Naive Donors Research Articles

The impact of the donors' COVID-19 status on the outcomes of allogeneic hematopoietic stem cell transplantation: a multi-center retrospective study.

To explore the impact of donors' COVID-19 status on allogeneic stem cell transplantation (allo-HSCT), we compared the transplant outcomes of 74 participants. This multi-center retrospective study included nine participants receiving grafts from COVID-19 positive donors (CPD), 45 from COVID-19 experienced donors (CED), and 20 from COVID-19 naive donors (CND). We evaluated engraftment, complications, and survival rates among the three groups. All apheresis procedures were successful with no significant differences in CD34+ cells or lymphocytes in grafts among the three groups. All patients achieved engraftment by day 30 post-HSCT. The incidence of grade II-IV acute graft-versus-host disease (aGVHD) was 55.6%, 20%, and 10% in the CPD, CED, and CND groups, respectively (p = 0.024). Multivariate analysis indicated that COVID-19 positivity in donors at the time of apheresis was an independent risk factor for II-IV aGVHD (p = 0.020, OR = 12.159, 95% CI 1.783 -135.760). No differences were observed among the groups in terms of chronic GVHD, viral infection, or sinusoidal obstruction syndrome. The 6-month overall survival and disease-free survival rates were also similar among the three groups. Our results suggest that the COVID-19-positive status of donors might not impact graft collection, engraftment, or short-term survival of allo-HSCT recipients but might increase the risk of aGVHD. Further research is needed to explore the influence of donors' COVID-19 status on long-term complications and survival in allo-HSCT recipients.

Donor T Cell Infusions Limit Novel Immune Checkpoint Therapy in Pancreatic Cancer: Implications for Adoptive T Cell Therapy

Background: We have previously published that pancreatic cancers over-express an immunosuppressive peptide, vasoactive intestinal polypeptide (VIP), and that a novel combination of antin-PD1 antibodies and small molecule antagonists to the VIP receptor can induce complete remission of pancreatic ductal adenocarcinoma (PDAC) in multiple mouse models (Ravibndranathan, Nat. Comm. 2022). To enhance the efficacy of this novel immunotherapy approach, we hypothesized that infusions of additional donor T cells to PDAC-bearing mice receiving the combination of anti-PD1/VIP receptor antagonists would lead to more intra-tumoral effector T cells and increased tumor regression rates. To investigate the potential impact of adoptive splenocyte infusion on checkpoint immunotherapy, we established sub-cutaneous KPC PDAC tumors in C57Bl/6 mice. As previously described, we treated them with a combination of ANT308 (VIP-receptor antagonists) and anti-PD1. We tested whether adding splenocyte infusions from donor mice that were immunologically naïve or donor mice previously engrafted with PDAC with regressed (positive control) or progressed tumors (negative control) would enhance the efficacy of the immune checkpoint blockade therapy. Methods: C57BL/6 B6 CD45.1 recipient mice were inoculated subcutaneously with 1 x 10 6 KPC-luc PDAC cell line cells on day -5. Recipient mice received anti-PD1 (200mg) every three days (totaling four doses) and 21 daily s.c. injections of 20 ug ANT308. Splenocytes from three groups of CD45.2+ C57BL/6 donor mice were injected into tumor-bearing recipients: naive mice, mice with a history of KPC regression post-ANT308/anti-PD1 treatment, and mice with progressed KPC tumors without any treatment. Anti-PD1 and ANT308 treatments were initiated simultaneously with adoptive splenocyte infusion (25 x 10 6 cells) on day 0. Tumor size was measured biweekly, and IVIS imaging was performed weekly. Blood was drawn weekly from recipient mice for flow cytometric analysis of immune cell subsets. A fraction of tumor-bearing recipient mice was euthanized ten days after donor splenocyte infusion and initiation of immune checkpoint therapy or at IAUCAC-defined endpoints. When tumor-bearing mice were euthanized, the spleen, lymph nodes, and tumors were harvested for further flow cytometric analysis. The remaining mice were monitored for tumor growth and survival kinetics. Results: Contrary to our original hypothesis, we observed substantially lower survival rates and faster tumor growth in the group that received splenocytes from immunologically naive donor mice versus the group treated with splenocytes from tumor-bearing mice (Figures 1A and 1B). Splenocytes from immunologically naive donors contained fewer Treg cells, whereas myeloid-derived suppressor cells (CD11b+Ly-6G+ Gr-1+) were increased in donor splenocytes from tumor-bearing mice. We next repeated the experiment utilizing the adoptive transfer of splenocytes from immunologically naïve donors separated into 8 x 10 6 MACS-purified T cell or 17 x 10 6 non-T cell fractions. Remarkably, the transfer of 8 x 10 6 immunologically naïve T cells abrogated the efficacy of immune checkpoint therapy leading to rapid tumor growth compared to tumor regression in mice treated with immune checkpoint therapy without added donor splenocytes or mice that received immune checkpoint therapy with 17 x 10 6 T-cell-depleted donor splenocytes. Flow cytometric and gene expression analyses of splenocytes from the recipient mice, and those not receiving donor T cell infusions, revealed that the tetramer population in the infusion group was significantly higher than in the group treated only with immune checkpoint inhibitors. Conclusion: Our findings indicated that natural suppressor/ Treg in immunologically naïve splenocytes could exert a profound negative regulatory effect on immune checkpoint therapy's efficacy in a murine PDAC model. Despite having fewer Treg than splenocytes from tumor-bearing donors, immunologically naïve natural Treg had increased potency in suppressing anti-cancer immunity. These findings suggest the importance of the depletion of natural Treg in adoptive cell therapy strategies to treat solid tumor malignancies. This research underscores the critical role of immune contexture in shaping therapeutic outcomes and proposes a novel biomarker for patient stratification in PDAC immunotherapy.

