Stimulation Of PBMC Research Articles

Immunosuppressive Treatment Does Not Prevent Humoral and Cellular Virus-Specific Immunity in Heart or Lung Recipients with SARS-CoV-2 Pneumonia

PurposeClinical characteristics of SARS-CoV-2 infection and virus-specific humoral and cellular response were analyzed in 4 heart (HR) and 3 lung (LR) Tx recipients in standard triple immunosuppressive regimen.MethodsSARS-CoV-2 infection was diagnosed by real-time PCR on naso-pharingeal swabs (NPS). T-cell response to structural antigens Spike (S), Envelope (E), Membrane (M) and Nucleocapsid (N) was evaluated by PBMC stimulation with overlapping peptides spanning the entire viral proteins and subsequent detection of cell activation markers CD137 and CD25. Serum IgG antibody to S and N, and IgA antibody to S were determined by ELISA.ResultsThree patients developed SARS-CoV-2 infection early (<3 months) and four patients late (>3 years) after Tx. One HR was asymptomatic, one LR presented only gastrointestinal symptoms, and five patients developed dyspnea with radiologic signs of interstitial pneumonia (in one HR ICU admittance was necessary. All patients recovered from SARS-CoV-2 infection, with viral clearance from NPS within 3 weeks. However, two HR (one early and one late HR) died at 6 and 4 months after infection because of multi-organ failure and sudden death. Both deaths were considered as unrelated to SARS-CoV2 infection. Patients who had no lung involvement did not develop specific antibody response, while all the other five patients developed IgG and IgA antibodies to S, and IgG antibody to N, within 2 months after infection. All the symptomatic patients developed a detectable CD4+ T-cell response to two or more antigens. Four patients were subsequently examined >3 months after infection, showing the persistence of IgG and IgA antibodies to S and a decline of IgG antibody to N, while CD4+ T-cell response to N was maintained. Timing of Tx did not affect the occurrence of virus specific immunity.ConclusionAlthough this series is small, the data indicate that immunosuppression does not prevent the development of a specific humoral and cellular anti-SARS-CoV-2 response, more likely in patients who have experienced clinically relevant pneumonia. These preliminary data encourage the maintenance of regular follow-up to monitor the persistence of immune response and the potential occurrence of SARS-CoV-2-related sequelae in heart and lung recipients.

N-Glycan Modification in Covid-19 Pathophysiology: In vitro Structural Changes with Limited Functional Effects.

In 2014, we reported two siblings with a rare congenital disorder of glycosylation due to mutations in mannosyl-oligosaccharide glucosidase (MOGS). The glycan alteration derived from this disease resulted in an in vitro infection resistance to particular enveloped, N-glycosylation-dependent viruses as influenza and HIV. As part of the global effort to find safe and effective antiviral therapies for Covid-19, we assessed the in vitro activity of the FDA-approved α-glucosidase inhibitor miglustat against SARS-CoV-2. Expression plasmids encoding SARS-CoV-2 spike (S) and human ACE2 glycoproteins (GP) were tested to evaluate N-glycan modifications induced by α-glucosidase inhibition. Immunoprecipitation was used to assess binding between these two GP. Cell-to-cell fusion was assessed by immunofluorescence of cocultures of SARS-CoV-2 S and ACE2-expressing cells. Miglustat effect on immune response was tested by measuring cytokine release from PBMC exposed to purified SARS-CoV-2 S. In our overexpression system, miglustat successfully and specifically modified N-glycans in both SARS-CoV-2 S and its main receptor ACE2. Binding between these two GP was not affected by glycan modifications. A surrogate marker for viral cytopathic effect, measured as receptor-dependent SARS-CoV-2 S-driven cell-to-cell fusion, was not disrupted by miglustat treatment. This observation was further confirmed in MOGS-null transfected cells. Miglustat produced no statistically significant effects on cytokine production following SARS-CoV-2 S glycoprotein stimulation of PBMC. Our work shows that despite clear N-glycan alteration in the presence of miglustat, the functions of the Covid-19-related glycoproteins studied were not affected, making it unlikely that miglustat can change the natural course of the disease.

Abstract 3865: A translational immuno-oncology platform to model T cell activation and Tex recovery in the tumor microenvironment

Abstract The tumor microenvironment (TME) is a complex network, consisting of malignant tumor cells, stromal and immune cells, blood vessels, extracellular matrix and soluble factors. The immune system plays an important role in combating tumor growth, and multiple studies associate immune infiltrate with beneficial outcome. Various tractable immuno-oncology targets have been identified that influence the behavior of immune cells in the TME. These include Cytotoxic CD8+ T cells; which can directly kill tumour cells and produce pro-inflammatory cytokines such as IFNγ. However, CD8+ T cell activity and survival can be suppressed by the TME and chronic exposure leads to the generation of exhausted T cells (Tex) which have a lowered proliferative capacity and diminished capacity for pro-inflammatory cytokine production. Conversely, a regulatory subset of CD4+ T cells (Tregs) act to skew the TME towards immunosuppression. Here we describe human and murine models to assess both relative T cell subset activation and whether T cell exhaustion can be broken. Screening assays using T cell stimulants such as SEB or IL-2 can be utilised to select candidate therapeutics based on functional target engagement. SEB stimulation of PBMC reveals a robust additive effect on T cell activation by established and emerging checkpoint inhibitors. Use of more complicated flow based readouts allows the discrimination of immune subset responses. IL-2, a key lymphocyte activator, drives pSTAT5 expression with differing potency in PBMC subsets (regulatory T cells, conventional CD4 cells, CD8 T cells, NKT cells and NK cells). The ability to target specific subset activation could expand anti-tumour responses. Persistent antigenic stimulation, such as chronic exposure to the TME (MHC I restricted peptides), can lead to a CD8 Tex cell phenotype. Tex cell generation and reversal can be modelled using CEFT peptide pool and monitored via the expression of markers such as LAG-3, TIM-3, TOX and IFNγ. We show here that CD8 Tex cells respond positively to PD-1 blockade treatment, as seen by a restoration in cellular proliferation and increased IFNγ production. Therapeutic candidates identified through in vitro lead optimization can be assessed for their ability to activate T cells via a murine pharmacodynamic (PD) model. Transgenic OT-I CD8 T cells expressing TCR recognising OVA peptide (SIINFEKL) were transferred into syngeneic mice with SIINFEKL challenge in the presence of test therapeutic. Flow cytometry was used to determine OT-I T cell proliferation, activation and cytokine release. As seen in vitro, OT-I cell activation was increased by CPI treatment. We show here the powerful combination of using sophisticated in vitro and in vivo models to determine the effect of therapeutic intervention in an immuno-oncology context. Citation Format: Robert Nunan, Hannah Gill, Ilona Aylott, Michelle Yap, Tommaso Iannitti, Louise Harvey, Rhiannon Jenkinson. A translational immuno-oncology platform to model T cell activation and Tex recovery in the tumor microenvironment [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 3865.

