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Abstract Transposable elements (TEs) are often expressed at higher levels in tumor cells than normal cells, implicating these genomic regions as an untapped pool of tumor-associated antigens. In ovarian cancer (OC), protein from the TE ERV-K is frequently expressed by tumor cells. Here we determined whether the targeting of previously identified epitope in the envelope gene (env) of ERV-K resulted in target antigen specificity against cancer cells. We found that transducing healthy donor T cells with an ERV-K-Env-specific T cell receptor construct resulted in antigen specificity only when co-cultured with HLA-A*03:01 B lymphoblastoid cells. Furthermore, in vitro priming of several healthy donors with this epitope of ERV-K-Env did not result in target antigen specificity. These data suggest that the T cell receptor is a poor candidate for targeting this specific ERV-K-Env epitope and has limited potential as a T cell therapy for OC.

Abstract Viral infections remain a major risk in immunocompromised pediatric patients, and virus-specific T cell (VST) therapy has been successful for treatment of refractory viral infections in prior studies. We performed a phase II multicenter study (NCT03475212) for the treatment of pediatric patients with inborn errors of immunity and/or post allogeneic hematopoietic stem cell transplant with refractory viral infections using partially-HLA matched VSTs targeting cytomegalovirus, Epstein-Barr virus, or adenovirus. Primary endpoints were feasibility, safety, and clinical responses (>1 log reduction in viremia at 28 days). Secondary endpoints were reconstitution of antiviral immunity and persistence of the infused VSTs. Suitable VST products were identified for 75 of 77 clinical queries. Clinical responses were achieved in 29 of 47 (62%) of patients post-HSCT including 73% of patients evaluable at 1-month post-infusion, meeting the primary efficacy endpoint (>52%). Secondary graft rejection occurred in one child following VST infusion as described in a companion article. Corticosteroids, graft-versus-host disease, transplant-associated thrombotic microangiopathy, and eculizumab treatment correlated with poor response, while uptrending absolute lymphocyte and CD8 T cell counts correlated with good response. This study highlights key clinical factors that impact response to VSTs and demonstrates the feasibility and efficacy of this therapy in pediatric HSCT.

Abstract Virus-specific T cells (VST) from partially-HLA matched donors have been effective for treatment of refractory viral infections in immunocompromised patients in prior studies with a good safety profile, but rare adverse events have been described. Here we describe a unique and severe adverse event of VST therapy in an infant with severe combined immunodeficiency, who receives, as part of a clinical trial (NCT03475212), third party VSTs for treating cytomegalovirus viremia following bone marrow transplantation. At one-month post-VST infusion, rejection of graft and reversal of chimerism is observed, as is an expansion of T cells exclusively from the VST donor. Single-cell gene expression and T cell receptor profiling demonstrate a narrow repertoire of predominantly activated CD4+ T cells in the recipient at the time of rejection, with the repertoire overlapping more with that of peripheral blood from VST donor than the infused VST product. This case thus demonstrates a rare but serious side effect of VST therapy.

Abstract Prussian blue nanoparticle‐based photothermal therapy (PBNP‐PTT) is an effective tumor treatment capable of eliciting an antitumor immune response. Motivated by the ability of PBNP‐PTT to potentiate endogenous immune responses, we recently demonstrated that PBNP‐PTT could be used ex vivo to generate tumor‐specific T cells against glioblastoma (GBM) cell lines as an adoptive T cell therapy (ATCT). In this study, we further developed this promising T cell development platform. First, we assessed the phenotype and function of T cells generated using PBNP‐PTT. We observed that PBNP‐PTT facilitated CD8+ T cell expansion from healthy donor PBMCs that secreted IFNγ and TNFα and upregulated CD107a in response to engagement with target U87 cells, suggesting specific antitumor T cell activation and degranulation. Further, CD8+ effector and effector memory T cell populations significantly expanded after co‐culture with U87 cells, consistent with tumor‐specific effector responses. In orthotopically implanted U87 GBM tumors in vivo, PBNP‐PTT‐derived T cells effectively reduced U87 tumor growth and generated long‐term survival in >80% of tumor‐bearing mice by Day 100, compared to 0% of mice treated with PBS, non‐specific T cells, or T cells expanded from lysed U87 cells, demonstrating an enhanced antitumor efficacy of this ATCT platform. Finally, we tested the generalizability of our approach by generating T cells targeting medulloblastoma (D556), breast cancer (MDA‐MB‐231), neuroblastoma (SH‐SY5Y), and acute monocytic leukemia (THP‐1) cell lines. The resulting T cells secreted IFNγ and exerted increased tumor‐specific cytolytic function relative to controls, demonstrating the versatility of PBNP‐PTT in generating tumor‐specific T cells for ATCT.

