scholarly journals Interleukin-15 enhances proinflammatory T-cell responses in patients with MS and EAE

2020 ◽  
Vol 8 (1) ◽  
pp. e931
Author(s):  
Cyril Laurent ◽  
Gabrielle Deblois ◽  
Marie-Laure Clénet ◽  
Ana Carmena Moratalla ◽  
Negar Farzam-kia ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
T Lymphocytes ◽  
Disease Onset ◽  
T Cell Subsets ◽  
In Vitro Assays ◽  
Gm Csf ◽  
Interleukin 15 ◽  
The Impact

ObjectiveWe posit that interleukin-15 (IL-15) is a relevant contributor to MS pathobiology as this cytokine is elevated in the CNS and periphery of patients with MS. We aim to investigate (1) the impact of IL-15 on T lymphocytes from patients with MS and (2) the in vivo role of IL-15 using the experimental autoimmune encephalomyelitis (EAE) mouse model.MethodsWe compared the impact of IL-15 on T lymphocytes obtained from untreated patients with MS (relapsing-remitting, secondary progressive, and primary progressive) to cells from age/sex-matched healthy controls (HCs) using multiparametric flow cytometry and in vitro assays. We tested the effects of peripheral IL-15 administration after EAE disease onset in C57BL/6 mice.ResultsIL-15 triggered STAT5 signaling in an elevated proportion of T cells from patients with MS compared with HCs. This cytokine also enhanced the production of key proinflammatory cytokines (interferon γ, granulocyte-macrophage colony-stimulating factor [GM-CSF], IL-17, and tumor necrosis factor) by T cells from both MS and controls, but these effects were more robust for the production of IL-17 and GM-CSF in T-cell subsets from patients with MS. At the peak of EAE disease, the proportion of CD4+ and CD8+ T cells expressing CD122+, the key signaling IL-15 receptor chain, was enriched in the CNS compared with the spleen. Finally, peripheral administration of IL-15 into EAE mice after disease onset significantly aggravated clinical scores and increased the number of inflammatory CNS-infiltrating T cells long term after stopping IL-15 administration.ConclusionsOur results underscore that IL-15 contributes to the amplification of T-cell inflammatory properties after disease onset in both MS and EAE.

scholarly journals Quantitative assessment of the pool size and subset distribution of cytolytic T lymphocytes within human resting or alloactivated peripheral blood T cell populations.

1983 ◽  
Vol 158 (2) ◽  
pp. 571-585 ◽  
Author(s):  
A Moretta ◽  
G Pantaleo ◽  
L Moretta ◽  
M C Mingari ◽  
J C Cerottini
Keyword(s):  
Monoclonal Antibody ◽  
T Cells ◽  
T Cell ◽  
T Lymphocytes ◽  
Peripheral Blood ◽  
Great Majority ◽  
T Cell Subsets ◽  
Cytolytic T Lymphocytes ◽  
Subset Distribution

In order to directly assess the distribution of cytolytic T lymphocytes (CTL) and their precursors (CTL-P) in the two major subsets of human T cells, we have used limiting dilution microculture systems to determine their frequencies. The two subsets were defined according to their reactivity (or lack thereof) with B9.4 monoclonal antibody (the specificity of which is similar, if not identical, to that of Leu 2b monoclonal antibody). Both B9+ and B9- cells obtained by sorting peripheral blood resting T cells using the fluorescence-activated cell sorter (FACS) were assayed for total CTL-P frequencies in a microculture system that allows clonal growth of every T cell. As assessed by a lectin-dependent assay, approximately 30% of peripheral blood T cells were CTP-P. In the B9+ subset (which represents 20-30% of all T cells), the CTL-P frequency was close to 100%, whereas the B9- subset had a 25-fold lower CTL-P frequency. It is thus evident that 90% and 10% of the total CTL-P in peripheral blood are confined to the B9+ or B9- T cell subsets, respectively. Analysis of the subset distribution of CTL-P directed against a given set of alloantigens confirmed these findings. CTL-P frequencies were also determined in B9+ and B9- subsets derived from T cells that had been activated in allogenic mixed leucocyte cultures (MLC). Approximately 10% of MLC T cells were CTL-P. This frequency was increased 3.5-fold in the B9+ subset, whereas the B9- subset contained only a small, although detectable number of CTL-P. Moreover, the great majority of the (operationally defined) CTL-P in MLC T cell population were found to be directed against the stimulating alloantigens, thus indicating a dramatic increase in specific CTL-P frequencies following in vitro stimulation in bulk cultures.

