Understanding the Immunomodulatory Effect of Mesenchymal Stem Cell Infused In Transplanted Patients with Steroid-Refractory GvHD

Abstract Abstract 2306 In the last few years the usage of third party mesenchymal stem cells (MSC) as therapy for steroid-refractory Graft versus Host Disease (GvHD) is constantly increasing and holds big promises. Nevertheless, at our knowledge, studies on MSC efficacy have been scarcely corroborated by biological analysis of patient response to cell infusion. Here, we report the immunological monitoring of 8 patients (7 male, 1 female; aged 4 to 33 years), with steroid-refractory GvHD (grade II to III), who received MSCs, between August 2009 and June 2010. GvHD presented as acute in 6 cases and chronic in 2 cases. In 5 cases GvHD occurred as a single organ pathology (2 skin, 2 gut, 1 liver), while in 3 cases GvHD had multi-organ involvement (1 liver and oral mucosa, 1 skin and oral/ocular mucosa, 1 skin, gut and liver). All patients received 2 to 3 MSC infusions from third party donors aiming at 1 × 106/kg recipient body weight MSCs for each infusion. After MSC therapy, 2 patients showed complete response, 3 patients showed partial response, whereas 3 patients did not respond to MSC infusion. To better comprehend the immunomodulatory effects of MSC infusions, we studied GvHD plasmatic markers, inflammatory cytokines and CD4+ T-cell subsets circulating in the peripheral blood (PB) of enrolled patients before MSC infusion and at day 7, 14 and 28 after cell therapy. In accordance with clinical observations, in patients responding to MSC infusions, we observed a dramatic decrease of three validated GvHD plasmatic markers TNFRI, IL2Rα and elafin (Paczesny S et al. Blood 2009) to the mean levels of Healthy Donors (HD). In particular, at day 28 after therapy, TNFRI and IL2Rα levels decreased of 2 times (range=1.9-2.4 and range=1.4-2.8, respectively) and elafin levels decreased of 2.5 times (range=1.7-3.6). Partially responding patients showed a transient decrease of TNFRI, IL2Rα and elafin levels, while non responding patients showed stable or even increasing levels of all analysed markers. Moreover, we investigated the effect of MSC infusion on lymphocyte counts. We demonstrated that patients responding to MSC infusion, oppositely to non responders, strongly decreased total and CD4+ lymphocyte counts in the PB (mean total T-cell Fold Decrease (FD)=11.85, range=1.3-116; mean CD4+ T-cell FD=12, range=1.5-116). Interestingly, after MSC infusion, CD4+ T-cell subsets changed significantly: Tregs increased and Th1 and Th17 populations decreased, and a new CD4+ cell subset balance was observed starting from day 7 after therapy. In particular, the mean FD of Th1/Treg ratio was 4.1 (range=4-4.2) and the mean FD of Th17/Treg ratio was 4.7 (range=3.3-6). Correspondingly, patient symptoms also gradually improved, suggesting an association between GvHD clinical course and CD4+ T-cell imbalance, reverted by MSCs in responding patients. In partially responding patients Th1/Treg and Th17/Treg showed a transient decreased and even slightly increased in the case of non responding patients. In accordance with the decrease of Th1 CD4+ T cells in the PB of patients responding to MSC infusion, we observed a valuable decrease of IFNγ plasma concentrations (mean FD=48, range=30-65 in complete responders), which reached the levels typical of HD. In summary, despite its limited size, the present study suggests that MSCs, upon infusion, are able to convert an inflammatory environment to a more physiological one, both at a cellular level, promoting the expansion of circulating Tregs, and at a molecular level, diminishing inflammatory cytokines. Further studies on a larger group of patients, clarifying the mechanisms of action used in vivo by MSC to tune ongoing allo-reactions, will be fundamental to provide the rationale for improving current clinical trials. Disclosures: No relevant conflicts of interest to declare.

