Specific Cytotoxic T Lymphocytes Research Articles

PD-1 knockout on cytotoxic primary murine CD8+ T cells improves their motility in retrovirus infected mice.

Cytotoxic T lymphocyte (CTL) motility is an important feature of effective CTL responses and is impaired when CTLs become exhausted, e.g. during chronic retroviral infections. A prominent T cell exhaustion marker is programmed cell death protein 1 (PD-1) and antibodies against the interaction of PD-1 and PD-ligand 1 (PD-L1) are known to improve CTL functions. However, antibody blockade affects all PD-1/PD-L1-expressing cell types, thus, the observed effects cannot be attributed selectively to CTLs. To overcome this problem, we performed CRISPR/Cas9 based knockout of the PD-1 coding gene PDCD1 in naïve Friend Retrovirus (FV)-specific CTLs. We transferred 1,000 of these cells into mice where they proliferated upon FV-infection. Using intravital two-photon microscopy we visualized CTL motility in the bone marrow and evaluated cytotoxic molecule expression by flow cytometry. Knockout of PDCD1 improved the CTL motility at 14 days post infection and enhanced the expression of cytotoxicity markers. Our data show the potential of genetic tuning of naive antiviral CTLs and might be relevant for future designs of improved T cell-mediated therapies.

Co-infection dynamics between HIV-HTLV-I disease with the effects of Cytotoxic T-lymphocytes, saturated incidence rate and study of optimal control

The spreading of HIV or HTLV-I among the cells has received the great attention in recent modelling study to explore the virus infection dynamics. The co-infection of HIV and HTLV-I with the effect of Cytotoxic T-lymphocytes (CTLs) immune response is also important from epidemiological point of view. To identify the co-infection scenario of HIV and HTLV-I with the CTLs effect we proposed in this paper a six compartmental ODE-model with uninfected, HIV-infected, HTLV-I infected CD4+T cells and free HIV virus particles with HIV specific CTLs and HTLV-I specific CTLs. The rates of infection of the cases are considered here saturated type and proliferation rate of uninfected and HIV infected CD4+ T-cells are of logistic terms. To establish the well-posedness of the model we have shown that the solution of the proposed model is non-negative and bounded. We obtain the basic reproduction number which is the maximum of the HIV-related reproduction and the HTLV-I related reproduction number. Along with the disease free equilibrium point the system contains other seven endemic equilibrium points containing infection by single disease or both. Analytically, we establish the local and global stability conditions of the equilibrium points and also we establish that the system experiences transcritical bifurcation by the generation of only HIV or HTLV-I infected endemic equilibrium point. Using numerical simulations, we validate the theoretical results and found two infection paths, one initiating with HIV and other with HTLV-I, both cases ultimately become co-infected. Finally, using the optimal control analysis we found the optimal policy for treatment using AVR, RTI & PI for HIV or AZT for HTLV-I control and lastly concluded by some recommendations.

Tabelecleucel for EBV+ PTLD after allogeneic HCT or SOT in a multicenter expanded access protocol

AbstractPatients with Epstein-Barr virus (EBV)–positive posttransplant lymphoproliferative disease (EBV+ PTLD) in whom initial treatment fails have few options and historically low median overall survival (OS) of 0.7 months after allogeneic hematopoietic cell transplant (HCT) and 4.1 months after solid organ transplant (SOT). Tabelecleucel is an off-the-shelf, allogeneic EBV-specific cytotoxic T-lymphocyte immunotherapy for EBV+ PTLD. Previous single-center experience showed responses in patients with EBV+ PTLD after HCT or SOT. We now report outcomes from a multicenter expanded access protocol in HCT (n = 14) and SOT (n = 12) recipients treated with tabelecleucel for EBV+ PTLD that was relapsed/refractory (R/R) to rituximab with/without chemotherapy. The investigator-assessed objective response rate was 65.4% overall (including 38.5% with a complete and 26.9% with a partial response), 50.0% in HCT, and 83.3% in SOT. The estimated 1- and 2-year OS rates were both 70.0% (95% confidence interval [CI], 46.5-84.7) overall, both 61.5% (95% CI, 30.8-81.8) in HCT, and both 81.5% (95% CI, 43.5-95.1) in SOT (median follow-up: 8.2, 2.8, and 22.5 months, respectively). Patients responding to tabelecleucel had higher 1- and 2-year OS rates (94.1%) than nonresponders (0%). Treatment was well tolerated, with no reports of tumor flare, cytokine release syndrome, or rejection of marrow and SOT. Results demonstrate clinically meaningful outcomes across a broad population treated with tabelecleucel, indicating a potentially transformative and accessible treatment advance for R/R EBV+ PTLD after HCT or SOT. This trial was registered at www.ClinicalTrials.gov as #NCT02822495.

Identification of a new HLA-A*0201-restricted cytotoxic T lymphocyte epitope from TC2N.

