Effect of HIV-1 Infection on B Cells in Lamina Propria and Peyer's Patches of Terminal Ileum
First, immunohistologic analyses for apoptosis of B cells in lamina propria areas were performed using TUNEL assays. Apoptotic B cells were frequently seen in the lamina propria often coincident with infiltration of CD68+, CD11c+ macrophages ().
Effect of HIV-1 on B cells in terminal ileum lamina propria.
The most striking changes were noted in the germinal centers of Peyer's patches (). Initial analysis of terminal ileum tissues in AHI patients not on ART versus those on ART (mean duration of therapy 31 d, ) demonstrated no significant difference in the degree of Peyer's patch follicular lysis, characterized by follicle damage, CD8+ T cell and macrophage infiltration, and apoptosis (94.7% in AHI patients not on ART versus 82.6% in AHI patients on ART, ). In terminal ileum tissues from AHI patients not on ART, five of 19 (26.3%) follicles had a germinal center, and this was not significantly different from tissues from AHI patients on ART, in whom ten of 23 (43.4%) had discernable germinal centers. Since similar B cell–inductive microenvironment damage existed in AHI patients both on and not on ART, we combined all AHI patients into a single group for the analyses of terminal ileum using immunohistology and flow cytometry data.
Germinal center damage in acute HIV-1 infection.
Overall, in seven terminal ileum tissue samples from uninfected participants, only one of 32 germinal centers showed evidence of follicle cell apoptosis outside of tingible body macrophages, and 21 of 32 (65.6%) B cell follicles contained germinal centers (). In contrast, out of 14 AHI patients' tissues analyzed, only 15 of 42 (35.7%) B cell follicles contained germinal centers (p
0.018 for AHI versus uninfected, Fisher's exact test) ().
Morphology of uninfected terminal ileum Peyer's patch B cell–inductive microenvironments.
Nearly normal morphology of secondary follicle in AHI terminal ileum.
In the Peyer's patches of uninfected participants, TUNEL+
cells were rare, and when present, were phagocytosed by tingible body macrophages within germinal centers (). In contrast, in Peyer's patches from AHI patients, TUNEL+
cells were abundant within the CD20+
areas in 13 of 14 patients (, S1B
, and S3H
). Even in the occasional B cell follicles that contained relatively few TUNEL+
B cells, other morphological evidence of programmed cell death (apoptotic bodies, cells with nuclear fragmentation) was clearly apparent either in the follicles or at the follicular margins (, ). The immunohistology of most HIV-1+
terminal ileal follicles was associated with TUNEL+
Peyer's patch follicular cells and loss of follicle architecture (, S1
, and S3C–S3H
). Twelve of 14 (86%) AHI patients had follicular areas with T cell apoptosis (). In 10 of 14 AHI patients, B-cell follicles contained areas of both B and T cell apoptosis (), and all (14/14) AHI patients had areas of either T cell or B cell apoptosis. When CD205+
, and Langerin+
dendritic cells were seen, they were either localized in the submucosa below the epithelium (Figure S1A–S1H
), or within or around gut epithelium (Figure S2A–S2D
Stages of B cell apoptosis in terminal ileal Peyer's patches soon after HIV-1 transmission.
Infiltration of NK cells in apoptotic and nonapoptotic areas of AHI terminal ileum Peyer's patches of patient 018-4.
FDC destruction over the course of HIV-1 infection has been well documented, with initial FDC and germinal center hyperplasia followed by FDC destruction and germinal-center involution 
. To determine how early these changes occurred after HIV-1 infection, staining for FDCs was performed in 12 of 14 terminal ileum samples. We found that 53% of germinal centers had evidence of FDC damage (). In contrast, the FDC network in five control tissue samples showed the fine reticular pattern of normal FDCs using monoclonal antibodies to CD21, CD35, and the low-affinity nerve growth factor receptor p75 
Zhang et al. have demonstrated SIV-induced B cell germinal center loss in lymph nodes from rhesus macaques that were poor antibody responders and were rapid progressors to AIDS; there was a concomitant loss of Ki-67+
germinal center B cells in samples from these animals 
. In the AHI participants in our study, whether they were on ART or not, Ki-67+
B cells were frequently depleted (), reflecting some degree of germinal center disruption by HIV-1 in most patients studied. When typical germinal centers were present in AHI gut, Ki-67+
germinal center B cells were similarly present ().