C-reactive protein binds to short phosphoglycan repeats of Leishmania secreted proteophosphoglycans and activates complement.

Human C-reactive protein (CRP) binds to lipophosphoglycan (LPG), a virulence factor of Leishmania spp., through the repeating phosphodisaccharide region. We report here that both major components of promastigote secretory gel (PSG), the filamentous proteophosphoglycan (fPPG) and the secreted acid phosphatase (ScAP), are also ligands. CRP binding was mainly associated with the flagellar pocket when LPG deficient Leishmania mexicana parasites were examined by fluorescent microscopy, consistent with binding to secreted material. ScAP is a major ligand in purified fPPG from parasite culture as demonstrated by much reduced binding to a ScAP deficient mutant fPPG in plate binding assays and ligand blotting. Nevertheless, in sandfly derived PSG fPPG is a major component and the major CRP binding component. Previously we showed high avidity of CRP for LPG ligand required multiple disaccharide repeats. ScAP and fPPG only have short repeats but they retain high avidity for CRP revealed by surface plasmon resonance because they are found in multiple copies on the phosphoglycan. The fPPG from many species such as L. donovani and L. mexicana bound CRP strongly but L. tropica and L. amazonensis had low amounts of binding. The extent of side chain substitution of [-PO4-6Galβ1-4Manα1-] disaccharides correlates inversely with binding of CRP. The ligand for the CRP on different species all had similar binding avidity as the half maximal binding concentration was similar. Since the PSG is injected with the parasites into host blood pools and phosphoglycans (PG) are known to deplete complement, we showed that CRP makes a significant contribution to the activation of complement by PSG using serum from naive donors.

CD4 Effector TCR Avidity for Peptide on APC Determines the Level of Memory Generated.

Initial TCR affinity for peptide Ag is known to impact the generation of memory; however, its contributions later, when effectors must again recognize Ag at 5-8 d postinfection to become memory, is unclear. We examined whether the effector TCR affinity for peptide at this "effector checkpoint" dictates the extent of memory and degree of protection against rechallenge. We made an influenza A virus nucleoprotein (NP)-specific TCR transgenic mouse strain, FluNP, and generated NP-peptide variants that are presented by MHC class II to bind to the FluNP TCR over a broad range of avidity. To evaluate the impact of avidity invivo, we primed naive donor FluNP in influenza A virus-infected host mice, purified donor effectors at the checkpoint, and cotransferred them with the range of peptides pulsed on activated APCs into second uninfected hosts. Higher-avidity peptides yielded higher numbers of FluNP memory cells in spleen and most dramatically in lung and draining lymph nodes and induced better protection against lethal influenza infection. Avidity determined memory cell number, not cytokine profile, and already impacted donor cell number within several days of transfer. We previously found that autocrine IL-2 production at the checkpoint prevents default effector apoptosis and supports memory formation. Here, we find that peptide avidity determines the level of IL-2 produced by these effectors and that IL-2Rα expression by the APCs enhances memory formation, suggesting that transpresentation of IL-2 by APCs further amplifies IL-2 availability. Secondary memory generation was also avidity dependent. We propose that this regulatory pathway selects CD4 effectors of highest affinity to progress to memory.