Identification of cytotoxic T lymphocyte (CTL) epitopes from tumor related antigens is a promising approach for malignant tumor immunotherapy. TC2N, a recently identified tumor associated antigen from human glioblastoma, is regarded as a promising target of tumor-specific immunotherapy. As one of the most widely used histocompatibility molecules in Chinese is HLA-A*0201, we were able to identify the TC2N peptides that are provided by this molecular type. A panel of antigenic peptides produced from TC2N were predicted by using a computer tool. The binding affinities of three peptides with the highest predicted score to the HLA-A*0201 molecule were evaluated after synthesis. In vitro and in vivo stimulation of the main T-cell response against the predicted peptides. The results demonstrated that TC2N (152-160) was able to release IFN-γ and lyse U251 cells in vitro as well as in vivo by eliciting peptide-specific CTLs. Our results indicated that peptide TC2N (152-160) (RLYGSVCDL) was a novel HLA-A2.1-restricted CTL epitope capable of inducing TC2N specific CTLs in vitro. As TC2N might qualify as a viable target for immunotherapeutic approaches for patients with GBM, we speculated that the newly identified epitope RLYGSVCDL would be of potential use in peptide-based, cancer-specific immunotherapy against GBM.

Adenoviral Cellular Therapy with Adenovirus (ADV)-Specific Cytotoxic T-Lymphocytes (CTLs) for Resistant Infections in Children, Adolescent, and Young Adult (CAYA) Patients Following Allogeneic Hematopoietic Stem Cell Transplantation (AlloHSCT): A Promising Approach

Adenoviral Cellular Therapy with Adenovirus (ADV)-Specific Cytotoxic T-Lymphocytes (CTLs) for Resistant Infections in Children, Adolescent, and Young Adult (CAYA) Patients Following Allogeneic Hematopoietic Stem Cell Transplantation (AlloHSCT): A Promising Approach

In vivo assembly of epitope-coated biopolymer particles that induce anti-tumor responses

There is an unmet need for antigen delivery systems that elicit efficient T cell priming to prevent infectious diseases or for treatment of cancers. Here, we explored the immunogenic potential of biologically assembled biopolymer particles (BPs) that have been bioengineered to display the antigenic MHC I and MHC II epitopes of model antigen ovalbumin (OVA). Purified dendritic cells (DCs) captured BP-OVA and presented the associated antigenic epitopes to CD4+ T cells and CD8+ T cells. Vaccination with BP-OVA in the absence of adjuvant elicited antigen presentation to OVA-specific CD8+ and CD4+ T cells and cross-primed effective cytotoxic T lymphocyte (CTL) killers. BP-OVA induction of CTL killing did not require CD4+ T cell help, with active CTLs generated in BP-OVA vaccinated I-Ab−/− and CD40−/− mice. In contrast, IL-15 and type I IFN were required, with abrogated CTL activity in vaccinated IL-15−/− and IFNAR1−/− mice. cDC1 and/or CD103+ DCs were not essential for BP-OVA specific CTL with immunization eliciting responses in Batf3−/− mice. Poly I:C, but not LPS or CpG, co-administered as an adjuvant with BP-OVA boosted CTL responses. Finally, vaccination with BP-OVA protected against B16-OVA melanoma and Eμ-myc-GFP-OVA lymphoma inoculation. In summary, we have demonstrated that epitope-displaying BPs represent an antigen delivery platform exhibiting a unique mechanism to effectively engage T cell immune responses.

Combined treatment with allogeneic Epstein–Barr- and human polyomavirus 1 specific T-cells in progressive multifocal leukoencephalopathy and EBV infection: a case report

Opportunistic viral infections in individuals with severe immunodeficiency can lead to fatal conditions such as progressive multifocal leukoencephalopathy (PML), for which treatment options are limited. These infections pose significant risks, especially when co-infections with other viruses occur. We describe a combined therapy approach using directly isolated allogeneic Human Polyomavirus 1 (also known as BKV) and Epstein–Barr virus (EBV) specific cytotoxic T-cells for the treatment of PML in conjunction with identified EBV in the cerebrospinal fluid (CSF) of a male patient infected with human immunodeficiency virus (HIV). A 53-year-old HIV-positive male, recently diagnosed with PML, presented with rapidly worsening symptoms, including ataxia, tetraparesis, dysarthria, and dysphagia, leading to respiratory failure. The patient developed PML even after commencing highly active antiretroviral therapy (HAART) 3 months prior. Brain magnetic resonance imaging (MRI) revealed multifocal demyelination lesions involving the posterior fossa and right thalamus suggestive of PML. In addition to the detection of human polyomavirus 2 (also known as JCV), analysis of CSF showed positive results for EBV deoxyribonucleic acid (DNA). His neurological condition markedly deteriorated over the following 2 months. Based on MRI, there was no evidence of Immune Reconstitution Inflammatory Syndrome contributing to this decline. The patient did not have endogenous virus-specific T-cells. We initiated an allogeneic, partially human leukocyte antigen-matched transfer of EBV and utilizing the cross-reactivity between BKV and JCV–BKV specific T-cells. This intervention led to notable neurological improvement and partial resolution of the MRI lesions within 6 weeks. Our case of a patient with acquired immune deficiency syndrome demonstrates that PML and concurrent EBV co-infection can still occur despite undergoing HAART treatment. This innovative experimental therapy, involving a combination of virus-specific T-cells, was demonstrated to be an effective treatment option in this patient.

