Expression Of LAG-3 And PD-1 In Diffuse Large B-Cell Lymphoma And Their Effect On Cell Function

Objective: This study intends to observe the expression of LAG-3 and PD-1 at the tissue,peripheral blood and cell levels of DLBCL.The question of whether the combined use of LAG-3 and PD-1 inhibitors can improve the efficacy of monotherapy is of significant clinical interest and also offers a new idea for individualized treatment.Methods: In the first part,1)146 patients diagnosed with DLBCL by paraffin-embedded histopathology were selected as the experimental group and 30 patients with reactive hyperplasia of the lymph nodes were selected as the control group.2)Epidemiological,clinicopathological and morphological characteristics of DLBCL were analyzed retrospectively,and the relationship between the clinicopathological characteristics of GCB and non-GCB in DLBCL was analyzed in combination with immunohistochemical results.3)The expression of LAG-3 and PD-1 in DLBCL group and reactive hyperplasia lymph node group were detected by immunohistochemical method,and the correlation between the expression and clinicopathologic features was analyzed.4)Kaplan-Meier curve was plotted to estimate the overall survival and prognosis distribution,and Cox regression model was used for multivariate survival analysis.The second part: 1)Peripheral blood samples of 92 patients with DLBCL and 30 patients with normal control were collected.Flow cytometry was used to detect the fraction of T lymphocyte subsets and the correlation between differential expression of LAG-3 and PD-1 on T lymphocytes between the experimental and normal control groups,as well as clinical characteristics.2)70 peripheral blood serum samples of DLBCL patients and 30 peripheral blood serum samples of normal control group were collected.CBA was used to detect correlations between differences in cytokine levels and expression of LAG-3 and PD-1 in T lymphocytes in peripheral blood serum samples as described above,as well as clinicopathological features.Part Three: Peripheral blood CD8+T cells and tumor cells(SU-DHL6,OCI-LY3)of DLBCL patients were co-cultured by magnetic beads,and LAG-3 and PD-1 blockers were added,respectively.After culture,the CD8+T cells were again sorted by magnetic beads,and the expression of LAG-3 and PD-1 on the surface of the CD8+T cells was detected by flow cytometry.CD8+T perforin and granzyme B expression were detected by intracellular staining,tumor apoptosis was detected by Annexin V/7-AAD flow cytometry,and IFN-γ expression was detected by CBA supernatant.Results: Part I: There were 146 DLBCL patients,mostly middle-aged males with an average age of 57 years.The first clinical symptoms are enlargement of the cervical lymph nodes accompanied by fever.Most of the lesions were in the brain and gastrointestinal tract,with a few involving the mouth,spine,mammary glands,kidneys and testes.Peripheral blood tests were frequently combined with decreased lymphocyte counts and increased LDH.There were 51 cases(34.9%)of the GCB subtype and 95 cases(65.1%)of the non-GCB subtype of DLBCL.CD10 expression rate was 24%,BCL-6expression rate was 80.8%,MUM-1 expression rate was 82.2%,BCL-2 expression rate was 37%,C-MYC expression rate was 28.8%,Ki-67 expression rate was 82.2%,non-GCB subtype in DLBCL was correlated with IPI > 3 of the patient’s score.The prognosis for non-GCB patients is poor.At the tissue level,LAG-3 and PD-1 were widely expressed in DLBCL TILs,with positive expression rate of LAG-3(n=71/144,49.3%)and positive expression rate of PD-1(n=75/142,52.8%).The positive co-expression rate of LAG-3 and PD-1 accounted for 32.1%(n=45/141),and the positive expression rate of LAG-3 or PD-1 alone accounted for 34.3%(n=48/141).Analysis of clinical and pathological features showed that the positive expression of LAG-3 was correlated with the invasion of tumor nodular,the positive expression of PD-1 was correlated with the age>60 years old and the positive expression of BCL-2,and the positive co-expression of LAG-3 and PD-1 was correlated with the age > 60 years old.Combined with clinical characteristics and expressions of LAG-3 and PD-1,a univariate analysis was performed and statistically significant single factors were included in a multivariate survival analysis.Age over 60,decreased lymphocyte count,and positive LAG-3 expression are independent prognostic risk factors for OS in DLBCL patients.Part II: Peripheral blood samples from DLBCL patients confirmed a decrease in the proportion of CD4+T cells,an increase in the proportion of CD8+T cells,a shift in the subset of T cells to the subset of CD8+T cells,and a decrease in the ratio of CD4+/CD8+T cells.PD-1 is highly expressed in CD8+T cells,LAG-3 is highly expressed in both CD4+T and CD8+T cells,and LAG-3 and PD-1 are highly expressed in both CD8+T cells.Additional analysis of the correlation between LAG-3+CD4+T,PD-1+CD8+T,LAG-3+CD8+T,and LAG-3+CD8+T cells and clinicopathological features showed that LAG-3+CD8+T cells were associated with IPI> 3scores and LDH≥250(U/L).Peripheral blood cytokines of DLBCL patients were selected to detect IL-6,IL-10,IL-2 and IL-4 levels increased,while TNF-α and IFN-γlevels decreased.Moreover,the decreased expression level of IFN-γ was correlated with age>60 years,III and IV clinical stages,number of extranodal invasion ≥2,LDH≥250(U/L),and IPI>3 scores.Furthermore statistical correlation analysis of IL-6,IL-10,IL-2,IL-4,TNF-α,IFN-γ,LAG-3+CD4+T,PD-1+CD8+T,and LAG-3+PD-1+CD8+T cells and cytokine levels showed that IFN-γ level and the proportion of LAG-3+CD8+T cells were negative related.Part III: This study continued to confirm at the cellular level that CD8+T cells and SU-DHL6/OCI-LY3 in DLBCL patients were co-cultured in vitro,and the expression ratio of perforin and granzyme B and IFN-γ secretion level of CD8+T cells were increased after the addition of LAG-3 or PD-1 inhibitors alone,and the total apoptosis ratio of tumor cells was increased.The combination of LAG-3 and PD-1inhibitors,the expression ratio of perforin and granzyme B and the secretion level of IFN-γ were better than those of LAG-3 or PD-1 inhibitors alone,and the total apoptosis of tumor cells was significantly increased.Conclusion: LAG-3 and PD-1 are widely and deeply expressed in TILs and peripheral blood of DLBCL tissue,and are correlated with prognosis and stage.The abnormal expression of immunosuppressive receptors on the surface of T lymphocytes may lead to blocked T cell proliferation and differentiation,imbalanced subpopulation proportions and abnormal cell function.CD8+T may be one of the critical cell subpopulations subject to immunosuppression.The combination of LAG-3and PD-1 inhibitors restored CD8+T secreting cytokine function better than the use of the inhibitors alone.Elevated expression of LAG-3 and PD-1has a strong inhibitory effect on the function of CD8+T cells,leading to diminished function in killing tumor cells.Combined blocking of LAG-3 and PD-1 will help restore the function of immune cells and provide a new direction for personalized cellular immunotherapy of DLBCL.