In Vivo Induction of Type 1-Like Regulatory T Cells Using Genetically Modified B Cells Confers Long-Term IL-10-Dependent Antigen-Specific Unresponsiveness

BackgroundUnmethylated CpG-DNA is a potent ligand for the endosomal Toll-like-receptor-9, important for the immune activation to pathogen-associated molecules.1 CpG and other TLR-ligands show effective immunotherapeutic capacities in cancer treatment by inducing an antitumorigenic immunity.2 They are able to reduce tumor progression by reduction of intratumoral secretion of the immunoregulating chemokine CCL223 and subsequent recruitment of immunosuppressive regulatory T cells (Treg), which express CCR4 the only so far known receptor for CCL22.4 Our recent work has shown that CCL22 secretion by dendritic cells (DC) in the lymph node, mediates tolerance by inducing DC-Treg contacts.5 Indeed, in the absence of CCL22, immune responses to vaccination were stronger and resulted in tumor rejection.6 Therefore, we are aiming to investigate the effects of TLR-ligands on systemic CCL22 levels, elucidating all involved mechanisms to identify new targets for cancer immunotherapy.Materials and MethodsT, B and CD11c+ DCs of wildtype (wt) and RAG1-/- mice were isolated from splenocytes by magnetic-activated cell sorting for in vitro assays. Different co-cultures were incubated with CpG and GM-CSF, known as an CCL22 inducer.5 For in vivo experiments, wt mice were treated with CpG, R484 or poly(I:C) alone and in combination with GM-CSF. CCL22-levels in a number of organs were analyzed.ResultsAnalyzing the different immune cell compartments in vitro, we found that DCs in whole splenocytes secrete CCL22 during culture while DC cultured alone showed no CCL22 secretion. When treated with CpG, CCL22-levels were reduced in splenocytes, while it was induced in DC culture alone. The same results were seen when RAG splenocytes, that lack functional B and T cells, were cultured with CpG. CpG treated B cells were able to suppress CCL22 secretion by DC unlike T cells alone. Co-cultures of T and B cells treated with CpG, however, induced the strongest CCL22 suppression in DC. In vivo, we could show that all TLR ligands tested reduced CCL22 in a number of organs significantly. Furthermore, CpG showed the strongest suppression of CCL22 even in the presence of the CCL22 inducer GM-CSF.5ConclusionsWe could show that B cells with T cells mediate CCL22 suppression by TLR ligands. The fact that CpG was able to reduce CCL22 levels even in the presence of the inducer GM-CSF demonstrates the potent CCL22 suppressive capacity of TLR ligands.ReferencesO’Neill LA, et al. The history of toll-like receptors – redefining innate immunity. Nat Rev Immunol 2013;13(6):453–60.Rothenfusser S, et al. Recent advances in immunostimulatory CpG oligonucleotides. Curr Opin Mol Ther 2003;5(2):98–106.Wang S, et al. Intratumoral injection of a CpG oligonucleotide reverts resistance to PD-1 blockade by expanding multifunctional CD8+ T cells. Proc Natl Acad Sci U S A 2016;113(46): E7240–E7249.Rapp M, et al. CCL22 controls immunity by promoting regulatory T cell communication with dendritic cells in lymph nodes. J Exp Med 2019;216(5):1170–1181.Piseddu I, et al. Constitutive expression of CCL22 is mediated by T cell-derived GM-CSF. J Immunol 2020;205(8):2056–2065.Anz D, et al. Suppression of intratumoral CCL22 by type i interferon inhibits migration of regulatory T cells and blocks cancer progression. Cancer Res 2015;75(21):4483–93.Disclosure InformationJ. Grün: None. I. Piseddu: None. C. Perleberg: None. N. Röhrle: None. S. Endres: None. D. Anz: None.

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In vivo induction of regulatory T cells for immune tolerance in hemophilia

Cellular Immunology ◽

10.1016/j.cellimm.2015.10.001 ◽

2016 ◽

Vol 301 ◽

pp. 18-29 ◽

Cited By ~ 19

Author(s):

Xiaomei Wang ◽

Cox Terhorst ◽

Roland W. Herzog

Keyword(s):

T Cells ◽

Regulatory T Cells ◽

Immune Tolerance ◽

In Vivo Induction

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Changes in the T and B- Cell Subsets Following Treatment with a CD25-Antibody in Patients with Steroid Refractory GvHD

Blood ◽

10.1182/blood.v124.21.2497.2497 ◽

2014 ◽

Vol 124 (21) ◽

pp. 2497-2497

Author(s):

Geothy Chakupurakal ◽

Maria Garcia- Marquez ◽

Alexander Shimabukuro- Vornhagen ◽

Hans Anton Schloesser ◽

Udo Holtick ◽

...