An extended SARS-CoV-2 mRNA vaccine prime-boost interval enhances B cell immunity with limited impact on T cells

Spacing the first two doses of SARS-CoV-2 mRNA vaccines beyond 3-4weeks raised initial concerns about vaccine efficacy. While studies have since shown that long-interval regimens induce robust antibody responses, their impact on B and Tcell immunity is poorly known. Here, we compare SARS-CoV-2 naive donors B and Tcell responses to two mRNA vaccine doses administered 3-4 versus 16weeks apart. After boost, the longer interval results in a higher magnitude and a more mature phenotype of RBD-specific B cells. While the two geographically distinct cohorts present quantitative and qualitative differences in Tcell responses at baseline and after priming, the second dose led to convergent features with overall similar magnitude, phenotype, and function of CD4+ and CD8+ Tcell responses at post-boost memory time points. Therefore, compared to standard regimens, a 16-week interval has a favorable impact on the B cell compartment but minimally affects Tcell immunity.

272 - Cellular and Humoral Responses from Sars-Cov-2 Specific CD45RA- Memory T Cells from Convalescent and Naive Donors. Clinical Application As an Adoptive Cell Therapy for COVID-19

272 - Cellular and Humoral Responses from Sars-Cov-2 Specific CD45RA- Memory T Cells from Convalescent and Naive Donors. Clinical Application As an Adoptive Cell Therapy for COVID-19

Visceral sensitivity modulation by faecal microbiota transplantation: the active role of gut bacteria in pain persistence.

Recent findings linked gastrointestinal disorders characterized by abdominal pain to gut microbiota composition. The present work aimed to evaluate the power of gut microbiota as a visceral pain modulator and, consequently, the relevance of its manipulation as a therapeutic option in reversing postinflammatory visceral pain persistence. Colitis was induced in mice by intrarectally injecting 2,4-dinitrobenzenesulfonic acid (DNBS). The effect of faecal microbiota transplantation from viscerally hypersensitive DNBS-treated and naive donors was evaluated in control rats after an antibiotic-mediated microbiota depletion. Faecal microbiota transplantation from DNBS donors induced a long-lasting visceral hypersensitivity in control rats. Pain threshold trend correlated with major modifications in the composition of gut microbiota and short chain fatty acids. By contrast, no significant alterations of colon histology, permeability, and monoamines levels were detected. Finally, by manipulating the gut microbiota of DNBS-treated animals, a counteraction of persistent visceral pain was achieved. The present results provide novel insights into the relationship between intestinal microbiota and visceral hypersensitivity, highlighting the therapeutic potential of microbiota-targeted interventions.

Hiv Specific T Cells Generated from HIV Naive Adult and Cord Blood Donors Target a Range of Novel Viral Epitopes: Implications for a Cure Strategy after Allogeneic HSCT and CBT

