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

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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MOG extracellular domain (p1–125) triggers elevated frequency of CXCR3+ CD4+ Th1 cells in the CNS of mice and induces greater incidence of severe EAE

Multiple Sclerosis Journal ◽

10.1177/1352458514524086 ◽

2014 ◽

Vol 20 (10) ◽

pp. 1312-1321 ◽

Cited By ~ 5

Author(s):

Jyothi T Mony ◽

Reza Khorooshi ◽

Trevor Owens

Keyword(s):

T Cells ◽

T Cell ◽

Cell Response ◽

Cd4 T Cells ◽

Cd8 T Cell ◽

Interferon Γ ◽

T Cell Response ◽

Receptor Expression ◽

Myelin Proteins ◽

Mhc Ii

Background: Myelin-specific T cells are implicated in multiple sclerosis (MS) and drive experimental autoimmune encephalomyelitis (EAE). EAE is commonly induced with short peptides, whereas in MS, whole myelin proteins are available for immune response. We asked whether immunization with the immunoglobulin-like domain of myelin oligodendrocyte glycoprotein (MOGIgd, residues 1–125) might induce distinct CD4+ T-cell response and/or a stronger CD8+ T-cell response, compared to the 21 amino acid immunodominant MHC II-associating peptide (p35–55). Objectives: Compare both EAE and T-cell responses in C57BL/6 mice immunized with MOGIgd and MOG p35–55. Methods: Cytokine production, and chemokine receptor expression by CD4+ and CD8+ T cells in the mouse central nervous system (CNS), were analyzed by flow cytometry. Results: MOGIgd triggered progression to more severe EAE than MOG p35–55, despite similar time of onset and overall incidence. EAE in MOGIgd-immunized mice was characterized by an increased percentage of CXCR3+ interferon-γ-producing CD4+ T cells in CNS. The CD8+ T-cell response to both immunogens was similar. Conclusions: Increased incidence of severe disease following MOGIgd immunization, accompanied by an increased percentage of CD4+ T cells in the CNS expressing CXCR3 and producing interferon-γ, identifies a pathogenic role for interferon-γ that is not seen when disease is induced with a single Major Histocompatibility Complex (MHC) II-associating epitope.

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The critical role of CD4+ T cells in PD-1 blockade against MHC-II–expressing tumors such as classic Hodgkin lymphoma

Blood Advances ◽

10.1182/bloodadvances.2020002098 ◽

2020 ◽

Vol 4 (17) ◽

pp. 4069-4082

Author(s):

Joji Nagasaki ◽

Yosuke Togashi ◽

Takeaki Sugawara ◽

Makiko Itami ◽

Nobuhiko Yamauchi ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Cd4 T Cells ◽

Critical Role ◽

Cd4 T Cell ◽

Antitumor Effects ◽

Cell Axis ◽

Mhc I ◽

Mhc Ii

Abstract Classic Hodgkin lymphoma (cHL) responds markedly to PD-1 blockade therapy, and the clinical responses are reportedly dependent on expression of major histocompatibility complex class II (MHC-II). This dependence is different from other solid tumors, in which the MHC class I (MHC-I)/CD8+ T-cell axis plays a critical role. In this study, we investigated the role of the MHC-II/CD4+ T-cell axis in the antitumor effect of PD-1 blockade on cHL. In cHL, MHC-I expression was frequently lost, but MHC-II expression was maintained. CD4+ T cells highly infiltrated the tumor microenvironment of MHC-II–expressing cHL, regardless of MHC-I expression status. Consequently, CD4+ T-cell, but not CD8+ T-cell, infiltration was a good prognostic factor in cHL, and PD-1 blockade showed antitumor efficacy against MHC-II–expressing cHL associated with CD4+ T-cell infiltration. Murine lymphoma and solid tumor models revealed the critical role of antitumor effects mediated by CD4+ T cells: an anti-PD-1 monoclonal antibody exerted antitumor effects on MHC-I−MHC-II+ tumors but not on MHC-I−MHC-II− tumors, in a cytotoxic CD4+ T-cell–dependent manner. Furthermore, LAG-3, which reportedly binds to MHC-II, was highly expressed by tumor-infiltrating CD4+ T cells in MHC-II–expressing tumors. Therefore, the combination of LAG-3 blockade with PD-1 blockade showed a far stronger antitumor immunity compared with either treatment alone. We propose that PD-1 blockade therapies have antitumor effects on MHC-II–expressing tumors such as cHL that are mediated by cytotoxic CD4+ T cells and that LAG-3 could be a candidate for combination therapy with PD-1 blockade.