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CD4+ T-Cell Plasticity in Non-Infectious Retinal Inflammatory Disease

International Journal of Molecular Sciences ◽

10.3390/ijms22179584 ◽

2021 ◽

Vol 22 (17) ◽

pp. 9584

Author(s):

Yi-Hsing Chen ◽

Sue Lightman ◽

Virginia L. Calder

Keyword(s):

T Cells ◽

T Cell ◽

Cd4 T Cells ◽

Th17 Cells ◽

Cell Subset ◽

T Cell Subsets ◽

Cd4 T Cell ◽

Dual Function ◽

Interleukin 17 ◽

Th1 Cells

Non-infectious uveitis (NIU) is a potentially sight-threatening disease. Effector CD4+ T cells, especially interferon-γ-(IFNγ) producing Th1 cells and interleukin-17-(IL-17) producing Th17 cells, are the major immunopathogenic cells, as demonstrated by adoptive transfer of disease in a model of experimental autoimmune uveitis (EAU). CD4+FoxP3+CD25+ regulatory T cells (Tregs) were known to suppress function of effector CD4+ T cells and contribute to resolution of disease. It has been recently reported that some CD4+ T-cell subsets demonstrate shared phenotypes with another CD4+ T-cell subset, offering the potential for dual function. For example, Th17/Th1 (co-expressing IFNγ and IL-17) cells and Th17/Treg (co-expressing IL-17 and FoxP3) cells have been identified in NIU and EAU. In this review, we have investigated the evidence as to whether these ‘plastic CD4+ T cells’ are functionally active in uveitis. We conclude that Th17/Th1 cells are generated locally, are resistant to the immunosuppressive effects of steroids, and contribute to early development of EAU. Th17/Treg cells produce IL-17, not IL-10, and act similar to Th17 cells. These cells were considered pathogenic in uveitis. Future studies are needed to better clarify their function, and in the future, these cell subsets may in need to be taken into consideration for designing treatment strategies for disease.

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TH17 Pathway and Associated Pro-Inflammatory Cytokines Promote Immune Dysfunction in Myeloma.

Blood ◽

10.1182/blood.v110.11.3517.3517 ◽

2007 ◽

Vol 110 (11) ◽

pp. 3517-3517

Author(s):

Rao Prabhala ◽

Dheeraj Pelluru ◽

Paola Neri ◽

Mariateresa Fulciniti ◽

James J. Driscoll ◽

...

Keyword(s):

T Cell ◽

Immune Function ◽

Inflammatory Cytokines ◽

Th17 Cells ◽

Cell Subset ◽

Immune Dysfunction ◽

T Cell Subsets ◽

Cytokine Network ◽

High Level ◽

Pro Inflammatory Cytokines

Abstract Multiple myeloma (MM) is associated with significant immune dysfunction. Although various mechanisms mediating immune dysregulation in MM have been studied, its molecular and cellular basis is ill defined. IL-6, TGF-β and IL-1β have been implicated in this process, but their mechanism of effects on immune function have not been studied in MM. Together, IL-6 and TGF-β enhance the generation of TH17 cells, important in the development of immunity and auto-immunity. Additionally, TH17 cells are differentiated by number of inflammatory cytokines including, IL-21, IL-22, IL-23, and IL-27. Therefore, we evaluated the immune dysfunction and the role of TH17 cells and associated pro-inflammatory cytokines in myeloma. We have previously characterized that the production of TH1 mediated cytokines including IFN-γ following anti-CD3-mediated activation is significantly lower in myeloma PBMC compared to normal PBMC. We hypothesize that this may be regulated via skewing the immune system towards TH17 pathway. We observed that TH17 cells, measured by intra-cellular flow cytometry, are significantly increased in number in myeloma (16.9%) and MGUS (6.2%) compared to normal (3.3%). Furthermore, we analysed supporting pro-inflammatory cytokine network for the generation of TH17 cells in myeloma, which may be responsible for the observed TH17 skewing of T cell subsets. Sera from MGUS (n=12) and myeloma (n=17) patients were evaluated for the presence of these pro-inflammatory cytokines compared with normal sera (n=6) using ELISA. We observed significant increase in serum IL-21, IL-22 and IL-23 in MGUS (373 pg/ml, 14 pg/ml and 147 pg/ml respectively; p<0.05) and myeloma (296 pg/ml, 12 pg/ml and 215 pg/ml respectively; p<0.05) compared with normal (63 pg/ml, 1.5 pg/ml and 39 pg/ml respectively). In addition, we also observed that the myeloma PBMC stimulated in the presence of IL-6 and TGF-β, both of the cytokines present at a high level in myeloma, induced significant IL-23 production compared with normal. Importantly, IL-23 levels were 10 fold higher in myeloma BM samples compared with matching blood samples. These results indicate that the cytokines from myeloma BM microenvironment may be responsible for the observed T cell subset abnormality by favouring TH17 cells via IL-23/IL-21 production. These cytokines thus may be targets to modulate immune responses in myeloma to enhance immune function and devise effective vaccination strategies in the future.

