In vitro assays for effector T cell functions and activity of immunomodulatory antibodies

Abstract Abstract 4738 Introduction IVIg is known to have immunosuppressive effects in a variety of inflammatory and autoimmune diseases, which may be caused by modulations of T cell functions in treated patients. The mechanisms responsible for these modulations have been mostly investigated using in vitro stimulated T cells. These studies revealed that IVIg inhibited the proliferation of activated T cells possibly by interfering with the secretion of cytokines important for T cell proliferation, such as IL-2. In the present study, we sought to determine the precise mechanism by which IVIg inhibited cytokine secretion by stimulated T cells. Methods Human PBMC and Jurkat T cells were stimulated with PHA. Human T cells purified from PBMC were stimulated with CD3/CD28 T cell expander beads. Cells were cultured in presence or not of 10 mg/ml of IVIg for 24 hours. IL-2 secretion was measured in the culture supernatants by ELISA. IVIg was depleted of PHA-reactive IgG by passage on a PHA-Sepharose column. The extent of depletion was evaluated by ELISA using PHA as capture antigen. The role of F(ab')2 fragments in the inhibitory effect of IVIg on IL-2 secretion was determined using pepsin-generated fragments. Results IVIg inhibited IL-2 secretion by PHA-stimulated T cells, as previously reported. However, the use of increasing concentrations of PHA for T cell stimulation led to a decreased ability of IVIg to inhibit IL-2 production, suggesting that IVIg acted by neutralizing PHA or by competition for receptor occupancy on the cell surface. Pre-incubation of T cells with IVIg followed by washing and addition of PHA did not result in inhibition of IL-2 secretion, indicating that competition for receptor was not involved in this IVIg-mediated inhibition. In contrast, inhibition of IL-2 production by IVIg was completely abrogated using IVIg depleted from PHA-reactive IgG, indicating that IVIg-mediated inhibition of IL-2 secretion was the consequence PHA neutralization. Testing of F(ab')2 fragments of IVIg showed that these fragments bound to PHA and inhibited IL-2 secretion as efficiently as IVIg. Using another activation strategy, we showed that IVIg could also decrease IL-2 secretion by purified human T cells following anti-CD3/CD28 stimulation. Preliminary data using light microscopy indicated that IVIg interfered with the binding of CD3/CD28 beads on T cells, therefore reducing cell activation as evaluated by IL-2 secretion. Conclusion Altogether, our results suggest that the inhibition of T cell responses by IVIg occurs during the cell activation step, by preventing the binding of mitogens or antibody-coated beads on the cell surface. These observations emphasize the importance of ruling out the possible interactions of IVIg with culture medium additives, mitogens or activating agents before deriving strong conclusions on the mechanisms of action of IVIg based on their apparent immunomodulatory effects observed in vitro assays. Disclosures: No relevant conflicts of interest to declare.

Download Full-text

Immune reconstitution following bone marrow transplantation: comparison of recipients of T-cell depleted marrow with recipients of conventional marrow grafts

Blood ◽

10.1182/blood.v73.5.1340.1340 ◽

1989 ◽

Vol 73 (5) ◽

pp. 1340-1350 ◽

Cited By ~ 177

Author(s):

CA Keever ◽

TN Small ◽

N Flomenberg ◽

G Heller ◽

K Pekle ◽

...

Keyword(s):