GPC3 Promotes Lung Squamous Cell Carcinoma Progression and HLA-A2-Restricted GPC3 Antigenic Peptide-Modified Dendritic Cell-Induced Cytotoxic T Lymphocytes to Kill Lung Squamous Cell Carcinoma Cells

Lung squamous cell carcinoma (LUSC) is associated with poor clinical prognosis and lacks available targeted agents. GPC3 is upregulated in LUSC. Our study aimed to explore the roles of GPC3 in LUSC and the antitumor effects of HLA-A2-restricted GPC3 antigenic peptide-sensitized dendritic cell (DC)-induced cytotoxic T lymphocytes (CTLs) on LUSC. LUSC cells with GPC3 knockdown and overexpression were built using lentivirus packaging, and cell viability, clone formation, apoptosis, cycle, migration, and invasion were determined. Western blotting was used to detect the expression of cell cycle-related proteins and PI3K-AKT pathway-associated proteins. Subsequently, HLA-A2-restricted GPC3 antigenic peptides were predicted and synthesized by bioinformatic databases, and DCs were induced and cultured in vitro. Finally, HLA-A2-restricted GPC3 antigenic peptide-modified DCs were co-cultured with T cells to generate specific CTLs, and the killing effects of different CTLs on LUSC cells were studied. A series of cell function experiments showed that GPC3 overexpression promoted the proliferation, migration, and invasion of LUSC cells, inhibited their apoptosis, increased the number of cells in S phase, and reduced the cells in G2/M phase. GPC3 knockdown downregulated cyclin A, c-Myc, and PI3K, upregulated E2F1, and decreased the pAKT/AKT level. Three HLA-A2-restricted GPC3 antigenic peptides were synthesized, with GPC3522-530 FLAELAYDL and GPC3102-110 FLIIQNAAV antigenic peptide-modified DCs inducing CTL production, and exhibiting strong targeted killing ability in LUSC cells at 80 : 1 multiplicity of infection. GPC3 may advance the onset and progression of LUSC, and GPC3522-530 FLAELAYDL and GPC3102-110 FLIIQNAAV antigenic peptide-loaded DC-induced CTLs have a superior killing ability against LUSC cells.

Treatment of Refractory Adenovirus (ADV) Infections Using Adv-Specific Cytotoxic T-Lymphocytes (CTLs) in Children, Adolescents and Young Adults (CAYA) Post Allogeneic Hematopoietic Stem Cell Transplantation (AlloHSCT), Solid Organ Transplantation (SOT), or with Primary Immunodeficiency (PID)

