Helper T Cell Response to Factor VIII In Vivo Requires Several Anatomically Distinct Types of Antigen Presenting Cells

Abstract Inhibitor formation is the most serious complication of factor (F)VIII replacement therapy for hemophilia A. It has long been clear that FVIII inhibitors arise in a CD4 + T cell-dependent manner, but early events in the immune response leading to MHC-II presentation and CD4 + T cell activation remain obscure. Several types of antigen presenting cells (APCs) have been implicated in the uptake of FVIII, but their relevance in T cell activation has been unclear. This study aimed to pinpoint the roles of APCs in priming FVIII-specific CD4 + T cells in vivo. Several transgenic strains of mice on the C57BL/6J background were employed to perform in vivo antigen presentation assays involving one intravenous (IV) injection of 5 µg FVIII-OVA fusion protein (FOVA), which contained an MHC-II I-A b peptide epitope of chicken ovalbumin in place of the B domain of FVIII, and an adoptive transfer (via IV injection on the following day) of 5E6 transgenic OT-II CD4 + T cells, which express chicken ovalbumin-specific T cell receptor (TCR). Before adoptive transfers, OT-II T cells were stained with CellTrace Violet (CTV). Spleens were collected for flow cytometry analyses four days after the adoptive transfer. FOVA shows identical specific activity to that of B domain deleted FVIII. CD11c-DTR/GFP mice (which express diphtheria toxin receptor and green fluorescent protein under the CD11c promoter) (n=3) received two intraperitoneal (IP) injections of 100 ng diphtheria toxin (DT) or PBS only (n=3) one day before and on the day of FVIII-OVA injection to deplete CD11c high cells (i.e. dendritic cells, DCs, marginal zone, sinusoidal and metallophilic macrophages, MFs). DT treatment completely abrogated T cell proliferation in the animals (p=0.03), while on average 11.1% of CD4 +CTV + cells from the control animals were progenies of the transferred cells. XCR1-DTRvenus mice (which express DT receptor and the Venus variant of yellow fluorescent protein under the XCR1 promoter) were similarly treated with DT or PBS (n=4/group) one day before FOVA injection and one day after adoptive transfer to deplete type I conventional DCs (which make up ~80% of XCR1 + cells). XCR1 + cell depletion reduced T cell proliferation by ~50% (p=0.02). CD4 +CTV + cells from the DT-treated and control mice included 19.4% and 38.6% progenies, respectively. Hemophilia A (F8e16-/-) mice received 100 µg of marginal zone (MZ) B cell-depleting antibodies anti-CD11a and anti-CD49d (n=4) or isotype control antibodies (n=3) four and two days before FOVA injection. MZ B cell depletion completely abrogated T cell proliferation (p=0.02), while on average 31% of CD4 +CTV + cells from the control animals were progenies of the transferred cells. In another experiment, hemophilia A mice were injected with GdCl 3 (n=4), which inactivates MFs, or PBS (n=3) one day before and on the day of FOVA administration. MF inactivation completely abrogated T cell proliferation in all but 1 animal (p=0.03), while on average 15.9% of CD4 +CTV + cells from the control animals were progenies of the transferred cells. To visualize APC-T cell interactions in vivo, we performed multiphoton (MP) intravital microscopy (IVM) of inguinal lymph nodes (LNs) in CD11c-DTR/GFP mice. The animals received adoptive transfers of 1E7 CTV-stained OT-II CD4 + T cells IV ~24 hours and 5 µg FOVA 20, 5 or 1 hour before IVM delivered intradermally (ID) to target the skin-draining LNs. Control animals received adoptive cell transfer only. For IVM, the LNs were surgically exposed in live, anesthetized animals. Five hours after FOVA injection, multiple CTV + OT-II T cells formed clusters around GFP + cells throughout the T cell zone with several motile T cells surveying the B cell follicle. Twenty hours after FOVA injection, CTV + T cells densely populated and demarcated the T-B border in the LNs. CTV + T cells were nearly absent from the inguinal LNs in the control animals. We propose that activation of CD4 + T cells in response to FVIII requires a complex interaction of multiple types of APCs, which occupy distinct compartments in the secondary lymphoid organs that FVIII antigen traverses en route to cognate CD4 + T cells. In the spleen, where response to FVIII primarily takes place, antigens larger than 60 kDa (such as FVIII at 280 kDa) do not freely flow into the white pulp and instead need to be ferried by APCs. While DCs ultimately activate CD4 + T helper cells, MFs shuttle the antigen to DCs and may also provide innate activation immune signals. Disclosures No relevant conflicts of interest to declare.

