Preferential infection shortens the life span of human immunodeficiency virus-specific CD4+ T cells in vivo

Abstract

CD4(+) T-cell help is essential for effective immune responses to viruses. In human immunodeficiency virus (HIV) infection, CD4(+) T cells specific for HIV are infected by the virus at higher frequencies than other memory CD4(+) T cells. Here, we demonstrate that HIV-specific CD4(+) T cells are barely detectable in most infected individuals and that the corresponding CD4(+) T cells exhibit an immature phenotype compared to both cytomegalovirus (CMV)-specific CD4(+) T cells and other memory CD4(+) T cells. However, in two individuals, we observed a rare and diametrically opposed pattern in which HIV-specific CD4(+) T-cell populations of large magnitude exhibited a terminally differentiated immunophenotype; these cells were not preferentially infected in vivo. Clonotypic analysis revealed that the HIV-specific CD4(+) T cells from these individuals were cross-reactive with CMV. Thus, preferential infection can be circumvented in the presence of cross-reactive CD4(+) T cells driven to maturity by coinfecting viral antigens, and this physical proximity rather than activation status per se is an important determinant of preferential infection based on antigen specificity. These data demonstrate that preferential infection reduces the life span of HIV-specific CD4(+) T cells in vivo and thereby compromises the generation of effective immune responses to the virus itself; further, this central feature in the pathophysiology of HIV infection can be influenced by the cross-reactivity of responding CD4(+) T cells.

FIG. 1.

Low frequencies of CD4⁺ T cells respond to HIV antigenic stimulation. PBMC from the HIV-infected subjects in the cohort were stimulated with peptides 15 amino acids in length and overlapping by 11 amino acids and grouped according to antigen. Peptides encompassing HIV Gag, Env, Pol, and Nef were used as stimulants. PBMC from all individuals were also stimulated with CMV whole antigen. In addition, for six individuals, CMV responses after stimulation with overlapping peptides encompassing CMV pp65 and IE1 are included for completeness. Following stimulation for 5 h in the presence of brefeldin A, cells were extracellularly stained for CD4, CD3, and CD57 and were intracellularly stained for TNF, IL-2, and IFN-γ. The percentages of CD4⁺ T cells that responded with cytokine secretion are displayed. HIV-specific CD4⁺ T-cell responses represent summation of responses to HIV Gag, Nef, Env, and Pol. Filled symbols represent results for subjects 1 and 21. Horizontal bars represent medians.

FIG. 2.

CD4⁺ T cells that respond to HIV-specific stimulation do not reach terminal differentiation in vivo. PBMC from individuals in the cohort were stimulated with HIV peptides and/or CMV whole antigen. The percentage of responding antigen-specific CD4⁺ T cells that reached terminal differentiation was determined based upon expression of CD57. HIV-specific CD4⁺ T-cell responses represent summation of responses to HIV Gag, Nef, Env, and Pol. Filled symbols represent results for subjects 1 and 21. Horizontal bars represent medians.

FIG. 3.

Physically identified HIV-specific CD4⁺ T cells do not reach terminal differentiation in vivo. PBMC from subject 9 were surface stained with soluble HLA DR 15*01 tetramers bound to either irrelevant peptide or HIV p24 DRFYKTLRAEQASQ. PBMC were then washed and surface stained with anti-CD3, anti-CD4, anti-CD27, and anti-CD57. CD4⁺ CD3⁺ events are shown.

FIG. 4.

HIV-specific CD4⁺ T cells can be present at high frequencies and composed of terminally differentiated CD4⁺ T cells. PBMC from subjects 1 and 21 were stimulated with Gag peptides or CMV whole antigen. Following stimulation for 5 h in the presence of brefeldin A, cells were extracellularly stained with CD4, CD3, and CD57 and were intracellularly stained for TNF, IL-2, and IFN-γ. CD4⁺ CD3⁺ events are shown.

FIG. 5.

Clonotypic analysis of HIV- and CMV-specific CD4⁺ T cells from subject 1. PBMC from subject 1 were stimulated with Gag EQIGWMTNNPPIPVG or CMV whole antigen in the presence of a CD45/IFN-γ bispecific antibody. Following stimulation for 5 h, cells were extracellularly stained with CD4, CD3, CD57, and IFN-γ. IFN-γ-producing cells were sorted based upon CD57 expression into RNAlater followed by unbiased anchored template switch PCR to identify all expressed TCRB genes. freq, frequency.

FIG. 6.

Clonotypic analysis of HIV- and CMV-specific CD4⁺ T cells from subject 21. PBMC from subject 21 were stimulated with overlapping Gag peptides or CMV whole antigen in the presence of a CD45/IFN-γ bispecific antibody. Following stimulation for 5 h, cells were extracellularly stained with CD4, CD3, and IFN-γ. IFN-γ-producing cells were sorted into RNAlater followed by unbiased anchored template switch PCR to identify all expressed TCRB genes. freq, frequency.