HIV Destroys Gut Immunity-new study -
Gut Flora Influences HIV Immune Response
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DURHAM, N.C.-Normal microorganisms in the intestines appear to play a pivotal role in how the HIV virus foils a successful attack from the body's immune system, according to new research from Duke Medicine.
The study, published Aug. 13, 2014, in the journal Cell Host & Microbe, builds on previous work from researchers at the Duke Human Vaccine Institute that outlined a perplexing quality about HIV: The antibodies that originally arise to fight the virus are ineffective.
These initial, ineffective antibodies target regions of the virus's outer envelope called gp41 that quickly mutates, and the virus escapes being neutralized. It turns out that the virus has an accomplice in this feat-the natural microbiome in the gut.
"Gut flora keeps us all healthy by helping the immune system develop, and by stimulating a group of immune cells that keep bacteria in check," said senior author Barton F. Haynes, MD, director of the Duke Human Vaccine Institute. "But this research shows that antibodies that react to bacteria also cross-react to the HIV envelope."
Haynes said the body fights most new infections by deploying what are known as naïve B cells, which then imprint a memory of the pathogen so the next time it encounters the bug, it knows how to fight it.
But when the HIV virus invades and begins replicating in the gastrointestinal tract, no such naïve B cells are dispatched. Instead, a large, pre-existing pool of memory B cells respond-the same memory B cells in the gut that fight bacterial infections such as E coli.
This occurs because the region of the HIV virus that the immune system targets, the gp41 region on the virus's outer envelope, appears to be a molecular mimic of bacterial antigens that B cells are primed to target.
HIV-1 Envelope gp41 Antibodies Can Originate from Terminal Ileum B Cells that Share Cross-Reactivity with Commensal Bacteria
The plasma cell and memory B cell pools in intestine contain a normal subset of B cells reactive with intestinal commensal bacteria (Benckert et al., 2011). In acute HIV-1 infection (AHI), virus replication is prominent in the gastrointestinal tract, with early depletion of CD4+ T cells (Brenchley et al., 2004, Guadalupe et al., 2003, Mehandru et al., 2006, Pope and Haase, 2003, Veazey et al., 1998, Veazey et al., 2001) as well as early destruction of B cell germinal centers (Levesque et al., 2009). Initial plasma (Tomaras et al., 2008) and mucosal fluid (Yates et al., 2013) antibody response in AHI is targeted to HIV-1 Env gp41. The AHI gp41 antibody response is nonneutralizing and does not select viral escape mutants (Tomaras et al., 2008). Rather, it is the initial autologous gp120 neutralizing antibody response that is the first Env antibody shown to select viral escape mutants (Moore et al., 2009, Richman et al., 2003, Wei et al., 2003).
Recombinant monoclonal antibodies (mAbs) isolated from blood plasmablasts and/or plasma cells (hereafter termed plasma cells) of individuals with AHI were predominantly targeted to Env gp41 and were polyreactive with both host and environmental antigens including commensal bacteria (Liao et al., 2011). These observations raised the hypothesis that a component of the peripheral blood HIV-1 Env gp41 response in blood originates from polyreactive memory B cells activated prior to transmission by environmental antigens (Liao et al., 2011).
Here we have used single B cell sorting and recombinant antibody technology to probe the plasma cell and memory B cell repertoire of the terminal ileum in early and chronic HIV-1 infection. We found that the terminal ileum plasma cell and memory B cell repertoire was comprised of predominantly polyclonally activated, non-HIV-1-reactive antibodies, and the dominant early HIV-1 B cell response in the terminal ileum was targeted to Env gp41. Remarkably, 82% of HIV-1 gp41-reactive terminal ileum antibodies cross-reacted with intestinal commensal bacterial antigens, and mutated antibodies cross-reactive with Env gp41 and intestinal commensal bacteria were isolated from HIV-1 uninfected individuals. Thus, the antibody response to HIV-1 may be shaped by intestinal B cells stimulated by microbiota to develop a preinfection pool of memory B cells cross-reactive with HIV-1 gp41.
In this study we have demonstrated that the dominant plasma cell antibody population to HIV-1 in both EHI and CHI in the terminal ileum was nonneutralizing, directed to Env gp41, and cross-reactive with intestinal commensal bacterial antigens. One such bacterial antigen identified was the α subunit of E. coli RNA polymerase. Similar specificities of gp41 commensal bacteria cross-reactive mutated antibodies could be isolated from HIV-1 uninfected individuals. Moreover, we demonstrated sharing of terminal ileum clonal lineage members with the blood compartment, providing support for the hypothesis that blood B cells cross-reactive with intestinal bacteria and gp41 are derived from the intestinal tract.
