CD4+ T lymphocytes are the principal target of human immunodeficiency virus (HIV), but infected macrophages also contribute to viral pathogenesis. The killing of infected cells by CD8+ cytotoxic T lymphocytes (CTLs) leads to control of viral replication. Here we found that the killing of macrophages by CTLs was impaired relative to the killing of CD4+ T cells by CTLs, and this resulted in inefficient suppression of HIV. The killing of macrophages depended on caspase-3 and granzyme B, whereas the rapid killing of CD4+ T cells was caspase independent and did not require granzyme B. Moreover, the impaired killing of macrophages was associated with prolonged effector cell–target cell contact time and higher expression of interferon-γ by CTLs, which induced macrophage production of pro-inflammatory chemokines that recruited monocytes and T cells. Similar results were obtained when macrophages presented other viral antigens, suggestive of a general mechanism for macrophage persistence as antigen-presenting cells that enhance inflammation and adaptive immunity. Inefficient killing of macrophages by CTLs might contribute to chronic inflammation, a hallmark of chronic disease caused by HIV.
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We thank S. Buus (University of Copenhagen) for monomers; G. Mylvaganam, G. Gaiha, T. Diefenbach and A. Balazs for comments; J. Trapani for discussions about granzymes; A. Piechocka-Trocha for experimental help; and the Flow Cytometry and Sample Processing Cores at the Ragon Institute for help with instrumentation and processing of the samples. Supported by the Howard Hughes Medical Institute (D.R.C. and B.D.W.), the Ragon Institute of MGH, MIT and Harvard (B.D.W.), the Canadian Institute of Health Research (K.L.C.), the US National Institutes of Health (R01 AI118544 to B.D.W.), amfAR (109326-59-RGRL to K.L.C. and B.D.W.) and the Harvard University Center for AIDS Research (P30 AI060354 to B.D.W.), which is supported by the following institutes and centers co-funded by and participating with the US National Institutes of Health: NIAID, NCI, NICHD, NHLBI, NIDA, NIMH, NIA, FIC and OAR.
Integrated supplementary information
Schematic of the experimental process to coordinate maturation, activation, and infection of target cells with the creation of HIV-specific CTL lines and setup of ex vivo CTLs. Details are described in the Methods.
Following a 4-hour co-culture between CellTrace Violet-stained CTLs and either infected autologous CD4+ T cells or macrophages, cells were stained with antibodies for CD3 (T cell only cultures), CD14 (macrophage cultures) and CD4, with a LIVE/DEAD fixable stain, and an antibody for intracellular Gag p24. Flow cytometry analysis for all elimination assay samples was performed as shown in these plots.
Supplementary Figure 3 Ex vivo CTL elimination of human-serum differentiated macrophages, peptide-loaded targets, and resting CD4+ T cells related to Fig. 1.
(a) Human-serum derived macrophages are relatively resistant to CTL-mediated killing. Monocytes were isolated from PBMCs as described in the Materials and Methods and differentiated into macrophages using 10% human serum in RPMI-1640. CD4+ T cells were activated in parallel followed by HIV infection (strain JRSCF) of both cell types five days after activation/differentiation. Two days after infection, HIV-infected macrophages and CD4+ T cells were co-cultured with autologous CellTrace Violet-stained ex vivo CTLs for 4 hours at multiple effector to target (E:T) ratios and elimination of infected (HIV Gag-p24+) populations was assessed via flow cytometry. Shown is the summary of elimination assays from HIV-infected patients (n = 18 individual samples from six independent experiments). Elimination assays using CTLs from an HIV- donor was used as a control (dotted lines). Shown are the means +/- SEM. Statistical analysis, two-sided unpaired t-test, *p<0.0001. (b) Ex vivo CTL elimination of peptide-loaded targets. CellTrace Violet-stained ex vivo CTLs were co-cultured with 50% peptide-loaded CellTrace FarRed-stained CD4+ T cells or macrophages for 4 hours followed by flow cytometry analysis of live targets. Shown is the summary from HIV+ patients (n = 4 individual samples from one experiment). Shown are the means +/− SEM. Statistical analysis, two-sided unpaired t-test; *p = 0.001. (c) CellTrace Violet-stained expanded CTLs were co-cultured with 50% peptide-loaded CellTrace FarRed-stained resting (ex vivo) CD4+ T cells, activated CD4+ T cells, or macrophages for 4 hours followed by flow cytometry analysis of live targets by flow cytometry. Shown is the summary of CEF responses from HIV- donors for activated CD4+ T cells and macrophages (n = 6 individual samples from three independent experiments) and for resting CD4+ T cells (n = 3 distinct samples from one experiment). Shown are the means +/− SEM. Statistical analysis, two-sided unpaired t-test; * p = 0.0317, ** p = 0.0011.
CellTrace Violet-stained expanded CTLs were co-cultured with peptide-loaded or unloaded CellTrace FarRed-stained CD4+ T cells or macrophages for 15 min, 1 h, 4 h or 12 h followed by flow cytometry analysis of live targets. Shown is the summary from HIV+ patients (n = 8 individual samples from two independent experiments). Shown are the means +/− SEM. Statistical analysis, two-sided unpaired t test; *p = 0.0286, ** p = 0.0004, ***p<0.0001.
(a) FMO for tetramer staining of CD8+ T cells. (b) Perforin, granzyme A, granzyme B, granzyme H, granzyme K, and granzyme M staining of naïve CD8+ T cells as negative controls for staining. (c) Perforin and granzyme staining of CD8+ T cell, CMV-tetramer+ cells as a comparison for Fig. 4a. (d) Perforin and granzyme FMO staining controls for the antigen-specific CD8+ T cells in Fig. 4d. (e) Cytolytic capacity of HIV-specific CD8+ T cells responding to infected CD4+ T cells and macrophages. Ex vivo or expanded CTL effector cells were incubated with HIV-infected CD4+ T cells or macrophages at an E:T of 2 for 6 hours followed by flow cytometry-based analysis of degranulation (surface CD107a expression) and perforin and granzyme expression. Shown are the summaries of the perforin and granzyme phenotyping of CTLs responding to infected CD4+ T cells or macrophages for HIV+ patients (n = 8 individual samples for two independent experiments). Box elements, center line, limits and whiskers are the median, 25th-75th percentiles and min-max, respectively. Statistical analysis, two-sided paired t-test.
Supplementary Figure 6 ImageStream-based assessment of MHC-I surface density and control recognition assay related to Fig. 5.
(a) Representative image of CD4+ T cell and macrophage samples acquired on the ImageStream. CD4+ T cells and macrophages were stained for surface MHC-I (clone W6/32) and LIVE/DEAD Near-IR followed by fixing and staining for actin. (b) Representative dot plots of the gating strategy used to gate on live cells followed by assessment of cell size (diameter) versus MHC-I intensity. (c) HIV- donor recognition assay responses. Ex vivo CTL effector cells were incubated with mock or HIV-infected CD4+ T cells or macrophages at an E:T of 2 for 6 hours followed by flow cytometry-based analysis of degranulation (surface CD107a expression). Shown is a representative analysis of CTL effector cells degranulation responses from an HIV- donor.
Cells used for chemotaxis assays were phenotyped for chemokine receptor expression. CXCR3 is the receptor for CXCL9, CXCL10 and CXCL11. CCR2 is the receptor for CCL2. CCR5 is the receptor for MIP-1α, MIP-1β, and RANTES.