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Allergen-Induced C5a/C5aR1 Axis Activation in Pulmonary CD11b+ cDCs Promotes Pulmonary Tolerance through Downregulation of CD40

Cells ◽

10.3390/cells9020300 ◽

2020 ◽

Vol 9 (2) ◽

pp. 300 ◽

Cited By ~ 4

Author(s):

Konstantina Antoniou ◽

Fanny Ender ◽

Tillman Vollbrandt ◽

Yves Laumonnier ◽

Franziska Rathmann ◽

...

Keyword(s):

Cell Proliferation ◽

T Cells ◽

T Cell ◽

T Cell Activation ◽

Cd4 T Cells ◽

Cell Activation ◽

T Cell Proliferation ◽

C5a Receptor ◽

The Impact

Activation of the C5/C5a/C5a receptor 1 (C5aR1) axis during allergen sensitization protects from maladaptive T cell activation. To explore the underlying regulatory mechanisms, we analyzed the impact of C5aR1 activation on pulmonary CD11b+ conventional dendritic cells (cDCs) in the context of house-dust-mite (HDM) exposure. BALB/c mice were intratracheally immunized with an HDM/ovalbumin (OVA) mixture. After 24 h, we detected two CD11b+ cDC populations that could be distinguished on the basis of C5aR1 expression. C5aR1− but not C5aR1+ cDCs strongly induced T cell proliferation of OVA-reactive transgenic CD4+ T cells after re-exposure to antigen in vitro. C5aR1− cDCs expressed higher levels of MHC-II and CD40 than their C5aR1+ counterparts, which correlated directly with a higher frequency of interactions with cognate CD4+ T cells. Priming of OVA-specific T cells by C5aR1+ cDCs could be markedly increased by in vitro blockade of C5aR1 and this was associated with increased CD40 expression. Simultaneous blockade of C5aR1 and CD40L on C5aR1+ cDCs decreased T cell proliferation. Finally, pulsing with OVA-induced C5 production and its cleavage into C5a by both populations of CD11b+ cDCs. Thus, we propose a model in which allergen-induced autocrine C5a generation and subsequent C5aR1 activation in pulmonary CD11b+ cDCs promotes tolerance towards aeroallergens through downregulation of CD40.

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Interleukin 4 production by CD4+ T cells from allergic individuals is modulated by antigen concentration and antigen-presenting cell type.

Journal of Experimental Medicine ◽

10.1084/jem.181.3.1081 ◽

1995 ◽

Vol 181 (3) ◽

pp. 1081-1089 ◽

Cited By ~ 169

Author(s):

H Secrist ◽

R H DeKruyff ◽

D T Umetsu

Keyword(s):

