Rhodococcus equi-infected macrophages are recognized and killed by CD8+ T lymphocytes in a major histocompatibility complex class I-unrestricted fashion

Abstract

The goal of this research was to examine the role of cytotoxic T lymphocytes (CTL) in the control of Rhodococcus equi and specifically to determine if R. equi-specific CD8+ CTL occurred in the blood of immune horses. Equine peripheral blood mononuclear cells stimulated with antigen-presenting cells either infected with R. equi or exposed to soluble R. equi antigen lysed R. equi-infected target cells. Lysis was decreased to background by depletion of either CD2+ or CD3+ cells, indicating that the effector cell had a T-lymphocyte, but not NK cell, phenotype. Stimulation induced an increased percentage of CD8+ T cells in the effector population, and depletion of CD8+ T cells resulted in significantly decreased lysis of infected targets. Killing of R. equi-infected macrophages by effector cells was equally effective against autologous and equine leukocyte antigen A (classical major histocompatibility complex [MHC] class I) mismatched targets. To evaluate potential target antigens, target cells were infected with either virulent (80.6-kb plasmid-containing) or avirulent (plasmid-cured) R. equi. The degree of lysis was not altered by the presence of the plasmid, providing evidence that the virulence plasmid, which is required for survival within macrophages, was not necessary for recognition and killing of R. equi-infected cells. These data indicate that immunocompetent adult horses develop R. equi-specific CD8+ CTL, which may play a role in immunity to R. equi. The apparent lack of restriction via classical MHC class I molecules suggests a novel or nonclassical method of antigen processing and presentation, such as presentation by CD1 or other nonclassical MHC molecules.

FIG. 1.

Specific lysis of PBAC targets increased as the E:T ratio increased. PBMC from H71 were stimulated in vitro for 5 days with virulent live R. equi and rested for 48 h. Stimulated effectors were then added, at increasing E:T ratios, to autologous and ELA-A mismatched infected and uninfected target cells. Lysis of infected targets was compared to uninfected negative controls. Increasing the number of effectors per infected target increased the percent specific lysis. An asterisk indicates significant increase compared to the corresponding uninfected control at the same E:T ratio.

FIG. 2.

Classical MHC class I-unrestricted killing of R. equi-infected targets. PBMC from four horses were stimulated for 5 days with either (A) live virulent R. equi, (B) no antigen (medium alone), or (C) 10-μg/ml SRA. PBAC targets were infected with live virulent R. equi for 12 h or were uninfected. Four and a half hours prior to collection of the supernatants, effectors were added to targets at an E:T of 5:1 (A), 5:1 (B), or 20:1 (C). An asterisk indicates where lysis of infected targets is significantly increased (>3 standard errors compared to the corresponding uninfected control).

FIG. 3.

Depletion of CD2⁺ and CD3⁺ effectors resulted in decreased lysis of infected targets. PBMC from two horses were stimulated for 5 days ex vivo with live virulent R. equi and then rested for 48 h. Stimulated PBMC were then depleted of (A) CD2⁺ cells or (B) CD3⁺ cells and used as effectors. Autologous or ELA-A mismatched PBACs were infected for a total of 12 h with virulent R. equi 33701 and exposed to effectors for 4.5 h (E:T ratio of 10:1). Uninfected PBAC targets served as negative controls. Representative data are shown from H68. Similar results were obtained for H71. a, depletion results in a significant decrease in killing (>3 standard errors relative to the infected PBMC control). b, following depletion, lysis of infected targets was significantly different from the lysis of the uninfected controls in only one horse.

FIG. 4.

Depletion of CD8⁺ CTL decreases lysis of infected targets. PBMC from four horses were stimulated for 5 days with live virulent R. equi and then rested for 48 h. Stimulated PBMC were then depleted of CD4⁺ cells or CD8⁺ cells and used as effectors. (A) Autologous or (B) ELA-A mismatched PBACs infected with virulent R. equi 33701 (E:T ratio of 5:1). Uninfected PBAC targets served as negative controls. a, following CD8⁺ T-lymphocyte depletion of effectors, lysis significantly decreased (>3 standard errors) relative to the infected PBMC (undepleted) control.

FIG. 5.

Equivalent lysis of targets infected with either the plasmid-cured (avirulent) or the plasmid-bearing (virulent) strain. PBMC effectors were stimulated in vitro for 5 days with live virulent R. equi. (A) Autologous and (B) ELA-A mismatched targets were infected with either virulent or avirulent (PC) R. equi. Uninfected PBACs served as controls. Representative data from one animal (H44) are presented in the figure. Similar results were obtained from H81. ELA-A-unrestricted lysis of PBAC targets infected with either plasmid-bearing R. equi 33701 or the PC derivative were not significantly different. However, in all cases, the lysis of infected targets was significantly different from that of uninfected targets at the same E:T ratio.

FIG. 6.

Equivalent lysis of infected targets was mediated by lymphocytes stimulated with either the plasmid-bearing (virulent) or the plasmid-cured (avirulent) strain. PBMC effectors were stimulated with either the PL+ or the PC strain of R. equi. Representative data from horses H71 and H68 at an E:T ratio of 9:1 are presented in the figure. ELA-A-unrestricted lysis of PBAC targets infected with the plasmid-bearing strain (virulent) of R. equi 33701 was equivalent for either stimulated lymphocyte population. The asterisks indicate significant killing compared to the corresponding uninfected control.