MR1-Restricted Immunity in Hematologic Malignancy

Introduction: Immunotherapy has revolutionized the treatment of many cancer types, yet approved agents such as immune checkpoint inhibitors have shown inconsistent efficacy against blood cancers. There are inherent limitations to targeting conventional peptide-specific T cells as they produce variable, patient-specific responses dependent on diverse HLA molecules. In contrast, innate T cells like the highly abundant mucosal-associated invariant T (MAIT) cell in humans, recognize conserved non-peptide antigens presented by an evolutionarily conserved oligomorphic MHC class I-related protein (MR1). Recent studies have identified self-reactive MR1-restricted T cells with broad targeting against cancers, providing the rationale to study their application in donor-unrestricted, off-the-shelf immunotherapy; however, the frequency and phenotype of tumor-reactive MAIT cells in cancer patients and the self-antigens responsible for their induction remain unknown. We hypothesize that distinct MAIT cell subpopulations exercise anti-tumor helper/regulatory and cytotoxic functions that synergize to control malignant proliferation.

Methods: We enrolled 30 subjects with newly diagnosed hematologic malignancy (lymphoid, n=25; myeloid, n=5) and collected longitudinal biospecimens (blood, bone marrow, and/or tissue) over the course of treatment. Healthy donors were also recruited as controls (n=26). MAIT cell abundance (CD161⁺/TCRVa7.2⁺ of CD3⁺ cells), surface activation receptors (CD69, CD25, PD1), and intracellular effector molecules/transcription factors (GZB, IFNγ, FOXP3) were measured by spectral flow cytometry. We conducted in vitro stimulation assays to evaluate the activity of CD4- or CD8-expressing MAIT cell subpopulations, including responses to purified MR1 ligands derived from riboflavin (5-OP-RU) and folic acid (6-FP) biosynthesis pathways.

Results: We found that the peripheral blood frequency of CD8⁺ MAIT cells were significantly decreased in patients with myeloid (31%±25% of total MAIT; p=0.002) or lymphoid (56%±27%; p=0.02) malignancy compared to healthy donors (81±9%), while the frequency of CD4⁺ MAIT cells was unchanged. CD4^-CD8^- double negative (DN) MAIT cells were significantly decreased in myeloid leukemia relative to healthy donors (2±2% vs 10±5%; p=0.02) but unchanged in lymphoid cancer. There was also a higher baseline of CD4⁺CD8⁺ double positive (DP) MAIT cells in lymphoid cancer compared to controls (14±27% vs 2±1%; p=0.04). These DP MAIT cells exhibited high levels of baseline activation and did not expand in culture following stimulation with purified MR1 ligands. In donors responding to treatment, there was a progressive re-expansion of the CD8⁺ subset. Further, both myeloid and lymphoid cancers had more robust MR1 ligand-inducible MR1 expression on B cells relative to healthy donors with similar trends in monocytes, T cells and natural killer cells, suggesting that multiple immune lineages may act as antigen presenting cells.

Conclusions: Our study found significant alterations in MAIT cell CD4- and CD8-expressing subpopulations in patients with hematologic malignancies relative to healthy donors with normalization of their distribution during treatment. This supports our ongoing work to understand the roles of these distinct subsets in anti-tumor immunity through application of single cell genomics technologies in study volunteers and direct MR1 ligand capture from primary cancer cells using human platform MR1 baculoviral expression systems. Defining conserved MR1 ligands in these blood cancers and the MAIT cell subsets that they induce will inform transformative approaches to MR1-directed cancer immunotherapy.

Lee:Kite Pharma: Consultancy.