scholarly journals High Dimensional Analyses of Circulating Immune Cells in Psoriatic Arthritis Detects Elevated Phosphorylated STAT3

2022 ◽  
Vol 12 ◽  
Author(s):  
Claudia Macaubas ◽  
Shamma S. Rahman ◽  
Idit Lavi ◽  
Amir Haddad ◽  
Muna Elias ◽  
...  
Keyword(s):  
T Cells ◽  
Psoriatic Arthritis ◽  
Cd4 T Cells ◽  
Immune Cell ◽  
Ex Vivo ◽  
Active Form ◽  
Constitutive Expression ◽  
T Cell Subsets ◽  
High Dimensional ◽  
Phosphorylated Stat3

Psoriatic arthritis (PsA) is a chronic inflammatory arthritis, affecting up to 40% of patients with psoriasis. Constitutive expression by CD4+ T cells of an active form of STAT3, a signal transducer and transcription factor, has been shown to induce many of the major features of PsA in an animal model. We used high dimensional mass cytometry (CyTOF) to probe ex-vivo levels of phosphorylated STAT3 (pSTAT3) in circulating immune cell subpopulations from PsA patients during active and inactive states. We evaluated the frequency of 16 immune cell populations and the levels of the activated forms of STAT3 (pSTAT3) and, for comparison, STAT1 (pSTAT1) and Src (pSrc) in whole blood fixed shortly after collection. In addition to PsA patients, we studied active rheumatoid arthritis (RA) patients. Increased levels of pSTAT3 were found in all the CD4+ T cell subsets analyzed, specifically, Th1, Th2, Th17, T follicular helper (Tfh) and T regulatory (Treg) as well as in CD14+CD16- (classical) monocytes from active PsA patients compared to inactive patients. After correcting for body mass index (BMI), smoking and conventional disease modifying antirheumatic drugs (c-DMARDs), levels of pSTAT3 levels remained increased in Th1 and Tfh CD4+ T cells, and in CD14+CD16- monocytes from active patients compared to inactive patients. No differences between the patient groups were observed for pSTAT1 or pSrc. No differences were found between the active PsA and active RA groups after correction for multiple testing. During active PsA, circulating Th1 and Tfh CD4+ T cells, and CD14+CD16- monocytes expressing high levels of pSTAT3 may play a role in PsA pathophysiology, perhaps by migration to inflamed sites.

Ex Vivo Fludarabine Selectively Depletes Human Naive T-Cell Subsets: A Step Toward Modifying Donor Lymphocyte Infusion.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 1071-1071
Author(s):  
Melody M. Smith ◽  
Cynthia R. Giver ◽  
Edmund K. Waller ◽  
Christopher R. Flowers
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cd8 T Cells ◽  
Cd4 T Cells ◽  
Memory T Cells ◽  
Ex Vivo ◽  
T Cell Subsets ◽  
Effector Memory ◽  
Naïve T Cell ◽  
Naive T Cell

