Implication of ICOSLG on Relapse in Infant T(4;11) Acute Lymphoblastic Leukemia

Infant t(4;11) acute lymphoblastic leukemia (ALL) is associated with a high relapse rate with 4-year event-free survival (EFS) of only 36%. Relapse has been shown to be the major cause of death as 83% of relapsed infant t(4;11) ALL patients die within three years of diagnosis. Therefore, it is of utmost therapeutic interest to elucidate molecular mechanisms of relapse. Here we show that t(4;11) ALL cells upregulate the inducible T-cell costimulator ligand (ICOSLG) in an early growth response 3 (EGR3) dependent manner thereby promoting the development of regulatory T-cells (T regs). We propose that short EFS and high ICOSLG expression are causally linked, in that ICOSLG-mediated induction of T regs interferes with immune recognition of leukemia cells. According to that hypothesis, ICOSLG would not only be a novel and independent prognostic biomarker but a potential therapeutic target. We investigated the function of EGR3 in infant ALL since EGR3 has been described as an indirect target of the iroquois homeobox 1 (IRX1) transcription factor. For that purpose we created a stable sleeping beauty transposon-based SEM cell line expressing EGR3 in a Doxycycline-inducible manner. Gene expression and western blot analysis revealed strong upregulation of ICOSLG 48h after Doxycycline induction when compared to an empty vector control. Additionally, chromatin immunoprecipitation (ChIP) experiments depicted that EGR3 directly binds to the promoter of the ICOSLG gene. The expression of ICOSLG in mesenchymal stem cells has been shown to foster the induction of T regs and ICOSLG-mediated T reg expansion has been identified as a driver of acute myeloid leukemia and glioblastoma. In the bone marrow (BM) hematopoietic stem cells (HSC) colocalize with T regs which provide an immune privilege to the stem cell niche. Based on these observations, we hypothesize that ICOSLG expressing ALL cells could create an immune privileged niche appearing to be necessary for HSC maintenance and sanctuary from immune attack. This could create independence from the BM immune privilege enabling migration of the ALL cells. If so, this could contribute to minimal residual disease (MRD) formation after induction therapy and subsequently to higher probability of relapse. To evaluate our hypothesis, we cocultured the EGR3-SEM and control cells with primary human CD4 + and CD8 + T-cells. The T-lymphocytes were isolated from the peripheral blood mononuclear cells (PBMC) of healthy donors and stimulated with coated α-CD2, -CD3 and -CD28 beads. We observed 16% more CD4 +CD25 ++FOXP3 + T regs after 48h of coculture with EGR3-SEM cells compared to the control. The addition of a neutralizing monoclonal α-ICOSLG antibody to the coculture led to a reduction of the T reg frequency in EGR3-SEM coculture. Taken together, these results strongly suggest that ALL cells expressing EGR3 induce the formation of T regs via ICOSLG expression. To confirm our results with patient-derived material, we investigated the gene expression of 50 infant t(4;11) pro-B phenotypic ALL patients. Pearson correlation testing confirmed that ICOSLG expression strongly correlates with EGR3 and IRX1 expression. Furthermore, we were able to classify the patients considering their ICOSLG expression level into an ICOSLG-high (ICOSLG-hi) and an ICOSLG-low (ICOSLG-lo) group. Outcome data for a 5-year follow-up were available for 35 of 50 patients, n=5 ICOSLG-hi and n=30 ICOSLG-lo. 100% (5/5) of the ICOSLG-hi patients failed within 17 months of diagnosis whereas 53% (16/30) of the ICOSLG-lo patients failed within 56 months of diagnosis. The remaining 14 ICOSLG-lo patients were alive after 60 months. These data underscore the role of overexpressed ICOSLG in relapse development. However, a verification of these findings in a larger cohort is needed. In conclusion, our study identifies ICOSLG as a promising prognostic marker and novel therapeutic target in infant t(4;11) ALL. Furthermore, our findings implicate the interaction between T-cells and leukemia stem cells as contributory to disease progression. Disclosures Cario: Novartis: Other: Lecture Fee.

