Background We previously developed Cell Distance Mapping (CDM) – a computerized algorithm to analyze confocal microscopy images - to analyze the tubulointerstitial inflammation (TII) present in human lupus nephritis (LuN) biopsies. Applying CDM, we showed that TII is a usual feature of LuN with T follicular helper (T_FH) cells participating in close conjugates with B cells. T_FH cells that participated in conjugates displayed an activated phenotype with elevated levels of IL-21 and bcl6 by quantitative PCR. Thus, CDM was able to translate proximal distance measurements of cell pairs to their functional state for the first time in human tissue.

Objectives A criticism of our previous work was that it was performed only using human tissue and could be descriptive. To address this, we set out to use a manipulatable model as our first objective, where the antigen specificity of cells can be controlled to test the utility and limits of CDM. Our second objective was to expand CDM to the study of innate and adaptive immune responses, chief among which are T cell:DC interactions. As DC populations tend to be more complex and require multiple phenotypic markers to identify, this required expanding our staining protocols.

Methods We used an adoptive mouse model that included 3 cell populations: antigen pulsed and LPS-activated dendritic cells (DCs), antigen-specific (5CC7) T cells, and wild type (WT) T cells. Each population was labeled with blue, red, and green cell trackers, respectively. After transfer, lymph nodes from five animals were subjected to two-photon excitation microscopy (TPEM) analysis. Lymph nodes were then frozen at -80°C and subjected to CDM analysis. A total of 37 images from 5 lymph nodes, each representing a separate animal, were used for CDM. To expanding our current staining, we used a new fluorochrome – Alexa 430 – in combination with SYTOX Blue – a unique nucleic acid-binding stain – along with our previously utilized fluorochromes (Alexas 488, 546, 594, and 647) to generate simultaneous 6-channel confocal microscopy images of human tonsil, normal kidney, and lupus nephritis samples.

Results Results revealed statistically significant differences at <0.27μm (8.70 vs 3.22%, p=0.028), <1 μm (11.7 vs 3.70%, p=0.01), and <2μm (13.1 vs 5.26%, p=0.031) distance cutoffs comparing antigen specific T cell:DC interactions versus WT T cell:DC interactions. Our results compared favorably with arrest coefficient calculation of the two populations performed on TPEM data (mean of 0.06 vs 0.26, respectively; p<0.01). For testing our new staining, we were successfully able to simultaneously visualize all 6 channels (5 antibody stains and SYTOX Blue for nuclei) with no bleed-through or artifact across all human tissue types. Utilization of this protocol allowed us to map plasmacytoid (CD123⁺BDCA1^-CD11c^-) and myeloid (CD123^-BDCA1⁺CD11c⁺) DCs, with concurrent staining for CD20+ B cells and CD3 across all tissues studied.

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Conclusions Our data shows that CDM is able to reliably identify cognate interactions on par with TPEM. We plan to further iteratively develop this technique in combination with our new 6 channel immunofluorescent staining of human TII to glean further insight into the organization of the in situ inflammatory response and disease pathogenesis.

Disclosure of Interest None declared