Abstract Ex vivo modification of donor lymphocytes with purine analogs (mDL) may help to minimize graft versus host disease (GvHD) while providing beneficial graft versus leukemia (GvL) effects. In a murine model system, we have shown that allogeneic donor splenocytes, treated with fludarabine ex vivo have significantly reduced GvHD activity when transferred to irradiated recipient mice, and retain anti-viral and GvL activities (Giver, 2003). This effect appears to be mediated by relative depletion of donor CD4 CD44low, “naive” T-cells. As a first step toward developing mDL for use in patients, we sought to evaluate the effects of ex vivo fludarabine exposure on human T-cell subsets, and to determine the minimum dose of fludarabine required to achieve this effect. Methods: Peripheral blood mononuclear cell samples from 6 healthy volunteers were evaluated at 0, 24, 48, and 72 hour time points after ex vivo incubation in varying dosages of fludarabine: 2, 5, and 10(n=3) mcg/ml. Fludarabine incubated samples were compared to samples that received no fludarabine (untreated). The total viable cell number was determined and the fractions and absolute numbers of viable CD4 and CD8 naïve and memory T-cells were determined using flow cytometry after incubation with 7-AAD (dead cell stain), CD4, CD8, CD45RA, CD62L, and CCR7 antibodies, and measuring the total viable cells/ml. Results: The numbers of viable CD4 and CD8 T-cells remained relatively stable in control cultures. Without fludarabine, the average viability at 72 hr of naive and memory T-cells were 92% and 77% for CD4 and 86% and 63% for CD 8 (Fig. 1A). Naive CD4 T-cells were more sensitive to fludarabine-induced death than memory CD4 cells. At 72 hr, the average viability of fludarabine-treated naive CD4 T-cells was 33% at 2 mcg/ml (8.2X the reduction observed in untreated cells) and 30% at 5 mcg/ml, while memory CD4 T-cells averaged 47% viability at 2 mcg/ml (2.3X the reduction observed in untreated cells) (Fig. 1B) and 38% at 5 mcg/ml. The average viability of naive CD8 T-cells at 72 hr was 27% at 2 mcg/ml and 20% at 5 mcg/ml, while memory CD8 T-cell viability was 22% at 2 mcg/ml and 17% at 5 mcg/ml. Analyses on central memory, effector memory, and Temra T-cells, and B-cell and dendritic cell subsets are ongoing. The 5 and 10 mcg/ml doses also yielded similar results in 3 initial subjects, suggesting that 2 mcg/ml or a lower dose of fludarabine is sufficient to achieve relative depletion of the naive T-cell subset. Conclusions: Future work will determine the minimal dose of fludarabine to achieve this effect, test the feasibility of using ex vivo nucleoside analog incubation to reduce alloreactivity in samples from patient/donor pairs, and determine the maximum tolerated dose of mDL in a phase 1 clinical trial with patients at high risk for relapse and infectious complications following allogeneic transplantation. Figure Figure

scholarly journals Blood-Based Immune Profiling Combined with Machine Learning Discriminates Psoriatic Arthritis from Psoriasis Patients

2021 ◽  
Vol 22 (20) ◽  
pp. 10990
Author(s):  
Michelle L. M. Mulder ◽  
Xuehui He ◽  
Juul M. P. A. van den Reek ◽  
Paulo C. M. Urbano ◽  
Charlotte Kaffa ◽  
...  
Keyword(s):  
Machine Learning ◽  
T Cells ◽  
Psoriatic Arthritis ◽  
Immune Cell ◽  
Joint Damage ◽  
Classification Model ◽  
T Cell Subsets ◽  
Correct Identification ◽  
Random Forest Classification ◽  
Disease Specific

Psoriasis (Pso) is a chronic inflammatory skin disease, and up to 30% of Pso patients develop psoriatic arthritis (PsA), which can lead to irreversible joint damage. Early detection of PsA in Pso patients is crucial for timely treatment but difficult for dermatologists to implement. We, therefore, aimed to find disease-specific immune profiles, discriminating Pso from PsA patients, possibly facilitating the correct identification of Pso patients in need of referral to a rheumatology clinic. The phenotypes of peripheral blood immune cells of consecutive Pso and PsA patients were analyzed, and disease-specific immune profiles were identified via a machine learning approach. This approach resulted in a random forest classification model capable of distinguishing PsA from Pso (mean AUC = 0.95). Key PsA-classifying cell subsets selected included increased proportions of differentiated CD4+CD196+CD183-CD194+ and CD4+CD196-CD183-CD194+ T-cells and reduced proportions of CD196+ and CD197+ monocytes, memory CD4+ and CD8+ T-cell subsets and CD4+ regulatory T-cells. Within PsA, joint scores showed an association with memory CD8+CD45RA-CD197- effector T-cells and CD197+ monocytes. To conclude, through the integration of in-depth flow cytometry and machine learning, we identified an immune cell profile discriminating PsA from Pso. This immune profile may aid in timely diagnosing PsA in Pso.

