Acquisition of direct antiviral effector functions by CMV-specific CD4+ T lymphocytes with cellular maturation

Abstract

The role of CD4+ T cells in the control of persistent viral infections beyond the provision of cognate help remains unclear. We used polychromatic flow cytometry to evaluate the production of the cytokines interferon (IFN)-gamma, tumor necrosis factor (TNF)-alpha, and interleukin (IL)-2, the chemokine macrophage inflammatory protein (MIP)-1beta, and surface mobilization of the degranulation marker CD107a by CD4+ T cells in response to stimulation with cytomegalovirus (CMV)-specific major histocompatibility complex class II peptide epitopes. Surface expression of CD45RO, CD27, and CD57 on responding cells was used to classify CD4+ T cell maturation. The functional profile of virus-specific CD4+ T cells in chronic CMV infection was unique compared with that observed in other viral infections. Salient features of this profile were: (a) the simultaneous production of MIP-1beta, TNF-alpha, and IFN-gamma in the absence of IL-2; and (b) direct cytolytic activity associated with surface mobilization of CD107a and intracellular expression of perforin and granzymes. This polyfunctional profile was associated with a terminally differentiated phenotype that was not characterized by a distinct clonotypic composition. Thus, mature CMV-specific CD4+ T cells exhibit distinct functional properties reminiscent of antiviral CD8+ T lymphocytes.

Figure 1.

Polyfunctional CMV-specific CD4⁺ T cell responses. (A) Representative plots from subject 7 showing functional profiles in response to cognate antigen (top) and background responses in the absence of cognate antigen (bottom). (B) Total frequency of individual peptide-specific CD4⁺ T cell functions. The frequencies of surface mobilization of CD107a and production of IFN-γ, IL-2, MIP-1β, and TNF-α are shown for subject 1 (◯), subject 2 (⋄), subject 3 (▪), subject 4 with peptide PPWQAGILARNLVPMV (×), subject 4 with peptide IIKPGKISHIMLDVA (+), subject 5 (♦), subject 6 (□), and subject 7 (•) in the total CD4⁺ population using box plots with the data for each individual superimposed. The line in the middle of the box represents the median, the top box represents the second quartile, and the box below represents the third quartile. (C) Frequency of normalized functional species in subjects shown in B. The mean percentage of the total CD4⁺ response for each of 31 functional species is shown ± SD.

Figure 2.

Mapping functional CMV-specific CD4⁺ T cell responses to maturational phenotype. (A) Individual responses for subject 1. The 31 possible response profiles are shown on the x axis, and the total frequency of each response profile is shown on the y axis. The dominant response profiles are color coded. (B) The maturational phenotypes of the CD4⁺ T cells expressing each of the dominant response profiles shown in A are overlaid on CD27 versus CD57 plots in which the phenotype of the total CD4⁺ T cell population is shown in gray. Responding cells are represented as 10% probability contour plots. (C) Functional responses for subject 1 (□), subject 2 (▪), subject 3 (⋄), subject 4 with peptide PPWQAGILARNLVPMV (◯), subject 4 with peptide IIKPGKISHIMLDVA (▵), subject 5 (▴), subject 6 (•), and subject 7 (♦) are shown with medians represented by a horizontal bar. To facilitate comparison between different CD4⁺ T cell subsets (CD45RO⁺CD27⁺ and CD45RO⁻CD27⁻; and CD45RO⁺CD27⁻CD57⁻ and CD45RO⁺CD27⁻CD57⁺), data were normalized based on the total frequency of responding cells within each maturational subset.

Figure 3.

Frequency of granzyme A, granzyme B, and perforin in CD57⁻ and CD57⁺ CD27⁻ memory CD4⁺ T cells. The frequency of granzyme A, granzyme B, and perforin in CD57⁻ and CD57⁺ CD27⁻ memory CD4⁺ T cells is shown using box plots, with individual data values superimposed for subject 1 (□), subject 2 (▪), subject 3 (⋄), subject 4 (◯), subject 5 (▴), subject 6 (•), and subject 7 (♦).