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Complementary Costimulation of Human T Cell Subpopulations by CD28 and CD81

Blood ◽

10.1182/blood.v118.21.1124.1124 ◽

2011 ◽

Vol 118 (21) ◽

pp. 1124-1124

Author(s):

Shoshana Levy ◽

Yael Sagi

Keyword(s):

Signal Transduction ◽

T Cells ◽

T Cell ◽

T Cell Activation ◽

Cell Activation ◽

Conflicts Of Interest ◽

Cell Subset ◽

T Cell Subsets ◽

T Cell Subset ◽

Human T Cell

Abstract Abstract 1124 CD81 is a widely expressed tetraspanin molecule that physically associates with CD4 and CD8 on the surface of human T cells. Coengagement of CD81 and CD3 results in the activation and proliferation of T cells. CD81 also costimulated mouse T cells that lack CD28, suggesting either a redundant or a different mechanism of action. Here we show that CD81 and CD28 have a preference for different subsets of T cells - primary human naïve T cells are better costimulated by CD81, while the memory T cell subsets and Tregs are better costimulated by CD28. The more efficient activation of naïve T cells by CD81 was due to prolonged signal transduction compared to that by CD28. We found that IL-6 played a role in the activation of the naïve T cell subset by CD81. Combined costimulation through both CD28 and CD81 resulted in an additive effect on T cell activation. Thus, these two costimulatory molecules complement each other both in the strength of signal transduction and in T cell subset inclusions. Costimulation via CD81 might be useful for expansion of T cells for adoptive immunotherapy to allow the inclusion of naïve T cells with their broad repertoire. Disclosures: No relevant conflicts of interest to declare.

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THU0039 DECIPHERING DISEASE-RELEVANT T CELL SUBSETS IN RHEUMATOID ARTHRITIS IDENTIFIES A NOVEL CELLULAR SUBSET OF PATHOGENETIC IMPORTANCE IN THERAPEUTIC RESISTANCE.

Annals of the Rheumatic Diseases ◽

10.1136/annrheumdis-2020-eular.5525 ◽

2020 ◽

Vol 79 (Suppl 1) ◽

pp. 232.1-233

Author(s):

M. Nyirenda ◽

I. Mcinnes ◽

C. Goodyear

Keyword(s):

