Studies assessing blood derived immune cells in the response to the immunotherapy for Type 1 diabetes have a high risk of false negative results, as the frequency of cells responding to any given self-antigen is very low.1 The standard approach in animal studies is to study responses in lymphoid organs.2 In humans, lymph nodes (LN) are accessible via ultrasound guided fine needle aspiration biopsy (FNA).3 Here we aimed to explore whether this technique, not previously tested for this purpose, could be used to improve the detection of antigen specific immune responses.
To test the feasibility of this concept we assessed microneedle delivered Tuberculin PPD response in healthy volunteers. Participants underwent pre-treatment axillary LN FNA, followed by intradermal injection of Tuberculin PPD and post-treatment axillary LN FNA 48 hrs later. The response was measured by the change in the number of IFN-gamma producing, spot-forming cells (SFC) before and after Tuberculin PPD injection, in the draining LN and peripheral blood mononuclear cells (PBMCs) detected by ELISPOTS.
We obtained on average 2.14x106±1.1x106 live white cells per biopsy (n=11 biopsies). The CD4+/CD8+ cells ratio was 6.4±2.5 in the LN cells and 2.2±0.01 in the respective PBMCs (n=2). The number of SFC/1x106 LN derived lymphocytes significantly increased after Tuberculin PPD (3.3±7.5vs.119.1±114.8;n=5,p=0.03), whilst there was no significant difference in the number of SFC/1x106 PBMCs after Tuberculin PPD (517.3±401.5vs.491.0±385.8,n=5).
LN cells showed higher, although non-significant, response to in-vitro PHA, following Tuberculin PPD (SFC/1x106:1130.0±883.9vs.3274.0±1444.0,n=5). Response to in-vitro tetanus-toxoid remained unchanged (SFC/1x106:3.3±3.5vs.4.3±5.1,n=3) suggesting non-specific response to PHA.
LN FNA provides live and functional lymphocytes capable of specifically responding to antigens injected into the skin in vivo. This approach shows promising superiority over standard methods that use blood-derived immune cells for detection and monitoring of antigen-specific immune responses. Future studies will examine its application to autoimmune responses.