte subsets in the alveolar and blood compartments
We then investigated the similarities and differences of HIV-associated changes in cell composition between BAL fluid and peripheral blood. In agreement with BAL fluid, the proportions of CD8 + T cells in peripheral blood were higher in HIV infected adults compared to HIV-uninfected controls (Median 47% vs. 24%, p<0.0001) (Supplementary Figure 1). The proportions of CD4 + T cells in peripheral blood were lower in HIV-infected adults compared  to HIV-uninfected controls (Median 20% vs. 46%, p<0.0001) (Supplementary Figure 1). In contrast with BAL fluid, the proportion of B cells in peripheral blood was lower in HIV-infected adults compared to HIV-uninfected controls (Median 5.8% vs. 9.4%, p=0.0472) (Supplementary Figure 1). The proportion of NK T cells in peripheral blood was lower in HIV-infected adults compared to HIV-uninfected controls (Median 0.03% vs. 0.09%, p=0.0386) (Supplementary Figure 1). The proportion of CD8 γδ T cells in peripheral blood was higher in HIV-infected adults compared to HIV-uninfected controls (Median 1.9% vs. 0.76%, p=0.0229) (Supplementary Figure 1). The findings show that HIV infection differentially impacts lymphocyte populations in the alveolar space and peripheral blood compartments.
Differential impact of HIV infection on monocyte subsets in the alveolar and blood compartments Next, we investigated the impact of HIV infection on the monocyte population in BAL fluid compared to peripheral blood. The gating strategy is illustrated in Figure 4. First we determined the composition of the monocyte cell population in BAL fluid in comparison to peripheral blood. We found that irrespective of HIV status CD14 + CD16 + intermediate monocytes were the predominant subset in BAL fluid, followed by CD14 ++ CD16 lo classical monocytes and then CD14 lo CD16 + non-classical monocytes (HIV-, Median 63% vs. 33% vs. 5%; HIV+, Median 81% vs. 13% vs. 9%) ( Figure 5A and 5C). In blood, irrespective of HIV status, CD14 ++ CD16 lo classical monocytes were the predominant monocyte subset, followed by CD14 lo CD16 + non-classical monocytes and then CD14 + CD16 + intermediate monocytes (HIV-, median 74% vs. 18% vs. 9%; HIV+, median 73% vs. 23% vs. 8%) ( Figure 5B and 5D).

Discussion
We report the broad impact of HIV infection on immune cell populations in the alveolar space beyond the well-characterised CD8 + T cell alveolitis observed in previous studies 5, 6,9,10 . We show that in addition to CD8 + T cells, B cells and γδ T cells are increased, while classical monocytes are decreased in BAL fluid from ARTnaïve HIV-infected adults compared to HIV-uninfected individuals. We further show generalised disruption in the proportions of immune cell subsets including alveolar macrophages, CD4 + T cells,

myeloid dendritic cells, intermediate monocytes and NK cells in BAL fluid of asymptomatic chronic HIV-infected adults.
Although HIV-infection was associated with accumulation of B cells and γδ T cells in BAL fluid, their contribution to pulmonary immunity during chronic HIV infection is incompletely understood. However, previous studies have reported HIV-associated impairment of function of these two cell subsets in peripheral blood [19][20][21] . Consistent with what has been observed in the systemic circulation, hyperglobulinemia has been reported in BAL fluid of HIV-infected adults 22,23 , but the antibodies have impaired opsonic function 24 . It is plausible that the HIV-associated increase in B cells in the lung results in increased antibody production and BAL fluid hyperglobulinemia. Furthermore, the increase in γδ T cells that we found in the present study supports the findings of Agostini et. al. 25 , who showed that HIV-infected individuals with CD8 + T cell alveolitis had increased γδ T cells in BAL fluid, which were predominantly of the Vδ2 subset. However, HIV infection was also associated with anergic γδ T cells that were characterised by their substantially deficient response to phosphoantigens [26][27][28] . Taken together, the findings of previous studies lead us to postulate that despite the increase in numbers, lung B cells and γδ T cells from HIV-infected individuals have impaired function as their blood counterparts.
HIV infection is associated with massive depletion of mucosal CD4 + T cells in the gut 29,30 and gradual decline in peripheral blood CD4 + T cells 15 . We have shown preserved mucosal CD4 + T cells in BAL fluid from chronic HIV-infected adults, even in those with depleted peripheral blood CD4 + T cells. Our findings are consistent with previous work that showed lung CCR5 + CD4 + T cells are not massively depleted during HIV infection 31 . The mechanisms behind this preservation of alveolar CD4 + T cells is unclear and warrants further investigation. However, Mahlknecht et. al. has shown that macrophages can prevent CD4 + T cell apoptosis in vitro via cell to cell contact using a mechanism that involves stimulation of nef-expressing CD4+ T cells with macrophage membrane-bound TNF 32 . Nef in presence of TNF stimulation promotes activation of anti-apoptotic transcription factor NF-κB, resulting in blockade of caspase-8 activation and subsequent apoptosis 32 . It is therefore plausible that alveoalar macrophages could promote survival of CD4 + T cells in the lung through similar mechanisms, but this warrants further investigation. However, although alveolar CD4 + T cells are not massively depleted during chronic HIV infection, their functional capacity is perturbed 5-7 .
Consistent with others [33][34][35] , we have showed that CD16 + CD14 + intermediate monocytes were the predominant subset in BAL fluid. CD16 + monocytes and AM have been shown to be permissive to HIV infection 8, 36 . The abundance of intermediate monocytes and AM in BAL fluid increases potential cellular targets for HIV. Our findings that AM are preserved during chronic HIV infection, may partly be attributed to the long life span of these cells 37,38 , as well as their resistance to the cytopathic effects of HIV 39,40 . In contrast, we observed a depletion in classical monocytes in BAL fluid from HIV-infected individuals. The mechanism for the selective depletion of classical monocytes is unclear, but might involve HIVinduced apoptosis 41 or loss/downregulation of surface CD14 42 . Alveolar macrophages originate from erythro-myeloid progenitors (EMPs), while monocytes originate from haematopoietic stem cells (HSCs) 43 , hence the differential impact of HIV on these subsets might be due to the distinct nature of their source of origin. Presence of a wide array of HIV-permissive cells in the lung, including recruited and resident cells, could contribute to maintenance of local viral production and subsequent disruption of immune cell populations and homeostasis in this compartment.
A potential limitation of the study is that the numbers of BAL cell subsets are extremely difficult to measure with a very high degree of accuracy due to the variations in the dilution factor of epithelial lining fluid and differences in BAL fluid volume return. However, using a method utilised in previous studies 5,6 , we calculated numbers of cell subsets using the BAL cell count obtained from a haemocytometer combined with proportions obtained by immunophenotyping. We have confidence in the reliability of this method to measure the numbers for the other cell subsets, as we have replicated the observation that the absolute number of CD8 + T cells is higher in HIV-infected adults compared with 6,9,10 .
In conclusion, our findings show that HIV infection is associated with broad alteration of immune cell populations in the lung. Disruption in immune homeostasis has been shown to lead to increased susceptibility to both infectious and non-infectious diseases. The broad alteration of immune cell populations in the lung in part explain the propensity to LRTI in HIV-infected individuals. However, the degree to which successful anti-retroviral therapy restores the composition of immune cells in the lung warrants further investigation.