Adoptive T cell therapy has been successful in boosting viral-specific immunity post-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 with allogeneic HSCT, there is only one case of functional HIV cure post-HSCT, the Berlin Patient. Hence, we hypothesized that broadly HIV-specific CD8 and CD4 T-cells (HXTCs) could be expanded from patients on ARVs, as well as HIV negative adult (dHXTCs) and cord blood donors (cbHXTCs), employing a non-HLA restricted approach for the treatment of HIV+ individuals after autologous or allogeneic HSCT.We have expanded autologous HXTCs from HIV+ subjects under NCT02208167. To extend this approach to the allogeneic HSCT setting, we generated dHXTCs from HIV naive adults (n=8) and cbHXTCs from cord blood donors (n=8). IFN-gamma ELISPOT showed dHXTCs were specific against Gag (mean=331.25 SFC/105cells) and Nef (mean=242.63) versus irrelevant antigen (mean=13 SFC) (n=8). Similarly, we are able to produce cbHXTCs (n=8) that showed specificity to Gag (mean=103.252 SFC/105cells), Nef (mean=109.43), or Pol (mean=130.56), (p<0.0001 compared to controls) in IFN-gamma ELISPOT. dHXTC products displayed polyfunctionality, producing proinflammatory TNFα, IL2, IL6, IL8, and perforin responses (p<0.05) to HIV stimulation. Importantly, dHXTCs (p=0.0004) and cbHXTCs (p=0.0003) were able to suppress HIV replication compared with nonspecific CD8+ T cells when co-cultured with autologous CD4+ T cells infected with HIV SF162 at an Effector-to-Target ratio of 20 to 1. Exhaustion marker analysis of cbHXTCs products revealed minimal expression of PD1, TIM3, LAG3, KLRG1, and CD57 (n=3). Epitope mapping revealed that dHXTC and cbHXTC products contained T cells recognizing unique epitopes not typically identified in HIV positive individuals, which may be critical in overcoming viral immune escape post-HSCT.In summary, HIV-specific T cells can be expanded from HIV positive and HIV negative donors for clinical use. Focusing on donors with HLA types that are associated with well characterized HIV responses (HLA A02) or associated with delayed progression to AIDS (HLA B27, B51, B57) may allow us to identify HLA restricted epitopes critical for the successful development of a potent HIV-specific T cell therapeutic. Hence, the administration of HXTCs derived from virus naive donors could offer a unique curative strategy post-allogeneic HSCT and cord blood transplant. [Display omitted] DisclosuresBollard:Torque: Membership on an entity's Board of Directors or advisory committees; Cellectis: Membership on an entity's Board of Directors or advisory committees; Neximmune: Consultancy, Membership on an entity's Board of Directors or advisory committees.

Protective T cell receptor identification for orthotopic reprogramming of immunity in refractory virus infections

Viral infections cause life-threatening disease in immunocompromised patients and especially following transplantation. Tcell receptor (TCR) engineering redirects specificity and can bring significant progress to emerging adoptive Tcell transfer (ACT) approaches. Tcell epitopes are well described, although knowledge is limited on which TCRs mediate protective immunity. In this study, refractory adenovirus (AdV) infection after hematopoietic stem cell transplantation (HSCT) was treated with ACT of highly purified Hexon5-specific Tcells using peptide major histocompatibility complex (pMHC)-Streptamers against the immunodominant human leukocyte antigen (HLA)-A∗0101-restricted peptide LTDLGQNLLY. AdV was successfully controlled through this oligoclonal ACT. Novel protective TCRs were isolated exvivo and preclinically engineered into the TCR locus of allogeneic third-party primary Tcells by CRISPR-Cas9-mediated orthotopic TCR replacement. Both TCR knockout and targeted integration of the new TCR in one single engineering step led to physiological expression of the transgenic TCR. Reprogrammed TCR-edited Tcells showed strong virus-specific functionality such as cytokine release, effector marker upregulation, and proliferation capacity, as well as cytotoxicity against LTDLGQNLLY-presenting and AdV-infected targets. In conclusion, exvivo isolated TCRs with clinical proven protection through ACT could be redirected into Tcells from naive third-party donors. This approach ensures that transgenic TCRs are protective with potential off-the-shelf use and widened applicability of ACT to various refractory emerging viral infections.

Shedding Light on Drug-Induced Liver Injury: Activation of T Cells From Drug Naive Human Donors With Tolvaptan and a Hydroxybutyric Acid Metabolite

Exposure to tolvaptan is associated with a significant risk of liver injury in a small fraction of patients with autosomal dominant polycystic kidney disease. The observed delayed onset of liver injury of between 3 and 18 months after commencing tolvaptan treatment, along with rapid recurrence of symptoms following re-challenge is indicative of an adaptive immune attack. This study set out to assess the intrinsic immunogenicity of tolvaptan and pathways of drug-specific T-cell activation using in vitro cell culture platforms. Tolvaptan (n = 7), as well as oxybutyric (DM-4103, n = 1) and hydroxybutyric acid (DM-4107, n = 18) metabolite-specific T-cell clones were generated from tolvaptan naive healthy donor peripheral blood mononuclear cells. Tolvaptan and DM-4103 T-cell clones could also be activated with DM-4107, whereas T-cell clones originally primed with DM-4107 were highly specific to this compound. A signature cytokine profile (IFN-γ, IL-13, granzyme B, and perforin) for almost all T-cell clones was identified. Mechanistically, compound-specific T-cell clone activation was dependent on the presence of soluble drug and could occur within 4 h of drug exposure, ruling out a classical hapten mechanism. However, antigen processing dependence drug presentation was indicated in many T-cell clones. Collectively these data show that tolvaptan-associated liver injury may be attributable to an adaptive immune attack upon the liver, with tolvaptan- and metabolite-specific T cells identified as candidate effector cells in such etiology.

