Evaluation of costimulatory molecules in dogs with B cell high grade lymphoma

B cell high grade lymphoma is the most common hematopoietic malignancy in dogs. Although the immune checkpoint molecules, programmed death-1 (PD-1) and cytotoxic T-lymphocyte-associated antigen-4 (CTLA-4), and immune checkpoint inhibitors have been evaluated for the treatment of various human lymphoid malignancies, the expression of those molecules and their relationship with prognosis remain unknown in canine lymphoma. The objective of this study was to evaluate the expression of costimulatory molecules on peripheral blood lymphocytes and tumor infiltrating lymphocytes, in addition to associated ligand expression in the lymph nodes of patients with B cell multicentric high grade lymphoma. Eighteen patients diagnosed with B cell high grade lymphoma and nine healthy control dogs were enrolled. Flow cytometric analysis revealed that the expression of PD-1 on CD4+ peripheral and tumor infiltrating lymphocytes and CTLA-4 on CD4+ peripheral lymphocytes was significantly higher in the lymphoma group than in the control group. The expression level of CD80 mRNA was significantly lower in the lymphoma group than in the control group. In contrast, there were no significant differences in PD-L1, PD-L2, and CD86 expression between the groups. Dogs with CTLA-4 levels below the cutoff values, which were determined based on receiver operating characteristic curves, on peripheral CD4+, CD8+, and tumor infiltrating CD4+ lymphocytes had significantly longer survival than dogs with values above the cutoff. Although it is uncertain whether the expression of immune checkpoint molecules affect the biological behavior of canine lymphoma, one possible explanation is that PD-1 and CTLA-4 might be associated with the suppression of antitumor immunity in dogs with B cell high grade lymphoma, particularly through CD4+ T cells.

Introduction blood lymphocytes and tumor infiltrating lymphocytes, as well as the expression of their ligands on tumor cells, from patients with B cell multicentric high grade lymphoma to assess immune status in these dogs.

Study population
Eighteen dogs with B cell multicentric high grade lymphoma (lymphoma group) and nine clinically healthy controls (control group) were enrolled in this prospective study. For patients in the lymphoma group, lymphoma was cytologically or histologically confirmed in a veterinary medical center at Obihiro University of Agriculture and Veterinary Medicine between October 2015 and August 2017. Staging of lymphoma was performed according to the WHO staging system [22] including thoracic radiography based on three views, abdominal ultrasound, and hematological examinations. Substage 'a' was defined as no clinical signs and substage 'b' was defined as systemic signs related to lymphoma including mild-to-moderate severity of gastrointestinal disorders or weight loss. The immunophenotype was determined by PCR antigen receptor rearrangement using fine needle aspirate (FNA) samples. All dogs received no prior chemotherapy treatment except for one relapsed case. Dogs in the control group were agematched healthy patients who came to the hospital for medical examinations. This study was approved by the Institutional Animal Care and Use Committees at the Obihiro University of Agriculture and Veterinary Medicine (Permission number: 27-143, 28-7, and 29-3).

Cell preparation
Heparinized peripheral blood was obtained from all patients and healthy controls. All heparinized blood samples were diluted with an equal volume of 0.9% saline, and peripheral blood mononuclear cells (PBMCs) were separated by Ficoll-Paque (Lymphoprep; Axis-Shield PoC AS, Oslo, Norway) density gradient centrifugation [23]. Following centrifugation at 800 × g for 20 min at room temperature, PBMCs were collected and washed twice with 0.9% saline. FNA samples were taken from palpable lymph nodes, and lymph nodes cells (LNCs) were suspended in 500 μl of saline. LNCs were centrifuged for 15 min at 1,500 rpm, and the supernatant was discarded. Cells were resuspended in 1 ml of VersaLyse (Beckman Coulter, Marseille Cedex, France) and incubated for 5 min at room temperature in the dark. Following centrifugation, LNCs were washed and resuspended in 0.9% saline.

RNA extraction and cDNA synthesis
Total RNA was isolated from LNCs using TRIzol 1 Reagent (Thermo Fisher Scientific Inc., Waltham, MA, USA) according to the manufacturer's protocol. After the isolation of total RNA, genomic DNA contamination was removed from the isolated RNA using recombinant DNase I (TURBO DNA-free™ Kit; Thermo Fisher Scientific Inc.) according to the manufacturer's recommended procedure. The RNA concentration was measured using a NanoDrop Lite (Thermo Fisher Scientific Inc.), and the purity of the extracted RNA was in the range of 1.8-2.0, as determined by absorbance ratios of A 260 /A 280 . The purified RNA was eluted in nuclease-free water and stored at −80˚C until further use.
cDNA was synthesized from 500 ng of total RNA using PrimeScript™ RT Master Mix containing Oligo dT primers and random primers (Takara Bio Inc., Kusatsu, Japan) according to the manufacturer's protocol.