Rheumatoid Arthritis ◽

T Cells ◽

T Cell ◽

Inflammatory Cytokines ◽

Regulatory T Cell ◽

Cell Subset ◽

T Cell Subsets ◽

Healthy Controls ◽

T Cell Subset ◽

Pro Inflammatory Cytokines

Background:Aberrant T cell responses are key in driving autoimmunity and are commonly associated with rheumatoid arthritis (RA). Unravelling pathways of importance in therapeutic partial response and failure is of critical importance, as this will potentially provide new insights into key drivers of immune-mediated pathogenesis.Objectives:To delineate disease-relevant T cell subsets in RA and assess their potential to act as cellular markers amenable to precision medicine approaches, particularly in the context of therapeutic partial or non-response.Methods:FACS-based immunophenotyping and ex-vivo functional response profiles of CD4+CD161+CCR2+CCR5+T cells were performed in peripheral blood mononuclear cells (PBMC) obtained from patients with RA and healthy controls, using previously characterised methodologies. RA patients fulfilled the 2010 ACR/EULAR criteria for RA. All samples were obtained after written consent, with the appropriate ethical approvals in place.Results:RA patients harboured a higher frequency of CCR2+CCR5+cells within the CD4+CD161+T cell compartment compared with healthy controls. In RA patients this T cell subset had a higher proportion of cells that secrete pro-inflammatory cytokines such as IL-17A, GM-CSF, IFN-γ, and TNF. Importantly, the CD4+CD161+CCR2+CCR5+T cell subset was significantly increased in DMARD non-responders compared to both responders and healthy controls. Moreover, in DMARD non-responders, these cells had a propensity to express increased proportions of pro-inflammatory cytokines. Notably, there was also a significant increase in the ratio of effector: regulatory T cell (Teff: Treg) compared to both responders and healthy controls. In addition, the CD4+CD161+CCR2+CCR5+T cell subset was less responsive to suppression by Tregs. In further support of a role for this T cell population in disease pathogenesis, the frequency of CD4+CD161+CCR2+CCR5+T cells significantly correlated with disease activity, as measured by the DAS28 (R2= 0.65; p = 0.003; n=11).Conclusion:Combined, our findings suggest that the CD4+CD161+CCR2+CCR5+T cell subset represents a substantially abnormal T cell subset in RA, exhibiting exaggerated pro-inflammatory responses, numerical abundance relative to Tregs, and resistant to regulation by Tregs. The CD4+CD161+CCR2+CCR5+T cell subset appears to be a marker of therapeutic response status in RA, via its contribution to disease pathology and highlights this subset as a potential therapeutic target in RA.References:[1]McInnes IB, Schett G. The pathogenesis of rheumatoid arthritis.N Engl J Med. 2011;365(23):2205-19.[2]Mexhitaj I, Nyirenda MH, Li R, O’Mahony J, Rezk A, Rozenberg A,et al. Abnormal effector and regulatory T cell subsets in paediatric-onset multiple sclerosis.Brain. 2019;142(3):617-32.[3]Cosmi L, Cimaz R, Maggi L, Santarlasci V, Capone M, Borriello F,et al. Evidence of the transient nature of the Th17 phenotype of CD4+CD161+T cells in the synovial fluid of patients with juvenile idiopathic arthritis.Arthritis Rheum. 2011;63(8):2504-15.Disclosure of Interests:Mukanthu Nyirenda: None declared, Iain McInnes Grant/research support from: Bristol-Myers Squibb, Celgene, Eli Lilly and Company, Janssen, and UCB, Consultant of: AbbVie, Bristol-Myers Squibb, Celgene, Eli Lilly and Company, Gilead, Janssen, Novartis, Pfizer, and UCB, Carl Goodyear: None declared

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Interaction of Human Mesenchymal Stem Cells with Alloantigen-Induced T Lymphocyte Activation Favors the Differentiation of CD4+ T Cell Subsets Expressing CD25 and/or CTLA4 Molecules.

Blood ◽

10.1182/blood.v104.11.2123.2123 ◽

2004 ◽

Vol 104 (11) ◽

pp. 2123-2123

Author(s):

