Detailed Phenotypic Characterization of T-Cell Populations in Reactive, Normal and Follicular Lymphoma Nodes: Elevation of Follicular B Helper T-Cell Subsets in Follicular Lymphoma.
Authors: Hilchey Shannon P. , Secor-Socha Shelley , Cochran Matthew R. , Hyrien Ollivier , Burack W. Richard , Wang Jyh-Chiang E. , Quataert Sally A. , Bernstein Steven H. 
Published: October 2019

Abstract
Abstract 2948
Poster Board II-924
The tumor microenvironment of follicular lymphoma (FL) has been shown to play a critical role in the biology of this disease, and to be predictive of treatment outcome for FL patients. To gain further insight into the biology of the FL microenvironment, we asked whether differences exist between the T-cell populations within FL involved lymph nodes (FLN; n=13), as compared to that seen in either reactive (RLN; n= 10) or normal lymph nodes (NLN; n=11; obtained from patients undergoing vascular surgery whereby lymph nodes are removed to gain access to the vascular structures), using 11-color flow cytometry. Interestingly, the proportions of the T-cell populations shown in Table 1 were not statistically different between FLN and RLN, whereas they were statistically different between FLN and NLN. Specifically, the FLN demonstrate higher proportions of CD4+CD45RA−CCR7− T-effector memory cells (TEM) and lower proportions of both CD4+CD45RA−CCR7+ T-central memory (TCM) and CD4+CD45RA+ naïve T-cell populations, as compared to that of the NLN. In addition, within the FLN the TCM subpopulations demonstrate a higher proportion of CXCR5+ non-polarized T-cells as compared to the NLN. In contrast, when the proportions of the TEM subpopulations were examined, there were no significant differences between the FLN, RLN and NLN.TABLE 1CD8+ (%CD3+)CD4+ (%CD3+)Naïve (%CD4+)TCM (%CD4+)TEM (%CD4+)non-preTH1 (%TCM)non-polarized (%TCM)pre-TH1 (%TCM) FLN18.2±2.570.8±3.215.0±2.115.5±1.764.0±3.030.5±3.749.2±5.020.2±2.2 RLN14.4±2.879.9±3.423.6±5.215.0±2.452.6±5.839.8±3.435.4±3.224.3±3.2 NLN10.5±1.4*82.9±2.1*28.7±4.7*21.9±1.345.4±4.9*39.0±1.9*34.2±3.0*25.8±1.7Statistical analysis was performed using Wilcoxon rank sum tests (distribution free).*p<0.05, pairwise comparison (bold text).
We next examined the proportion of the total CD45RA− memory CD4+ T-cells that were CXCR5+, a phenotype consistent with Follicular B Helper T-cells (TFH). Such TFH cells are crucial for the elicitation of B-cell memory and the generation of high affinity antibody responses. Whereas there was no significant difference in the mean percent of the memory T-cells that have a TFH phenotype in the FLN (64.1±5.0%) as compared to that seen in the RLN (55.8±4.6%), the proportion of TFH in the FLN was significantly higher than that seen in the NLN (48.0±4.0%). As discrete TFH populations have been identified based on their immunophenotype and anatomical localization, we next evaluated the distribution of TFH subpopulations, as defined by their surface expression of CD25 and CD57.TABLE 2Germinal Center (GC)Light zoneOuter zoneDual CD25+CD57+FLN16.4±5.022.5±2.754.4±4.16.72±1.06RLN9.4±0.5*15.5±3.272.9±3.6*2.21±0.50*NLN9.3±1.29.4±1.3*80.0±2.5*1.24±0.13*Statistical analysis was performed using Wilcoxon rank sum tests (distribution free).*p<0.05, pairwise comparison (bold text).
In FLN there appears to be a skewing of TFH subsets away from the CD25−CD57− outer zone subset (a component of which is reported to contain pre-formed CD40L and thus likely provides help to GC-B cells) and towards the CD25+CD57− GC subset (reported to suppress TFH cell help to GC B-cells and inhibit AID expression); and towards the CD25−CD57+ light zone subset (reported to provide help to GC B-cells by inducing expression of AID, as well as inducing immunoglobulin production and class switching). Finally, we identified a potentially unique “dual” CD25+CD57+ population of TFH cells, with a higher frequency seen in FLN (6.72±1.06%) compared to both RLN (2.21±0.50) and NLN (1.24±0.13), with frequencies as high as 12% seen in some FLN samples. Taken together our findings suggest that; (a) the T-cell subset distribution of FL is overall similar to that of RLN but different than that of NLN suggesting that the FLN microenvironment is driven in part by the same immune reactivity and inflammatory signals as seen in RLN and; (b) FLN have a higher proportion of TFH cells then that of NLN, and a different profile of TFH subsets than in both RLN and NLN. Given the critical role that TFH cells play in normal GC-B cell biology, we speculate that differences in the TFH populations in FLN compared to that of RLN and NLN may in part regulate the malignant transformation and/or survival of FL B-cells.

Disclosures:
No relevant conflicts of interest to declare.