Fresh PBMC Research Articles

Parallel recovery of chromatin accessibility and gene expression dynamics from frozen human regulatory T cells

Epigenetic features such as DNA accessibility dictate transcriptional regulation in a cell type- and cell state- specific manner, and mapping this in health vs. disease in clinically relevant material is opening the door to new mechanistic insights and new targets for therapy. Assay for Transposase Accessible Chromatin Sequencing (ATAC-seq) allows chromatin accessibility profiling from low cell input, making it tractable on rare cell populations, such as regulatory T (Treg) cells. However, little is known about the compatibility of the assay with cryopreserved rare cell populations. Here we demonstrate the robustness of an ATAC-seq protocol comparing primary Treg cells recovered from fresh or cryopreserved PBMC samples, in the steady state and in response to stimulation. We extend this method to explore the feasibility of conducting simultaneous quantitation of chromatin accessibility and transcriptome from a single aliquot of 50,000 cryopreserved Treg cells. Profiling of chromatin accessibility and gene expression in parallel within the same pool of cells controls for cellular heterogeneity and is particularly beneficial when constrained by limited input material. Overall, we observed a high correlation of accessibility patterns and transcription factor dynamics between fresh and cryopreserved samples. Furthermore, highly similar transcriptomic profiles were obtained from whole cells and from the supernatants recovered from ATAC-seq reactions. We highlight the feasibility of applying these techniques to profile the epigenomic landscape of cells recovered from cryopreservation biorepositories.

CAR-T manufactured from frozen PBMC yield efficient function with prolonged in vitro production.

Chimeric antigen receptor (CAR)-T cells are engineered to identify and eliminate cells expressing a target antigen. Current manufacturing protocols vary between commercial CAR-T cell products warranting an assessment of these methods to determine which approach optimally balances successful manufacturing capacity and product efficacy. One difference between commercial product manufacturing methods is whether T cell engineering begins with fresh (unfrozen) patient cells or cells that have been cryopreserved prior to manufacture. Starting with frozen PBMC material allows for greater manufacturing flexibility, and the possibility of collecting and storing blood from patients prior to multiple lines of therapy. We prospectively analyzed if second generation anti-CD19 CAR-T cells with either CD28 or 4-1BB co-stimulatory domains have different phenotype or function when prepared side-by-side using fresh or cryopreserved PBMCs. We found that cryopreserved PBMC starting material is associated with slower CAR-T cell expansion during manufacture but does not affect phenotype. We also demonstrate that CAR-T cell activation, cytokine production and in vitro anti-tumor cytotoxicity were not different when CAR-T cells were manufactured from fresh or cryopreserved PBMC. As CAR-T cell therapy expands globally, the need for greater flexibility around the timing of manufacture will continue to grow. This study helps support the concept that cryopreservation of PBMCs could be the solution to these issues without compromising the quality of the final CAR-T product.

Persistent expression of activation markers on Mycobacterium tuberculosis-specific CD4 T cells in smear negative TB patients.

T cell activation (HLA-DR, CD-38), proliferation (KI-67), and functional (IFN-γ, TNF-α) markers have recently been shown to be useful in predicting and monitoring anti-TB responses in smear positive TB, but previous research did not characterize the activation and proliferation profiles after therapy of smear negative TB. In this study, we used polychromatic flow cytometry to assess selected PPD-specific T cell markers using fresh PBMC of smear negative and positive pulmonary tuberculosis (PTB) patients, recruited from health facilities in Addis Ababa. Levels of activation (HLA-DR, CD38) and proliferation (Ki-67) among total unstimulated CD4 T cells decreased significantly after therapy, particularly at month 6. Similarly, levels of PPD-specific T cell activation markers (HLA-DR, CD-38) were significantly lower in smear positive PTB patients following treatment, whereas a consistent decline in these markers was less apparent among smear negative PTB patients at the sixth month. After six months of standard anti-TB therapy, persistent levels of activation of HLA-DR and CD-38 from PPD specific CD4+T cells in this study could indicate that those markers have little value in monitoring and predicting anti-TB treatment response in smear negative pulmonary TB patients in Ethiopian context.

Abstract 1070: AUTO7: Anti-PSMA humanized CAR T-cell with improved persistence and resistance to tumor microenvironment for metastatic castration resistant prostate cancer (mCRPC)