Rita Maccario ◽

Marina Podestà ◽

Antonia Moretta ◽

Angela Cometa ◽

Patrizia Comoli ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

T Cell Activation ◽

Cell Activation ◽

Human Mesenchymal Stem Cells ◽

Lymphocyte Activation ◽

Third Party ◽

T Cell Subsets ◽

Cd4 T Cell ◽

Dose Dependent

Abstract Experimental evidence and preliminary clinical studies have demonstrated that human mesenchymal stem cells (MSCs) display important immune modulatory function of potential relevant interest in the setting of allogeneic hematopoietic stem cell (HSC) transplantation. Effectiveness of MSCs in controlling severe GVHD seems to be related to the immune-regulatory role they play in suppressing alloantigen-specific T-cell activation. Aim of the present study was to extend the analysis of the mechanisms responsible for the immune regulatory effect of interaction between MSCs and alloantigen-specific immune response elicited in vitro in primary and in secondary mixed lymphocyte culture (MLC). At difference with most previously reported studies, we decided to employ non-irradiated MSCs, reasoning that irradiation might impair, beside the proliferative capacity, also the differentiation capability of MSCs and, consequently, alter their interaction pattern with lymphocyte subsets. MSC were added to primary MLC at different doses (MLC-responder-PBMC:MSC ratios = 1:1 and 10:1). Dendritic cell (DC) differentiation, lymphocyte proliferation, alloantigen-specific cytotoxic activity and differentiation of CD4+ T-cell subsets expressing CD25 and/or CTLA4 antigens were assessed in primary and secondary MLC, comparing the effect observed using third-party MSCs with that obtained employing autologous to the MLC-responder (autologous) MSCs. Results demonstrated that human MSCs: (1) strongly inhibit alloantigen-induced DC1 differentiation; (2) down-regulate, in a dose-dependent manner, alloantigen-induced lymphocyte expansion, especially that of CD8+ T cells and of NK lymphocytes; (3) favor the differentiation of CD4+ T cells co-expressing CD25 and/or CTLA4, a phenotype associated with regulatory/suppressive function of immune response; (4) cause a dose-dependent reduction of alloantigen-specific cytotoxic capacity mediated by either cytotoxic T lymphocytes or NK cells; (5) exert more effective suppressive activity on MLC-induced T-cell activation when they are allogeneic rather than autologous with respect to responder cells. In particular, higher percentages of CD4+ and of CD4+CD25+ T cells co-expressing CTLA4+ were detected when third-party, rather than autologous, MSCs were added to MLC. These data suggest that T-cell recognition of alloantigens expressed by MSCs may further facilitate the preferential differentiation of activated CD4+ T cells expressing CTLA4, a glycoprotein, known to deliver an inhibitory signal to T cells and to mediate apoptosis of previously activated T lymphocytes. Several studies previously demonstrated that MSCs exert inhibitory effect on lymphocyte activation through the release of soluble factors. Our data suggest that the preferential differentiation of CD4+CD25+ regulatory T-cell subsets may be favored by other mechanisms of MSC-mediated inhibition of alloantigen-induced effector cell activation and expansion, and, in turn, these CD4+CD25+ cells contribute to propagate and extend suppressor activity. Altogether, our results provide immunological support to the use of MSCs for prevention of immune complications related to both HSC and solid organ transplantation and to the theory that MSCs are “universal” suppressors of immune reactivity.

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Murine thymic CD4+ T cell subsets: a subset (Thy0) that secretes diverse cytokines and overexpresses the V beta 8 T cell receptor gene family.

Journal of Experimental Medicine ◽

10.1084/jem.176.1.269 ◽

1992 ◽

Vol 176 (1) ◽

pp. 269-274 ◽

Cited By ~ 64

Author(s):

K Hayakawa ◽

B T Lin ◽

R R Hardy

Keyword(s):

T Cell ◽

Gene Family ◽

T Cell Receptor ◽

Receptor Gene ◽

Cell Subset ◽

Cell Receptor ◽

T Cell Subsets ◽

Cd4 T Cell ◽

Clonal Deletion ◽

T Cell Selection

We demonstrate here the presence of a distinct mature CD4+8- T cell subset in mouse thymus. This subset, termed "Thy0," is delineated by the absence of 3G11 expression from about half of the 6C10-/HSAlow/- fraction of CD4+8- thymic cells. Thy0 is detectable from the neonatal period and largely contributes the Th0-type diverse cytokine production previously reported for the HSAlow/-CD4+ thymic population. Further, cells expressing the T cell receptor V beta 8 gene family are found at increasing frequency in Thy0 with age, comprising 40-60% of Thy0 in adult BALB/c mice. This alteration of V beta 8+ cell frequency is unique to Thy0, since no other CD4+ subset in thymus or spleen shows such V beta 8 overusage. All functional CD4+ T cell subsets, including Thy0, show appropriate V beta clonal deletion associated with endogenous superantigens. Thus, it appears that Thy0 is an intrathymically generated secondary cell subset produced after CD4+ T cell selection.