Quantification of PD-L1, PD-L2, CD80, and CD86 mRNA by real-time RT-PCR
RT-PCR amplification was performed to measure the levels of PD-L1, PD-L2, CD80, and CD86 mRNA. Primers used for quantitative PCR were designed using Primer3Plus (http://www. bioinformatics.nl/cgi-bin/primer3plus/primer3plus.cgi/) based on canine GenBank sequences or previous reports ( Table 1). All primer sets were designed to specifically amplify a region encompassing two exons of each gene. The NormFinder algorithm (https://www.moma.dk/ normfinder-software) was used to identify the most stable gene among three candidate reference genes (ACTB, RPL13A, and RPL32) [26], and RPL13A was chosen as the reference gene. PCR products were subjected to Hokkaido System Science (Sapporo, Japan), and the specificities of these primers were confirmed by sequencing analysis. Nucleotide sequence results were confirmed using the BLAST search program (http://www.ncbi.nlm.nih.gov/blast/Blast.cgi) for comparison of other known sequences. Each real-time RT-PCR reaction was performed in a 20-μl total volume, containing 200 nM of each primer, 1 μl of cDNA, and GoTaq qPCR Master Mix (Promega Corporation, Madison, WI, USA), using a StepOne Real-Time PCR System (Applied Biosystems, South San Francisco, CA, USA). Initial incubation at 95˚C for 2 min was followed by 40 cycles consisting of denaturation at 95˚C for 3 sec and annealing/extension at 60˚C for 30 sec. A melt curve (60-95˚C) was generated at the end of each run to verify specificity. Amplification efficiency for each reaction was tested by the series dilution method. Absolute quantification of each mRNA was performed by converting sample cycle threshold values to a concentration (copies/μl), based on standard curves, which were generated using 10-fold serial dilutions (10 7 -10 3 copies/μl) of each gene amplicon. The target amount was then divided by RPL13A levels to obtain a normalized target value. All samples were evaluated in triplicate.

Statistical analysis
Sexes within each group were compared using the chi-squared test. Continuous variables such as age and PD-1 and CTLA-4 parameters were analyzed by performing Mann-Whitney U tests. Survival time was measured as the interval between the sampling day, usually the start of treatment, and death due to lymphoma. Dogs alive at the end of the study were censored for survival analysis. The survival curves were examined by the Kaplan-Meier method, which were compared using the log-rank test. Receiver operating characteristic (ROC) curve analysis was used to determine the optimal cutoff values of continuous variables used for the prediction of a survival time exceeding a median value. A minimum area under the curve (AUC) of 0.7 was required for the ROC model to be considered. X 2 Fisher's exact test was used to evaluate categorical values. All analyses were performed using JMP 13 (SAS Institute Inc., Cary, NC, USA). Differences were considered statistically significant if the P value was less than 0.05.

Characteristics of the study population
In the lymphoma group, the median age at sampling was 9.5 years (range, 3-14 years). Eleven dogs were male (five were castrated) and seven dogs were female (five were spayed). In the control group, the median age at sampling was 8.5 years (range, 1-12 years). Three dogs were male (one was castrated) and six dogs were female. There were no significant differences in terms of age or sex between the two groups. According to WHO staging, one dog was classified as stage II, five as III, six as IV, and six as V. Eleven dogs were classified as substage a and seven as b. Only two dogs were treated with prednisolone before sampling. Ten dogs were treated with a CHOP-based protocol and five dogs with prednisolone alone. The others were treated with L-asparaginase (as outlined in Table 2). Euthanasia was performed only for one dog, which was in extremis, and all other cases died naturally. The overall median survival time was 121 days (range, 1-340 days). Two dogs were still alive at the end of the study, representing survival of 191 and 208 days.
Regarding, PD-1 expression in CD8+ lymphocytes obtained from PBMCs and LNCs, there were no significant differences between the groups (Fig 1B and 1D). The proportion of CTLA-4+ cells in CD4+ lymphocyte populations obtained from PBMCs was significantly higher in the lymphoma group (1.40% ± 0.92%) than in the control group (0.62% ± 0.26%, P = 0.018, Fig 1E). Regarding CTLA-4 expression in CD8+ lymphocytes obtained from PBMCs and CD4 + and CD8+ lymphocytes obtained from LNCs, there were no significant differences between the groups (Fig 1F, 1G and 1H Fig 2C). In contrast, there were no significant differences in terms of PD-L1, PD-L2, and CD86 expression between the groups (Fig 2A, 2B and 2D).