Keyword(s):

T Cells ◽

B Cells ◽

B Cell ◽

Interleukin 2 ◽

Unrelated Donors ◽

B Cell Subsets ◽

Regulatory Type ◽

Steroid Refractory

Abstract Allogeneic stem cell transplantation is the therapeutic option for a variety of malignant and non-malignant haematological diseases. Graft versus Host Disease (GvHD) is a common post transplant complication. In 40% of these patients, GvHD is steroid refractory and associated with a mortality of around 60%. Basiliximab is a chimeric murine –human antibody also selective for interleukin -2 receptor (IL-2R) with a half life of 7 days. It is routinely used as part of the induction therapy in renal transplant recipients to prevent acute rejection following successful phase III studies. Phase 2 studies have demonstrated its superior efficacy in treating patients with steroid refractory GvHD (1). We administered Basiliximab in 14 patients with steroid refractory GvHD with a median age of 41 (range 20-69). M: F 7:7. All patients but one 13/14 received PBSC from unrelated donors and 6/13 had mismatched unrelated donors. Overall response was in the order of 12/14 (85%). One patient could not be assessed. 7/14 (50%) achieved a complete response to treatment. We aimed to study the in vivo T- and B-cell changes following Basiliximab administration as this would be an ideal platform to monitor the alterations in the regulatory T and B-cell compartment. PBMCs were obtained from all donors after informed consent, Immucan (Nr 11-116) approved by our local ethics committee, prior to and after weekly administration of Basiliximab 40mg for 4 weeks. Control samples were obtained from patients with steroid responsive acute GvHD. The total number of CD3+ as well as CD4+ and CD8+ T-cells remained constant during treatment and no change was observed on comparison with the controls. Gagliani et al (2) demonstrated that regulatory type 1 T-cells can be identified by the co-expression of CD49b and Lag3. No difference was observed between the % CD49d+, Lag3+ T-cells in the control cohort and the treatment cohort prior to therapy, ie day 0. The % CD49d+, Lag3+ T-cells decreased during the treatment period (statistically significant) in comparison to the control cohort. Despite the use of the CD25-antibody, a small population of CD25+, CD127+ cells could be detected and this population correlated to the % CD49d+, Lag3+ T-cells. Figure 1 Figure 1. Figure 2 Figure 2. The % CD19+, CD20+ B-cells were similar prior to treatment in the treatment group and control. Following the first administration a rise was observed followed by a decline over the next 3 weeks. No changes were seen in the activated (CD20+, CD86+) and anergic B-cell subsets (CD20+, CD21-) during the observation period. The % of CD24high, CD27+ regulatory B-cells were found to be twice that seen in the controls. With treatment a decrease was seen in this population. The CD24high, CD38high transitional B-cells were also found to be higher than that seen in the controls. No change was observed in this subset with treatment. Figure 3 Figure 3. This is the first attempt to study the in-vivo changes induced by a CD25 antibody in patients with steroid refractory GvHD. We conclude that this antibody not only depletes the alloreactive CD25+ T and B-cell population but also alters the regulatory T and B-cell subsets in comparison to patients with steroid responsive GvHD. Our clinical data supports the efficacy of this drug in patients with steroid refractory GvHD. Contrary to the current knowledge that regulatory T-cells are required for GvHD suppression our data suggests that Basiliximab facilitates regulatory T-cell depletion. The reduction of the regulatory T-cell subset observed in patients responding to anti CD25 treatment suggests a complex regulation and potential dichotomous role of these cells in acute GvHD. Schmidt-Hieber M, Fietz T, Knauf W, Uharek L, Hopfenmuller W, Thiel E, et al. Efficacy of the interleukin-2 receptor antagonist basiliximab in steroid-refractory acute graft-versus-host disease. Br J Haematol. 2005 Aug;130(4):568-74.Gagliani N, Magnani CF, Huber S, Gianolini ME, Pala M, Licona-Limon P, et al.Coexpression of CD49b and LAG-3 identifies human and mouse T regulatory type 1 cells. Nat Med. 2013 Jun;19(6):739-46 Disclosures No relevant conflicts of interest to declare.

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Expression of CD10 and CD7 Defines Distinct In Vivo Lymphoid Reconstituting Capacity within Human Cord Blood CD34+CD38-Cells.

Blood ◽

10.1182/blood.v108.11.3184.3184 ◽

2006 ◽

Vol 108 (11) ◽

pp. 3184-3184

Author(s):

Shuro Yoshida ◽

Fumihiko Ishikawa ◽

Leonard D. Shultz ◽

Noriyuki Saito ◽

Mitsuhiro Fukata ◽

...

