Autoreactive T Cells from Patients with Myasthenia Gravis Are Characterized by Elevated IL-17, IFN-γ, and GM-CSF and Diminished IL-10 Production

Abstract

Myasthenia gravis (MG) is a prototypical autoimmune disease that is among the few for which the target Ag and the pathogenic autoantibodies are clearly defined. The pathology of the disease is affected by autoantibodies directed toward the acetylcholine receptor (AChR). Mature, Ag-experienced B cells rely on the action of Th cells to produce these pathogenic Abs. The phenotype of the MG Ag-reactive T cell compartment is not well defined; thus, we sought to determine whether such cells exhibit both a proinflammatory and a pathogenic phenotype. A novel T cell library assay that affords multiparameter interrogation of rare Ag-reactive CD4(+) T cells was applied. Proliferation and cytokine production in response to both AChR and control Ags were measured from 3120 T cell libraries derived from 11 MG patients and paired healthy control subjects. The frequency of CCR6(+) memory T cells from MG patients proliferating in response to AChR-derived peptides was significantly higher than that of healthy control subjects. Production of both IFN-γ and IL-17, in response to AChR, was also restricted to the CCR6(+) memory T cell compartment in the MG cohort, indicating a proinflammatory phenotype. These T cells also included an elevated expression of GM-CSF and absence of IL-10 expression, indicating a proinflammatory and pathogenic phenotype. This component of the autoimmune response in MG is of particular importance when considering the durability of MG treatment strategies that eliminate B cells, because the autoreactive T cells could renew autoimmunity in the reconstituted B cell compartment with ensuing clinical manifestations.

Figure 1. Schematic representation of the amplified T cell library assay

Naïve, CCR6⁻ memory and CCR6⁺ memory CD4⁺ T cells from patients with MG or healthy controls (HC) were sorted, seeded into 96-well plates, and stimulated by PHA and IL-2 in the presence of irradiated allogeneic feeder cells for 2 weeks. After the stimulation cocktail was washed out, the cells from each well were re-stimulated by autologous monocytes and antigen-derived peptides. T cell proliferation (day 5) and cytokine production (day 7) were then measured by ³H-thymidine incorporation and ELISA respectively.

Figure 2. Sorting strategy for the isolation and analysis of CD4⁺ T cell subpopulations

The sorting (A) first involved enriching total CD4⁺ T cells from PBMCs by negative selection. Untouched CD4⁺ T cells were then stained with monoclonal antibodies against CD45RA, CD45RO, CD25 and CCR6 for T cell subpopulation isolation. The following subpopulations were gated and sorted: naïve (CD45RA⁺CD45RO⁻CD25⁻), CCR6⁻ memory (CD45RA⁻ CD45RO⁺CD25⁻CCR6⁻), and CCR6⁺ memory (CD45RA⁻CD45RO⁺CD25⁻CCR6⁺). (B) The frequency of each T cell subpopulation was calculated and is shown as a percentage of the total CD4⁺ T cells (n = 11). No statistically significance difference between the frequencies in the MG and healthy control (HC) cohorts was detected (unpaired t-test).

Figure 3. Cell proliferation of AChR-reactive, oligoclonal CD4⁺ T libraries from a patient with MG and a healthy control subject

(A) Naïve, (B), CCR6⁻ memory, and (C) CCR6⁺ memory CD4⁺ T cells were sorted and amplified by PHA and IL-2 in the presence of feeder cells for two weeks. The culture was then re-stimulated with irradiated autologous monocytes with or without AChR-P1 (195–212) and AChR-P2 (257–269). Cell proliferation was measured by ³H-thymidine incorporation on day 5. Each data point (288 total) represents a single oligoclonal library. There were 12 libraries for the no-peptide condition and 18 for each AChR peptide (AChR1 and AChR2). These conditions were repeated for each of the three T cell subsets and the MG and healthy control (HC) pair. This representative experiment shows subject MG-02 and the paired HC subject.

Figure 4. Phenotypic analysis of AChR-reactive, oligoclonal CD4⁺ T libraries from patients with MG and healthy control (HC) subjects

(A) Naïve, (B), CCR6⁻ memory, and (C) CCR6⁺ memory CD4⁺ T cells were sorted and amplified by PHA and IL-2 in the presence of feeder cells for two weeks. The culture was then re-stimulated with irradiated autologous monocytes with or without AChR-P1 (195–212), AChR-P2 (257–269), or S. aureus. Culture supernatants were collected and measured on day 7 for IFN-γ, IL-17, GM-CSF and IL-10 production by ELISA. Multiple oligoclonal libraries were prepared for the no-peptide, AChR peptides (AChR-P1 and AChR-P2) and positive control conditions. These conditions were repeated for each of the three T cell subsets and each MG and HC pair totaling 2880 oligoclonal libraries. Each data point represents one of the single oligoclonal libraries from the ten MG and healthy control pairs.

Figure 5. Consolidated phenotypic analysis of oligoclonal CD4⁺ T cell libraries from patients with MG and healthy control (HC) subjects

Heat map comparing functional responses of naïve, CCR6⁻ memory, and CCR6⁺ memory CD4⁺ T cells isolated from HC and MG patients following library generation by T cell amplification. The values show the average Z-score normalized response over multiple replicates (n = 10 replicates for CPM, IFN-γ, and IL-17; n = 7 replicates for GM-CSF and IL-10).

Figure 6. Single cell clonal analysis of AChR-reactive CD4⁺ memory T cells in a patient with MG and a healthy control subject

CCR6⁺ memory CD4⁺ T cells (240 oligoclonal libraries) from an MG patient and a healthy control were amplified by PHA and IL-2, then labeled with CFSE, and re-stimulated with autologous monocytes and AChR peptides. Proliferating (CFSE^low) cells were sorted into 96-well culture plates for single-cell clone generation. Single cell clones were re-stimulated with autologous monocytes and AChR peptides for 5 days. (A) ³H-thymidine was added 16 hours before harvest and the cell proliferation of each clone was measured. (B) Cytokine production from AChR-specific single cell clones was measured by intracellular staining on day 7. Golgi stop was added to the culture 4 hours before harvest. Then cells were fixed, permeabilized and stained with antibodies specific for IFN-γ and IL-17.