Three-color flow cytometry detection of virus-specific CD4+ and CD8+ T cells in the cat

Abstract

We describe a three-color flow cytometry assay for the detection of virus-specific CD4+ and CD8+ T cells in the cat. The assay is based upon detection of intracellular TNFalpha using the cross-reactive mAb 6401.1111, raised against the human cytokine. Allophycocyanin-conjugated mAb 6401.1111 specifically stained feline TNFalpha-producing murine cells and also Staphylococcus aureus Enterotoxin B-stimulated feline T cells, thus providing formal evidence for cross-reactivity. By using the anti-TNFalpha mAb in combination with PE- and FITC-conjugated mAbs against feline CD4 and CD8, respectively, antiviral CD4+ and CD8+ T cells could be identified in the peripheral blood and in the spleens of feline infectious peritonitis virus-infected cats. Moreover, feline calicivirus (FCV)-specific CD4+ T cells were detected in the spleens of FCV-vaccinated cats. As antigen-presenting cells (APCs), we used immortalized autologous fibroblast cell lines, PBMC or splenocytes. A straightforward protocol, in which splenocyte preparations served both as APCs and effector cells, consistently yielded best results. The assay will permit further studies of the cellular immune responses in cats during natural and experimental viral infections. It will contribute to vaccine development against feline viruses by facilitating the identification of T cell antigens and epitopes, and by allowing the quantitative detection of virus-specific T cells after vaccination. Furthermore, the assay will add to the value of those systems in which viral infections of the cat serve as models for human disease.

Natural History of a Recurrent Feline Coronavirus Infection and the Role of Cellular Immunity in Survival and Disease

“…FIPV-specific T-cell responses in persistently infected, partially protected cats and in a long-term survivor. Splenocytes were stimulated with autologous fibroblast cultures (10), which had been infected with rVVs expressing the various FIPV proteins. An rVV with lacZ inserted into the TK locus (rVV-TK) served as a negative control.…”

“…Single-cell spleen suspensions, prepared as previously described (10), were resuspended in 25 mM HEPES-buffered RPMI 1640 (Invitrogen Corporation), supplemented with 10% heat-inactivated fetal calf serum, Glutamax I (Invitrogen Corporation), 50 M 2-mercaptoethanol, 100 IU of penicillin/ml, and 100 g of streptomycin/ ml. To detect FIPV-specific T cells, 0.5 ϫ 10 6 to 1 ϫ 10 6 splenocytes were stimulated for 6 h in the presence of 10 M brefeldin A with autologous immortalized fibroblasts (5 ϫ 10 4 ) (10), which had been infected with rVV for 16 h. Three-color flow cytometry analysis of virus-specific CD4 ϩ and CD8 ϩ T cells, based upon detection of intracellular tumor necrosis factor alpha (TNF-␣), was performed as previously described (10).…”

“…4; Table 2). Autologous fibroblast cultures were infected with these recombinant viruses and were used to stimulate splenocytes in our intracellular cytokine (TNF-␣) staining assay (10). We analyzed T-cell responses in the spleen because it is both the major lymphoid organ harboring memory T cells and a prime target site for FIPV replication.…”

Vaccination with vif-deleted feline immunodeficiency virus provirus, GM-CSF, and TNF-α plasmids preserves global CD4 T lymphocyte function after challenge with FIV

“…Antibodies against human TNF-α and IL-2 were utilized for this protocol due to the lack of feline cytokine-specific monoclonal antibodies. Cross-reactivity of the anti-human TNF-α monoclonal antibody used for detection of feline TNF-α by flow cytometry was previously described [58]. To validate cross-reactivity of a human IL-2 monoclonal antibody (clone MQ1-17H12) for feIL-2, immunoprecipitates were generated by this human IL-2 monoclonal antibody (clone MQ1-17H12) from mitogen-stimulated feline PBMC culture supernatants.…”

Testes fluorimétricos na sorologia da toxoplasmose humana: detecção simultânea de anticorpos de IgG e IgM específicos

“…Alguns fluorocromos derivados destes compostos originais foram produzidos para melhorar a solubilidade e fotoestabilidade e para possibilitar a bioconjugação, tais como o isotiocianato de fluoresceína (FITC), o N-hidroxisuccinimidil fluoresceína (NHS-Fuorescein), o isotiocianato de tetrametil rodamina (TRITC), Nhidroxisuccinimidil éster rodamina (NHS-Rhodamine), e sulfonil cloridre rodamina (Rhodamine B) (Wisdom, 2005). Além disso, outros fluorocromos estão disponíveis comercialmente para o uso em imunoensaios, como as indocarbocianinas (Cy2, Cy3 e Cy5), aloficocianina (APC) e ficoeritrina (PE) (Fu et al, 2012, Groot-Mijnes et al, 2004, Iijima et al, 2011. E ainda, atualmente, novos compostos fluorescentes sintéticos mais sensíveis tem sido produzidos, como o violeta brilhante (BV421).…”