Quantitative Assessment of the Impact of Ex Vivo Manipulation on the In Vivo Functionality of Human T Cells in RAG2−/− γc−/− mice.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 450-450
Author(s):  
Rozemarijn S. van Rijn ◽  
Elles R. Simonetti ◽  
Gert Storm ◽  
Mark Bonyhadi ◽  
Anton Hagenbeek ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Ex Vivo ◽  
T Cell Subsets ◽  
Critical Parameters ◽  
In Vivo Evaluation ◽  
Human T Cells ◽  
The Impact

Abstract T cells retrovirally modified to express therapeutic genes encoding cytokines, exogenous TCRs or suicide molecules represent a novel class of immune therapeutics of great potency. However, recent clinical trials using retrovirally-modified T cells have indicated that T cells exhibit a diminished reactivity upon ex vivo manipulation. In addition, virus-specific memory T cells seem to be lost during gene transfer. In a BNML rat model we have shown that the culture procedure is one of the critical parameters. To preserve T cell reactivity, reliable models are required which permit readout of human T cell activity. We recently developed a huPBMC-RAG2−/−γc−/− mouse model for xenogeneic graft-versus-host disease (xGVHD), in which iv injection of 15 x 106 human T cells into RAG2−/−γc−/− mice consistently leads to high level engraftment and lethal xGVHD within 3 weeks in 80% of mice (van Rijn et al, Blood 2003). We have now used this model to analyze in vivo functionality of human T cells following different ex vivo culture procedures. For this, we cultured human T cells for 7 days with either of the two currently available clinically applicable stimulation conditions: 1) via CD3 and 2) via CD3/CD28. In addition, we included CD3/CD28/4-1BB stimulation to explore the effect of extensive costimulation. Mice were injected with escalating doses T cells. HuCD45+ cells in peripheral blood were measured by FACS. Lethal xGVHD occurred at only 6 times (90.106) the dose of fresh cells for CD3-stimulated T cells and 3 times for CD3/28- or CD3/28/4-1BB-stimulated cells. About 20% of surviving mice developed chronic xGVHD, independent of culture method. While lethal xGVHD was always associated with very high levels of engraftment (up to 95%) engraftment levels in chronic mice ranged from 1–75%. To compare the impact of the different culture conditions on in vivo T cell function, we analyzed engraftment potential. The fraction of huCD45+ cells was plotted against the time and the areas under the curves were compared. Based on a total of 68 mice, statistical analysis showed a 2-fold improvement of engraftment potential for C28-costimulated human T cells compared to CD3-stimulated cells (P<0.0001). Additional ligation of 4-1BB did not increase engraftment potential. In addition, different T cell subsets (naïve, memory, effector) were monitored based on the combined expression of CD45RA, CD27 and CCR7. For all primary T cells and variably cultured T cells, a strikingly similar pattern was observed in vivo. After 3 weeks mainly effector and memory effector T cells (both CD4+ and CD8+) could be detected, suggesting a (xeno-)antigen-driven survival and expansion. This was a very consistent observation independent of donor, culture condition, engraftment level or severity of disease. In conclusion, in vitro costimulation preserves in vivo functionality of human T cells and should therefore be included in future clinical protocols for ex vivo manipulation of T cells. These data show the feasibility to use the huPBMC-RAG2−/−γc−/− model for in vivo evaluation of in vitro effects on human T cells. This model is the most sensitive to date for in vivo evaluation of human T cells and will be a promising new tool for the study of human T cells in, for instance, autoimmune disease, cancer and infectious diseases like AIDS.

scholarly journals Ex vivo modelling of PD-1/PD-L1 immune checkpoint blockade under acute, chronic, and exhaustion-like conditions of T-cell stimulation

