Abstract
We report here the utility of major histocompatibility complex (MHC) class II dextramers for in situ detection of self-reactive CD4 T cells in two target organs, the brain and heart. We optimized the conditions for in situ detection of antigen-specific CD4 T cells using brain sections obtained from SJL mice immunized with myelin proteolipid protein (PLP) 139–151; the sections were costained with IAs/PLP 139–151 (specific) or Theiler's murine encephalomyelitis virus (TMEV) 70–86 (control) dextramers and anti-CD4. Analysis of sections by laser scanning confocal microscope revealed detection of cells positive for PLP 139–151 but not for TMEV 70–86 dextramers to be colocalized with CD4-expressing T cells, indicating that the staining was specific to PLP 139–151 dextramers. Further, we devised a method to reliably enumerate the frequencies of antigen-specific T cells by counting the number of dextramer+ CD4+ T cells in the ‘Z’ serial images acquired sequentially. We next extended these observations to detect cardiac myosin-specific T cells in autoimmune myocarditis induced in A/J mice by immunizing with cardiac myosin heavy chain-α (Myhc) 334–352. Heart sections prepared from immunized mice were costained with Myhc 334–352 (specific) or bovine ribonuclease 43–56 (control) dextramers together with anti-CD4; the sections showed the infiltrations of Myhc-specific CD4 T cells. The data suggest that MHC class II dextramers are useful tools for enumerating the frequencies of antigen-specific CD4 T cells in situ by direct staining without having to amplify the fluorescent signals, an approach commonly employed with conventional MHC tetramers.
Highlights
Limiting dilution analysis, enzyme-linked immunosorbent spot assay, intracellular cytokine staining, and cytokinesecretion assay have been used to enumerate frequencies of antigen-specific T cells [1–7]
Using three different autoimmune disease models 2 myelin proteolipid protein (PLP) 139-151-induced experimental autoimmune encephalomyelitis (EAE) in SJL mice; myelin oligodendrocyte glycoprotein (MOG) 35-55-induced EAE in C57Bl/6 mice; and cardiac myosin heavy chain-a (Myhc) 334-352-induced experimental autoimmune myocarditis (EAM) in A/J mice 2 we demonstrated that the major histocompatibility complex (MHC) class II dextramers were at least fivefold more sensitive than the tetramers, and their specificity was superior [19]
In this study, using PLP 139-151-induced EAE and Myhc 334-352-induced EAM models, we report that MHC class II dextramers can be successfully used to detect autoreactive CD4 T cells in situ with a high degree of specificity by direct staining without the need to amplify the signals with fluorophore antibodies, which is generally required with tetramers
Summary
Limiting dilution analysis, enzyme-linked immunosorbent spot assay, intracellular cytokine staining, and cytokinesecretion assay have been used to enumerate frequencies of antigen-specific T cells [1–7]. In this study, using PLP 139-151-induced EAE and Myhc 334-352-induced EAM models, we report that MHC class II dextramers can be successfully used to detect autoreactive CD4 T cells in situ with a high degree of specificity by direct staining without the need to amplify the signals with fluorophore antibodies, which is generally required with tetramers. The quantitative analysis was performed in each mouse using three sets of paired sections from the cerebrum or eight sets of paired sections from the heart; one section in each set was stained with specific or control dextramers together with anti-CD4, and the sections were examined by LSCM.
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