Identification of immunogenic T-cell peptides of Mycobacterium tuberculosis PE_PGRS33 protein

The development of a more efficient vaccine is needed to improve tuberculosis control. One of the current approaches is to identify immunogenic T-cell peptides that can elicit a protective and specific immune response. These peptides come from immunogenic proteins of the pathogen. The PE_PGRS33 protein of Mycobacterium tuberculosis has been proved immunogenic. However, little is known about immunogenic T-cell peptides of PE_PGRS33 and their interactions with MHC-II molecules. Therefore, we used the SYFPHEITHI database to determine the immunogenic PE_PGRS33 T-cell peptides. Next, we built homology models by using MOE v2018.1 software in order to obtain information about the specific interactions between the peptides and I-Ak. The AlgPred server was employed to look for allergenic sites in PE_PGRS33. We developed a sequence alignment between PE_PGRS33 and all the human proteins by using BLAST. Three peptides were commercially synthesized, and their activity was evaluated in vitro by the stimulation of PBMC from household contacts of TB patients. Our in silico results showed five immunogenic T-cell peptides. BLAST analysis showed low homology of PE_PGRS33 with human proteins and AlgPred did not reveal allergenic sites in PE_PGRS33. The three peptides triggered the activation of CD4+ T cells from the households contacts, showed by the production of IFN-γ. We identified three immunogenic peptides of PE_PGRS33 that demonstrated activity in vitro which allows to deepen into the immune response towards mycobacterial antigens, moving forward to the identification of new vaccine candidates.

Assessing Anti-HCMV Cell Mediated Immune Responses in Transplant Recipients and Healthy Controls Using a Novel Functional Assay.

HCMV infection, reinfection or reactivation occurs in 60% of untreated solid organ transplant (SOT) recipients. Current clinical approaches to HCMV management include pre-emptive and prophylactic antiviral treatment strategies. The introduction of immune monitoring to better stratify patients at risk of viraemia and HCMV mediated disease could improve clinical management. Current approaches quantify T cell IFNγ responses specific for predominantly IE and pp65 proteins ex vivo, as a proxy for functional control of HCMV in vivo. However, these approaches have only a limited predictive ability. We measured the IFNγ T cell responses to an expanded panel of overlapping peptide pools specific for immunodominant HCMV proteins IE1/2, pp65, pp71, gB, UL144, and US3 in a cohort of D+R– kidney transplant recipients in a longitudinal analysis. Even with this increased antigen diversity, the results show that while all patients had detectable T cell responses, this did not correlate with control of HCMV replication in some. We wished to develop an assay that could directly measure anti-HCMV cell-mediated immunity. We evaluated three approaches, stimulation of PBMC with (i) whole HCMV lysate or (ii) a defined panel of immunodominant HCMV peptides, or (iii) fully autologous infected cells co-cultured with PBMC or isolated CD8+ T cells or NK cells. Stimulation with HCMV lysate often generated non-specific antiviral responses while stimulation with immunodominant HCMV peptide pools produced responses which were not necessarily antiviral despite strong IFNγ production. We demonstrated that IFNγ was only a minor component of secreted antiviral activity. Finally, we used an antiviral assay system to measure the effect of whole PBMC, and isolated CD8+ T cells and NK cells to control HCMV in infected autologous dermal fibroblasts. The results show that both PBMC and especially CD8+ T cells from HCMV seropositive donors have highly specific antiviral activity against HCMV. In addition, we were able to show that NK cells were also antiviral, but the level of this control was highly variable between donors and not dependant on HCMV seropositivity. Using this approach, we show that non-viraemic D+R+ SOT recipients had significant and specific antiviral activity against HCMV.

P0924IMMUNE DISTURBANCES INDUCED BY HYPERVOLEMIA DO NOT ACTIVATE THE NLRP3 INFLAMMASOME IN HAEMODIALYSIS PATIENTS

Abstract Background and Aims Hypervolemia is frequent in haemodialysis (HD) patients and is linked to inflammation, which may be further enhanced by endotoxemia. The impact of hypervolemia on immune cells i.e. a main monocytic defence mechanism - the NLRP3 inflammasome, has not been studied in detail. Therefore, we hypothesized that endotoxinemia putatively present in hyper- but normovolemic patients can trigger the activation of caspase-1 and thus increasing IL-1ß secretion. Method 45 HD patients were included in the observational study, measuring immune parameters (cell counts, caspase-1, oxidative stress, cytokine gene expression and serum analysis (IL-1ß) at two time points (directly after dialysis BS1) and three days later after the long dialysis free interval (BS2). Fluid status was evaluated by electrical bioimpedance vector analysis, defining hypervolemia (H) as &amp;gt;75 vector percentile. Data collected after the long dialysis interval is presented in this paper. Results Seventeen patients were classified as hyper-(H-HD), 19 as normovolemic (N-HD), 9 failed to meet the inclusion criteria. Monocytes were elevated and lymphocytes decreased by hypervolemia. Oxidative stress was slightly elevated in H-HD. However, neither caspase-1 frequency (H: 77.9±4.2% vs. 79.2±6.9%, p=0.916) nor caspase-1 expression (MFI, H: 42.8±13.3 vs. 43.6±17.1, p=0.880) were different. In vitro stimulation of PBMC’s by specific NLRP3 inflammasome stimulation significantly increased caspase activity in both cohorts to the same extent (89.9±22.6 vs. 88.0±23.3, p=0.900). As a result of it both IL-1ß mRNA expression (1.7±1.9 vs N: 1.1±0.8, 0.306) and serum IL-1ß (pg/ml) did not differ between the groups (H: 1.99±0.2 vs. N: 1.98±0.6, p&amp;lt;0.135). Conclusion The NLRP3 inflammasome is not activated thus refuting the hypothesis that endotoxemia is a main driver for inflammation in H-HD.