The preponderance of gp41 antibodies in terminal ileum plasma cell and memory B cell pools now potentially explains the mechanism of induction of gp41 antibody immunodominance in plasma and mucosal fluid studies (Tomaras et al., 2008, Yates et al., 2013). Liao et al. (2011)) showed a predominance of blood gp41 antibodies from HIV-1 plasma cell-derived mAbs from AHI and found them to be a minority of the plasma cell pool 17-46 days after HIV-1 transmission. The polyclonal pool of non-HIV-1-reactive B cells is likely due to the massive cytokine storm that occurs early on after HIV-1 transmission (Stacey et al., 2009) and prompted us to ask if the plasma cell and memory B cell pools in terminal ileum would be a location of a more robust HIV-1 Env antibody response.
Instead, we found in both EHI and CHI that terminal ileum contained primarily non-HIV-1-reactive polyclonal plasma and memory B cells, and the few HIV-1-reactive B cells that were present were targeted to Env gp41.
Host-specific bacterial colonization of the gastrointestinal tract is required for normal development of the intestinal immune system (Chung et al., 2012, Erturk-Hasdemir and Kasper, 2013, Hooper et al., 2012). Germ-free mice have numerous immunological deficiencies, including small Peyer's patches and mesenteric lymph nodes, reduced secretory IgA, fewer plasma cells, CD4+ T cells and CD8+ T cells, and diminished antimicrobial peptide production (Erturk-Hasdemir and Kasper, 2013, Hooper et al., 2012, Round and Mazmanian, 2009). Recolonization of germ-free mice with host-specific commensal bacteria ameliorates these defects (Chung et al., 2012, Smith et al., 2007). The presence of intestinal commensal bacteria induces immune maturation that is not only required for gut homeostasis, but helps generate a pool of mature adaptive immune cells prepared to protect the host from infections. The pre-HIV-1 infection presence of B cells within the intestine cross-reactive with both bacterial antigens and HIV-1 gp41 is evidence of molecular mimicry between HIV-1 antigens and bacteria antigens and suggests an explanation for why the initial antibody response to AHI in the plasma and mucosal fluids is to gp41 (Fujinami et al., 1983, Liao et al., 2011, Oldstone, 1998, Srinivasappa et al., 1986, Tomaras et al., 2008, Yates et al., 2013).
Isolation of mutated gp41 and gut flora cross-reactive antibodies from terminal ileum HIV-1 uninfected individuals directly suggests that commensal or pathogenic bacteria or other cross-reactive environmental antigens can trigger gp41 cross-reactive responses before HIV-1 infection. These data provide evidence in support of the hypothesis that the dominant HIV-1 gp41 antibody response after HIV-1 transmission is mediated by previously activated memory B cells that are present before HIV-1 infection and cross-reactive with intestinal bacteria. Once HIV-1 infection occurs, then gp41 would begin to trigger the previously activated bacterial-driven lineages toward affinity maturation to gp41-specific antibodies. A critical test of this notion would be to demonstrate that reactivity in commensal bacteria-gp41 lineage begins with a gut flora-reactive UCA followed by acquisition of gp41 reactivity upon affinity maturation. In the present study, we provide three examples of gp41-reactive antibodies, DH306, DH308, and DH309, that showed affinity maturation to autologous and/or heterologous Env (Figure 4). In the case of antibody DH306, reactivity of the UCA with gp41, but not the T/F Env gp140, may well be an example of cross-reactive stimulation of the UCA by an environmental gp41 cross-reactive antigen before transmission that gave rise to the affinity mature antibody that, after infection, reacted with the autologous T/F Env. Both DH308 and DH308 UCA bound to MN gp41 with nanomolar affinity but did not bind to the autologous T/F gp140 (Figure 4). It is important to note that antibody DH308 was isolated from individual 004-0 3 years into infection. Thus, it is likely that a T/F Env variant selected by antibodies over time initiated the DH308 lineage, given the high level of affinity maturation to gp41 from ~10 nM in DH308 UCA to 0.4 M in the mature antibody DH308 (Figure 4D). DH305 UCA and DH319 UCA are examples of naturally paired, unmutated VHDHJH and VLJL with high affinities for viral antigens and B cell clonal lineages reaching an affinity ceiling prior to accumulation of the mutations found in the mature mAbs as previously described (Batista and Neuberger, 1998). We have also previously shown that in a reconstructed blood gp41 clonal lineage, the UCA and the first intermediate antibody in the lineage were commensal bacteria reactive, but not gp41-reactive (Liao et al., 2011). Instead, gp41 reactivity only occurred later in clonal lineage development, and after gp41 reactivity occurred, there was affinity maturation to HIV-1 env gp41 in the clonal lineage. The presence of CD4+ memory T cells cross-reactive with both HIV-1 antigens and microbial peptides in uninfected adults (Campion et al., 2014, Su et al., 2013), and the 5.2% and 11.9% VH mutation frequencies of DH306 and DH309, suggested that the affinity maturation of gp41 commensal bacteria cross-reactive B cells to gp41 is T cell dependent.
