Canine B cell lymphoma shows no deficit of cytotoxic T cells relative to thymic Tregs.

<p>When expressed as a proportion of all cells aspirated from the lymph node, the frequency of CD8⁺ T cells (A,B) was decreased in BCL (and MCT), again attributed to neoplastic effacement (p = 0.0005). The frequency of CD8⁺ cells within the CD5⁺ T cell population also showed differences between groups (C; p = 0.04). The ratio of CD8⁺:FOXP3⁺ cells (D) was not significantly different between groups (p = 0.06), though dogs with BCL showed the six highest ratios of the cohort, and a trend for higher values in the BCL group was observed. Indeed, the ratio of CD8⁺:FOXP3⁺Helios⁺ cells (E) was higher in the BCL than RH cases (p = 0.04); only two data points for the MCT (3.48; 3.95) were available for this variable and were not therefore represented. Representative dot plots for all the groups are shown alongside their respective isotype controls, followed by box-and-whisker plots summarising the data. (For key to symbols, refer to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0105027#pone-0105027-g002" target="_blank">Figure 2</a>).</p

Clinical, cytological and treatment details of dogs in the study.

<p><u>Abbreviations</u>: Me, entire male; Mn, neutered male; Fe, entire female; Fn, neutered female; WHO, World Health Organization; N/A, not applicable; DLBCL, diffuse large B cell lymphoma; PTCL, peripheral T cell lymphoma; CHOP, cyclophosphamide, doxorubicin, vincristine, prednisolone protocol; COP, cyclophosphamide, vincristine, prednisolone protocol; Pred, prednisolone alone; NSAIDS, non-steroidal anti-inflammatory drugs.</p><p><u>Notes</u>: ¹: Age when presenting signs were first observed. ²: Body condition score was assessed by different clinicians on either a 5 or 9 point scale: ‘under-conditioned’ was defined by a score of 1–2/5 or 1–3/9; ‘optimal’ was defined by a score of 2.5–3/5 or 4–5/9; while ‘over-conditioned’ was defined by a score of 3.5–5/5 or 6–9/9. ³: Serum or plasma calcium concentration was not measured in one of the T cell lymphoma cases prior to initiation of therapy. ⁴: The initial treatment protocol is listed. ‘Other’ treatments included cytarabine, L-asparaginase and lomustine (B cell lymphoma) and L-asparaginase, lomustine, prednisolone, masitinib and chlorambucil (T cell lymphoma).</p

Gating strategies used to interrogate the neoplastic and non-neoplastic cell populations.

<p>Following exclusion of dead cells and debris, two different gating strategies were employed to evaluate the cells isolated from lymph node aspirates. For the purposes of generating an immunophenotyping profile, an upper 30% gate was applied to identify the population of largest events, enriched in neoplastic cells. The immunophenotype was then determined using a 60% expression threshold within this population. For the purposes of evaluating frequencies of T cells within fine needle aspirates of B cell lymphoma (BCL), reactive hyperplasia (RH) and mast cell tumour (MCT) cases, an ‘All’ gate was applied and then the populations subsequently delineated by means of CD5 expression. Non-neoplastic cells were identified as CD5⁺ and neoplastic populations CD5⁻ in the context of BCL; TCL cases were excluded from such comparisons on account of their CD5 expression by both neoplastic and non-neoplastic cells.</p

FOXP3⁺ Treg frequency is no higher in canine B cell lymphoma than control samples.

<p>The frequency of FOXP3⁺ cells in fine needle aspirates harvested from the lymph nodes of dogs with B cell lymphoma (BCL), T cell lymphoma (TCL), reactive hyperplasia (RH) and mast cell tumors (MCT) is shown, expressed as a proportion of all cells (A, B, showing the effect of neoplastic effacement of normal lymphoid tissue; p = 0.00005), gated CD5⁺ cells (C, showing the resident T cells; p = 0.41), or sequentially gated CD5⁺ and then CD4⁺ T cells (D, showing specifically the resident CD4⁺ T cells; p = 0.74). Representative dot plots are shown for each of the groups, followed by box-and-whisker plots summarising the data. In each of the dot plots, the respective isotype control stain is shown above the specific stain. In this and subsequent figures, red symbols correspond to data from dogs ‘pre-treated’ with corticosteroids or any chemotherapeutic drug within three weeks of sampling. In the case of RH cases, square symbols (□) represent data from dogs with dermatitis; diamonds (◊) represent data from dogs with distant neoplasia; triangles (Δ) represent data from dogs with immune-mediated disease; and inverted triangles (▾) represent data from dogs with systemic infection. The expression of FOXP3 by PBMCs (E; p = 0.21) or by the CD5⁺ (F; p = 0.53) or specific CD5⁺CD4⁺ (G; p = 0.15) T cells within PBMCs similarly showed no differences between groups.</p

FOXP3, MHC class II, age at presentation and rescue therapy are independent prognostic factors in canine B cell lymphoma.

<p><u>Abbreviations</u>: TTR, time to remission; PFS, progression-free survival; OS, overall survival; CI, confidence interval; FOXP3⁺ (% CD5⁺CD4⁺), frequency of FOXP3⁺ cells within the sequential CD5⁺ and CD4⁺ gates; FOXP3⁺ (%), frequency of FOXP3⁺ cells within the total harvested population; median ratio =  ratio of median remission or survival time (TTR/PFS/OS) of the sub-group of dogs with a value of the variable greater than the median of the group to that of the sub-group of dogs with a value of the variable less than or equal to the median of the group.</p

Staining antibodies, isotype controls and fluorochromes.

<p><u>Abbreviations</u>: FITC, fluorescein isothiocyanate; PE, phycoerythrin; APC, allophycocyanin.</p

Canine T cell lymphoma is associated with high-frequency Helios expression.

<p>Helios expression by cells harvested by fine needle aspiration from the lymph nodes of dogs with B cell lymphoma (BCL), T cell lymphoma (TCL), reactive hyperplasia (RH) and mast cell tumors (MCT) was examined as a marker for thymic Tregs, but the unexpectedly high frequency of Helios⁺ events in a few B cell and several T cell lymphoma cases suggested neoplastic expression of this marker. Median frequency of Helios expression was significantly different between the groups (p = 0.00003). Representative dot plots for BCL and TCL cases, with their respective isotype control stains, is shown (A), followed by box-and-whisker plots summarising the data (B). (For key to symbols, refer to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0105027#pone-0105027-g002" target="_blank">Figure 2</a>).</p

Expression of T cell FOXP3 and Helios, and tumor cell MHC class II, all impact prognosis in dogs with B cell lymphoma.

<p>The frequency of FOXP3⁺ cells within the ‘All’ gate (A), or within a cascaded All→CD5⁺→CD4⁺ gate (B), influenced survival times. Overall survival times were shorter in those dogs with frequencies of FOXP3⁺ T cells higher than the median value (A: p = 0.02); a similar pattern in progression-free survival (PFS) was observed (B: p = 0.01). The frequency of Helios⁺ cells within the ‘All’ gate also influenced PFS (C: p = 0.01). The intensity of MHC class II expression by the lymphoma cells, expressed as the ratio of geometric mean fluorescence intensity of MHC II⁺:MHC II⁻ cells within the CD5⁻ subset of the ‘All’ gate, was also of prognostic significance: time to remission was longer in those cases with less MHC class II expression per cell (D: p = 0.02).</p