The Pattern of Haematological Changes in the Baseline Blood Cell Counts and the CD4&amp;amp;#8314; T Lymphocyte Levels among Antiretroviral Therapy Na&amp;#239;ve Adult HIV Positive Patients in a Nigerian Hospital

Background: Haematological abnormalities are strong independent predictors of morbidity and mortality in HIV infection. Objectives of the Study: This study was carried out to assess the pattern of the changes in the baseline peripheral blood cell counts among adult HIV positive patients. To also determine the CD4+ T cell levels and its correlation with the changes in the baseline cell counts of the patients using HIV negative blood donors as controls. Methods: This was a case controlled prospective study. The subjects were antiretroviral therapy naive adult HIV positive patients and HIV negative blood donor controls. Five milliliters (5 mls) of venous blood was collected from the cubitus of every consecutive consenting subject. Blood sample was analysed for baseline complete blood cell counts and the CD4+ T lymphocyte levels using Sysmex and Cyflow R Counter autoanalysers respectively. Obtained data was analysed with the statistical package for the social scientist (SPSS version 20.0). The Erythrocyte sedimentation rate of subjects was measured by the manual standard Westergreen method. Results: Of 300 subjects, there were 139 (46.3%) males and 161 (53.7%) females in the study. Anaemia was found in 72 (29.2%), leucopenia in 20 (8%) and thrombocytopenia in 6 (2.4%) of the patients. The mean erythrocyte sedimentation rates of the subjects were 81.88 mm/hr and 9.46 mm/hr (p = 0.000) for the patients and the controls respectively. The mean CD4+ T lymphocyte cell counts were 293 cells/μl and 750 cells/μl (p = 0.000) for the patients and controls respectively. Conclusion: Anaemia, leucopenia and thrombocytopenia were more prevalent among the studied HIV positive patients.

Comprehensive viromewide antibody responses by systematic epitope scanning after hematopoietic cell transplantation

AbstractFurther insight into humoral viral immunity after hematopoietic cell transplantation (HCT) could have potential impact on donor selection or monitoring of patients. Currently, estimation of humoral immune recovery is inferred from lymphocyte counts or immunoglobulin levels and does not address vulnerability to specific viral infections. We interrogated the viral antibody repertoire before and after HCT using a novel serosurvey (VirScan) that detects immunoglobulin G responses to 206 viruses. We performed VirScan on cryopreserved serum from pre-HCT and 30, 100, and 365 days after myeloablative HCT from 37 donor-recipient pairs. We applied ecologic metrics (α- and β-diversity) and evaluated predictors of metrics and changes over time. Donor age and donor/recipient cytomegalovirus (CMV) serostatus and receipt systemic glucocorticoids were most strongly associated with VirScan metrics at day 100. Other clinical characteristics, including pre-HCT treatment and conditioning, did not affect antiviral repertoire metrics. The recipient repertoire was most similar (pairwise β-diversity) to that of donor at day 100, but more similar to pre-HCT self by day 365. Gain or loss of epitopes to common viruses over the year post-HCT differed by donor and recipient pre-HCT serostatus, with highest gains in naive donors to seropositive recipients for several human herpesviruses and adenoviruses. We used VirScan to highlight contributions of donor and recipient to antiviral humoral immunity and evaluate longitudinal changes. This work builds a foundation to test whether such systematic profiling could serve as a biomarker of immune reconstitution, predict clinical events after HCT, or help refine selection of optimal donors.