Abstract Background. Prostate cancer (PCa) is the most common cancer in men and metastatic disease has no curative options. Here we describe a combinatorial/multi-modular CAR T therapeutic for prostate cancer termed AUTO7. The therapeutic consists of 6 functional modules. The encoded modules are: 1) an anti-PSMA CAR based on a novel humanized binder; 2) the safety switch; 3) dominant negative TGFβRII to induce TGFβ1 resistance (dnTGFβRII); 4) truncated SHP2 (dSHP2) for PD1/PD-L1 pathway inhibition; 5) a constitutively active IL-7 receptor (IL7R_CCR) to induce proliferation and finally 6) an IL-12 module (IL12) for lymphocyte recruitment/activation. Methods. PSMA-binder was generated by CDR grafting of an anti-PSMA antibody derived from genetically vaccinated rats. CAR T-cells were generated by transduction of T-cells with vectors containing the respective modules. CAR T-cells were evaluated in vitro in co-culture assays with PSMA positive cell lines for redirected lysis, cytokine secretion, T cell proliferation, resistance to immunosuppression and IL-12 expression. Antitumor activity of AUTO7 containing the CAR and all 6 modules was assessed in vivo in an established human xenograft in NSG mice. Results. AUTO7 CAR T-cells were highly potent in cytotoxicity assays against PSMA positive tumor cell lines showing no difference to a CAR alone control. AUTO7 CAR T cells encoding the dnTGFβRII and the dSHP2 showed resistance to both TGFβ1 and PD1/PDL1 mediated immunosuppression in vitro whilst the IL7R_CCR module conferred cytokine independent viability and proliferation. Finally, a module designed to enable the CAR T-cells to secrete IL-12 showed potent efficacy by its ability to activate fresh PBMC to release IFNγ. We demonstrated AUTO7 efficacy in an in-vivo model in which human PC3-PSMA PCa cells were engrafted into NSG mice for 3 weeks, prior to i.v. infusion of CAR T-cells. Although T cells transduced with CAR alone was unable to control tumor growth, AUTO7 CAR T-cells completely eradicated tumor with no signs of toxicity. Furthermore, examination of the IL12 module in immunocompetent models also showed no signs of cytokine mediated toxicity. Conclusions. Our results demonstrate the feasibility and efficacy of the multi-modular AUTO7 product. Addition of IL7R_CCR, IL-12, dnTGFβRII and dSHP2 modules to our anti-PSMA CAR product enhanced T cell functions by extending persistence, proliferation, activation and resistance to TGFβ1 and PD1/PDL1 driven immune inhibition. Citation Format: Marco Della Peruta, Victoria Koullourou, Mohamed El-Kholy, Mathew Robson, Philip Wu, Matteo Righi, Mathieu Ferrari, Giulia Agliardi, James Sillibourne, Simon Thomas, Shimobi Onuoha, Shaun Cordoba, Martin Pule. AUTO7: Anti-PSMA humanized CAR T-cell with improved persistence and resistance to tumor microenvironment for metastatic castration resistant prostate cancer (mCRPC) [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1070.

Process optimization for CD4+ and CD8+ T cell formulation and cryopreservation

Background & Aim Cellular therapies are living drugs that have proven highly effective in the treatment of a broad range of human ailments. Logistical considerations for manufacturing living drugs necessitate incorporation of strategies to preserve their potency and efficacy. Many commercialization models employ a frozen cell product to be delivered to the clinic and stored, thawed and infused into patients on demand. Factors influencing post-cryopreservation cell viability and function include, but are not limited to, the selection of an appropriate biopreservation solution and cryoprotective agents, pre-freeze processing time, the temperature of cryomedia addition, ice nucleation temperature, and freezing and warming rates. Methods, Results & Conclusion In this study, CD4+ and CD8+ human T-cells from healthy donors were isolated from fresh PBMC (isolated by using CD4- or CD8-magnetically labelled microbeads) and cryopreserved separately at ∼ 20e6 cells/mL in either a home-brew formulation (10% DMSO, 20% Normosol, and 20% of a 25% Human Serum Albumin solution), or in commercially-available, serum-free, protein-free CryoStor® (BioLife Solutions, Bothell, WA) with 5% v/v or 10% v/v DMSO (CryoStor CS5, and CryoStor CS10, respectively). The cells were subject to varying pre-freeze incubation times at 2-8°C before the freezing process. The vials containing 1 mL of the cell suspension were placed inside a Mr. Frosty type device and were placed at -80°C for a minimum of 2 hours before transfer to LN2. On thaw, cells were processed using different thawing practices (fast vs. slow, and cold vs. warm dilution, among others). Post-thaw cellular viability and count was assessed using the NC-3000 imaging cytometer (ChemoMetec, Denmark) immediately post-thaw. To investigate delayed onset cell death, thawed samples were cultured in complete growth medium (RPMI+ 10%FBS) and cell viability was assessed at 24 h and 48 h post-thaw. Mitochondrial membrane potential was assessed to investigate the potential of this assay as a predictive analytic for delayed onset cell death. Our results suggest that cryopreservation media formulation and post-thaw dilution practice can significantly influence cell viability and recovery, and demonstrate how these parameters contribute to increased variability in observed post-thaw results. Our findings indicate that optimization of the cryopreservation process should be a major focus during early development as a practical means to improve the clinical efficacy of cellular therapies.