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Alexander Roberts ◽  
Lindsay Bentley ◽  
Tina Tang ◽  
Fay Stewart ◽  
Chiara Pallini ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
T Cell Activation ◽  
Immune Checkpoint ◽  
Inflammatory Cytokine ◽  
Cell Activation ◽  
Ex Vivo ◽  
In Vitro Assays ◽  
The Impact

AbstractBlockade of PD-1/PD-L1 interactions is proving an exciting, durable therapeutic modality in a range of cancers whereby T cells are released from checkpoint inhibition to revive their inherent anti-tumour activity. Here we have studied various ways to model ex vivo T cell function in order to compare the impact of the clinically utilised anti-PD-1 antibody, pembrolizumab (Keytruda) on the activation of human T cells: focussing on the release of pro-inflammatory IFNγ and anti-inflammatory IL-10 to assess functionality. Firstly, we investigated the actions of pembrolizumab in an acute model of T-cell activation with either immature or mature allogeneic dendritic cells (DCs); pembrolizumab enhanced IFNγ and IL-10 release from purified CD4+ T-cells in the majority of donors with a bias towards pro-inflammatory cytokine release. Next, we modelled the impact of pembrolizumab in settings of more chronic T-cell activation. In a 7-day antigen-specific response to EBV peptides, the presence of pembrolizumab resulted in a relatively modest increase in both IFNγ and IL-10 release. Where pembrolizumab was assessed against long-term stimulated CD4+ cells that had up-regulated the exhaustion markers TIM-3 and PD-1, there was a highly effective enhancement of the otherwise exhausted response to allogeneic DCs with respect to IFNγ production. By contrast, the restoration of IL-10 production was considerably more limited. Finally, to assess a direct clinical relevance we investigated the consequence of PD-1/PD-L1 blockade in the disease setting of dissociated cells from lung and colon carcinomas responding to allogeneic DCs: here, pembrolizumab once more enhanced IFNγ production from the majority of tumour preparations whereas, again, the increase in IL-10 release was modest at best. In conclusion, we have shown that the contribution of PD-1—revealed by using a canonical blocking antibody to interrupt its interaction with PD-L1—to the production of an exemplar pro- and anti-inflammatory cytokine, respectively, depends in magnitude and ratio on the particular stimulation setting and activation status of the target T cell. We have identified a number of in vitro assays with response profiles that mimic features of dissociated cell populations from primary tumours thereby indicating these represent disease-relevant functional assays for the screening of immune checkpoint inhibitors in current and future development. Such in vitro assays may also support patient stratification of those likely to respond to immuno-oncology therapies in the wider population.

scholarly journals Post-thymic T lymphocyte maturation during ontogenesis.

1981 ◽  
Vol 154 (3) ◽  
pp. 581-593 ◽  
Author(s):  
P F Piguet ◽  
C Irle ◽  
E Kollatte ◽  
P Vassalli
Keyword(s):  
T Cells ◽  
T Cell ◽  
T Lymphocytes ◽  
Lectin Binding ◽  
T Cell Subsets ◽  
Proliferating Cells ◽  
Peanut Lectin ◽  
Peripheral T Cells

Peripheral T lymphocytes from newborn (4-6-d-old) mice, isolated from the spleen or lymph nodes, show phenotypic features of immature cortical thymocytes, such as high frequencies of proliferating cells and of peanut lectin-binding cells. These are features of peripheral T cells of recent thymic origin, as shown by in situ labeling of thymocytes and subsequent observation of the migrants to the spleen, which were mainly peanut lectin-binding cells. The function of newborn peripheral T cells was compared, on a per T cell basis, with that of thymocytes and of fully mature peripheral T cells of the adult, using preparations of newborn lymph node cells containing approximately 80% of T lymphocytes. They were strikingly (about 10-fold) less competent than adult T cells in their phytohemagglutinin responsiveness, their capacities to induce a graft vs. host reaction, to proliferate in the mixed lymphocyte reaction, and to help B lymphocytes in a humoral response in vivo and in vitro. In contrast, newborn T lymphocytes were comparable to those of adults in their capacity to generate cytotoxic T lymphocytes. No suppressive effect of newborn T lymphocytes could be demonstrated in several of these assays. These results argue for an asynchronous maturation of two T cell subsets during ontogeny and demonstrate that at least some T lymphocytes leave the thymus as immature T cells resembling cortical thymocytes and further mature at the periphery. Investigation of mice submitted to thymectomy of 5 d of age showed that these incompetent post-thymic T lymphocytes are capable of considerable expansion and maturation in the peripheral lymphoid organs in the absence of a thymic influence.