Relationship between expression of immune checkpoints and survival time
Optimal cutoff values were determined based on each ROC curve (Table 3). Regarding CTLA-4 expression on CD4+ and CD8+ lymphocytes, dogs with levels below the cutoff value had significantly longer survival time than the dogs with values above the cutoff (CD4+ CTLA-4+ in PBMCs, median survival times below and above the cutoff = 138 days and 11 days, respectively, P < 0.001, Fig 3A; CD8+ CTLA-4+ in PBMCs, 138.5 days and 15.5 days, respectively, P = 0.002, Fig 3B; CD4+ CTLA-4+ in LNCs, 138 days and 16.5 days, respectively, P = 0.026, Fig 3C). In addition, for CTLA-4+ cells in CD8+ populations and PD-1+ cells in CD4+ lymphocytes obtained from LNCs, dogs with levels below the cutoff value had significantly longer survival time than dogs with values above the cutoff (CD8+ CTLA-4+ in LNCs, 121 days and 44 days, respectively, P = 0.048; CD4+ PD-1+ in LNCs, 127.5 days and 13 days, respectively, P = 0.023); however, their AUC values were below 0.7 (data not shown). Except for CD4+ PD-1+ in LNCs, the expressions of PD-1+ on lymphocytes did not correlate with survival time.
Regarding mRNA expression, no correlation was found with survival time (data not shown).