Keyword(s):

Bone Marrow ◽

T Cells ◽

B Cells ◽

Cord Blood ◽

Nk Cells ◽

Progenitor Cells ◽

Human Cord Blood ◽

Cd34 Cells

Abstract Human cord blood (CB) CD34+ cells are known to contain both long-term hematopoietic stem cells (LT-HSCs) and lineage-restricted progenitor cells. In the past, in vitro studies suggested that CD10, CD7 or CD127 (IL7Ra) could be candidate surface markers that could enrich lymphoid-restricted progenitor cells in human CB CD34+ cells (Galy A, 1995, Immunity; Hao QL, 2001, Blood; Haddad R, 2004, Blood). However, in vivo repopulating capacity of these lymphoid progenitors has not been identified due to the lack of optimal xenogeneic transplantation system supporting development of human T cells in mice. We aim to identify progenitor activity of human CB CD34+ cells expressing CD10/CD7 by using newborn NOD-scid/IL2rgKO transplant assay that can fully support the development of human B, T, and NK cells in vivo (Ishikawa F, 2005, Blood). Although LT-HSCs exist exclusively in Lin-CD34+CD38- cells, not in Lin-CD34+CD38+ cells, CD10 and CD7 expressing cells are present in Lin-CD34+CD38- cells as well as in Lin-CD34+CD38+ cells (CD10+CD7+ cells, CD10+CD7- cells, CD10-CD7+ cells, CD10-CD7- cells accounted for 4.7+/−2.7%, 10.5+/−1.9%, 7.6+/−4.4%, and 77.1+/−5.2% in Lin-CD34+CD38- CB cells, respectively). We transplanted 500–6000 purified cells from each fraction into newborn NOD-scid/IL2rgKO mice, and analyzed the differentiative capacity. CD34+CD38-CD10-CD7- cells engrafted long-term (4–6 months) in recipient mice efficiently (%hCD45+ cells in PB: 30–70%, n=5), and gave rise to all types of human lymphoid and myeloid progeny that included granulocytes, platelets, erythroid cells, B cells, T cells, and NK cells. Successful secondary reconstitution by human CD34+ cells recovered from primary recipient bone marrow suggested that self-renewing HSCs are highly enriched in CD34+CD38–CD10–CD7- cells. CD10–CD7+ cells were present more frequently in CD34+CD38+ cells rather than in CD34+CD38- cells. Transplantation of more than 5000 CD34+CD38+CD10–CD7+ cells, however, resulted in less than 0.5% human cell engraftment in the recipients. Within CD34+CD38–CD10+ cells, the expression of CD7 clearly distinguished the distinct progenitor capacity. At 8 weeks post-transplantation, more than 70% of total human CD45+ cells were T cells in the CD10+CD7+ recipients, whereas less than 30% of engrafted human CD45+ cells were T cells in the CD10+CD7– recipients. In the CD10+CD7- recipients, instead, more CD19+ B cells and HLA–DR+CD33+ cells were present in the peripheral blood, the bone marrow and the spleen. Both CD34+CD38–CD10+CD7+ and CD34+CD38–CD10+CD7- cells highly repopulate recipient thymus, suggesting that these progenitors are possible thymic immigrants. Taken together, human stem and progenitor activity can be distinguished by the expressions of CD7 and CD10 within Lin-CD34+CD38- human CB cells. Xenotransplant model using NOD-scid/IL2rgKO newborns enable us to clarify the heterogeneity of Lin-CD34+CD38- cells in CB by analyzing the in vivo lymphoid reconstitution capacity.

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Naive B cells generate regulatory T cells in the presence of a mature immunologic synapse

Blood ◽

10.1182/blood-2006-10-053793 ◽

2007 ◽

Vol 110 (5) ◽

pp. 1519-1529 ◽

Cited By ~ 119

Author(s):

Peter Reichardt ◽

Bastian Dornbach ◽

Song Rong ◽

Stefan Beissert ◽

Faikah Gueler ◽

...

Keyword(s):

T Cells ◽

B Cells ◽

Regulatory T Cells ◽

Immune Responses ◽

B Cell ◽

Dependent Manner ◽

Naive T Cells ◽

Naïve T Cells ◽

Immunologic Synapse

Abstract Naive B cells are ineffective antigen-presenting cells and are considered unable to activate naive T cells. However, antigen-specific contact of these cells leads to stable cell pairs that remain associated over hours in vivo. The physiologic role of such pairs has not been evaluated. We show here that antigen-specific conjugates between naive B cells and naive T cells display a mature immunologic synapse in the contact zone that is absent in T-cell–dendritic-cell (DC) pairs. B cells induce substantial proliferation but, contrary to DCs, no loss of L-selectin in T cells. Surprisingly, while DC-triggered T cells develop into normal effector cells, B-cell stimulation over 72 hours induces regulatory T cells inhibiting priming of fresh T cells in a contact-dependent manner in vitro. In vivo, the regulatory T cells home to lymph nodes where they potently suppress immune responses such as in cutaneous hypersensitivity and ectopic allogeneic heart transplant rejection. Our finding might help to explain old observations on tolerance induction by B cells, identify the mature immunologic synapse as a central functional module of this process, and suggest the use of naive B-cell–primed regulatory T cells, “bTregs,” as a useful approach for therapeutic intervention in adverse adaptive immune responses.

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