scholarly journals Supplementing the diet with sodium propionate suppresses the severity of viral immuno-inflammatory lesions

2020 ◽  
Author(s):  
Deepak Sumbria ◽  
Engin Berber ◽  
Barry T Rouse
Keyword(s):  
T Cells ◽  
T Cell ◽  
Inflammatory Cells ◽  
T Cell Subsets ◽  
Sodium Propionate ◽  
Fatty Acid Salt ◽  
Inflammatory Lesions ◽  
Simplex Virus ◽  
The Impact

This report evaluates a dietary manipulation approach to suppress the severity of ocular infections caused by herpes simplex virus infection. The virus causes chronic damage to the cornea that results from a T cell orchestrated inflammatory reaction to the infection. Lesion severity can be limited if cells with regulatory activity predominate over proinflammatory T cells and non-lymphoid inflammatory cells. In this report, we show that this outcome can be achieved by including the short chain fatty acid salt sodium propionate (SP) in the drinking water. Animals given the SP supplement developed significantly reduced ocular lesions than those receiving no supplement. Corneas and lymphoid organs contained fewer CD4 Th1 and Th17 T cells, neutrophils and macrophages than controls, but a higher frequency of regulatory T cells (Treg) was present. The inclusion of SP in cultures to induce CD4 T cell subsets in-vitro reduced the magnitude of Th1 and Th17 responses but expanded Treg induction. Diet manipulation was an effective approach to limit the severity of viral immuno-inflammatory lesions and may be worth exploring as a means to reduce the impact of herpetic lesions in humans. IMPORTANCE Herpetic lesions are a significant problem in several persons and they are difficult to control with therapeutics. Our studies show that the severity of herpetic lesions in a mouse model can be diminished by changing the diet to include increased levels of SCFA which acts to inhibit the involvement of inflammatory T cells. We suggest that changing the diet to include higher levels of SCFA might be a useful approach to reduce the impact of recurrent herpetic lesions in humans.

scholarly journals Donor/Recipient HLA Molecular Mismatch Scores Predict Primary Humoral and Cellular Alloimmunity in Kidney Transplantation

2021 ◽  
Vol 11 ◽  
Author(s):  
Maria Meneghini ◽  
Elena Crespo ◽  
Matthias Niemann ◽  
Alba Torija ◽  
Nuria Lloberas ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
De Novo ◽  
Human Leukocyte ◽  
Hla Typing ◽  
T Cell Subsets ◽  
Kidney Transplant Recipients ◽  
Indirect Pathway ◽  
The Impact

