B220 expression as an immunological marker for differentiation of Feline Leukemia Virus carrying cats

ABSTRACT: Feline leukemia virus (FeLV) causes an infection in cats that, in some cases, can also be reported with other pathologies, such as infection with feline immunodeficiency virus (FIV), feline infectious peritonitis (FIP), and lymphoma. Although, a compromised immune response is reported in these animals, little is known about the immunological state of their cells. To shed some light in this area, we studied peripheral blood samples from both infected and non-infected cats with FeLV, with or without FIV, FIP, and lymphoma. We tested a panel of monoclonal antibodies (n=11) against mouse and human antigens and we reported that cat leukocytes can be stained with anti-mouse B220 monoclonal antibody; therefore, percentages of B cells were evaluated in different cat groups. Our results showed that cats with FeLV and FIP, or with leukemia, presented a large decrease in B220+ mononuclear cells. However, FeLV+ cats without clinical signs, or with unspecific clinical signs, had the same amount of B220+ mononuclear cells as healthy cats (control cats). Since the expression of B220 is exclusively restricted to the naïve B cell population, we inferred that the absence of these B cells in FeLV+ cats is related to other conditions that affect B cell numbers, such as viral infections and leukemias. Therefore, the amount of naïve B cells in peripheral blood (i.e., B220+ cells) can be used to identify FeLV+ cats concomitantly carrying FIP or leukemia, from FeLV+ cats with lymphoma or without any clinical signs.

Frota et al. JÚNIOR, 2011), sometimes with high lymphoma association (CRISTO et al., 2019), while in other countries these indexes are low, e.g., 5.6% in the USA (BURLING et al., 2017;SHAHRANI et al., 2011;STAVISKY et al., 2017). However, in endemic areas, transmission rates can reach nearly 30% by vertical or horizontal transmissions (HARDY JR. et al., 1976) The most common method for detecting FeLV is an ELISA for p27 antigen (LEVY et al., 2017), and the most efficient disease control measurements is immunization of FeLV-negative cats and isolation of FeLV-positive cats (BURLING et al., 2017;GROSENBAUGH et al., 2017).
An important factor in disease outcome is the cat immune response. It is assumed that some cats efficiently suppress the onset of infection, either by completely deleting the first infected cells (no viremia), or by establishing a protective immunity after a period of positive viremia (virus carriers for the rest of their lives). It is also possible that FeLV+ animals can become viremia-negative, with no signs of disease (TORRESA et al., 2015). Symptomatic cats present non-specific symptoms, for which the most common is weight loss (BURLING et al., 2017;RUDAN et al., 2017). However, some adult cats can develop lymphomas, nonregenerative anemia, myeloproliferative diseases, panleukopenia, and septicemia; some FeLV infected cats can even become infected with other viruses and develop feline infectious peritonitis (FIP) (HARDY JR., 1976). Therefore, it is important to develop new protocols for evaluating the immunological state of these FeLV+ cats. Taking this under consideration, we tested a panel of monoclonal antibodies (MAbs) in peripheral blood of FeLV+ cats, which were concomitantly carrying or affected by other diseases. Our findings showed that anti-B220 MAb can be used to identify a positive cell population (possibly naïve B cells), which in turn could be an indicator of their immunological status.
We The cats used in this study were from the institutional veterinary hospital, from a local pet clinic, and from a local illness-free cattery.
All data were analyzed with Cell Quest Pro software (BD Biosciences Inc., USA). We presented the variables in terms of mean ± standard deviation (SD) and results were plot and statistically evaluated in a GraphPad Prism 5.0 Software (GraphPad Software Inc., USA). The differences between groups were tested by ANOVA, followed by pair-wise comparison with Tukey post-hoc test. Significant differences were observed when P<0.05.
Our results showed that anti-mouse B220 antibody was the only one capable of labeling cat leukocytes (Figure 1). When we used it to study the amount of naïve B cell population in cat leukocytes, we observed that the average of this cell proportion in Control cats was 42.6% (Figure 1), which is similar to the values described by other authors (WILLETT et al., 2007). FeLV+ cats with lymphoma (Lymphoma group) or with no clinical signs (NCS group) had similar levels of B cells as well as control cats. Although, FeLV/FIV+ cats may not seem to represent a high percentage of sick cats in Brazil (BIEZUS et al., 2019;LEITE-FILHO et al., 2019), since our NCS group also gather these FeLV/FIV+ cats with "normal" naïve B cell amounts (Figure 1), it is interesting to know if these cats could active them after an immunization. With respect to the naïve B cell amounts (Figure 1), cats from Leukemia group or form FIP group had fewer B cells than cats from other groups (ANOVA: F 4,22 = 8.85, P<0.001) ( Figure  1). In these two cat groups, it is possible that these animals could have a decreased capacity to produce antibodies and compromised humoral immunity against other pathogens. As described before by other authors (PEDERSEN, 2009), FIP infection can lead to a decrease in immune system cells, which can be aggravated by FeLV infection (ANDERSON et al., 1971); in fact, these FIP+ animals died in less than 3 days after being tested, with clinical signs of severe respiratory or intestinal problems. Other recent study has shown that a FeLV/FIP+ cat developed a severe form of toxoplasmosis (ZANDONÀ et al., 2018). In our leukemia+ cats, one died a month after testing, and the other was still alive (for 7 months, according to the owner last communication), making it hard to derive any conclusion about their immunological state.
In summary, our results indicated that anti-mouse B220 MAbs can be used to identify a cell population (possibly naïve B cell) in FeLV+ cats, which are concomitantly carrying other debilitating conditions, being an indicator of their immunological status.

ACKNOWLEDGMENTS
We would like to thank all staff working at Hospital Veterinário UVV -Prof. Ricardo Alexandre Hippler for their help to tracking all FeLV+ patients and Centro de Tecnologia Animal (CTA) for providing all control patients used in this work. We also would like to thank Fundação de Amparo à Pesquisa e Inovação do Espírito Santo (FAPES) for partially supporting this research (grant process # 100/2019). This work was financed in part by the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES), Brasil -Finance code 001.

FUNDING
This work was partially supported by a FAPES grant (process # 100/2019) and IDSF received a CAPES PROSUP