Abstract 614: Resiquimod, a Toll-like receptor agonist promotes melanoma regression by enhancing plasmacytoid dendritic cells and T cytotoxic activity as a vaccination adjuvant and by direct tumor application

Abstract Introduction: Cancer immunotherapy is a modern strategy aiming at restoring the capacity of the immune system to target tumors in cancer patients. Toll-like receptor (TLR) agonists may enhance vaccination or direct immune activation at the tumor microenvironment. This clinical trial evaluated the biologic effects of Resiquimod, a TLR agonist that can activate both myeloid (TLR 8) and plasmacytoid (TLR 7) dendritic cells, on advanced stage melanoma. Methods: Subjects with in-transit melanoma metastases or high risk for recurrence and appropriate HLA were treated with peptide vaccination (class 1 restricted peptide GP100(g209-2m) and, if HLA-DP4+, class 2 restricted peptide MAGE-3243-258). Half of the patients were randomized to receive Resiquimod as an adjuvant applied to the GP100 vaccination site. Subjects with in-transit disease were then treated with resiquimod topically on half of the target lesions. To evaluate the T cell function, fresh PBMC and single cell tumor suspension were analyzed by flow cytometry using gp100-specific dextramer staining. RNA from the vaccination site was also analyzed using real-time PCR. Results: All patients (n=47) underwent GP100(g209-2m) vaccination, a majority (39) also received the MAGE-3243-258 peptide. Type 1 interferon pathway protein profiles of vaccination sites showed activation of plasmacytoid dendritic cells in patients with Resiquimod, but not in its absence. Nineteen subjects had in-transit disease at entry into the trial. In response to peptide vaccination alone, tumor regression was more likely in patients who received Resiquimod (group A) compared to those who did not (group B). (4/9 vs 0/10). In group A, 5 patients continued treatment with Resiquimod topically on the tumors, and all had tumor response (4PR, 1CR). In group B, 5 continued to tumoral resiquimod and 3 had regression (3 PR). Type I interferon (as measured by MxA and IRF7) IFN-gamma and TNF-alpha increased at the vaccination site 24 hrs after vaccination only at the sites where Resiquimod was applied. In blood, Resiquimod increased gp100-specific CD8 T cells frequency at week 8 (p=0.03) only in patients who received Resiquimod at the vaccination site. Conclusions: Resiquimod activates plasmacytoid dendritic cells at a peptide vaccination site and augments peptide vaccination sufficiently to mediate regression of in-transit melanoma metastasis. Resiquimod on in-transit melanoma, in vaccinated hosts, drives regression of metastases, regardless of previous exposure at vaccination site. An increased amount of cytokines such type I interferon, IFN-gamma, TNF-alpha, and T specific cytotoxic frequency were increased at the vaccination site after patients received Resiquimod. Citation Format: Jorge A. Borin Scutti, Luis M. Vence, Richard E. Royal, Tara C. Wray, Janice N. Cormier, Jeffrey E. Lee, Anthony Lucci, Jeffrey E. Gershenwald, Merrick I. Ross, Jennifer Wargo, Karen A. Millerchip, Rodabe N. Amaria, Michael A. Davis, Adi Diab, Isabella C. Glitza, Wen Hwu, Sapna Patel, Scott E. Woodman, Willem W. Overwijk, Patrick Hwu. Resiquimod, a Toll-like receptor agonist promotes melanoma regression by enhancing plasmacytoid dendritic cells and T cytotoxic activity as a vaccination adjuvant and by direct tumor application [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 614.

Fast and easy immunomagnetic isolation of untouched human naïve T cells in less than 15 minutes

Abstract Peripheral blood circulating T cells can be subdivided into either antigen experienced effector and memory T cells or naïve T cells that have not yet encountered their cognate antigen. Naïve T cells are valuable tools when studying mechanisms of T cell activation and are required for foundational research into infectious diseases, cancer and transplantation. We describe here a simple immunomagnetic cell isolation protocol for the isolation of untouched human naïve T cells from fresh or previously frozen PBMC or leukapheresis samples. The column-free, negative selection procedure involves labelling and removing unwanted cells using bispecific antibody complexes that crosslink cell surface antigens to magnetic particles. The labelling cocktail is composed of antibodies that target non-T cells, memory T cells, and an optional cocktail for depleting gamma delta T cells. Briefly, the procedure involves a five minute incubation with the antibody cocktail followed by the addition of magnetic particles and two three minute magnetic separations in a hand-held EasySep™ magnet. Following the second magnetic separation, the untouched naïve T cells are simply poured off into a new tube and are ready for use. We define naïve T cells phenotypically as CD3+CD45RA+CD45RO-CD197+. Starting from fresh PBMC, we were able to obtain purities of 96.1 ± 2.3% (mean ± SD, n=14) and recoveries of 62.6% ± 24.5% (mean ± SD, n=14). Additionally, isolated naïve T cells are functional and respond by upregulating the expression of CD25 and CD69 upon stimulation. Our EasySep™ Human Naïve Pan T Cell Isolation Kit offers a fast and easy isolation method for naïve T cells that is suitable for downstream applications such as flow cytometry, cell culture or DNA/RNA extraction.

