PD-1⁺ T Cell Subsets in Follicular Lymphoma Tumor Microenvironment and Their Implications for Prognosis and Therapy

Programmed death (PD)-1, a coinhibitory receptor expressed by effector T cells (Teffs) is highly expressed on intratumoral T cells (mean 61%, range 34–86% for CD4⁺ T cells and mean 44%, range 31–69% for CD8⁺ T cells) in follicular lymphoma (FL), a finding associated with impaired ability to recognize autologous tumor (Nattamai et al, ASH 2007). Hence, PD-1 expression would be expected to confer an unfavorable prognosis in FL. However, correlation of PD-1 with clinical outcome in FL has been inconsistent with two studies showing favorable (Carreras et al, J Clin Oncol 2009; Wahlin et al, Clin Cancer Res 2010) and one study showing unfavorable (Richendollar et al, Hum Pathol 2011) outcome. While differences in method of analysis and type of treatment may explain the disparate results, a more complex model may be necessary to understand the prognostic impact of PD-1 in FL as PD-1 is expressed not only on antitumor Teffs but also on protumor follicular helper T cells (Tfh) and regulatory T cells (Tregs).

To determine the nature of PD-1⁺ T cells in FL we performed comprehensive genomic and immunologic studies. By flow cytometry, we observed that the intratumoral CD4⁺ T cells in FL may be categorized into 3 subsets based on PD-1 expression - PD-1 high (PD-1^hi), intermediate (PD-1^int), and low (PD-1^lo). The intratumoral CD8⁺ T cells consisted of PD-1^int and PD-1^lo subsets. The 3 CD4⁺ T cell subsets were FACSorted from FL tumors (n=3) and whole genome gene expression profiling (GEP) was performed. T cell subsets sorted similarly from tonsils served as controls for reactive follicular hyperplasia (FH) (n=3). Differentially expressed genes in GEP studies were confirmed at the mRNA level by real-time PCR (n=5) and at the protein level by flow cytometry when antibodies were available (n=5–10).

Our results suggested that CD4⁺PD-1^hi T cells are Tfh cells (CXCR5^hiBcl6^hi ICOS^hiCD40L^hiSAP^hiPRDM1^loIL-4^hiIL-21^hi); the CD4⁺PD-1^int T cells consisted of a mixture of activated Teffs (CD45RO⁺CD45RA⁻) including Th1 (Tbet⁺IFNg⁺), Th2 (IL-10⁺), and Th17 cells (RORc⁺IL-17⁺), and Tregs (Foxp3⁺CD25^hiCD127^lo); and the CD4⁺PD-1^lo T cells consisted of a mixture of activated Teffs (CD45RO⁺CD45RA⁻ but IFNg⁻IL-4⁻IL-10⁻IL-17⁻), Tregs, and naïve T cells (CD45RO⁻CD45RA⁺CCR7⁺). Although these subsets were present in both FL and FH, there were important differences. IL-4 expression was significantly higher in Tfh in FL vs. FH and may play a role in the pathogenesis of FL. IL-17 expression was low and expression of coinhibitory molecules BTLA and CD200 was high in CD4⁺PD-1^int T cells in FL vs. FH. BTLA and CD200 were also increased in CD8⁺PD-1^int T cells in FL vs. FH. However, other coinhibitory molecules (LAG-3, Tim-3, CD160, CTLA-4, CD244, KLRG1) were not significantly different between FL and FH. CD4⁺PD-1^int T cells also had higher expression of BATF, a transcription factor associated with T cell exhaustion in FL vs. FH. Together, these results suggest that the CD4⁺PD-1^int T cells in FL may be in a state of T cell exhaustion whereas the CD4⁺PD-1^int T cells in FH may represent recently activated Teffs. Consistent with this, blocking PD-1 with anti-PD-1 blocking antibody significantly enhanced proliferation and the production of Th1 (IFNg, TNFa) but not Th2 (IL-4, IL-5, IL-10, IL-13) cytokines by intratumoral CD4⁺ and CD8⁺ T cells in response to stimulation with autologous FL tumor cells (n=3). As expected, Tregs were increased in number in FL vs. FH and were present in the PD-1^int and PD-1^lo T cell subsets. We found 74% (range 40–97%) of FL Tregs expressed PD-1. Among the CD4⁺PD-1^lo and CD8⁺PD-1^lo T cells, there were more activated Teffs and fewer naïve T cells in FL vs. FH.

Our results suggest that the PD-1⁺ T cells in FL are comprised of a mixture of antitumor Teffs and protumor Tfh and Tregs. The prognostic impact of PD-1⁺ T cells in FL may dependent on the relative frequency of these subsets as ligation of PD-1 may produce favorable (inhibition of protumor Tfh and Tregs) or unfavorable (inhibition of antitumor Teffs) outcomes by inhibiting or promoting tumor growth, respectively. Conversely, our results imply that agents that block PD-1/PD-ligand pathway may have the opposite effect on these T cell subsets and enumeration of the intratumoral PD-1⁺ T cell subsets may serve as biomarker to predict response to these agents in FL and possibly other B-cell malignancies.

Dong:GSK: Consultancy; Genentech: Honoraria; Tempero: Consultancy; Ono: Consultancy; AnaptysBio: Consultancy. Neelapu:Cure Tech Ltd: Research Funding.