We now directly demonstrate the intestinal tract origin for commensal bacteria-gp41 cross-reactive antibodies found in the blood. Moreover, we demonstrated that 21% of commensal bacteria-reactive B cells were not gp41 reactive, adding additional support to the idea that, in HIV-1-infected individuals, the gp41-reactive plasma cells and memory B cells represented a response to HIV-1. The proportion of these control non-HIV-1-reactive antibodies isolated from the terminal ileum of HIV-1-infected individuals that reacted with gut flora (21%) is greater than the ~12% of plasma cells from the terminal ileum of HIV-1 uninfected individuals determined to be reactive with specific gut flora by Benckert et al. (2011)). Microbial translocation that occurs in HIV-1 infection may account for this higher level of commensal bacteria-reactive terminal ileum B cells in our study (Brenchley et al., 2006).
A critical test of the hypothesis that blood gp41 commensal bacteria-reactive B cells arise in the intestine was to determine if commensal bacteria-gp41 clonal lineages shared members with blood B cells. Indeed, we have now found evidence for three such intestinal commensal bacteria-gp41 clonal lineages shared by both terminal ileum and peripheral blood compartments (Figures 7 and S6 and Table S7).
In summary, these data provide evidence for the hypothesis that the postinfection B cell response to HIV-1 is shaped by the preinfection B cell repertoire to environmental antigens. Env gp41 antibodies cross-react with human intestinal commensal bacteria, suggesting that commensal bacteria play critical roles in shaping the preinfection response to HIV-1, and demonstrate a major role for the memory B cell pool in contributing to the initial antibody response to HIV-1. These data also raise the hypothesis that the human B cell response to a wide variety of other infectious agents may similarly be affected by cross-reactivity to environmental antigens.
HIV-1 gp41-Reactive Antibodies in Terminal Ileum in Early and Chronic HIV-1 Infection Individuals
We investigated the plasma cell response to HIV-1 infection within the terminal ileum of six early HIV-1 infection (EHI) individuals (Table S1). We expressed 114 mAbs from plasma cells and 140 mAbs from memory B cells recovered from terminal ileum. Of the 254 total mAbs isolated from EHI individuals, only 5 (2.0%) reacted with gp41 and none (0.0%) with gp120 (Figure 1 and Table S2).
HIV-1-reactive mAbs primarily utilized heavy-chain variable gene segments from VH family 3. VH mutation frequencies ranged from 0.0% to 10.4%, and HCDR3 lengths ranged from 11 to 25 amino acids. There were no statistical differences between the mean VH mutation frequencies and HCDR3 lengths of the HIV-1-reactive antibodies compared to non-HIV-1-reactive antibodies isolated from terminal ileum plasma cells from EHI individuals (Figures 1B and 1C). All recombinant HIV-1 mAbs were expressed with an immunoglobulin G1 (IgG1) backbone; their original isotypes were IgA1, IgA2, and IgG3 (Table S2). IgA2 and IgG3 only made up 6.7% and 5.1% of total terminal ileum mAbs isolated from EHI, respectively (Table S3). Four of the five gp41-reactive mAbs were low affinity, with effective antibody binding 50% concentrations (EC50s) of >100 μg/ml. DH300 had the highest apparent affinity, with an EC50 of only >25 μg/ml (Figure 1D and Table S2). With a VH mutation frequency of 10.4%, the heavy chain of DH300 was also the most mutated of the EHI terminal ileum HIV-1-reactive mAbs isolated (Table S2). These HIV-1-reactive mAbs were tested for neutralization against the easy-to-neutralize (tier 1) viruses, ADA, MN, and SF162, and the difficult-to-neutralize virus (tier 2) DU156, and all were nonneutralizing when assayed in the TZM-bl pseudovirus infection assay. Thus, the plasma cell and memory B cell response in EHI was polyclonal, and the HIV-1-reactive mAbs were targeted to Env gp41 and were nonneutralizing.