Immune effects of combinatorial repurposing drugs in refractory ovarian and colorectal cancer patients.

Abstract Background Some refractory cancer patients have a good Karnofsky despite refractory disease. We studied the potential immunomodulatory properties of approved drugs. We used fresh PBMC from refractory patients to study the immune effects of several drugs such as metformin, pidotimod, erdostein, esomeprazole, colchicine, bortezomib, gemcitabine, zoledronic acid and naproxen. Methods 25 high-grade serous ovarian cancer (HGSOC) and 25 colorectal cancer (CRC) subjects were included after approved protocol. The inclusion criteria include Karnofsky 80–100%, complete CT scan, acute phase proteins, granzyme B ELISPOT and antigen-specific ELISA. Results We found after 24 months of treatment in HGSOC 80% of complete response (CR), 15% of stable disease (SD) and 5 % of pseudo-progressive disease (PPD). In CRC we had 50% of CR, 30% of SD and 20% of PPD. In the patients with CR there was an immunological correlation of CD8 persistent immune response by granzyme B ELISPOT in HGSOC and CRC (p=0.0001) and PR (p=0.001), respectively. The combination was well tolerated and after 10 months of stopping the treatment some patients have persistent CD8 specific immune response by ELISPOT and ELISA. Discussion The combination of daily oral metformin, colchicine, pidotimod, erdostein, and naproxen plus weekly low dose of intravenous (IV) esomeprazole (80 mg), bortezomib (0.5 mg/m2), gemcitabine (0.5 mg/m2) and zoledronic acid (100 mg) is clinically feasible, with limited clinical toxicity. We now understand the importance of preserving the CD8 immune response and the use of immune biomarkers such as antigen specific ELISA to improve the rational and generate new combinations to improve clinical outcomes in patients with refractory HGSOC and CRC.

High TCR Vbeta Skewing in CD4+ and Low in CD8+ Is Associated with Development of Chronic Graft-Versus-Host Disease

IntroductionChronic graft-versus-host disease (cGVHD) remains a major cause of morbidity and mortality after allogeneic hematopoietic stem cell transplantation (allo-HSCT). Perturbations in immune reconstitution following allo-HSCT are believed to be integral to the pathophysiology of cGVHD. We hypothesized that skewing in T cell repertoire around day 80 would be predictive of cGVHD, while maintenance of a broad repertoire would be protective. One method used to assess skewing is the Gini Index, a conventional measure of income inequality. In this prospective study, we utilized a rapid FACS based method of TCR Vβ repertoire analysis within CD4+ and CD8+ in patients at day 80-110 post allo-HSCT to determine whether preferential TCR skewing predicts the development of cGVHD.MethodsPeripheral blood samples were obtained from 79 consented patients at day 80-110 following allo-HSCT at The University of Texas MD Anderson Cancer Center from October 2012 to November 2015. All patients were in remission of their disease at the time of blood sampling. Fresh PBMC were analyzed by FACS after staining for CD3, CD4, CD8 and IOTest Beta Mark 24 TCR Vβ kit. The TCR Gini Index scores (GS) were calculated for the T cell subsets, and ranged from 0 to 100. Low GS indicate more equal distribution of TCR-Vβ families (i.e. broad repertoire), whereas high GS reflect unequal distribution of TCR-Vβ families (i.e. repertoire skewing). Predictive thresholds of GS were identified in quartiles analysis and confirmed by Receiver Operating Characteristic (ROC) curve.The association between GS and the rate of cGVHD since the date of blood sampling was evaluated using competing-risks regression analysis. The diagnosis of cGVHD was based on the 2014 National Institutes of Health guidelines.ResultsThe median age at transplant was 53 years (range 21-75) and 60% of patients were male. The most common transplant indication was AML/MDS (52%) followed by lymphoma (16%), ALL (14%), and other (18%). Graft source was peripheral blood (54%), bone marrow (43%) and umbilical cord blood (UCB) (3%) with donors being matched unrelated (65%), matched-related (24%), mismatch related (9%) and UCB (3%). Forty three (54%) patients were not in remission at the time of transplant. At the time of enrollment, 47% had history of grade II-IV acute GVHD and 44% were on systemic steroids. At a median follow up of 2 years since blood sampling, 16 patients developed cGVHD (cumulative incidence (CI) 23%; 95% Confidence Interval: 15-35). The median time from HSCT to cGVHD was 174 days (range 111-551 days). None of the clinical or demographic characteristics listed above, except history of acute GVHD, was significantly associated with the incidence of cGVHD. GS were significantly predictive of the incidence of cGVHD. The 2 year %CI of cGVHD was 47% in patients who had CD4+ TCR GS >55% (n=19) compared with 16% in those with CD4+ GS ≤55% (n=60) (hazard ratio (HR) =3.4, P=0.01). In contrast to CD4+, lower CD8+ GS predicted for higher incidence of cGVHD. The 2 year %CI of cGVHD was 39% in patients (n=34) with CD8+ GS ≤60% compared with 10% in patients (n=45) with CD8+ GS >60% (HR=4.5, P=0.009). When both CD4+ and CD8+ GS were considered in combination, the 2 year incidence of cGVHD was highest in patients (n=6) with both low CD8+ (≤60%) and high CD4+ (>55%) GS, and lowest in patients (n=32) with high CD8+ (>60%) and low CD4+ (≤55%) GS (%CI: 83% vs 4%, P=0.001). The combined effect of high CD4+ and low CD8+ TCR skewing was more pronounced in patients with a history of grade II-IV acute GVHD. Low CD8+ (≤60%, n=28) or high CD4+ (>55%, n=13) GS occurring separately were associated with comparable rates of cGVHD (%CI: 31% and 27%, p=0.8), irrespective of history of grade II-IV acute GVHD (Figure).ConclusionsHigh CD4+ and low CD8+ TCR Gini indices were independent predictors of cGVHD. Our results indicated that the higher skewing of CD8+ TCR appeared to be protective of cGVHD and the opposite for CD4+. Our study is the first to test the usefulness of FACS-based TCR measurement and Gini Index score in a clinical setting to help predict the development of cGVHD and provide greater insights into the pathogenesis of cGVHD. Using this convenient FACS-based method to identify patients who are at risk of developing cGVHD could be beneficial from development of effective preventive or therapeutic strategies. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Reduced and skewed TCR Vβ repertoire in CD8 compared to Foxp3+ regulatory T cells in acute gastrointestinal graft versus host disease (TRAN1P.947)

