Anti-α4β7 therapy targets lymphoid aggregates in the gastrointestinal tract of HIV-1-infected individuals

Abstract

Gut homing CD4+ T cells expressing the integrin α4β7 are early viral targets and contribute to HIV-1 pathogenesis, likely by seeding the gastrointestinal (GI) tract with HIV. Although simianized anti-α4β7 monoclonal antibodies have shown promise in preventing or attenuating the disease course of simian immunodeficiency virus in nonhuman primate studies, the mechanisms of drug action remain elusive. We present a cohort of individuals with mild inflammatory bowel disease and concomitant HIV-1 infection receiving anti-α4β7 treatment. By sampling the immune inductive and effector sites of the GI tract, we have discovered that anti-α4β7 therapy led to a significant and unexpected attenuation of lymphoid aggregates, most notably in the terminal ileum. Given that lymphoid aggregates serve as important sanctuary sites for maintaining viral reservoirs, their attrition by anti-α4β7 therapy has important implications for HIV-1 therapeutics and eradication efforts and defines a rational basis for the use of anti-α4β7 therapy in HIV-1 infection.

Conflict of interest statement

Competing interests: S.M. and J.-F.C. have an unrestricted, investigator-initiated grant from Takeda Pharmaceuticals to examine novel homing mechanisms to the GI tract. All other authors declare that they have no competing interests.

Copyright © 2018 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.

Figures

Fig. 1.. Anti-α4β7 therapy decreases the frequency of B cell subsets in the TI.

(A to C) Frequency of B cells and plasma cells in the GI tract before and after VDZ therapy. (A) Representative flow cytometry plots showing the expression of CD19 and CD38 among live, CD45+ cells derived from the TI (top panels) and LC (bottom panels) of subject 583–017, before VDZ and at week 30 after VDZ. (B) Plots comparing the change in frequency of nonplasma cell (CD19+CD38−) B cells and plasma cells (CD45+CD38++CD27+) in the TI (top) and LC (bottom) between before and after VDZ treatment. (C) Plots demonstrating changes in the frequency of naïve (CD45+CD19+CD10−CD38−IgM+IgD+) and SM B cells (CD45+CD19+CD10−CD38−IgM−IgD−) in TI (top) and LC (bottom). (D) Change in the frequency of nonplasma cell B cells, B cell subsets, and plasma cells in the blood after VDZ therapy. In (A) to (D), each of the patients is represented with a unique color code. (E) Group comparisons in the frequency of B cell subsets and plasma cells within the TI and the LC. (F) Group comparisons in the frequency of B cell subsets and plasma cells in the peripheral blood. In (E) and (F), healthy volunteers (n = 11) are shown in red, HIV controls (n = 10) in blue, HIV-IBD subjects (n = 5) before VDZ in green, and HIV-IBD subjects after VDZ in gray. Two-tailed t test was performed to compare the different groups, and two-tailed paired t test was used to compare pre- and post-VDZ time points in the HIV-IBD patients. Statistical values are as indicated. *P < 0.05. NS, not significant.

Fig. 2.. Anti-α4β7 therapy results in a significant attenuation of lymphoid aggregates, most pronounced in the TI.

(A) Representative ×10 magnification images of TI-derived biopsies immunostained for CD20 expression (brown) in two subjects (583–013 and 583–024) before (top panels) and after (bottom panels) VDZ. (B) Quantitative analyses of CD20+ B cells in the TI (top) and LC (bottom). Cell frequency was determined separately in lymphoid aggregates (left) and in lamina propria (right). (C) Representative images from subject 583–017 showing dual immunohistochemical staining with CD19 (pink) and CD4 (brown) before (top) and after (bottom) VDZ. Original magnification, ×4 (left panel) and ×20 (right panel). (D) Percentage of tissue covered by lymphoid aggregates in the TI (top) and LC (bottom) in each of the subjects. (E) Representative immunofluorescence image showing the expression of CD3 (red), CD20 (green), and 4′,6-diamidino-2-phenylindole (blue) from the TI of subject 583–024, before (left panel) and after VDZ (right panel). Original magnification, ×10. (F) Cumulative data showing size of lymphoid aggregates between before and after VDZ treatment. In (B), (D), and (F), each of the patients is represented with a unique color code. Statistical values are as indicated. Wilcoxon matched-pairs signed-rank test and two-tailed t test (F) were used for statistical comparisons. Statistical values are as indicated. ***P < 0.0005.

