Shabnum Patel

Conjugating Prussian blue nanoparticles onto antigen-specific T cells as a combined nanoimmunotherapy.

AIM To engineer a novel nanoimmunotherapy comprising Prussian blue nanoparticles (PBNPs) conjugated to antigen-specific cytotoxic T lymphocytes (CTL), which leverages PBNPs for their photothermal therapy (PTT) capabilities and Epstein-Barr virus (EBV) antigen-specific CTL for their ability to traffic to and destroy EBV antigen-expressing target cells. MATERIALS & METHODS PBNPs and CTL were independently biofunctionalized. Subsequently, PBNPs were conjugated onto CTL using avidin-biotin interactions. The resultant cell-nanoparticle construct (CTL:PBNPs) were analyzed for their physical, phenotypic and functional properties. RESULTS Both PBNPs and CTL maintained their intrinsic physical, phenotypic and functional properties within the CTL:PBNPs. CONCLUSION This study highlights the potential of our CTL:PBNPs nanoimmunotherapy as a novel therapeutic for treating virus-associated malignancies such as EBV+ cancers.

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.

Latent HIV reservoirs exhibit inherent resistance to elimination by CD8+ T cells

The presence of persistent, latent HIV reservoirs in CD4+ T cells obstructs current efforts to cure infection. The so-called kick-and-kill paradigm proposes to purge these reservoirs by combining latency-reversing agents with immune effectors such as cytotoxic T lymphocytes. Support for this approach is largely based on success in latency models, which do not fully reflect the makeup of latent reservoirs in individuals on long-term antiretroviral therapy (ART). Recent studies have shown that CD8+ T cells have the potential to recognize defective proviruses, which comprise the vast majority of all infected cells, and that the proviral landscape can be shaped over time due to in vivo clonal expansion of infected CD4+ T cells. Here, we have shown that treating CD4+ T cells from ART-treated individuals with combinations of potent latency-reversing agents and autologous CD8+ T cells consistently reduced cell-associated HIV DNA, but failed to deplete replication-competent virus. These CD8+ T cells recognized and potently eliminated CD4+ T cells that were newly infected with autologous reservoir virus, ruling out a role for both immune escape and CD8+ T cell dysfunction. Thus, our results suggest that cells harboring replication-competent HIV possess an inherent resistance to CD8+ T cells that may need to be addressed to cure infection.

Toward a Rapid Production of Multivirus-Specific T Cells Targeting BKV, Adenovirus, CMV, and EBV from Umbilical Cord Blood

Umbilical cord blood (CB) has emerged as an effective alternative donor source for hematopoietic stem cell transplantation. Despite this success, the prolonged duration of immune suppression following CB transplantation and the naiveté of CB T cells leave patients susceptible to viral infections. Adoptive transfer of ex vivo-expanded virus-specific T cells from CB is both feasible and safe. However, the manufacturing process of these cells is complicated, lengthy, and labor-intensive. We have now developed a simplified method to manufacture a single culture of polyclonal multivirus-specific cytotoxic T cells in less than 30 days. It eliminates the need for a live virus or transduction with a viral vector, thus making this approach widely available and GMP-applicable to target multiple viruses. The use of overlapping PepMixes as a source of antigen stimulation enable expansion of the repertoire of the T cell product to any virus of interest and make it available as a third party “off the shelf” treatment for viral infections following transplantation.

T-cell therapies for HIV: Preclinical successes and current clinical strategies.

Although antiretroviral therapy (ART) has been successful in controlling HIV infection, it does not provide a permanent cure, requires lifelong treatment, and HIV-positive individuals are left with social concerns such as stigma. The recent application of T cells to treat cancer and viral reactivations post-transplant offers a potential strategy to control HIV infection. It is known that naturally occurring HIV-specific T cells can inhibit HIV initially, but this response is not sustained in the majority of people living with HIV. Genetically modifying T cells to target HIV, resist infection, and persist in the immunosuppressive environment found in chronically infected HIV-positive individuals might provide a therapeutic solution for HIV. This review focuses on successful preclinical studies and current clinical strategies using T-cell therapy to control HIV infection and mediate a functional cure solution.

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.

Beyond Chemotherapy: Checkpoint Inhibition and Cell-Based Therapy in Non-Hodgkin Lymphoma

Immune-based treatment strategies, such as checkpoint inhibition and chimeric antigen receptor (CAR) T cells, have started a new frontier for treatment in non-Hodgkin lymphoma (NHL). Checkpoint inhibition has been most successful in Hodgkin lymphoma, where higher expression of PD-L1 is correlated with better overall response rate. Combinations of checkpoint inhibition with various chemotherapy or biologics are in clinical trials, with initially promising results and manageable safety profiles. CAR T-cell therapies that target CD19 are a promising and attractive therapy for B-cell NHLs, with a product approved by the US Food and Drug Administration in 2017. Changes in the target, hinge, or costimulatory domain can dramatically alter the persistence and efficacy of the CAR T cells. The ZUMA trials from Kite used CD19-(CD28z) CAR T cells, whereas the TRANSCEND studies from Juno and the JULIET studies from Novartis used CD19-(4-1BBz) CARs. Despite the recent successes with CAR T-cell clinical trials, major concerns associated with this therapy include cytokine release syndrome, potential neurotoxicities, B-cell aplasia, loss of tumor antigen leading to relapse, and cost and accessibility of the treatment. Although first-generation CAR T-cell therapies have failed in solid malignancies, newer second- and third-generation CAR T cells that target antigens other than CD19 (such as mesothelin or B-cell maturation antigen) are being studied in clinical trials for treatment of lung cancer or multiple myeloma. Overall, immune-based treatment strategies have given oncologists and patients hope when there used to be none, as well as a new basket of tools yet to come with further research and development.

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.