Abstract It is unclear if skewing of TCR repertoire in CD4, CD8 and Foxp3+Tregs plays a role in acute gastrointestinal (GI) GVHD. This prospective clinical study examined the TCR Vβ diversity among CD4, CD8 and Foxp3+Tregs from 18 adult patients (median age 53) post allogeneic stem cell transplant (SCT) at day 80 with acute GI GVHD (giGVHD, >grade 1, n=9) vs. nonGVHD (n=9). Fresh PBMC were analyzed by FACS using IOTest Beta Mark 24 TCR Vβ and Foxp3 (236A/E7)/Helios eBioscience kits. Mann Whitney test was used. Mean %CD4 was lower in giGVHD vs. nonGVHD (44vs72%, p=0.02) while no difference in %CD8 (51vs55%), %Tregs (16vs16%) and %Helios+Tregs (72vs76%). %Total Vβ were similar for CD4 (60), CD8 (60) and Tregs (64) in nonGVHD. In giGVHD, %CD8 (40) was lower than %Tregs (69), p<0.001 and %CD4 (56), p=0.08. Between giGVHD vs. nonGVHD, there was a difference for %TCR in CD8 (p=0.02) but none for CD4 or Tregs. In nonGVHD, there was a direct correlation between %CD8 TCR vs. %CD8 and %CD4 TCR vs. %CD4 but no correlation for %Treg TCR vs. %Tregs. In giGVHD, there was an inverse correlation between %CD8 TCR vs. %CD8 and %CD4 TCR vs. %CD4 but no correlation for %Treg TCR vs. %Tregs. These results indicate a restricted, reduced and skewed TCR repertoire within the CD8 as compared to Tregs and CD4 in giGVHD. These CD8 might be the pathogenic cells escaping Treg suppression. In conclusion, a reduced and skewed TCR diversity in CD8 at day 80 might be predictive for the development of chronic GVHD.

PD-1 expression on peripheral blood cells increases with stage in renal cell carcinoma patients and is rapidly reduced after surgical tumor resection (TUM2P.881)

Abstract In this study, we analyzed expression of numerous immune cell markers on fresh PBMC from 90 RCC patients preoperatively and 25 age-matched healthy controls by 10-color flow cytometry. Postoperative blood samples were also analyzed from 23 members of the RCC patient cohort. The most striking phenotypic immune biomarker in RCC patients was a significant increase in PD-1 expression on certain PBMC in a subset of patients. Increased PD-1 expression on CD14bright myelomonocytic cells, effector T cells, and NK cells correlated to disease stage. Importantly, we also observed that expression was significantly reduced on all cell types in virtually all patients within 2 weeks after surgical resection of the primary tumor. Based upon our results, we conclude that PD-1 expression on fresh peripheral blood leukocytes may provide a useful indicator of RCC disease progression. Furthermore, measuring PD-1 levels in peripheral blood may assist in identifying patients likely to respond to PD-1 blocking antibodies, and these therapies may be most effective before and immediately after surgical resection of the primary tumor, when PD-1 expression is most prominent. Finally, we conclude that long-term PD-1 blocking therapy after nephrectomy may only be effective in stage 3 and stage 4 patients that retain PD-1 expression, presumably maintained by the presence of residual metastatic tumor.