Bone Marrow ◽

Bone Marrow Transplantation ◽

T Cell ◽

Marrow Transplantation ◽

Lymphoid Cells ◽

In Vitro Assays ◽

Normal Numbers ◽

Cell Functions ◽

The Absolute

Abstract The reconstitution of hematopoietic cells and in vitro assays of immunologic function have been followed in leukemic patients after conventional bone marrow transplantation (BMT) (N = 34) and T-cell depleted BMT (N = 52) from human leukocyte antigen (HLA)-identical sibling donors. No effects of the T-cell depletion could be seen on the recovery of myeloid or lymphoid cells as measured by the day to engraftment or by the absolute number of cells through day 100. Normal numbers of lytically active natural killer cells returned the earliest and were rapidly followed in both groups of patients by the appearance of circulating B cells and normalization of the responses to B-cell mitogens. However, the recovery of normal T-cell proliferative responses were more delayed in recipients of T-cell depleted grafts. Significant quantitative differences were seen only during the first 3 months after transplantation. Neither the number of CD3+ T cells nor the ratio of CD4:CD8 positive cells differed markedly between the two transplant groups. Mitogen-induced immunoglobulin production by peripheral blood lymphocytes (PBL) from patients following T-cell depleted BMT was quantitatively less than that of conventional marrow recipients through the first year, with low normal IgM production reached by 4 to 6 months in both groups. IgG production reached low normal 7 to 9 months after conventional BMT but did not remain at this level until 1 year following either type of transplant. Assessment of the incidence of infections from the day the absolute neutrophil count reached 500 until day 180 after transplant revealed no significant differences between the two groups; indeed, the overall nonleukemic mortality was higher in the recipients of conventional bone marrow. Thus, in our series, the removal of mature cells from the marrow graft did not affect the rate or degree of recovery of myeloid and lymphoid cells but did affect the regeneration of in vitro T-cell dependent functions. We noted early quantitative differences and a delay in the normalization of the T-cell functions measured rather than prolonged absolute deficiencies. The in vitro deficiencies did not result in significant clinically apparent differences between the two groups.

Download Full-text

Immune reconstitution following bone marrow transplantation: comparison of recipients of T-cell depleted marrow with recipients of conventional marrow grafts

Blood ◽

10.1182/blood.v73.5.1340.bloodjournal7351340 ◽

1989 ◽

Vol 73 (5) ◽

pp. 1340-1350 ◽

Cited By ~ 2

Author(s):

CA Keever ◽

TN Small ◽

N Flomenberg ◽

G Heller ◽

K Pekle ◽

...

Keyword(s):

Bone Marrow ◽

Bone Marrow Transplantation ◽

T Cell ◽

Marrow Transplantation ◽

Lymphoid Cells ◽

In Vitro Assays ◽

Normal Numbers ◽

Cell Functions ◽

The Absolute

The reconstitution of hematopoietic cells and in vitro assays of immunologic function have been followed in leukemic patients after conventional bone marrow transplantation (BMT) (N = 34) and T-cell depleted BMT (N = 52) from human leukocyte antigen (HLA)-identical sibling donors. No effects of the T-cell depletion could be seen on the recovery of myeloid or lymphoid cells as measured by the day to engraftment or by the absolute number of cells through day 100. Normal numbers of lytically active natural killer cells returned the earliest and were rapidly followed in both groups of patients by the appearance of circulating B cells and normalization of the responses to B-cell mitogens. However, the recovery of normal T-cell proliferative responses were more delayed in recipients of T-cell depleted grafts. Significant quantitative differences were seen only during the first 3 months after transplantation. Neither the number of CD3+ T cells nor the ratio of CD4:CD8 positive cells differed markedly between the two transplant groups. Mitogen-induced immunoglobulin production by peripheral blood lymphocytes (PBL) from patients following T-cell depleted BMT was quantitatively less than that of conventional marrow recipients through the first year, with low normal IgM production reached by 4 to 6 months in both groups. IgG production reached low normal 7 to 9 months after conventional BMT but did not remain at this level until 1 year following either type of transplant. Assessment of the incidence of infections from the day the absolute neutrophil count reached 500 until day 180 after transplant revealed no significant differences between the two groups; indeed, the overall nonleukemic mortality was higher in the recipients of conventional bone marrow. Thus, in our series, the removal of mature cells from the marrow graft did not affect the rate or degree of recovery of myeloid and lymphoid cells but did affect the regeneration of in vitro T-cell dependent functions. We noted early quantitative differences and a delay in the normalization of the T-cell functions measured rather than prolonged absolute deficiencies. The in vitro deficiencies did not result in significant clinically apparent differences between the two groups.

