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Angioimmunoblastic T-cell lymphoma with predominant CD8+ tumor-infiltrating T-cells is a distinct immune pattern with an immunosuppressive microenvironment

Background Angioimmunoblastic T-cell lymphoma (AITL) has a rich tumor microenvironment (TME) that typically harbors plenty of CD4+tumor infiltrating lymphocytes, (TIL)-T-cells (so called common AITL). Nonetheless, AITL with large numbers of CD8+TIL-Ts that outnumber CD4+cells have been observed (CD8-predominant AITL). However, detailed comparison of CD8-predominant AITL and common AITL are still lacking. Methods We compared clinicopathological features, TIL subsets, TME T cell receptor-β (TRB), and immunoglobulin heavy chain (IGH) repertoires, and gene expression profiles in six CD8-predominant and 12 common AITLs using case-control matching (2014 to 2019). Results Comparing with common AITLs, CD8-predominant AITLs showed more frequent edema (P = 0.011), effusion (P = 0.026), high elevated plasma EBV-DNA (P = 0.008), and shorter survival (P = 0.034). Moreover, they had more pronounced eosinophil increase (P = 0.004) and a higher Ki67 index (P = 0.041). Flow cytometry revealed an inverted CD4/CD8 ratio in TIL-Ts and lower TIL-B proportions (P = 0.041). TRB repertoire metrics deteriorated, including lower productive clones (P = 0.014) and higher clonality score (P = 0.019). The IGH repertoire was also narrowed, showing a higher proportion of the top 10 clones (P = 0.002) and lower entropy (P = 0.027). Gene expression analysis showed significant enrichment for upregulated negative regulation of immune system processes and downregulated T-cell activation and immune cell differentiation. Conclusion Our findings demonstrated that CD8-predominant AITL is a distinct immune pattern of AITL characterized by anti-tumor immunity impairment and an immunosuppressive microenvironment. These characteristics can interpret its severe clinical manifestations and poor outcomes.


Introduction
Angioimmunoblastic T-cell lymphoma (AITL) is a subtype of peripheral T-cell lymphoma (PTCL) with a T follicular helper (TFH) phenotype (1). In our previous analysis, it was the fourth most common mature T-cell lymphoma, accounting for 11% of all patients with PTCL (2). Patients with AITL frequently exhibit clinical symptoms, including skin rash, arthritis, effusion, and positive autoimmune tests, indicating an aberrant immune response (3,4). Finally, patients exhibit immunodeficiency secondary to neoplastic development. Even following an intensive treatment regimen, the prognosis is generally poor, with most studies reporting a median survival of < 3 years (5). As a result, immunological dysregulation characterizes the entire course of AITL and has been identified as a distinguishing feature of this malignancy.
AITL possesses a complex tumor microenvironment (TME) composed of irregularly proliferating follicular dendritic cells in the meshwork, arborizing high endothelial venules (HEVs) and a variety of reactive immune cells, the most obvious of which are B-immunoblasts, plasma cells, and tumor-infiltrating lymphocyte (TIL) -T cells (TIL-Ts) (6). Immunostaining has revealed that AITL TIL-Ts often have a significantly larger proportion of CD4+ cells than CD8+ cells (defined as common AITL in our study), which is due to CD4 expression in neoplastic T-cells and a normal CD4+/CD8+ ratio in TIL-Ts (CD4+TIL-Ts outnumber CD8+TIL-Ts) (7,8). Nonetheless, we observed a few cases of AITL with an extremely inverted CD4/ CD8 ratio (defined as CD8-predominant AITL in our study), indicating the significant increase of CD8+TIL-Ts. However, CD8-predominant AITL was rare, accounting for only 1.5% of AITL cases in daily practice (Time: 2014-2019).
The different CD8+ TIL-T proportions in the TMEs of CD8predominant AITL and common AITL implied that TME immune function differed between the two AITL groups (7). Furthermore, the immune function of the TME not only correlated with the patient's clinical manifestations related to inflammation and immune response but also reflects anti-tumor immunity, which has a direct impact on tumor development and progression. However, detailed comparison of CD8-predominant AITL and common AITL are still lacking. Therefore, we used a case-control matching approach to include cohorts of CD8-predominant AITL and common AITL cases for clinicopathological analysis, flow cytometry testing, T cell receptor-b (TRB), immunoglobulin heavy chain (IGH) repertoire sequencing, and RNA sequencing. Data were compared to gain a better understanding of the TME immune function of this uncommon CD8-predominant AITL and its connection with clinicopathological findings.