Resistance to HIV-1 infection among HIV-exposed seronegative partners in HIV-discordant couples is associated with higher frequency of CD8+ T cells expressing CD107a and b molecules

Methods Ten HIV-1 ESN in HIV-discordant couples were enrolled at the Fann University hospital, Dakar, Senegal. Thirty HIV-1 infected patients (10 HIV-1 non transmitted partners in discordant coupes and 20 HIV-1 infected partners in HIV-concordant couples) and 10 HIV-negative unexposed subjects were included as controls. Levels of CD107a and b, and CD107a/b+IFN-g+ in CD8+ T cells sub-set were measured in fresh PBMC by flow cytometry, in the presence or absence of stimulation with SEB.

Effects of cryopreservation on effector cells for antibody dependent cell-mediated cytotoxicity (ADCC) and natural killer (NK) cell activity in 51Cr-release and CD107a assays

Freshly isolated PBMC are broadly used as effector cells in functional assays that evaluate antibody-dependent cell mediated cytotoxicity (ADCC) and NK activity; however, they introduce natural-individual donor-to-donor variability. Cryopreserved PBMC provide a more consistent source of effectors than fresh cells in cytotoxicity assays. Our objective was to determine the effects of cryopreservation of effector PBMC on cell frequency, and on the magnitude and specificity of ADCC and NK activity. Fresh, frozen/overnight rested and frozen/not rested PBMC were used as effector cells in 51Cr-release and CD107a degranulation assays. Frozen/overnight rested PBMC had higher ADCC and NK activity in both assays when compared to fresh PBMC; however, when using frozen/not rested PBMC, ADCC and NK activities were significantly lower than fresh PBMC. Background CD107a degranulation in the absence of target cell stimulation was greater in PBMC that were frozen/not rested when compared to fresh PBMC or PBMC that were frozen overnight and rested. The percentages of CD16+CD56dim NK cells and CD14+ monocytes were lower in PBMC that were frozen and rested overnight than in fresh PBMC. CD16 expression on CD56dim NK cells was similar for all PBMC treatments. PBMC that were frozen and rested overnight were comparable to fresh PBMC effectors. PBMC that were frozen and used immediately when evaluating ADCC or NK activity using either a 51Cr-release assay or a CD107a degranulation assay had the lowest activity. Clinical studies of antibodies that mediate ADCC would benefit from using effector cells that have been frozen, thawed and rested overnight prior to assay.

Ex Vivo-Expanded Cynomolgus Macaque Regulatory T Cells Are Resistant to Alemtuzumab-Mediated Cytotoxicity

Alemtuzumab (Campath-1H) is a humanized monoclonal antibody (Ab) directed against CD52 that depletes lymphocytes and other leukocytes, mainly by complement-dependent mechanisms. We investigated the influence of alemtuzumab (i) on ex vivo-expanded cynomolgus monkey regulatory T cells (Treg) generated for prospective use in adoptive cell therapy and (ii) on naturally occurring Treg following alemtuzumab infusion. Treg were isolated from PBMC and lymph nodes and expanded for two rounds. CD52 expression, binding of alemtuzumab and both complement-mediated killing and Ab-dependent cell-mediated cytotoxicity (ADCC) were compared between freshly isolated and expanded Treg and effector T cells. Monkeys undergoing allogeneic heart transplantation given alemtuzumab were monitored for Treg and serum alemtuzumab activity. Ex vivo-expanded Treg showed progressive downregulation of CD52 expression, absence of alemtuzumab binding, minimal change in complement inhibitory protein (CD46) expression and no complement-dependent killing or ADCC. Infusion of alemtuzumab caused potent depletion of all lymphocytes, but a transient increase in the incidence of circulating Treg. After infusion of alemtuzumab, monkey serum killed fresh PBMC, but not expanded Treg. Thus, expanded cynomolgus monkey Treg are resistant to alemtuzumab-mediated, complement-dependent cytotoxicity. Furthermore, our data suggest that these expanded monkey Treg can be infused into graft recipients given alemtuzumab without risk of complement-mediated killing.

T cell cytokine profile of early tuberculosis infection and disease in Cynomolgus macaque model (P3335)

Abstract Mycobacterium tuberculosis (Mtb) remains one of the world’s most successful human pathogen. Addressing questions on immunological events soon after Mtb infection in human is challenging. Non-human primate model (Cynomolgus macaques) provide the necessary platform to understand the early immunological events in Mtb infection. We infected 28 Cynomolgus macaques with a low dose of Mtb (Erdman strain, ~25-50 CFU) and monitored clinically for signs and symptoms of tuberculosis up to six months post infection and classified to have either latent TB infection or active disease based on chest X-ray and Mtb culture. Peripheral blood was collected at time-points: pre Mtb infection; and at days: 10, 20, 30, 42, 56, 90, 120, 150 and 180 post infection. PBMC were isolated and multi-parametric intracellular flow cytometry was performed in fresh PBMC to track the early CD4 and CD8 T cell cytokine profiles (CD107a, GMCSF, IFN-g, IL-2, IL-4, IL-6, IL-10 and IL-17) during the course of Mtb infection. Preliminary analysis shows difference in the cytokine response in the early phase of infection between those who developed active disease or latent infection. In addition, we find a spectrum of cytokine response, which overlap between active and latent tuberculosis.

