Systematic assessment of LCMV based vaccine vectors expressing melanocyte differentiation antigens in human in vitro assays and in mouse melanoma models.

2019 ◽  
Vol 37 (15_suppl) ◽  
pp. e14299-e14299
Author(s):  
Lukas Flatz ◽  
Jovana Cupovic ◽  
Sarah Schmidt ◽  
Lucas Onder ◽  
Marie Therese Abdou ◽  
...  
Keyword(s):  
T Cell ◽  
Cancer Vaccine ◽  
Intratumoral Injection ◽  
Lymphocytic Choriomeningitis ◽  
In Vitro Assays ◽  
Systematic Assessment ◽  
Differentiation Antigens ◽  
Mouse Melanoma ◽  
Melanoma Patients

e14299 Background: Antibodies blocking the immune checkpoint pathways represent an important milestone in the treatment of patients with metastatic melanoma. However, individuals presenting cold tumors do usually not show a clinical benefit. New cancer vaccine approaches are needed to cover this gap. Methods: A novel replication attenuated vaccine vector based on lymphocytic choriomeningitis virus expressing full length melanocyte differentiation antigens is evaluated in mouse melanoma models and in PBMCs and T cell cultures from melanoma patients. Results: Here, we demonstrate that intratumoral but not intravenous injection of a recombinant propagating LCMV vector expressing the melanoma-associated antigen TRP2 leads to T cell-dependent eradication of established s.c. melanoma. Importantly, intratumoral vaccination shows an abscopal effect on distant lung metastasis and protects from a rechallenge with melanoma. Confocal microscopy and flow cytometry reveal that intratumoral injection of rLCMV vectors reprograms the tumor microenvironment resulting in sustained T cell fitness. In addition, we demonstrate that rLCMV vectors can efficiently transduce human antigen presenting cells. Moreover, in vitro data confirm that rLCMV efficiently induces T cells against various melanoma-associated antigens in PBMCs from melanoma patients. Conclusions: Preclinical assessment of propagating rLCMV vectors shows unique features of this cancer vaccine resulting in a profound and multistep activation of the cancer immunity cycle resulting in eradication of established melanomas in the B16F10 mouse model after one single immunization. Positive proof of principle experiments using PBMCs from melanoma patients suggest a rapid evaluation in clinical trials.

scholarly journals Slow viral propagation during initial phase of infection leads to viral persistence in mice

2021 ◽  
Vol 4 (1) ◽  
Author(s):  
Haifeng C. Xu ◽  
Ruifeng Wang ◽  
Prashant V. Shinde ◽  
Lara Walotka ◽  
Anfei Huang ◽  
...  
Keyword(s):  
T Cell ◽  
Immune Evasion ◽  
Protein Transport ◽  
Adaptive Immune System ◽  
Viral Persistence ◽  
Lymphocytic Choriomeningitis ◽  
Type I ◽  
Viral Propagation

AbstractImmune evasion of pathogens can modify the course of infection and impact viral persistence and pathology. Here, using different strains of the lymphocytic choriomeningitis virus (LCMV) model system, we show that slower propagation results in limited type I interferon (IFN-I) production and viral persistence. Specifically, cells infected with LCMV-Docile exhibited reduced viral replication when compared to LCMV-WE and as a consequence, infection with LCMV-Docile resulted in reduced activation of bone marrow derived dendritic cells (BMDCs) and IFN-I production in vitro in comparison with LCMV-WE. In vivo, we observed a reduction of IFN-I, T cell exhaustion and viral persistence following infection of LCMV-Docile but not LCMV-WE. Mechanistically, block of intracellular protein transport uncovered reduced propagation of LCMV-Docile when compared to LCMV-WE. This reduced propagation was critical in blunting the activation of the innate and adaptive immune system. When mice were simultaneously infected with LCMV-Docile and LCMV-WE, immune function was restored and IFN-I production, T cell effector functions as well as viral loads were similar to that of mice infected with LCMV-WE alone. Taken together, this study suggests that reduced viral propagation can result in immune evasion and viral persistence.

scholarly journals 112 Tumor-specific reactivity and effector function of chimeric antigen receptor engineered macrophages targeting MUC1

2021 ◽  
Vol 9 (Suppl 3) ◽  
pp. A122-A122
Author(s):  
Seth Eisenberg ◽  
Amy Powers ◽  
Jason Lohmueller ◽  
James Luketich ◽  
Rajeev Dhupar ◽  
...  
Keyword(s):  
Breast Cancer ◽  
T Cell ◽  
Tumor Cell ◽  
Tumor Cells ◽  
Cancer Vaccine ◽  
Chimeric Antigen Receptor ◽  
Effector Function ◽  
Specific Antibodies ◽  
The University

