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 Rhodococcus equi is a gram-positive, weakly acid-fast, facultative intracellular bacterium, closely related to Mycobacterium species (18). The cell wall structure and intracellular location during infection by these nocardioform actinomycetes are similar. Immunocompromised humans, notably AIDS patients, are susceptible to R. equi infection, which manifests as a severe pyogranulomatous pneumonia mimicking tuberculosis (7,29). R. equi is also an important cause of mortality in 2-to 6-monthold horses, where it produces a similar pyogranulomatous lesion in the lungs (43). As R. equi is ubiquitous in the equine environment, virtually all horses are exposed early in life. Most horses ultimately develop protective immune responses which mediate clearance of the bacteria from the lung and provide lifelong immunity (16,17).Immunity to R. equi has been most extensively studied in murine systems. As with Mycobacterium tuberculosis, gamma interferon (IFN-␥) secretion by CD4 ϩ Th1 lymphocytes appears to be critical for immune control (21, 23, 24). However, mice which lack major histocompatibility complex (MHC) class II-restricted CD4 ϩ T lymphocytes also significantly curtail the pathogen load following challenge (22). These experiments and others suggest that both CD4 ϩ and CD8 ϩ T lymphocytes are involved in the clearance of R. equi (34,39). In immune horses, studies of pulmonary recall responses have shown that clearance of a virulent pulmonary challenge is associated with antigen-specific proliferation by both CD4 ϩ T lymphocytes and CD8 ϩ T lymphocytes (16). Clearance of R. equi in immune horses also associates with increased numbers of IFN-␥-positive CD4 ϩ and IFN-␥-positive CD8 ϩ T lymphocytes at the site of pulmonary challenge (17). CD8 ϩ T lymphocytes clearly play a significant role in protective immune responses to M. tuberculosis (26,40). Both CD8 knockout mice and ␤ 2 -microglobulin knockout mice, which lack MHC class I-restricted CD8 ϩ T lymphocytes, are more susceptible to infection than syngeneic controls (35). The effect may reflect, in part, the contribution of CD8 ϩ T lymphocytes to IFN-␥ secretion and, as a result, macrophage activation. However, human and murine CD8 ϩ T-lymphocyte lines, such as Lyt-2 ϩ murine T lymphocytes, have also been shown to lyse M. tuberculosis-infected macrophages in an MHC class I-restricted and antigen-specific fashion, thus supporting the role of CD8 ϩ T-cell cytotoxicity in the control of M. tuberculosis infection (11). In addition, several MHC class I binding motifs have been characterized in M. tuberculosis proteins ...

Rhodococcus equi-Specific Cytotoxic T Lymphocytes in Immune Horses and Development in Asymptomatic Foals

“…Two healthy adult horses, one Quarterhorse (H71) and one Hanoverian (H68), presently maintained in the Washington State University (WSU) Veterinary Teaching Hospital herd, were selected based on mismatched ELA-A haplotypes and previous demonstration of CTL activity against R. equi-infected targets (32). For the experiments involving foals, six Arabian foals were selected based on age.…”

“…Pellets were consolidated and washed three times with complete medium containing decreasing amounts of antibiotic/ antimycotic (wash 1, 1 g of amphotericin/ml [Gibco]; wash 2, 2 U of penicillin/ ml, 2 U of streptomycin/ml, and 0.1 g of amphotericin/ml [Gibco]; wash 3, antibiotic/antimycotic free). Resultant cells were plated at 4 ϫ 10 6 cells/ml on 150-cm 2 collagen/serum-coated petri dishes and incubated for 1 h at 37°C with 5% CO 2 as previously described (32). Following incubation, nonadherent cells were collected by vigorous pipetting of the supernatant 10 times and washing again with PBS via pipetting 10 ml of PBS over the plate 10 times.…”

“…More recently, research has examined the role of CTL in the immune control of R. equi (32). Equine PBMC stimulated with antigen-presenting cells (APC) either infected with R. equi or exposed to soluble R. equi antigen (SRA) lysed R. equi-infected target cells but not uninfected targets.…”

Nonhealing Wound Due toRhodococcus equiin an Apparently Immunocompetent Patient, Revealing CD8+T-Lymphocyte Deficiency

“…CD4 ϩ T lymphocytes and CD8 ϩ T lymphocytes are the major cells involved in bacterial clearance in both murine and equine models (11,16,17), and CD8 ϩ T lymphocytes play a major role in immunity to R. equi (16,17). In our case, the CD8 ϩ T-lymphocyte deficit could partly explain the persistence of R. equi in the skin.…”

Cloning and large-scale expansion of epitope-specific equine cytotoxic T lymphocytes using an anti-equine CD3 monoclonal antibody and human recombinant IL-2

“…Cytotoxic T lymphocytes are involved in the control of several important intracellular pathogens of horses, including equine herpes virus-1 (1-7), equine arteritis virus (8), and Rhodococcus equi (9,10). In horses infected with equine infectious anemia virus (EIAV), a lentivirus with a world-wide distribution, CTL are critical in the control of viral replication and clinical disease (11)(12)(13)(14)(15)(16)(17)(18)(19)(20)(21)(22).…”