Donor/recipient molecular human leukocyte antigen (HLA) mismatch predicts primary B-cell alloimmune activation, yet the impact on de novo donor-specific T-cell alloimmunity (dnDST) remains undetermined. The hypothesis of our study is that donor/recipient HLA mismatches assessed at the molecular level may also influence a higher susceptibility to the development of posttransplant primary T-cell alloimmunity. In this prospective observational study, 169 consecutive kidney transplant recipients without preformed donor-specific antibodies (DSA) and with high resolution donor/recipient HLA typing were evaluated for HLA molecular mismatch scores using different informatic algorithms [amino acid mismatch, eplet MM, and Predicted Indirectly Recognizable HLA Epitopes (PIRCHE-II)]. Primary donor-specific alloimmune activation over the first 2 years posttransplantation was assessed by means of both dnDSA and dnDST using single antigen bead (SAB) and IFN-γ ELISPOT assays, respectively. Also, the predominant alloantigen presenting pathway priming DST alloimmunity and the contribution of main alloreactive T-cell subsets were further characterized in vitro. Pretransplantation, 78/169 (46%) were DST+ whereas 91/169 (54%) DST−. At 2 years, 54/169 (32%) patients showed detectable DST responses: 23/54 (42%) dnDST and 31/54 (57%) persistently positive (persistDST+). 24/169 (14%) patients developed dnDSA. A strong correlation was observed between the three distinct molecular mismatch scores and they all accurately predicted dnDSA formation, in particular at the DQ locus. Likewise, HLA molecular incompatibility predicted the advent of dnDST, especially when assessed by PIRCHE-II score (OR 1.014 95% CI 1.001–1.03, p=0.04). While pretransplant DST predicted the development of posttransplant BPAR (OR 5.18, 95% CI=1.64–16.34, p=0.005) and particularly T cell mediated rejection (OR 5.33, 95% CI=1.45–19.66, p=0.012), patients developing dnDST were at significantly higher risk of subsequent dnDSA formation (HR 2.64, 95% CI=1.08–6.45, p=0.03). In vitro experiments showed that unlike preformed DST that is predominantly primed by CD8+ direct pathway T cells, posttransplant DST may also be activated by the indirect pathway of alloantigen presentation, and predominantly driven by CD4+ alloreactive T cells in an important proportion of patients. De novo donor-specific cellular alloreactivity seems to precede subsequent humoral alloimmune activation and is influenced by a poor donor/recipient HLA molecular matching.

696 Brentuximab vedotin, a CD30-directed antibody-drug conjugate, selectively depletes activated Tregs in vitro and in vivo

2020 ◽  
Vol 8 (Suppl 3) ◽  
pp. A738-A738
Author(s):  
Bryan Grogan ◽  
Reice James ◽  
Michelle Ulrich ◽  
Shyra Gardai ◽  
Ryan Heiser ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Efflux Pump ◽  
Apoptotic Cell ◽  
T Cell Subsets ◽  
Memory Cells ◽  
Antibody Drug Conjugate ◽  
Selective Depletion

BackgroundRegulatory T cells (Tregs) play an important role in maintaining immune homeostasis, preventing excessive inflammation in normal tissues. In cancer, Tregs hamper anti-tumor immunosurveillance and facilitate immune evasion. Selective targeting of intratumoral Tregs is a potentially promising treatment approach. Orthogonal evaluation of tumor-infiltrating lymphocytes (TILs) in solid tumors in mice and humans have identified CCR8, and several tumor necrosis family receptors (TNFRs), including TNFSFR8 (CD30), as receptors differentially upregulated on intratumoral Tregs compared to normal tissue Tregs and other intratumoral T cells, making these intriguing therapeutic targets.Brentuximab vedotin (BV) is approved for classical Hodgkin lymphoma (cHL) across multiple lines of therapy including frontline use in stage III/IV cHL in combination with doxorubicin, vinblastine, and dacarbazine. BV is also approved for certain CD30-expressing T-cell lymphomas. BV is comprised of a CD30-directed monoclonal antibody conjugated to the highly potent microtubule-disrupting agent monomethyl auristatin E (MMAE).The activity of BV in lymphomas is thought to primarily result from tumor directed intracellular MMAE release, leading to mitotic arrest and apoptotic cell death.The role CD30 plays in normal immune function is unclear, with both costimulatory and proapoptotic roles described. CD30 is transiently upregulated following activation of memory T cells and expression has been linked to highly activated/suppressive IRF4+ effector Tregs.MethodsHere we evaluated the activity of BV on CD30-expressing T cell subsets in vitro and in vivo.ResultsTreatment of enriched T cell subsets with clinically relevant concentrations of BV drove selective depletion of CD30-expressing Tregs > CD30-expressingCD4+ T memory cells, with minimal effects on CD30-expressing CD8+ T memory cells. In a humanized xeno-GVHD model, treatment with BV selectively depleted Tregs resulting in accelerated wasting and robust T cell expansion. The observed differential activity on Tregs is likely attributable to significant increases in CD30 expression and reduced efflux pump activity relative to other T cell subsets. Interestingly, blockade of CD25 signaling prevents CD30 expression on T cell subsets without impacting proliferation, suggesting a link between CD25, the high affinity IL-2 receptor, and CD30 expression.ConclusionsTogether, these data suggest that BV may have an immunomodulatory effect through selective depletion of highly suppressive CD30-expressing Tregs.AcknowledgementsThe authors would like to thank Michael Harrison, PharmD for their assistance in abstract preparation.Ethics ApprovalAnimals studies were approved by and conducted in accordance with Seattle Genetics Institutional Care and Use Committee protocol #SGE-024.