Abstract 550: Tumor-conditional anti-CTLA-4 uncouples anti-tumor efficacy from immunotherapy-related toxicity

While immune checkpoint blockade leads to potent anti-tumor efficacy, it also leads to immune-related adverse events in cancer patients. These toxicities stem from systemic immune activation resulting in multi-organ inflammation including the gastrointestinal tract, lung, and endocrine organs. We developed a dual variable domain immunoglobulin of anti-CTLA-4 antibody (anti-CTLA-4 DVD) possessing an outer tumor-specific antigen-binding site engineered to shield the inner anti-CTLA-4 binding domain. Upon reaching the tumor, the outer domain is cleaved by membrane type-serine protease 1 (MT-SP1) present in the tumor microenvironment, leading to enhanced localization of CTLA-4 blockade. Anti-CTLA-4 DVD significantly reduced multi-organ immune toxicity by preserving tissue-resident regulatory T cells (Tregs) in Rag 1-/- mice that received naive donor CD4+ T cells from wild-type C57BL/6j mice. Moreover, anti-CTLA-4 DVD induced potent antitumor effects by decreasing tumor-infiltrating Tregs and increasing the infiltration of antigen-specific CD8+ T lymphocytes in TRAMP-C2 bearing C57BL/6j mice. Tregs depletion was mediated through the antibody-dependent cellular cytotoxicity (ADCC) mechanism, as anti-CTLA-4 without the FcγR binding portion (anti-CTLA-4 DANA) spared Tregs, preventing treatment-induced toxicities. In summary, our results demonstrated a new approach to anti-CTLA-4 blockade that depletes tumor-infiltrating, but not tissue-resident Tregs, preserving anti-tumor effects while minimizing toxicity. Thus, our tumor-conditional anti-CTLA-4 DVD provides an avenue to uncouple anti-tumor efficacy from immunotherapy-induced toxicities. Citation Format: Steven C. Pai, Donald M. Simons, Xiaoqing Lu, Wendy Ritacco, Michael Evans, Gillian Kingsbury, Lawrence Fong. Tumor-conditional anti-CTLA-4 uncouples anti-tumor efficacy from immunotherapy-related toxicity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 550.

TCRαβ-depleted Progenitor Cell Graft With Additional Memory T-cell DLI, Plus Selected Use of Blinatumomab, in Naive T-cell Depleted Haploidentical Donor Hematopoietc Cell Transplantation for Hematologic Malignancies

TCRαβ-depleted Progenitor Cell Graft With Additional Memory T-cell DLI, Plus Selected Use of Blinatumomab, in Naive T-cell Depleted Haploidentical Donor Hematopoietc Cell Transplantation for Hematologic Malignancies

Generation of Zika Virus-Specific T-Cells for Adoptive Immunotherapy

Background Zika virus (ZIKV) infection during pregnancy can cause severe birth defects, with no effective treatment. Adoptive transfer of virus-specific T-cells has proven to be safe and effective for the prevention or treatment of many viral infections, and could represent a novel treatment approach for ZIKV infection. However, this approach for ZIKV infection has been hampered by limited data on immunogenic T-cell antigens within ZIKV. Hence, we have generated ZIKV-specific T-cells and characterized the cellular immune responses against ZIKV antigens. Methods T-cell products were generated from blood of ZIKV-exposed donors (n=3), ZIKV-naive adult donors (n=6), and umbilical cord blood (n=7) by stimulation with overlapping peptide libraries (15mers overlapping by 11 amino acids) spanning four ZIKV polyproteins (C, M, E, and NS1) using a GMP-compliant protocol. Results We successfully generated T-cells targeting ZIKV antigens with a mean 56.0-fold T cell expansion at 17 days for ZIKV-exposed donors and 21 days for ZIKV-naive donors and cord blood donors. The resultant products specifically recognized C, M, E, and NS1 proteins by IFN-γ ELISpot, with mean 142.5 spot forming cells (SFCs)/1 × 105 cells, 81.7 SFCs/1 × 105 cells, 132.1 SFCs/1 × 105 cells, and 180.7 SFCs/1 × 105 cells, respectively, compared to 5.4 SFCs/1 × 105 cells for irrelevant antigen (Fig 1). ZIKV-specific T-cells were polyclonal (16.2±2.8% CD3+CD4+ and 32.4±2.4% CD3+CD8+ T-cells) and expressed markers for both central and effector memory phenotype (3.5±0.6% CD45RO+CD62L+ and 48.1±1.9% CD45RO+CD62L–), with minimal expression of PD-1, Tim-3, Lag-3, and CTLA-4. Notably, both CD4+ and CD8+ subsets were polyfunctional comprising ZIKV-directed T-cell populations, which secreted multiple cytokines (16.7±0.6% CD4+IFN-γ+TNF-α+ and 7.4±0.4% CD8+IFN-γ+TNF-α+) as measured by intracellular cytokine staining. Moreover, multiplex cytokine assay demonstrated specific production of IFN-γ (928.7±239.8 pg/mL), TNF-α (1495.5±453.3 pg/mL), IL-2 (38.2±8.4 pg/mL), and GM-CSF (67.0±22.2 pg/mL) in response to ZIKV antigens. Importantly, ZIKV-specific T-cells selectively killed autologous monocytes infected with ZIKV confirming functionality of this unique T cell therapeutic. Epitope mapping using peptide arrays in IFN-γ ELISpot identified several novel HLA class I or class II-restricted epitopes within NS1, which is essential for viral replication and immune evasion. Conclusions Our findings demonstrate that it is feasible to generate potent ZIKV-specific T-cells from a variety of cell sources including virus naive donors for future clinical use in an “off the shelf’ setting.