Cord blood (CB)-derived natural killer (NK) cells that are genetically engineered to express a chimeric antigen receptor (CAR) are an attractive off-the-shelf therapy for the treatment of cancer, demonstrating a robust safety profile in vivo. For poor prognosis brain tumors such as glioblastoma multiforme (GBM), novel therapies are urgently needed. Although CAR-T cells demonstrate efficacy in preclinical GBM models, an off-the-shelf product may exhibit unwanted side effects like graft-versus-host disease. Hence, we developed an off-the-shelf CAR-NK cell approach using a B7H3 CAR and showed that CAR-transduced NK cells have robust cytolytic activity against GBM cells in vitro. However, transforming growth factor (TGF)-β within the tumor microenvironment has devastating effects on the cytolytic activity of both unmodified and CAR-transduced NK cells. To overcome this potent immune suppression, we demonstrated that co-transducing NK cells with a B7H3 CAR and a TGF-β dominant negative receptor (DNR) preserves cytolytic function in the presence of exogenous TGF-β. This study demonstrates that a novel DNR and CAR co-expression strategy may be a promising therapeutic for recalcitrant CNS tumors like GBM.

Adoptive T cell immunotherapy has been used to restore immunity against multiple viral targets in immunocompromised patients after bone-marrow transplantation and has been proposed as a strategy for preventing coronavirus 2019 (COVID-19) in this population. Ideally, expanded severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-virus-specific T cells (CSTs) should demonstrate marked cell expansion, T cell specificity, and CD8+ T cell skewing prior to adoptive transfer. However, current methodologies using IL-4 + IL-7 result in suboptimal specificity, especially in CD8+ cells. Using a microexpansion platform, we screened various cytokine cocktails (IL-4 + IL-7, IL-15, IL-15 + IL-4, IL-15 + IL-6, and IL-15 + IL-7) for the most favorable culture conditions. IL-15 + IL-7 optimally balanced T cell expansion, polyfunctionality, and CD8+ T cell skewing of a final therapeutic T cell product. Additionally, the transcriptomes of CD4+ and CD8+ T cells cultured with IL-15 + IL-7 displayed the strongest induction of antiviral type I interferon (IFN) response genes. Subsequently, microexpansion results were successfully translated to a Good Manufacturing Practice (GMP)-applicable format where IL-15 + IL-7 outperformed IL-4 + IL-7 in specificity and expansion, especially in the desirable CD8+ T cell compartment. These results demonstrate the functional implications of IL-15-, IL-4-, and IL-7-containing cocktails for therapeutic T cell expansion, which could have broad implication for cellular therapy, and pioneer the use of RNA sequencing (RNA-seq) to guide viral-specific T cell (VST) product manufacturing.

HIV-specific T cells are necessary for control of HIV-1 replication but are largely insufficient for viral clearance. This is due in part to these cells’ recognition of immunodominant but variable regions of the virus, which facilitates viral escape via mutations that do not incur viral fitness costs. HIV-specific T cells targeting conserved viral elements are associated with viral control but are relatively infrequent in people living with HIV (PLWH). The goal of this study was to increase the number of these cells via an ex vivo cell manufacturing approach derived from our clinically-validated HIV-specific expanded T-cell (HXTC) process. Using a nonhuman primate (NHP) model of HIV infection, we sought to determine i) the feasibility of manufacturing ex vivo-expanded virus-specific T cells targeting viral conserved elements (CE, CE-XTCs), ii) the in vivo safety of these products, and iii) the impact of simian/human immunodeficiency virus (SHIV) challenge on their expansion, activity, and function. NHP CE-XTCs expanded up to 10-fold following co-culture with the combination of primary dendritic cells (DCs), PHA blasts pulsed with CE peptides, irradiated GM-K562 feeder cells, and autologous T cells from CE-vaccinated NHP. The resulting CE-XTC products contained high frequencies of CE-specific, polyfunctional T cells. However, consistent with prior studies with human HXTC and these cells’ predominant CD8+ effector phenotype, we did not observe significant differences in CE-XTC persistence or SHIV acquisition in two CE-XTC-infused NHP compared to two control NHP. These data support the safety and feasibility of our approach and underscore the need for continued development of CE-XTC and similar cell-based strategies to redirect and increase the potency of cellular virus-specific adaptive immune responses.

