Disruption of intestinal CD4+ T cell homeostasis is a key marker of systemic CD4+ T cell activation in HIV-infected individuals

Abstract

HIV infection is associated with depletion of intestinal CD4(+) T cells, resulting in mucosal immune dysfunction, microbial translocation, chronic immune activation, and progressive immunodeficiency. In this study, we examined HIV-infected individuals with active virus replication (n = 15), treated with antiretroviral therapy (n = 13), and healthy controls (n = 11) and conducted a comparative analysis of T cells derived from blood and four gastrointestinal (GI) sites (terminal ileum, right colon, left colon, and sigmoid colon). As expected, we found that HIV infection is associated with depletion of total CD4(+) T cells as well as CD4(+)CCR5(+) T cells in all GI sites, with higher levels of these cells found in ART-treated individuals than in those with active virus replication. While the levels of both CD4(+) and CD8(+) T cell proliferation were higher in the blood of untreated HIV-infected individuals, only CD4(+) T cell proliferation was significantly increased in the gut of the same patients. We also noted that the levels of CD4(+) T cells and the percentages of CD4(+)Ki67(+) proliferating T cells are inversely correlated in both blood and intestinal tissues, thus suggesting that CD4(+) T cell homeostasis is similarly affected by HIV infection in these distinct anatomic compartments. Importantly, the level of intestinal CD4(+) T cells (both total and Th17 cells) was inversely correlated with the percentage of circulating CD4(+)Ki67(+) T cells. Collectively, these data confirm that the GI tract is a key player in the immunopathogenesis of HIV infection, and they reveal a strong association between the destruction of intestinal CD4(+) T cell homeostasis in the gut and the level of systemic CD4(+) T cell activation.

FIGURE 1

Frequency and activation status of T cells in HIV uninfected individuals. A, The average frequency of CD4⁺ T cells (pregated on CD3) in the blood (PBMCs) and from four sites along the intestinal tract (TI, RC, LC, and SC) in HIV uninfected individuals. B, The average frequency of CD8⁺ T cells (pregated on CD3) in PBMCs and from the TI, RC, LC, and SC in HIV uninfected individuals. C (top panel), Representative flow cytometric plots showing the expression of CD45RO and CD27 in cells from blood and the intestines. T cells that express CD45RO and are either CD27⁺ or CD27⁻ are considered memory cells, while cells that are CD45RO⁻ and CD27⁺ are naive. The proportions of naive and memory CD4 and CD8 T cells in the blood and various intestinal sites in HIV uninfected individuals are shown (bottom panel). D, The frequency of CD4⁺ T cells expressing the nuclear Ag Ki67 in the blood and the intestines. E, The frequency of CD8⁺ T cells expressing the nuclear Ag Ki67 in the blood and the intestines. A one-way ANOVA with a Dunn multiple comparison posttest was used to determine p values.

FIGURE 2

HIV infection causes a depletion of CD4⁺ T cells. A, Representative flow plots showing the frequency of CD4⁺ and CD8⁺ T cells (pregated on CD3) in the blood (PBMCs) and from four sites along the intestinal tract (TI, RC, LC, and SC) in one HIV uninfected individual and two HIV-infected individuals, one of whom is on ART. B, Average frequency of CD4⁺ T cells from the blood and the intestines in HIV uninfected individuals (white), HIV infected individuals on ART (black), and HIV infected individuals that are not on therapy (red). The p values were determined by a two-way ANOVA with a Dunn multiple comparison posttest. C, Representative immunohistochemical staining for CD4 (brown, original magnification ×20) in the intestines of one HIV uninfected individual and two HIV infected individuals, one of whom is on ART. D, The frequency of CD4⁺ T cells at each intestinal site studied (TI, RC, LC and SC) is significantly correlated to the number of CD4⁺ T cells in the blood. A Spearman rank correlation was used to determine the R and p values.

FIGURE 3

CD4⁺ T cells expressing the coreceptor for HIV, CCR5, are significantly depleted from all intestinal sites. A, Representative flow cytometric plots showing CCR5 staining on CD4⁺ T cells in the blood and the RC. B, Average frequency of CD4⁺ T cells expressing CCR5 in the blood and from four sites along the intestinal tract (TI, RC, LC, and SC). HIV uninfected patients are shown in white, HIV-infected patients on ART are shown in black, and those not on therapy are shown in red. C, The frequency of memory CD4⁺ T cells expressing CCR5 in the blood and the intestines. D, The frequency of CD4⁺ T cells expressing CCR5 in the blood is inversely related to the viral load in the plasma. A Spearman rank correlation was used to determine R and p values.

FIGURE 4

HIV induces generalized T cell activation in the blood and all sites of the GI tract. A, Average frequency of CD4⁺ T cells expressing Ki67 in the blood and from four sites along the intestinal tract (TI, RC, LC, and SC). HIV uninfected patients are shown in white, HIV-infected patients on ART are shown in black, and those not on therapy are shown in red. B, Average frequency of CD8⁺ T cells expressing Ki67 in the blood and the intestines. A two-way ANOVA with the Dunn multiple comparison posttest was used to determine the p values. C, Representative immunohistochemical staining for Ki67 (brown, original magnification ×20) in intestinal tissues from an HIV uninfected individual and from two HIV-infected individuals, one of whom is on ART. D, The frequency of Ki67 expression on CD4⁺ T cells from the intestines correlates with the frequency of Ki67 expression on CD4⁺ T cells from the blood. E, The frequency of Ki67 expression on CD8⁺ T cells from the colon correlates with the frequency of Ki67 expression on CD8⁺ T cells from the blood. A Spearman rank correlation was used to determine the R and p values.

FIGURE 5

Systemic immune activation is a correlate of CD4⁺ T cell loss in the colon and the loss of IL-17–producing cells from the intestine. A, The frequency of CD4⁺ T cells in the colon is inversely related to the level of CD4⁺ T cells expressing Ki67 in the blood. B, The frequency of CD4 T cells expressing Ki67 in the blood is inversely related to the frequency of intestinal memory cells that express the cytokine IL-17.