Background: AlloHSCT induces long-term, cure in CAYA with malignant and non-malignant disorders, including those with primary immunodeficiencies (PID). However, post-transplant complications secondary to immunosuppression and slow or delayed immune reconstitution remains a cause of morbidity and mortality amongst AlloHSCT recipients (Satwani/Cairo, BBMT, 2009). Major complications include refractory viral infections, most importantly adenovirus (ADV), which is secondary to absent or decreased adenovirus specific T-cell immunity which occurs due to delayed immune reconstitution and/or continued immunosuppression (George/Cairo, BJH, 2012). The use of virus-specific (vs) CTLs in immunocompromised patients offer an opportunity to restore temporary virus specific immunity. From the Viral Cytotoxic T-Lymphocyte Consortium (VIRCTLC), we present safety and efficacy results on immune compromised, AlloHSCT, or SOT patients treated with familial ADV specific CTLs. Objective: Determine the safety and efficacy of familial ADV specific CTLs for the treatment of refractory systemic ADV infections. Design/Methods: Patient were deemed eligible if they had a history of AlloHSCT, SOT, or PID and had evidence of ADV infections refractory to anti-viral agents. Refractory ADV infection was defined by increasing serum RT-PCR DNA (by 1 log) after 7 days or persistent quantitative RT-PCR DNA copies after 14 days of appropriate anti-viral therapy, and/or known resistance/intolerance to anti-viral agents. Related donors were required to be matched ≥3 HLA (A, B, or DRB1) loci and have an adequate T-cell response to viral specific MACS ADV PepTivators®. We chose a direct selection method to facilitate more rapid generation of cellular products in a point of care setting (and with 14 hrs eligibility determination). Peripheral blood mononuclear cells were collected from eligible family donors using non-mobilized apheresis. vsCTLs were isolated using the CliniMACS ® Prodigy following stimulation with viral specific MACS® GMP PepTivator® AdV5 Hexon, generously provided by Miltenyi Biotec®. ADV specific CTLs were enriched using a Cytokine Capture System (CCS). CD4+ and CD8+ vsCTLs were quantified by flow cytometry (Feuchtinger et al, Blood, 2010). The target cell dose was 0.5x10 4 CD3+ cells/kg (recipient weight) for haploidentical related donors and 2.5x10 4 CD3+ cells/kg for matched related donors. Repeated doses were permitted every 2 weeks to a maximum of doses in the absence of a complete response (CR) and adverse events. The following were used to define response: CR - undetected ADV PCR, partial response (PR) - at least one log decrease from baseline, progressive disease (PD) - at least one log increase from baseline, and patients with stable disease. Results: Fifteen patients were enrolled: 10 females and 5 males; aged 1-19 years. All patients were post-AlloHSCT. All vsCTL donors were haploidentical: 8 were the original HSCT donors and 7 were third party donors (5 maternal, 2 paternal). The mean number of ADV CTL infusions was 3.7 (range: 1-16). One patient with underlying aplastic anemia had progressive disease complicated by increased adenoviral load despite 5 infusions of vsCTLs. Patient received FDA approval for additional CTLs, totaling 16 doses. Thirteen patients achieved CR as defined and 2 achieved PR. The overall response (OR) was 100% and the CR was 88%. The average time to OR was 33 days (range 6-112 days) and time to CR was 34 days (range 6-112 days). Day 100 and 365 overall survival (OS) post-HSCT was 86.1% (CI95: 49.4-95.7) and 70.5% (CI95: 38.9-87.8), respectively (Figure 1). For patients who achieved CR, the Day 100 OS post AlloHSCT was 83.9% (CI95: 55.0-94.3) and Day 365 post AlloHSCT was 74.6% (CI95: 39.8-91.1). Day 100 and 365 probability of ADV related mortality was 0% (Figure 2). One patient developed acute grade 2 skin GVHD possibly related to infusion, which resolved. Conclusion: Our preliminary data demonstrate that haploidentical ADV specific CTLs manufactured by direct selections using the CCS with the CliniMACS® Prodigy and ADV specific peptivators are safe, well-tolerated, and efficacious in patients with refractory/persistent ADV infections after AlloHSCT. Manufacturing is rapid, reproducible, and effective. Accrual is ongoing. This research is supported by FDA RO1006301A1.

Mathematical Modeling Reveals Mechanisms of Cancer-Immune Interactions Underlying Hepatocellular Carcinoma Development

Understanding the relationship between hepatocellular carcinoma (HCC) and immunity is crucial for HCC immunotherapy. However, the existing research has solely focused on a novel population of primary tumor-induced non-leukocytes called Ter-cells and their circulating components in distant organs, neglecting the examination of immunity’s impact on cancer. In order to thoroughly examine the dynamics of Ter cells, HCC, and the known regulatory elements in the immunological milieu, we used a mathematical model in the form of a system of differential equations in this work. According to simulation studies, tumor cells cannot be completely eliminated by either the effective killing of HCC by cytotoxic T lymphocytes (CTL) or the inhibition of tumor cell proliferation. Nonetheless, continuous CTL activation and TGF-β-induced differentiation of CTL facilitated a transition from a high steady-state of HCC quantity to an unstable state, followed by a low state of HCC quantity, aligning with the three phases of the cancer immunoediting concept (escape, equilibrium, and elimination). Our survival study revealed that the ratio of CTL proliferation to CTL killing and relative TGF-β-induced differentiation of CTL have a significant impact on cancer-free survival. Sensitivity and bifurcation analysis of these parameters demonstrated that the rate of CTL proliferation, as well as the number of HCCs when the production rate reaches half of one, strongly affects the number of HCCs. Our findings highlight the critical role of immune system activation in cancer therapy and its potential impact on HCC treatment.

Single-cell transcriptome analysis and protein profiling reveal broad immune system activation in IgG4-related disease.