Fig. 3.. Anti-α4β7 therapy results in a decrease in naïve CD4+ T cells in the TI.

(A and B) Frequency of T cells in the GI tract before and after VDZ therapy. (A) Frequency of total CD3+ T cells and (B) CD4/CD8 T cell ratio in the TI (top panels) and LC (bottom panels). Each of the patients is represented with a unique color code, and statistical values are indicated. (C and D) Group comparisons between the frequency of T cells (C) and CD4/CD8 ratio (D) in the GI tract (top panels) and peripheral blood (bottom panels). Healthy volunteers (n = 12) are shown in red, HIV alone controls (n = 10) in blue, HIV-IBD subjects before VDZ (n = 5) in green, and HIV-IBD subjects after VDZ (n = 5) in gray. (E and F) Frequency of naïve and memory CD4+ T cells in the GI tract before and after VDZ. (E) Representative flow cytometry plots showing the expression of CD45RA on CD45+CD3+CD4+ T cells derived from the TI (top panels) and LC (bottom panels) of subject 583–024 before VDZ and at week 26 after VDZ. (F) Cumulative data showing changes in CD45RA+ (left) and CD45RA−CD4+ T cell subsets (right) within the TI (top panels) and LC (bottom panels). Notably, CD45RA+ staining on CD4+ T cells was available on four of five patients. (G) Cumulative data showing changes in the frequency of circulating total CD3+ T cells, CD4/CD8 ratio, and total CD4+ T cells during VDZ therapy for each patient. In (F) and (G), each of the patients is represented with a unique color code, and statistical values are indicated. *P < 0.05, **P < 0.005. Two-tailed t test was performed to compare the different groups, and two-tailed paired t test was used to compare pre- and post-VDZ time points in the HIV-IBD patients.

Fig. 4.. Anti-α4β7 therapy is associated with alterations in the number and phenotype of β7+ cells in circulation.

(A and B) Frequency of β7hi memory T cell subsets in the peripheral blood before and after VDZ as measured by flow cytometry. (A) Representative flow cytometry plots from subject 583–017 comparing the expression of β7 integrin and CD45RA on circulating CD4+ T cells before VDZ and at week 30 after VDZ. Three distinct populations are defined: β7hiCD45RA−, β7−CD45RA−, and β7intCD45RA+. (B) Cumulative data showing changes in circulating β7hiCD45RA−CD4+ T cells during VDZ therapy for each patient. (C to E) CyTOF analyses to define alterations in the frequency of immune cell subsets after VDZ. (C) t-distributed stochastic neighbor embedding (tSNE) analyses showing the major T cell subsets including naïve, CM, EM, EMRA, Treg cells, and αE+ (CD103) cells. These populations were manually gated on the basis of the expression of canonical markers as shown in the heat map on the right. (D) β7 integrin expression on each of the immune populations defined in (C). The expression of CD29, CD38, and CD161 on each of the cellular subsets is shown by a heat map. (E) Frequency of the indicated cell populations by CyTOF for each of the patients at baseline, week 2, and week 30. (F and G) Flow cytometric evaluation of αE+ (CD103) cells after VDZ. (F) Representative flow cytometry plots from subject 583–017 comparing the expression of αE (CD103) and CD45RA on circulating CD4+ T cells before VDZ and at week 30 after VDZ. (G) Cumulative data showing changes in circulating αEβ7+CD45RA−CD4+ T cells during VDZ therapy for each patient. In (E) and (G), each of the patients is represented with a unique color code. Significance values are as indicated in the figure. Two-tailed t test was performed to compare the different groups, and two-tailed paired t test was used to compare pre- and post-VDZ time points in the HIV-IBD patients.

