Improved Protocol for Establishing CD4⁺ Hybridomas Specific for Human Class II MHC/Peptide Complex.

Area of Science:

• Immunology
• Genetics

Background:

• Autoreactive CD4+ T cells play a crucial role in autoimmune diseases.
• HLA-DQ8 is a significant genetic risk factor for type 1 diabetes and celiac disease.
• Understanding HLA-DQ8's influence on T-cell responses is vital for developing treatments.

Purpose of the Study:

• To investigate how HLA-DQ8 shapes the autoreactive CD4+ T-cell repertoire.
• To overcome limitations in existing mouse models for studying human HLA-DQ8-restricted T-cell responses.
• To develop an improved hybridoma system for accurate assessment of T-cell signaling.

Main Methods:

• Generated T-cell hybridomas from HLA-DQ8 humanized mice using a modified BW5147 Nur77-GFP (BW-GFP) platform.
• Engineered a BW-GFP fusion partner with optimized human CD4 (hCD4) to enhance CD4-HLA-DQ8 interactions.
• Compared responses of hybridomas with and without hCD4 to HLA-DQ8/peptide complexes.

Main Results:

• The modified BW-GFP platform with hCD4 demonstrated enhanced responses of CD4+ T cells to HLA-DQ8/peptide complexes.
• This improvement was observed in both regulatory (Treg) and conventional (non-Treg) CD4+ T cells.
• The engineered system showed improved reactivity and physiological accuracy in screening T-cell responses.

Conclusions:

• The optimized hCD4-BW-GFP system enhances CD4-HLA class II compatibility in HLA-DQ8 humanized hybridoma models.
• This improved system accurately reflects HLA-DQ8-restricted autoimmunity.
• The approach is adaptable for studying other HLA-transgenic mouse models and self/foreign antigens.