EBV-specific T cells for therapeutic use demonstrate differences in phenotype, transcriptional profile and function depending upon manufacturing process

Background & Aim Adoptive immunotherapy with Epstein-Barr virus (EBV)-specific T cells (VST) has proven effective for patients with refractory EBV-related malignancies. Two principal manufacturing approaches have been utilized to generate sufficient allogeneic T cells for therapy; sequential stimulation of donor PBMC with autologous EBV-positive lymphoblastic cell lines (LCLs), or in vitro expansion of interferon-gamma selected EBV-VST identified using EBV peptides. The Scottish National Blood Transfusion Service has a LCL-based EBV-VST bank which has been used to treat refractory PTLD patients for the last ten years on a Specials basis. This bank is being replaced by a next-generation bank based on EBV peptide selection and expansion in GMP-compliant closed-process manufacture. We have investigated the therapeutic material from both manufacturing processes and compared the phenotypic and functional characteristics of both to determine comparability. Methods, Results & Conclusion VST from both manufacturing processes were assessed for surface and intracellular markers by flow cytometry, which indicated that there were no significant differences in most parameters including CD3 content, CD4:CD8 ratio, and differentiation status. Peptide-generated products did demonstrate a significantly increased central memory compartment and mean IFNg expression. Further transcriptional analysis was undertaken using a custom TaqMAN low density array for relevant chemokine receptors, and indicated that EBV peptide-isolated VST had significantly increased levels of CCR7, CXCR4 and CXCR5, which correlate strongly with enhanced expansion and retention of central memory T cells. Transcriptional and phenotypic results were correlated with functional cytokine and migration assay to confirm responses. LCL-generated VST products showed significantly increased levels of CD38, indicating an accumulation of terminally differentiated functional T cells with reduced proliferative potential. The results from this study indicate that both EBV-VST manufacturing methods generate effective functional cells, but peptide-based VST demonstrate enhanced central memory cell retention which may predicate towards improved persistence in PTLD recipients.

Abstract P3-09-15: A phase 1b study of PVX-410 (PVX) vaccine plus durvalumab (DUR) as adjuvant therapy in HLA-A2+ early stage triple negative breast cancer (eTNBC) to assess safety and immune response

Abstract Background: eTNBC remains at high risk for recurrence despite modern (neo)adjuvant chemotherapy. Immunotherapy with checkpoint inhibition is active in early and metastatic TNBC, and vaccines may further induce host immune response. PVX-410 (PVX, OncoPep) is a novel tetra-peptide vaccine with 3 of the 4 antigens (XBP1 [2 splice variants] and CD138) commonly overexpressed in TNBC. Here we present results from a phase1b study evaluating safety, tolerability and immune response of PVX and durvalumab (DUR) as adjuvant therapy in eTNBC. Methods: Eligibility for this phase 1b multi-center, single arm study included: HLA-A2+ female patients (pts) with TNBC and tumor size at least 1 cm or node positive; treatment with at least 4 cycles of adjuvant or neoadjuvant chemotherapy; completion of all planned therapy 1-6 months prior to study entry. Pts with local-regional recurrence without evidence of distant metastases treated with curative were also eligible. Pts received 6 doses of 800ug PVX (subcutaneously) emulsified in Montanide and co-administered with Hiltonol (IM) every 2 weeks, and 2 doses of 1500mg DUR (IV) concurrent with the 4th and 6th vaccine treatments. The study included a 6 pt run-in to assess for dose limiting toxicity (DLT), followed by a 14 pt expansion. Blood samples were collected for immune response assessment via flow cytometry at week 0 (Baseline) and at weeks 6, 10, 14, and 24 post-treatment. The primary objective was safety and tolerability. The key secondary objective was PVX specific immune response assessment defined at week 14 as a 2-fold or greater change over baseline in the proportion of CD3+CD8+ T cells that expressed IFNγ and the proportion of CD3+CD8+ T cells positive for PVX tetramers following an in vitro stimulation of PBMC with PVX peptides and a flow cytometric determination. Other immune response assessments included the proportion of CD3+CD8+ T cells expressing TNFα, IL-2, and CD137 following the in vitro peptide stimulation. Results: Among 22 pts enrolled, median age was 48 yrs (range 34-68) and anatomical stage at diagnosis was: Stage 1 = 1 (4.5%), Stage 2 = 14 (64%), and Stage 3 = 7 (32%). Among 20 pts evaluable for week 14 immune response, all planned PVX doses were given, and 1 DUR dose was held due to toxicity (tox). No DLTs were observed in the 6 pt run-in. The most common adverse events (all grades) include: injection site reaction (96%), flu-like symptoms, fatigue (41% each), arthralgia, pruritis (36% each), ALT elevation (32%), myalgia (27%), pain, diarrhea, AST and amylase elevation (23% each). One pt had grade 3 diarrhea and 1 pt had grade 3 hyponatremia, AST and ALT elevation. There were no grade 4 or 5 events. Immune response assessment for the first 12 pts are available at the time of abstract submission. Comparing baseline to week 14, 10 of 12 patients demonstrated a PVX specific immune response (as defined above). Immune response persisted in all patient samples tested at 6 months. Additionally, most pts tested to date had increases over baseline in the proportions of CD3+CD8+ T cells that expressed TNFα, IL-2, and CD137 following the in vitro stimulation with PVX peptides. Final immune response assessment in all 20 evaluable patients will be updated for presentation. At the time of data cut off (4/29/19), with a median follow up of 15.4 months, 4 of 22 (18%) pts had a local (1) or metastatic (3) recurrence and 2 (9%) have died. Conclusions: PVX plus DUR is safe and tolerable in pts with eTNBC with no new unexpected toxicities identified. Immune response data demonstrate that PVX induces antigen-specific T cell expansion and activation in these pts as observed by increases in PVX tetramer, IFNγ, TNFα, IL-2 and CD137 positive T cells. These immune responses to PVX persisted for up to 6 months in most patients, indicating a prolonged immune response. Citation Format: Steven J Isakoff, Sylvia Adams, Hatem H Soliman, Nadine Tung, William T Barry, Jiani Hu, Lorenzo Trippa, Rachel Deering, Joanne Parker, Hannah Park, Elena F Brachtel, Leif W Ellisen, Mariano Severgnini, Doris Peterkin, Sara M Tolaney. A phase 1b study of PVX-410 (PVX) vaccine plus durvalumab (DUR) as adjuvant therapy in HLA-A2+ early stage triple negative breast cancer (eTNBC) to assess safety and immune response [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P3-09-15.