IgG4-related disease (IgG4-RD) is a systemic autoimmune disease with unclear pathogenesis. We performed single-cell RNA-seq and surface proteome analyses on 61,379 PBMCs from 9 treatment-naive IgG4-RD patients and 7 age- and sex-matched healthy controls. Integrative analyses were performed for altered gene expression in IgG4-RD, and flow cytometry and immunofluorescence were used for validation. We observed expansion of plasmablasts with enhanced protein processing and activation, which correlated with the number of involved organs in IgG4-RD. Increased proportions of CD4+ cytotoxic T lymphocytes (CTLs), CD8+ CTLs-GNLY (granulysin), and γδT cells with enhanced chemotaxis and cytotoxicity but with suppressed inhibitory receptors characterize IgG4-RD. Prominent infiltration of lymphocytes with distinct compositions were found in different organs of IgG4-RD patients. Transcription factors (TFs), including PRDM1/XBP1 and RUNX3, were upregulated in IgG4-RD, promoting the differentiation of plasmablasts and CTLs, respectively. Monocytes in IgG4-RD have stronger expression of genes related to cell adhesion and chemotaxis, which may give rise to profibrotic macrophages in lesions. The gene activation pattern in peripheral immune cells indicated activation of multiple interaction pathways between cell types, in part through chemokines or growth factors and their receptors. Specific upregulation of TFs and expansion of plasmablasts and CTLs may be involved in the pathogenesis of IgG4-RD, and each of these populations are candidate targets for therapeutic interventions in this disease.

Abstract 2995: Cross-presented serine proteases as immunotherapy targets for lung cancer and osteosarcoma

Abstract Background: Immunotherapy is the standard of care for many solid tumor malignancies. However, due to a paucity of known tumor antigens in non-small cell lung cancer (NSCLC) and osteosarcoma (OS), identification of novel immunogenic antigens is needed. Our group has experience with neutrophil serine proteases (NSP) derived from the azurophilic granules of polymorphonuclear leukocytes (PMN), specifically, cathepsin (CG), proteinase 3 (P3), and neutrophil elastase (NE). We have identified two HLA-A2 (HLA-A*0201)-restricted nonameric peptides: CG1 (FLLPTGAEA), derived from CG, and PR1 (VLQELNVTV), derived from NE and P3. We have previously shown that CG1 and PR1 are effective targets in hematological malignancies and that PR1 can be a target for some solid tumor malignancies. Here, we extend our findings to demonstrate that CG1 and PR1 can be immunotherapeutic targets for NSCLC and OS. Methods: Healthy donor PMN were isolated from buffy coats using double Ficoll gradient centrifugation. To evaluate uptake, malignant cells were pulsed with PMN lysates, harvested, permeabilized, and stained with anti-CG, anti-NE or anti-P3. To evaluate cross-presentation, cells were cultured with PMN lysates, harvested and stained with CG1 TCR-like antibody, or PR1 TCR-like antibody (8F4). Flow cytometry was performed using the Fortessa X-20 Cell Analyzer. CG1- or PR1-specific cytotoxic T-lymphocyte (CTL) were expanded from healthy donor peripheral blood mononuclear cells. Standard cytotoxicity assays were used to evaluate the lysis of target cells co-cultured with CG1- or PR1-specific CTL. Results: We have previously demonstrated that NE and P3 are taken up and cross-presented by NSCLC and become targets for PR1-CTL, thus in this study, we investigate if NSCLC cells take up CG, and whether OS cells take up NE and P3, resulting in enhanced cytotoxicity by peptide-specific CTLs. We first employed The Cancer Cell Line Encyclopedia to show the absence of NSP’s in NSCLC and OS cell lines. RT-PCR confirmed lack of endogenous CG mRNA expression in NSCLC and OS, and lack of NE and P3 in OS cell lines. Next, we analyzed antigen cross-presentation and showed that NSCLC cells take up and cross-present CG, leading a 13.3-fold increase in CG1-CTL killing of NSCLC cells pulsed with CG versus non-pulsed cells. In addition, OS cells take up and cross-present NE and P3, with a 13.1-fold increase in PR1-CTL killing of OS cells that were pulsed with NE and P3 vs. non-pulsed cells. Conclusion: Our data define two novel HLA-A2 restricted peptides that can be targeted in NSCLC and OS. Specifically, CG1 and PR1 are presented on the surfaces of NSCLC and OS, increasing their susceptibility to killing by CG1 and PR1 specific CTL. Our work represents a novel link between innate and adaptive immune responses in solid tumors and identifies potential antigen targets for CTL, peptide vaccines, and TCR-like antibodies in the setting of NSCLC and OS. Citation Format: Amber Gibson, Pariya Sukhumalchandra, Celine Kerros, Na Qiao, Anne Philips, Mao Zhang, Sami Alkoutami, Scott Semelsberger, Yoshimi Lu, Lauren Byers, Ze Tian, Chunhua Shi, Jun Yan, Dongxing Zha, Alejandro Marinos, Anne Sergeeva, Hong He, Lisa St. John, Jeffrey J. Molldrem, Gheath Alatrash. Cross-presented serine proteases as immunotherapy targets for lung cancer and osteosarcoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2995.

Altered HLA-A2-restricted TP53 epitope induces specific CTL cytotoxicity against hepatocellular carcinoma.