Cell Proliferation ◽

T Cells ◽

T Cell ◽

B Cell ◽

Cd4 T Cells ◽

Cd4 T Cell ◽

Interleukin 4 ◽

T Cell Proliferation ◽

High Concentrations ◽

Antigen Presenting

We have previously shown that CD4+ T cells from allergic individuals are predisposed to produce interleukin (IL)-4 in response to allergens, and that allergen immunotherapy greatly reduced IL-4 production in an allergen-specific fashion. The mechanism that results in the reduction of IL-4 synthesis in treated individuals is unknown, but because clinical improvement during immunotherapy is associated with the administration of the highest doses of allergen, we hypothesized that high concentration of allergen results in the downregulation of IL-4 synthesis in CD4+ T cells. In this report, we demonstrated that CD4+ T cells from allergic donors produced high levels of IL-4 when stimulated with low concentrations of allergen (0.003-0.01 micrograms/ml), particularly when B cell-enriched populations presented the antigen. In contrast, the same responding CD4+ T cell population produced little IL-4 when stimulated with high concentrations of allergen (10-30 micrograms/ml), especially when monocytes were used as antigen-presenting cells (APC). The quantity of IL-4 produced was also found to be inversely related to the extent of proliferation of the CD4+ T cells in response to allergen/antigen; maximal proliferation of CD4+ T cells occurred in response to high concentrations of antigen when IL-4 production was minimal. Antigen presentation by B cell-enriched populations, instead of monocytes, induced less CD4+ T cell proliferation, but induced much greater IL-4 synthesis. Moreover, the addition of increasing numbers of APC (either B cells or monocytes) to cultures containing a constant number of responder T cells resulted in increased T cell proliferation and decreased IL-4 production. These results indicate that the circumstances under which memory T cells are activated, as well as the strength of the proliferative signal to T cells, greatly affect the quantity of IL-4 produced. Thus, our observations that the cytokine profile of allergen-specific memory CD4+ T cells can indeed be modulated by the antigen dose and APC type suggest that methods that preferentially enhance allergen uptake by monocytes and that enhance T cell proliferation will improve the clinical efficacy of immunotherapy in the treatment of allergic disease.

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Critical roles of mTOR Complex 1 and 2 for T follicular helper cell differentiation and germinal center responses

eLife ◽

10.7554/elife.17936 ◽

2016 ◽

Vol 5 ◽

Cited By ~ 48

Author(s):

Jialong Yang ◽

Xingguang Lin ◽

Yun Pan ◽

Jinli Wang ◽

Pengcheng Chen ◽

...

Keyword(s):

Cell Proliferation ◽

T Cells ◽

T Cell ◽

B Cell ◽

Germinal Center ◽

Cd4 T Cells ◽

Cell Formation ◽

T Cell Proliferation ◽

Akt Activation ◽

Follicular Helper

T follicular helper (Tfh) cells play critical roles for germinal center responses and effective humoral immunity. We report here that mTOR in CD4 T cells is essential for Tfh differentiation. In Mtorf/f-Cd4Cre mice, both constitutive and inducible Tfh differentiation is severely impaired, leading to defective germinal center B cell formation and antibody production. Moreover, both mTORC1 and mTORC2 contribute to Tfh and GC B cell development but may do so via distinct mechanisms. mTORC1 mainly promotes CD4 T cell proliferation to reach the cell divisions necessary for Tfh differentiation, while Rictor/mTORC2 regulates Tfh differentiation by promoting Akt activation and TCF1 expression without grossly influencing T cell proliferation. Together, our results reveal crucial but distinct roles for mTORC1 and mTORC2 in CD4 T cells during Tfh differentiation and germinal center responses.

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ATG5 Dependent Autophagy Uncouples T Cell Functions and Modulates Experimental Graft-Versus-Host Disease

Blood ◽

10.1182/blood.v126.23.149.149 ◽

2015 ◽

Vol 126 (23) ◽

pp. 149-149

Author(s):

Katherine Oravecz-Wilson ◽

Corinne Rossi ◽

Chen Liu ◽

Tomomi Toubai ◽

Hiroya Tamaki ◽

...

Keyword(s):