Data availability
The data underlying the results presented in this manuscript are available from OSF: osf.io/ykve4.

Supplementary Figure 1. Proportions of CD4 + T cells, CD8 + T cells, CD19 + B cells, γδ T cells and NK in peripheral blood from ART-naïve HIV-infected compared to HIV-uninfected individuals.
PBMC were stained with fluorochrome-conjugated antibodies. A) Proportion of CD4 + and CD8 + T cells in peripheral blood. B) Proportion of B cells in peripheral blood. C) Proportion of γδ T cell subsets in peripheral blood. D) Proportion of NK cell subsets in peripheral blood. The horizontal bars represent median and 95% confidence intervals. Data were analyzed using Mann Whitney U test. (HIV-, n=16; HIV+ ART-, n=14).
Click here to access the data.

Supplementary Table 1. Details of fluorochrome-conjugated antibodies used in the study.
Click here to access the data. 1.

2.
3. Mwale and co-workers examine the cellular composition of BAL asymptomatic HIV-positive Malawians. They show increased numbers of CD8 T-cells, B-cells and gd T-cells, while also identifying a reduction in classical monocytes in HIV which resulted in an increased proportion but not absolute number of intermediate monocytes.

Open Peer Review
The data is important and adds a significant contribution to the literature, in particular through providing clinically relevant samples in HIV in patients naïve to ART. While the data makes a valuable contribution to the literature some areas of further detail would be informative.
Background demographic data shows the control group are well matched in terms of age and gender. More details on the HIV-positive group are required. What steps were taken to exclude TB. Is there any data on baseline CXR or TB screening or is the assumption that patients were negative purely based on lack of symptoms? Is there any data on baseline HIV viral load? Some further methodological details would aid interpretation. While the differences in proportions of cell subsets are clear interpretation of absolute numbers requires evidence that volumes of instillation and BAL recovery are similar. Can the authors report their standard methodology involved instillation of 200 mls in four aliquots, into the right middle lobe, or whatever? Have they specific data on the volume recovered ad its variability. Can they confirm there were low (<5%) numbers of bronchial epithelial cells or squamous cells?
Although the focus is analysis of cellular components have they any information on the permeability of the alveolar space in the two groups through measurement of albumin or a related marker? The authors appropriately remark in the discussion that these considerations limit interpretation of absolute numbers so this comment is meant only to provide detail not as a significant criticism.
The absolute number of monocytes in BAL should also be reported. In the abstract, the primary finding is presented as an increase in the proportion of intermediate monocytes in HIV BAL but in reality, the main finding appears to be a reduction in absolute number of classical monocytes which results in a relative rather than absolute increase in intermediate monocytes in HIV. Subsets of intermediate monocytes may also be defined by HLA-DR and the authors appear to have also used antibodies against HLA-DR. Did they find any differences in intermediate subsets by HLA-DR in their HIV positive population?
While reductions in non-classical monocytes are described by many groups the magnitude of the reduction in numbers of non-classical monocytes in the BAL is a little surprising since these cells are thought to be a source of alveolar macrophages. The authors suggest they can detect very few classical cells in BAL in 4.
contrast to the blood. Some more discussion of this point seems needed. Have the authors any data with alternative markers e.g. CCR2, CX3CR1 to confirm such low numbers? Or may they be missing some non-classical monocytes? This finding should be developed further and discussed a little further and related to other BAL-specific lung data.
In the discussion the authors highlight the different origins of monocytes and lung macrophages but some qualification of the differences in origin of lung macrophages in inflammatory settings and the potential for classical monocytes to contribute to lung macrophage numbers in inflammation may be pertinent.
In Figure 5D the HIV + dot has been labelled HIV -and needs to be altered.

If applicable, is the statistical analysis and its interpretation appropriate? Yes
Are all the source data underlying the results available to ensure full reproducibility? Yes

Are the conclusions drawn adequately supported by the results? Partly
No competing interests were disclosed.

Competing Interests:
I have read this submission. I believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard, however I have significant reservations, as outlined above.