Follicular lymphoma-protective HLA class II variants correlate with increased HLA-DQB1 protein expression

Abstract Major histocompatibility complex (MHC) class I and class II are similar in function since they present peptides at the cell surface to CD8+ and CD4+ T cells, respectively. There is mounting evidence indicating that MHC genes play a role in the etiology and clinical course of non-Hodgkin lymphomas (NHL), especially in diffuse large B-cell lymphoma (DLBCL). We investigated the expression of human leukocyte antigen-G (HLA-G) and HLA class II protein in DLBCL among 148 patients and related their expression to the clinical course of the disease. Negative HLA-G expression was associated with a lower probability of achieving a complete remission (P= 0.04). There was no impact of HLA-G or HLA class II expression on the probability of progression-free survival (PFS). Patients with positive HLA-G expression presented a trend towards a higher 3-year overall survival (OS) rate compared to those with negative expression of HLA-G (P= 0.08). The estimated 3-year OS rate of patients with positive HLA class II expression was 59.4% (95%CI 49-69) in comparison to the 37.4% (95%CI 22-56; P= 0.04) in subjects with negative expression. In a multivariate Cox analysis adjusted for the IPI factors, we found that both the intermediate high/high IPI risk group (P= 0.001) and the loss of HLA class II expression (P= 0.05) independently increased the risk of death in the study group. We also investigated whether the impact of HLA class II expression on OS may be related to the subtypes of DLBCL. In the subgroup of 58 patients (39%) with GCB-type pattern, the patients with the loss of HLA class II expression presented a significantly lower 3-year OS rate than those with its positive expression (26% [95% CI 10-53] vs 68.2% [95% CI 51-81], P= 0.02). In contrast, in the subgroup of 90 non-GCB patients (61%), HLA class II expression did not influence OS. To further explore the unexpected favorable effect of positive HLA-G expression on the clinical course of DLBCL, we performed an additional analysis that considered the type of treatment (chemotherapy with or without rituximab). In the group of patients treated with immunochemotherapy, a more pronounced effect of the positive HLA-G expression on OS was revealed. The estimated 3-year OS rate of patients with the positive HLA-G expression was 73.3 % (95% CI 49-88) compared to 47.5% (95% CI 35-60, P= 0.03) in subjects with the negative HLA-G expression. In contrast, in the group treated with CHOP-like regimens, no significant impact of HLA-G expression on OS was observed: the 3-year OS rate for HLA-G positivity was 20% (95% CI 4-62) vs 55.3% (95% CI 37-72; P= 0.08) for the absence of HLA-G. Additionally, the prognostic value of HLA class II expression was also shown to depend on the use of rituximab as a part of first line treatment. In the patients receiving immunochemotherapy, those that had positive HLA class II expression demonstrated a 3-year OS rate of 65.3% (95% CI 52-76) compared to 29.6% (95% CI 13-53, P= 0.04) in subjects with the loss of HLA class II expression. However, HLA class II expression did not have a prognostic impact on OS in the patients treated with chemotherapy alone: the 3-year OS rate was 49.5% (95% CI 32- 67) in the subjects with positive expression in comparison to 50% (95% CI 15-85, P= 0.8) in those with the loss of HLA class II expression. In conclusion, we demonstrated for the first time expression of HLA-G protein in DLBCL and its association with the clinical course of the disease as well as we confirming the association of the loss of HLA class II protein expression with poor survival in patients treated with immunochemotherapy. Although the clinical significance of the loss of HLA class II protein expression seems to be well understood, the contribution of HLA-G to the prognosis of B-cell malignancies deserves further study, especially its immunoregulatory functions in relation to treatment with rituximab, which remains an open question. Disclosures Robak: MorphoSys AG: Research Funding.