The early Th17/Treg ratio predicts the immune activation set point in patients with primary HIV infection

BACKGROUND: Persistent systemic immune activation plays a central role in the pathogenesis of HIV disease. Impairment of the intestinal barrier and subsequent microbial translocation might be involved in chronic immune activation. Th17 cells are important in the maintenance of intact epithelium and host defense against extracellular pathogens. The ratio between the two closely related CD4 subsets Th17 and Tregs has been recently found to shrink with HIV/SIV disease progression. The aim of the study was to analyze, in patients with early primary HIV infection (PHI), the relationship between Th17/Treg ratio and the immune activation set point, known to predict disease progression. METHODS: 27 patients with early PHI were included in a prospective longitudinal study and followed-up for 6 months. T-cell activation and CD4+CD25+CD127lowFoxp3+ Treg frequency were assessed on fresh PBMC. Th17 cells were quantified by intracellular cytokine staining on sorted peripheral CD4 T cells stimulated with PMA/ionomycin for 5h. Correlations were assessed using spearman non-parametric tests. Plasma I-FABP, a marker of mucosal damages and soluble CD14 (sCD14) were measured by ELISA. RESULTS: A strong negative relationship was found at baseline between the Th17/Treg ratio and the proportion of activated CD8 T cells expressing CD38/HLA-DR (p=0.008) or Ki-67 (P=0.001). At baseline, Th17/Treg ratios also negatively correlated with sCD14 plasma levels (p=0.003). I-FABP levels, which were similar to controls at baseline, increased at month 6. The Th17/Treg ratio at baseline (but not the proportion of Th17 cells) negatively correlated with the frequency of HLA-DR+CD38+ or Ki-67+ CD8 T cells at month 6, defining the immune activation set point (p=0.02 and p=0.0005 respectively). sCD14 plasma levels were also found to predict the immune set point (p=0.02). CONCLUSION: Our data do not support early mucosal damages in PHI. However, the early Th17/Treg balance correlates with sCD14 levels and predicts the immune activation set point.

Comparative analysis of myeloid-derived suppressor cell (MDSC) subsets in patients with gastrointestinal (GI) malignancies.

10566 Background: Immunotherapy represents a potential new treatment option for patients with cancer. Myeloid derived suppressor cells (MDSCs) constitute a heterogenous cell population, which inhibit the host anti-tumor immune response, limiting the effectiveness of both immune and non-immune treatments. MDSC are known to be increased in patients with GI cancer. Characterization of MDSC in humans has been difficult with heterogeneous phenotype classification. Differences in processing are also potentially important. The aim of this study was to perform a comparative analysis of MDSC subsets in patients with GI cancer. Methods: Fluorescence activated cell sorting (FACS) analysis was performed on PBMC and whole blood (WB) from healthy adults (N=44) and patients with GI cancer (N=41). MDSC subsets were enumerated using the following antibodies: anti-CD14; anti-CD15; anti-HLA-DR; anti-CD33; anti-CD11b; 7-AAD. Suppressor function of MDSC subsets was tested using FACS sorted MDSC subpopulations co-cultured with CD3/CD28-stimulated autologous effector cells. Proliferation and IFN-g were measured. Results: The frequencies of Monocytic (Def: CD14+, HLA-DR -/low) and Granulocytic (Def: CD15+,CD14-CD33+CD11b+) MDSC are shown in the table below. Monocytic MDSC were increased in the blood of patients (vs control; P<.0001) and frozen (vs fresh PBMC; P<.05). While CD15+ cells were increased in cancer patients we did not find an increase in granulocytic MDSC. Functional studies will also be presented. Conclusions: Monocytic MDSC are more frequent in patients with advanced GI cancer compared to healthy volunteers. An increase in this population was also found in WB and frozen PBMC compared to fresh PBMC reflecting the need for homogenous preparation in studies evaluating this population in humans. While CD15+CD14- cells were also increased in cancer patients we did not find an increase in CD14-CD15+CD33+CD11b+ cells. [Table: see text]

Phase I trial of extended-dose anti-PD-1 antibody BMS-936558 with a multipeptide vaccine for previously treated stage IV melanoma.