We next characterized the plasma cell response in the terminal ileum of three chronically HIV-1-infected (CHI) individuals, 038-7, 004-0, and 071-8 (Table S1). From these individuals, we expressed 158 mAbs from terminal ileum plasma cells; 14 (8.8%) of these mAbs reacted with HIV-1 antigens, 9 (5.7%) with Env gp41, 4 (2.5%) with HIV-1 capsid protein (p24), and 1 (0.6%) with Env gp120 (Figure 2 and Table S2). Similar to the gp41 antibodies from EHI individuals, the HIV-1-reactive mAbs isolated from CHI individual 071-8 predominantly used VH family 3 gene segments; mutation frequencies ranged from 1.3% to 7.0%, and the majority of the original mAbs were of IgA isotype (Table S2). In contrast, 8 of the 9 (89%) HIV-1-reactive mAbs from plasma cells of CHI individual 004-0 used VH1-69; all these VH1-69 antibodies were originally IgG1 (Table S2). Both individuals, 071-8 and 004-0, have three genomic copies of VH1-69 as determined by digital PCR (Table S4). The number of B cells utilizing the gene segment VH1-69 has been reported to be proportional to the gene copy number of certain VH1-69 alleles (Sasso et al., 1996). However, this was not seen at the terminal ileum single B cell level in our study, where both 071-8 and 004-0 had three genomic copies of VH1-69, yet only 004-0 predominately used VH1-69 to respond to HIV-1 infection (Table S2).
The VH mutation frequencies of antibodies from individual 004-0 ranged from 3.3% to 11.9%, and HCDR3 lengths ranged from 12 to 23 amino acids (Table S2). There were no statistical differences between the mean VH mutation frequencies and HCDR3 lengths of the HIV-1-reactive mAbs compared to non-HIV-1-reactive mAbs isolated from terminal ileum plasma cells from CHI individuals (Figures 2B and 2C). The estimated EC50s for gp41 binding of these antibodies ranged from <0.1 μg/ml to >100 μg/ml (Table S2). DH306 and DH309 had high apparent affinities to gp41 (EC50s of <0.1 μg/ml). DH310, DH311, DH312, and DH314 had high affinities to Gag p24 (EC50s of <1 μg/ml - <0.1) (Figure 2D and Table S2). These HIV-1-reactive mAbs were also tested for neutralization against viruses, ADA, MN, SF162, and DU156 in TZM-bl assays and were nonneutralizing.
Because the HIV-1 antigen-specific terminal ileum mAbs account for such a small proportion of the plasma cell and memory B cell response as measured by single-cell sorting, we next quantified the Env-specific memory B cell pool by an alternative method. We assayed paired peripheral blood mononuclear cells (PBMCs) and terminal ileum samples from four CHI individuals (078-2, 067-8, 072-3, and 076-4) (Table S1) by flow cytometry analysis of HIV-1 Env-specific memory B cells with a fluorescent-labeled consensus group M gp140 Env, consensus-S (CON-S) previously shown to bind to clade B-reactive antibodies (Liao et al., 2006, Tomaras et al., 2008). We found means of 0.04% ± 0.02%, 0.26% ± 0.24%, and 0.20% ± 0.29% IgM, IgG, and IgA CON-S gp140-reactive memory B cells, respectively, in blood (Table S5). The mean percentage of IgM, IgG, and IgA CON-S gp140-reactive memory B cells in terminal ileum were 0.01% ± 0.02%, 0.05% ± 0.1%, and 0.03% ± 0.06%, respectively (Table S5). Thus, by flow cytometry with a fluorophor-labeled Env, there was also a relative dearth of HIV-1 Env-reactive memory B cells in terminal ileum compared to blood in CHI.