Autoimmune inner ear disease patient-associated 28-kDa proinflammatory IL-1β fragment results from caspase-7-mediated cleavage in vitro.

Interleukin-1β (IL-1β) is a key proinflammatory cytokine involved in the progression of many autoinflammatory and autoimmune diseases, including autoimmune inner ear disease (AIED). IL-1β inhibition has been shown to result in clinical hearing improvement in a small cohort of corticosteroid-resistant patients with AIED. Canonical processing of pro-IL-1β by caspase-1 generates an active 17-kDa fragment, capable of instigating a proinflammatory microenvironment. However, in response to LPS, PBMCs from patients with AIED uniquely express a 28-kDa IL-1β fragment, as compared with PBMCs from control subjects. We synthesized and compared the biologic activity of the 28-kDa fragment to the 17-kDa IL-1β product and the pro-IL-1 31-kDa protein. The 28-kDa IL-1β fragment induces IL-6, TNF-α, and CCL3 in PBMCs. Uniquely, only caspase-7 treatment showed a dose- and time-dependent increase in 28-kDa band generation. Mass spectrometry confirmed the putative caspase-7 cleavage site of pro-IL-1β, which was used to generate the 28-kDa fragment used for PBMC stimulation studies. Collectively, these results provide insight into the function of a poorly understood, processed 28-kDa form of IL-1β in patients with AIED that is uniquely generated by caspase-7 and is capable of activating further downstream proinflammatory cytokines. Further investigation may provide novel pharmacologic targets for the treatment of this rare disease.

The Safety and Efficacy of Targeted Virus Specific Cytotoxic T-Lymphocytes (VST) Manufactured By the IFN-g Cytokine Capture System (CCS) for the Treatment of Refractory Adenovirus (ADV), Cytomegalovirus (CMV), Epstein Barr Virus (EBV) and BK Virus (BKV) in Children, Adolescents and Young Adults (CAYA) after Allogenic Hematopoietic Stem Cell Transplantation (Allo-HSCT), Solid Organ Transplantation (SOT), or with Primary Immunodeficiency (PID) (IND#

Viral infection remains a major cause of morbidity and mortality after allo-HSCT (Bollard/Heslop <i>Blood</i> 2016). Antibiotics for treatment of viral infection in immunocompromised patients are limited in efficacy and associated with toxicity (Gerdemann <i>BBMT</i> 2004; Sili <i>Cytother</i> 2012). Increasing evidence suggests that use of VST for immunocompromised patients with viral infections can provide therapeutic benefit and improve OS (Bollard/Heslop <i>Blood</i> 2016; Sutrave <i>Cytother</i> 2017). Methods of VST production include ex-vivo expansion and direct selection (Gottlieb <i>Cytother</i> 2017). Ex-vivo expansion requires prolonged manufacturing time, is associated with exhaustion, and provides a limited donor pool. Direct selection results in rapid production and allows for expanded HLA matching. A multicenter consortium (VIRCTLC) was created to investigate the safety and efficacy of VST (Figure 1) manufactured by the IFN- CCS process automated on the CliniMACS® Prodigy device (Miltenyi Biotec) for immunocompromised patients with viral infection. To determine the safety and efficacy of VST therapy for immunocompromised CAYA patients with viral infection. CAYA patients after allo-HSCT, SOT or with PID with refractory ADV, CMV, EBV or BKV infections despite 2 weeks of appropriate anti-viral therapy, and/or resistance to antibiotics and/or intolerance to antibiotics were eligible. Donors were screened with viral specific antigen (PepTivator®) to predict successful VST manufacturing. Eligible donor PBMC were collected with non-mobilized apheresis. VST were isolated using the CliniMACS® Prodigy following specific stimulation of PBMC with specific viral MACS PepTivator® pools, generously provided by Miltenyi Biotec. Production of CD4+ and CD8+ VST was performed as previously described (Feucthinger <i>Blood</i> 2010). Target cell dose was 0.5 × 10⁴ CD3⁺/kg for HLA mismatched related donors and 2.5 × 10⁴ CD3⁺/kg for matched related donors. Based on response and safety, additional doses of VST were given every 2 weeks, for a maximum of 5 infusions. Thirteen patients were treated under IND 17449 and other single INDs. Median number of VST infusions was 2 (1-5). Mean±SEM CD3⁺ cell dose infused was 0.48±0.03 × 10⁴. There were 10 CR (PCR negative), 2 PR (PCR≥1 log decrease), and 1 too early to assess (ORR 100%, CRR 83%). Median time to maximal response was 24 days (range 3-111). No patient developed aGVHD, extensive cGVHD, infusion reaction or CRS associated with VST infusions. One patient experienced recurrence of aGVHD, possibly related to VST. (Table 1) Preliminary results of this pilot study demonstrate that VST are safe and efficacious in CAYA with refractory viral infections after alloHSCT, SOT or with PID. VST manufacturing utilizing the CliniMACS® Prodigy device is rapid (24 hours), reproducible and effective. Accrual is ongoing.

