Comparative Analysis of T Cell Profiles in Splenic Small B-Cell Malignancies

Primary splenic small B-cell malignancies are rare disorders with still understudied biology, mainly due to the paucity of relevant primary spleen samples. A particularly uncharacterized aspect concerns the interactions of the malignant cells with their microenvironment. Here we sought to address this knowledge gap through interrogating T cell subset composition and T-cell receptor (TR) gene repertoires in single-cell suspensions from splenectomy samples of patients with splenic marginal zone lymphoma (SMZL; n=9), splenic diffuse red pulp lymphoma (SDPRL; n=6) and hairy cell leukemia variant (HCLv; n=8). Multi-color flow cytometry revealed that CD4⁺ cells were the predominant T-cell subpopulations in all entities, with significant (p<0.05) differences between CD4⁺ and CD8⁺ cells in SMZL (60.5% vs 31%) and SDRPL (47.3% vs 37.7%). CD4⁺ central memory (Tcm, CD45RA⁺CCR7⁺) and effector memory (Tem, CD45RO⁺CCR7^-) T cells were significantly (p<0.05) increased over the naïve (CD45RA⁺CCR7⁺) and effector memory CD45RA⁺ (CD45RA⁺CCR7^-) subpopulations in SMZL (35.2% & 48.4%) and SDRPL (25.4% & 56.3%). HCLv exhibited significantly (p<0.05) higher numbers of CD8⁺ Tem cells and, in contrast, lower numbers of exhausted (PD-1⁺CTLA-4⁺) CD8⁺ cells. Notably, a rare subpopulation of transitional double-positive (CD45RA⁺CD45RO⁺) T cells expressing CCR7 represented the dominant T-cell subset in 5/8 HCLv samples, whereas it was observed in only 2 SMZL cases and none of the SDRPL cases. TR beta chain gene repertoire analysis by NGS was performed in selected CD4⁺ and CD8⁺ cells from 12 cases (4 from each entity). Clonotypes (i.e. TRBV-TRBD-TRBJ gene rearrangements with unique pairs of TRBV genes and identical CDR3 sequences) were calculated and clonality was estimated as the median cumulative frequency of the 10 most frequent clonotypes per sample (MCF-10). CD8⁺ cells were more clonal (MCF-10: 24.5% in SMZL, 37.8% in SDRPL, 24.9% in HCLv) compared to CD4⁺ cells (MCF-10: 3.9% in SMZL, 4.9% in SDRPL, 8.9% in HCLv), with CD4⁺ cells in SMZL being significantly (p<0.05) more polyclonal compared to the other two entities. Finally, in-silico analysis was performed to identify specific T cell clones that may recognize neoantigens (nAgs) derived from the B-cell receptor immunoglobulin (BcR IG) expressed by the malignant cells, similar to the case in other B-cell malignancies (e.g. CLL and multiple myeloma). The heavy and light CDR3 sequences from the clonotypic BcR IG were dissected into k-mers of 9-15 amino acids length, representing the putative nAg. HLA haplotypes were determined from whole exome data using OptiType, while high-affinity nAg-MHC pairs, likely to be naturally presented on cell surfaces, were selected based on NetMHCpan4.1 results. ERGO-II was employed to identify tumor-specific T cells recognizing these nAgs by selecting TR-nAg-MHC complexes with binding probability >0.9. The BcR IG of SMZL cases generated the largest number of immunogenic nAgs (median of 12 from the heavy and 8 from the light CDR3 vs 8 heavy and 4 light CDR3 nAgs in the other entities). A varying number of mostly low-frequent nAg-specific CD8⁺ clones were identified, with medians ranging from 6 in SMZL to 17 in SDPRL and 33 in HCLv. In conclusion, we report distinct T-cell profiles in different primary splenic small B-cell malignancies with substantial numbers of functionally exhausted CD8⁺ cells in SDRPL; higher levels of CD4⁺ Tem and Tcm in SMZL, indicative of chronic antigen exposure; and, increased numbers of CD8⁺ Tem cells along with frequent occurrence of a population of transitional T cells capable of lymphoid trafficking in HCLv. Moreover, we offer immunogenetic evidence that the clonotypic BcR IG can serve as a source of neoantigens shaping the TR repertoire of these entities.

Chu:Pfizer: Current equity holder in private company. Zent:Hairy Cell Leukemia Foundation: Research Funding; Acerta/AstraZeneca: Research Funding; GenMab: Research Funding. Stamatopoulos:AbbVie: Honoraria, Research Funding; AstraZeneca: Honoraria, Research Funding; Janssen: Honoraria, Research Funding; Novartis: Research Funding; Roche: Research Funding; Bristol Myers Squibb: Honoraria; Lilly: Honoraria.