Page 136 - Haematologica - Vol. 105 n. 6 - June 2020

W. Townsend et al.
results, most likely because of differences in patient popu- lations studied, therapy administered and technical limita- tions of single parameter immunohistochemistry (IHC) that preclude accurate identification of cell subsets.
In normal germinal centers (GC), B cells are critically dependent on interactions with CD4pos follicular helper T cells (TFH),17-20 which are characterized by expression of PD-1, ICOS, CXCR5, CXCL13, IL-21 and IL-4 and the transcription factor BCL6.19,21,22 TFH provide signals neces- sary for the survival and proliferation of GC B cells and induce expression of activation induced cytidine deami- nase (AID), a DNA modifying enzyme that initiates somatic hypermutation (SHM) and class switch recombi- nation (CSR) leading to a class-switched, high-affinity antibody response.17,19,20,23
FL follicles and normal GC share a number of features; FL B cells have a similar phenotype and GEP as their nor- mal counterparts and neoplastic follicles contain both fol- licular dendritic cells (FDC) and T cells. Studies performed on disaggregated FL lymph nodes (LN) have previously demonstrated an enrichment of IL-4-producing TFH in FL with a distinct gene expression profile and the ability to support FL B-cell growth and modify stromal cell function in vitro.24-28 The anatomic relationship between TFH and FL B cells and how closely this mimics the situation in normal GC has, however, not previously been studied.
In this study we compared the architecture of normal GC and neoplastic follicles, specifically focusing on the spatial relationship between B cells and TFH using multipa- rameter confocal immunofluorescence microscopy and semi-automated image analysis. We found that TFH - as identified by surface expression of CD4, PD1, and ICOS - constitute a similar proportion of CD4pos T cells in FL as they do in normal GC. They colocalize and form synapses with proliferating neoplastic B cells, which also express the DNA modifying enzyme AID. Finally, T-cell receptor (TCR) repertoire analysis revealed that T cells in neoplas- tic follicles are significantly more clonal than those in interfollicular areas, suggesting a role for antigen stimula- tion in this process. Overall, our findings further highlight the role of the microenvironment in FL and are relevant to the mode of action of new therapies such as those target- ing antigen receptor signaling and the PD1/PDL1 axis.29-32
Methods
Patient samples
Formalin fixed paraffin embedded (FFPE) LN biopsies were obtained from 25 patients with histologically confirmed untreated or relapsed FL including three cases of grade IIIb FL, and eight patients with reactive lymphadenopathy. Patients with relapsed FL had not received any treatment for at least 12 months. Clinical details are presented in the Online Supplementary Tables S1-2. Ethical approval was obtained from the UK national research ethics committee, reference 13/NW/0040.
Immunofluorescent confocal microscopy
FFPE LN sample preparation steps including deparaffinization, antigen retrieval, and staining are described in the Online Supplementary Materials and Methods. All images were acquired on a Nikon Eclipse Ti-E microscope and analyzed using Nikon ele- ments NIS Advanced Research software. Full descriptions of imag- ing and analysis techniques including the use of binary layers for image analysis are presented in the Online Supplementary Materials
and Methods and further explained in the Online Supplementary Figure S1.
Laser micro-dissection, DNA extraction, and TCR sequencing
Follicles were highlighted by conventional IHC staining for BCL6. Follicular and interfollicular areas were dissected from sequential 10 μm FL sections using a laser capture microscope (PALM, Carl Zeiss MicroImaging, Jena, Germany). After DNA extraction, TCR sequences were subject to multiplex PCR ampli- fication prior to next generation sequencing (Adaptive Biotechnologies, Seattle, WA, USA).33 TCRV CDR3 regions and their component V, D and J segments were identified using the IMGT definitions.34 Sequences not corresponding to a CDR3 were discarded and unique clones defined by the presence of more than one identical productive CDR3 DNA sequence. The number and size of each clone was determined and the richness, clonality and overlap of the follicular and interfollicular TCR repertoires deter- mined (see the Online Supplementary Materials and Methods).
Statistical analysis
Statistical analysis was performed using GraphPad Prism soft- ware v5 (GraphPad Software Inc, La Jolla, CA, USA). Normally distributed values are presented as the mean (+/- standard devia- tion), non-normally distributed values are presented as median (+/- interquartile range). Further details of the statistical analysis are presented in the Online Supplementary Materials and Methods.
Results
Normal and neoplastic follicles contain similar numbers of TFH
CD4pos T cells were predominantly located in the inter- follicular areas of reactive and FL LN but discrete popula- tions were also present within the GC and malignant fol- licles. We investigated the phenotype of these by staining for CD4, PD-1 and ICOS simultaneously. Within GC of reactive LN 33.05% (24.7-43.7) of CD4pos T cells co- expressed PD-1 and ICOS (TFH phenotype) and these were distributed predominantly in the light zones (Figure 1A). In FL, 25.0% (18.5-28.7) of follicular CD4pos T cells expressed both PD-1 and ICOS and were located at the follicular border or diffusely distributed within the folli- cles (Figure 1B). The proportion of CD4pos cells co-express- ing PD-1 and ICOS was not significantly different between FL follicles and GC (Figure 1C). CD4posPD- 1posICOSpos cells were tightly restricted to the GC of reac- tive LN and FL follicles with only 0.34% (0.26-1.13) and 3.63% (1.89-6.15) of non-GC or interfollicular FL CD4pos cells co-expressing PD-1 and ICOS respectively.
In FL, although 46.9% (34.7-51.9) of follicular CD4pos cells expressed PD-1, only about 50% of these co- expressed ICOS indicating that there are at least two dis- tinct populations of CD4posPD-1pos cells within FL follicles, highlighting the importance of using all three parameters for identification of TFH. There was no difference in the proportion of CD4pos cells that co-expressed PD-1 and ICOS by histological grade in FL (Online Supplementary Figure S8), however, as the number and size of neoplastic follicles increase with histological grade, so must the absolute number of TFH.
To investigate differences in T cells located in the folli- cles and interfollicular areas of FL, the intensity of CD4 and PD-1 expression were measured. CD4 expression was
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