HLA Typing Service

Leveraging Full-Resolution HLA-Genotyping from Creative Biolabs in GAD65 Limbic Encephalitis Research

Background

GAD65-LE is a chronic, progressive autoimmune condition characterized by limbic encephalitis, typically presenting with mesial temporal lobe seizures and memory disturbances. While autoantibodies against GAD65 are a hallmark of this condition, the intracellular location of GAD65 has led researchers to hypothesize a pathogenic role for T cells. This study aimed to explore the involvement of T cells in GAD65-LE and to identify specific HLA alleles that might contribute to the presentation of GAD65 peptides to T cells.

Methodology

The study included ten patients with long-standing GAD65-LE. The following methodologies were employed:

1. Clinical Assessment: Comprehensive clinical, neuropsychological, and imaging assessments were conducted.
2. Flow Cytometry: Peripheral blood (PB) and cerebrospinal fluid (CSF) samples were analyzed using multi-parameter flow cytometry to assess T cell activation.
3. Histopathological Analysis:Selective hippocampectomy specimens from two patients were examined for T cell infiltrates and neuronal damage.
4. HLA-Genotyping: Genomic DNA was extracted from PB samples, and next-generation sequencing-based HLA-genotyping was performed by Creative Biolabs.

Results

The study found significant findings that advance the understanding of GAD65-LE:

1. T Cell Activation: Flow cytometry of PB and CSF samples demonstrated increased fractions of activated HLADR+ CD4+ T cells (PB: p = 0.0003, CSF: p < 0.0001) and activated HLADR+ CD8+ T cells (PB: p = 0.0046, CSF: p = 0.0027) in both compartments compared to controls. This indicated elevated T cell activation in patients with GAD65-LE.
2. Correlation with Neurodegeneration: Intrathecal fractions of CD8+ T cells negatively correlated with hippocampal volume (r = -0.782, p = 0.008) and memory function (r = -0.818, p = 0.004), while intrathecal fractions of CD4+ T cells showed positive correlations with hippocampal volume (r = 0.782, p = 0.008) and memory function (r = 0.782, p = 0.008). This suggests that CD8+ T cells have a pathogenic role, whereas CD4+ T cells may have a regulatory role.
3. Neuronal Damage: Histopathological analysis of hippocampectomy specimens from two patients revealed loss of neurons in the hippocampus, with strong parenchymal T cell infiltrates. CD8+ T cells were found in close proximity to small interneurons, suggesting an antigen-specific attack on inhibitory neuronal networks.
4. Cytotoxic Effector Molecules: Peripheral and parenchymal CD8+ T cells expressed cytotoxic effector molecules, such as perforin and granzyme B. This was observed in the antigen-experienced memory CD8+ T cell population but not in naïve CD8+ T cells.
5. HLA-A*02:01 Prevalence: Genotyping revealed that six out of ten patients carried the HLA-A*02:01 allele, known to present the immunodominant GAD65114–123 peptide. This allele is prevalent in about 30% of the general population in Germany.

Conclusion

The study concluded that CD8+ T cells likely play a pathogenic role in GAD65-LE by targeting neurons presenting the GAD65114–123 peptide via the HLA-A*02:01 molecule. This antigen-specific attack by CD8+ T cells contributes to hippocampal neurodegeneration and cognitive impairment. Conversely, CD4+ T cells appear to have a regulatory effect, positively correlating with hippocampal volume and memory function. These findings underscore the importance of detailed genetic analysis in understanding the mechanisms of autoimmune diseases and highlight the potential for targeted therapeutic strategies to modulate T cell responses in GAD65-LE.

Role of Creative Biolabs

Creative Biolabs provided full-resolution HLA-genotyping services, which were critical for identifying the HLA alleles present in the study participants. This genotyping included the HLA-A, -B, -C, DRB1, DQB1, and DPB1 molecules at an 8-digit resolution, enabling a comprehensive understanding of the genetic predisposition of the patients.