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Intratumoral and Peripheral Blood T Cells Recognize Mutated Neo-Antigens in Follicular Lymphoma

Blood ◽

10.1182/blood.v124.21.809.809 ◽

2014 ◽

Vol 124 (21) ◽

pp. 809-809

Author(s):

Seung Tae Lee ◽

Elena Percivalle ◽

Xiaoyun Cheng ◽

Greg Lizee ◽

Nathan Fowler ◽

...

Keyword(s):

T Cells ◽

Follicular Lymphoma ◽

Cd8 T Cells ◽

Peripheral Blood ◽

Class Ii ◽

Hla Class Ii ◽

Adoptive T Cell Therapy ◽

Class I ◽

Wild Type ◽

Binding Peptides

Abstract Introduction: The tumor microenvironment of follicular lymphoma is heavily infiltrated with CD4+ and CD8+ T cells that are tumor-reactive and produce cytokines such as IFN-g, TNF-a, and GM-CSF that may play a role in tumor control or regression. However, the nature of the tumor antigen(s) recognized by these T cells is unknown. Here, we investigated the possibility that intratumoral T cells recognize mutated tumor antigens, which may be recognized as neo-antigens by the host immune system as opposed to non-mutated proteins that are recognized as self-antigens. Methods: Whole exome sequencing was performed on three follicular lymphoma tumors and their corresponding germline (peripheral blood mononuclear cells, PBMC) samples obtained at initial diagnosis. Nonsynonymous mutations were identified. The binding affinities of the peptides spanning each mutation to their respective MHC class I and class II molecules were determined in silico by MHC binding algorithms. Peptides predicted to have high binding affinity (IC50<500 nM) were selected for synthesis. For MHC Class I binding peptides, nonamer peptides predicted to bind to HLA-A*0201 (all three follicular lymphoma patients expressed HLA-A*0201) were synthesized and for MHC Class II binding peptides, 40-mer polypeptides were synthesized with the mutated amino acid in the middle. Corresponding wild type peptides were synthesized where necessary. Autologous T cells isolated from PBMC or tumors obtained at initial diagnosis were used to test reactivity to mutated and/or wild type peptides. Autologous PBMCs depleted of T cells were used as antigen-presenting cells in co-culture assays with peripheral blood or intratumoral T cells. IFN-g was assessed in culture supernatants by ELISA. Results: In patient 1, of 61 nonsynonymous mutations, 9 nonamer peptides and 16 pentadecamer peptides were predicted to bind with high affinity to HLA-A*0201 and HLA Class II alleles expressed in the patient, respectively. Of the 9 nonamer HLA-A*0201-binding peptides, two were recognized by autologous peripheral blood and intratumoral T cells. Of the 16 HLA Class II-binding peptides, autologous peripheral blood and intratumoral T cells recognized four. HLA Class II and HLA-DR blocking antibodies but not HLA Class I and HLA-DP or DQ blocking antibodies inhibited production of IFN-g in response to the HLA Class II-binding peptides suggesting that they were recognized by CD4+ T cells. More importantly, the T cells were not reactive against the wild type peptides tested suggesting that the mutations converted the non-immunogenic wild type proteins into immunogenic neo-antigens. Testing of mutated peptides from additional patients is ongoing. Conclusions: Our results suggest that peripheral blood and intratumoral T cells recognize mutated neo-antigens in follicular lymphoma. These results are consistent with recent results from solid tumors that showed intratumoral CD4+ and CD8+ T cells primarily recognize mutated neo-antigens. More importantly, administration of such neo-antigen-reactive CD4+ and CD8+ T cells as adoptive T-cell therapy resulted in long-term tumor regressions in humans. Thus, identification of immunogenic neo-antigens may facilitate development of novel personalized forms of cancer immunotherapy including therapeutic vaccines and adoptive T-cell therapy strategies. Moreover, characterization of such cancer regression antigens will be valuable to understand the effects of immune checkpoint antagonists and other novel immunotherapies. Disclosures No relevant conflicts of interest to declare.

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