Human CD8(+) T cells clear Cryptosporidium parvum from infected intestinal epithelial cells

Abstract

Intracellular protozoans of the genus Cryptosporidium are a major cause of diarrheal illness worldwide, especially in immunocompromised individuals. CD4(+) T cells and interferon-gamma are key factors in the control of cryptosporidiosis in human and murine models. Previous studies led us to hypothesize that CD8(+) T cells contribute to clearance of intestinal epithelial Cryptosporidium infection in humans. We report here that antigen expanded sensitized CD8(+) T cells reduce the parasite load in infected intestinal epithelial cell cultures and lyse infected intestinal epithelial cells. These effects are most likely mediated by the release of cytotoxic granules. Elimination of parasites seems to require antigen presentation through both human leukocyte antigen (HLA)-A and HLA-B. These data suggest that cytotoxic CD8(+) T cells play a role in clearing Cryptosporidium from the intestine, a previously unrecognized feature of the human immune response against this parasite.

Figure 1.

CD8⁺ cells from a sensitized donor clear Cryptosporidium parvum messenger RNA (mRNA) from infected cells. Antigen expanded sensitized CD8⁺ T cells reduced the amount of C. parvum mRNA in infected intestinal epithelial cell cultures. CD8⁺ T cells were isolated from a sensitized and a non-sensitized donor (HLA-A and HLA-B matched with the CaCo-2 cells) and used as effector cells in co-cultures with infected CaCo-2 target cells (effector:target ratio of 25:1) in complete RPMI supplemented with IL-7. After 24 hrs, cell pellets were harvested, and C. parvum RNA was quantified by real-time polymerase chain reaction (rtPCR). The experiment was repeated two times. The results of one representative experiment are shown. * P < 0.05

Figure 2.

Antigen expanded sensitized CD8⁺ T cells lyse intestinal epithelial cells from a culture infected with Cryptosporidium parvum. CD8⁺ T cells were isolated from sensitized Donor 1 and non-sensitized Donor 4 (HLA-A and HLA-B matched with CaCo-2 cells) and used as effector cells in co-cultures with ⁵¹Cr-labeled CaCo-2 target cells at various ratios of effector CD8⁺ T cells to CaCo-2 target cells (E:T ratio). Four hours after incubation, lysis of target cells was determined by ⁵¹Cr release. The experiment was repeated at least three times. The results of one representative experiment are shown. ▲ = expanded sensitized; • = non-expanded sensitized; Δ = expanded non-sensitized; ○ = non-expanded non-sensitized.

Figure 3.

Parasite clearance is blocked by antibody to HLA-A or HLA-B/C. CD8⁺ T cells were isolated from gp15 antigen expanded peripheral blood mononuclear cells (PBMCs) from sensitized Donor 1 (HLA-A and HLA-B matched with the CaCo-2 cells) and used as effector cells in co-cultures with C. parvum-infected CaCo-2 target cells at an effector:target ratio of 25:1 in complete RPMI supplemented with IL-7. Before co-culture, CaCo-2 cells were incubated with either anti-HLA-A or anti-HLA-B/C-antibody. After 24 hrs, cell pellets were harvested and C. parvum RNA was quantified by real-time polymerase chain reaction (rtPCR). * P < 0.05

Figure 4.

CD8⁺ lymphocytes bind infected epithelial cells. Isolated, gp15 expanded CD8⁺ T cells from sensitized donor 1 were co-cultured with Cryptosporidium parvum-infected CaCo-2 cells. Cells were analyzed with a Zeiss Axiovert 200M inverted microscope using a stage incubator. Cryptosporidium parvum sporozoites were stained yellow (CFSE), CaCo-2 cell membrane stained red (PKH-26), CD8⁺ T cell nuclei stained blue (Hoechst 33342), and CD8⁺ T cell cytolytic granules stained red (LysoTracker). Panel A: CD8⁺ T cells associated with infected target cells (1.5 hrs of co-culture). Panel B: three-dimensional reconstruction of effector cell releasing granules into an infected target cell (1.5 hrs of co-culture). This figure appears in color at www.ajtmh.org.

Figure 5.

Parasite clearance is blocked by Concanamycin A or antibody to Fas ligand. CD8⁺ T cells were isolated from gp15 antigen expanded peripheral blood mononuclear cells (PBMCs) from sensitized Donor 1 and used as effector cells in co-cultures with Cryptosporidium parvum-infected CaCo-2 target cells at an effector:target ratio of 25:1. Before co-culture CD8⁺ T cells were incubated with either Concanamycin A or antibody to Fas ligand. After 24 hrs of co-culture in complete RPMI supplemented with IL-7 cell pellets were harvested and C. parvum RNA was quantified by real-time polymerase chain reaction (rtPCR). * P < 0.05