Discussion
In this study, we evaluated the expression of immune checkpoint molecules including PD-1 and CTLA-4 and their ligands in canine lymphoma patients. In these lymphoma samples, significantly higher expression of CTLA-4 was observed on CD4+ lymphocytes obtained from PBMCs. In addition, the expression of PD-1 on CD4+ lymphocytes obtained from PBMCs and LNCs was also significantly higher in the lymphoma group. CTLA-4, a costimulatory receptor expressed on the surface of activated T cells, is a negative regulator of T cell activation.  PD-1 is also an immunoinhibitory receptor expressed by chronically stimulated CD4+ and CD8+ T cells after T cell activation [8,9]. Up-regulation of the expression of checkpoint regulators plays an important role in the evasion of immune surveillance by malignant cells [17]. In humans, PD-1 expression has been evaluated in different lymphoma types including B cell lymphoma [27,28,29,30]. CTLA-4 is highly expressed on tumor-infiltrating lymphocytes in lymphoid malignancies [31,32]. There have also been some reports on the PD-1/PD-L1 axis in dogs with different malignancies [13,14,33]. Recently, high expression of PD-1 on tumor infiltrating T cells was reported in dogs with B and T cell lymphoma [34]. In this study, significantly higher expression of PD-1 was observed on CD4+ T cells obtained from LNCs from dogs with lymphoma, and CD8+ T cells tended to up-regulate these molecules. In addition, PD-1 expression on peripheral CD4+ T cells obtained from dogs with lymphoma was significantly higher compared to that in control animals. In veterinary medicine, although previous studies revealed high expression of CTLA-4 in dogs with oligodendroglioma and histiocytic sarcoma [15,16], the expression of this marker in lymphoma had not previously been reported. We found that CTLA-4 expression on CD4+ T cells obtained from PBMCs was significantly higher in the lymphoma group than in the control group. Lymphocytes including T and B cells are the major cells of the adaptive immune system. CD8+ T cells destroy virusinfected cells and tumor cells. CD4+ T lymphocytes have no cytotoxic or phagocytic activity and cannot kill infected cells or clear pathogens; however, they do manage the immune response by directing other cells to perform these tasks [35]. This study suggested that PD-1 and CTLA-4 might be associated with the suppression of antitumor immunity in dogs with B cell lymphoma, and particularly through CD4+ T cells.
To further evaluate the immune checkpoint pathway, mRNA expression levels of PD-L1, PD-L2, CD80, and CD86 were measured in LNCs. Although there was no significant difference, PD-L1 expression was variable between the lymphoma group and the control group. Maekawa et al. observed that canine DLBCL samples did not express PD-L1 [13]. Shosu and Kumar et al. reported the expression of PD-L1 in few lymphoma tissues and cell lines [14,33]. Our results are in agreement with those from the previous reports. In humans, the proportion of DLBCL tumor cells that express PD-L1 was ranged from 10.0 to 49.0% [36,37]. Further studies are needed to clarify the expression of PD-L1 and the role of the PD-1/PD-L1 axis in canine B cell lymphoma using a large sample size. Interestingly, low expression of CD80 was observed in canine lymphoma samples. CD80, also known as B7.1, is a coregulatory receptor expressed on the surface of antigen presenting cells, activated T cells, and myeloid-derived suppressor cells. Full activation of T cells requires binding of the CD28 receptor by CD80 or CD86 on antigen-presenting cells [38]. It was reported that CD80 is expressed in the majority of human DLBCL cases and is also present on nonmalignant stromal cells [39]. In contrast, one study revealed that acute lymphoblastic leukemia cells have low expression of costimulatory molecules including CD80 and suggested that this probably contributes to the absence of a host T cell-stimulated immune response [40]. This is the first report of the evaluation of CD80 expression in canine B cell lymphomas. Further understanding of the expression of this marker, as well as other immune coregulatory molecules that control immune cell function, will be needed to better understand the complexity and functional implications of the tumor microenvironment in canine B cell high grade lymphoma.
In this study, CTLA-4 expression on peripheral CD4+ and CD8+ lymphocytes and CD4 + lymphocytes obtained from LNCs was found to be associated with poor prognosis in canine B cell high grade lymphomas. Especially, high expression of CTLA-4 in PBMCs was significantly correlated with substage b, which has been identified as an indicator of poor prognosis. Although the prognostic role and clinical application of CTLA-4 in tumors are still controversial, increased CTLA-4 expression on T cells contributes to poor prognosis in various cancers including DLBCL [41,42]. In addition, CTLA-4 is a negative immunomodulatory factor that is known to be expressed by effector regulatory T cells, which mainly suppress antitumor immunity, but not by non-Tregs [41]. It was thought that expression of CTLA-4 on T cells might be associated with a worse prognosis and might represent a prognostic factor for canine B cell high grade lymphoma. In contrast, PD-1 expression on peripheral and tumor infiltrating T cells was not associated with prognosis. In humans, the relationship between PD-1 expression and prognosis in lymphoid malignancies is also complex [43]. To the best of our knowledge, this is the first report to explore the relationship between immune checkpoint molecules and B cell lymphoma prognosis in dogs. However, we did not control for treatment and stage/ substage for each analysis, and the small sample size of this study might affects the reliability of the results. Further studies using larger cohorts and controlled patients are needed to evaluate the association between immune checkpoint molecule expression and prognosis in canine B cell high grade lymphomas.
PD-1-and CTLA-4-blocking antibodies (ipilimumab and nivolumab) were shown to produce robust responses and improve overall survival in human patients with various lymphoid malignancies [43]. Interestingly, in canine visceral leishmaniasis, high level of PD-1 on T cells and in vitro efficacy of monoclonal antibodies that block PD-1 and its ligands have been reported [24,25]. Recently, in veterinary medicine, a pilot study demonstrated promising anticancer activity with tolerable toxicity profiles using an anti-PD-L1 antibody for canine malignancies [44]. The results of the present study indicated that such immunotherapy regimens might also be effective for patients with canine B cell high grade lymphoma.
Several limitations should be noted when interpreting the results of this study. First, the small sample size for each analysis might limit the power to detect differences between groups. Therefore, our findings should be verified in a larger population, as this study represents a pilot study. Second, the treatment modality for the dogs with lymphoma was not unified. A future prospective study using standardized treatment is thus necessary for adequate evaluation. Another limitation of our method is the lack of protein quantification of the immune checkpoint ligands. Finally, it remains unclear whether the expression of immune checkpoint molecules on tumor cells or other nonmalignant cells is associated with the anti-tumor immune response and clinical outcome in canine lymphoma patients. Future functional analyses are necessary to elucidate the complex roles of this pathway.
In summary, this study indicates that PD-1 expression is up-regulated on both peripheral and tumor infiltrating T cells and that CTLA-4 expression is up-regulated on CD4+ T cells from peripheral blood obtained from dogs with B cell high grade lymphoma. CTLA-4 expression on T cells was also associated with a poor prognosis. Our findings support the hypothesis that these molecules might represent new therapeutic targets for the treatment of canine B cell high grade lymphoma, and could serve as prognostic factors.