TNF-Producing Th1 Cells Are Selectively Expanded in Liver Infiltrates of Patients with Autoimmune Hepatitis.

Autoimmune hepatitis (AIH) is a chronic inflammatory liver disease that is believed to be driven by a CD4+ T cell response to liver Ags. However, the pathogenic function of CD4+ effector T cells in AIH is not fully understood. To characterize liver-infiltrating lymphocytes in AIH, we determined the cytokine production of infiltrating cells obtained from biopsy material by quantitative RT-PCR and flow cytometry. A cytokine quantitiative RT-PCR array of AIH specimens revealed that TNF was the most strongly upregulated cytokine, as compared with control livers. To confirm this finding, we determined the frequencies of TNF-producing CD4+ T cells in peripheral blood and in liver biopsy specimens in comparison with those of CD4+ T cells producing IFN-γ or IL-17. In AIH, TNF-producing CD4+ T cells were significantly expanded, both in blood and liver, whereas IL-17-producing CD4+ T cells were not. However, the majority of the TNF-producing CD4+ T cells in AIH also produced IFN-γ, suggesting that TNF producers might represent a pathogenic activation state of Th1 cells. Ag-specific stimulation of PBMC from AIH patients with the AIH-associated autoantigen SEPSECS resulted in significant TNF production only in patients manifesting SLA/LP autoantibodies targeting SEPSEC but not in healthy individuals who do not manifest this reactivity. Taken together, our findings indicated that TNF-producing CD4+ T cells are expanded in AIH, both in blood and in liver. TNF-producing CD4+ T cells in AIH seem to be aberrantly activated Th1 cells. Our findings provide a rationale for therapeutic efforts using TNF blockade in AIH.

In Vitro Evaluation of CMV Specific CD8+T Cells Function in CMV+ Colorectal Cancer Patients Compared to Healthy Controls.

The oncogenic role of human cytomegalovirus (HCMV) has been recently shown in different cancers like colorectal cancer (CRC). According to the recent immunotherapy approach to target the CMV-expressing tumor cells, we investigated the CMV peptide-stimulated CD8+T cells functions in CRC patients compared to healthy individuals.All sixteen patients and seven controls were CMV seropositive. Blood samples were obtained from patients without chemotherapy and radiotherapy before surgery. Cytotoxic CD8+ T cells were generated using 14-day culture of PBMCs in the presences of CMV peptide epitopes and rhIL-2. In addition to the supernatant evaluations for TNF-α and IFN-γ, the functionality of CD8+ T cells was examined by detecting CD107a and intracellular IFN-γ using flow cytometry. CMV DNA was detected in tissues by Real Time PCR. CMV DNA was found in 31% of tumor tissues, while it was not seen in the adjacent non-tumor tissues. There was a close association between CMV in tumor tissue and tumor grade. Surface expression of CD107a and intracellular IFN-γ in CMV-stimulated CD8+T cells and the level of IFN-γ production in patient and control groups increased significantly after culture. The number of functions increased in patients (p<0.05) and healthy individuals after culture. Followingstimulation, expressions of CD107a and intracellular IFN-γ were elevated in tumor CMV positive patients while the TNF-α secretion was decreased. In vitro stimulation of PBMC in the presence of CMV peptide epitopes and IL-2 can be an applicable method to generate cytotoxic CD8+ T cells in CRC patients for future T cell therapy.

TOLLIP deficiency is associated with increased resistance to Legionella pneumophila pneumonia.

Legionella pneumophila (Lp) is a flagellated, intracellular bacterium that can cause Legionnaires’ disease (LD). Lp activates multiple innate immune receptors, and TOLLIP dampens MyD88-dependent signaling and may influence susceptibility to LD. We evaluated the effect of TOLLIP on innate immunity, pneumonia severity, and LD susceptibility in mouse lungs and human populations. To accomplish this, we evaluated the effect of TOLLIP on lung-specific Lp control and immune response and associated a common functional TOLLIP variant with Lp-induced innate immune responses and LD susceptibility in humans. After aerosol Lp infection, Tollip−/− mice demonstrated significantly fewer bacterial CFU and increased cytokine responses from BAL fluid. Tollip−/− macrophages also suppressed intracellular Lp replication in a flagellin-independent manner. The presence of a previously characterized, functionally active SNP associated with decreased TOLLIP mRNA transcript in monocytes was associated with increased TNF and IL-6 secretion after Lp stimulation of PBMC ex vivo. This genotype was separately associated with decreased LD susceptibility (309 controls, 88 cases, p = 0.008, OR 0.36, 95% CI 0.16 – 0.76) in a candidate gene association study. These results suggest that TOLLIP decreases lung-specific TLR responses to increase LD susceptibility in human populations. Better understanding of TOLLIP may lead to novel immunomodulatory therapies.

Tissue Cytokine IL-33 Modulates the Cytotoxic CD8 T Lymphocyte Activity During Nutrient Deprivation by Regulation of Lineage-Specific Differentiation Programs.