Mechanism for the Immuno-Suppresion Activity of Pentostatin: Selective Apoptosis of Naive T Cell Subsets.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 5160-5160
Author(s):  
Melody M. Smith ◽  
William Powers ◽  
Cindy R. Giver ◽  
Ned Waller ◽  
Christopher Flowers
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cd4 T Cells ◽  
Ex Vivo ◽  
T Cell Subsets ◽  
Purine Analogs ◽  
In Vitro Exposure ◽  
Selective Depletion ◽  
Naive T Cell

Abstract Background: Nucleoside analogs have potent anti-tumor activity, especially against lymphoid malignancies, with immuno-suppression being a significant toxicity. We have previously described the immunosuppressive effects of fludarabine on murine T-cells, and found selective cytotoxicity against naïve subsets that reduced their allo-reactivity in a GvHD model of murine BMT (Giver et al 2003 BBMT 9:616). In this study we tested the cytotoxicity of fludarabine and pentostatin against human and murine T-cell subsets to determine their immune-suppressive activity, mechanism of action, and their potential utility as agents to decrease the allo-reactivity of allogeneic donor lymphocyte infusions. Methods: Peripheral blood mononuclear cell samples from normal donors were incubated ex vivo ex vivo treatment with Flu at doses of 5, 50, and 250 mcg/ml or pentostatin at doses of 1, 10, and 100 mcg/ml at 0, 4, 24, and 48-hour time points following 1 or 24 hour drug exposure. All samples that obtained dCF also received 25 mcg/ml of deoxyadenosine (dAdo), which enables dCF to mediate cytotoxicity against lymphocytes. Control samples for fludarabine and pentostatin treatment were untreated or treated with dAdo only, respectively. The total number of viable cells and the fractions of the CD4 and CD8 memory and naive T cell subsets that survived nucleoside exposure were determined by multi-parameter flow cytometry following staining with antibodies to CD3, CD4, CD8, CD45RA, CD6L, Annexin, and 7-AAD. Results: The decrease in the viability of the human naïve CD4 T cells, whether treated with fludarabine or pentostatin, was rapid and most notably observed after the 24-hour time point. On the other hand, the human memory CD4 T cells displayed a more gradual decline in percent viability in both treatment with fludarabine and pentostatin. Furthermore, the potency of pentostatin was exhibited in that it achieved a comparable effect to fludarabine while at substantially lower doses. Data obtained from the murine model also demonstrated a relative a relative sensitivity of the CD4 naïve T cells as oppesed to the CD4 memory to purine analog exposure. Conclusions: Future work will determine the optimal dosage and period of incubation for pentostatin and fludarabine that can utilized to induce immunosuppresion ex vivo in donor lymphocytes. SELECTIVE DEPLETION OF NAÏVE CD4+ T-CELLS FOLLOWING IN VITRO EXPOSURE TO PURINE ANALOGS SELECTIVE DEPLETION OF NAÏVE CD4+ T-CELLS FOLLOWING IN VITRO EXPOSURE TO PURINE ANALOGS

scholarly journals Distinctive CD26 Expression on CD4 T-Cell Subsets

Biomolecules ◽  
2021 ◽  
Vol 11 (10) ◽  
pp. 1446
Author(s):  
Oscar J. Cordero ◽  
Carlos Rafael-Vidal ◽  
Rubén Varela-Calviño ◽  
Cristina Calviño-Sampedro ◽  
Beatriz Malvar-Fernández ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cell Surface ◽  
Cd4 T Cells ◽  
Ex Vivo ◽  
T Cell Subsets ◽  
Effector Memory ◽  
T Helper ◽  
Cd26 Expression