Fig. 5.. Anti-α4β7 therapy is associated with a decrease in activated CD4+ T cells in the TI.

(A and B) Frequency of activated T cells in the GI tract before and after VDZ. (A) Representative flow cytometry plots showing the expression of CD38 on live CD45+CD3+CD4+ T cells derived from TI (top) and LC (bottom) in subject 583–013 before VDZ and at week 26 after VDZ. (B) Cumulative data showing changes in the number of CD4+CD38+ T cells (left) and CD8+CD38+ T cells (right) in the TI (top panels) and LC (bottom panels) in each of the study subject after VDZ. (C) Frequency of CD4+CD38+ T cells (top) and of CD8+CD38+ T cells (bottom) in the peripheral blood throughout the study. (D) CyTOF analyses showing the median intensity of expression of CD38, compared for the indicated cell population on each of the patients at baseline, week 2, and week 30. In (B) to (D), each of the patients is represented with a unique color code. Significance values are as indicated in the figure. (E) Group comparisons between the frequency of CD4+CD38+ T cells (top left) and CD8+CD38+ cells (top right) in the GI tract and in the peripheral blood (bottom panels). Healthy volunteers (n = 12) are shown in red, HIV controls (n = 10) in blue, HIV-IBD subjects before VDZ (n = 5) in green, and HIV-IBD subjects after VDZ (n = 5) in gray. Two-tailed t test was performed to compare the different groups, and two-tailed paired t test was used to compare pre- and post-VDZ time points in the HIV-IBD patients.

Fig. 6.. Anti-α4β7 therapy results in the activation of circulating NK cell subsets.

(A) NK cell phenotype at weeks 0, 2, and 30 of therapy with VDZ. After exclusion of dead cells, monocytes, B cells, and T cells, cytolytic NK cells were gated as CD56dimCD16high NK cells, cytokine-secreting NK cells were gated as CD56brightCD16low NK cells, and CD56null cells were gated as CD56lowCD16high NK cells. (B to E) Composite graphs representing frequency of cytokine-producing (B), cytolytic (C), and CD56null NK cells (D), as well as CD56+CD8+ NK cells (E) from each of the five subjects (color-coded) at weeks 0, 2, and 30 of therapy with VDZ. (F and G) Change in the expression of HLA-DR (F) and PD-1 (G) on cytolytic CD56dimCD16high NK cells from five subjects (color-coded) at weeks 0, 2, and 30 of therapy with VDZ.

Fig. 7.. Impact of anti-α4β7 therapy on HIV-1 levels in the peripheral blood and in the GI tract.

(A) Estimated copies per million cells of total and integrated HIV DNA in sorted CD4+ T cells derived from PBMCs before and after VDZ. (B) Total and integrated HIV DNA in whole biopsies derived from the TI (top panels) and LC (bottom panels) before and after VDZ. DNA copy number was normalized per housekeeping gene (CD3) copy number. (C) HIV-1 long terminal repeat (LTR)–gag RNA in unstimulated bead-selected, circulating CD4 cells before VDZ and at week 30 after VDZ (left panel). In the right panel, frequency of cells with inducible multiply spliced RNA (msRNA; tat/rev), measured with the TILDA assay, is compared before and after VDZ. (D) Plots showing the linear regression line between various parameters in the TI mucosa. Left panel shows correlation between HIV total DNA within the TI and the percentage of tissue covered by lymphoid aggregates (LA). Middle panel shows the correlation between the frequency of CD8+CD38+ activated cells and the surface covered by lymphoid aggregates in the TI. The right panel represents the correlation between the magnitude of week 0 to week 30 changes in total HIV-1 DNA levels and the frequency of CD8+CD38+ cells. A positive number is representative of a decrease from week 0 to week 30. Correlation factors and P values were estimated with the Spearman correlation test. Two-tailed paired t test was used to compare before and after VDZ values. Statistical values are as indicated.