High-frequency mutation of the TP53 tumor suppressor gene is observed in multiple human cancers, which promotes cancer progression. However, the mutated gene-encoded protein may serve as a tumor antigen to elicit tumor-specific immune responses. In this study, we detected widespread expression of shared TP53-Y220C neoantigen in hepatocellular carcinoma with low affinity and low stability of binding to HLA-A0201 molecules. We substituted the amino acid sequences VVPCEPPEV with VLPCEPPEV in the TP53-Y220C neoantigen to yield a TP53-Y220C (L2) neoantigen. This altered neoantigen was found to increase affinity and stability and induce more cytotoxic T lymphocytes (CTLs), indicating improvements in immunogenicity. In vitro assays showed the cytotoxicity of CTLs stimulated by both TP53-Y220C and TP53-Y220C (L2) neoantigens against multiple HLA-A0201-positive cancer cells expressing TP53-Y220C neoantigens; however, the TP53-Y220C (L2) neoantigen showed higher cytotoxicity than the TP53-Y220C neoantigen against cancer cells. More importantly, in vivo assays demonstrated greater inhibition of hepatocellular carcinoma cell proliferation by TP53-Y220C (L2) neoantigen-specific CTLs relative to TP53-Y220C neoantigen in zebrafish and nonobese diabetic/severe combined immune deficiency mouse models. The results of this study demonstrate enhanced immunogenicity of the shared TP53-Y220C (L2) neoantigen, which has the potential as dendritic cells or peptide vaccines for multiple cancers.

Systems Biology Approaches for the Improvement of Oncolytic Virus-Based Immunotherapies.

Oncolytic virus (OV)-based immunotherapy is mainly dependent on establishing an efficient cell-mediated antitumor immunity. OV-mediated antitumor immunity elicits a renewed antitumor reactivity, stimulating a T-cell response against tumor-associated antigens (TAAs) and recruiting natural killer cells within the tumor microenvironment (TME). Despite the fact that OVs are unspecific cancer vaccine platforms, to further enhance antitumor immunity, it is crucial to identify the potentially immunogenic T-cell restricted TAAs, the main key orchestrators in evoking a specific and durable cytotoxic T-cell response. Today, innovative approaches derived from systems biology are exploited to improve target discovery in several types of cancer and to identify the MHC-I and II restricted peptide repertoire recognized by T-cells. Using specific computation pipelines, it is possible to select the best tumor peptide candidates that can be efficiently vectorized and delivered by numerous OV-based platforms, in order to reinforce anticancer immune responses. Beyond the identification of TAAs, system biology can also support the engineering of OVs with improved oncotropism to reduce toxicity and maintain a sufficient portion of the wild-type virus virulence. Finally, these technologies can also pave the way towards a more rational design of armed OVs where a transgene of interest can be delivered to TME to develop an intratumoral gene therapy to enhance specific immune stimuli.

Current approaches to evaluate the function of cytotoxic T-cells in non-human primates

Cytotoxic T-lymphocytes (CTL) are a subset of T-cells that play a critical role in protecting against intracellular infections and cancer, and have the ability to identify and kill infected or transformed cells expressing non-self peptides associated with major histocompatibility (MHC) Class I molecules. Conversely, aberrant CTL activity can contribute to immune-related pathology under conditions of overwhelming infection or autoimmunity. Disease-modifying therapeutics can have unintended effects on CTL, and a growing number of therapeutics are intended to either suppress or enhance CTL or their functions. The susceptibility of CTL to unintended effects from common therapeutic modalities underscores the need for a better understanding of the impact that such therapies have on CTL function and the associated safety implications. While there are reliable ways of quantifying CTL, notably via flow cytometric analysis of specific CTL markers, it has been a greater challenge to implement fit-for-purpose methods measuring CTL function in the context of safety studies of therapeutics. This review focuses on methods for measuring CTL responses in the context of drug safety and pharmacology testing, with the goals of informing the reader about current approaches, evaluating their pros and cons, and providing perspectives on the utility of these approaches for safety evaluation.

Mycobacterium bovis BCG Given at Birth Followed by Inactivated Respiratory Syncytial Virus Vaccine Prevents Vaccine-Enhanced Disease by Promoting Trained Macrophages and Resident Memory T Cells.