Download Full-text

Evidence for Associations Between Th1/Th17 “Hybrid” Phenotype and Altered Lipometabolism in Very Severe Graves Orbitopathy

The Journal of Clinical Endocrinology & Metabolism ◽

10.1210/clinem/dgaa124 ◽

2020 ◽

Vol 105 (6) ◽

pp. 1851-1867 ◽

Cited By ~ 2

Author(s):

Sijie Fang ◽

Shuo Zhang ◽

Yazhuo Huang ◽

Yu Wu ◽

Yi Lu ◽

...

Keyword(s):

T Cell ◽

Th17 Cell ◽

Th17 Cells ◽

T Cell Subsets ◽

Th1 Cell ◽

Effector T Cell ◽

Cell Lineages ◽

Graves Orbitopathy ◽

Ifn Γ

Abstract Purpose The purpose of this article is to investigate the characteristics of Th1-cell and Th17-cell lineages for very severe Graves orbitopathy (GO) development. Methods Flow cytometry was performed with blood samples from GO and Graves disease (GD) patients and healthy controls, to explore effector T-cell phenotypes. Lipidomics was conducted with serum from very severe GO patients before and after glucocorticoid (GC) therapy. Immunohistochemistry and Western blotting were used to examine orbital-infiltrating Th17 cells or in vitro models of Th17 polarization. Results In GD, Th1 cells predominated in peripheral effector T-cell subsets, whereas in GO, Th17-cell lineage predominated. In moderate-to-severe GO, Th17.1 cells expressed retinoic acid receptor-related orphan receptor-γt (RORγt) independently and produced interleukin-17A (IL-17A), whereas in very severe GO, Th17.1 cells co-expressed RORγt and Tbet and produced interferon-γ (IFN-γ). Increased IFN-γ–producing Th17.1 cells positively correlated with GO activity and were associated with the development of very severe GO. Additionally, GC therapy inhibited both Th1-cell and Th17-cell lineages and modulated a lipid panel consisting of 79 serum metabolites. However, in GC-resistant, very severe GO, IFN-γ–producing Th17.1 cells remained at a high level, correlating with increased serum triglycerides. Further, retro-orbital tissues from GC-resistant, very severe GO were shown to be infiltrated by CXCR3+ Th17 cells expressing Tbet and STAT4 and rich in triglycerides that promoted Th1 phenotype in Th17 cells in vitro. Conclusions Our findings address the importance of Th17.1 cells in GO pathogenesis, possibly promoting our understanding of the association between Th17-cell plasticity and disease severity of GO.

Download Full-text

Flow cytometric quantitation of calcium-dependent and -independent mitogen-stimulation of T cell functions in whole blood: inhibition by immunosuppressive drugs in vitro

Journal of Immunological Methods ◽

10.1016/s0022-1759(01)00369-6 ◽

2001 ◽

Vol 253 (1-2) ◽

pp. 95-112 ◽

Cited By ~ 64

Author(s):

Markus J Barten ◽

Jan F Gummert ◽

Teun van Gelder ◽

Randi Shorthouse ◽

Randall E Morris

Keyword(s):

T Cell ◽

Whole Blood ◽

Immunosuppressive Drugs ◽

Mitogen Stimulation ◽

Calcium Dependent ◽

Cell Functions ◽

Flow Cytometric ◽

Stimulation Of

Download Full-text

Modulation of T-Cell Functions in KIR2DL3 (CD158b) Transgenic Mice

Blood ◽

10.1182/blood.v94.7.2396.419k17_2396_2402 ◽

1999 ◽

Vol 94 (7) ◽

pp. 2396-2402 ◽

Cited By ~ 26

Author(s):

Anna Cambiaggi ◽

Sylvie Darche ◽

Sophie Guia ◽

Philippe Kourilsky ◽

Jean-Pierre Abastado ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Transgenic Mice ◽