Case selection
The CD8-predominant AITL cohort was identified from the database of the Department of Pathology of West China Hospital, Sichuan University from January 2014 to October 2019 according to the following criteria: 1) Diagnosed with lymph node sample and fulfilled the WHO-classified diagnostic criteria of AITL (4th edition, 2008/Revised 4th edition, 2017); 2) identification of neoplastic T-cells with aberrant expression of T cell markers by flow cytometry (9); 3 ) id en t ifi c a t i o n o f C D 8 + c e l l s / C D 4 + c e l l s > 1 b y immunostaining; and 4) de novo cases. For comparison, we employed case-control matching to randomly choose de novo common AITL cases (diagnosed with lymph node sample) from a list of potential matched controls throughout the same period (2014-2019, Supplementary Method in detail). In order to eliminate the influence of confounding factors on the prognosis, we matched the CD8-predominant and common AITLs based on the neoplastic T-cell proportion (detected by flow cytometry) and treatment strategy. Two common AITL cases were matched to each CD8-predominant AITL case. Case-control matching was performed using SPSS (v24.0; SPSS Corp., Chicago, IL, USA). Pathological data were gathered via slide review by four expert hematopathologists (Z.C., S. Z., W. Z., and W. L.). The pathological analysis, flow cytometry, and the following molecular analysis were all performed on the same lymph node sample of each case. Detailed clinical data were collected from medical records. Follow-up data were obtained via telephone interviews and/or medical records. Overall survival (OS) was calculated from the date of diagnosis to the date of death or the last follow-up. Histological, immunohistochemical, and Epstein-Barr virus (EBV) status assessment Sections (4 µm) were cut from paraffin blocks and stained with hematoxylin and eosin for histological examination. Immunohistochemical staining was performed using the following antibodies: cytoplasmic CD3 (cCD3), CD20, CD5, CD4, CD8, CD10, CD21, Bcl-6, CXCL13, PD1, CD30, and Ki-67. The basic information of the antibodies is summarized in the Supplementary Method (Supplementary Material Data Sheet 1). In situ hybridization with a digoxin-labeled oligonucleotide probe complementary to EBER-1 and EBER-2 (EBER1/2; Dako, NO. Y520001) was used to assess EBV status.

Flow cytometry
The process for sample preparation, staining, acquisition, data analysis, and identification of neoplastic T-cells and TILs was performed in the same manner as previously described (9)(10)(11). The fluorescent-labeled antibodies used in this study are listed in Supplementary Method (Supplementary Material Data Sheet 1). Flow cytometry data were collected using the BD FACSCanto II equipped with two lasers (blue and red) and analyzed with FACSDiva software. The proportion of neoplastic T-cells and various TIL subsets, including TIL-Bs (CD20 + ), activated TIL-Bs (CD38 dim+ CD20 + ), non-activated TIL-Bs (CD38 -CD20 + ), TIL-Ts (CD3 + ), CD4+TIL-Ts, CD8 +TIL-Ts, and plasma cells (CD38 bight+ CD20 -) were defined as counts of each type of cell/total counts of lymphocytes.

TRB and IGH sequencing
DNA was isolated from formalin-fixed paraffin-embedded tissues using the QIAamp DNA FFPE Tissue Kit (Qiagen Inc., Valencia, CA, USA). For TRB and IGH repertoire sequencing, the LymphoTrack kits (TRB Assay and IGH Assay) were used following the manufacturer's instructions with 50 ng of AITL specimen DNA as a template. Primers in the LymphoTrack assays were designed using Illumina adapters. Each amplicon was purified using AMPure XP beads (Beckman Coulter, Brea, CA, USA) and quantified using an Agilent 2100 Bioanalyzer (Santa Clara, CA, USA). Subsequently, the samples were sequenced on an Ion PGM (Thermo Fisher Scientific, Loughborough, UK). Sequencing reads were then aligned using MiXCR (v3.0.13), and only productive rearrangements were included in further analyses.
Lymphoma-derived clone were identified based on TRB clonal rearrangement analysis following the manufacturer's instructions and removed to obtain the TRB repertoire of the AITL TME (Supplementary Method in detail). The proportion of the top 10 clones ["% top 10 maximal frequency clones"; Frequency of each clone in AITL TME = reads of clone/(Total reads -Reads of lymphoma clone)], Shannon's entropy (entropy), and the clonality score were calculated. The numbers of productive clones in the samples were identified by calculating the number of "productive rearrangements". This is a measure of the number of functional T or B cells with a distinct TR or IGH rearrangement which represents the "richness." Entropy measures both the sample richness and the degree of unevenness in clone frequencies. The clonality score describes the "evenness" of the distribution of TR or IGH clones in the repertoire, that is, how much of the TRB or IGH repertoire is composed of expanded clones independent of sample size. The top 10 clones is the frequency of the top 10 dominant clones identified in each sample. The distribution of V-gene segment usage by both TRB and IGH in CD8-predominant AITL and common AITL were compared. Both shared and differential clones with a specific complementarity determining region 3 (CDR3) sequence between the two AITL groups were identified. These analyses were performed on R using the "LymphoSeq" and "tcR" packages (12-15).

