Testing the NKT cell hypothesis of human IDDM pathogenesis

Abstract

Defects in IL-4-producing CD1d-autoreactive NKT cells have been implicated in numerous Th1-mediated autoimmune diseases, including diabetes, multiple sclerosis, rheumatoid arthritis, lupus, and systemic sclerosis. Particular attention has been focused on autoimmune insulin-dependent diabetes mellitus (IDDM) because nonobese diabetic (NOD) mice and humans with IDDM are both reported to express severe deficiencies in the frequency and Th2 functions of NKT cells. Furthermore, experimental manipulations of the NKT defect in the NOD mouse induced corresponding changes in disease. Taken together, these converging studies suggested a general role of NKT cells in natural protection against destructive autoimmunity. However, in previous reports the identification of NKT cells was based on indirect methods. We have now devised a direct, highly specific CD1d tetramer-based methodology to test whether humans with IDDM have associated NKT cell defects. Surprisingly, although we find marked and stable differences in NKT cells between individuals, our study of IDDM patients and healthy controls, including discordant twin pairs, demonstrates that NKT cell frequency and IL-4 production are conserved during the course of IDDM. These results contradict previous conclusions and refute the hypothesis that NKT cell defects underlie most autoimmune diseases.

Figure 1

Specific identification of human NKT cells in PBLs. (a) NKT cells in PBLs were identified by double staining with CD1d-αGalCer tetramers followed by anti-Vα24 mAb (left panels). The CD4 distribution and Vβ11 usage among these cells were identified in the same staining (right panels). (b) Vα24/Vβ11 double-positive PBLs (left panels) were stained with CD1d-αGalCer tetramers (right panels). Numbers above the boxes represent percentages of cells among total PBLs, whereas numbers in the quadrants represent percentages among boxed cells. Representative FACS plots of donors expressing high or low frequency of NKT cells are shown. (c) Double staining with CD1d-αGalCer and empty CD1d (left panels) identified the same percentage of NKT cells as double staining with CD1d-αGalCer and Vα24 (right panels).

Figure 2

Stability of NKT cell frequency in PBLs. Vα24⁺ CD1d-αGC⁺ NKT cell frequencies were repeatedly measured over a period of 10 months in seven individual donors. Asterisks denote concomitant seasonal rhinovirus or influenza-like infection.

Figure 3

Conserved expression of NKT cells in individuals with IDDM. (a) Summary plot showing individual results in controls, subdivided in age- and DQ-matched individuals (cohort 1) and healthy volunteers (cohort 2), at-risk individuals (separated into low [L], medium [M], and high [H] risk), and IDDM new onset (NO) and long-standing (LS) individuals. (b) Representative FACS plots of healthy or IDDM individuals expressing high or low frequency of NKT cells in PBLs. (c) Summary plot of the relative proportion of CD4⁺ cells among Vα24 NKT cells in control, at-risk, and diabetic individuals.

Figure 4

Frequency of NKT cells in twins with discordant IDDM status. Healthy twins did not show evidence for circulating anti-islet Ab’s or altered intravenous glucose tolerance test. One of three twin pairs expressed DQ8, as shown.

Figure 5

Cytokine production by fresh NKT cells. Summary plots show frequencies of IL-4 (left panels) and IFN-γ (right panels) producers among CD4 NKT cells (upper panels) and DN NKT cells (middle panels) in control, at-risk, and IDDM individuals. Representative FACS staining of IL-4 and IFN-γ are shown in control and IDDM individuals (bottom panels).

Figure 6

Frequency of different populations of innate-like lymphocytes in PBLs. The γδ T cells expressing (a) Vγ9 or (b) Vδ1 and (c) NK cells were measured in control, at-risk, and IDDM individuals. Mean ± SD in each group were compared using Student t test.