Impact of Cryopreservation on Viability, Phenotype, and Functionality of Porcine PBMC

The use of frozen peripheral blood mononuclear cells (PBMC) is common in immunological studies. The impact of freezing PBMC has been assessed using human and mice cells, but little information is available regarding domestic animals. In the present study, the phenotype and functionality of frozen porcine PBMC were examined. In a preliminary experiment, three freezing media: fetal bovine serum plus 10% dimethyl sulfoxide, PSC cryopreservation kit, and Cryostor CS10, were compared regarding the preservation of cell viability and the response of PBMC to mitogens after thawing. After being stored one month in liquid nitrogen, cell viability was above 89% for all freezing media. The ELISPOT IFN-gamma (IFN-γ) results in response to PHA and of IgG ELISPOT in response to R848+IL-2 were similar to those obtained using fresh PBMC. In the second set of experiments, PBMC were obtained from five pigs vaccinated against Porcine reproductive and respiratory syndrome virus (PRRSV) and then frozen using Cryostor CS10. Recovered cells were phenotyped by flow cytometry using anti-CD3, CD4, CD8, and CD21 antibodies and were used to assess the PRRSV-specific responses in a proliferation experiment, an IFN-γ ELISPOT, and an IgG ELISPOT, and compared to the results obtained with fresh cells. The antigen-specific responses of frozen cells were significantly (p<0.05) impaired in the proliferation assay, particularly for CD4/CD8 double-positive T-cells and for CD21+ cells. Freezing resulted in decreased proliferation when Con A, but not PHA, was used. In ELISPOT, cryopreservation resulted in a decreased frequency of IFN-γ-secreting cells in response to PRRSV (p<0.05) but the response to PHA was not affected. No differences were observed in the IgG ELISPOT after polyclonal activation. Taken together, cryopreservation of porcine PBMC had a significant impact on the magnitude of recall antigen responses and therefore, it may affect the response of effector/memory cells but seems not to have a major impact on naïve T-cells. These results may help to the better use of frozen porcine PBMC, and to the interpretation of the results obtained from them.

739 Intratumor childhood vaccine-specific CD4+ T cell recall helps antitumor CD8 T cells

BackgroundPVSRIPO, a recombinant poliovirus derived from the live-attenuated Sabin oral polio vaccine strain, is being tested in multi-institutional phase II clinical trials for recurrent glioblastoma (NCT04479241) and unresectable, PD-1 refractory melanoma (NCT04577807) in combination with PD1 blockade. PVSRIPO capsid is identical to the Sabin vaccine strain and >99% identical to the inactivated Polio vaccine (IPOL, Salk), against which public health mandated childhood vaccination is near universal. In non-vaccinated mice, PVSRIPO mediates antitumor efficacy in a replication-dependent manner via engaging innate inflammation and antitumor T cells. Accordingly, it is anticipated that pre-existing immunity to PVSRIPO impedes antitumor therapy. However, recent evidence indicates that immunological 'recall', or reactivation of memory T cells, may mediate anti-tumor effects.MethodsThe impact of prior polio vs control (KLH) vaccination on intratumor viral replication, tumor inflammation, and overall tumor growth after intratumor PVSRIPO therapy was assessed in murine tumor models. The role of polio capsid and tetanus recall antigens in mediating intratumor inflammation and antitumor efficacy was similarly studied in mice non-permissive to PVSRIPO infection. To mechanistically define antitumor effects of polio recall, B cell and CD8 T cell knockout mice were used, in addition to adoptive transfer of CD4+ T cells from vaccinated mice. Intratumor polio or tetanus recall antigen therapy was performed after OT-I transfer (OVA-specific T cells) in the B16-OVA melanoma model to gauge antitumor T cell activity. Lastly, the inflammatory effects of polio and tetanus antigens was tested in human peripheral blood mononuclear cells (PBMCs).ResultsDespite curtailing intratumor viral replication, prior polio vaccination in mice potentiated subsequent antitumor efficacy of PVSRIPO. Intratumor recall responses induced by polio and tetanus antigens also delayed tumor growth. Recall antigen therapy was associated with marked intratumor influx of eosinophils, conventional CD4+ T cells, and increased expression of IFN-g, TNF, and Granzyme B in tumor infiltrating T cells. The antitumor efficacy of polio recall antigen was mediated by CD4+ T cells, partially depended upon CD8+ T cells, and was impaired by B cells. Both polio and tetanus recall antigen therapy bolstered the antitumor function of tumor-specific OT-I CD8+ T cells. Polio and tetanus antigens induced CXCL10 and type I/II/III IFNs in PBMCs in vitro.ConclusionsChildhood vaccine-specific CD4+ T cells hold cancer immunotherapy potential. In the context of PVSRIPO therapy, antitumor and inflammatory effects of polio vaccine-specific CD4+ T cell recall supersedes inhibitory effects of attenuated intratumor viral replication, and represents a novel mechanism of action.Ethics ApprovalThe animal work described in this study was approved by the Duke University IACUC.

Characterization of anti-interferon-γ antibodies in HIV-negative immunodeficient patients infected with unusual intracellular microorganisms

A major characteristic of immunodeficiency associated with life-threatening intracellular infection in adults is the presence of anti-interferon-γ antibodies. Although little is known about the mechanism underlying this syndrome, it is believed that the antibodies inhibit the activity of downstream signaling pathway of interferon-γ. In this study, the characteristics of these antibodies in patients who presented, or have a history of, intracellular infection and were positive to anti-interferon-γ antibodies were investigated. The antibodies exhibited mainly the IgG1 and the IgG4 subtypes and recognized the C-terminal of the interferon-γ linear epitope containing the KRKR motif, which is required for the biological activity of interferon-γ. The antibodies bound to recombinant interferon-γ with significantly lower avidity than antibodies to a recall antigen, tetanus toxoid, suggesting that the antibodies might have not undergone affinity maturation. The data from this study may provide fundamental information to better understand the properties of anti-interferon-γ antibodies, which can be useful for future studies. Impact statement An increase in the number of immunodeficient patients related to autoantibodies to interferon (IFN)-γ has been observed particularly in East Asian adults. These patients are often presented with opportunistic infections caused by intracellular pathogens, including non-tuberculous mycobacteria (NTM), Cryptococcus neoformans, Penicillium marneffei (now called Talaromyces marneffei), and non-typhoidal Salmonella spp. The mortality rate for this syndrome is relatively high with 32% patients dying at the median time of 25 months after diagnosis. Characterization of these autoantibodies may promote better understanding of the syndrome, an emerging health problem affecting East Asia populations and impeding their welfare and economic development.