GAD-Alum immunization induces de novo priming and expansion of bi-functional/hybrid CD4 T cell responses

Antigen-specific therapy aims to modify inflammatory T cell responses in type 1 diabetes (T1D) and restore immune tolerance. One strategy employs GAD-Alum to induce a TH2 response to regulate pathological TH1 autoimmunity. We explored the cellular/molecular mechanism of GAD-Alum action in the setting of TrialNet’s Phase II efficacy study.

T1D patients were immunised with 20µg GAD-Alum (twice or thrice) or Alum alone. At day 91 post-immunization, we detected GAD-specific IL-13 responses in PBMCs of 73% of GAD-Alum immunised patients compared to 23% of those given Alum alone (p=0.0003) accompanied by high levels of IL-13, IL-4 and IL-5, confirming a GAD-specific, GAD-Alum induced TH2 response.

To characterise this response phenotype further, we generated GAD-specific CD4 T cell lines from selected patients. We noted bi-functionality of T cell lines, which displayed a hybrid TH1/Th2 phenotype. Single cell gene-expression analysis identified cells positive for both IL-13 and IFN-g production and the canonical TH2 and TH1 transcription factor genes (GATA3 and TBX21, respectively). GAD-Alum induced bi-functional/hybrid T cell responses were confirmed as only present after immunization using direct ex vivo dual (IL-13/ IFN-g) fluorospot analysis. To examine whether expanded bi-functional responses derive from activation of antigen-inexperienced, naïve CD4 T cells or immune-diversion of existing, non-hybrid CD4 T cell responses, we sequenced TCRBV CDR3 regions of GAD-specific hybrid T cells and searched our repository of TCR sequences  from circulating naïve, central memory, regulatory and stem cell-like memory CD4+ T cell subsets (>2 × 10⁸ total sequences from 14 T1D patients and 14 controls). GAD-specific CDR3s were predominant within the circulating naïve CD4 T cell pool, suggesting they are generated de novo by GAD-Alum immunization. In summary, GAD-Alum immunization activates and propagates GAD-specific naïve CD4 T cells with a distinctive bi-functional/hybrid phenotype, the functional analysis of which might be important in understanding clinical responsiveness.