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International Congress of Immunology 2016
Abstract Book
GPI glycoprotein, CD24, in the presence of the damage-
associated molecular pattern (DAMP) protein, high-mobility
group box 1 (HMGB1) (2).
Objective:
To determine the role of HMGB1 in Siglec-10 binding
and action of CD52.
Methods:
Binding of recombinant CD52-Fc to HMGB1 and Siglec-
10-Fc proteins was performed in microtiter plates and by surface
plasmon resonance. Native proteins associated with CD52-Fc
were analysed by immunoprecipitation and Western blotting of
lysed human Jurkat line and human blood CD4
+
T cells.
Results:
T-cell suppression by CD52-Fc
in vitro
depended on
the presence of native HMGB1 in serum and did not occur in
serum-free medium. Intact HMGB1 or its cytokine-inducing
Box B domain, but not its anti-inflammatory Box A domain,
bound to soluble CD52 with a Kd of ~ 70nM. Binding to HMGB1
significantly enhanced binding of CD52 to Siglec-10. This
interaction impaired phosphorylation of T-cell receptor (TCR)-
associated tyrosine kinases Lck and Zap70, after TCR crosslinking
by anti-CD3 monoclonal antibody. CD52-Fc was recovered from
T cells in a complex with HMGB1 and Siglec-10, and the SH2
domain-containing tyrosine phosphatase
SHP1
.
Conclusion:
CD52 exerts a concerted immunosuppressive
effect first by capturingHMGB1, abrogating its pro-inflammatory
effect, followed by binding to the Siglec-10 receptor to inhibit
TCR phosphorylation.
1) Bandala-Sanchez et al (2013).
Nat Immunol
14: 741-8.
2) Chen et al (2009).
Science
323: 1722-5.
3090
Role of Galectin-3 in influenza virus infection by regulating
host immune responses and IL-1
β
production
Chen, Y.-J.
1
, Wang, S.-F.
2
, Chen, H.-Y.
1,3
, Liu, F.-T.
1,3
1
IBMS, Academia Sinica, Taipei, Taiwan, Republic of China,
2
Kaohsiung Medical University, Department of Medical Laboratory
Science and Biotechnology, Kaohsiung, Taiwan, Republic of
China,
3
UC Davis Medical Center, Department of Dermatology,
Sacramento, United States
H5N1 Virus induces cytokine production and excessive
inflammatory responses that contribute to the pathogenesis
of human H5N1 disease. Galectin-3 is a β-galactoside-binding
animal lectin and has been reported to participate in the host
response to microbial infections. Here we investigated the role
of endogenous galectin-3 in H5N1 virus-induced host immune
responses. We observed that galectin-3 knockout (Gal3KO)
mice were less susceptible to H5N1 infection compared to
wild-type (WT) mice, whereas the viral loads in the lungs were
comparable between the two genotypes. Moreover, we found
that H5N1-infected Gal3KO mice exhibited a lower degree
of lung inflammation, including neutrophil infiltration. In
addition, the levels of the proinflammatory cytokine IL-1β in
both bronchoalveolar larvage fluid and lungs were significantly
reduced in H5N1-infected Gal3KO mice. It has been reported
that influenza virus induces IL-1β production through the
NLRP3 inflammasome pathway. We showed that NLRP3 and
IL-1β mRNA levels and NLRP3 inflammasome components and
pro-IL-1β protein levels were comparable between Gal3KO
and WT bone marrow-derived macrophages (BMMs) infected
with H5N1. However, IL-1β secretion and ASC oligomerization,
an indicator of inflammasome activation, were decreased in
Gal3KO BMMs compared to WT BMMs, in response to H5N1
infection. In addition, the presence of galectin-3 enhanced
the IL-1β processing in HEK293T cells containing reconstituted
NLRP3 inflammasome complex and thus autonomously
secreting IL-1β. Combined, our results suggest that galectin-3
may enhance the pathological effects of H5N1 virus infection by
promoting the host inflammatory response and that galectin-3
may positively regulate IL-1β production by promoting NLRP3
inflammasome activation.
1457
Themis regulates cytokine signals in mature CD8+ T cells
Brzostek, J., Zhao, X., Mehta, M., Gascoigne, N.
National University of Singapore, Department of Microbiology and
Immunology, Singapore, Singapore
Themis plays a critical role in regulation of T cell receptor
(TCR) signal strength during thymic development, through
interaction with phosphatase Shp1
1
. Germline deletion of
Themis leads to severe perturbation of mature T cell numbers
and phenotype, but this is likely to result from the abnormal
thymic development. We have developed a conditional
knockout mouse to investigate the role of Themis in peripheral
T cells.
Post-selectiondeletionofThemis reduces CD8+T cell numbers in
the periphery. Themis-deficient lymphocytes show unimpaired
responses to TCR stimulation. However, Themis-deficient CD8+
T cells show reduction in proliferative responses to common γ
chain cytokines. Since Themis interacts with Shp1, a negative
regulator of IL-4 signalling in peripheral T cells
2
, we investigated
the role Shp1 in controlling common γ chain cytokine responses
in CD8+ T cells. T-cell specific deletion of Shp1 enhances CD8+
T cell responses to common γ chain cytokines. This opposing
effect of Themis and Shp1 deficiency on cytokine responses
suggests that Themis acts as a positive regulator of cytokine
signals, though binding to and sequestering the negative
regulator Shp1 from the cytokine signalling networks. We are
currently investigating this proposedmolecular mechanism and
the physiological significance of Themis-dependent regulation
of cytokine signals the context of T cell responses to bacterial
infections.
References:
1. Fu et al. (2013) Themis sets the signal threshold for positive
and negative selection in T cell development. Nature 504, 441-
445.
2. Johnson et al. (2013) Shp1 regulates T cell homeostasis by
limiting IL-4 signals. JEM 210, 1419-1451.
3310
Interactomics and structure-function analysis of the RLTPR protein
underpin its essential role for costimulation via CD28 in mouse and
human T cells
Malissen, M., Roncagalli, R., Cucchetti, M., Bergot, E., Jarmuzynski,
N., Gregoire, C., Goncalves Menoita, M., Liang, Y., Malissen, B.
CIML, Marseille, France