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Single cell sequencing reveals expanded cytotoxic CD4+ T cells and two states of peripheral helper T cells in synovial fluid of ACPA+ RA patients

10.1101/2021.05.28.21255902 ◽

2021 ◽

Author(s):

Alexandra Argyriou ◽

Marc H Wadsworth ◽

Adrian Lendvai ◽

Stephen M Christensen ◽

Aase Hensvold ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Synovial Fluid ◽

Single Cell ◽

Cd4 T Cells ◽

Cell Subset ◽

T Cell Subsets ◽

Cd4 T Cell ◽

T Cell Subset ◽

Single Cell Sequencing

Rheumatoid arthritis is an autoimmune disease affecting the synovial joints where different subsets of CD4+ T cells are suspected to play a pathogenic role. So far, our understanding of the contribution of cytotoxic CD4+ T cells is incomplete, particularly in the context of the recently described peripheral helper T-cell subset (TPH). Here, using single cell sequencing and multi-parameter flow cytometry, we show that cytotoxic CD4+ T cells are enriched in synovial fluid of anti-citrullinated peptides antibody (ACPA)-positive RA patients. We identify two distinct TPH states differentially characterized by the expression of CXCL13 and PRDM1, respectively. Our data reveal that the adhesion G-Protein Coupled Receptor 56 (GPR56), a marker of circulating cytotoxic cells, delineates the synovial TPH CD4+ T-cell subset. At the site of inflammation, GPR56+CD4+ T cells expressed the tissue-resident memory markers LAG-3, CXCR6 and CD69. Further, TCR clonality analysis revealed that most expanded clones in SF are contained within the cytotoxic and the CXCL13+ TPH CD4+ T-cell populations. Finally, the detection of common TCRs between the two TPH and cytotoxic CD4+ T-cell clusters suggest a shared differentiation. Our study provides comprehensive immunoprofiling of the heterogenous T-cell subsets at the site of inflammation in ACPA+ RA and suggests GPR56 as a therapeutic target to modulate TPH cells and cytotoxic CD4+ T cell function

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CD127 expression inversely correlates with FoxP3 and suppressive function of human CD4+ T reg cells

Journal of Experimental Medicine ◽

10.1084/jem.20060772 ◽

2006 ◽

Vol 203 (7) ◽

pp. 1701-1711 ◽

Cited By ~ 1718

Author(s):

Weihong Liu ◽

Amy L. Putnam ◽

Zhou Xu-yu ◽

Gregory L. Szot ◽

Michael R. Lee ◽

...

Keyword(s):

T Cell ◽

Cell Subset ◽

T Cell Subsets ◽

Cd4 T Cell ◽

Surface Markers ◽

Cell Surface Markers ◽

Low Levels ◽

Cd127 Expression ◽

Number Of Cells

Regulatory T (T reg) cells are critical regulators of immune tolerance. Most T reg cells are defined based on expression of CD4, CD25, and the transcription factor, FoxP3. However, these markers have proven problematic for uniquely defining this specialized T cell subset in humans. We found that the IL-7 receptor (CD127) is down-regulated on a subset of CD4+ T cells in peripheral blood. We demonstrate that the majority of these cells are FoxP3+, including those that express low levels or no CD25. A combination of CD4, CD25, and CD127 resulted in a highly purified population of T reg cells accounting for significantly more cells that previously identified based on other cell surface markers. These cells were highly suppressive in functional suppressor assays. In fact, cells separated based solely on CD4 and CD127 expression were anergic and, although representing at least three times the number of cells (including both CD25+CD4+ and CD25−CD4+ T cell subsets), were as suppressive as the “classic” CD4+CD25hi T reg cell subset. Finally, we show that CD127 can be used to quantitate T reg cell subsets in individuals with type 1 diabetes supporting the use of CD127 as a biomarker for human T reg cells.

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