Follicular lymphoma tumor-infiltrating T-helper (T(H)) cells have the same polyfunctional potential as normal nodal T(H) cells despite skewed differentiation

Abstract

The follicular lymphoma (FL) T-cell microenvironment plays a critical role in the biology of this disease. We therefore determined the lineage, differentiation state, and functional potential of FL-infiltrating CD4(+) T-helper cells (T(H)) compared with reactive and normal lymph node (NLN) T(H) cells. Relative to NLNs, FL cells have decreased proportions of naive and central memory but increased proportions of effector memory T(H) cells. We further show differences in the distribution and anatomical localization of CXCR5(+) T(H) populations that, on the basis of transcription factor analysis, include both regulatory and follicular helper T cells. On Staphylococcus enterotoxin-B stimulation, which stimulates T cells through the T-cell receptor, requires no processing by APCs, and can overcome regulator T cell-mediated suppression, the proportion of uncommitted primed precursor cells, as well as T(H)2 and T(H)17 cells is higher in FL cells than in reactive lymph nodes or NLNs. However, the proportion of T(H)1 and polyfunctional T(H) cells (producing multiple cytokines simultaneously) is similar in FL cells and NLNs. These data suggest that, although T(H)-cell differentiation in FL is skewed compared with NLNs, FL T(H) cells should have the same intrinsic ability to elicit antitumor effector responses as NLN T(H) cells when tumor suppressive mechanisms are attenuated.

Figure 1

Flow cytometric gating strategy for discerning T_H subsets. (A) Shown is a representative healthy donor PBMC analysis, whereby gated populations (from left to right) are indicated defining viable CD3⁺CD4⁺ T cells. (B) As indicated, viable CD3⁺CD4⁺ T cells, defined in panel A, are differentiated into T_CM (CD45RA⁻CCR7⁺), T_EM (CD45RA⁻CCR7⁻), and T_N (CD45RA⁺) populations. T_CM and T_EM populations are further gated on the basis of CXCR3 expression with CXCR3⁺ T_CM and T_EM populations being associated with a pre-T_H1 and T_H1 population, respectively, and CXCR5⁺ T_CM and T_EM populations being associated with non–pre-T_H1 and non-T_H1 populations, respectively. (C) Total CD45RA⁻ memory CD4⁺ T cells are gated based on CXCR5 expression. CXCR5⁺ cells are further divided into CD25 and CD57 subsets: germinal center (GC; CD57⁻CD25⁺), Dual (CD57⁺CD25⁺), light zone (LZ; CD57⁺CD25⁻), and outer zone (OZ; CD57⁻CD25⁻).

Figure 2

Skewed proportions of CD4⁺ T_N, T_CM, and T_EM cells in FL. (A) Shown are representative plots of healthy donor PBMCs, reactive TN, NLN, RLN, and FL defining CD4⁺ T_N (CD45RA⁺), T_CM (CD45RA⁻CCR7⁺), and T_EM (CD45RA⁻CCR7⁻) populations (numbers above each gate are representative of the percentage of total CD4⁺ T cells). Proportions of T_N, (B), T_CM (C), and T_EM (D) are expressed as the percentage of the total CD4⁺ T cells in the indicated tissues. Also shown are the proportions of T_CM that are either CXCR3⁻ or CXCR3⁺ (E) or the proportions of T_EM that are either CXCR3⁻ or CXCR3⁺ (F). Triangles represent values for individual samples, and the rectangles represent averages for each tissue type expressed as a percentage of the total CD4⁺ T cells (B-D); T_CM cells (E-F), or T_EM cells (G-H). *P < .05, **P < .01, and ***P < .001 compared with FL.

Figure 3

Skewed proportions of CD4⁺ CXCR5⁺ memory T-cell subsets in FL. (A) Proportions of CXCR5⁺ memory (CD45RA⁻) CD4⁺ T cells in the indicated tissues are expressed as the percentage of the total CD45RA⁻ memory T-cell population. (B) Shown are representative plots of healthy donor PBMCs, reactive TN, NLN, RLN, and FL defining CD4⁺CD45RA⁻CXCR5⁺ subsets defined by CD57 and CD25 expression. (C) Proportions of GC (CD57⁻CD25⁺), LZ (CD57⁺CD25⁻), OZ (CD57⁻CD25⁻), and the dual (dual, CD57⁺CD25⁺) CD4⁺CXCR5⁺ memory T-cell in the indicated tissues expressed as the percentage of the total CXCR5⁺CD45RA⁻ memory T-cell population. Triangles represent values for individual samples, and rectangles represent averages for each tissue type. *P < .05, **P < .01, and ***P < .001 compared with FL. (D) Quantitative RT-PCR analysis of sorted CXCR5⁺ T_FH subsets defined in panel B, from FL or NLN (BCL-6 or FOXP3 expression normalized to GAPDH). Color indicates the number of cycles more (red) or less (blue) than the GAPDH control. (E) Further characterizing the FL CD57⁺CD25⁺ dual population (first plot) into PD-1 and ICOS populations (second plot) defining a ICOS⁺PD-1^DIM and a ICOS⁺PD-1^BRIGHT population that are predominately FoxP3⁺ (third plot) and Bcl-6⁺ (forth plot), respectively. Shown is a representative analysis of one FL specimen, with similar results obtained when analyzing an additional 3 FL patient specimens (total n = 4).