Association of Peyer's Patch CD8+ T Cells, CD11c+ Macrophages, and Natural Killer Cells with Follicular B Cell Apoptosis in Terminal Ileum of AHI Patients
In association with follicular B cell apoptosis, we found infiltration of B cell follicles with CD11c+
macrophages (, S1C
, and S3E
), and CD8+
T cells (, and S3F
), and loss of normal follicle architecture 
. Overall, there were increased CD8+
T cells and CD11c+
myeloid cell infiltrations in areas of apoptosis in 10 of 14 patients.
Activated natural killer (NK) cells are expanded in AHI, and their cytolytic activity is correlated with the level of plasma viremia, with a decrease in NK activity after the initiation of ART 
. While total NK cell numbers are expanded in AHI, there is early depletion of CD56hi
NK cells that secrete cytokines and chemokines 
; continued viremia causes the remaining cytolytic CD56dim
NK cells to become anergic. Thus, activated NK cells could be a mediator of B cell death by direct cytolysis or by antibody-dependent cellular cytotoxicity (ADCC), and it was of interest to evaluate B cell follicles for CD16+
NK cell infiltrations.
In tissues from uninfected participants, Peyer's patches were found to have CD16+
NK cells intermixed with CD68+
macrophages and CD83+
dendritic cells at the follicular dome just under the site of follicle-associated epithelial cells (M cells) specialized for antigen transport () 
, consistent with a recent report by Cella, et al. 
. B cell areas with massive apoptosis contained only scattered cytolytic CD16+
NK cells 
, but did contain areas of infiltrations of CD56+
NK cells ( and S1F
). Thus, while some degree of B cell apoptosis may be mediated by NK cells, the majority is likely mediated either directly or indirectly by CD8+
T cells that were consistently seen infiltrating apoptotic B cell areas (). CD16+
as well as CD57+
NK cells were scattered throughout the lamina propria in similar patterns in both uninfected and infected participants (unpublished data).
Effects of HIV-1 Infection on Numbers of Gastrointestinal Tract Lamina Propria B Cells Using Quantitative Image Analysis
We next evaluated the status of B cells in acute HIV-1 infection in effector (lamina propria) areas in situ in terminal ileum using immunohistochemistry. and Table S2
show the acute HIV-1 induced changes in lamina propria B cell effector sites of terminal ileum. In the lamina propria, there were significant elevations of CD3+
T cells (p
0.008) and CD8+
T cells (p
-test) in AHI patients. Lamina propria CD4+
T cells were decreased in six of 12 biopsies compared to uninfected terminal ileum biopsies (Table S2
Effect of HIV-1 on Terminal Ileum Immune Cells.
We studied lamina propria plasma cells for Ig isotype and κ/λ light chain (LC) ratios to better understand plasma cell populations during AHI. AHI patient number 003-7 had the highest number of lamina propria IgG plasma cells and the second highest number of IgA plasma cells, and had a κ/λ LC ratio of 1.7 (Table S2
). AHI patient number 025-7 had the highest number of IgA plasma cells and a κ/λ LC ratio of 2.6; show the lamina propria profile of this participant (Table S2
). Overall, after a mean of 82 d after transmission the AHI terminal ileum group had an elevated κ/λ LC ratio (1.64±0.14) compared to uninfected terminal ileum (κ/λ
-test) (). Similar κ/λ LC skewing was observed in select Peyer's patch follicles from AHI patients (Figures S3A and S3B
Effects of HIV-1 Infection on Percentages of Gastrointestinal Tract B Cells Using Flow Cytometry
We next used flow cytometry to determine the percentages of total terminal ileum B cells in tissues from AHI patients compared to uninfected control tissues. In mononuclear cells, we found an increase in the percentage of B cells in AHI terminal ileum (26.2%) compared to control uninfected terminal ileum (12.5%) (p
-test). There were no differences between AHI patients who were on or not on ART (25.3% versus 27.9%, respectively; mean duration of therapy 31 d, range 14–63 d; ). There was no significant difference in naïve B cells between AHI and uninfected participants () but the percentages of gut memory B plus plasmablasts/plasma cells (CD19+
) was elevated in AHI versus uninfected participants () (p
-test). There were no differences in naïve, memory, or plasma cell subsets between AHI patients who were on or not on ART (unpublished data).
