Kaylor Wright

Functionally Active HIV-Specific T Cells that Target Gag and Nef Can Be Expanded from Virus-Naïve Donors and Target a Range of Viral Epitopes: Implications for a Cure Strategy after Allogeneic Hematopoietic Stem Cell Transplantation

Allogeneic hematopoietic stem cell transplantation (HSCT) can potentially cure human immunodeficiency virus (HIV) by eliminating infected recipient cells, particularly in the context of technologies that may confer HIV resistance to these stem cells. But, to date, the Berlin patient remains the only case of HIV cure despite multiple attempts to eradicate infection with HSCT. One approach to improve this is to administer virus-specific T cells, a strategy that has proven success in preventing other infections after transplantation. Although we have reported that broadly HIV-specific T cells can be expanded from HIV+ patients, allogeneic transplantations only contain virus-naïve T cells. Modifying this approach for the allogeneic setting requires a robust, reproducible platform that can expand HIV-specific cells from the naïve pool. Hence, we hypothesized that HIV-specific T cells could be primed ex vivo from seronegative individuals to effectively target HIV. Here, we show that ex vivo-primed and expanded HIV-specific T cells released IFNγ in response to HIV antigens and that these cells have enhanced ability to suppress replication in vitro. This is the first demonstration of ex vivo priming and expansion of functional, multi-HIV antigen-specific T cells from HIV-negative donors, which has implications for use of allogeneic HSCT as a functional HIV cure.

Human parainfluenza virus-3 can be targeted by rapidly ex vivo expanded T lymphocytes

BACKGROUND AIMS Human parainfluenza virus-3 (HPIV) is a common cause of respiratory infection in immunocompromised patients and currently has no effective therapies. Virus-specific T-cell therapy has been successful for the treatment or prevention of viral infections in immunocompromised patients but requires determination of T-cell antigens on targeted viruses. METHODS HPIV3-specific T cells were expanded from peripheral blood of healthy donors using a rapid generation protocol targeting four HPIV3 proteins. Immunophenotyping was performed by flow cytometry. Viral specificity was determined by interferon (IFN)-γ ELISpot, intracellular cytokine staining and cytokine measurements from culture supernatants by Luminex assay. Cytotoxic activity was tested by 51Cr release and CD107a mobilization assays. Virus-specific T cells targeting six viruses were then produced by rapid protocol, and the phenotype of HPIV3-specific T cells was determined by immunomagnetic sorting for IFN-γ-producing cells. RESULTS HPIV3-specific T cells were expanded from 13 healthy donors. HPIV3-specific T cells showed a CD4+ predominance (mean CD4:CD8 ratio 2.89) and demonstrated specificity for multiple HPIV3 antigens. The expanded T cells were polyfunctional based on cytokine production but only had a minor cytotoxic component. T cells targeting six viruses in a single product similarly showed HPIV3 specificity, with a predominant effector memory phenotype (CD3+/CD45RA-/CCR7-) in responder cells. DISCUSSION HPIV3-specific T cells can be produced using a rapid ex vivo protocol from healthy donors and are predominantly CD4+ T cells with Th1 activity. HPIV3 epitopes can also be successfully targeted alongside multiple other viral epitopes in production of six-virus T cells, without loss of HPIV3 specificity. These products may be clinically beneficial to combat HPIV3 infections by adoptive T-cell therapy in immune-compromised patients.

A New Method for Reactivating and Expanding T Cells Specific for Rhizopus oryzae

Mucormycosis is responsible for an increasing proportion of deaths after allogeneic bone marrow transplantation. Because this disease is associated with severe immunodeficiency and has shown resistance to even the newest antifungal agents, we determined the feasibility of reactivating and expanding Rhizopus oryzae-specific T cells for use as adoptive immunotherapy in transplant recipients. R. oryzae extract-pulsed monocytes were used to stimulate peripheral blood mononuclear cells from healthy donors, in the presence of different cytokine combinations. The generated R. oryzae-specific T cell products were phenotyped after the third stimulation and further characterized by the use of antibodies that block class I/II molecules, as well as pattern recognition receptors. Despite the very low frequency of R. oryzae-specific T cells of healthy donors, we found that stimulation with interleukin-2 (IL-2)/IL-7 cytokine combination could expand these rare cells. The expanded populations included 17%–83% CD4+ T cells that were specific for R. oryzae antigens. Besides interferon-γ (IFN-γ), these cells secreted IL-5, IL-10, IL-13, and tumor necrosis factor alpha (TNF-α), and recognized fungal antigens presented by HLA-II molecules rather than through nonspecific signaling. The method described herein is robust and reproducible, and could be used to generate adequate quantities of activated R. oryzae-specific T cells for clinical testing of safety and antifungal efficacy in patients with mucormycosis.