Bone Marrow ◽

Cell Proliferation ◽

T Cells ◽

T Cell ◽

T Cell Activation ◽

Cell Activation ◽

T Cell Proliferation ◽

Apoptotic Proteins

Abstract ATG5 is a key protein that regulates autophagy, a vital cellular process whose role in various immune cells is poorly understood. A recent report showed that the deficiency of autophagy gene Atg16l1 in host DCs increased graft-vs-host disease (GVHD). Nevertheless, the direct role of autophagy in regulating T cell alloreactivity after bone marrow transplant (BMT) is unknown. In order to investigate the role of autophagy in T cells, we first analyzed the changes in autophagosome marker LC3 upon WT T cell activation. TCR stimulation with anti-CD3/CD28, increased cytosolic LC3-I and its membrane-bound LC3-II form. Interestingly, we found that the upregulation of LC3 was predominant in dividing cells, which lead us to hypothesize that autophagy is essential for T cell proliferation. Therefore we next explored if the deficiency in autophagy impaired T cell proliferation utilizing B6 T cell-specific ATG5 knockout (ATG5 KO T cell) mouse and hydroxychloroquine (CQ, a known inhibitor of autophagy). As hypothesized, when compared with WT controls, both CQ treated WT T cells and the ATG5 KO T cells, in vitro, demonstrated a significant decrease in proliferation as demonstrated by 3H-thymidine incorporation and CFSE staining (p<0.0001) and were associated with a failure to upregulate LC3. These effects were observed after anti-CD3/CD28 TCR stimulation as well as following allogenic stimulation in a mixed lymphocyte reaction (MLR) with bone marrow-derived dendritic cells (DCs) from BALB/c mice. The reduction in T cell proliferation was accompanied by a significant increase in apoptosis (p<0.0001). However it was not associated with a decrease in T-helper (TH) signature cytokines (IFNγ, IL-2, IL-17, IL-4) suggesting no impact on T cell differentiation. Furthermore ATG5 deficiency also did not alter T cell activation as determined by upregulation of NFAT and ZAP70. Thus lack of autophagy lead to the decrease survival of T cell along with decreased proliferation after TCR stimulation but did not affect TH differentiation and T cell activation. Given the in vitro observations, we hypothesized that ATG5 KO T cells would also induce less GVHD following allogenic bone marrow transplantation (BMT). Utilizing clinically relevant MHC-mismatched B6 → BALB/c BMT model, we lethally irradiated (800cGy) WT-BALB/c mice and transplanted 5x106 T cell-depleted bone marrow from WT-B6 mice along with 0.5x106 splenic T cells purified from ATG5 KO or WT- B6 mice. WT-BALB/c TCD BM and T cells were used for syngeneic controls. Consistent with in vitro results, ATG5 KO T cells showed decreased proliferation in vivo but showed no difference in Th1/Th17 differentiation. Allogenic animals transplanted with ATG5 KO T cells also showed a significantly improved survival (p=0.001) and reduced GVHD severity (p=0.03). Phenotypic analyses prior to BMT showed that ATG5 KO T cells show decreased CD62L and an increased expression of CD44. Because naïve (CD62L+CD44-) T cells are critical for GVHD, we next explored if the observed improvement in GVHD could be due to a decreased population of these naïve T cells in the transplant inoculum of ATG5 KO animals. Therefore, using the same BMT model and design, we transplanted WT-BALB/c mice 0.5x106 isolated splenic CD62L+ T cells only from either ATG5 KO or WT-B6 animals and observed that GVHD mortality was reduced in the allo-recipients of ATG5 KO T cells compared with WT T cells (p=0.005). To determine the potential molecular mechanism, we next hypothesized that upon activation ATG KO T cells may show alterations in pro and anti-apoptotic proteins. We observed that ATG5 KO failed to increase Bcl-2 level and showed a decrease in Bcl-XL level upon TCR stimulation compared to WT T cells. In contrast to the relative lack of anti-apoptotic proteins, they displayed similar levels of the pro-apoptotic proteins BIM, Bak and Bax. These results suggest that an imbalance between pro- and anti-apoptotic factors is likely a cause for the reduced T cell expansion by ATG5 KO T cells. Our results collectively demonstrate that inhibition of autophagy decreases T cell expansion but not its differentiation in vitro and in vivo. Furthermore contrary to the aggravation of GVHD when autophagy is targeted in host DCs, it mitigated GVHD when targeted in donor T cells. Thus the net impact of manipulating autophagy after allogeneic BMT on GVHD is dependent on which immune cell subsets are being targeted. Figure 1. Figure 1. Figure 2. Figure 2. Disclosures No relevant conflicts of interest to declare.

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Prenatal cadmium exposure alters proliferation in mouse CD4+ T cells via LncRNA Snhg7

10.1101/2021.06.04.446938 ◽

2021 ◽

Author(s):

Jamie L McCall ◽

Melinda E Varney ◽

Sebastian A. Dziadowicz ◽

Casey Hall ◽

Kathryn E Blethen ◽

...