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Self major histocompatibility complex class I antigens expressed solely in lymphoid cells do not induce tolerance in the CD4+ T cell compartment.

Journal of Experimental Medicine ◽

10.1084/jem.184.4.1573 ◽

1996 ◽

Vol 184 (4) ◽

pp. 1573-1578 ◽

Cited By ~ 11

Author(s):

R Schulz ◽

A L Mellor

Keyword(s):

T Cells ◽

T Cell ◽

Cd4 T Cells ◽

Lymphoid Cells ◽

Cd4 T Cell ◽

Cell Compartment ◽

Mhc I ◽

Mhc Ii ◽

Histocompatibility Complex ◽

Self Peptides

Transgenic mice expressing self major histocompatibility complex (MHC) class I (H-2Kb) antigen solely in lymphoid cell lineages do not acquire tolerance to H-2Kb expressed on skin grafts. H-2Kb-specific cytotoxic T cell responses were completely abrogated in these mice, even after they had rejected skin grafts. Moreover, thymocytes expressing T cell receptors that confer H-2Kb reactivity on cytotoxic CD8+ T cells were eliminated. The ability to reject grafts correlated with the presence of a novel population of H-2Kb-reactive CD4+ T cells. At least some of these CD4+ T cells recognize peptides derived from H-2Kb by processing. We conclude that self MHC I antigens induce tolerance in the CD8 T cell compartment via negative selection when expressed exclusively by lymphoid cells. In contrast, tolerance to MHC class II-restricted self peptides derived by processing of such MHC I antigens is not induced in the CD4 T cell compartment. This suggests that effective transfer of self antigens from lymphoid cells to MHC II-positive cells that can process and present them as self peptides to thymocytes or CD4+ T cells does not take place in vivo. Thus, sequestration of self antigens and MHC II molecules in distinct cell types in the thymic microenvironment allows potentially autoreactive and functionally competent CD4+ T cells that recognize cryptic MHC II-restricted self peptides to mature into the peripheral T cell repertoire under normal physiological circumstances.

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CD4+ T Cells Are Able to Promote Tumor Growth through Inhibition of Tumor-Specific CD8+ T-Cell Responses in Tumor-Bearing Hosts

Cancer Research ◽

10.1158/0008-5472.can-04-3344 ◽

2005 ◽

Vol 65 (15) ◽

pp. 6984-6989 ◽

Cited By ~ 22

Author(s):

Annemieke Th. den Boer ◽

Geertje J.D. van Mierlo ◽

Marieke F. Fransen ◽

Cornelis J.M. Melief ◽

Rienk Offringa ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Tumor Growth ◽

Cd4 T Cells ◽

T Cell Responses ◽

Cd8 T Cell ◽

Cell Responses ◽

Tumor Bearing ◽

Promote Tumor Growth

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In Situ Characterization of Human Lymphoid Tissue Immune Cells by Multispectral Confocal Imaging and Quantitative Image Analysis; Implications for HIV Reservoir Characterization

Frontiers in Immunology ◽

10.3389/fimmu.2021.683396 ◽

2021 ◽

Vol 12 ◽

Author(s):

Eirini Moysi ◽

Perla M. Del Rio Estrada ◽

Fernanda Torres-Ruiz ◽

Gustavo Reyes-Terán ◽

Richard A. Koup ◽

...

Keyword(s):

T Cells ◽

B Cells ◽

Immune Cells ◽

Cd4 T Cells ◽

Lymphoid Tissue ◽

Immune Cell ◽

Hiv Reservoir ◽

Human Lymphoid Tissue

CD4 T cells are key mediators of adaptive immune responses during infection and vaccination. Within secondary lymphoid organs, helper CD4 T cells, particularly those residing in germinal centers known as follicular helper T cells (Tfh), provide critical help to B-cells to promote their survival, isotype switching and selection of high affinity memory B-cells. On the other hand, the important role of Tfh cells for the maintenance of HIV reservoir is well documented. Thus, interrogating and better understanding the tissue specific micro-environment and immune subsets that contribute to optimal Tfh cell differentiation and function is important for designing successful prevention and cure strategies. Here, we describe the development and optimization of eight multispectral confocal microscopy immunofluorescence panels designed for in depth characterization and immune-profiling of relevant immune cells in formalin-fixed paraffin-embedded human lymphoid tissue samples. We provide a comprehensive library of antibodies to use for the characterization of CD4+ T-cells -including Tfh and regulatory T-cells- as well as CD8 T-cells, B-cells, macrophages and dendritic cells and discuss how the resulting multispectral confocal datasets can be quantitatively dissected using the HistoCytometry pipeline to collect information about relative frequencies and immune cell spatial distributions. Cells harboring actively transcribed virus are analyzed using an in-situ hybridization assay for the characterization of HIV mRNA positive cells in combination with additional protein markers (multispectral RNAscope). The application of this methodology to lymphoid tissues offers a means to interrogate multiple relevant immune cell targets simultaneously at increased resolution in a reproducible manner to guide CD4 T-cell studies in infection and vaccination.