Summary: The synovial sarcoma X breakpoint 2 (SSX2) belongs to a multigene family of cancer-testis antigens and can be found overexpressed in multiple malignancies. Its restricted expression in immune-privileged normal tissues suggest that SSX2 may be a relevant target antigen for chimeric antigen receptor (CAR) therapy. We have developed a T cell receptor (TCR)-like antibody (Fab/3) that binds SSX2 peptide 41-49 (KASEKIFYV) in the context of HLA-A∗-0201. The sequence of Fab/3 was utilized to engineer a CAR with the CD3 zeta intra-cellular domain along with either a CD28 or 4-1BB costimulatory endodomain. Human T cells from HLA-A2+ donors were transduced to mediate anti-tumor activity against acute myeloid leukemia (AML) tumor cells. Upon challenge with HLA-A2+/SSX2+ AML tumor cells, CAR-expressing T cells released interferon-γ and eliminated the tumor cells in a long-term co-culture assay. Using the HLA-A2+ T2 cell line, we demonstrated a strong specificity of the single-chain variable fragment (scFv) for SSX2 p41-49 and the closely related SSX3 p41-49, with no response against the others SSX-homologous peptides or unrelated homologous peptides. Since SSX3 has not been observed in tumor cells and expression cannot be induced by pharmacological intervention, SSX241-49 represents an attractive target for CAR-based cellular therapy to treat multiple types of cancer.

Targeting tumor-associated antigens in paediatric medulloblastomas (MB) is challenging due to their low mutational burden. Here, the authors develop a sensitive proteogenomic approach to identify tumour specific neoantigens, which may enable personalised T cell immunotherapy in paediatric MB.

While antiretroviral therapy (ART) can completely suppress viremia, it is not a cure for HIV. HIV persists as a latent reservoir of infected cells, able to evade host immunity and re-seed infection following cessation of ART. Two promising immunotherapeutic strategies to eliminate both productively infected cells and reactivated cells of the reservoir are the adoptive transfer of potent HIV-specific T cells and the passive administration of HIV-specific broadly neutralizing antibodies also capable of mediating antibody-dependent cellular cytotoxicity (ADCC). The simultaneous use of both as the basis of a single therapeutic has never been explored. We therefore sought to modify HIV-specific T cells from HIV-naive donors (to allow their use in the context of allotransplant, a promising platform for sterilizing cures) so they are able to secrete a broadly neutralizing antibody (bNAb) directed against the HIV envelope to elicit ADCC. We designed an antibody construct comprising bNAb 10-1074 heavy and light chains, fused to IgG3 Fc to elicit ADCC, with truncated cluster of differentiation 19 (CD19) as a selectable marker. HIV-specific T cells were expanded from HIV-naive donors by priming with antigen-presenting cells expressing overlapping HIV antigens in the presence of cytokines. T cells retained specificity against Gag, Nef, and Pol peptides (218.55 ± 300.14 interferon γ [IFNγ] spot-forming cells [SFC]/1 × 105) following transduction (38.92 ± 25.30) with the 10-1074 antibody constructs. These cells secreted 10-1074 antibodies (139.04 ± 114.42 ng/mL). The HIV-specific T cells maintained T cell function following transduction, and the secreted 10-1074 antibody bound HIV envelope (28.13% ± 19.42%) and displayed ADCC activity (10.47% ± 4.11%). Most critically, the 10-1074 antibody-secreting HIV-specific T cells displayed superior in vitro suppression of HIV replication. In summary, HIV-specific T cells can be engineered to produce antibodies mediating ADCC against HIV envelope-expressing cells. This combined innate/adaptive approach allows for synergy between the two immune arms, broadens the target range of the immune therapy, and provides further insight into what defines an effective anti-HIV response.