Keyword(s):

Flow Cytometry ◽

Cell Proliferation ◽

T Cells ◽

T Cell ◽

T Cell Activation ◽

Cd4 T Cells ◽

Cell Activation ◽

T Cell Proliferation ◽

Protein Levels ◽

Cd Exposure

Objective: Prenatal cadmium (Cd) exposure leads to immunotoxic phenotypes in the offspring affecting coding and non-coding genes. Recent studies have shown that long non-coding RNAs (lncRNAs) are integral to T cell regulation. Here, we investigated the role of long non-coding RNA small nucleolar RNA hostgene 7 (lncSnhg7) in T cell proliferation. Methods: RNA sequencing was used to analyze the expression of lncRNAs in splenic CD4+ T cells with and without CD3/CD28 stimulation. Next, T cells isolated from offspring exposed to control or Cd water throughout mating and gestation were analyzed with and without stimulation with anti-CD3/CD28 beads. Quantitative qPCR and western blotting were used to detect RNA and protein levels of specific genes. Overexpression of a miR-34a mimic was achieved using nucleofection. Apoptosis was measured using flow cytometry and luminescence assays. Flow cytometry was also used to measure T cell proliferation in culture. Results: We identified 23 lncRNAs that were differentially expressed in stimulated versus unstimulated T cells, including lncSnhg7. LncSnhg7 and a downstream protein, GALNT7, are upregulated in T cells from offspring exposed to Cd during gestation. Overexpression of miR-34a, a regulator of lncSnhg7 and GALNT7, suppresses GALNT7 protein levels in primary T cells, but not in a mouse T lymphocyte cell line. The T cells isolated from Cd-exposed offspring exhibit increased proliferation after activation in vitro, but Treg suppression and CD4+ T cell apoptosis are not affected by prenatal Cd exposure. Conclusion: Prenatal Cd exposure alters the expression of lncRNAs during T cell activation. The induction of lncSnhg7 is enhanced in splenic T cells from Cd offspring resulting in the upregulation of GALNT7 protein and increased proliferation following activation. miR-34a overexpression decreased GALNT7 expression suggesting that the lncSnhg7/miR-34a/GALNT7 is an important pathway in primary CD4+ T cells. These data highlight the need to understand the consequences of environmental exposures on lncRNA functions in non-cancerous cells as well as the effects in utero.

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CAR T Cells Secreting IL18 Augment Antitumor Immunity and Increase T Cell Proliferation and Costimulation

10.1101/111260 ◽

2017 ◽

Cited By ~ 2

Author(s):

Biliang Hu ◽

Jiangtao Ren ◽

Yanping Luo ◽

Brian Keith ◽

Regina M. Young ◽

...

Keyword(s):

Cell Proliferation ◽

T Cells ◽

T Cell ◽

Cd8 T Cells ◽

Cd4 T Cells ◽

Ectopic Expression ◽

T Cell Proliferation ◽

Antitumor Effects

AbstractInterleukin 18 (IL18) is known to induce the expression of interferon-γ (IFNG), but its effects on T cell proliferation and costimulation are not completely understood. In this study, we demonstrate that ectopic expression of IL18 in CART cells caused significant T cell proliferationin vitroandin vivo,and enhanced antitumor effects in xenograft models. Moreover, IL18 mediated T cell expansion required neither tumor antigen nor CAR expression, and produced severe GVHD in NSG mice. Furthermore, recombinant IL18 costimulated IFNG secretion and proliferation of anti-CD3 beads treated T cells. Interestingly, IL18 costimulation could expand purified CD4 T cells, but not CD8 T cells. However, CD8 T cells proliferated greater than CD4 T cells in magnitude within bulk T cells, suggesting CD4 help effect was involved. Using CRISPR/Cas9 gene editing, we confirmed that IL18-driven expansion was both TCR and IL18 receptor (IL18R) dependent. Importantly, we demonstrated that TCR-deficient, IL18-expressing CD19 CART cells exhibited remarkable proliferation and persistent antitumor activity against CD19-expressing tumor cellsin vivo, without eliciting any detectable GVHD symptom. Finally, we describe APACHE T cells, a novel strategy for coupling IL18 expression in CART cells to antigen stimulation, thereby limiting potential toxicity associated with persistent IL18 production. In sum, our study supports human IL18 as a T cell costimulatory cytokine for fueling CART therapy.