scholarly journals Immune function of the blood-brain barrier: incomplete presentation of protein (auto-)antigens by rat brain microvascular endothelium in vitro.

1990 ◽  
Vol 110 (5) ◽  
pp. 1757-1766 ◽  
Author(s):  
W Risau ◽  
B Engelhardt ◽  
H Wekerle
Keyword(s):  
T Cells ◽  
Endothelial Cells ◽  
T Cell ◽  
T Lymphocytes ◽  
Rat Brain ◽  
Blood Brain Barrier ◽  
T Cell Activation ◽  
Cell Activation ◽  
Ifn Gamma

The endothelial blood-brain barrier (BBB) has a critical role in controlling lymphocyte traffic into the central nervous system (CNS), both in physiological immunosurveillance, and in its pathological aberrations. The intercellular signals that possibly could induce lymphocytes to cross the BBB include immunogenic presentation of protein (auto-)antigens by BBB endothelia to circulating T lymphocytes. This concept has raised much, though controversial, attention. We approached this problem by analyzing in vitro immunospecific interactions between clonal rat T lymphocyte lines with syngeneic, stringently purified endothelial monolayer cultures from adult brain micro-vessels. The rat brain endothelia (RBE) were established from rat brain capillaries using double collagenase digestion, density gradient fractionation and selective cytolysis of contaminating pericytes by anti-Thy 1.1 antibodies and complement. Incubation with interferon-gamma in most of the brain-derived endothelial cells induced Ia-antigens in the cytoplasm and on the cell surface in some of the cells. Before the treatment, the cells were completely Ia-negative. Pericytes were unresponsive to IFN-gamma treatment. When confronted with syngeneic T cell lines specific for protein (auto-)antigens (e.g., ovalbumin and myelin basic protein, MBP), RBE were completely unable to induce antigen-specific proliferation of syngeneic T lymphocytes irrespective of pretreatment with IFN-gamma and of cell density. RBE were inert towards the T cells, and did not suppress T cell activation induced by other "professional" antigen presenting cells (APC) such as thymus-derived dendritic cells or macrophages. IFN-gamma-treated RBE were, however, susceptible to immunospecific T cell killing. They were lysed by MBP-specific T cells in the presence of the specific antigen or Con A. Antigen dependent lysis was restricted by the appropriate (MHC) class II product. We conclude that the interaction of brain endothelial cells with encephalitogenic T lymphocytes may involve recognition of antigen in the molecular context of relevant MHC products, but that this interaction per se is insufficient to initiate the full T cell activation program.

In vitro responses of human CD45R0brightRA− and CD45R0−RAbright T cell subsets and their relationship to memory and naive T cells

1997 ◽  
Vol 27 (9) ◽  
pp. 2383-2390 ◽  
Author(s):  
Joyce L. Young ◽  
Judith M. Ramage ◽  
J. S. Hill Gaston ◽  
Peter C. L. Beverley
Keyword(s):  
T Cells ◽  
T Cell ◽  
T Cell Subsets ◽  
Naive T Cells ◽  
Naïve T Cells

scholarly journals P01.02 TLR-mediated suppression of the CCL22-CCR4 axis as a new target for tumor immunotherapy

2021 ◽  
Vol 9 (Suppl 1) ◽  
pp. A3.2-A4
Author(s):  
J Grün ◽  
I Piseddu ◽  
C Perleberg ◽  
N Röhrle ◽  
S Endres ◽  
...  
Keyword(s):  
T Cells ◽  
Dendritic Cells ◽  
T Cell ◽  
B Cells ◽  
Regulatory T Cells ◽  
Type I ◽  
List Type ◽  
Gm Csf

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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