Figure 4

Increased proportions and altered anatomical localization of CD57⁺ T cells in FL. (A) Proportions of CD57^BRIGHT CD4⁺ T cells in the indicated tissue are expressed as the percentage of the total CD4⁺ T cells. Triangles represent values for individual samples, and rectangles represent averages for each tissue type expressed as a percentage of the total CD4⁺ T cells. *P < .05, **P < .01, and ***P < .001 compared with FL. (B) ICH analysis of CD57⁺ cells within RLNs and FL. In RLNs, CD57⁺ cells are highly restricted to the GC and show polarization, with a relative abundance in the LZ (Bi-iii). This stereotypic distribution was seen in 10 of 10 RLN specimens, even when the node showed other features of inflammation such as necrotizing granulomata (* in Biii). In contrast, the distribution of CD57⁺ cells varied widely among FL specimens. Random microscope fields were selected (original magnification, ×100) from 20 cases of FL, and the location of 200 CD57⁺ cells was noted as either follicular or otherwise. Three of 20 cases had more than three-quarters of the CD57⁺ cells within follicles (Bvi), 11 of 20 showed more than three-quarters of CD57⁺ cells outside follicles (Bv, in which the CD57⁺ cells appear associated with a mantle zone), and 6 of 20 showed a mixed distribution of cells (Biv). (C) IHC analysis of CD57 and CD4 shows CD57⁺ cells colocalized to areas of CD4⁺ cells in a FL nodal section.

Figure 5

Unsupervised cluster analysis of CD4⁺ T_H populations. (A) Clustered heat map showing samples (columns) and T-cell subsets (rows). For each subset, the frequency data (parent population is in braces) is expressed as the number of SDs above (yellow) and below (cyan) the mean across all samples, after log-transformation. Samples are clustered (within each tissue type) on the basis of Euclidean distance; subsets clustered by correlation coefficient. Dendrograms were constructed with complete linkage. (B) Same as panel A, but all samples are clustered together to show how the samples group independent of the tissue type. (C) PC analysis of dataset. The first 2 PCs of the 21 original variables (normalized cell subset frequencies) are shown. Each symbol represents a NLN (green circle), FL (purple triangle), or RLN (red star) sample. Most of the variance in the data is explained by the first 3 PCs (see pareto plot, inset, which shows that the first 3 PC account for ∼ 80% of the overall variance), indicating that several of the cell subsets are correlated with each other. Vectors emanating out from the center show how the original variables (cell subsets) are related to the first 2 PCs. The horizontal and vertical projections of a vector indicate the relative contributions of that variable to the first and second PC, respectively. The color of the cell subset label for each vector indicates the parent population used to compute the frequencies (see the key, top right corner of plot).

Figure 6

Higher proportions of CD69⁺ CD4⁺ T cells in FL before SEB stimulation. Activation status, as measured by CD69, before and after SEB stimulation of CD4⁺ T cells from (A) NLN, (B) RLN, (C) FL, and (D) healthy donor PBMCs are shown expressed as the percentage of the total CD4⁺ T cells. Triangles represent values for individual samples, and rectangles represent averages for each tissue type expressed as a percentage of the total CD4⁺ T cells. *P < .05, **P < .01, and ***P < .001 compared with FL, either before or after SEB stimulation.

Figure 7

Proportions of cytokine producing CD4⁺ T cells after SEB stimulation. (A) The T_H subsets defined by their cytokine secretion profile after SEB stimulation are expressed as the percentage of the total CD4⁺ T cells; T_H1 (IL-2⁺IFN⁻γ⁺IL-4⁻), T_H2 (IL-2⁺IFN-γ⁻IL-4⁺), T_H17 (IL-17⁺), or T_Hpp (IL-2⁺IFN-γ⁻IL-4⁻). (B) The T_H subsets defined by their cytokine profile as above, T_H1, T_H2, T_H17, and T_Hpp, after stimulation with soluble anti-CD3 and anti-CD28 mAbs. (C) Proportions of CD4⁺ T cells producing either 3 or 4 cytokines simultaneously after stimulation with SEB, polyfunctional T cells 3 (PFT 3; IL-2⁺IFN-γ⁺TNF-α⁺) or PFT 4 (IL-2⁺IFN-γ⁺TNF-α⁺MIP-1β⁺), respectively. For all plots, triangles represent values for individual samples, and the rectangles represent averages for each tissue type expressed as a percentage of total CD4⁺ T cells. *P < .05, **P < .01, and ***P < .001 compared with FL.