Although anti-retroviral therapy (ART) is successful in suppressing HIV-1 replication, HIV latently infected reservoirs are not eliminated, representing a major hurdle in efforts to eradicate the virus. Current strategies to eradicate HIV involve two steps: (1) the reactivation of latently infected cells with latency reversing agents (LRAs) to expose persisting HIV, and (2) the elimination of these cells with immune effectors while continuing ART to prevent reinfection. HIV-specific T cells (HSTs) can kill reactivated HIV-infected cells and are currently being evaluated in early-stage immunotherapy trials. HIV can mutate sequences in T cell epitopes and evade T cell-mediated killing of HIV-infected cells. However, by directing T cells to target multiple conserved, non-escaped HIV epitopes, the opportunity for viral escape can be reduced. Using a good manufacturing practice (GMP)-compliant platform, we manufactured HSTs against non-escape epitope targets (HST-NEETs) from HIV+ and HIV-seronegative donors. HST-NEETs expanded to clinically relevant numbers, lysed autologous antigen-pulsed targets, and showed a polyfunctional pro-inflammatory cytokine response. Notably, HST-NEETs recognized multiple conserved, non-escaped HIV epitopes and their common variants. We propose that HST-NEETs could be used to eliminate reactivated virus from latently infected cells in HIV+ individuals following LRA treatment. Additionally, HST-NEETs derived from HIV-negative individuals could be used post-transplant for HIV+ individuals with hematologic malignancies to augment anti-viral immunity and destroy residual infected cells. Keywords: HIV-specific T cells, immunotherapy, latent reservoir, viral escape, conserved epitopes

BackgroundDespite similar rates of infection, adults and children have markedly different morbidity and mortality related to severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2). Compared to adults, children have infrequent severe manifestations of acute infection but are uniquely at risk for the rare and often severe Multisystem Inflammatory Syndrome in Children (MIS-C) following infection. We hypothesized that these differences in presentation are related to differences in the magnitude and/or antigen specificity of SARS-CoV-2-specific T cell (CST) responses between adults and children. We therefore set out to measure the CST response in convalescent adults versus children with and without MIS-C following SARS-CoV-2 infection.MethodsCSTs were expanded from blood collected from convalescent children and adults post SARS-CoV-2 infection and evaluated by intracellular flow cytometry, surface markers, and cytokine production following stimulation with SARS-CoV-2-specific peptides. Presence of serum/plasma antibody to spike and nucleocapsid was measured using the luciferase immunoprecipitation systems (LIPS) assay.FindingsTwenty-six of 27 MIS-C patients, 7 of 8 non-MIS-C convalescent children, and 13 of 14 adults were seropositive for spike and nucleocapsid antibody. CST responses in MIS-C patients were significantly higher than children with uncomplicated SARS-CoV-2 infection, but weaker than CST responses in convalescent adults.InterpretationAge-related differences in the magnitude of CST responses suggest differing post-infectious immunity to SARS-CoV-2 in children compared to adults post uncomplicated infection. Children with MIS-C have CST responses that are stronger than children with uncomplicated SARS-CoV-2 infection and weaker than convalescent adults, despite near uniform seropositivity.

Abstract Background Medulloblastoma (MB), the most common pediatric brain cancer, presents with a poor prognosis in a subset of patients with high risk disease, or at recurrence, where current therapies are ineffective. Cord blood (CB) natural killer (NK) cells may be promising off-the-shelf effector cells for immunotherapy due to their recognition of malignant cells without the need for a known target, ready availability from multiple banks, and their potential to expand exponentially. However, they are currently limited by immune suppressive cytokines secreted in the MB tumor microenvironment including Transforming Growth Factor β (TGF-β). Here, we address this challenge in in vitro models of MB. Methods CB-derived NK cells were modified to express a dominant negative TGF-β receptor II (DNRII) using retroviral transduction. The ability of transduced CB cells to maintain function in the presence of medulloblastoma-conditioned media was then assessed. Results We observed that the cytotoxic ability of nontransduced CB-NK cells was reduced in the presence of TGF-β-rich, medulloblastoma-conditioned media (21.21 ± 1.19% killing at E:T 5:1 in the absence vs. 14.98 ± 2.11% in the presence of medulloblastoma-conditioned media, n = 8, p = 0.02), but was unaffected in CB-derived DNRII-transduced NK cells (21.11 ± 1.84% killing at E:T 5:1 in the absence vs. 21.81 ± 3.37 in the presence of medulloblastoma-conditioned media, n = 8, p = 0.85. We also observed decreased expression of CCR2 in untransduced NK cells (mean CCR2 MFI 826 ± 117 in untransduced NK + MB supernatant from mean CCR2 MFI 1639.29 ± 215 in no MB supernatant, n = 7, p = 0.0156), but not in the transduced cells. Finally, we observed that CB-derived DNRII-transduced NK cells may protect surrounding immune cells by providing a cytokine sink for TGF-β (decreased TGF-β levels of 610 ± 265 pg/mL in CB-derived DNRII-transduced NK cells vs. 1817 ± 342 pg/mL in untransduced cells; p = 0.008). Conclusions CB NK cells expressing a TGF-β DNRII may have a functional advantage over unmodified NK cells in the presence of TGF-β-rich MB, warranting further investigation on its potential applications for patients with medulloblastoma.