RNA sequencing
The procedures of RNA sequencing, quality control, and normalization of data were performed by Shanghai Rightongene Biotechnology Co. Ltd (Shanghai, China), and the differentially expressed genes were identified using the R package "limma" (v3.38.3).

Statistical analysis
Continuous variables were analyzed using the Mann-Whitney test, while categorical variables were compared using Fisher's exact test. Survival data were calculated using the Kaplan-Meier estimator. The log-rank test was used to compare survival functions. Tests were considered significant at two-sided probability values less than 0.05 (P <0.05). All results were analyzed using GraphPad Prism (v8.0.2; GraphPad Software Inc., USA) or SPSS (v24.0; SPSS Corp., Chicago, IL, USA).

IGH repertoires
IGH sequencing was performed on the same samples as the TRB sequencing. The productive clones were not different between CD8-predominant AITLs and common AITLs ( Figure 4D). CD8-   The clonality score did not differ between the two groups ( Figure 4D).
The global usage of IGHV segments did not differ between CD8-predominant AITLs and common AITLs, with IGHV3-7 being the most frequently used segment in both AITL groups Comparison of TRB and IGH repertoires between CD8-predominant AITLs and matched common AITLs. Comparison between two AITL groups TME in statistical metrics including production clones, V-gene family usage, top 10 clones' proportion, clonality score, and entropy of TRB repertoire (A) and IGH repertoire (D), in global usage of V-gene of TRB repertoire (B) and IGH repertoire (E), and in the CDR3 sequence frequency of the TRB (C) and IGH repertoires (F). "ns" indicates "no significance".

Comparative gene expression profiling between CD8-predominant and common AITL for inflammation and immune response
RNA sequencing was performed on three CD8-predominant AITLs and four common AITLs. To compare these two groups for inflammation and immune response, we created a panel of 772 genes based on the gene ontology biological process annotation (Supplementary Method in detail, the gene expression profile data were provided in Supplementary Table  S4). Comparative analysis identified 36 genes in this panel with significant differential expression between CD8-predominant AITL and common AITL through the linear model fit with pvalue correction (Padj < 0.05 and log2fc > 1), of which 20 genes were upregulated and 16 genes were downregulated in CD8predominant AITLs ( Figure 5A). Functional annotation analysis revealed predominant enriched differentially expressed genes in T-cell activation, negative regulation of immune system process, regulation of leukocyte activation, and regulation of lymphocyte activation ( Figure 5B). CD8-predominant AITL was considerably enriched for upregulated negative regulation of immune system processes ( Figure 5C) and downregulated T-cell activation and immune cell differentiation ( Figure 5D) compared with common AITL, which suggested suppressed immune function.

Survival analysis
Survival data were obtained from all six patients with CD8predominant AITL and 11 of the 12 patients with common AITL. Four patients with CD8-predominant AITL (4/6, 67%) and three patients with common AITL (3/11, 27%) died of the disease. Besides tumor proportion and treatment strategy, the other recognized prognostic factors were also comparable between the two AITL groups ( Table 1). The prognosis of patients with CD8-predominant AITL (median overall survival: 5.0 months, 95%CI: 0-14.4 months) was significantly worse than that for those with common AITL (median overall survival not reached, P = 0.034, Figure 5E).

