Severe CD4+ T-cell depletion in gut lymphoid tissue during primary human immunodeficiency virus type 1 infection and substantial delay in restoration following highly active antiretroviral therapy

Abstract

Gut-associated lymphoid tissue (GALT) harbors the majority of T lymphocytes in the body and is an important target for human immunodeficiency virus type 1 (HIV-1). We analyzed longitudinal jejunal biopsy samples from HIV-1-infected patients, during both primary and chronic stages of HIV-1 infection, prior to and following the initiation of highly active antiretroviral therapy (HAART) to determine the onset of CD4(+) T-cell depletion and the effect of HAART on the restoration of CD4(+) T cells in GALT. Severe depletion of intestinal CD4(+) T cells occurred during primary HIV-1 infection. Our results showed that the restoration of intestinal CD4(+) T cells following HAART in chronically HIV-1-infected patients was substantially delayed and incomplete. In contrast, initiation of HAART during early stages of infection resulted in near-complete restoration of intestinal CD4(+) T cells, despite the delay in comparison to peripheral blood CD4(+) T-cell recovery. DNA microarray analysis of gene expression profiles and flow-cytometric analysis of lymphocyte homing and cell proliferation markers demonstrated that cell trafficking to GALT and not local proliferation contributed to CD4(+) T-cell restoration. Evaluation of jejunal biopsy samples from long-term HIV-1-infected nonprogressors showed maintenance of normal CD4(+) T-cell levels in both GALT and peripheral blood. Our results demonstrate that near-complete restoration of mucosal immune system can be achieved by initiating HAART early in HIV-1 infection. Monitoring of the restoration and/or maintenance of CD4(+) T cells in GALT provides a more accurate assessment of the efficacy of antiviral host immune responses as well as HAART.

FIG. 1.

(A) Onset of severe depletion of CD4⁺ T-cell subsets in GALT occurs in primary HIV-1 infection. Prevalence of CD4⁺, CD8⁺, and CD4⁺CD8⁺ T-cell subsets in gut T lymphocytes and peripheral blood mononuclear cells (PBMC) of two HAART-naïve individuals (patients 134 and 116) during primary HIV-1 infection was analyzed by flow cytometry and compared with that in uninfected healthy controls. (B) Increased proliferation of intestinal CD4⁺ T cells in response to primary HIV-1 infection. Flow-cytometric analysis was performed to determine Ki67 expression in CD4⁺ T cells in peripheral blood and gut T lymphocytes 4 weeks after a suspected high-risk exposure to HIV-1 infection (patient 134), in comparison to a healthy uninfected control.

FIG. 2.

Alterations in CD4⁺ and CD8⁺ T-cell subsets in GALT of HIV-1-infected individuals with diverse clinical outcomes. Flow-cytometric analysis was performed to determine changes in percentages of CD3-gated CD4⁺, CD4⁺CD8⁺, and CD8⁺ T cells in gut T lymphocytes and peripheral blood mononuclear cells (PBMC) from HAART-naïve chronically HIV-1-infected individuals (n = 11), LTNPs (n = 3), and uninfected healthy controls (n = 6). Absolute CD4⁺ T-cell numbers, viral loads, and length of infection at the time of sample collection are presented in Table 1.

FIG. 3.

Delayed and incomplete restoration of CD4⁺ T-cell subsets in GALT compared to peripheral blood in chronically HIV-1-infected patients receiving HAART. (A) Longitudinal analysis of viral loads and absolute CD4⁺ T-cell numbers in peripheral blood of HIV-1-infected patients (n = 4) prior to and following HAART. (B) Flow-cytometric analysis was performed to determine alterations in percentages of CD3-gated CD4⁺, CD8⁺, and CD4⁺CD8⁺ T cells in longitudinal jejunal biopsy samples from HIV-1-infected patients prior to and following HAART.

FIG. 4.

Flow-cytometric and immunohistochemical analysis of CD4⁺ T-cell depletion in GALT of HIV-1-infected individuals. (A) Representative dot plots of CD3-gated CD4⁺ and CD8⁺ T cells in peripheral blood and GALT; (B) immunohistochemical detection of CD4⁺ cells of an HIV-negative healthy control, an LTNP, a HAART-naïve chronically HIV-1-infected patient, and an HIV-1-infected patient who had received HAART for 5 years.

FIG. 5.

Initiation of HAART during primary HIV-1 infection results in near-complete restoration of CD4⁺ T cells in GALT. (A) Time line, plasma viral loads, and absolute CD4⁺ T-cell numbers in peripheral blood of patient 116 during early HIV-1 infection and following the initiation of HAART. ▪, viral RNA copies; •, CD4⁺ cells. (B) Alterations in percentages of CD4⁺, CD4⁺CD8⁺, and CD8⁺ T cells in longitudinal jejunal biopsy samples and peripheral blood samples of patient 116, prior to and following HAART. −, HIV-1 seronegative; +, plasma HIV-1 RNA positive.

FIG. 6.

Increased expression of lymphocyte homing marker on repopulating CD4⁺ T cells in GALT following HAART. Flow-cytometric analysis of integrin β₇ expression was performed on CD4⁺ T cells in longitudinal jejunal and peripheral blood samples of three healthy uninfected controls and three HIV-1-infected patients following HAART.

FIG. 7.

DNA microarray analysis of gene expression profiles in longitudinal jejunal biopsy samples of HIV-1-infected patients prior to and following HAART. Red indicates increased gene expression compared to five HIV-1-negative controls, green indicates decreased gene expression, and grey indicates no change in the level of gene expression.