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690 CD122-selective IL-2 complexes treat ovarian carcinomas, induce Treg fragility and promote T cell stem cells
  1. Yilun Deng1,
  2. Justin Drerup2,
  3. Xinyue Zhang1,
  4. Ryan Reyes1,
  5. Jenny Mendez1,
  6. Aravind Kancharla1,
  7. Myrna Garcia1,
  8. Haiyan Bai1,
  9. Alvaro Padron1,
  10. Harshita Gupta1 and
  11. Tyler Curiel1
  1. 1UT Health San Antonio, San Antonio, TX, USA
  2. 2UT Southwestern, Dallas, TX, USA


Background Ovarian cancer (OC) responds poorly to immunotherapies. Regulatory T cells (Treg) engage IL-2 by high-affinity CD25 for differentiation and function,1 and anti-tumor effector T cells (Teff) use intermediate affinity CD122. We studied IL-2 complexes (IL-2c) that selectively activate CD122 (Teff) over CD25 (Tregs).

Methods Orthotopic ID8agg-luc mouse OC burden was measured by in vivo imaging. Tumor, ascites and draining lymph nodes (TDLN) were analyzed by flow and tSNE. IL-2c was complexed using 1.5 µg/mouse IL-2 and 7.5 µg/mouse aIL-2 (clone JES6-5H4) before i.p. injection every other day x 4 starting at day 7. antiPD-L1 was given at 100ug/mouse every 3 days x 4 starting from Day 11. FIR mice2 were used to sort live Tregs.

Results IL-2c but not antiPD-L1 potently inhibits ID8agg (figure 1). IL-2c decreased ascites Treg functional markers (e.g., CD25, granzymeB) while upregulating the same markers on Teffs (figure 2). IL-2c inhibited Treg suppression in ascites while TDLN Tregs were unaffected (figure 3). tSNE showed great similarity of TDLN Tregs treated with isotype and IL-2c while ascites Tregs after IL-2c showed a fragile phenotype (e.g., increased PD-1, T-bet, and IFNgamma with maintained FoxP3 expression [figure 4]) which is known to contribute to better response to cancer immunotherapy.3 4 We observed a complete reduction of tumor bioluminescence with IL-2c and antiPD-L1 combo treatment in nearly all subjects significantly exceeding effect of IL-2c alone (figure 5). A CD8+CXCR5+TCF-1+ T cell stem cell (TCSC) population reportedly improves immune checkpoint blockade efficacy.5 6 Since CD122 is regulated by TCF-1,7 we explored the effect of IL-2c on these TCSC. IL-2c significantly induced a CD8+TCF-1+ TCSC population in ID8agg tumors (figure 6), possibly through a positive feedback loop by further enhancing CD122 expression on TCF-1+, but not TCF-1- cells (figure 7). tSNE analysis of detailed immune phenotype of IL-2c induced TCSC revealed that these TCSC differed from those induced by antiPD-L1. In ID8agg, antiPD-L1-induced TCSC are mostly CXCR5+ and PD1+, consistent with previous reports in other cancers3 4 while IL-2c-induced TCSC were PD1- (figure 8), expressed CCR2 and CXCR3, and produced TNFalpha (figure 9).

Abstract 690 Figure 1

IL-2c but not aPD-L1 treats ID8aggLuciferase signal of ID8agg-luc tumors treated with isotype, ?PD-L1, or IL-2c measured by in vivo imaging. Arrows indicate treatments.

Abstract 690 Figure 2

IL-2c inhibits functional markers on tregs and promotes teffExpression of CD25 and granzymeB were measured by flow cytometry in indicated population from isotype or IL-2c treated ascites 3 weeks after final IL-2c dose.

Abstract 690 Figure 3

IL-2c reduces ascites Treg suppressive functionTregs sorted from ascites or TDLN of isotype or IL-2c treated FIR mice co-cultured with Teff cells. Percent suppression of Teff cells proliferation shown.

Abstract 690 Figure 4

IL-2c induces Treg fragility in ascites but not TDLN tSNE analysis on CD45+CD3+CD4+FoxP3+ cells from ascites and TDLN of isotype and IL-2c treated ID8agg-luc challenged mice. Right, representative bar graphs of flow data.

Abstract 690 Figure 5

IL-2c and aPD-L1 combo treatment effectively cures ID8aggLuciferase signal of ID8agg-luc tumors treated with isotype, ?PD-L1, IL-2c or combo measured by in vivo imaging. Arrows indicate treatments.

Abstract 690 Figure 6

IL-2c induces a CD8+TCF-1+ population in ID8aggID8agg-luc tumors analyzed by flow cytometry gated on CD45+CD3+CD8+ cells.

Abstract 690 Figure 7

IL-2c induces CD122 on CD8+TCF-1+ but not TCF-1- cellsCD122 expression was measured by flow cytometry in CD8+TCF-1+ and CD8+ TCF-1- cells from tumors treated with isotype or IL-2c.

Abstract 690 Figure 8

CD8+TCF1+ cells from ID8agg analyzed by flow cytometry plus tSNE. Cells expressing CXCR5 (blue) and PD1 (red) indicated in overlay

Abstract 690 Figure 9

CD8+TCF1+ cells from ID8agg analyzed by flow cytometry

Conclusions We define two novel IL-2c effects: inducing Treg fragility therefore reducing immunosuppression while promoting TCSC that could enhance effective anti-tumor immunity. Current work tests if effects are related and help efficacy, and mechanisms for IL-2c Treg effects. We also show that elicited TCSC differ by treatment and tumor, requiring additional investigations.

Acknowledgements This work is supported by CPRIT Research Training Award (RP 170345), Ovarian Cancer Research Alliance Ann and Sol Schreiber Mentored Investigator Award to YD and R01 CA205965to TC.

Ethics Approval All mice studies were approved by UT Health San Antonio Institutional Animal Care and Use Committee (IACUC). Approval number 20150093AR, 20140001AR, 20170035AR, 20140039AR, 20140027AR, 20090128AR, 20120071AR, 20180021AR.


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