BackgroundChimeric antigen receptors (CAR) have demonstrated remarkable efficacy in licensing T cells for antitumor responses against hematopoietic malignancies but have had limited success against solid tumors. Macrophages, both archetypic phagocytes and professional antigen presenting cells, may exert profound effector functions which complement adaptive cellular immunity.1 Recently, it was shown that human macrophages engineered to express CARs (CAR-Ms) demonstrated antigen-specific phagocytosis, inhibited solid xenograph tumors, and induced an inflammatory tumor microenvironment boosting antitumor T cell responses.2 Kimura et al. previously completed the first prophylactic cancer vaccine trial based on a non-viral antigen, tumor-associated hypoglycosylated Mucin 1 (MUC1).3 A panel of fully-human affinity-matured MUC1-specific antibodies raised in healthy subjects following immunization was identified from these patients.4 Using these MUC1-specific scFv domains for CAR generation, we have now engineered MUC1-targeting CAR-Ms that may potentially possess reduced off-target specificities.MethodsLentiviral CAR expression vectors containing the scFv domains of three unique hypoglycosylated MUC1-specific antibodies or a CD20-specific antibody, the CD3zeta signaling domain, and CD28 and OX40 co-stimulatory domains were constructed. The human monocyte/macrophage U937, SC, and THP-1 lines were stably transduced and flow-sort purified to generate MUC1- or CD20-specific CAR-Ms. CAR-Ms were differentiated into macrophages via 48 hour PMA treatment, and subsequently evaluated for antigen-specific function against MUC1- and/or CD20-expressing K562, ZR-75-1, and Raji cells or cancer cells isolated from solid lung tumors or malignant pleural effusions. CAR-M phenotype was evaluated by flow cytometry following in vitro differentiation and polarization with conventional ‘M1’ and ‘M2’ stimuli. Phagocytosis and lysosomal processing of phagocytosed cargo were evaluated by fluorescence microscopy of GFP/CellTrace labeled targets or detection of pH-sensitive pHrodo expression following CAR-M and tumor cell co-culture, respectively. Antigen-specific cytokine production was determined via cytometric bead array following co-culture of CAR-Ms with MUC1- or CD20-expressing tumor cells or 100mer MUC1 peptide.ResultsDifferentiated CAR-Ms possessed an inflammatory phenotype expressing IL-8 and CD86 which was further enhanced by IFNgamma or LPS treatment and was resistant to ‘M2’ polarization with conventional stimuli. CAR-Ms exhibited phagocytosis and subsequent lysosomal processing in an antigen-specific manner, with minimal reactivity against tumor cell targets in the absence of the corresponding MUC1 or CD20 antigen. MUC1-specific CAR-Ms stimulated with MUC1 peptide or MUC1+ tumor cells secreted robust levels of pro-inflammatory IL-8, TNFa, and IL-1beta, but not immunosuppressive IL-10.ConclusionsMUC1-targeting CAR-Ms exert potent tumor-restricted effector function in vitro and may provide a novel treatment strategy either alone or in potential synergistic combination with T cell-mediated immunotherapies.AcknowledgementsThe authors would like to thank Dr. Olivera J. Finn for generously providing reagents and guidance and Dr. Michael T. Lotze for his mentorship. This study was supported by funding from the University of Pittsburgh’s Department of Cardiothoracic Surgery to ACS and RD.ReferencesWilliams CB, Yeh ES, Soloff AC. Tumor-associated macrophages: unwitting accomplices in breast cancer malignancy. Npj Breast Cancer [Internet]. Breast Cancer Research Foundation/Macmillan Publishers Limited; 2016;2:15025. Available from: http://dx.doi.org/10.1038/npjbcancer.2015.25Klichinsky M, Ruella M, Shestova O, Lu XM, Best A, Zeeman M, et al. Human chimeric antigen receptor macrophages for cancer immunotherapy. Nat Biotechnol 2020;38:947–53.Kimura T, McKolanis JR, Dzubinski LA, Islam K, Potter DM, Salazar AM, et al. MUC1 Vaccine for Individuals with Advanced Adenoma of the Colon: A Cancer Immunoprevention Feasibility Study. Cancer Prev Res [Internet] 2013;6:18–26. Available from: http://cancerpreventionresearch.aacrjournals.org/content/6/1/18.abstractLohmueller JJ, Sato S, Popova L, Chu IM, Tucker MA, Barberena R, et al. Antibodies elicited by the first non-viral prophylactic cancer vaccine show tumor-specificity and immunotherapeutic potential. Sci Rep 2016;6:31740.Ethics ApprovalThe study was approved by the University of Pittsburgh’s Institutional Review Board approval number CR19120172-005.