Abstract: Adoptive transfer of virus-specific T cells (VSTs) has been shown to be safe and effective in stem cell transplant recipients. However, the lack of virus-experienced T cells in donor cord blood (CB) has prevented the development of ex vivo expanded donor-derived VSTs for recipients of this stem cell source. Here we evaluated the feasibility and safety of ex vivo expansion of CB T cells from the 20% fraction of the CB unit in pediatric patients receiving a single CB transplant (CBT). In 2 clinical trials conducted at 2 separate sites, we manufactured CB-derived multivirus-specific T cells (CB-VSTs) targeting Epstein-Barr virus (EBV), adenovirus, and cytomegalovirus (CMV) for 18 (86%) of 21 patients demonstrating feasibility. Manufacturing for 2 CB-VSTs failed to meet lot release because of insufficient cell recovery, and there was 1 sterility breach during separation of the frozen 20% fraction. Delayed engraftment was not observed in patients who received the remaining 80% fraction for the primary CBT. There was no grade 3 to 4 acute graft-versus-host disease (GVHD) associated with the infusion of CB-VSTs. None of the 7 patients who received CB-VSTs as prophylaxis developed end-organ disease from CMV, EBV, or adenovirus. In 7 patients receiving CB-VSTs for viral reactivation or infection, only 1 patient developed end-organ viral disease, which was in an immune privileged site (CMV retinitis) and occurred after steroid therapy for GVHD. Finally, we demonstrated the long-term persistence of adoptively transferred CB-VSTs using T-cell receptor-Vβ clonotype tracking, suggesting that CB-VSTs are a feasible addition to antiviral pharmacotherapy.

Human Parainfluenza Virus-3 (HPIV3) causes severe respiratory illness in immunocompromised patients and lacks approved anti-viral therapies. A phase I study of adoptively transferred virus-specific T-cells (VSTs) targeting HPIV3 following bone marrow transplantation is underway (NCT03180216). We sought to identify immunodominant epitopes within HPIV3 Matrix protein and their cross-reactivity against related viral proteins. VSTs were generated from peripheral blood of healthy donors by ex-vivo expansion after stimulation with a 15-mer peptide library encompassing HPIV3 matrix protein. Epitope mapping was performed using IFN-γ ELIspot with combinatorial peptide pools. Flow cytometry was used to characterize products with intracellular cytokine staining. In 10 VST products tested, we discovered 12 novel immunodominant epitopes. All products recognized an epitope at the C-terminus. On IFN-γ ELISpot, individual peptides eliciting activity demonstrated mean IFN-γ spot forming units per well (SFU)/1x105 cells of 115.5 (range 24.5–247.5). VST products were polyfunctional, releasing IFN-γ and TNF-α in response to identified epitopes, which were primarily HLA Class II restricted. Peptides from Human Parainfluenza Virus-1 corresponding to the HPIV3 epitopes showed cross-reactivity for HPIV1 in 11 of 12 tested epitopes (mean cross reactivity index: 1.19). Characterization of HPIV3 epitopes may enable development of third-party VSTs to treat immune suppressed patients with HPIV infection.