325 Eliminating tumor immune privilege through immune checkpoint cytoreduction

BackgroundAnti-checkpoint antibodies blocking T cell co-inhibitory molecules (e.g. αPD-1, αCTLA-4) allow immune effector cells to persist, expand, and maintain cytotoxic function in the tumor microenvironment (TME). Despite being effective in immune ”hot” tumors that are infiltrated by effector anti-tumor cells, immune ”cold” tumors fail to respond to checkpoint blockade. ”Cold” tumors are populated with immune suppressive cells including regulatory T cells, M2 macrophages, and myeloid derived suppressor cells, which inhibit immune effector infiltration and function. These suppressive populations, along with tumor cells, express co-inhibitory checkpoints already targeted with current immunotherapeutics, but also some checkpoints in need of further investigation. We hypothesized that by targeting these checkpoints with cytoreductive antibodies which selectively deplete suppressive populations and tumor cells via ADCC/ADCP, we will compromise ”cold” immune privilege and restore an efficient anti-tumor immune response.MethodsTo identify novel targets to produce checkpoint cytoreductive antibodies we conducted in silico analysis that prioritized immune-inhibitory targets with tumor-specific or tumor-selective expression on cell surface. We cross-referenced a previously published list of transmembrane proteins against publicly sourced datasets including TCGA, HPA, GTEx, BioGPS, and SAGE.1 We then characterized the expression profile of each selected target on tumor cells in vitro and on cell populations in the TME ex vivo via multiparameter flow cytometry. Finally, we assessed the impact of existing checkpoint-targeting cytoreductive antibodies on survival and tumor growth in murine ”hot” and ”cold” tumors.ResultsVISTA and DLL3 were identified via in silico analysis as co-inhibitory surface proteins specifically and selectively in the TME and not in healthy tissues. DLL3 is mainly expressed on tumor cells while VISTA was described mostly on immunosuppressive myeloid cells. An anti-DLL3 antibody was produced by a previous laboratory as an IgG1 antibody, and we engineered a version in the depletive (IgG2a) isotype, which will enable us to target this checkpoint with either a blocking or a depleting antibody. Flow cytometry analysis identified VISTA on multiple myeloid cell populations in ”cold” 4T1 murine mammary tumors while its expression was low in spleen. To start assessing the efficiency of depleting versus non-depleting antibodies, ”hot” CT26 murine tumors and 4T1 tumors were treated with an αCTLA-4-IgG2a or αCTLA-4-IgG1. Groups treated with depleting antibodies showed increased survival compared to groups treated with non-depleting antibodies.ConclusionsNovel immune-inhibitory checkpoints can be identified and targeting them with cytoreductive antibodies leads to a higher anti-tumor immune response. This investigation opens the door to more efficient combination therapies.AcknowledgementsSupported by a training fellowship from The University of Texas Health Science Center at Houston Center for Clinical and Translational Sciences TL1 Program (Grant No. TL1 TR003169).ReferencesWang J, et al. Siglec-15 as an immune suppressor and potential target for normalization cancer immunotherapy. Nat Med 25,(2019).

Corneal Allografts: Factors for and against Acceptance

Cornea is one of the most commonly transplanted tissues worldwide. However, it is usually omitted in the field of transplantology. Transplantation of the cornea is performed to treat many ocular diseases. It restores eyesight significantly improving the quality of life. Advancements in banking of explanted corneas and progressive surgical techniques increased availability and outcomes of transplantation. Despite the vast growth in the field of transplantation laboratory testing, standards for corneal transplantation still do not include HLA typing or alloantibody detection. This standard practice is based on immune privilege dogma that accounts for high success rates of corneal transplantation. However, the increasing need for retransplantation in high-risk patients with markedly higher risk of rejection causes ophthalmology transplantation centers to reevaluate their standard algorithms. In this review we discuss immune privilege mechanisms influencing the allograft acceptance and factors disrupting the natural immunosuppressive environment of the eye. Current developments in testing and immunosuppressive treatments (including cell therapies), when applied in corneal transplantation, may give very good results, decrease the possibility of rejection, and reduce the need for retransplantation, which is fairly frequent nowadays.