scholarly journals IL4I1 Accelerates the Expansion of Effector CD8+ T Cells at the Expense of Memory Precursors by Increasing the Threshold of T-Cell Activation

2020 ◽  
Vol 11 ◽  
Author(s):  
Marie-Line Puiffe ◽  
Aurélie Dupont ◽  
Nouhoum Sako ◽  
Jérôme Gatineau ◽  
José L. Cohen ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
T Cell Activation ◽  
Cd8 T Cells ◽  
Cell Activation ◽  
Cell Clone ◽  
Cd8 T Cell ◽  
Lymphocytic Choriomeningitis ◽  
T Cell Clone

IL4I1 is an immunoregulatory enzyme that inhibits CD8 T-cell proliferation in vitro and in the tumoral context. Here, we dissected the effect of IL4I1 on CD8 T-cell priming by studying the differentiation of a transgenic CD8 T-cell clone and the endogenous repertoire in a mouse model of acute lymphocytic choriomeningitis virus (LCMV) infection. Unexpectedly, we show that IL4I1 accelerates the expansion of functional effector CD8 T cells during the first several days after infection and increases the average affinity of the elicited repertoire, supporting more efficient LCMV clearance in WT mice than IL4I1-deficient mice. Conversely, IL4I1 restrains the differentiation of CD8 T-cells into long-lived memory precursors and favors the memory response to the most immunodominant peptides. IL4I1 expression does not affect the phenotype or antigen-presenting functions of dendritic cells (DCs), but directly reduces the stability of T-DC immune synapses in vitro, thus dampening T-cell activation. Overall, our results support a model in which IL4I1 increases the threshold of T-cell activation, indirectly promoting the priming of high-affinity clones while limiting memory T-cell differentiation.

Anatomic location and T-cell stimulatory functions of mouse dendritic cell subsets defined by CD4 and CD8 expression

Blood ◽  
2002 ◽  
Vol 99 (6) ◽  
pp. 2084-2093 ◽  
Author(s):  
Alexander D. McLellan ◽  
Michaela Kapp ◽  
Andreas Eggert ◽  
Christian Linden ◽  
Ursula Bommhardt ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cd8 T Cells ◽  
Antigen Expression ◽  
Cell Receptor ◽  
In Vitro Assays ◽  
Peptide Complexes ◽  
Marginal Zones

Abstract Mouse spleen contains CD4+, CD8α+, and CD4−/CD8α− dendritic cells (DCs) in a 2:1:1 ratio. An analysis of 70 surface and cytoplasmic antigens revealed several differences in antigen expression between the 3 subsets. Notably, the Birbeck granule–associated Langerin antigen, as well as CD103 (the mouse homologue of the rat DC marker OX62), were specifically expressed by the CD8α+ DC subset. All DC types were apparent in the T-cell areas as well as in the splenic marginal zones and showed similar migratory capacity in collagen lattices. The 3 DC subtypes stimulated allogeneic CD4+ T cells comparably. However, CD8α+ DCs were very weak stimulators of resting or activated allogeneic CD8+ T cells, even at high stimulator-to-responder ratios, although this defect could be overcome under optimal DC/T cell ratios and peptide concentrations using CD8+ F5 T-cell receptor (TCR)–transgenic T cells. CD8α− or CD8α+DCs presented alloantigens with the same efficiency for lysis by cytotoxic T lymphocytes (CTLs), and their turnover rate of class I–peptide complexes was similar, thus neither an inability to present, nor rapid loss of antigenic complexes from CD8α DCs was responsible for the low allostimulatory capacity of CD8α+ DCs in vitro. Surprisingly, both CD8α+ DCs and CD4−/CD8− DCs efficiently primed minor histocompatibility (H-Y male antigen) cytotoxicity following intravenous injection, whereas CD4+ DCs were weak inducers of CTLs. Thus, the inability of CD8α+ DCs to stimulate CD8+ T cells is limited to certain in vitro assays that must lack certain enhancing signals present during in vivo interaction between CD8α+ DCs and CD8+ T cells.

scholarly journals Antiviral cytotoxic T cell response induced by in vivo priming with a free synthetic peptide.