Immune system CD4 T-cells with high cell-surface CD26 expression show anti-tumoral properties. When engineered with a chimeric antigen receptor (CAR), they incite strong responses against solid cancers. This subset was originally associated to human CD4 T helper cells bearing the CD45R0 effector/memory phenotype and later to Th17 cells. CD26 is also found in soluble form (sCD26) in several biological fluids, and its serum levels correlate with specific T cell subsets. However, the relationship between glycoprotein sCD26 and its dipeptidyl peptidase 4 (DPP4) enzymatic activity, and cell-surface CD26 expression is not well understood. We have studied ex vivo cell-surface CD26 and in vitro surface and intracellular CD26 expression and secretome’s sCD26 in cultured CD4 T cells under different polarization conditions. We show that most human CD26negative CD4 T cells in circulating lymphocytes are central memory (TCM) cells while CD26high expression is present in effector Th1, Th2, Th17, and TEM (effector memory) cells. However, there are significant percentages of Th1, Th2, Th17, and Th22 CD26 negative cells. This information may help to refine the research on CAR-Ts. The cell surface CD45R0 and CD26 levels in the different T helper subsets after in vitro polarization resemble those found ex vivo. In the secretomes of these cultures there was a significant amount of sCD26. However, in all polarizations, including Th1, the levels of sCD26 were lower (although not significantly) compared to the Th0 condition (activation without polarization). These differences could have an impact on the various physiological functions proposed for sCD26/DPP4.

targetSCAPE and ultraSCAPE: Simultaneous identification and deep profiling of human antigen-specific T cells and other immune cell subsets by mass cytometry.

2020 ◽  
Vol 38 (15_suppl) ◽  
pp. e15204-e15204
Author(s):  
Brian Abel ◽  
Faris Kairi ◽  
Alessandra Nardin ◽  
Evan Newell ◽  
Michael Fehlings
Keyword(s):  
T Cells ◽  
T Cell ◽  
Immune Cell ◽  
T Cell Subsets ◽  
Single Sample ◽  
High Dimensional ◽  
Cell Populations ◽  
Mass Cytometry ◽  
Immune Cell Subsets

e15204 Background: During clinical trial immune monitoring, especially in the field of immunotherapy, it is critical to collect in-depth phenotypic information from multiple immune cell populations in order to assess the biological activity of the immunotherapy, to identify biomarkers of response or disease progression, and/or to identify new drug targets. However, patient samples such as peripheral blood mononuclear cells or tissues, are often scarce and current methods face limitations in either achieving a sufficient depth of analysis and/or cell throughput. Methods: In order to identify therapy-relevant antigens and to facilitate a concurrent in-depth characterization of T cells directed towards these targets, immunoSCAPE leverages the high-dimensional immune profiling capabilities of mass cytometry and a unique methodology allowing the identification and characterization of rare antigen-specific T-cell subsets (targetSCAPE). By implementing a new cutting-edge technology that combines flow and mass cytometry in parallel with a combinatorial live cell barcoding strategy, we further increased the high-dimensional phenotyping capacities to over 100 different marker molecules on up to four different immune cell subsets simultaneously within the same sample. Results: We isolated 4 different immune cell populations from a single sample and combined 3 different phenotypic panels consisting of 35 makers each together with a combinatorial tetramer multiplex and phenotyping panel for deep profiling of myeloid cells, NK cells, B cells and T cells. We demonstrate the potential of this novel immuno-phenotyping method, by tracking virus-specific T cells while simultaneously characterizing 4 immune cell subsets with over 100 distinct phenotypic markers from a single sample, which is currently impossible employing modern flow cytometers or classical mass cytometry methods. Conclusions: With its ability to provide an unprecedented picture of the immune status within a single sample, including T cell specificity information and in-depth profiling of relevant immune cell subsets, ultraSCAPE in combination with targetSCAPE can provide detailed insights on the effects of immunotherapy on the immune cell population. Information learned from in-depth immune phenotyping of several immune cell subsets such as T, B, NK and myeloid cell subsets can be leveraged for the development of novel diagnostics, biomarker discovery and monitoring therapeutic strategies in immunotherapy clinical trials.

scholarly journals High-dimensional characterization of IL-10 production and IL-10 dependent regulation during primary gammaherpesvirus infection

