Expansion of T Follicular Helper Cells in NLC Co-Cultures Reinforces the Concept of Co-Evolution of CLL and Supportive T Helper Cell Clones
Authors: Vaca Alicia , Ioannou Nikolaos , Sivina Mariela , Vlachonikola Elisavet , Clise-Dwyer Karen , Kim Ekaterina , Li Dan , Ma Qing , Ferrajoli Alessandra , Estrov Zeev E. , Wierda William G. , Patten Piers E.M. , Ramsay Alan G. , Burger Jan A. 
Published: November 2021

Abstract
CLL cells proliferate in secondary lymphatic tissues where the CLL cells interact with T cells, monocyte-derived nurselike cells and mesenchymal stromal cells, collectively referred to as the tissue microenvironment. The CLL lymph node morphology with proliferation centers resembles normal B cell follicles, suggesting that mechanisms regulating the expansion of antigen-selected normal B cells during the germinal center (GC) reaction also regulate CLL cell expansion. Normal GC reactions require specialized T follicular helper (Tfh) cells, which provide T cell help to antigen-stimulated B cells. Another important T cell subset are Tregs which regulate immune tolerance and homeostasis. NLC co-culture have been used to dissect cellular and molecular interactions between CLL cells and the lymph node microenvironment in vitro. To gain insight into the role of T cell subsets in CLL, we utilized the NLC model to characterize T cell subsets and their dynamics in NLC co-cultures. We quantified CD4 + and CD8 + T cells in co-cultures from 18 different patients, observing that CD4 + T cell counts remained stable over time (449±62 at baseline to 443±80 cells/µl after 14 days). In contrast, numbers of CD8 + T cells declined from 180±42 cells/µl to 98±16 cells/µl on day 14 (p&lt;0.05). T helper cell subset analyses in 28 different samples revealed an increase in Tfh cells (from 1.7±0.3% to 6.0±0.9%, p&lt;0.001) and Tregs (from 3.3±0.5% to 9.6±1.7%, p&lt;0.001) after 14 days. In contrast, Th17 cell frequency remained unchanged. PD-1 expression levels on Tfh cells were tested to distinguish memory (PD-1 +) from germinal (PD-1 +++) Tfh cells. NLC co-culture resulted in an increase of germinal (0.2±0.1% to 2.4±0.3%, p&lt;0.0001) and memory Tfh cells (10.2±1.5% to 18.8±1.7%, p&lt;0.0001). In addition, germinal Tfh cells expressed high levels of BCL6 (39.4±4.4 to 179.0±5.9, n=8, p&lt;0.001), ICOS (119.0±68.0 to 574.0±154.0, n=6, p&lt;0.05), and IL-21 after co-culture (10.3±2.6% to 29.5±6.5%, p&lt;0.01, n=12). In contrast, CD40L was downregulated on memory Tfh cells from 24.4±8.6% to 8.7±2.4% (p=0.05, n=9). Tregs showed an increase of TGFß levels (4.3±1.4% to 25.3±8.5%, p&lt;0.05), and IL-10 expression (2.2±0.4% to 7.4±2.0%, p&lt;0.05, n=12), indicating activation during co-culture. Interestingly, NLC co-cultures also induced Ki-67 in germinal (13.0±4.9% to 39.5±5.7%, p&lt;0.001) and memory Tfh cells (1.7±0.3% to 13.0±4.9%, p&lt;0.05), and Tregs (2.5±0.6% to 18.0±3.3%, p&lt;0.001, n=13). To examine whether the expansion of Tfh and Treg cells in NLC co-cultures correlated with changes in T cell receptor (TR) gene repertoire we performed TR beta chain immunosequencing. Using the beta-binomial method, we analyzed the abundance of TR gene rearrangements, identifying clonotypes that were differentially abundant at 14 days. Next, to assess the similarity/overlap between the CD4 + TR gene repertoires at baseline and after co-culture, we utilized the Morisita overlap index (MOI). We found that the 6 samples with increased clonality had MOIs ranging from 0.12 to 0.53. Then, we compared the top 20 most expanded CDR3 sequences from each sample with low MOI with CDR3 sequences obtained in previous TR immunoprofiling studies from untreated CLL patients. Interestingly, we found numerous shared TR beta CDR3 sequences between NLC co-cultures and unrelated CLL patient samples. Furthermore, we correlated the Tfh expansion at 14 days normalized to baseline with MOI, a significant indirect correlation was noted (R 2=0.5, p=0.02, n=10). Interactions between CLL cells and Tfh or Tregs were evaluated in six LN CLL samples using multispectral microscopy. High magnification imaging and 3D Z-stack reconstructions revealed that there was a close spatial relationship between clusters of Ki-67 +CLL and Tfh cells within CLL LNs. In contrast, FOXP3 +CD4 + cells were generally found in cell groups in close proximity to other CD4 + cells. We next assessed whether the ICOS +CD4 + cell expansion was correlated with CLL proliferation and found a correlation between the percentage of ICOS +CD4 + cells with proliferating CLL cells in situ (R 2=0.75, p=0.03, n=6), whereas, no correlation was found with FOXP3 +CD4 + cell frequency. Collectively, these data provide new insight into the cellular and molecular cross-talk between CLL and T cell subsets, resulting in clonal expansion of T helper cells and interaction of Tfh cells with proliferating CLL cells opening new avenues for therapeutic targeting.

Disclosures
Ferrajoli: Janssen: Other: Advisory Board ; AstraZeneca: Other: Advisory Board, Research Funding; BeiGene: Other: Advisory Board, Research Funding. Wierda: Oncternal Therapeutics, Inc.: Research Funding; Sunesis: Research Funding; Gilead Sciences: Research Funding; GSK/Novartis: Research Funding; Acerta Pharma Inc.: Research Funding; Genentech: Research Funding; Karyopharm: Research Funding; KITE Pharma: Research Funding; Juno Therapeutics: Research Funding; Pharmacyclics LLC, an AbbVie Company: Research Funding; Cyclacel: Research Funding; Miragen: Research Funding; Loxo Oncology, Inc.: Research Funding; Janssen: Research Funding; Xencor: Research Funding; AstraZeneca: Research Funding; Genzyme Corporation: Consultancy; AbbVie: Research Funding. Patten: ABBVIE: Honoraria; ASTRA ZENECA: Honoraria; GILEAD SCIENCES: Honoraria, Research Funding; JANSSEN: Honoraria; NOVARTIS: Honoraria; ROCHE: Research Funding. Burger: Novartis: Other: Travel/Accommodations/Expenses, Speakers Bureau; Beigene: Research Funding, Speakers Bureau; Pharmacyclics LLC: Consultancy, Other: Travel/Accommodations/Expenses, Research Funding, Speakers Bureau; Gilead: Consultancy, Other: Travel/Accommodations/Expenses, Research Funding, Speakers Bureau; TG Therapeutics: Other: Travel/Accommodations/Expenses, Research Funding, Speakers Bureau; AstraZeneca: Consultancy; Janssen: Consultancy, Other: Travel/Accommodations/Expenses, Speakers Bureau.