Terminal Ileum HIV-1-Reactive Antibodies Were Cross-Reactive with Commensal Bacterial Antigens
We tested HIV-1-reactive mAbs isolated from terminal ileum of EHI for reactivity to antigens in anaerobic commensal bacteria whole-cell lysates (WCLs) by surface plasmon resonance (SPR) and to both anaerobic and aerobic commensal bacteria WCLs by western blot analysis. Of the six gp41-reactive antibodies from EHI, all were reactive to anaerobic intestinal commensal bacteria by both SPR and western blot (Figures 3A-3C and S1 and Table S6). Similarly, 11 of the 16 HIV-1-reactive mAbs isolated from the terminal ileum of CHI cross-reacted with anaerobic commensal bacteria by SPR and western blot (Figures 3A, 3B, 3E, and S1 and Table S6). Antibody reactivity to aerobic and anaerobic commensal bacteria was also tested in Luminex-based binding antibody multiplex assays (BAMAs) (Figure S2A). Of 17 antibodies positive in western blot and SPR, 14 could also be confirmed in BAMA (Figures 3D, 3F, S1, and S2 and Table S6).
To determine if HIV-1 and commensal bacteria cross-reactive mAbs were polyreactive/autotreactive, we tested the HIV-1-reactive mAbs in Luminex AtheNA ANA II and HEp-2 immunofluorescence ANA assays. Four of the six gp41- commensal bacteria cross-reactive mAbs from EHI terminal ileum were not reactive with additional antigens by these assays (Figure S1B and Table S6). Of the 11 HIV-1 and commensal bacteria cross-reactive mAbs isolated from CHI terminal ileum plasma cells, six were not reactive in either assay (Figure S2B and Table S6).
In addition to HIV-reactive mAbs, we produced and purified 19 terminal ileum mAbs that did not bind HIV-1 epitopes by ELISA or BAMA (Table S2). Of these, four antibodies (21%) were reactive with intestinal bacterial WCLs by both western blot and BAMA (Figures S1A and S3 and Table S6). Three of these four antibodies were not reactive in AtheNA ANA II or HEp-2 ANA assays (Table S6). Therefore, not all commensal bacteria-reactive antibodies from intestine were cross-reactive with gp41.
Affinity Maturation of Commensal Bacteria Cross-Reactive Antibodies to Autologous Envelope
To determine if HIV-1 gp41-reactive antibodies that were cross-reactive with commensal bacteria underwent affinity maturation to gp41, we inferred the heavy- and light-chain unmutated common ancestors (UCAs) of five gp41-reactive mAbs, DH306, DH309, DH308, DH305, and DH319, and produced their UCAs, termed DH306 UCA, DH309 UCA, DH308 UCA, DH305 UCA, and DH319 UCA, respectively. For mAbs isolated from 004-0, DH306, DH309, and DH308, we determined UCA and mature antibody affinities to autologous HIV-1 004-0 gp140 and heterologous HIV-1 MN gp41, as well as relative binding to commensal bacterial antigens. Affinity for the autologous Env increased from undetectable binding to 0.62 nM when comparing the UCA DH306 UCA and the mature antibody DH306 and similarly increased from 4.44 nM to 0.34 nM for DH309 UCA and DH309 (Figures 4A, 4B, and 4D ). The mature antibody DH306 also had a greater reactivity to commensal bacteria compared to its UCA (Figure 4A). Binding to the 004-0 T/F gp140 was undetectable for DH308 UCA and DH308; however, affinity to MN gp41 increased from 9.97 nM to 0.41 nM (Figures 4C and 4D). In contrast, DH305 UCA and DH319 UCA had high affinities of 3.55 nM and 0.41 nM to MN gp41, respectively, and affinity did not increase upon accumulation of mutations in the mature mAbs, DH305 and DH319 (Figures 4E and 4F). Therefore, in three commensal bacterial antigen cross-reactive gp41 clonal lineages, affinity maturation to gp41 could be demonstrated from UCAs to mature antibodies.
HIV-1 gp41 Commensal Bacterial Cross-Reactive Antibodies Isolated from the Terminal Ileum of Uninfected Individuals
If preinfection terminal ileum antibodies cross-reactive with intestinal commensal bacteria and gp41 are responsible for the initial antibody response to HIV-1 Env gp41 following HIV-1 infection, mutated gp41 and gut flora cross-reactive antibodies should exist in the terminal ileum of uninfected individuals. To investigate this hypothesis, we sorted single plasma cells and memory B cells from three HIV-1 uninfected individuals (Table S1) and identified two low-affinity gp41-reactive antibodies, DH366 and DH367, both of which also reacted with intestinal commensal bacteria (Figures 5 and S1B and Table S2). Both antibodies used VH gene segments from family 3 and were class-switched to IgG; the VH mutation frequencies of these antibodies were 5.2% and 9.7% (Table S2). Therefore, commensal bacteria-reactive mutated B cells that are cross-reactive with Env gp41 can be found in the intestinal B cell repertoire of HIV-1 uninfected individuals, supporting the notion that the initial gp41 antibody response to HIV-1 derived from preexisting commensal bacterial cross-reactive memory B cells.