2018 ◽  
Author(s):  
Abigail K. Kimball ◽  
Lauren M. Oko ◽  
Rachael E. Kaspar ◽  
Linda F. van Dyk ◽  
Eric T. Clambey
Keyword(s):  
T Cells ◽  
Cd4 T Cells ◽  
Fluorescent Protein ◽  
Acute Infection ◽  
Cell Types ◽  
T Cell Subsets ◽  
High Dimensional ◽  
Murine Gammaherpesvirus ◽  
Green Fluorescent

Interleukin (IL)-10 is a potent immunomodulatory cytokine produced by multiple cell types to restrain immune activation. Many herpesviruses use the IL-10 pathway to facilitate infection, but how endogenous IL-10 is regulated during primary infection in vivo remains poorly characterized. Here, we infected mice with murine gammaherpesvirus 68 (γHV68) and analyzed the production, and genetic contribution, of IL-10 using mass cytometry (cytometry by time-of-flight,CyTOF) analysis. γHV68 infection elicited a breadth of effector CD4 T cells in the lungs of acutely infected mice, including a highly activated effector subset that co-expressed IFNγ, TNFα, and IL-10. By using IL-10 green fluorescent protein (gfp) transcriptional reporter mice, we identified that IL-10 was primarily expressed within CD4 T cells during acute infection in the lungs. IL10gfp expressing CD4 T cells were highly proliferative and characterized by the expression of multiple co-inhibitory receptors including PD-1 and LAG-3. When we analyzed acute γHV68 infection of IL-10 deficient mice, we found that IL-10 limits the frequency of both myeloid and effector CD4 T cell subsets in the infected lung, with minimal changes at a distant mucosal site. These data emphasize the unique insights that high-dimensional analysis can afford in investigating antiviral immunity, and provide new insights into the breadth, phenotype and function of IL-10 expressing effector CD4 T cells during acute virus infection.

scholarly journals 602 STING agonist-based treatment promotes vascular normalization and tertiary lymphoid structure formation in the therapeutic melanoma microenvironment

2020 ◽  
Vol 8 (Suppl 3) ◽  
pp. A637-A637
Author(s):  
Manoj Chelvanambi ◽  
Ronald Fecek ◽  
Jennifer Taylor ◽  
Walter Storkus
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cd8 T Cells ◽  
Immune Cell ◽  
Ex Vivo ◽  
In Vitro Assays ◽  
Type I ◽  
Vascular Normalization ◽  
S 100 ◽  
Transcriptional Changes

BackgroundThe degree of immune infiltration in tumors, especially CD8+ T cells, greatly impacts patient disease course and response to interventional immunotherapy. Hence, enhancement of TIL prevalence is a preferred clinical endpoint, one that may be achieved via administration of agents that normalize the tumor vasculature (VN) leading to improved immune cell recruitment and/or that induce the development of local tertiary lymphoid structures (TLS) within the tumor microenvironment (TME).MethodsLow-dose STING agonist ADU S-100 (5 μg/mouse) was delivered intratumorally to established s.c. B16.F10 melanomas on days 10, 14 and 17 post-tumor inoculation under an IACUC-approved protocol. Treated and control, untreated tumors were isolated at various time points to assess transcriptional changes associated with VN and TLS formation via qPCR, with corollary immune cell composition changes determined using flow cytometry and immunofluorescence microscopy. In vitro assays were performed on CD11c+ BMDCs treated with 2.5 μg/mL ADU S-100 (vs PBS control) and associated transcriptional changes analyzed via qPCR or profiled using DNA microarrays. For TCRβ-CDR3 analyses, CDR3 was sequenced from gDNA isolated from enzymatically digested tumors and splenocytes.ResultsWe report that activation of STING within the TME leads to slowed melanoma growth in association with increased production of angiostatic factors including Tnfsf15 (Vegi), Cxcl10 and Angpt1, and TLS inducing factors including Ccl19, Ccl21, Lta, Ltb and Tnfsf14 (Light). Therapeutic responses from intratumoral STING activation were characterized by increased vascular normalization (VN), enhanced tumor infiltration by CD8+ T cells and CD11c+ DCs and local TLS neo-genesis, all of which were dependent on host expression of STING. Consistent with a central role for DC in TLS formation, ex vivo ADU S-100-activated mCD11c+ DCs also exhibited upregulated expression of TLS promoting factors including lymphotoxin-α (LTA), IL-36, inflammatory chemokines and type I interferons. TLS formation was associated with the development of a therapeutic TIL TCR repertoire enriched in T cell clonotypes uniquely detected within the tumor but not the peripheral circulation in support or local T cell cross-priming within the TME.ConclusionsOur data support the premise that i.t. delivery of STING agonist promotes a pro-inflammatory TME in support of VN and TLS formation, leading to the local expansion of unique TIL repertoire in association with superior anti-melanoma efficacy.