Flow cytometric analysis of B cell populations.
Effect of HIV-1 on Peripheral Blood B Cell Subsets
We next used the same flow cytometric method to study B cell subsets in peripheral blood in AHI versus uninfected participants. The percentages and absolute numbers of total blood B cells in AHI patients not on ART (n
12) versus AHI patients on ART (n
9) were not different (4.0%, 195 B cells/µl versus 5.5%, 326 B cells/µl, respectively). Similarly, no differences were seen in total blood B cell absolute numbers/µl in AHI versus uninfected participants (3.7%, 227 B cells/µl in uninfected participants) (Table S3
In contrast to reports of elevated numbers of blood naïve and transitional B cells in the blood of patients with chronic HIV-1 infection 
, naïve B cells in the blood of patients with acute and early HIV-1 infection were markedly decreased as a percentage of the total B cell compartment (p
-test) (). Calculated as absolute number of circulating naïve B cells/µl of blood, we found no significant difference in naïve B cell number between uninfected and AHI participants (). The combined percentages and absolute numbers of memory B cells and plasmablasts/plasma cells were elevated in the blood of AHI patients (p
0.0003 and p
0.03, respectively, t
-test) (). Elevations of terminally differentiated B cells in AHI were observed at the earliest time points studied, 17–36 d after transmission, where the percentage of memory B and plasma cells was 23.7%±3.3% (range 12.4%–37.9%) compared to 9.4%±1.9% in the uninfected control participants. Analyzed separately, the percentages of both memory B cells and plasmablasts/plasma cells in the blood of AHI patients were elevated compared to uninfected control participants (p
0.005 and p
0.005, respectively, t
-test) (Figure S4B and S4E
). Absolute number of circulating memory B cells were elevated in AHI patients (p
-test), whereas circulating plasmablasts/plasma cells were similar to uninfected controls (Figure S4C and S4F
Analysis was also performed for all of the B cell subsets described above by separating participants into high plasma VL and low VL groups. No significant differences were found for B cell subsets between the two groups (t-test).
Recently, a population of CD19+
B cells has been described as IgM+
memory B cells and/or circulating human marginal zone B cells 
cells were included in the above memory B cell analysis, but we did not analyze the CD19+
B cell subset. A separate analysis of this population showed that the percentage of CD19+
B cells in blood was similar in both uninfected and AHI cohorts (7.0%±1.5% in uninfected, 6.2%±1.5% in AHI, not significantly different, t
-test). In terminal ileum, CD19+
B cell percentages also were not different between infected and uninfected participants (1.9%±0.7% in uninfected, 1.4%±0.3% in infected, not significantly different, t
-test). Finally, including CD19+
B cell populations in our analyses did not alter our conclusion that acute HIV-1 infection results in increased percentages of memory cells in blood or gut (unpublished data).
EBV Transformation of Terminal Ileum Memory B Cells for Analysis of Specificity of Antibodies Produced
EBV preferentially transforms recently primed B cells 
. The amount of IgM, IgG, or IgA produced in 96-well cultures of EBV-transformed B cells was determined in six AHI terminal ileum samples versus terminal ileum from six uninfected participants. If HIV-1 infection promoted class-switching there should be an increased number of primary EBV-transformed cultures producing supernatant IgG and IgA after EBV transformation compared to terminal ileum B cell cultures from uninfected individuals. In addition, if there are more primed IgM+
B cells present in terminal ileum, there should be more B cell cultures with elevated IgM. shows that this was the case with more IgM-, IgG-, and IgA-producing B cell cultures in AHI terminal ileum 2 wk after EBV stimulation versus uninfected EBV-transformed B cell cultures.
Total immunoglobulin (Ig) levels and specificities in the EBV-transformed B cell cultures.
