Polyclonal B cell differentiation and loss of gastrointestinal tract germinal centers in the earliest stages of HIV-1 infection

Abstract

Background: The antibody response to HIV-1 does not appear in the plasma until approximately 2-5 weeks after transmission, and neutralizing antibodies to autologous HIV-1 generally do not become detectable until 12 weeks or more after transmission. Moreover, levels of HIV-1-specific antibodies decline on antiretroviral treatment. The mechanisms of this delay in the appearance of anti-HIV-1 antibodies and of their subsequent rapid decline are not known. While the effect of HIV-1 on depletion of gut CD4(+) T cells in acute HIV-1 infection is well described, we studied blood and tissue B cells soon after infection to determine the effect of early HIV-1 on these cells.

Methods and findings: In human participants, we analyzed B cells in blood as early as 17 days after HIV-1 infection, and in terminal ileum inductive and effector microenvironments beginning at 47 days after infection. We found that HIV-1 infection rapidly induced polyclonal activation and terminal differentiation of B cells in blood and in gut-associated lymphoid tissue (GALT) B cells. The specificities of antibodies produced by GALT memory B cells in acute HIV-1 infection (AHI) included not only HIV-1-specific antibodies, but also influenza-specific and autoreactive antibodies, indicating very early onset of HIV-1-induced polyclonal B cell activation. Follicular damage or germinal center loss in terminal ileum Peyer's patches was seen with 88% of follicles exhibiting B or T cell apoptosis and follicular lysis.

Conclusions: Early induction of polyclonal B cell differentiation, coupled with follicular damage and germinal center loss soon after HIV-1 infection, may explain both the high rate of decline in HIV-1-induced antibody responses and the delay in plasma antibody responses to HIV-1. Please see later in the article for Editors' Summary.

Figure 1. Effect of HIV-1 on B cells in terminal ileum lamina propria.

Lamina propria B cell apoptosis with CD11c⁺ macrophage infiltration in participant 015-5, 55 d after transmission, on ART 15 d. A cluster of lamina propria B cells (A; arrows), TUNEL stain of the same area with arrows pointing out brown apoptotic nuclei (B), and the same area with infiltrating CD11c⁺ macrophages (C) (100×).

Figure 2. Morphology of uninfected terminal ileum Peyer's patch B cell–inductive microenvironments.

Serial sections of the same Peyer's patch follicle in an uninfected terminal ileum (20×). (A) CD20⁺ B cells in a secondary follicle with germinal center (g). (B) The same follicle with TUNEL staining showing that the predominant apoptotic cells are within tingible body macrophages (insert). (C) Ki-67⁺ dividing B cells. (D) CD11c⁺ follicular dendritic cells and macrophages. (E) CD68⁺ macrophages and dendritic cells in the subepithelial zone below follicular associated epithelial cells (arrow, top of photomicrograph) at the dome of the Peyer's patch, and CD68⁺ tingible body macrophages in the germinal center (insert). (F) CD16⁺ NK cells clustered in the areas below follicle associated epithelial cells (arrow).

Figure 3. Nearly normal morphology of secondary follicle in AHI terminal ileum.

A near-normal terminal ileal secondary follicle in patient 018-4, 74 d after transmission, on ART 29 d (20×). (A) CD20⁺ B cells; (B) Ki-67⁺ dividing germinal center B cells; (C) subepithelial CD3⁺ T cells and a small area of infiltrating CD3⁺ T cells in the marginal zone around the germinal center (arrow); (D) normal-appearing CD11c⁺ macrophages and dendritic cells (arrow). TUNEL stain was negative for apoptotic nuclei (unpublished data).

Figure 4. Stages of B cell apoptosis in terminal ileal Peyer's patches soon after HIV-1 transmission.

(A–F) Early follicular lysis in a primary follicle of patient 019-2, 47 d after transmission, not on ART; arrows in each photomicrograph show same mantle zone T cell area (A–P, 40×). (A) Hematoxylin and eosin stain. (B) CD20. (C) TUNEL stain with apoptosis in the mantle zone. (D) Rim of CD4⁺ T cells in apoptotic mantle zone. (E) Infiltrating CD8⁺ T cells. (F) Infiltrating CD11c⁺ macrophages. (G–P) Serial sections of a small germinal center remnant in patient 023-3, 66 d after transmission, not on ART (plasma viral load 203 copies/ml, and the only participant in this study who spontaneously controlled plasma viremia). (G) Hematoxylin and eosin stain. (H) CD20⁺ B cells. (I) TUNEL stain of apoptotic B cells surrounding a small germinal center. (J) Follicular CD4⁺ depletion with weakly stained CD4⁺ macrophages. (K) CD8⁺ T cells in follicle areas around the germinal center. (L) CD68⁺ tingible body macrophages in the germinal center (arrows). (M) Ki-67⁺ dividing germinal center cells in serial sections with same area outlined in dotted line; (N) CD20⁺ B cells in same area. (O) TUNEL-stained apoptotic cells surrounding the Ki-67⁺, nonapoptotic germinal center B cells. (P) CD11c⁺ myeloid cells. (Q) Brown CD8⁺ T cells in close apposition to apoptotic and fragmented cells (arrows) in patient 017-9, 56 d after transmission and not on ART (100×).