Figure 4.

Degranulation of epitope-specific CD4⁺ T cells. (A) Concurrent loss of granzyme A with surface mobilization of CD107a in response to peptide QEFFWDANDIYRIFA during a 5-h incubation (right) compared with the αCD28/49d control stimulation in the absence of peptide (left) from TCRVβ12⁺ CD4⁺ T cells from subject 1. (B) Concurrent loss of granzyme B with surface mobilization of CD107a in response to peptide QEFFWDANDIYRIFA during a 5-h incubation (right) compared with the αCD28/49d control stimulation in the absence of peptide (left) from TCRVβ12⁺ CD4⁺ T cells from subject 1.

Figure 5.

Killing of antigen-loaded autologous B-LCLs by epitope-specific CD4⁺ T cells. (A) Mapping of CD4⁺ T cells from subject 7 that degranulate in response to peptide QEFFWDANDIYRIFA (red dots) to the CD45RO⁺CD27⁻ CD4⁺ T cell compartment and the CD57⁺ compartment. (B) Mapping of perforin to the CD27⁻CD45RO⁺ and CD27⁻CD45RO⁻ compartment and the CD57⁺ compartments. (C) Loss of antigen-loaded B-LCLs during incubation with PBMCs. Killing of B-LCLs was determined by loss of antigen-loaded, dye-stained B-LCLs as a percentage of the total amount of CFSE-stained antigen-loaded and CMTMR-stained unloaded B-LCLs in incubations containing none (•) and 1E06 PBMCs (▪), 2E06 PBMCs (♦), and 4E06 PBMCs/ml (▴). Lines are the least square fits of the data forced to intersect the y axis at the average of the initial percentage of CFSE-stained B-LCLs for all four incubations. Rate of decrease per million PBMCs is shown in the inset. (D) Killing of peptide-loaded B-LCLs in incubations containing none (•), 3E06 PBMCs after the depletion of CD4⁺ T cells (▪), 3E06 PBMCs (▴), and 1E06 CD4⁺ enriched PBMCs (♦). CD4-depleted PBMC CD4⁺ T cells contained >15× less CD4⁺ T cells compared with CD8⁺ T cells than nondepleted PBMCs. CD4⁺-enriched PBMC CD4⁺ T cells represented >99% of CD3⁺ cells. Both PBMCs and B-LCLs are from subject 7. Lines are the least square fits of the data forced to intersect the y axis at the average of the initial percentage of CFSE-stained B-LCLs for all four incubations. (E) Production of IFN-γ by CD4⁺-enriched T cells after a 6-h incubation with 30,000 B-LCLs either without peptide or loaded with peptide QEFFWDANDIYRIFA. IFN-γ–producing CD3⁺ T cells are overlaid on a histograph showing CD4⁺ and CD8⁺ T cells. These assays were performed without costimulation and run in parallel with the killing experiments shown in C.

Figure 6.

Functional response patterns for CD4⁺ T cells with different viral specificities. PBMCs from (A) subjects with chronic CMV stimulated with overlapping pp65 peptides (n = 5); (B) HIV-infected long-term nonprogressors stimulated with overlapping Gag peptides (n = 11); and (C) smallpox vaccinees stimulated with whole vaccinia virus 1 mo after vaccination (n = 6). In each panel, the 31 possible response profiles are shown on the x axis, and the percentage of the total response is shown on the y axis. The mean percentage of the total CD4⁺ response for each of 31 functional species is shown ± SD.

Figure 7.

Clonotypic composition of peptide-responsive CD4⁺CD107a⁺CD154⁺ and CD4⁺CD154⁺ T cell populations. Functional responses to cognate antigen stimulation and sort gates are shown in the left panels for subject 7 (A), subject 1 (B), and subject 2 (C). The corresponding TCRBV usage, CDR3 amino acid sequence, and TCRBJ usage of the depicted CD4⁺ T cell populations are shown in the right panels with the percentage frequency of each clonotype and the total number of clones sequenced.