8582 Background: BMS-936558, a fully human IgG4 anti-PD-1 antibody, has shown clinical activity in melanoma and renal cancer. Methods: 30 HLA A*0201 positive patients with unresectable, previously treated stage IV melanoma received MART-1/gp100/NY-ESO-1 peptides with adjuvant Montanide ISA 51 injected subcutaneously with BMS-936558 at 1, 3 or 10 mg/kg intravenously every 2 weeks for 24 weeks, followed by BMS-936558 alone every 3 months. No patient had prior ipilimumab. Endpoints were toxicity, correlative studies of immunity measured by ELISPOT and T-cell phenotype assays, and response by RECIST. Results: Median age was 62, with 14 men and 16 women. 73% of patients had M1c disease. One patient had dose limiting optic neuritis; one patient had dose limiting grade 3 interstitial pneumonitis. Of 10 patients at 1 mg/kg, 2 had confirmed partial responses (PR), both one year from initiation of study. 13 patients were treated at the 3 mg/kg cohort; 3 patients did not complete more than 3 doses secondary to progressive disease and one withdrew consent. Of 12 evaluable patients, 3 had confirmed PRs and 2 unconfirmed PRs. In the 10 mg/kg cohort, thus far 6 patients completed one cycle with 2 unconfirmed PRs and one patient stable. Only 2 of the 9 responders after one cycle have progressed with a median follow-up of 7 months. 2/6 patients failing BMS-936558 responded to ipilimumab. At week 12, patients in all cohorts had decreased PD-1 on CD4 and CD8 T-cells (p<0.0001), increased CD4 CTLA-4 levels (p=0.01) and dose related decreases in CD8 T-cells whereas CD4 T-cells increased (both p<0.05). ELISPOT assays showed that patients at all doses had no increased responses in fresh PBMC to MART-1/gp100, but showed dose related decreased responses to viral (CMV, EBV, FLU) peptides over time (p=0.04). Conclusions: BMS-936558 in combination with a peptide vaccine is well tolerated at 1 and 3mg/kg. Accural to the 10mg/kg cohort is on-going. Responses have been observed at all dose levels. PD-1 levels on T-cells decreased, and CTLA-4 levels increased in CD4 T cells in all cohorts. There was a dose-related decrease in CD8 T-cells and in viral-specific T-cells. Further study of BM-936558 is warranted to determine its optimal dose.

A trivalent recombinant Ad5 gag/pol/nef vaccine fails to protect rhesus macaques from infection or control virus replication after a limiting-dose heterologous SIV challenge

It has been suggested that poor immunogenicity may explain the lack of vaccine efficacy in preventing or controlling HIV infection in the Step trial. To investigate this issue we vaccinated eight Indian rhesus macaques with a trivalent replication-incompetent adenovirus serotype 5 vaccine expressing SIV Gag, Pol, and Nef using a regimen similar to that employed in the Step trial. We detected broad vaccine-induced CD8+ (2–7 pool-specific responses) and CD4+ (5–19 pool-specific responses) T-cell responses in IFN-γ ELISPOT assays at one week post-boost using fresh PBMC. However, using cryopreserved cells at one and four weeks post-boost we observed a reduction in both the number and magnitude of most vaccine-induced responses. This demonstrates that the time points and conditions chosen to perform immune assays may influence the observed breadth and frequency of vaccine-induced T-cell responses. To evaluate protective efficacy, we challenged the immunized macaques, along with naïve controls, with repeated, limiting doses of the heterologous swarm isolate SIVsmE660. Vaccination did not significantly affect acquisition or control of virus replication in vaccinees compared to naïve controls. Post-infection we observed an average of only two anamnestic CD8+ T-cell responses per animal, which may not have been sufficiently broad to control heterologous virus replication. While the trivalent vaccine regimen induced relatively broad T-cell responses in rhesus macaques, it failed to protect against infection or control viral replication. Our results are consistent with those observed in the Step trial and indicate that SIV immunization and challenge studies in macaque models of HIV infection can be informative in assessing pre-clinical HIV vaccines.

Inherent baseline levels of phosphorylated IRF7 impact pDC IFNα response to TLR7 stimulation (108.8)

Abstract Background: We previously showed that plasmacytoid dendritic cells (pDCs) derived from females can produce significantly more IFNα in response to HIV-1 and HIV-1-encoded TLR7/8 ligands than pDCs derived from males, resulting in stronger secondary activation of CD8+ T cells. Given the crucial role of interferon regulatory factor 7 (IRF7) in the regulation of type I IFN production by pDCs, we hypothesized that IRF7is involved in the differences observed in IFNα production. Methods: Fresh PBMC were isolated from HIV-1-negative subjects enrolled at Massachusetts General Hospital. Levels of IRF7 were measured in pDCs by phospho-flow cytometry, In-Cell Western blotting and qRT-PCR at baseline and at different time-points after TLR7 stimulation. IFNα levels were quantified by flow cytometry, ELISA and qRT-PCR Results: We observed a higher percentage of pDCs expressing phosphorylated IRF7 at baseline in females than in males using flow cytometry (p<0.05). This difference persisted after TLR7 stimulation and was associated with higher IFNα production by females. Conclusion: These data show that sex differences in the baseline level of phosphorylated IRF7 in pDCs might account for described higher IFNα production upon TLR7 stimulation, and might provide new insights into the mechanisms underlying faster HIV-1 disease progression in females compared to males after controlling for viral load.