1990 ◽  
Vol 171 (5) ◽  
pp. 1815-1820 ◽  
Author(s):  
P Aichele ◽  
H Hengartner ◽  
R M Zinkernagel ◽  
M Schulz
Keyword(s):  
T Cell ◽  
Cell Response ◽  
Lymphocytic Choriomeningitis ◽  
Effector T Cells ◽  
T Cell Response ◽  
Class I ◽  
Cytotoxic T Cell ◽  
Effector T Cell

Induction in vivo of antiviral cytotoxic T cell response was achieved in a MHC class I-dependent fashion by immunizing mice three times with a free unmodified 15-mer peptide derived from the nucleoprotein of lymphocytic choriomeningitis virus in IFA. The effector T cells are CD8+, restricted to the class I Ld allele of the analyzed mouse strain, and are specific both at the level of secondary restimulation in vitro and at the effector T cell level. These results suggest that cocktails of viral peptides may be used as antiviral T cell vaccines.

scholarly journals Interleukin-1R Signaling Is Essential for Induction of Proapoptotic CD8 T Cells, Viral Clearance, and Pathology during Lymphocytic Choriomeningitis Virus Infection in Mice

2012 ◽  
Vol 86 (16) ◽  
pp. 8713-8719 ◽  
Author(s):  
Lars T. Joeckel ◽  
Reinhard Wallich ◽  
Sunil S. Metkar ◽  
Christopher J. Froelich ◽  
Markus M. Simon ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Interleukin 1 ◽  
Lymphocytic Choriomeningitis ◽  
Lymphocytic Choriomeningitis Virus ◽  
Viral Clearance ◽  
T Cell Immunity ◽  
Cell Immunity ◽  
Deficient Mice

The T cell granule exocytosis pathway is essential to control hepatotropic lymphocytic choriomeningitis virus strain WE (LCMV-WE) but also contributes to the observed pathology in mice. Although effective antiviral T cell immunity and development of viral hepatitis are strictly dependent on perforin and granzymes, the molecular basis underlying induction of functionally competent virus-immune T cells, including participation of the innate immune system, is far from being resolved. We demonstrate here that LCMV-immune T cells of interleukin-1 receptor (IL-1R)-deficient mice readily express transcripts for perforin and granzymes but only translate perforin, resulting in the lack of proapoptotic potentialin vitro. LCMV is not cleared in IL-1R-deficient mice, and yet the infected mice develop neither splenomegaly nor hepatitis. These results demonstrate that IL-1R signaling is central to the induction of proapoptotic CD8 T cell immunity, including viral clearance and associated tissue injuries in LCMV infection.

scholarly journals Antivirally protective cytotoxic T cell memory to lymphocytic choriomeningitis virus is governed by persisting antigen.

1992 ◽  
Vol 176 (5) ◽  
pp. 1273-1281 ◽  
Author(s):  
S Oehen ◽  
H Waldner ◽  
T M Kündig ◽  
H Hengartner ◽  
R M Zinkernagel
Keyword(s):  
T Cell ◽  
Lymphocytic Choriomeningitis ◽  
Lymphocytic Choriomeningitis Virus ◽  
T Cell Response ◽  
Cytotoxic T Cell ◽  
Protective Capacity ◽  
Activated T Cells ◽  
Recombinant Vaccinia ◽  
Activated State

The basis of antiviral protection by memory cytotoxic T lymphocytes (CTL) was investigated in vivo and in vitro using lymphocytic choriomeningitis virus (LCMV) and recombinant vaccinia viruses expressing the LCMV-glycoprotein (vacc-GP) or -nucleoprotein (vacc-NP). The widely replicating LCMV with a tendency to persist induced solid long-term protective memory. The poorly replicating vaccinia recombinant viruses revealed in the vaccinated host that the antiviral capacity of the secondary immune T cell response and the protection against lethal LCM was dependent upon the immunizing antigen and its dose. Protection against lethal choriomeningitis is less sensitive to assess memory because it depends upon high levels of CTL precursors (p) and/or on an activated state of memory CTL. In contrast, antiviral protection measured as the capacity of the primed host to reduce virus titers after challenge infection correlated with elevated CTLp frequencies after immunization with live LCMV or recombinant vaccinia virus-expressing the major LCMV epitope. CTLp frequencies were constantly increased up to 70 d for LCMV immune mice, but rapidly decreased a few weeks after immunization with low dose vaccinia recombinant virus. For example, mice primed with 2 x 10(6) plaque-forming units (PFU) of vacc-NP, or 2 x 10(2) PFU, or 2 x 10(6) PFU of vacc-GP were antivirally protected on day 7 but not after day 30 when CTLp could not be measured any longer in vitro. However, greater priming doses of vacc-NP (10(4) or 2 x 10(6) PFU) as well as LCMV (2 x 10(2) PFU) induced elevated levels of CTLp and antiviral protection for 60 d or longer. Adoptive transfer experiments of immune spleen cells into syngeneic recipients without addition of antigen demonstrated that maintenance of the antiviral protective capacity of the transferred cells depended on the presence of viral antigen. Thus, antiviral protection by memory CTL may be rather short-lived since it is based on activated T cells continuously stimulated by persisting antigen. This is best achieved by high immunizing antigen doses yielded either by widely replicating viruses or high doses of poorly replicating recombinant vaccines.

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