IL-1 family member IL-33 exerts a variety of immune activating and regulating properties and has recently been proposed as a prognostic biomarker for cancer diseases, although its precise role in tumor immunity is unclear. Here we analyzed in vitro conditions influencing the function of IL-33 as an alarmin and a co-factor for the activity of cytotoxic CD8+ T cells in order to explain the widely discussed promiscuous behavior of IL-33 in vivo. Circulating IL-33 detected in the serum of healthy human volunteers was biologically inactive. Additionally, bioactivity of exogenous recombinant IL-33 was significantly reduced in plasma, suggesting local effects of IL-33, and inactivation in blood. Limited availability of nutrients in tissue causes necrosis and thus favors release of IL-33, which—as described before—leads to a locally high expression of the cytokine. The harsh conditions however influence T cell fitness and their responsiveness to stimuli. Nutrient deprivation and pharmacological inhibition of mTOR mediated a distinctive phenotype characterized by expression of IL-33 receptor ST2L on isolated CD8+ T cells, downregulation of CD8, a transitional CD45RAlowROlow phenotype and high expression of secondary lymphoid organ chemokine receptor CCR7. Under nutrient deprivation, IL-33 inhibited an IL-12 induced increase in granzyme B protein expression and increased expression of GATA3 and FOXP3 mRNA. IL-33 enhanced the TCR-dependent activation of CD8+ T cells and co-stimulated the IL-12/TCR-dependent expression of IFNγ. Respectively, GATA3 and FOXP3 mRNA were not regulated during TCR-dependent activation. TCR-dependent stimulation of PBMC, but not LPS, initiated mRNA expression of soluble IL-33 decoy receptor sST2, a control mechanism limiting IL-33 bioactivity to avoid uncontrolled inflammation. Our findings contribute to the understanding of the compartment-specific activity of IL-33. Furthermore, we newly describe conditions, which promote an IL-33-dependent induction of pro- or anti-inflammatory activity in CD8+ T cells during nutrient deprivation.

Abstract 539: A bispecific Fc-silenced IgG1 antibody (MCLA-145) requires PD-L1 binding to activate CD137

Abstract CD137 (4-1BB) is a transmembrane costimulatory receptor on T and NK cells that enhances adaptive immune responses and is a critical mediator of antitumor immunity. The development of CD137 targeted agents for cancer therapy has been hampered by on-target off-tumor toxicity in the case of agonist monospecific, bivalent mAbs or limited antitumor activity in the case of crosslinking mAbs. Here we have developed an Fc-silenced bispecific IgG1 antibody to CD137 and PD-L1 with monovalent binding specificity to each target. MCLA-145 drives transactivation of CD137 in the vicinity of cells expressing PD-L1, such as in the immunosuppressive tumor microenvironment. The degree of CD137 agonistic activity in T cells correlated with the expression level of PD-L1 on neighboring cells, as demonstrated in transactivation assays whereby reporter T cells were co-cultured with cells expressing different levels of PD-L1. PD-L1 expression as low as 6000 receptors per cell was sufficient to activate CD137 in neighboring T cells. In contrast, MCLA-145 blocked PD-1 signaling without requirement for CD137 binding in a PD-1/PD-L1 reporter assay. CD137 signaling was induced by MCLA-145 in multiple primary human immune cell assays including the mixed lymphocyte reaction, human PBMC, and whole blood SEB stimulation assays. MCLA-145 reversed T cell suppression mediated by M2 macrophages or Tregs, in vitro. In addition, MCLA-145 enhanced Ag-specific expansion and differentiation of human naïve CD8+ T cells in vitro. In vivo, MCLA-145 treatment resulted in significant tumor immune activation and antitumor responses in two separate humanized mouse tumor models. In one model, human T cells expressing NY-ESO specific TCR were adoptively transferred to mice bearing A549 tumors which expressed NY-ESO antigen and human PD-L1. MCLA-145 treatment at 5 mg/kg resulted in 54% tumor growth inhibition (TGI) as compared to T cell only treated mice. In the tumors of MCLA-145 treated mice, the percentage of NY-ESO specific CD8+ T cells were significantly increased compared to controls. In a second model, mice engrafted with human CD34+ cells were implanted with the breast tumor cell line MDA-MB-231. MCLA-145 at 0.5 mg/kg and 5 mg/kg induced significant tumor growth inhibition (55 and 57% respectively) as compared to vehicle control or Fc-silenced huIgG1 controls. Additionally, two out of nine animals in the 5 mg/kg MCLA-145-treated group had complete tumor regression. MCLA-145 increased the number of infiltrating CD8+ T cells, as well as the percentage of central memory CD8+ T cells. The cured animals were then re-challenged with MDA-MB-231 tumor cells, and tumors of previously cured mice were rejected as compared to no growth inhibition in treatment-naïve CD34+ NSG mice. In conclusion, these data support the clinical evaluation of MCLA-145 as a novel, PD-L1 dependent CD137 agonist immune therapy. Citation Format: Patrick Mayes, Paul Tacken, Steve Wang, Pieter-Fokko van Loo, Thomas Condamine, Hans van der Maaden, Eric Rovers, Steef Engels, Floris Fransen, Ashwini Kulkarni, Yao-bin Liu, Arpita Mondal, Leslie Hall, Soyeon Kim, Marina Martinez, Shaun O'Brien, Edmund Moon, Steven Albelda, Peggy Scherle, Gregory Hollis, Reid Huber, Mark Throsby, Cecile A. Geuijen. A bispecific Fc-silenced IgG1 antibody (MCLA-145) requires PD-L1 binding to activate CD137 [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 539.

Abstract 3244: Improved Fc-mediated effector functions by an anti-CTLA-4 multivalent Fc agent