Tumor-conditional anti-CTLA4 uncouples antitumor efficacy from immunotherapy-related toxicity.

While immune checkpoint blockade leads to potent antitumor efficacy, it also leads to immune-related adverse events in cancer patients. These toxicities stem from systemic immune activation resulting in inflammation of multiple organs, including the gastrointestinal tract, lung, and endocrine organs. We developed a dual variable domain immunoglobulin of anti-CTLA4 antibody (anti-CTLA4 DVD, where CTLA4 is defined as cytotoxic T lymphocyte-associated antigen-4) possessing an outer tumor-specific antigen-binding site engineered to shield the inner anti-CTLA4-binding domain. Upon reaching the tumor, the outer domain was cleaved by membrane type-serine protease 1 (MT-SP1) present in the tumor microenvironment, leading to enhanced localization of CTLA4 blockade. Anti-CTLA4 DVD markedly reduced multiorgan immune toxicity by preserving tissue-resident Tregs in Rag 1-/- mice that received naive donor CD4+ T cells from WT C57BL/6j mice. Moreover, anti-CTLA4 DVD induced potent antitumor effects by decreasing tumor-infiltrating Tregs and increasing the infiltration of antigen-specific CD8+ T lymphocytes in TRAMP-C2-bearing C57BL/6j mice. Treg depletion was mediated through the antibody-dependent cellular cytotoxicity (ADCC) mechanism, as anti-CTLA4 without the FcγR-binding portion (anti-CTLA4 DANA) spared Tregs, preventing treatment-induced toxicities. In summary, our results demonstrate an approach to anti-CTLA4 blockade that depletes tumor-infiltrating, but not tissue-resident, Tregs, preserving antitumor effects while minimizing toxicity. Thus, our tumor-conditional anti-CTLA4 DVD provides an avenue for uncoupling antitumor efficacy from immunotherapy-induced toxicities.

Novel Cellular Therapeutic Approaches for the Prevention and Management of Graft-Versus-Host Disease

Pharmacologic immunosuppression is the mainstay of treatment for GVHD, but there is a crucial need for new therapeutic approaches. Our review focuses on the preclinical and clinical evidence supporting immune regulatory cell infusions, allograft engineering, and suicide-gene-transduced donor T cells to mitigate GVHD. We also review potential challenges to the adoption and use of these therapies. Mesenchymal stromal cells and regulatory T cells appear effective in preventing and treating GVHD in preliminary studies, although methods of isolating and expanding these cells differ and warrant further investigation. Elimination of naive or host-reactive donor T cells from allografts can prevent GVHD but may increase infection risk. Donor T cells engineered with human caspase 9 are rapidly eliminated after inducible gene activation, preventing GVHD. Cellular therapeutics are a promising approach to mitigate GVHD. Clinical trials are underway domestically and internationally to establish the safety and efficacy of these approaches.

Isolation and Characterization of Cross-Neutralizing Human Anti-V3 Single-Chain Variable Fragments (scFvs) Against HIV-1 from an Antigen Preselected Phage Library.