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In Situ Peptide-MHC-II Tetramer Staining of Antigen-Specific CD4+ T Cells in Tissues

PLoS ONE ◽

10.1371/journal.pone.0128862 ◽

2015 ◽

Vol 10 (6) ◽

pp. e0128862 ◽

Cited By ~ 2

Author(s):

Thamotharampillai Dileepan ◽

Hyeon O. Kim ◽

P. Patrick Cleary ◽

Pamela J. Skinner

Keyword(s):

T Cells ◽

Cd4 T Cells ◽

Mhc Ii ◽

Tetramer Staining

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Assessing the Role of Tissue Infiltrating APCs in Graft-Versus-Host Disease Through Two Photon Intravital Microscopy

Blood ◽

10.1182/blood.v120.21.342.342 ◽

2012 ◽

Vol 120 (21) ◽

pp. 342-342

Author(s):

Christian Wysocki ◽

Sarah Morin-Zorman ◽

David Gonzalez ◽

Ann Haberman ◽

Warren Shlomchik

Keyword(s):

Bone Marrow ◽

T Cells ◽

T Cell ◽

Cd8 T Cells ◽

Cd4 T Cells ◽

Fluorescent Protein ◽

Cd8 T Cell ◽

Skin Lesions ◽

Two Photon

Abstract Abstract 342 Graft-versus-host disease (GVHD) limits the broader application of allogeneic bone marrow transplantation. While initial T cell activation in GVHD occurs predominantly in secondary lymphoid organs, we have consistently observed MHCII+ donor-derived APCs in histopathologic GVHD lesions in tissues such as skin and intestine, frequently adjacent to infiltrating T cells. We hypothesized that productive interactions occur between donor APCs and T cells in situ in GVHD target tissues, which propagate disease. To address this hypothesis we utilized two photon intravital microscopy to analyze interactions between fluorescently labeled donor CD8+ T cells and tissue infiltrating donor dendritic cells (DCs), within skin lesions of living mice in a murine GVHD model. Lethally irradiated 129 mice received T cell-depleted (TCD) marrow from C57BL6 (B6) mice expressing yellow fluorescent protein (YFP) driven by the CD11c promoter (B6-CD11cYFP), B6 red fluorescent protein (RFP)+ CD8+ T cells and WT (unlabeled) B6 CD4+ T cells. The skin of the ear was imaged in anesthetized, living mice, between days 21 and 28 post-transplant, using a LaVision two photon laser scanning microscope, scanning 60uM thick z-stacks every 30 seconds for 1 hour. Individual RFP+ CD8+ T cells were tracked over time throughout the 3 dimensional image. Detailed surface maps of the YFP+ dendritic cell (DC) network were generated. A distance transformation to calculate the distance of each RFP+ CD8+ T cell from the surface of the YFP+ DC network at all times was performed. Through these analyses, we observed both highly motile donor CD8+ T cells making contact with donor DCs (defined as <=2μM between RFP+ CD8+ T cell and YFP+ DC surface), and a proportion which make long term, stable contact (continuous contact between RFP+ CD8+ T cell and YFP+ DC for at least 30 minutes, during which the RFP+ CD8+ T cell speed is below 5.5μM/min). To test whether CD8+ T cell:DC interactions required cognate TCR:MHCI interactions, lethally irradiated 129 mice received a 1:1 mixture of WT B6 and MHCI-deficient B6 BM. In group 1, labeled marrow was from B6-CD11cYFP, and unlabeled marrow from B6-β2-microglobulin (β2m)−/− donors. In group 2, labeled marrow was from B6-CD11cYFP/β2m−/− mice, and unlabeled marrow from B6. In addition to these bone marrow mixtures, all mice received B6 RFP+ CD8+ T cells and unlabeled B6 CD4+ T cells. Mice were imaged as above. Long lasting contacts between donor RFP+ CD8+ T cells and YFP+ donor DCs in skin lesions were less frequent when YFP+ DCs were β2m−/−, and therefore MHCI-deficient. We have also examined whether MHCII-dependent interactions occur between CD4+ T cells and DCs in situ in skin lesions. In preliminary experiments 129 mice received B6-CD11cYFP bone marrow, B6 RFP+ CD4+ T cells, and B6 unlabeled CD8+ T cells. After 1 hour of imaging, mice received anti-MHCII antibody or isotype control and imaging was continued for 2 hours thereafter. RFP+ CD4+ T cell motility increased after anti-MHCII but not after isotype control antibody treatment. Because GVL occurs primarily in BM and spleen, targeting of tissue-infiltrating APCs could represent a unique strategy to ameliorate GVHD while preserving GVL. Disclosures: No relevant conflicts of interest to declare.

