Phenotype And Characteristics Of Tumor-infiltrating Lymphocytes In Meningiomas

Objective:Infiltration of B lymphocytes into the tumor tissue of meningioma patients is a common occurrence, but till now, research on the phenotype, characteristics and possible function mechanism of these cells in the immune response is limited. We have therefore investigated the phenotype and immunoglobulin (Ig) variable region of heavy chain (VH) repertoire of B cells infiltrating22meningiomas.Methods:1. Immunohistochemistry was used to grossly characterize and enumerate infiltrating lymphocytes.2. A group of294rearranged Ig VH genes from micro dissected foci of meningioma-infiltrating B cells,34from normal single-sorted naive B cells and45from normal single-sorted antigen-experienced B cells were amplified, cloned, and sequenced to characterize their antigen experience.3. BASELINe algorithm to detect the antigen selection strength of TIL-Bs Ig VH genes.Results:1. There are different numbers of CD4+B cells and CD138+antibody-secreting plasma cells sporadically disperse through meningioma tissues. No particular region provided a preferential environment for the infiltrate.2. Neither the frequency nor the type of cells in the infiltrate correlated with tumor WHO grade.3. Compared with the human B cell Ig VH germ-line genes, there were19nucleotide mutations and11amino acid replacements on average in TIL-Bs Ig VH genes, which are about400nucleotides in length, and those numbers were consistent with single-sorted antigen-experienced B cells from normal subjects,15and9respectively. On contrary, nearly no nucleotide mutations were observed in single-sorted naive B cells from normal subjects (<1).4. We sequenced294TIL-Bs from22meningioma specimens. There were46%B cells belonged to clones, and the rest were not correlated with each other. On average, there were three clones of each meningioma, and there were six B cells in each clone. Of those six B cells, five were identified as intraclonal variants. No clonal expansion B cells in the PBMCs were observed.5.72%TIL-Bs and67%single-sorted antigen-experienced B cells from normal subjects underwent isotype switching from IgM+To IgG+, indicating that TIL-Bs were also antigen experienced.6. Antigen selection strength was evaluated, and we found positive selection within complementarity determining regions, and negative selection within framework regions.Conclusions:Analysis of the characteristics of Ig VH expressed by tumor-infiltrating B cells in meningioms shows that these cells are undergoing isotype switching, clonal expansion and intraclonal variants, somatic hypermutation and affinity maturation, indicating that TIL-Bs are meningioma-related antigen-experienced and activated effector/memory B cells. ABSTRACT:Objective:T lymphocytes play a key role in tumor immunity, including acting as effectors and regulators. There are T cells infiltrating meningioma tissues, but till now, there’s no research on the characteristics of those T cells. In order to explore the potential mechanism of the tumor infiltrating T cells (TIL-Ts) in meningiomas, we studied the subtypes and T cell receptor (TCR) Vβ repertoire within both the TIL-Ts and matched peripheral blood T cells.Methods:1. Immunohistochemistry was used to grossly characterize and enumerate infiltrating lymphocytes.2. Flow cytometry of fresh tissue homogenate and paired peripheral blood lymphocytes was used to identify T cell phenotypes and characterize the T cell repertoire.Results:1. There were more T cells infiltrating meningiomas than B cells. Some T cells often assembled in clusters in the perivascular space.2. The frequency of T cells in the infiltrate did not correlate with tumor WHO grade.3. More CD4+T cells (64%) than CD8+T (36%) were observed within the meningioma patients’peripheral blood, consistent with those within PBMCs of normal subjects. On the contrary, there were only22%CD4+T cells in TIL-Ts, and most of them (78%) were CD8+cytotoxic lymphocyte (CTL) cells.4. There were more antigen-experienced than naive CD4+/CD8+T cells in meningiomas, while the distribution was totally opposite in PBMCs of the same patient.5. Both PD-1and Tims-3were over expressed in meningiomas compared to matched PBMCs.6. Tregs were over expressed in meningiomas compared to matched PBMCs.7. The TCR Vβ gene usage was quite similar within TIL-Ts and matched peripheral blood within CD4+T cells. But the situation was totally different within CD8+T cells. Within one meningioma, Vβ3,Vβ2and VP21.3were selectively expressed within TIL-Ts, while Vβ2was only expressed within peripheral blood, indicating that the two different T cell populations underwent different activation pathways. Conclusions:1. The over-expression of suppressive markers PD-1and Tim-3, as well as T regulatory cells, maybe involved with the development of meningiomas.2. The analysis of TCR Vβ repertoire revealed that, the TCR Vβ gene usage is skewed, both within CD4+/CD8+TIL-Ts and peripheral blood T cell population. All our results indicate that TIL-Ts are meningioma-related antigen-experienced and activated effector/memory T cells.