T Cell Activation ◽

Cell Activation ◽

Killer Cell ◽

In Vitro Proliferation ◽

Cell Functions ◽

Double Transgenic Mice

In humans, a minor subset of T cells express killer cell Ig-like receptors (KIRs) at their surface. In vitro data obtained with KIR+ β and γδ T-cell clones showed that engagement of KIR molecules can extinguish T-cell activation signals induced via the CD3/T-cell receptor (TCR) complex. We analyzed the T-cell compartment in mice transgenic for KIR2DL3 (Tg-KIR2DL3), an inhibitory receptor for HLA-Cw3. As expected, mixed lymphocyte reaction and anti-CD3 monoclonal antibody (MoAb)-redirected cytotoxicity exerted by freshly isolated splenocytes can be inhibited by engagement of transgenic KIR2DL3 molecules. In contrast, antigen and anti-CD3 MoAb-induced cytotoxicity exerted by alloreactive cytotoxic T lymphocytes cannot be inhibited by KIR2DL3 engagement. In double transgenic mice, Tg-KIR2DL3 × Tg-HLA-Cw3, no alteration of thymic differentiation could be documented. Immunization of double transgenic mice with Hen egg white lysozime (HEL) or Pigeon Cytochrome-C (PCC) was indistinguishable from immunization of control mice, as judged by recall antigen-induced in vitro proliferation and TCR repertoire analysis. These results indicate that KIR effect on T cells varies upon cell activation stage and show unexpected complexity in the biological function of KIRs in vivo.

Download Full-text

In Vitro Comparison of Three Different Chimeric Receptor-modified Effector T-cell Populations for Leukemia Cell Therapy

Journal of Immunotherapy ◽

10.1097/cji.0b013e31821e763b ◽

2011 ◽

Vol 34 (6) ◽

pp. 469-479 ◽

Cited By ~ 14

Author(s):

Irene Pizzitola ◽

Valentina Agostoni ◽

Elisabetta Cribioli ◽

Martin Pule ◽

Raphael Rousseau ◽

...

Keyword(s):

T Cell ◽

Cell Therapy ◽

Leukemia Cell ◽

Cell Populations ◽

Chimeric Receptor ◽

Effector T Cell ◽

In Vitro Comparison

Download Full-text

The Ubiquitously Expressed Csk Adaptor Protein Cbp Is Dispensable for Embryogenesis and T-Cell Development and Function

Molecular and Cellular Biology ◽

10.1128/mcb.25.23.10533-10542.2005 ◽

2005 ◽

Vol 25 (23) ◽

pp. 10533-10542 ◽

Cited By ~ 59

Author(s):

Marc-Werner Dobenecker ◽

Christian Schmedt ◽

Masato Okada ◽

Alexander Tarakhovsky

Keyword(s):

T Cell ◽

Adaptor Protein ◽

Regulatory Function ◽

Loss Of Function ◽

Thymic Development ◽

Cell Functions ◽

Carboxy Terminal ◽

And Function

ABSTRACT Regulation of Src family kinase (SFK) activity is indispensable for a functional immune system and embryogenesis. The activity of SFKs is inhibited by the presence of the carboxy-terminal Src kinase (Csk) at the cell membrane. Thus, recruitment of cytosolic Csk to the membrane-associated SFKs is crucial for its regulatory function. Previous studies utilizing in vitro and transgenic models suggested that the Csk-binding protein (Cbp), also known as phosphoprotein associated with glycosphingolipid microdomains (PAG), is the membrane adaptor for Csk. However, loss-of-function genetic evidence to support this notion was lacking. Herein, we demonstrate that the targeted disruption of the cbp gene in mice has no effect on embryogenesis, thymic development, or T-cell functions in vivo. Moreover, recruitment of Csk to the specialized membrane compartment of “lipid rafts” is not impaired by Cbp deficiency. Our results indicate that Cbp is dispensable for the recruitment of Csk to the membrane and that another Csk adaptor, yet to be discovered, compensates for the loss of Cbp.

Download Full-text