Increased HLA-E Expression Correlates with Early Relapse in Multiple Myeloma
Authors: Lagana Alessandro , Ruan Dan Fu , Melnekoff David , Leshchenko Violetta , Perumal Deepak , Rahman Adeeb , Kim-Schultze Seunghee , Song Ying , Keats Jonathan J , Yesil Jennifer , Auclair Daniel , Madduri Deepu , Richter Joshua , Chari Ajai , Cho Hearn Jay , Barlogie Bart , Jagannath Sundar , Dudley Joel , Horowitz Amir , Parekh Samir 
Published: October 2019

Abstract
HLA-E is a non-classical major histocompatibility complex (MHC) class I molecule characterized by limited polymorphism in contrast to its class I counterparts HLA-A, -B, and -C. HLA-E elicits an inhibitory signal via its interactions with NKG2A on NK cells (and subsets of T cells) and is considered the most dominant inhibitory signal for an NK cell. Analyses of genetic variation in HLA-A, -B and -C genes indicate that human populations are split into two roughly equal groups with ~40% expressing high levels of HLA-E and the remaining individuals expressing low levels of HLA-E. A recent analysis of 9,763 treatment naïve, HIV-infected individuals demonstrated that higher levels of HLA-E expression profoundly correlates with immunosuppression and higher viral loads.
The objective of this study was to measure the prognostic power of HLA-E expression on tumor cells in newly diagnosed, treatment-naïve multiple myeloma (MM) patients. We hypothesized that expression of HLA-E will significantly influence NK cell and reactivity to tumors and their capacity to potentiate tumor-specific CD8 T cells as well as distinguish patients with enhanced natural ability to control the disease. This hypothesis is supported by recent evidence showing that knocking down HLA-E on B16 melanoma cells conferred 100% protection after challenge in mice treated with anti-PD-1 and the GM-CSF-secreting tumor vaccine (GVAX). In humans, however, emerging data suggest that the breadth and magnitude of NK cell reactivity vary strongly depending on education (HLA-E vs HLA-ABC). Thus, we further hypothesized that individuals with higher levels of HLA-E expression on their germline tissue would have repertoires of NK cells strongly educated/trained through interactions of HLA-E and NKG2A and a significantly higher threshold for NK cell activation in response to MM plasma cells.
We investigated the effect and prognostic impact of HLA-E expression in a cohort of 436 newly diagnosed, treatment naïve MM patients enrolled in the Multiple Myeloma Research Foundation (MMRF) CoMMpass study. We computationally inferred HLA-E expression based on HLA-A and -B genotypes determined using Optitype on Whole-Exome samples from matched germline and tumor tissue. To infer HLA-E expression, we used pre-determined expression scores for HLA-A that was generated from a large analysis of healthy and HIV-infected individuals determined by measuring the logistic regression of mean mRNA expression of HLA-A. We then determined whether the alleles of HLA-B encode a methionine (M) and/or threonine (T) at residue -21 of their leader sequence peptide. Using this approach, we then inferred whether an individual had low, medium or high expression of HLA-E. Finally, we stratified MM patients in three groups based on their HLA-E high (n=32), medium (n=296) and low (n=120) expression.
Using mass cytometry and a panel of 40 antibodies targeting broad phenotypes of major cell lineages known to reside in the circulation and in the bone marrow (BM), we performed ex vivo analyses of BM-derived CD138-negative cells from a subset of patients in our cohort.
Cox regression analysis showed that patients in the HLA-E high group had a significantly shorter duration of progression-free survival (PFS) compared to patients with medium and low HLA-E expression (Log-Rank p < 0.01, Fig. 1A).
Semi-supervised clustering analyses were performed on mass cytometry data using the Phenograph function (Fig. 1B and 1C). We clustered on 40 markers targeting major cell lineages, including B cells, T cells, NK cells, monocytes, macrophages, neutrophils and DCs. In the group with low/medium HLA-E expression, we observed expansions of effector memory CD4 T cells, CD57+ NK cells and three subsets of effector memory CD8 T cells. We also observed an opposite trend in the group with high HLA-E, where we found expansions of effector CD4 and CD8 T cells expressing the highest levels of PD-1 and TIGIT as well as a subset of macrophages and regulatory T cells (Tregs).
In conclusion, we demonstrated that HLA-E expression correlates with worse PFS in newly diagnosed MM patients. Our data suggests that HLA-E-mediated inhibition of NKG2A-expressing NK cells and T cells is a significant factor in host immune responses and clinical outcome in MM. We are currently analyzing a larger cohort of patient samples by mass cytometry to look more closely at phenotypes and functions of T cells, NK cells and myeloid cells.

Disclosures
Chari: Janssen: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Takeda: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Array Biopharma: Research Funding; Bristol Myers Squibb: Consultancy; Celgene: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Novartis: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Amgen: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Pharmacyclics: Research Funding; The Binding Site: Consultancy; Adaptive Biotechnology: Membership on an entity's Board of Directors or advisory committees; Seattle Genetics: Membership on an entity's Board of Directors or advisory committees. Cho:BMS: Membership on an entity's Board of Directors or advisory committees; F. Hoffmann-La Roche Ltd: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Genentech Inc: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Agenus Inc.: Research Funding; J & J: Consultancy; Janssen: Consultancy. Barlogie:Millenium: Consultancy, Research Funding; Myeloma Health, LLC: Patents & Royalties: : Co-inventor of patents and patent applications related to use of GEP in cancer medicine licensed to Myeloma Health, LLC; Multiple Myeloma Research Foundation: Other: travel stipend; ComtecMed- World Congress on Controversies in Hematology: Other: travel stipend; European School of Haematology- International Conference on Multiple Myeloma: Other: travel stipend; International Workshop on Waldenström's Macroglobulinemia: Other: travel stipend; Celgene: Consultancy, Research Funding; Dana Farber Cancer Institute: Other: travel stipend. Jagannath:Novartis: Consultancy; Merck: Consultancy; Bristol-Myers Squibb: Consultancy; Celgene: Consultancy; Medicom: Speakers Bureau; Multiple Myeloma Research Foundation: Speakers Bureau.