Discussion
AITL is highly heterogeneous, and exhibits complex TMEs (16). Our previous research has discovered that the ratio of the components, particularly TILs, varies in AITL cases (9). When CD8+TIL-Ts proliferated abundantly, they outnumbered CD4+ cells, formatting the CD8-predominant AITL. Because TME is directly associated with immune response, investigating TME immune activity in CD8-predominant AITL may facilitate immune-subtyping and prognostic stratification of AITL cases. We conducted an integrated analysis of the TME of CD8predominant AITLs and compared them to common AITLs. To reduce the impact of tumor cell proportion on the microenvironment and treatment effect on prognosis, we matched these features in two groups of AITL cases.
To compare TME functions between the two AITL groups, TIL subset proportion, TRB and IGH repertoires, and gene expression profiles were examined. TIL subsets and proportions directly reflected TME immune function and have been shown to be predictive of various malignancies (17-19). TR and IGH repertoires of TME dictate TIL-T and TIL-B variety and represent the ability to identify tumor antigens and combat tumor immunoevasion (13,14). Furthermore, gene expression profiling may reveal immunological activity by examining the regulation of related pathways. Our findings revealed that CD8predominant AITL differs significantly from common AITL, which appears to have an immunosuppressive TME.
Although CD8-predominant AITL shared histopathological characteristics with common AITL in diffuse growth patterns and arborizing HEVs, it showed an increase in eosinophil counts. High eosinophil counts have been reported in AITL and have been described to be associated with cytotoxic agents, which may partly imply the cause of our findings (20). Meanwhile, CD8-predominant AITL showed in CD8+ TIL-Ts and a decrease in both CD4+TIL-Ts and TIL-Bs, implying a dysimmune TME. Despite the increase of CD8+TIL-Ts in CD8predominant AITL, single-cell transcriptome analysis of AITL TME revealed that CD8+TIL-Ts expressed CD45RO, CD27, PD1, and TIGIT, indicating an exhausted phenotype (21). Therefore, CD8-predominant AITL may have reduced cytotoxicity in the TME. Furthermore, CD4+TIL-Ts are required for CD8+ TIL-T activation, and their depletion may result in CD8+ TIL-T impotence (22,23). Moreover, CD4+TIL-Ts exert anti-tumor effects by recognizing tumor antigens and releasing cytokines, which activate other tumoricidal immune cells (24). Therefore, the low level of CD4+TIL-Ts in CD8predominant AITL also indicated that anti-tumor immunity was downregulated. Additionally, neoplastic T cells in AITL still preserve the function of TFH cells that secrete CXCL13 and other cytokines to promote B cell recruitment, expansion, and activation, generally resulting in a B-cell-rich TME (6,7,25,26). Moreover, our previous study found that the expanded B-cells represent an efficient humoral response and enhance cellular immunity through T-cell and B-cell crosstalk in the TME, leading to good survival (9,27). As a result, the lower TIL-Bs in CD8-predominant AITL implied suppressed humoral response and immune dysfunction in the TME.
CD8-predominant AITLs displayed narrowed TRB and IGH repertoires in TME, showing a significant drop in productive clones and increased clonality score in the TRB repertoire, a decrease in entropy, and an increase in the proportion of the top 10 clones in the IGH repertoire, which also indicated the immunosuppressive TME and anti-tumor immunity impairment. TR and IGH repertoires, the essential determinants of the TME in both solid tumors and hematological malignancies, reflect the immune activity of the TME (12,15). Previous investigations have shown that deteriorated TR repertoire metrics could be caused by immunosuppression due to aging or inborn errors (28)(29)(30)(31). Accordingly, our findings in the TRB repertoire of the CD8predominant AITL TME may be interpreted by TME immunosuppression. Additionally, in keeping with knowledge from other tumors (13,32), the narrowed TR and IGH repertoires in the TME of CD8-predominant AITL may indicate downregulated anti-tumor immunity via inadequate immunosurveillance of tumor T-cell neoantigens.
The gene expression profile further supported the findings of flow cytometry analysis and TRB and IGH repertoire sequencing. RNA sequencing data were analyzed focusing on genes related to inflammation and immune response, revealing the increased negative regulation of immune system processes and decreased T-cell activation and immune cell differentiation compared to common AITL. These results directly demonstrate the immunosuppressive TME in CD8-predominant AITL.
Neoplastic T-cells in AITL shape the TME in terms of histological structure and immune pattern by secreting A B D E C FIGURE 5 Gene expression profiles and survival analysis in CD8-predominant AITLs and matched common AITLs. Heatmap analysis showed differences in inflammation and immune response-related gene expression between CD8-predominant AITLs and common AITLs (A). The top 20 enriched biological processes in gene oncology from all differential genes (B), upregulated differential genes (C), and downregulated differential genes (D). Kaplan-Meier estimates of overall survival rate for CD8-predominant AITL and matched patients with common AITL (E).
cytokines and producing tumor antigens (7,33). Therefore, TME similarity is closely related to tumor cell similarity. Neoplastic AITL cells retain TFH cell functions and harbor a recurrent RHOA G17V mutation (6). Furthermore, in our previous study, AITL tumor cells were found to have a biased usage of TRBV19-1 and a high level of identity in the CDR3 sequence (34). Thus, these commonalities of AITL tumor cells could explain our findings: CD8-predominant AITLs and common AITLs had similar histological structures and shared CDR3 sequences in the TME TRB and IGH repertoires. Neoplastic cell heterogeneity is associated with differences in the TME. We found that CD8-predominant AITLs and common AITLs had different V family segment usage and CDR3 sequence frequency in the TME of both TRB and IGH repertoires. Consistent with the literature on solid tumors and hematopoietic malignancies, these findings reflect the heterogeneity of the tumor antigen portfolio and further imply a heterogeneous mutational landscape of neoplastic cells between the two AITL groups (35)(36)(37). Thus, heterogeneity of neoplastic T-cells may be the cause of TME immune function differences between the two AITL groups, resulting in CD8predominant AITL having an immunosuppressive TME.
Studies from both lymphoma and other solid tumors demonstrated that the TME changes as tumor progresses; in general, the immune response of the TME gradually decreases and eventually leads to immune silence (38,39). Our results demonstrated that CD8-predominant AITL has an immunosuppressive TME, indicating that CD8-predominant AITL is at a later stage of tumor development and thus has significantly inferior survival compared to common AITLs.
Combined with the patient's clinical manifestations, we hypothesized that the immunosuppressed TME of CD8predominant AITL leads to insufficient anti-tumor immunity, manifesting as an increase in tumor proliferation index, which in turn leads to tumor dissemination (40), causing aggravation of the patient's clinical manifestations such as frequent effusion and edema. Furthermore, the patients' immune system collapses secondary to tumor development, resulting in EBV reactivation and increased plasma EBV-DNA (41). This eventually leads to a reduction in the patient's survival ( Figure 6). Therefore, future research should concentrate on enhancing the immune function of the TME of CD8predominant AITL, thereby improving patient prognosis.
This study has potential limitations. The main point is the small sample size, which is due to the rarity of CD8predominant AITL. To reduce the confounding factors and sampling error, we introduced the case-control matching. Nevertheless, our findings must be validated through the accumulation of cases.