Respiratory syncytial virus (RSV) infects more than 60% of infants in their first year of life. Since an experimental formalin-inactivated (FI) RSV vaccine tested in the 1960s caused enhanced respiratory disease (ERD), few attempts have been made to vaccinate infants. ERD is characterized by Th2-biased responses, lung inflammation, and poor protective immune memory. Innate immune memory displays an increased nonspecific effector function upon restimulation, a process called trained immunity, or a repressed effector function upon restimulation, a process called tolerance, which participates in host defense and inflammatory disease. Mycobacterium bovis bacillus Calmette-Guérin (BCG) given at birth can induce trained immunity as well as heterologous Th1 responses. We speculate that BCG given at birth followed by FI-RSV may alleviate ERD and enhance protection through promoting trained immunity and balanced Th immune memory. Neonatal mice were given BCG at birth and then vaccinated with FI-RSV+Al(OH)3. BCG/FI-RSV+Al(OH)3 induced trained macrophages, tissue-resident memory T cells (TRM), and specific cytotoxic T lymphocytes (CTL) in lungs and inhibited Th2 and Th17 cell immune memory, all of which contributed to inhibition of ERD and increased protection. Notably, FI-RSV+Al(OH)3 induced tolerant macrophages, while BCG/FI-RSV+Al(OH)3 prevented the innate tolerance through promoting trained macrophages. Moreover, inhibition of ERD was attributed to trained macrophages or TRM in lungs but not memory T cells in spleens. Therefore, BCG given at birth to regulate trained immunity and TRM may be a new strategy for developing safe and effective RSV killed vaccines for young infants. IMPORTANCE RSV is the leading cause of severe lower respiratory tract infection of infants. ERD, characterized by Th2-biased responses, inflammation, and poor immune memory, has been an obstacle to the development of safe and effective killed RSV vaccines. Innate immune memory participates in host defense and inflammatory disease. BCG given at birth can induce trained immunity as well as heterologous Th1 responses. Our results showed that BCG/FI-RSV+Al(OH)3 induced trained macrophages, TRM, specific CTL, and balanced Th cell immune memory, which contributed to inhibition of ERD and increased protection. Notably, FI-RSV+Al(OH)3 induced tolerant macrophages, while BCG/FI-RSV+Al(OH)3 prevented tolerance through promoting trained macrophages. Moreover, inhibition of ERD was attributed to trained macrophages or TRM in lungs but not memory T cells in spleens. BCG at birth as an adjuvant to regulate trained immunity and TRM may be a new strategy for developing safe and effective RSV killed vaccines for young infants.

Familial CD45RA- T cells to treat severe refractory infections in immunocompromised patients.

Immunocompromised patients are susceptible to high-risk opportunistic infections and malignant diseases. Most antiviral and antifungal drugs are quite toxic, relatively ineffective, and induce resistance in the long term. The transfer of pathogen-specific Cytotoxic T-Lymphocytes has shown a minimal toxicity profile and effectiveness in treating Cytomegalovirus, Adenovirus, Epstein - Barr virus, BK Virus and Aspergillus infections, but this therapy have the main limitations of regulatory issues, high cost, and absence of public cell banks. However, CD45RA- cells containing pathogen-specific memory T-cells involve a less complex manufacturing and regulatory process and are cheaper, feasible, safe, and potentially effective. We present preliminary data from six immunocompromised patients: four who had severe infectious diseases and two who had EBV lymphoproliferative disease. All of them underwent multiple safe familial CD45RA- T-cell infusions as adoptive passive cell therapy, containing Cytomegalovirus, Epstein - Barr virus, BK virus, and Aspergillus-specific memory T-cells. We also present the method for selecting the best donors for CD45RA- cells in each case and the procedure to isolate and store these cells. The infusions were safe, there was no case of graft-versus host disease, and they showed a clear clinical benefit. The patients treated for BK virus nephritis, Cytomegalovirus encephalitis, Cytomegalovirus reactivation, and disseminated invasive aspergillosis experienced pathogen clearance, complete resolution of symptoms in 4-6 weeks and a lymphocyte increase in 3 of 4 cases after 3-4 months. Donor T cell transient microchimerism was detected in one patient. The two patients treated for EBV lymphoproliferative disease underwent chemotherapy and several infusions of CD45RA- memory T-cells containing EBV cytotoxic lymphocytes. Donor T-cell microchimerism was observed in both patients. The viremia cleared in one of the patients, and in the other, despite the viremia not clearing, hepatic lymphoproliferative disease remained stable and was ultimately cured with EBV-specific Cytotoxic T-Lymphocytes. The use of familial CD45RA- T-cells containing specific Cytotoxic T-lymphocytes is a feasible, safe and potential effective approach for treating severe pathogen infections in immunocompromised patients through a third party donor. Furthermore, this approach might be of universal use with fewer institutional and regulatory barriers.

CD200+ cytotoxic T lymphocytes in the tumor microenvironment are crucial for efficacious anti-PD-1/PD-L1 therapy.

