Despite recent major clinical breakthroughs in human cancer immunotherapy including the use of checkpoint inhibitors and engineered T cells, important challenges remain, including determining the sub-populations of patients who will respond and who will experience at times significant toxicities. Although advances in cancer immunotherapy depend on preclinical testing, the majority of in-vivo testing currently relies on genetically identical inbred mouse models which, while offering critical insights regarding efficacy and mechanism of action, also vastly underrepresent the heterogeneity and complex interplay of human immune cells and cancers. Additionally, laboratory mice uncommonly develop spontaneous tumors, are housed under specific-pathogen free conditions which markedly impacts immune development, and incompletely model key aspects of the tumor/immune microenvironment. The canine model represents a powerful tool in cancer immunotherapy research as an important link between murine models and human clinical studies. Dogs represent an attractive outbred combination of companion animals that experience spontaneous cancer development in the setting of an intact immune system. This allows for study of complex immune interactions during the course of treatment while also directly addressing long-term efficacy and toxicity of cancer immunotherapies. However, immune dissection requires access to robust and validated immune assays and reagents as well as appropriate numbers for statistical evaluation. Canine studies will need further optimization of these important mechanistic tools for this model to fulfill its promise as a model for immunotherapy. This review aims to discuss the canine model in the context of existing preclinical cancer immunotherapy models to evaluate both its advantages and limitations, as well as highlighting its growth as a powerful tool in the burgeoning field of both human and veterinary immunotherapy.

Independent studies have demonstrated different cell tropisms for molecular clones of feline immunodeficiency virus (FIV). In this report, we examined three clones, FIV-pF34, FIV-14, and FIV-pPPR, for replication in Crandell feline kidney (CrFK) cells, feline peripheral blood mononuclear cells (PBMC), and feline macrophage cultures. Importantly, cell tropism for these three clones was also examined in vivo. FIV-pF34 replication was efficient in CrFK cells but severely restricted in PBMC, whereas replication of FIV-pPPR was vigorous in PBMC but severely restricted in CrFK cells. FIV-14 replication was productive in both CrFK cells and PBMC. Interestingly, all three molecular clones replicated with similar efficiencies in primary feline monocyte-derived macrophages. In vivo, FIV-pF34 proved least efficient for establishing persistent infection, and proviral DNA when detectable, was localized predominately to nonlymphoid cell populations (macrophages). FIV-pPPR proved most efficient for induction of a persistent viremia in vivo, and proviral DNA was localized predominately in CD4 ⁺ and CD8 ⁺ lymphocyte subsets. FIV-14 inoculation of cats resulted in an infection characterized by seroconversion and localization of proviral DNA in CD4 ⁺ lymphocytes only. Results of this study on diverse FIV molecular clones revealed that in vitro replication efficiency of an FIV isolate in PBMC directly correlated with replication efficiency in vivo, whereas proficiency for replication in macrophages in vitro was not predictive for replication potential in vivo. Also, infection of both CD4 ⁺ and CD8 ⁺ lymphocyte subsets was associated with higher virus load in vivo. Results of the studies on these three FIV clones, which exhibited differential cell tropism, indicated a correlation between in vitro and in vivo cell tropism and virus replication.

Feline immunodeficiency virus (FIV) is a lentivirus of cats that establishes a lifelong persistent infection with immunologic impairment.

The orf-A ( orf-2 ) gene of feline immunodeficiency virus (FIV) is a small open reading frame predicted to encode a 77-amino-acid protein that contains putative domains similar to those of the ungulate lentiviral Tat protein. Orf-A is reported to be critical for efficient viral replication in vitro and in vivo. A series of FIV-pPPR-derived proviruses with in-frame deletions and point mutations within orf-A were constructed and tested for replication in feline lymphoid cells. Orf-A mutant proviruses were also tested for viral gene and protein expression, viral particle formation, and virion infectivity. Deletions within orf-A severely restricted FIV replication in feline peripheral blood mononuclear cells (PBMC) and interleukin-2-dependent T-cell lines. In addition, substitutions of alanines for leucines in the putative leucine-rich domain, for cysteines in the putative cysteine-rich domain, and for a tryptophan at position 43 in Orf-A restricted the replication of FIV mutants. Deletions and point mutations in orf-A imposed a small effect or no effect on FIV long-terminal-repeat-driven viral gene expression and had no effect on viral protein expression. However, release of cell-free, virion-associated viral RNA in supernatants from cells transfected with orf-A mutant proviruses was severely restricted but was rescued by cotransfection with a wild-type Orf-A expression vector. In addition, virions derived from orf-A mutant proviruses expressed reduced infectivity for feline PBMC. Our findings suggest that Orf-A functions involve multiple steps of the FIV life cycle including both virion formation and infectivity. Furthermore, these observations suggest that Orf-A represents an FIV-encoded analog more similar to the accessory gene vpr , vpu , or nef than to the regulatory gene tat encoded by the primate lentiviruses.

Squamous cell carcinoma is the most common oral cancer in the cat and presents as a locally aggressive lesion for which an effective therapeutic protocol remains elusive. Feline oral squamous cell carcinoma (OSCC) shares many clinical characteristics with human head and neck squamous cell carcinoma (HNSCC). Accordingly, present studies were conducted to determine similarities for immune markers shared by feline OSCC and human HNSCC. Biopsies harvested from a feline patient cohort-1 (n = 12) were analyzed for lymphoid cell infiltrates by immunohistochemistry (IHC). Results revealed unique patterns of T cell infiltration involving both neoplastic epithelium and stroma that were detected in most patient tumor biopsies (92%) examined by IHC staining for CD3. Intratumoral B cell infiltrates were detected within tumor stroma only, based on IHC staining for CD79a and CD20 for all patients within the same cohort-1. Infiltration of tumors by a regulatory CD4 T cell subset (Tregs) defined by expression of the forkhead transcription factor FoxP3, was also detected in biopsies from 57% of patients and involved infiltration of neoplastic epithelium and stroma. Patient biopsies were also examined for expression of immunomodulator cyclooxygenase (COX)-2 and revealed positive but weak staining of neoplastic epithelium in a significant proportion of cases (75%). Interestingly, COX-2 expression was detected in both neoplastic epithelium and stroma. Blood collected from a second cohort of feline OSCC patients (n = 9) revealed an increased frequency of circulating CD4+FoxP3+ T cells when compared to healthy adult controls (n = 7) (P = 0.045), although frequencies of CD4+CD25+FoxP3+ T cells were comparable between patients and healthy pet cat controls. Lastly, biopsies from feline OSCC patients were characterized for histologic subtype using a classification scheme previously described for human HNSCC. This analysis revealed the conventional subtype as the predominant variant (75%) with conventional subtypes split evenly between well differentiated and moderately differentiated carcinomas. Two cases were classified as papillary and one case as basaloid subtypes. Correlations between subtype, immune marker scores or circulating Treg frequencies and clinical characteristics or outcome were not detected, most likely due to small patient numbers within patient cohorts. However, findings from these studies provide a preliminary step in the characterization of immune and histologic markers that will be critical to defining prognostic immune markers for feline OSCC and potential targets for testing of immunotherapeutics also relevant to human HNSCC in future studies.