Figure 5. Infiltration of NK cells in apoptotic and nonapoptotic areas of AHI terminal ileum Peyer's patches of patient 018-4.

(A and B) The same follicle with germinal center as in Figures 3A–3D; brown CD16⁺ cells (A) and CD56⁺ cells (B). These photomicrographs show normal accumulation of NK cells in subepithelial areas. (C and D) An apoptotic Peyer's patch area in patient 018-4 terminal ileum with brown CD16⁺ cells (C), and CD56⁺ cells (D). In all four panels arrows indicate positive cells (40×).

Figure 6. Changes in FDCs) in AHI.

(A–D) 20×; (E–H) 40×. (A and E) The fine reticular network of FDCs in an uninfected terminal ileum Peyer's patch (stained with p75 nerve growth factor receptor monoclonal antibody NGFR5). (B and F) Typical hyperplastic FDCs in patient 018-4, 74 d after transmission (stained with NGFR5 monoclonal antibody). (C) (stained with CD21 monoclonal antibody 1F8) and (G) (stained with CD35 monoclonal antibody BerMAC) show condensation and early loss of FDC in AHI terminal ileum 010-1, 52 d after transmission. (D) (stained with NGFR5 monoclonal antibody) and (H) (stained with CD35 monoclonal antibody BerMAC) show progressive FDC destruction in terminal ileum 025-7, 105 d after transmission.

Figure 7. Effect of HIV-1 on Terminal Ileum Immune Cells.

(A–B) Bars (A) and horizontal line (B) show mean values; error bars show standard error of the mean. (A) Quantitative image analysis of terminal ileum lamina propria immune cells in six uninfected participants, six AHI patients not on ART, and eight AHI patients on ART. CD3⁺ T cells were elevated in AHI (*p = 0.008, t-test) as were CD8+ T cells (^† p = 0.003, t-test). (B) Lamina propria κ/λ ratio skewing (p = 0.016, t-test). (C–H) (100×) Lamina propria of patient 025-7, 105 d after transmission, on ART 63 d, with predominant IgA, κ plasma cell expansion (κ/λ ratio = 2.53). (C) IgM, (D) IgG, (E) IgA, (F) Hematoxylin and Eosin stain, (G) κ light chains, and (H) λ light chains.

Figure 8. Flow cytometric analysis of B cell populations.

For all analyses, B cells were identified as CD3⁻ CD14⁻ CD16⁻ CD235a⁻ and CD19⁺. Horizontal lines show mean values; error bars show standard error of the mean. (A) Terminal ileal naïve B cell percentages. Naïve B cells were defined as B cells that were also surface IgD⁺ and CD27⁻. In cells from terminal ileal biopsies, uninfected participants had a mean±standard error of the mean 29.9%±7.0% of naïve B cells that were decreased to 15.7%±8.9% in AHI patients. (B) Terminal ileal memory and plasma cell percentages. Memory B and plasma cells were defined as B cells that were also surface IgD⁻ and CD27⁺, leaving out IgD⁺ IgM⁺ CD27⁺ B cells. In terminal ileum, uninfected participants had 17.1%±5.8% of memory B and plasma cells that were increased to 36.4%±6.9% in AHI patients (p = 0.047, t-test). (C) Peripheral blood naïve B cell percentages. Uninfected participants had 71.1%±2.9% of naïve B cells that were decreased to 49.7%±4.2% in AHI patients (p = 0.0001, t-test). (D) Peripheral blood IgD⁻ CD27⁺ memory and plasma cell percentages. Uninfected individuals had 9.4%±1.9% of memory B and plasma cells that were increased to 20.2%±2.6% in AHI patients (p = 0.0003, t-test). (E) Absolute numbers of circulating peripheral blood naïve B cells. Calculated as absolute naïve B cells per microliter of blood, uninfected participants had 169±23 cells/µl, while AHI patients had 148±48 cells/µl. (F) Absolute circulating memory B and plasma cells. Calculated as absolute number of cells per microliter of blood, uninfected individuals had 21.6±4.4 cells/µl, and 36.0±4.4 cells/µl in AHI participants (p = 0.03, t-test). All data in this figure were also analyzed using a two-tailed exact Wilcoxon test that resulted in similar p-values.

Figure 9. Total immunoglobulin (Ig) levels and specificities in the EBV-transformed B cell cultures.

Horizontal lines show mean values; error bars show standard error of the mean. Graph shows the levels of IgA, IgG, and IgM in the cell culture supernatants derived from terminal ileal samples of six acute/early HIV-infected and six uninfected participants determined by ELISA 14 d after EBV transformation. Each data point represents the geometric mean of IgA, IgG, and IgM levels in all wells plated from each terminal ileum sample.