Human immunodeficiency virus (HIV) has caused millions of deaths and continues to threaten the health of millions of people worldwide. Despite anti-retroviral therapy (ART) substantially alleviating severity and limiting transmission, HIV has not been eradicated and its persistence can lead to other health concerns such as cancer. The only two cases of HIV cure to date are HIV+ cancer patients receiving an allogeneic hematopoietic stem cell transplantation (allo-HSCT) from a donor with the CCR5 Δ32 mutation. While this approach has not led to such success in other patients and is not applicable to HIV+ individuals without cancer, the encouraging results may point toward a breakthrough in developing a cure strategy for HIV. Adoptive transfer of virus-specific T cells (VSTs) post HSCT has been effectively used to treat and prevent reactivation of latent viral infections such as cytomegalovirus (CMV) and Epstein-Barr virus (EBV), making VSTs an attractive therapeutic to control HIV rebound. Here we will discuss the potential of using adoptive T cell therapies in combination with other treatments such as HSCT and latency reversing agents (LRAs) to achieve a functional cure for HIV.

TCR-gamma delta (γδ) T-cells are considered important players in the graft-vs.-tumor effect following allogeneic hematopoietic cell transplantation (alloHCT) and have emerged as candidates for adoptive transfer immunotherapy in the treatment of both solid and hematological tumors. Systemic β-adrenergic receptor (β-AR) activation has been shown to mobilize TCR-γδ T-cells to the blood, potentially serving as an adjuvant for alloHCT and TCR-γδ T-cell therapy. We investigated if systemic β-AR activation, using acute dynamic exercise as an experimental model, can increase the mobilization, ex vivo expansion, and anti-tumor activity of TCR-γδ T-cells isolated from the blood of healthy humans. We also sought to investigate the β-AR subtypes involved, by administering a preferential β1-AR antagonist (bisoprolol) and a non-preferential β1 + β2-AR antagonist (nadolol) prior to exercise as part of a randomized placebo controlled cross-over experiment. We found that exercise mobilized TCR-γδ cells to blood and augmented their ex vivo expansion by ~182% compared to resting blood when stimulated with IL-2 and ZOL for 14-days. Exercise also increased the proportion of CD56+, NKG2D+/CD62L–, CD158a/b/e+ and NKG2A− cells among the expanded TCR-γδ cells, and increased their cytotoxic activity against several tumor target cells (K562, U266, 221.AEH) in vitro by 40–60%. Blocking NKG2D on TCR-γδ cells in vitro eliminated the augmented cytotoxic effects of exercise against U266 target cells. Furthermore, administering a β1 + β2-AR (nadolol), but not a β1-AR (bisoprolol) antagonist prior to exercise abrogated the exercise-induced enhancement in TCR-γδ T-cell mobilization and ex vivo expansion. Furthermore, nadolol completely abrogated while bisoprolol partially inhibited the exercise-induced increase in the cytotoxic activity of the expanded TCR-γδ T-cells. We conclude that acute systemic β-AR activation in healthy donors markedly augments the mobilization, ex vivo expansion, and anti-tumor activity of TCR-γδ T-cells and that some of these effects are due to β2-AR signaling and phenotypic shifts that promote a dominant activating signal via NKG2D. These findings highlight β-ARs as potential targets to favorably alter the composition of allogeneic peripheral blood stem cell grafts and improve the potency of TCR-γδ T-cell immune cell therapeutics.

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. Keywords: transplantation, immune reconstitution, cytokines, fungal, cytotoxic T cells, antigen presentation/processing

Abstract: T-cell therapy has emerged from the bench for the treatment of patients with lymphoma. Responses to T-cell therapeutics are regulated by multiple factors, including the patient's immune system status and disease stage. Outside of engineering of chimeric antigen receptors and artificial T-cell receptors, T-cell therapy can be mediated by ex vivo expansion of antigen-specific T cells targeting viral and/or nonviral tumor-associated antigens. These approaches are contributing to enhanced clinical responses and overall survival. In this review, we summarize the available T-cell therapeutics beyond receptor engineering for the treatment of patients with lymphoma.

Latently human immunodeficiency virus (HIV)-infected cells are transcriptionally quiescent and invisible to clearance by the immune system. To demonstrate that the latency reversing agent vorinostat (VOR) induces a window of vulnerability in the latent HIV reservoir, defined as the triggering of viral antigen production sufficient in quantity and duration to allow for recognition and clearance of persisting infection, we developed a latency clearance assay (LCA). The LCA is a quantitative viral outgrowth assay (QVOA) that includes the addition of immune effectors capable of clearing cells expressing viral antigen. Here we show a reduction in the recovery of replication-competent virus from VOR exposed resting CD4 T cells following addition of immune effectors for a discrete period. Take home message: VOR exposure leads to sufficient production of viral protein on the cell surface, creating a window of vulnerability within this latent reservoir in antiretroviral therapy (ART)-suppressed HIV-infected individuals that allows the clearance of latently infected cells by an array of effector mechanisms.

