Acute loss of intestinal CD4+ T cells is not predictive of simian immunodeficiency virus virulence

Abstract

The predictive value of acute gut-associated lymphoid tissue (GALT) CD4+ T cell depletion in lentiviral infections was assessed by comparing three animal models illustrative of the outcomes of SIV infection: pathogenic infection (SIVsmm infection of rhesus macaques (Rh)), persistent nonprogressive infection (SIVagm infection of African green monkeys (AGM)), and transient, controlled infection (SIVagm infection of Rh). Massive acute depletion of GALT CD4+ T cells was a common feature of acute SIV infection in all three models. The outcome of this mucosal CD4+ T cell depletion, however, differed substantially between the three models: in SIVsmm-infected Rh, the acute GALT CD4+ T cell depletion was persistent and continued with disease progression; in SIVagm, intestinal CD4+ T cells were partially restored during chronic infection in the context of normal levels of apoptosis and immune activation and absence of damage to the mucosal immunologic barrier; in SIVagm-infected Rh, complete control of viral replication resulted in restoration of the mucosal barrier and immune restoration. Therefore, our data support a revised paradigm wherein severe GALT CD4+ T cell depletion during acute pathogenic HIV and SIV infections of humans and Rh is necessary but neither sufficient nor predictive of disease progression, with levels of immune activation, proliferation and apoptosis being key factors involved in determining progression to AIDS.

FIGURE 1

A comparison of viral dynamics during acute and chronic pathogenic SIVsmm infection of Rh and SIVagm infection of AGMs and Rh. a, No significant difference between the three models during acute infection was shown. Important differences in viral replication were observed during the chronic phase: in SIVsmm-infected Rh, plasma viral load showed significant increases with the progression to AIDS, starting from day 100 p.i. In SIVagm-infected AGMs, set-point viral loads were maintained during the follow-up. In SIVagm-infected Rh, plasma viral load became undetectable at day 72 p.i. SIVagm RNA dynamics in the intestine paralleled that of plasma viral load. b, Intestinal viral loads were maintained in SIVagm-infected AGMs, whereas in Rh, SIVagm RNA became undetectable by day 100 p.i. BAL viral loads quantified during chronic infection in AGMs were lower than those from intestine (insert). The detection limit of the assay was 10² copies/ml of plasma and 10² copies per 10⁵ cells. Plots represent the average viral loads from the animals in each study group. Vertical lines represent SD. ISH showed that SIVagm replication is lower in SIVagm-infected AGMs (c) compared with SIVagm-infected Rh (d), in agreement with real-time PCR quantification and with the lower number of target cells in AGMs (47).

FIGURE 2

Immunohistochemistry for CD4 confirmed the loss of CD4⁺ cells in the SIVagm-infected AGM LNs. Loss was demonstrated by the paucity of CD4⁺ T cells in LNs at day 7 p.i. (b), and day 14 p.i. (c), compared with either preinfection (a) or day 410 p.i. (d), when CD4⁺ T cells had recovered despite persistent viremia. SIVagm-infected Rh showed rapid depletion of LN CD4⁺ T cells during acute infection. Immunohistochemistry for CD4 confirms the loss of CD4⁺ cells in the LNs by demonstrating the paucity of CD4⁺ T cells at day 14 p.i. (f) and 28 p.i. (g), compared with either preinfection (e) or day 410 (h), when CD4⁺ T cells had recovered. Original magnification, ×50.

FIGURE 3

Longitudinal flow cytometric analysis of the percentage of intestinal CD4⁺ T cells in SIVagm-infected AGMs inoculated with SIVagm. A similar magnitude of CD4⁺ T cell depletion in the intestine (a) was shown as in pathogenic infection (f). Plots represent mean percentages of CD4⁺ T cells from animals in each group. Vertical lines with cross marks represent the SEM. Immunohistochemistry for CD4 confirms the loss of CD4⁺ cells in the gut by demonstrating the paucity of CD4⁺ T cells in lamina propria at day 7 p.i. (c), and day 28 p.i. (d), compared with either preinfection (b) or day 410 p.i. (e) when CD4⁺ T cells had recovered despite persistent viremia. Rh inoculated with SIVagm (red line) showed rapid and profound depletion of intestinal CD4⁺ T cells during acute infection (f) of the same order of magnitude as Rh infected with SIVsm (black line) (f). Controls inoculated with HIV-1 group N showed no change in CD4⁺ T cells (green line) (f). Vertical lines represent SEM. Immunohistochemistry for CD4 in macaques infected with SIVagm confirms the loss of CD4⁺ cells in the gut by demonstrating the paucity of CD4⁺ T cells in the lamina propria at day 7 p.i. (h) and 28 p.i. (i) compared with either preinfection (g) or day 410 (j), when CD4⁺ T cells recover. Original magnification, ×400.