Conclusions
CD8-predominant AITL is an uncommon disease with more severe clinical manifestations and a shorter OS than that of common AITL. Although CD8-predominant AITL has histological features similar to those of common AITL, it has a lower TIL-B proportion, an inverted CD4+/CD8+ TIL-Ts ratio, deterioration of TR and IGH repertoire metrics, and altered gene expression profiles, indicating the anti-tumor immunity FIGURE 6 Hypothesized correlation between CD8-predominant AITL TME immune function and clinicopathological features. TIL, tumor infiltrating lymphocyte; HEV, high endothelial venule; EO, eosinophil; DC, dendritic cell; M∅, macrophage. This figure was drawn using ScienceSlides 2016, VisiScience, Inc. impairment in TME. According to the accumulated evidence, CD8-predominant AITL is a distinct immune pattern of AITL with an immunosuppressive TME that must be better identified and investigated to improve patient survival.

Data availability statement
TRB and IGH sequencing data have been deposited in the National Center for Biotechnology Information Sequence Read Archive under accession number PRJNA887199. The RNA-seq data were provided in the Supplementary Material.

Ethics statement
The studies involving human participants were reviewed and approved by Ethics Committee on Biomedical Research, West China Hospital of Sichuan University. Written informed consent for participation was not required for this study in accordance with the national legislation and the institutional requirements.
Author contributions ZC, YT, and SZ designed and conceived the study. ZC, QZ, WZ, WL, and SZ selected samples. ZC, QZ, and XD performed experiments. ZC, QZ, and WY analyzed the data. ZC wrote the manuscript. All authors contributed to the article and approved the submitted version.