Anti-PD-1/PD-L1 therapy, either by anti-PD-1 antibody or anti-PD-L1 antibody, has efficacy by reinvigorating tumor-infiltrating CD8+ T cells in a subset of patients with cancer, but it has unequal effects on heterogeneous CD8+ T cell populations. Hence, the subset crucial to efficacious PD-1 blockade therapy remains elusive. Here, we found an increase in tumor-infiltrating CD200+ cytotoxic T lymphocytes (CTLs) upon PD-1/PD-L1 blockade, with higher proportions of CD200+ T cells positively related to a favorable clinical outcome to anti-PD-1/PD-L1 therapy in three independent cohorts of patients with cancer. Using multiple mouse tumor models, we demonstrated that CD200+ CTLs are essential for efficacious anti-PD-L1 therapy. Mechanistically, we observed a unique chromatin landscape in CD200+ CTLs and found that these cells are enriched for tumor antigen-specific CTLs and have antitumor effector functions. Coinoculation of CD200+ CTLs with tumor cells led to robust tumor regression in two transplanted mouse models. Clinically, we found that infiltration of CD200+ CTLs into tumors could predict immunotherapy efficacy in six patient cohorts. Together, our findings reveal that CD200+ CTLs in the tumor microenvironment are crucial for efficacious anti-PD-1/PD-L1 therapy and could serve as a predictor of successful immunotherapy in the clinic.

Prognostic value of tumor-infiltrating T-lymphocytes density in the therapeutic response to initial platinum-based chemotherapy in patients with non-small cell lung cancer

Background/Aim. The fact that lung carcinomas, like other solid tumors, can be immunogenic may have a substantial prognostic value in non-small cell lung cancer (NSCLC). Specific cytotoxic T-lymphocytes (CTL) can be demonstrated in most patients with primary tumors of different histological types. Two main groups of T-lymphocytes participate in the coupled recognition of tumor-specific antigens ? CTL (CD8+) and helper T-lymphocytes (CD4+). The aim of the study was to assess the relationship between the tumor infiltration of T-lymphocytes and the therapeutic response to initial chemotherapy. Methods. Data were obtained from patients with NSCLC whose therapeutic response after four cycles of initial platinum-based chemotherapy was observed in relation to the density of tumor-infiltrating T-lymphocytes (CD4+ and CD8+) in small tumor biopsy samples. The therapeutic response was assessed in line with Response Evaluation Criteria in Solid Tumors (RECIST) 1.1 therapeutic response evaluation system. Based on the expected therapeutic response, the patients were divided into three groups: favorable therapeutic response patients (complete and partial regression), stable disease patients, and disease progression patients. To assess the density of CD4+ and CD8+ T-lymphocytes, the number of lymphocytes was determined at ?200 magnification (1.1 mm2). Three visual fields with the densest lymphocyte infiltrate were selected for counting, and the values of all individual fields were added up. Based on the mean value, the samples were classified into the following groups: score 0, score 1, score 2, and score 3. During statistical data processing, low infiltration density combined score 0 and score 1 groups, and high infiltration density combined score 2 and score 3 groups. Based on the collected data, a database was created in SPSS 22.0 software and used for further statistical analysis. Statistical analysis of the data included descriptive and analytical statistics methods. Results. There was no significant difference in the distribution of CD4+ T-lymphocytes and CD8+ T-lymphocytes in the epithelial component of the tumor between patients with a different therapeutic response (?2 = 2.977; p = 0.226 and ?2 = 1.329; p = 0.515, respectively). There was no significant influence of the infiltration density of CD4+ T-lymphocytes and CD8+ T-lymphocytes in the stromal component of the tumor on the therapeutic response (?2 = 0.606; p = 0.739 and ?2 = 5.167; p = 0.076, respectively). Conclusion. The research did not prove that patients with a high level of tumor-infiltrating CD4+ and CD8+ T-lymphocytes in the epithelial and stromal component of the NSCLC had a better therapeutic response to standard initial chemotherapy.

Exposure of Immunogenic Tumor Antigens in Surrendered Immunity and the Significance of Autologous Tumor Cell-Based Vaccination in Precision Medicine.

The mechanisms by which immune systems identify and destroy tumors, known as immunosurveillance, have been discussed for decades. However, several factors that lead to tumor persistence and escape from the attack of immune cells in a normal immune system have been found. In the process known as immunoediting, tumors decrease their immunogenicity and evade immunosurveillance. Furthermore, tumors exploit factors such as regulatory T cells, myeloid-derived suppressive cells, and inhibitory cytokines that avoid cytotoxic T cell (CTL) recognition. Current immunotherapies targeting tumors and their surroundings have been proposed. One such immunotherapy is autologous cancer vaccines (ACVs), which are characterized by enriched tumor antigens that can escalate specific CTL responses. Unfortunately, ACVs usually fail to activate desirable therapeutic effects, and the low immunogenicity of ACVs still needs to be elucidated. This difficulty highlights the significance of immunogenic antigens in antitumor therapies. Previous studies have shown that defective host immunity triggers tumor development by reprogramming tumor antigenic expressions. This phenomenon sheds new light on ACVs and provides a potential cue to improve the effectiveness of ACVs. Furthermore, synergistically with the ACV treatment, combinational therapy, which can reverse the suppressive tumor microenvironments, has also been widely proposed. Thus, in this review, we focus on tumor immunogenicity sculpted by the immune systems and discuss the significance and application of restructuring tumor antigens in precision medicine.