Genetic Mechanisms of Immune Escape in Diffuse Large B Cell Lymphoma

Diffuse large B cell lymphoma (DLBCL) is the most common form of B cell non-Hodgkin lymphoma (B-NHL), accounting for ~25-40% of all lymphoid tumors. DLBCL comprises genetically, phenotypically and clinically distinct subtypes, including the prognostically favorable germinal center B cell like (GCB)-DLBCL and the more aggressive activated B cell like (ABC)-DLBCL. We have shown that >60% of DLBCL, independent of molecular subtype, lack cell surface expression of HLA-class I (HLA-I), suggesting that these tumors may escape immune recognition by cytotoxic T cells (CTL) (Challa-Malladi, Lieu et al., Cancer Cell, 2011). HLA-I loss also represents a common lesion acquired at transformation of follicular lymphoma (FL) to DLBCL (Pasqualucci et al., Cell Reports 2014).

We have investigated the expression of HLA-I across the clinico-pathological spectrum of mature B cell tumors, and found that HLA-I loss is significantly less common in other mature B-NHL, including Burkitt lymphoma (13/43, 30.2%; p=.002), FL (12/60, 20.0%; p<.001), mantle cell lymphoma (1/38, 2.6%; p<.001), marginal zone lymphoma (0/39, 0%; p<.001), and chronic lymphocytic leukemia (1/36, 2.8%; p<.001). These results suggest that HLA-I loss and, thus, escape from recognition from CTL is an important pathogenetic feature of DLBCL.

One mechanism of HLA-I loss, identified by exome-sequencing and copy number analysis, is represented by genomic deletions and/or mutational inactivation of the B2M gene, which are found in ~50% of HLA-I negative cases (29% of all DLBCL). These lesions lead to the complete loss of B2-microglobulin, a required component for the assembly and cell surface expression of the HLA-I complex (Pasqualucci et al. Nat Genet, 2011; Challa-Malladi, Lieu et al. Cancer Cell, 2011). However, the remaining ~50% of patients lack surface HLA-I despite the absence of B2M genetic lesions, suggesting the existence of additional underlying mechanisms. In particular, a fraction of patients express an intact B2M protein, which is mislocalized to the cytoplasm. To investigate whether direct genetic disruption of the HLA-I genes could be responsible for the lack of surface HLA-I in these cases, we performed Sanger sequencing and SNP6.0 array analysis of the HLA-I heavy chain genes (HLA-A and HLA-B) in two DLBCL cell lines (Ly10 and RCK8) with wild-type B2M alleles, but cytoplasmic B2M protein. In both lines, we found the presence of biallelic mutations or deletions in the HLA-I loci. Accordingly, transduction with a retrovirus expressing either HLA-I gene was sufficient to restore cell surface B2M and HLA-I in both lines, documenting that DLBCL can exploit genetic disruption of HLA-I as an alternative mechanism to impair the assembly of a membrane HLA-I complex. The overall contribution of this mechanism to HLA-I loss is currently being determined by using a custom capture/next generation sequencing approach of the HLA-I loci in a large panel of paired tumor/normal biopsies with negative or mislocalized B2M/HLA-I.

We also examined the role of B2M (HLA-I) loss in lymphomagenesis in vivo. Particularly, since constitutional B2m deletion is not tumorigenic per se (Koller et al., Science 1990), and B2M loss is frequently acquired during FL transformation to DLBCL, we investigated whether the absence of major histocompatibility complex on the cell surface of mature B cells accelerates tumorigenesis in the presence of other oncogenic lesions. To this end, we generated a conditional knock-out mouse model in which the B2m gene is specifically deleted in germinal center B cells upon expression of a Cγ1-Cre allele, and crossed them with IµHABCL6 knock-in mice, which develop DLBCL due to deregulated expression of the BCL6 oncogene (Cattoretti, Pasqualucci et al., Cancer Cell 2006).