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Costimulation by B7 Modulates Specificity of Cytotoxic T Lymphocytes: A Missing Link That Explains Some Bystander T Cell Activation

Journal of Experimental Medicine ◽

10.1084/jem.186.10.1787 ◽

1997 ◽

Vol 186 (10) ◽

pp. 1787-1791 ◽

Cited By ~ 10

Author(s):

Pan Zheng ◽

Yang Liu

Keyword(s):

Cell Proliferation ◽

T Cells ◽

Influenza Virus ◽

T Cell ◽

T Cell Activation ◽

Cell Activation ◽

T Cell Proliferation ◽

Target Cells ◽

Cell Proliferation And Differentiation ◽

Proliferation And Differentiation

It has been proposed that some bystander T cell activation may in fact be due to T cell antigen receptor (TCR) cross-reactivity that is too low to be detected by the effector cytotoxic T lymphocyte (CTL). However, this hypothesis is not supported by direct evidence since no TCR ligand is known to induce T cell proliferation and differentiation without being recognized by the effector CTL. Here we report that transgenic T cells expressing a T cell receptor to influenza virus A/NT/68 nucleoprotein (NP) 366-374:Db complexes clonally expand and become effector CTLs in response to homologous peptides from either A/PR8/34 (H1N1), A/AA/60 (H2N2), or A/NT/68 (H3N2). However, the effector T cells induced by each of the three peptides kill target cells pulsed with NP peptides from the H3N2 and H2N2 viruses, but not from the H1N1 virus. Thus, NP366–374 from influenza virus H1N1 is the first TCR ligand that can induce T cell proliferation and differentiation without being recognized by CTLs. Since induction of T cell proliferation was mediated by antigen-presenting cells that express costimulatory molecules such as B7, we investigated if cytolysis of H1N1 NP peptide–pulsed targets can be restored by expressing B7-1 on the target cells. Our results revealed that this is the case. These data demonstrated that costimulatory molecule B7 modulates antigen specificity of CTLs, and provides a missing link that explains some of the bystander T cell activation.

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Siplizumab, an Anti-CD2 Monoclonal Antibody, Induces a Unique Set of Immune Modulatory Effects Compared to Alemtuzumab and Rabbit Anti-Thymocyte Globulin In Vitro

Frontiers in Immunology ◽

10.3389/fimmu.2020.592553 ◽

2020 ◽

Vol 11 ◽

Author(s):

Christian Binder ◽

Felix Sellberg ◽

Filip Cvetkovski ◽

Erik Berglund ◽

David Berglund

Keyword(s):

Cell Proliferation ◽

T Cells ◽

T Cell ◽

T Cell Activation ◽

Cell Activation ◽

Mixed Lymphocyte Reaction ◽

T Cell Proliferation ◽

Allogeneic Mixed Lymphocyte Reaction ◽

Dependent Cell

Antibodies are commonly used in organ transplant induction therapy and to treat autoimmune disorders. The effects of some biologics on the human immune system remain incompletely characterized and a deeper understanding of their mechanisms of action may provide useful insights for their clinical application. The goal of this study was to contrast the mechanistic properties of siplizumab with Alemtuzumab and rabbit Anti-Thymocyte Globulin (rATG). Mechanistic assay systems investigating antibody-dependent cell-mediated cytotoxicity, antibody-dependent cell phagocytosis and complement-dependent cytotoxicity were used to characterize siplizumab. Further, functional effects of siplizumab, Alemtuzumab, and rATG were investigated in allogeneic mixed lymphocyte reaction. Changes in T cell activation, T cell proliferation and frequency of naïve T cells, memory T cells and regulatory T cells induced by siplizumab, Alemtuzumab and rATG in allogeneic mixed lymphocyte reaction were assessed via flow cytometry. Siplizumab depleted T cells, decreased T cell activation, inhibited T cell proliferation and enriched naïve and bona fide regulatory T cells. Neither Alemtuzumab nor rATG induced the same combination of functional effects. The results presented in this study should be used for further in vitro and in vivo investigations that guide the clinical use of immune modulatory biologics.