Next, we determined the relative frequency of the specificities of antibodies in each EBV-transformed well of B cells to determine the relative percentages of B cells responding to HIV-1, either as HIV-1–specific or as polyclonally induced antibodies (). This method of analysis does not reflect the absolute percentage of memory B cells making various antibody specificities, because the gut mononuclear cells were cultured at 10,000 cells per well as opposed to single cell per well cultures. Rather this analysis provides the relative frequency of antibody specificities in the entire terminal ileum memory B cell population. In uninfected participants, 8/420 (1.9%) of EBV-transformed B cell cultures were positive for gp41 antibodies, and 1.9% were positive for gp120 antibodies (IgM only); none were positive for antibodies to HIV-1 Tat, while 1.4% and 0.2% were positive for anti-cardiolipin antibodies or rheumatoid factor, respectively (). Two percent of cultures from uninfected participants were also positive for reactivity with the 2007 inactivated influenza vaccine Fluzone (Sanofi Pasteur). In contrast, in AHI terminal ileum, 18.6% of EBV B cell cultures were positive for antibodies to HIV-1 gp41, 5.7% for antibodies to HIV-1 gp120, and 9.3% for antibodies to HIV-1 Tat. In addition, 6.2% of AHI B cell cultures were reactive with cardiolipin, 3.0% had rheumatoid factor activity and 16.2% were reactive with the 2007 inactivated influenza vaccine (). Taken together this pattern of relative specificities of EBV-transformed memory B cells indicated the presence of a large component of polyclonal B cell activation in addition to HIV-1–induced specific antibody responses.
Percentage of EB virus-stimulated cultures producing antibodies reactive with HIV and non-HIV antigens.
Relationship of Plasma and GALT HIV-1 Viral Load to Peyer's Patch Germinal Center Morphology
We identified Peyer's patch follicles containing germinal centers at various stages of damage in eight AHI patients (; ). Comparison of GALT germinal center depletion and plasma VL demonstrated that those with germinal center depletion had significantly higher viral loads (mean 34,065 copies/ml) than those with germinal centers (mean 1,711 copies/ml) (p
0.021, exact Wilcoxon test). Six of eight patients with germinal centers had initiated ART, while only two of six of the patients with no germinal centers were on ART. These data are in contrast to the work of Muro-Cacho et al. who showed that the intensity of lymph node apoptosis in advanced HIV-1 infection did not correlate with plasma VL 
. It is of interest that, of the 14 participants with gut biopsies, the only patient (023-3) that controlled plasma viremia in the absence of ART (203 copies/ml at the time of sampling 66 d after transmission) also had some degree of germinal center preservation (). GALT-associated HIV-1 RNA did not correlate with germinal center depletion () 
Comparison of AHI patients with terminal ileum germinal centers versus those without terminal ileum germinal centers.
Effect of Presence of Peyer's Patch Germinal Centers and Antiretroviral Treatment During Acute HIV-1 Infection on Plasma Rheumatoid Factor Levels and HIV-1 Protein Antibody Levels
Comparison of HIV-1 envelope and gag p55 antibodies in patients with germinal centers to those with no germinal centers showed no differences except for higher anti-gp41 IgG antibodies in those without germinal centers (p
-test) (). Similarly, there was no difference in antibody levels when the participants providing terminal ileum biopsies () were grouped as high- (>20,000 copies/ml) versus low plasma VL (<20,000 copies/ml) (unpublished data). We next determined the effect of early institution of ART on levels of plasma IgM, IgG and IgA antibodies against HIV-1 gp41, p55, gp120 and gp140, as well as plasma rheumatoid factor levels, in patients from whom terminal ileal biopsies were obtained (). The time from transmission in the treated versus the non-treated group was similar (82 d versus 79 d) (). As expected, the anti-gp41 IgA, anti-gp140 IgA, and anti-gp140 IgG levels were significantly lower in the AHI on ART group compared to the AHI on no ART group. Thus, even one month of ART significantly lowered plasma levels of antibody to HIV-1 Env following HIV transmission.
Antibody responses to HIV-1 proteins: Antibody to HIV-1 proteins in AHI with germinal centers versus no germinal centers.
Effect of antiretroviral treatment in AHI on HIV-1 antibody responses.