Initial increase in blood CD4(+) lymphocytes after HIV antiretroviral therapy reflects redistribution from lymphoid tissues

Abstract

Previous studies proposed a dynamic, steady-state relationship between HIV-mediated cell killing and T-cell proliferation, whereby highly active antiretroviral therapy (HAART) blocks viral replication and tips the balance toward CD4(+) cell repopulation. In this report, we have analyzed blood and lymph node tissues obtained concurrently from HIV-infected patients before and after initiation of HAART. Activated T cells were significantly more frequent in lymph node tissue compared with blood at both time points. Ten weeks after HAART, the absolute number of lymphocytes per excised lymph node decreased, whereas the number of lymphocytes in the blood tended to increase. The relative proportions of lymphoid subsets were not significantly changed in tissue or blood by HAART. The expression levels of mRNA for several proinflammatory cytokines (IFN-gamma, IL-1beta, IL-6, and macrophage inflammatory protein-1alpha) were lower after HAART. After therapy, the expression of VCAM-1 and ICAM-1 -- adhesion molecules known to mediate lymphocyte sequestration in lymphoid tissue -- was also dramatically reduced. These data provide evidence suggesting that initial increases in blood CD4(+) cell counts on HAART are due to redistribution and that this redistribution is mediated by resolution of the immune activation that had sequestered T cells within lymphoid tissues.

Figures

Figure 1

Absolute numbers of lymphocytes in blood and a single excision lymph node biopsy from level 3 or 4 in the posterior cervical chain before induction of HAART and 10 weeks after HAART in 7 subjects. Blood and lymph node biopsies were obtained on the same day. Different symbols represent individual subjects. Biopsies after HAART were taken from the opposite side of the neck in the same anatomical location as the prior biopsy. The decrease in number of lymphocytes per lymph node is significant (P < 0.05) by paired t test (median: 121 × 106 cells before HAART and 14.4 × 106 cells after HAART). The gross size of the measured lymph node specimens was also significantly decreased (P < 0.05), as determined by paired t test (median: 1.3 cm3 before HAART and 0.5 cm3 after HAART). The increase in lymphocytes in the blood does not reach formal statistical significance (P = 0.11; median: 989 cells/μL before HAART and 1,480 cells/μL after HAART). Note that subject 2 (filled circle; see Table 2), with the highest lymphocyte count and lowest viral load before HAART, showed decreased blood T cells but increased cells in lymph node. This subject confounds the general trend of increased cells in blood but is consistent with an inverse relationship between blood and tissue cells.

Figure 2

Comparison of relative frequency of total T cells and the CD4 and CD8 subsets of T cells by flow cytometry analysis in paired specimens of blood and lymph node obtained the same day before induction of HAART and 10 weeks after HAART in 7 subjects. The fraction of total T cells and CD8+ T cells among the total lymphocytes was significantly lower in lymph node than in blood (**P < 0.01; paired t test) both before and after HAART. There were no significant differences in the fraction of any subset of cells comparing the before/after HAART analysis within the same compartment.

Figure 3

Comparison of relative frequency (top) and absolute number (bottom) of lymphoid subsets in either lymph node or blood. The dark bars are data from baseline (before HAART), and the light bars are data from 10 weeks after HAART induction. The fall in lymphocytes in the lymph node biopsy (Figure 1) results in a calculated decrease in total T cells, CD8+ T cells, and B cells in the lymph node (*P < 0.05), although the relative fraction of these cells is not significantly changed by HAART (top). Although the increase in blood does not achieve formal statistical significance, a single subject with high initial lymphocyte count that fell after therapy confounds the trend of increased cells in blood.

Figure 4

Comparison of relative frequency of multiple T-cell subsets by three-color flow cytometry analysis in paired specimens of blood and lymph node before and after induction of HAART. In each set of analyses, the first set of bars represents the fraction cells positive for the first marker, and the second set represents the fraction cells positive for the second marker. The third set of bars (+/+) represents the fraction of cells that coexpress both markers. Top: CD4 T cells; bottom: CD8 T cells; left: data before HAART; right: results from 10 weeks after HAART. Differences between lymph node and blood for the same subset on the same day with statistical significance by a paired t test are indicated by asterisks: *P < 0.05, **P < 0.01. There were no statistically significant differences among these 7 subjects, comparing the data for any subset before versus after HAART.

Figure 5

Expression levels of various inflammatory cytokines determined by quantitative competitive RT-PCR in the lymph node biopsies of these 7 subjects before and after induction with HAART. Reduced expression of IFN-γ, IL-1β, IL-6, and MIP-1α is statistically significant (*P < 0.05) by a paired t test. The expression levels are normalized to the number of copies of each cytokine mRNA per 105 mRNA copies of the constitutive intracellular enzyme G3PDH, which corresponds to ∼103 lymphocytes. The ratio of G3PDH mRNA per volume of lymph node tissue analyzed did not change after HAART (data not shown).

Figure 6

Representative photomicrographs demonstrating immunohistochemical staining of lymph node sections before (left) and after (right) HAART with ICAM-1 (top) and VCAM-1 (bottom). Expression of these markers was decreased in each pair of biopsies examined. These sections represent analysis performed on the same day with the same staining conditions for biopsies obtained from an individual patient.