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.

Mycobacterial Infections can be severe in patients with T-cell deficiency or phagocyte disorders, and treatment is frequently complicated by antimicrobial resistance. Restoration of T-cell immunity via stem cell transplantation facilitates control of mycobacterial infections, but presence of active infections during transplantation is associated with a higher risk of mortality. Adoptive T cell immunotherapy has been successful in targeting viruses, but has not been attempted to treat mycobacterial infections. We sought to expand and characterize mycobacterial-specific T-cells derived from healthy donors in order to determine suitability for adoptive immunotherapy. Mycobacteria-specific T-cells (MSTs) were generated from 10 healthy donors using a rapid ex vivo expansion protocol targeting five known mycobacterial target proteins (AG85B, PPE68, ESXA, ESXB, and ADK). MSTs were compared to T-cells expanded from the same donors using lysate from M. tuberculosis or purified protein derivative from M. avium (sensitin). MST expansion from seven patients with primary immunodeficiency disorders (PID) and two patients with IFN-γ autoantibodies and invasive M. avium infections. MSTs expanded from healthy donors recognized a median of 3 of 5 antigens, with production of IFN-γ, TNF, and GM-CSF in CD4+ T cells. Comparison of donors who received BCG vaccine (n = 6) to those who did not (n = 4) showed differential responses to PPE68 (p = 0.028) and ADK (p = 0.015) by IFN-γ ELISpot. MSTs expanded from lysate or sensitin also recognized multiple mycobacterial antigens, with a statistically significant differences noted only in the response to PPE68 (p = 0.016). MSTs expanded from patients with primary immunodeficiency (PID) and invasive mycobacterial infections showed activity against mycobacterial antigens in only two of seven subjects, whereas both patients with IFN-γ autoantibodies recognized mycobacterial antigens. Thus, MSTs can be generated from donors using a rapid expansion protocol regardless of history of BCG immunization. Most tested PID patients had no detectable T-cell immunity to mycobacteria despite history of infection. MSTs may have clinical utility for adoptive immunotherapy in T-cell deficient patients with invasive mycobacterial infections.

In this study, we describe poly (lactic-co-glycolic) acid (PLGA)-based nanoparticles that combine photothermal therapy (PTT) with epigenetic therapy for melanoma. Specifically, we co-encapsulated indocyanine green (ICG), a PTT agent, and Nexturastat A (NextA), an epigenetic drug within PLGA nanoparticles (ICG-NextA-PLGA; INAPs). We hypothesized that combining PTT with epigenetic therapy elicits favorable cytotoxic and immunomodulatory responses that result in improved survival in melanoma-bearing mice. We utilized a nanoemulsion synthesis scheme to co-encapsulate ICG and NextA within stable and monodispersed INAPs. The INAPs exhibited concentration-dependent and near-infrared (NIR) laser power-dependent photothermal heating characteristics, and functioned as effective single-use agents for PTT of melanoma cells in vitro. The INAPs functioned as effective epigenetic therapy agents by inhibiting the expression of pan-histone deacetylase (HDAC) and HDAC6-specific activity in melanoma cells in vitro. When used for both PTT and epigenetic therapy in vitro, the INAPs increased the expression of co-stimulatory molecules and major histocompatibility complex (MHC) Class I in melanoma cells relative to controls. These advantages persisted in vivo in a syngeneic murine model of melanoma, where the combination therapy slowed tumor progression and improved median survival. These findings demonstrate the potential of INAPs as agents of PTT and epigenetic therapy for melanoma.

Patients with primary immunodeficiency disorders (PID) have an increased risk from acute and chronic Epstein–Barr Virus (EBV) viral infections and EBV-associated malignancies. Hematopoietic stem cell transplantation (HSCT) is a curative strategy for many patients with PID, but EBV-related complications are common in the immediate post-transplant period due to delayed reconstitution of T cell immunity. Adoptive T cell therapy with EBV-specific T cells is a promising therapeutic strategy for patients with PID both before and after HSCT. Here we review the methods used to manufacture EBV-specific T cells, the clinical outcomes, and the ongoing challenges for future development of the strategy.