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Activation Outcomes Induced in Naïve CD8 T-Cells by Macrophages Primed via “Phagocytic” and Nonphagocytic Pathways

Molecular Biology of the Cell ◽

10.1091/mbc.e07-07-0650 ◽

2008 ◽

Vol 19 (2) ◽

pp. 701-710 ◽

Cited By ~ 10

Author(s):

Isabel María Olazabal ◽

Noa Beatriz Martín-Cofreces ◽

María Mittelbrunn ◽

Gloria Martínez del Hoyo ◽

Balbino Alarcón ◽

...

Keyword(s):

Cell Proliferation ◽

T Cells ◽

T Cell ◽

T Cell Activation ◽

Cd8 T Cells ◽

Cell Activation ◽

T Cell Proliferation ◽

Microtubule Organizing Center ◽

Antigen Uptake ◽

Soluble Peptide

The array of phagocytic receptors expressed by macrophages make them very efficient at pathogen clearance, and the phagocytic process links innate with adaptive immunity. Primary macrophages modulate antigen cross-presentation and T-cell activation. We assessed ex vivo the putative role of different phagocytic receptors in immune synapse formation with CD8 naïve T-cells from OT-I transgenic mice and compared this with the administration of antigen as a soluble peptide. Macrophages that have phagocytosed antigen induce T-cell microtubule-organizing center and F-actin cytoskeleton relocalization to the contact site, as well as the recruitment of proximal T-cell receptor signals such as activated Vav1 and PKCθ. At the same doses of loaded antigen (1 μM), “phagocytic” macrophages were more efficient than peptide-antigen–loaded macrophages at forming productive immune synapses with T-cells, as indicated by active T-cell TCR/CD3 conformation, LAT phosphorylation, IL-2 production, and T-cell proliferation. Similar T-cell proliferation efficiency was obtained when low doses of soluble peptide (3–30 nM) were loaded on macrophages. These results suggest that the pathway used for antigen uptake may modulate the antigen density presented on MHC-I, resulting in different signals induced in naïve CD8 T-cells, leading either to CD8 T-cell activation or anergy.

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Mapping of the autophagosomal degradome identifies IL-7Rα as key cargo in proliferating CD4+ T-cells

10.1101/2021.12.08.471825 ◽

2021 ◽

Author(s):

Dingxi Zhou ◽

Mariana Borsa ◽

Daniel J. Puleston ◽

Susanne Zellner ◽

Jesusa Capera ◽

...

Keyword(s):

Cell Proliferation ◽

T Cells ◽

T Cell ◽

Cd4 T Cells ◽

Surface Expression ◽

T Cell Proliferation ◽

Gamma Chain ◽

Direct Identification ◽

The Common ◽

First Time

CD4+ T cells orchestrate both humoral and cytotoxic immune responses. While it is known that CD4+ T cell proliferation relies on autophagy, direct identification of the autophagosomal cargo involved is still missing. Here, we created a transgenic mouse model, which, for the first time, enables us to directly map the proteinaceous content of autophagosomes in any primary cell by LC3 proximity labelling. IL-7Rα, a cytokine receptor mostly found in naive and memory T cells, was reproducibly detected in autophagosomes of activated CD4+ T cells. Consistently, CD4+ T cells lacking autophagy showed increased IL-7Rα surface expression, while no defect in internalisation was observed. Mechanistically, excessive surface IL-7Rα sequestrates the common gamma chain, impairing the IL-2R assembly and downstream signalling crucial for T cell proliferation. This study provides proof-of-principle that key autophagy substrates can be reliably identified with this model to help mechanistically unravel autophagy's contribution to healthy physiology and disease.

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