FIGURE 4

Phenotyping of SIVagm-infected cells. Combined ISH (SIV) and immunohistochemistry staining (T lymphocytes-CD3 and macrophages-HAM56) at day 10 p.i. showed that SIVagm (red) replicated mainly in T lymphocytes (green) (a), with no macrophages (red-HAM56, immunohistochemical staining, blue-SIV, ISH) being infected at the peak viral production (b) in the intestine. c, All CD4⁺ T cell subsets, naive, central memory, and effector memory CD4⁺ T cells, were depleted in the intestine of SIVagm-infected AGMs, as determined by flow cytometry. The major depletion is observed in the memory CD4⁺ T cell compartment. Naive cells are defined as CD3⁺CD4⁺CD28⁺CD95⁻, central memory cells are defined as CD3⁺CD4⁺CD28⁺CD95⁺, and effector memory cells are defined as CD3⁺CD4⁺CD28⁻ CD95⁺. Combined ISH (SIV-red) and immunohistochemical staining (CD45RA-green) confirmed that the majority of infected cells in the lamina propria (d) and Peyer's patches (e) display a memory phenotype (lack of colocalization between the virus and CD45RA⁺ cells). Only a few naive cells are infected (double positive for SIV and CD45RA), shown by white arrow in e. The majority of SIVagm-infected cells (red) do not express Ki-67 (green) in lamina propria (f), similar to SIVmac-infected Rh (28). Few SIV⁺ cells coexpressed Ki-67 in the Peyer's patches (white arrow) (g). Original magnification: ×200 (a, top left); ×400 (a, top right); ×400 (a, bottom); ×400 (b, e, f, and g); and ×200 (d).

FIGURE 5

Mechanism of CD4⁺ T cell depletion in SIVagm infection. No significant contribution of apoptosis to intestinal CD4⁺ T cells was observed in SIVagm-infected AGMs (a) and transient increases in apoptosis occurred in SIVagm-infected Rh (b). a, Significant increase in necrotic cells, paralleling CD4⁺ T cell depletion suggests that the initial CD4⁺ T cell depletion in AGMs is caused by direct viral lysis. b, Apoptosis levels increased only in Rh and remained elevated even after viral clearance, paralleling the dynamics of T cell activation. Plots represent mean percentages of apoptotic and necrotic CD4⁺ T cells from the animals in each study group. Vertical lines represent the SEM. In AGMs, SIVagm replication in lamina propria and Peyer's patches (c) exceeded the amount of apoptotic cells (d). Infected cells were located in the interfollicular (IF) area (T cell zone), whereas apoptotic cells were more frequent in the germinal centers (GC) (B cell zone). Arrows point to apoptotic cells. Dual CD4 (green) and activated caspase 3 (red) staining showed nocolocalization of CD4⁺ and apoptotic cells in either lamina propria (e) or Peyer's patches (f).

FIGURE 6

Comparative dynamics of peripheral CD4⁺ and CD8⁺ T cell activation (DR). Unlike SIVagm infections in AGMs (b), pathogenic SIVsmm infection in Rh showed continuous increases in immune activation that correlated with progression to AIDS (a). The number represents the percentage of CD3⁺CD4⁺ and CD3⁺CD8⁺ T cells. c, SIVagm infection of Rh induced intermediate and transient levels of activation of both CD3⁺CD4⁺ and CD3⁺ CD8⁺ T cells. Plots represent the average expression for the animals in each study group. Vertical lines represent the SEM.

FIGURE 7

Comparative dynamics of peripheral CD3⁺CD4⁺ and CD3⁺ CD8⁺ T cell proliferation (Ki-67). Percentages are shown in SIVsmm-infected Rh (a), SIVagm-infected AGMs (b), and SIVagm-infected Rh (c). Unlike SIVagm infections in AGMs (b), pathogenic SIVsm infection in Rh showed sustained increases in cell proliferation that correlated with progression to AIDS (a). Data represent percentage of CD3⁺CD4⁺ and CD3⁺ CD8⁺ T cells. c, SIVagm infection of Rh induced intermediate and transient levels of cell proliferation of both CD3⁺CD4⁺ and CD3⁺CD8⁺ T cells. Plots represent the average expression for the animals in each study group. Vertical lines represent the SEM.

FIGURE 8

Longitudinal analysis of the plasma levels of LPS in SIVagm-infected models. Eight SIVagm-infected AGMs (a) and three SIVagm-infected Rh (b) are shown. No increase in the plasma LPS levels was observed in AGMs, with the exception of a blip in AGM EI42 following perforation of the small intestine during an endoscopic biopsy. Only transient increase was observed in SIVagm-infected Rh. Then, with the control of viral replication, the LPS levels returned to normal.