scholarly journals 413 GEN-009, a personalized neoantigen vaccine, elicits robust immune responses in individuals with advanced or metastatic solid tumors

2020 ◽  
Vol 8 (Suppl 3) ◽  
pp. A438-A438
Author(s):  
Mara Shainheit ◽  
Devin Champagne ◽  
Gabriella Santone ◽  
Syukri Shukor ◽  
Ece Bicak ◽  
...  
Keyword(s):  
Clinical Trial ◽  
T Cells ◽  
T Cell ◽  
Immune Responses ◽  
Solid Tumors ◽  
Ex Vivo ◽  
T Cell Responses ◽  
Checkpoint Blockade ◽  
T Cell Subsets ◽  
Cell Responses

BackgroundATLASTM is a cell-based bioassay that utilizes a cancer patient‘s own monocyte-derived dendritic cells and CD4+ and CD8+ T cells to screen their mutanome and identify neoantigens that elicit robust anti-tumor T cell responses, as well as, deleterious InhibigensTM.1 GEN-009, a personalized vaccine comprised of 4–20 ATLAS-identified neoantigens combined with Hiltonol®, harnesses the power of neoantigen-specific T cells to treat individuals with solid tumors. The safety and efficacy of GEN-009 is being assessed in a phase 1/2a clinical trial (NCT03633110).MethodsA cohort of 15 adults with solid tumors were enrolled in the study. During the screening period, patients received standard of care PD-1-based immunotherapies appropriate for their tumor type. Subsequently, patients were immunized with GEN-009 with additional doses administered at 3, 6, 12, and 24 weeks. Peripheral blood mononuclear cells (PBMCs) were collected at baseline, pre-vaccination (D1), as well as 29, 50, 92, and 176 days post first dose. Vaccine-induced immunogenicity and persistence were assessed by quantifying neoantigen-specific T cell responses in ex vivo and in vitro stimulation dual-analyte fluorospot assays. Polyfunctionality of neoantigen-specific T cells was evaluated by intracellular cytokine staining. Additionally, potential correlations between the ATLAS-identified profile and vaccine-induced immunogenicity were assessed.ResultsGEN-009 augmented T cell responses in 100% of evaluated patients, attributable to vaccine and not checkpoint blockade. Furthermore, neoantigen-induced secretion of IFNγ and/or TNFα by PBMCs, CD4+, and CD8+ T cells was observed in all patients. Responses were primarily from polyfunctional TEM cells and detectable in both CD4+ and CD8+ T cell subsets. Some patients had evidence of epitope spreading. Unique response patterns were observed for each patient with no apparent relationship between tumor types and time to emergence, magnitude or persistence of response. Ex vivo vaccine-induced immune responses were observed as early as 1 month, and in some cases, persisted for 176 days. Clinical efficacy possibly attributable to GEN-009 was observed in several patients, but no correlation has yet been identified with neoantigen number or magnitude of immune response.ConclusionsATLAS empirically identifies stimulatory neoantigens using the patient‘s own immune cells. GEN-009, which is comprised of personalized, ATLAS-identified neoantigens, elicits early, long-lasting and polyfunctional neoantigen-specific CD4+ and CD8+ T cell responses in individuals with advanced cancer. Several patients achieved clinical responses that were possibly attributable to vaccine; efforts are underway to explore T cell correlates of protection. These data support that GEN-009, in combination with checkpoint blockade, represents a unique approach to treat solid tumors.AcknowledgementsWe are grateful to the patients and their families who consented to participate in the GEN-009-101 clinical trial.Trial RegistrationNCT03633110Ethics ApprovalThis study was approved by Western Institutional Review Board, approval number 1-1078861-1. All subjects contributing samples provided signed individual informed consent.ReferenceDeVault V, Starobinets H, Adhikari S, Singh S, Rinaldi S, Classon B, Flechtner J, Lam H. Inhibigens, personal neoantigens that drive suppressive T cell responses, abrogate protection of therapeutic anti-tumor vaccines. J. Immunol 2020; 204(1 Supplement):91.15.

