A Reproducible Method for Isolation and <i>In Vitro</i> Culture of Functional Human Lymphoid Stromal Cells from Tonsils

<div><p>The stromal compartment of secondary lymphoid organs is classicaly known for providing a mechanical scaffold for the complex interactions between hematopoietic cells during immune activation as well as for providing a niche which is favorable for survival of lymphocytes. In recent years, it became increasingly clear that these cells also play an active role during such a response. Currently, knowledge of the interactions between human lymphoid stroma and hematopoietic cells is still lacking and most insight is based on murine systems. Although methods to isolate stromal cells from tonsils have been reported, data on stability in culture, characterization, and functional properties are lacking. Here, we describe a reproducible and easy method for isolation and <i>in vitro</i> culture of functional human lymphoid stromal cells from palatine tonsils. The cells isolated express markers and characteristics of T cell zone fibroblastic reticular cells (FRCs) and react to inflammatory stimuli by upregulating inflammatory cytokines and chemokines as well as adhesion molecules, as previously described for mouse lymphoid stroma. Also, cultured tonsil stromal cells support survival of human innate lymphoid cells, showing that these stromal cells can function as bone fide FRCs, providing a favorable microenvironment for hematopoietic cells.</p></div

Stimulation of TSC lines with pro-inflammatory cytokines.

<p>(A) TSC (passage 2 or 3, n = 6 individual donors) were cultured for 6 hr with either 5ng/mL rhTNFα or medium alone. Shown is expression relative to GAPDH. (B) TSC (passage 2 or 3, n = 6 individual donors) were cultured for 6 hr with either 300U/mL rhIFNγ or medium alone. Shown is expression relative to GAPDH. For all plots: *: p < 0.05, **: p < 0.01, ***: p < 0.001, ****: p < 0.0001, ratio-paired Student’s T-test.</p

Used rtPCR primer sequences.

<p>Used rtPCR primer sequences.</p

Transcript expression of stromal-associated genes by cultured TSC remains stable in culture.

<p>PCR analysis of cultured TSC harvested at passage 2, 4, 6 and 8 (P2,P4, P6 and P8, respectively) and PBMCs for IL-7, CCL21, COL1A1, COL5A2, FN1 and DCN (n = 3 individual donors for the stromal cells, n = 4 for PBMCs). *: p < 0.05, **: p < 0.01, Kruskall-Wallis test with Dunn’s multiple comparisons test. Error plots represent mean ± SD.</p

Transcript expression of stromal-associated genes by cultured TSC.

<p>(A) PCR analysis of cultured TSC (harvested at passage 2 or 3, n = 4 individual donors) as compared to PBMCs (n = 4 individual donors) for cytokines/ chemokines and their receptor, associated with lymphoid stromal cells. Shown is expression relative to GAPDH. (B) PCR analysis of cultured TSC (harvested at passage 2 or 3, n = 4 individual donors) as compared to PBMCs (n = 4 individual donors) for genes associated with extracellular matrix. Shown is expression relative to GAPDH. For all plots: *: p < 0.05, Two Tailed Mann-Whitney Test, error plots represent mean ± SEM.</p

Cross-Tissue Transcriptomic Analysis of Human Secondary Lymphoid Organ-Residing ILC3s Reveals a Quiescent State in the Absence of Inflammation

A substantial number of human and mouse group 3 innate lymphoid cells (ILC3s) reside in secondary lymphoid organs, yet the phenotype and function of these ILC3s is incompletely understood. Here, we employed an unbiased cross-tissue transcriptomic approach to compare human ILC3s from non-inflamed lymph nodes and spleen to their phenotypic counterparts in inflamed tonsils and from circulation. These analyses revealed that, in the absence of inflammation, lymphoid organ-residing ILC3s lack transcription of cytokines associated with classical ILC3 functions. This was independent of expression of the natural cytotoxicity receptor NKp44. However, and in contrast to ILC3s from peripheral blood, lymphoid organ-residing ILC3s express activating cytokine receptors and have acquired the ability to be recruited into immune responses by inflammatory cytokines. This comprehensive cross-tissue dataset will allow for identification of functional changes in human lymphoid organ ILC3s associated with human disease. Bar-Ephraim et al. describe a cross-tissue transcriptional comparison of human ILC3s and show that ILC3s in lymph nodes and spleen share a transcriptional profile that is distinct from that of tonsil ILC3s. Lymphoid organ ILC3s are a substantial pool of resting cells that can be recruited into immune responses upon local activation

CD62L Is a Functional and Phenotypic Marker for Circulating Innate Lymphoid Cell Precursors

Innate lymphoid cells (ILCs) guard epithelial tissue integrity during homeostasis, but can be potent immune effector cells during inflammation. Precursors to all ILC subsets (ILC precursors [ILCP]) have been identified in human peripheral blood (PB). We found that during homeostasis, ILCP in PB of mouse and human expressed homing receptors for secondary lymphoid organs, mainly CD62L. These ILCP entered mouse lymph nodes in a CD62L-dependent way and relied on S1P receptors for their exit. Importantly, CD62L expression was absent on human ILCs expressing NKp44 in tonsils and PB of Crohn disease patients, and relatively fewer CD62L+ ILCP were present in PB of Crohn disease patients. These data are in agreement with selective expression of CD62L on nonactivated ILCP. As such, we conclude that CD62L not only serves as a functional marker of ILCP, but has potential to be used in the clinic as a diagnostic marker in inflammatory disorders