Abstract Numerous therapeutic monoclonal antibodies (mAbs) rely on antibody-dependent cell cytotoxicity (ADCC), antibody-dependent cell phagocytosis (ADCP) and complement-dependent cytotoxicity (CDC) Fc effector functions to deplete target cells and achieve clinical efficacy. It has been previously shown that adding multiple Fc domains to Abs or afucosylating the Fc domain increases Fc effector functions. We have leveraged a proprietary Fc multimerization technology (SIF; selective immunomodulator of Fc receptors) to identify potential novel products designed to improve the immune system’s elimination of tumor and other pathogenic cells. These agents utilize the valency effect of Fc multimerization to increase the binding to Fc receptors and complement, enhancing immune-mediated cytotoxicity mechanisms. CTLA-4 is a clinically validated immune checkpoint inhibitor exemplified by the human IgG1 therapeutic mAb ipilimumab. CTLA-4 is induced upon activation on T cells and constitutively expressed on T regulatory cells (Tregs). Based on data from mouse models of cancer and clinical studies, the proposed mechanisms of action of anti-CTLA-4 mAbs, including ipilimumab, are to block the interaction of CTLA-4 with its ligands CD80 and CD86 resulting in T cell activation and to induce Fc-mediated ADCC of CTLA-4+ cells, mainly intratumoral Tregs. Therefore, we have produced an anti-CTLA-4 multivalent IgG1 Fc agent termed anti-CTLA-4 SIFbody with the purpose of enhancing binding to Fc gamma receptors (FcγRs) and complement and increasing Fc-mediated elimination of intratumoral Tregs. Data from avidity binding to low affinity FcγRs showed more than 100-fold increase with anti-CTLA-4 SIFbody as compared to ipilimumab. In in vitro functional assays using CTLA-4 transfected target cells and primary human effector cells, anti-CTLA-4 SIFbody showed more than 10-fold increase in potency in ADCC and more than 5-fold increase in ADCP as compared to ipilimumab. Anti-CTLA-4 SIFbody induced 80% cell lysis by CDC, whereas ipilimumab failed to show any activity. More importantly, we generated in vitro expanded Tregs with suppressive function and showed that anti-CTLA-4 SIFbody induced significant enhanced ADCC on these cells as compared to ipilimumab and to an afucosylated anti-CTLA-4 mAb. ADCP was also significantly increased on Tregs. Unexpectedly, Tregs were resistant to anti-CTLA-4 SIFbody induced CDC. Notably, the blocking activity of the SIFbody F(ab)s was not altered by the multivalent Fc structure as shown by similar CTLA-4/CD80 and CD86 blockade and induction of IL-2 production upon antigen stimulation of PBMC. In conclusion, these data demonstrate that our Fc multimerization technology applied to an anti-CTLA-4 mAb significantly improves Fc-dependent immune-mediated cytotoxicity and suggest that anti-CTLA-4 SIFbody may represent an optimized novel product to deplete intratumoral Tregs and enhance anti-tumor activity. Citation Format: Laura Rutitzky, Daniel F. Ortiz, Jonathan C. Lansing, Julia Brown, Joseph Paquette, Kevin Garofalo, Josephine D'Alessandro, Naveen Bhatnagar, Maurice Hains, Abhinav Gupta, Stan Lee, Radouane Zouaoui, Jason Wang, John Schaeck, Salvatore Marchese, Robin Meccariello, Nathaniel Washburn, Kimberly Holte, Carlos J. Bosques, Anthony M. Manning. Improved Fc-mediated effector functions by an anti-CTLA-4 multivalent Fc agent [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 3244.

Unusual Placement of an EBV Epitope into the Groove of the Ankylosing Spondylitis-Associated HLA-B27 Allele Allows CD8+ T Cell Activation.

The human leukocyte antigen HLA-B27 is a strong risk factor for Ankylosing Spondylitis (AS), an immune-mediated disorder affecting axial skeleton and sacroiliac joints. Additionally, evidence exists sustaining a strong protective role for HLA-B27 in viral infections. These two aspects could stem from common molecular mechanisms. Recently, we have found that the HLA-B*2705 presents an EBV epitope (pEBNA3A-RPPIFIRRL), lacking the canonical B27 binding motif but known as immunodominant in the HLA-B7 context of presentation. Notably, 69% of B*2705 carriers, mostly patients with AS, possess B*2705-restricted, pEBNA3A-specific CD8+ T cells. Contrarily, the non-AS-associated B*2709 allele, distinguished from the B*2705 by the single His116Asp polymorphism, is unable to display this peptide and, accordingly, B*2709 healthy subjects do not unleash specific T cell responses. Herein, we investigated whether the reactivity towards pEBNA3A could be a side effect of the recognition of the natural longer peptide (pKEBNA3A) having the classical B27 consensus (KRPPIFIRRL). The stimulation of PBMC from B*2705 positive patients with AS in parallel with both pEBNA3A and pKEBNA3A did not allow to reach an unambiguous conclusion since the differences in the magnitude of the response measured as percentage of IFNγ-producing CD8+ T cells were not statistically significant. Interestingly, computational analysis suggested a structural shift of pEBNA3A as well as of pKEBNA3A into the B27 grooves, leaving the A pocket partially unfilled. To our knowledge this is the first report of a viral peptide: HLA-B27 complex recognized by TCRs in spite of a partially empty groove. This implies a rethinking of the actual B27 immunopeptidome crucial for viral immune-surveillance and autoimmunity.

T cells responses during resolving Hepacivirus A infection, a surrogate animal model for human Hepatitis C

Abstract Hepacivirus A (HcV A) is a hepatotropic equine virus and the closest known relative of hepatitis C virus (HCV). HcV A is also highly similar to HCV in its replication kinetics and severity of associated acute hepatitis, but, unlike HCV, HcV A is typically cleared by the host before causing significant disease. As such, HcV A is considered a tenable model for defining the correlates of resolving hepaciviral infection and vaccine development. The pathogenesis of acute HcV A-hepatitis and the mechanism(s) that lead to viral clearance are unknown. In this study we aimed to characterize T cell responses in horses (n=2) following inoculation with plasma containing HcV A. Viremia and hepatic injury were monitored by RT-PCR and plasma liver enzyme activity [gamma glutamyl transferase (GGT) and sorbitol dehydrogenase (SDH)], respectively. HcV A-specific T-cell responses were evaluated every two weeks by interferon gamma Elispot following ex-vivo PBMC stimulation with peptides pools spanning the HcV A polyprotein. Liver biopsies were collected prior to infection, at the peak of liver enzyme activity, and following viral clearance to quantify CD4, CD8 and proinflammatory cytokine mRNA levels, and characterize CD3+ T cell infiltration by immunohistochemistry (IHC). No distinct HcV A specific peripheral T cell response was detected during infection, but when liver enzymes peaked, IHC revealed a mean 15 fold increase in CD3+ T cell infiltration. CD4, CD8 and proinflammatory cytokine mRNA levels were also significantly increased at the same time point. In conclusion, our findings suggest that acute HcV A-hepatitis is mediated by CD3+ T cells and that HcV A-specific T cell response are likely sequestered in the liver.