823 CD4 T cells are essential for an anti-tumor effect in a B78 murine melanoma tumor model

2020 ◽  
Vol 8 (Suppl 3) ◽  
pp. A873-A873
Author(s):  
Arika Feils ◽  
Mackenzie Heck ◽  
Anna Hoefges ◽  
Peter Carlson ◽  
Luke Zangl ◽  
...  
Keyword(s):  
T Cells ◽  
Tumor Growth ◽  
Cd4 T Cells ◽  
Tumor Response ◽  
Immune Cell ◽  
Cd8 T Cell ◽  
Mhc I ◽  
Mhc Ii ◽  
Flow Cytometric

BackgroundMice bearing B78 melanoma tumors can be cured using an in situ vaccine (ISV) regimen that includes radiation (RT) together with immunocytokine (tumor-targeting mAb conjugated to IL-2). B78 melanoma cells, derived from B16 cells, express minimal to no MHC-I but express MHC-II upon IFN-g/TNF-a stimulation. Although B78 cells are primarily MHC-I-deficient, an increased CD8 T cell infiltration into the tumor microenvironment (TME) has been shown following ISV.1 To further investigate the potential role of specific immune cell lineages in the B78 anti-tumor response to ISV, immune subset depletion studies and flow cytometric analyses were performed.MethodsC57BL/6 mice bearing B78 tumors were depleted of immune cell subsets with mAbs (anti-CD4, anti-CD8, anti-NK1.1, or Rat IgG control) for 3 weeks during the course of treatment. Treatment groups included no treatment, RT (12 Gy), or ISV (RT D0 and immunocytokine D5-D9). 6 mice/group (repeated three times) were followed for survival/tumor growth, and flow cytometry studies included 4 mice/group, sacrificed on D8 and D13 following the start of ISV.ResultsMice depleted of CD4 T cells during the course of ISV showed a significant reduction of anti-tumor effect as compared to mice treated with ISV/Rat IgG (pConclusionsThese studies suggest that CD4 T cells are essential for an anti-tumor response in the B78 melanoma model. In vivo depletion data show that CD4 T cells, but not CD8 or NK cells, are required for a decrease in tumor growth via ISV. Flow cytometric analyses suggest an interplay between CD4 and CD8 T cells as indicated by a decrease in CD8/IFN-g expression following ISV in the absence of CD4 T cells. The role that MHC-I and MHC-II expression plays in this CD4/CD8 T cell anti-tumor response is under investigation. In future studies, B78 melanoma may serve as a critical syngeneic model for development of more effective immunotherapy treatment regimens.Ethics ApprovalAll animal experiments were performed in accordance with protocols approved by Animal Care and Use Committees of the University of Wisconsin-Madison.ReferenceMorris Z, Guy E, Francis D, et al. In situ tumor vaccination by combining local radiation and tumor-specific antibody or immunocytokine treatments. Cancer Res 2016;76(13):3929-3941.

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