While progress has been made in the treatment of both hematologic cancers and solid tumors, chemorefractory or relapsed disease often portends a dismal prognosis, and salvage chemotherapy or radiation expose patients to intolerable toxicities and may not be effective. Hematopoietic stem cell transplant offers the promise of cure for many patients, and while mismatched, unrelated or haploidentical donors are increasingly available, the recipients are at higher risk of severe immunosuppression and immune dysregulation due to graft versus host disease. Viral infections remain a primary cause of severe morbidity and mortality in this patient population. Again, many therapeutic options for viral disease are toxic, may be ineffective or generate resistance, or fail to convey long-term protection. Adoptive cell therapy with virus-specific T cells (VSTs) is a targeted therapy that is efficacious and has minimal toxicity in immunocompromised patients with CMV and EBV infections in particular. Products have since been generated specific for multiple viral antigens (multi-VST), which are not only effective but also confer protection in 70–90% of recipients when used as prophylaxis. Notably, these products can be generated from either virus-naive or virus-experienced autologous or allogeneic sources, including partially matched HLA-matched third-party donors. Obstacles to effective VST treatment are donor availability and product generation time. Banking of third-party VST is an attractive way to overcome these constraints and provide products on an as-needed basis. Other developments include epitope discovery to broaden the number of viral antigens targets in a single product, the optimization of VST generation from naive donor sources, and the modification of VSTs to enhance persistence and efficacy in vivo.

Cytomegalovirus, of the Herpesviridae family, has evolved alongside humans for thousands of years with an intricate balance of latency, immune evasion, and transmission. While upwards of 70% of humans have evidence of CMV infection, the majority of healthy people show little to no clinical symptoms of primary infection and CMV disease is rarely observed during persistent infection in immunocompetent hosts. Despite the fact that the majority of infected individuals are asymptomatic, immunologically, CMV hijacks the immune system by infecting and remaining latent in antigen-presenting cells that occasionally reactivate subclinically and present antigen to T cells, eventually causing the inflation of CMV-specific T cells until they can compromise up to 10% of the entire T cell repertoire. Because of this impact on the immune system, as well as its importance in fields such as stem cell and organ transplant, the relationship between CMV and the immune response has been studied in depth. Here we provide a review of many of these studies and insights into how CMV-specific T cells are currently being used therapeutically.

Hematopoietic stem cell transplantation has revolutionized the treatment of hematologic malignancies, but infection, graft-versus-host disease and relapse are still important problems. Calcineurin inhibitors, T-cell depletion strategies, and immunomodulators have helped to prevent graft-versus-host disease, but have a negative impact on the graft-versus-leukemia effect. T cells and natural killer cells are both thought to be important in the graft-versus-leukemia effect, and both cell types are amenable to ex vivo manipulation and clinical manufacture, making them versatile immunotherapeutics. We provide an overview of these immunotherapeutic strategies following hematopoietic stem cell transplantation, with discussions centered on natural killer and T-cell biology. We discuss the contributions of each cell type to graft-versus-leukemia effects, as well as the current research directions in the field as related to adoptive cell therapy after hematopoietic stem cell transplantation.

Adoptive transfer of antigen-specific cytotoxic T lymphocytes (CTLs) has been successfully used to treat patients with different types of cancer. However, the long-term spatial-temporal dynamics of the distribution of systemically infused CTLs remains largely unknown. Noninvasive imaging of adoptively transferred CTLs using molecular-genetic reporter imaging with positron emission tomography and computed tomography (PET-CT) represents an innovative approach to understanding the long-term migratory patterns and therapeutic potential of adoptively transferred T cells. Here we report the application of repetitive PET-CT imaging with [ 18 F]fluoro-5-ethyl-1-beta-D-arabinofuranosyluracil ( 18 F-FEAU) in two nonhuman primates demonstrating that autologous polyclonal macaque T lymphocytes activated and transduced with a retroviral vector encoding for the sr39 mutant herpes simplex virus 1 thymidine kinase ( sr39HSV1-tk ) reporter gene can be detected after intravenous infusion in discrete lymphoid organs and in sites of inflammation. This study represents a proof of principle and supports the application of 18 F-FEAU PET-CT imaging for monitoring the distribution of intravenously administered sr39HSV1-tk gene-transduced CTLs in humans.