I-106 Early treatment initiation in children with vertical HIV infection influences HIV specific immune responses

Background: Despite successes in prevention of mother to child HIV transmission (MTCT), issues related to early diagnosis, ART initiation and adherence play a role in the disease course of HIV infected children. Early ART initiation in HIV infected infants leads to reduced size of HIV reservoirs and curtails clinical progression but role of the immune system in this process is not well understood. With early initiation of ART, HIV infected infants preserve their CD4+ T cells and in some instances remain seronegative for HIV due to limited exposure of B cells to HIV antigen. We are performing immunologic assessments in the HIV Exposed Pediatric Patients (HEPP) Project and discuss a group of children from Rome, Italy. The goal of the present study is to evaluate HIV-specific immunity of vertically HIV infected children in relation to timing of ART initiation. Methods: The study group consists of older vertically infected HIV+ children and adolescents who initiated ART at different ages after birth and have maintained durable viral control (plasma HIV-RNA &lt;50 cp/mL). Immunologic studies were performed in 27 children (at ages 2.1–15.7 yrs) of whom 22 had started ART early, at age &lt;6 mo [early treated (ET)] and 5 who started ART later, at age &gt;2 yrs [late treated (LT)] with ET experiencing longer duration of ART (P &lt; 0.05) at the time of study. ET children were also evaluated by HIV serology (Abbott Architect HIV Ag/Ab Combo Assay) to establish if they were seropositive (SP) or seronegative (SN) to HIV. Flow cytometry was performed using BD LSRFortessa for T cells and BD FACS Aria II for B cells. HIV-gag specific CD4+ and CD8+ T cells were investigated by flow cytometry in cryopreserved PBMC for the induction of intracellular cytokines (IL-2, IFN-g, TNF, IL-21) in CD40L+ CD4+ T cells and intracellular cytokines (IL-2, IFN-g, TNF) as well as Perforin, Granzyme B and CD107a in CD69+ CD8+ T cells following 12 hours stimulation with HIV gag PTE peptides. In addition, Env-specific CD4+ T cells were evaluated following 12 hours stimulation of PBMC with Env peptides. IgD+ and IgM+ B cell subsets were determined in gp140+ B cells. Moreover, env-specific and non-specific B cells were sorted to perform transcriptomic analysis with Fluidigm (Biomark). Differences between groups were determined by unpaired or Mann-Whitney t tests if the data were normally distributed or not, respectively. Results: In this group of HIV+ children, frequencies of HIV gag-specific CD4+ T cells and CD8+ T cells were similar between ET and LT but differed qualitatively. Gag stimulated CD4+ T cells of ET exhibited higher proportions of IL-21 and IFNg with enrichment of polyfunctional T cells defined by the simultaneous presence of 3 or more cytokines. Gag stimulated CD8+ T cells of ET exhibited higher frequencies of cytotoxic cells (CD107a+Perf+GrzB+). In ET, serologic evaluation of HIV Ab revealed 6/22 (27.3%) to be SN. The gp140-specific CD4+ T cell frequencies were similar in SN and SP but intracellular IL-21 expression was higher in SP. Frequencies of gp140+ CD19+ B cells were similar between SN and SP, but HIV-specific CD27+ memory B cells were predominantly IgM+ in SN while in SP, the gp140+CD27+ memory B cells were enriched in IgD-cells. Gene expression signatures in gp140+IgM+ cells revealed upregulation of genes involved in both plasma cell differentiation (PRDM1) and GC reaction (BCL6) only in SP after in-vitro stimulation. Conclusions: Time of ART initiation after birth has a long-term impact on the quality of the host HIV-specific immune responses in vertically infected HIV+ children with durable virus suppression. Earlier ART initiation is associated with preserved HIV gag-specific CD4+ and CD8+ T cells. Moreover, even in those who become seronegative for HIV, the HIV Env-specific CD4+ T cells and B cells persist, the latter demonstrating a blunted differentiation with enrichment of IgM+ B cells. ET children with consistent viral control are prime candidates for therapeutic approaches aimed at permanent remission of HIV. These studies are supported by ViiV Health Care grant to EPIICAL and NIH grant AI127347 to SP.

Cancer vaccines: designing artificial synthetic long peptides to improve presentation of class I and class II T cell epitopes by dendritic cells

ABSTRACTThere is now a consensus that efficient peptide vaccination against cancer requires that peptides should (i) be exclusively presented by professional APC and (ii) stimulate both CD4 and CD8-specific T cell responses. To this aim, in recent trials, patients were vaccinated with pools of synthetic long peptides (SLP) (15–30 aa long) composed of a potential class I epitope(s) elongated at both ends with native antigen sequences to also provide a potential class II epitope(s). Using MELOE-1 as a model antigen, we present an alternative strategy consisting in linking selected class I and class II epitopes with an artificial cathepsin-sensitive linker to improve epitope processing and presentation by DC. We provide evidence that some linker sequences used in our artificial SLPs (aSLPs) could increase up to 100-fold the cross-presentation of class I epitopes to CD8-specific T cell clones when compared to cross-presentation of the corresponding native long peptide. Presentation of class II epitopes were only slightly increased. We confirmed this increased cross-presentation after in vitro stimulation of PBMC from healthy donors with aSLP and assessment of CD8-specific responses and also in vivo following aSLP vaccination of HLA*A0201/HLA-DRB0101 transgenic mice. Finally, we provide some evidence that vaccination with aSLP could inhibit the growth of transplanted tumors in mice. Our data thus support the use of such aSLPs in future cancer vaccination trials to improve anti-tumor CD8 T cell responses and therapeutic efficacy.