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Immune classification of clear cell renal cell carcinoma

Scientific Reports ◽

10.1038/s41598-021-83767-z ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Sumeyye Su ◽

Shaya Akbarinejad ◽

Leili Shahriyari

Keyword(s):

T Cells ◽

Cd8 T Cells ◽

Renal Cell ◽

Cd4 T Cells ◽

Immune Cell ◽

Clear Cell ◽

P Value ◽

Cell Type ◽

Primary Tumors ◽

Immune Cell Type

AbstractSince the outcome of treatments, particularly immunotherapeutic interventions, depends on the tumor immune micro-environment (TIM), several experimental and computational tools such as flow cytometry, immunohistochemistry, and digital cytometry have been developed and utilized to classify TIM variations. In this project, we identify immune pattern of clear cell renal cell carcinomas (ccRCC) by estimating the percentage of each immune cell type in 526 renal tumors using the new powerful technique of digital cytometry. The results, which are in agreement with the results of a large-scale mass cytometry analysis, show that the most frequent immune cell types in ccRCC tumors are CD8+ T-cells, macrophages, and CD4+ T-cells. Saliently, unsupervised clustering of ccRCC primary tumors based on their relative number of immune cells indicates the existence of four distinct groups of ccRCC tumors. Tumors in the first group consist of approximately the same numbers of macrophages and CD8+ T-cells and and a slightly smaller number of CD4+ T cells than CD8+ T cells, while tumors in the second group have a significantly high number of macrophages compared to any other immune cell type (P-value $$<0.01$$ < 0.01 ). The third group of ccRCC tumors have a significantly higher number of CD8+ T-cells than any other immune cell type (P-value $$<0.01$$ < 0.01 ), while tumors in the group 4 have approximately the same numbers of macrophages and CD4+ T-cells and a significantly smaller number of CD8+ T-cells than CD4+ T-cells (P-value $$<0.01$$ < 0.01 ). Moreover, there is a high positive correlation between the expression levels of IFNG and PDCD1 and the percentage of CD8+ T-cells, and higher stage and grade of tumors have a substantially higher percentage of CD8+ T-cells. Furthermore, the primary tumors of patients, who are tumor free at the last time of follow up, have a significantly higher percentage of mast cells (P-value $$<0.01$$ < 0.01 ) compared to the patients with tumors for all groups of tumors except group 3.

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Cd40-Independent Pathways of T Cell Help for Priming of Cd8+ Cytotoxic T Lymphocytes

Journal of Experimental Medicine ◽

10.1084/jem.191.3.541 ◽

2000 ◽

Vol 191 (3) ◽

pp. 541-550 ◽

Cited By ~ 144

Author(s):

Zhengbin Lu ◽

Lingxian Yuan ◽

Xianzheng Zhou ◽

Eduardo Sotomayor ◽

Hyam I. Levitsky ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Cd4 T Cells ◽

Cytotoxic T Lymphocyte ◽

Cell Communication ◽

Cd8 T Cell ◽

Cd4 Help ◽

T Cell Help

In many cases, induction of CD8+ CTL responses requires CD4+ T cell help. Recently, it has been shown that a dominant pathway of CD4+ help is via antigen-presenting cell (APC) activation through engagement of CD40 by CD40 ligand on CD4+ T cells. To further study this three cell interaction, we established an in vitro system using dendritic cells (DCs) as APCs and influenza hemagglutinin (HA) class I and II peptide–specific T cell antigen receptor transgenic T cells as cytotoxic T lymphocyte precursors and CD4+ T helper cells, respectively. We found that CD4+ T cells can provide potent help for DCs to activate CD8+ T cells when antigen is provided in the form of either cell lysate, recombinant protein, or synthetic peptides. Surprisingly, this help is completely independent of CD40. Moreover, CD40-independent CD4+ help can be documented in vivo. Finally, we show that CD40-independent T cell help is delivered through both sensitization of DCs and direct CD4+–CD8+ T cell communication via lymphokines. Therefore, we conclude that CD4+ help comprises at least three components: CD40-dependent DC sensitization, CD40-independent DC sensitization, and direct lymphokine-dependent CD4+–CD8+ T cell communication.

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