Recently conducted human phase- I trials showed protective effect of anti-HIV-1 broadly neutralizing antibodies (bnAbs). The V3 region of the HIV-1 envelope is highly conserved as it is the co-receptor binding site, and is highly immunogenic. Recombinant single-chain antibody fragments (scFvs) can serve as potential tools for construction of chimeric/bispecific antibodies that can target different epitopes on the HIV-1 envelope. Previously, we have constructed a V3 specific human scFv phage recombinant library by a combinational approach of Epstein-Barr virus (EBV) transformation and antigen (V3) preselection, using peripheral blood mononuclear cells (PBMCs), from a subtype C HIV-1 infected antiretroviral naive donor. In the present study, by biopanning this recombinant scFv phage library with V3B (subtype B) and V3C (subtype C) peptides, we identified unique cross reactive anti-V3 scFv monoclonals. These scFvs demonstrated cross-neutralizing activity when tested against subtype A, subtype B, and subtype C viruses. Further, molecular modeling of the anti-V3 scFvs with V3C and V3B peptides predicted their sites of interaction with the scFvs, providing insights for future immunogen design studies. A large collection of such monoclonal antibody fragments with diverse epitope specificities can be useful immunotherapeutic reagents along with antiretroviral drugs to prevent HIV-1 infection and disease progression.

Detection of ABCB5 tumour antigen-specific CD8+ T cells in melanoma patients and implications for immunotherapy.

ATP binding cassette subfamily B member 5 (ABCB5) has been identified as a tumour-initiating cell marker and is expressed in various malignancies, including melanoma. Moreover, treatment with anti-ABCB5 monoclonal antibodies has been shown to inhibit tumour growth in xenotransplantation models. Therefore, ABCB5 represents a potential target for cancer immunotherapy. However, cellular immune responses against ABCB5 in humans have not been described so far. Here, we investigated whether ABCB5-reactive T cells are present in human melanoma patients and tested the applicability of ABCB5-derived peptides for experimental induction of human T cell responses. Peripheral blood mononuclear cells (PBMNC) isolated from blood samples of melanoma patients (n = 40) were stimulated with ABCB5 peptides, followed by intracellular cytokine staining (ICS) for interferon (IFN)-γ and tumour necrosis factor (TNF)-α. To evaluate immunogenicity of ABCB5 peptides in naive healthy donors, CD8 T cells were co-cultured with ABCB5 antigen-loaded autologous dendritic cells (DC). ABCB5 reactivity in expanded T cells was assessed similarly by ICS. ABCB5-reactive CD8+ T cells were detected ex vivo in 19 of 29 patients, melanoma antigen recognised by T cells (MART-1)-reactive CD8+ T cells in six of 21 patients. In this small, heterogeneous cohort, reactivity against ABCB5 was significantly higher than against MART-1. It occurred significantly more often and independently of clinical characteristics. Reactivity against ABCB5 could be induced in 14 of 16 healthy donors in vitro by repeated stimulation with peptide-loaded autologous DC. As ABCB5-reactive CD8 T cells can be found in the peripheral blood of melanoma patients and an ABCB5-specific response can be induced in vitro in naive donors, ABCB5 could be a new target for immunotherapies in melanoma.

Virus-Specific T Cells for Hematopoietic Stem Cell Transplantation

During the period of T cell immune recovery after hematopoietic stem cell transplantation (HSCT), viral infections and reactivation are a major cause of morbidity and mortality. Adoptive immunotherapy using virus-specific T cells (VSTs) derived from virus-exposed or naive donors has emerged as an effective strategy to prevent viral complication early after HSCT. Early-phase clinical trials show that such VSTs can both prevent and treat viral infections in 70–90% of patients, are safe, and are well tolerated with no infusion-related adverse events and/or worsening of graft-versus-host disease. These clinical trials have a tremendous potential in preventing and treating viral infections post HSCT. The field of adoptive T cell therapy has made huge strides to overcome some of the challenges such as cumbersome manufacturing strategies, targeting multiple viral antigens, and making the T cell product widely available through third-party banks. A collaborative effort between academic centers and industry is likely to facilitate the broader implementation and accessibility of these novel therapies. It will also enable randomized phase 3 clinical trials to prove the efficacy of these T cell products in comparison to often expensive and ineffective pharmacotherapies. Here we review the current methods of VST generation, clinical results using VSTs in the HSCT setting, and applications of VSTs to treat viral-associated malignancies.