Human papilloma virus-specific T cells can be generated from naïve T cells for use as an immunotherapeutic strategy for immunocompromised patients.

Human papilloma virus (HPV) is a known cause of cervical cancer, squamous cell carcinoma and laryngeal cancer. Although treatments exist for HPV-associated malignancies, patients unresponsive to these therapies have a poor prognosis. Recent findings from vaccine studies suggest that T-cell immunity is essential for disease control. Because Epstein-Barr Virus (EBV)-specific T cells have been highly successful in treating or preventing EBV-associated tumors, we hypothesized that the development of a manufacturing platform for HPV-specific T cells from healthy donors could be used in a third-party setting to treat patients with high-risk/relapsed HPV-associated cancers. Most protocols for generating virus-specific T cells require prior exposure of the donor to the targeted virus and, because the seroprevalence of high-risk HPV types varies greatly by age and ethnicity, manufacturing of donor-derived HPV-specific T cells has proven challenging. We, therefore, made systematic changes to our current Good Manufacturing Practice (GMP)-compliant protocols to improve antigen presentation, priming and expansion for the manufacture of high-efficacy HPV-specific T cells. Like others, we found that current methodologies fail to expand HPV-specific T cells from most healthy donors. By optimizing dendritic cell maturation and function with lipopolysaccharide (LPS) and interferon (IFN)γ, adding interleukin (IL)-21 during priming and depleting memory T cells, we achieved reliable expansion of T cells specific for oncoproteins E6 and E7 to clinically relevant amounts (mean, 578-fold expansion; n = 10), which were polyfunctional based on cytokine multiplex analysis. In the third-party setting, such HPV-specific T-cell products might serve as a potent salvage therapy for patients with HPV-associated diseases.

763. HIV-Specific T Cells Can Be Expanded from Virus-Naive Donors to Target a Range of Viral Epitopes: Implications for a Cure Strategy After Allogeneic HSCT

Background: Adoptive T cell therapy has been successful in boosting viral-specific immunity post-hematopoietic stem cell transplant (HSCT), preventing viral rebound of CMV and EBV. However, the therapeutic use of T cells to boost HIV-specific T cell immunity in HIV+ patients has been met with limited success. Despite multiple attempts to eradicate HIV infection with allogeneic HSCT, the Berlin patient remains the only case of functional HIV cure. Previous infusions of HIV-specific T cells have resulted in immune escape from single epitope specificity and limited persistence of the T cell product. Our approach to address these limitations is to expand HIV-specific T cells derived from virus-naive donors including umbilical cord blood, employing a non-HLA restricted approach for HIV+ patients receiving allogeneic HSCT for HIV-associated hematologic malignancies. Design: We have developed a robust, reproducible platform that can expand HIV-specific T cells (HXTCs) from the naive pool in the allogeneic setting. Peripheral blood mononuclear cells isolated from virus-naive donors are used to generate dendritic cells and T cells. T cells are stimulated with antigen presenting cells pulsed with HIV-pepmix and a combination of cytokines that promote proliferation and differentiation. T cells were tested for: (1) specificity against HIV antigens and individual peptides, (2) pro-inflammatory cytokine secretion in response to stimulation with HIV peptides, and (3) ability to suppress HIV replication in vitro. Results: We successfully expanded (75.705 mean fold expansion) HXTCs recognizing HIV antigens from virus naive donors. IFNg ELISPOT showed HXTCs (n=8) were specific against Gag (mean=331.25 SFC/1e5 cells) and Nef (mean=242.63 SFC/1e5 cells) vs Irrelevant (mean=13 SFC/1e5 cells). HXTCs produced significantly pro-inflammatory responses (p less than 0.05) to stimulation by gag/nef, as determined by levels of TNF-alpha, IL-2, IL-6, IL-8, and perforin (n=3). Importantly, HXTCs (n=4) were able to suppress HIV replication more than non-specific CD8+ T cells when co-cultured with autologous CD4+ T cells infected with HIV SF162 (HXTC 78.62% viral suppression compared to CD8+ T cell 34.19% viral suppression). HXTCs showed both HLA Class I or II specificity for individual HIV epitopes, as determined by HLA blocking and IFNg ELISPOT. Conclusion: This is the first report demonstrating generation of functional, multi-HIV antigen specific T-cells from HIV-negative donors, which has implications for using allogeneic HSCT as a functional HIV cure. The low frequency of circulating HXTCs post-infusion suggests these HXTCs could have a significant effect on preventing viral rebound. The generation of HXTCs from cord blood could provide a further advantage to increase the donor pool.