Abstract
Idiopathic multicentric Castleman disease (iMCD) is an atypical hematologic disorder with significant morbidity and mortality. Defined by enlarged lymph nodes (LN) with characteristic histopathology, there are a range of clinical subtypes with the most severe form having symptoms including thrombocytopenia, anasarca, fever, renal dysfunction, and organomegaly (iMCD-TAFRO). Though not well understood, iMCD is often described as a lymphoproliferative disorder assumed to be due to expansion of lymphocytes in LNs. However, our prior findings indicated that lymphocyte mobilization, rather than proliferation, contributed to the lymphadenopathy phenotype of iMCD. We showed that chemokines associated with lymphocyte trafficking to the LN, including CXCL13, CCL19, and CCL21, were highly elevated in the serum of iMCD patients; however, their significance and whether their expression is associated with dysregulated lymphocyte mobilization is not understood. Here, we identified the LN-derived sources of homing chemokines and investigated whether dysregulated chemokine-driven trafficking of lymphocytes to and within LNs may explain the lymphadenopathy observed clinically.
To determine what cell types expressed CXCL13, CCL19, and CCL21 in iMCD-TAFRO lymph nodes, we performed 10X Genomics' FLEX single-cell RNA (scRNA) sequencing on dissociated formalin-fixed paraffin-embedded LN tissue from iMCD-TAFRO patients (n=4) and reactive controls (n=4). We defined major cell types using unbiased clustering followed by manual evaluation of the top differentially expressed genes per cluster. The major producers of CXCL13, CCL19, and CCL21 were stromal populations in both control and iMCD-TAFRO LNs, however each was significantly elevated in iMCD-TAFRO LNs. We next examined which stromal populations produced these chemokines using differential gene expression analysis. We discovered that CXCL13, CCL19, and CCL21 were among the most up-regulated genes in iMCD-TAFRO follicular dendritic cells, while perivascular reticular cells in iMCD-TAFRO expressed significantly higher levels of CXCL13 and CCL21. Thus, stromal cells are the primary producers of LN-homing chemokines, which are elevated in iMCD-TAFRO LNs.
Since T follicular helper (Tfh) cells, a specialized T cell subset critical for providing help to B cells, express CXCR5, the cognate receptor of CXCL13, we interrogated whether Tfh cells were dysregulated in number or location in iMCD-TAFRO LNs. Using our scRNA sequencing dataset, we did not identify a significant change in the overall proportion of Tfh cells between iMCD-TAFRO LNs and controls. These data were perhaps not surprising since Tfh cells provide help to B cells within germinal centers (GC) but can also be found in other regions of the lymph node. Differential gene expression analysis in the Tfh cell cluster between groups revealed that the most down-regulated genes in iMCD-TAFRO Tfh cells were genes typically associated with GC Tfh, including PDCD1, TIGIT, and TOX2. Together, these data indicate that while the overall numbers of Tfh cells in iMCD LNs is typical, their gene expression programs suggest that their localization within the LN may be dysregulated.
To directly examine the spatial location of Tfh cells in iMCD-TAFRO LNs, we performed immunocytochemistry/immunofluorescence microscopy on iMCD-TAFRO (n=8) and reactive control LNs (n=6). We used staining of Tfh markers CD3, PD-1, and BCL-6 followed by quantification by scikit-image color-deconvolution and subsequently classified two intensity-based categories using a 2-component Gaussian Mixture Model. We discovered significantly fewer CD3+, PD-1+, BCL6+ Tfh cells per area within GCs of iMCD-TAFRO LNs compared to controls. Interestingly, we did not observe any significant difference in the number of CD3+, PD-1+, BCL6+ Tfh cells per area outside of GCs in iMCD-TAFRO. These data suggest that Tfh cell trafficking to GCs is restricted and might contribute to B cell dysregulation in iMCD-TAFRO.
Together, these data demonstrate that in iMCD-TAFRO LNs, Tfh cells are mis-localized and do not appropriately traffic to GCs despite elevated CXCL13 levels by LN stromal cells. Indeed, the sub-tissue organization of iMCD-TAFRO patient LNs is broadly dysregulated. These data suggest multiple emerging treatments, including targets that promote CXCL13-CXCR5 chemotaxis and Tfh activation, might be efficacious in the treatment of iMCD-TAFRO patients.