E. coli RNA Polymerase Is One Intestinal Bacterial Antigen Cross-Reactive with HIV-1 gp41 Antibodies
To identify antigens in commensal bacteria cross-reactive with gp41 mAbs, we used the AHI blood-derived HIV-1 gp41, gut bacterial WCL-reactive antibody 558_2 previously reported to bind to an ~520 kDa band of both aerobic and anaerobic commensal bacteria WCLs (Liao et al., 2011) (Figure 6A). The large molecular weight fraction of bacterial WCL was isolated by size exclusion chromatography (SEC) (Figure 6B), and isoelectric zoom fractionation showed that the protein reactive with mAb558_2 migrated to the gel compartment with pH 7-10 (Figure S4A). E. coli RNA polymerase subunits ß, ß', and α were identified by liquid chromatography-tandem mass spectrometry (LC-MS/MS) of the 520 kDa excised bands from two lanes of the SEC-enriched >500 kDa fraction analyzed on a NativePAGE gel (Figures 6B, 6C, and S4B-S4D). We determined that mAb558_2 binding was specific for the core enzyme of E. coli RNA polymerase (Figures 6D, S4E, and S4F). By western blot, we mapped the specificity of mAb558_2 to the 37 kDa α subunit of recombinant E. coli RNA polymerase (Figure 6D).
We found that 2 of 14 (14.3%) Env gp41 and intestinal commensal bacteria cross-reactive EHI and CHI terminal ileum antibodies also reacted with rRNA polymerase by BAMA. (Figures 6E-6G) Moreover, the gp41 commensal bacterial cross-reactive antibody DH367 isolated from the terminal ileum of an uninfected individual also reacted with rRNA polymerase by BAMA (Figure 6H).
Terminal Ileum HIV-1-Reactive Antibody Clonal Lineage Members Shared by Terminal Ileum and Peripheral Blood Compartments
We next asked if HIV-1 and commensal bacteria cross-reactive B cells recirculate in the terminal ileum and peripheral blood. We studied paired blood samples of three of the individuals (042-8, 004-0, and 071-8) from whom we had isolated terminal ileum plasma cell and memory B cell mAbs (Table S1). We sorted single plasma cells and memory B cells from PBMCs and identified 13 antibodies with HIV-1 reactivity (Figure S5 and Table S2). By single-cell PCR, we were able to identify four clonal lineages within the terminal ileum and one clonal lineage within the blood (Table S7). However, by these methods we were unable to identify any clonal lineages with members shared between the terminal ileum and blood.
We next conducted pyrosequencing of genomic DNA isolated from PBMCs taken at the same time as terminal ileum samples from chronically infected individuals 004-0 and 071-8 and searched the sequences for VHDHJH members clonally related to the 149 terminal ileum VHDHJH sequences isolated from these same two individuals by single-cell sorting. By this method we identified a total of 18 clonal lineages that had members in both terminal ileum and blood compartments (Figures 7 and S6 and Table S7). Thus, 12% of terminal ileum B cells isolated by single-cell PCR had cross-compartment clonal lineage members in the blood. Of these 18 cross-compartment clonal lineages, we determined that two clonal lineages were cross-reactive with Env gp41 and intestinal commensal bacteria, and one lineage was cross-reactive with HIV-1 Gag p24 and commensal bacteria (Figures 1D, 2D, 7, and S6 and Table S2).
To determine if the cross-compartmentalization of B cell clonal lineages identified in 004-0 and 071-8 was due to contamination of the terminal ileum tissue biopsies with blood B cells trafficking through the ileum vasculature without entering the tissue, we performed quantitative image analysis of B cells in the terminal ileum of HIV-1-infected individuals and found that of the 12 terminal ileum biopsies studied, only 0.2% of the CD20+ cells within the tissue samples were found within blood vessels (Figure S7). Thus, blood contamination of the biopsy could not explain the 12% of terminal ileum B cells isolated by single-cell PCR as contaminating B cells from the blood compartment.