Neuroantigen-Specific CD4 Cells Expressing Interferon-γ (IFN-γ), Interleukin (IL)-2 and IL-3 in a Mutually Exclusive Manner Prevail in Experimental Allergic Encephalomyelitis (EAE).

Experimental allergic encephalomyelitis (EAE) is mediated by neuroantigen-specific pro-inflammatory T cells of the Th1 and Th17 effector class. Th-17 cells can be clearly defined by expression of IL-17, but not IFN-γ, IL-2 or IL-3. Th1 cells do not express IL-17, but it is unclear presently to what extent they co-express the cytokines canonically assigned to Th1 immunity (i.e., IFN-γ, IL-2 and IL-3) and whether CD4 cells producing these cytokines indeed belong to a single Th1 lineage. It is also unclear to what extent the Th1 response in EAE entails polyfunctional T cells that co-express IFN-γ and IL-2. Therefore, we dissected the Th1 cytokine signature of neuroantigen-specific CD4 cells studying at single cell resolution co-expression of IFN-γ, IL-2 and IL-3 using dual color cytokine ELISPOT analysis. Shortly after immunization, in the draining lymph nodes (dLN), the overall cytokine signature of the neuroantigen-specific CD4 cells was highly type 1-polarized, but IFN-γ, IL-2, and IL-3 were each secreted by different CD4 cells in a mutually exclusive manner. This single cell - single cytokine profile was stable through the course of chronic EAE-polyfunctional CD4 cells co-expressing IL-2 and IFN-γ presented less than 5% of the neuroantigen-specific T cells, even in the inflamed CNS itself. The neuroantigen-specific CD4 cells that expressed IFN-γ, IL-2 and IL-3 in a mutually exclusive manner exhibited similar functional avidities and kinetics of cytokine production, but showed different tissue distributions. These data suggest that Th1 cells do not belong to a single lineage, but different Th1 subpopulations jointly mediate Th1 immunity.

IL-2 absorption affects IFN-gamma and IL-5, but not IL-4 producing memory T cells in double color cytokine ELISPOT assays.

Cytokine assays are gaining increasing importance for human immune monitoring because they reliably detect antigen-specific T cells in primary PBMC, even at low clonal sizes. Double color ELISPOT assays permit the simultaneous visualization of cells producing two different cytokines. Permitting the simultaneous assessment of type 1 and 2 immunity and due to the limited numbers of PBMC available from human study subjects, double color assays should be particularly attractive for clinical trials. Since the performance of double color assays has not yet been validated, we set out to compare them to single color measurements. Testing the recall antigen-induced cytokine response of PBMC, we found that double color assays regularly provided lower numbers of IFN-gamma and IL-5 spots than single color measurements when IL-2 detection was part of the double color assay. We showed that the inhibitory effect resulted from IL-2 absorption and could be overcome by either antibody free preactivation cultures or by inclusion of anti-CD28 antibody. In contrast, the simultaneous detection of IL-2 did not affect the numbers of IL-4 spots. Therefore, unlike IL-2/IL-4 and IFN-gamma/IL-5 assays, IL-2/IFN-gamma, and IL-2/IL-5 assays require compensation for the IL-2 capture to provide accurate numbers for the frequencies of cytokine producing memory T cells.

Islet-cell antigen-reactive T cells show different expansion rates and Th1/Th2 differentiation in type 1 diabetic patients and healthy controls.

The low frequency of islet-cell antigen-reactive T cells in type 1 diabetes makes their direct measurement difficult. Commonly used in vitro expansion could alter in vivo frequencies and Th1/Th2 differentiation states. Using IFN-gamma/IL-4 double color ELISPOT, we tested longitudinally the reactivity of PBMC from HLA-matched diabetic patients and healthy controls to GAD65, IA-2, and proinsulin peptides ex vivo and after in vitro culture. The peptide-reactive T cells showed IFN-gamma bias in the patients' PBMC in the primary assay. During in vitro culture, both IFN-gamma- and IL-4-producing cells were induced in controls, suggesting that the precursor cells were uncommitted naive T cells in vivo. In contrast, in diabetic patients, the ex vivo IFN-gamma response was conserved during culture, suggesting their Th1 commitment. Using CFSE-dye-dilution, we demonstrate that naive T cells expand in vitro at a faster rate than memory cells, which might account for the differences in expansion rates between diabetic patients and controls.

Increased in vivo frequency of IA-2 peptide-reactive IFNgamma+/IL-4- T cells in type 1 diabetic subjects.

Active T cell recognition of islet antigens has been postulated as the pathogenic mechanism in human type 1 diabetes, but evidence is scarce. If T cells are engaged, they are expected to display increased clonal size and exhibit a T helper (Th)1/Th2 differentiation state. We used a peptide library that covers tyrosine phosphatase IA-2, a target antigen expressed in pancreatic beta cells, to probe 8 diabetic patients and 5 HLA-matched controls. When tested in a high resolution IFNgamma/IL-4 double color ELISPOT assay directly ex vivo, the number of IA-2-reactive IFNgamma producing cells was 17-fold higher in patients than in controls and IL-4 producing cells were not present. An average of 9 peptides was recognized in the patients vs. one in the controls. Determinant recognition primarily involved CD4+ cells and showed high variability among the patients. Furthermore, anti-CD28 antibody signal enhances quantitative assessment of effector T cells in T1D patients. In vitro expansion with peptides and IL-2 results in detection of responding cells in the controls and loss of disease specificity of the T cell response. Together these data provide strong evidence for the active targeting of IA-2 by Th1 memory effector cells in human type 1 diabetes.