Rhodococcus equi-specific cytotoxic T lymphocytes in immune horses and development in asymptomatic foals

Abstract

Rhodococcus equi is an important cause of pneumonia in young horses; however, adult horses are immune due to their ability to mount protective recall responses. In this study, the hypothesis that R. equi-specific cytotoxic T lymphocytes (CTL) are present in the lung of immune horses was tested. Bronchoalveolar lavage (BAL)-derived pulmonary T lymphocytes stimulated with R. equi lysed infected alveolar macrophages and peripheral blood adherent cells (PBAC). As with CTL obtained from the blood, killing of R. equi-infected targets by pulmonary effectors was not restricted by equine lymphocyte alloantigen-A (ELA-A; classical major histocompatibility complex class I), suggesting a novel or nonclassical method of antigen presentation. To determine whether or not CTL activity coincided with the age-associated susceptibility to rhodococcal pneumonia, CTL were evaluated in foals. R. equi-stimulated peripheral blood mononuclear cells (PBMC) from 3-week-old foals were unable to lyse either autologous perinatal or mismatched adult PBAC targets. The defect was not with the perinatal targets, as adult CTL effectors efficiently killed infected targets from 3-week-old foals. In contrast, significant CTL activity was present in three of five foals at 6 weeks of age, and significant specific lysis was induced by PBMC from all foals at 8 weeks of age. As with adults, lysis was ELA-A unrestricted. Two previously described monoclonal antibodies, BCD1b3 and CD1F2/1B12.1, were used to examine the expression of CD1, a nonclassical antigen-presenting molecule, on CTL targets. These antibodies cross-reacted with both foal and adult PBAC. However, neither antibody bound alveolar macrophages, suggesting that the R. equi-specific, major histocompatibility complex-unrestricted lysis is not restricted by a surface molecule identified by these antibodies.

FIG. 1.

PBMC from immune adult horses lyse both pulmonary- and blood-derived R. equi-infected targets. PBMC from H71 (left) and H68 (right) 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 (A) PBAC- or (B) BAL-derived targets. The asterisks indicate significant lysis compared to that of the uninfected control. Vertical bars represent the standard errors of the mean specific lysis of the replicate wells.

FIG. 2.

Pulmonary T lymphocytes stimulated with R. equi lyse both pulmonary- and blood-derived targets. Either PBMC or BAL effectors from H71 (left panels) and H68 (right panels) were stimulated in vitro for 5 days with virulent live R. equi and rested for 48 h (PBMC-RE and BAL-RE, respectively). PBMC effectors were also cultured for 7 days with no antigen (medium alone [PBMC-NA]). Effectors were then added, at an E:T ratio of 1:1, to autologous and ELA-A-mismatched infected and uninfected (A) PBAC- or (B) BAL-derived targets. The data presented is representative of two separate assays performed for each horse. The asterisk indicates significant lysis compared to that of the uninfected control. Vertical bars represent the standard error of the mean specific lysis of the replicate wells.

FIG. 3.

Stimulated PBMC effectors from adult horses, but not perinatal foals, lyse both adult horse and perinatal foal PBAC. PBMC effectors from (A) adult (H71) and (B) perinatal foals (A2197 and A2198) 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 adult and foal infected and uninfected PBAC targets. In addition, all foals were ELA-A mismatched compared to the adult control. The data presented are representative of assays involving two of the four foals. The asterisk indicates significant lysis compared to that of the uninfected control. Vertical bars represent the standard error of the mean specific lysis of the replicate wells.

FIG. 4.

Development of R. equi CTL activity in 3- to 8-week-old foals. PBMC effectors from (A) 3-week-old, (B) 6-week-old, and (C) 8-week-old foals were stimulated in vitro for 5 days with virulent live R. equi and rested for 48 h. In all experiments, PBMC effectors from H71 were simultaneously stimulated to serve as an internal positive control. Stimulated effectors were then added at increasing E:T ratios to autologous (foal) and ELA-A-mismatched (adult H71) infected and uninfected PBAC targets. Foals A2196 and A2197 were experimentally challenged with a small dose (10⁴ CFU) of virulent R. equi by intrabronchial inoculation at 6 weeks of age. The data presented are at an E:T ratio of 27:1 for all foals, except A2197 at the 3-week time point, for which the E:T ratio is 64:1. (C) The adult horse data (H71; E:T, 27:1) were representative of the 14 assays performed for this study using H71 (8-week-old foal A2196 served as the ELA-A mismatch and foal PBAC target cell donor in this example). The spontaneous lysis in all cases was <19%. The asterisk indicates significant lysis compared to that of the uninfected control. Vertical bars represent the standard error of the mean specific lysis of the replicate wells.

FIG. 5.

PBMC effector CD8⁺ T-lymphocyte populations in 6- and 8-week-old foals expand in vitro, similar to adult PBMC following R. equi stimulation. Prestimulation and after R. equi stimulation, PBMC populations from 3-, 6-, and 8-week-old foals were evaluated and compared for surface expression of CD8 and CD4 molecules. Four 3-week-old foals, five 6-week-old foals, and four 8-week-old foals were evaluated. The asterisk indicates significant difference at P < 0.05 compared to the prestimulation control (6-week-old foal CD8⁺, P = 0.0007; 8-week-old foal CD8⁺, P = 0.0014; 8-week-old foal CD4⁺, P = 0.0402). Vertical bars represent the standard deviation.

FIG. 6.

CD1 expression on equine PBAC. CD1 expression on target cells was assessed by flow cytometry using MAb BCD1b3 and MAb CD1F2. (A) PBAC cultures from four adult horses and six 6-week-old foals as well as BAL macrophage cultures from two adult horses were evaluated for expression of the CD1 markers as well as DH59B, a MAb to CD172a (monocyte specific in PBAC). An example of CD1 expression on the surface of 6-week-old foal A2196 PBAC is shown in panel B. The asterisk indicates a significant difference in CD1 expression compared to adult PBAC. Vertical bars represent the standard deviation.