The Journal of Experimental Medicine
JEM © The Rockefeller University Press
Vol. 202, No. 7, October 3, 2005 955–965
TIM-2 is expressed on B cells and in liver
and kidney and is a receptor for
Thomas T. Chen,
Suzy V. Torti,
Frances M. Brodsky,
William E. Seaman,
Jason G. Cyster,
Eréne C. Niemi,
and Michael R. Daws
Christopher D.C. Allen,
Mary C. Nakamura,
Frank M. Torti,
Veterans Administration Medical Center, San Francisco, CA 94121
Department of Medicine, Department of Microbiology and Immunology,
The G.W. Hooper Foundation and the Departments of Biopharmaceutical Sciences and Pharmaceutical Chemistry, University
of California San Francisco, CA 94143
Department of Cancer Biology, Wake Forest University Health Sciences, Wake Forest University, Winston-Salem, NC 27157
Howard Hughes Medical Institute,
T cell immunoglobulin-domain and mucin-domain (TIM) proteins constitute a receptor family
that was identified first on kidney and liver cells; recently it was also shown to be expressed
on T cells. TIM-1 and -3 receptors denote different subsets of T cells and have distinct
regulatory effects on T cell function. Ferritin is a spherical protein complex that is formed by
24 subunits of H- and L-ferritin. Ferritin stores iron atoms intracellularly, but it also
circulates. H-ferritin, but not L-ferritin, shows saturable binding to subsets of human T and B
cells, and its expression is increased in response to inflammation. We demonstrate that
mouse TIM-2 is expressed on all splenic B cells, with increased levels on germinal center B
cells. TIM-2 also is expressed in the liver, especially in bile duct epithelial cells, and in renal
tubule cells. We further demonstrate that TIM-2 is a receptor for H-ferritin, but not for
L-ferritin, and expression of TIM-2 permits the cellular uptake of H-ferritin into endosomes.
This is the first identification of a receptor for ferritin and reveals a new role for TIM-2.
T cell immunoglobulin-domain and mucin-
domain (TIM) proteins constitute a receptor
family that was identified first on kidney and
liver cells; recently it was also shown to be ex-
pressed on T cells (1–5). In humans, the TIM
receptor family seems to include only three
receptors, TIM-1, -3, and -4, whereas in the
mouse it may include as many as eight (4).
Human TIM-3 and -4 have apparent orthologs
in mice, based on sequence homology, but
human TIM-1 is almost equally homologous to
mouse TIM-1 (41%) and mouse TIM-2 (36%),
which are 66% homologous to each other.
In the mouse, the TIM gene family is
linked to a locus (
hypersensitivity and the production of Th2
cytokines (6). In accord with a role for TIM
receptors in immunity, TIM-1 is expressed
preferentially by Th2 cells, and polymorphisms
) that regulates airway
in human TIM-1 are associated with atopy,
asthma, and rheumatoid arthritis (5–10). Mouse
TIM-1 binds to TIM-4, which is expressed on
antigen-presenting cells, and ligation of TIM-1
potentiates T cell activation (7, 11, 12). In
contrast to TIM-1, TIM-3 is expressed prefer-
entially on Th1 cells. Blockade or loss of
this receptor in mice accelerates autoimmunity,
which suggests that ligation of the receptor is
Although TIM receptors are of evident im-
portance in immunity, their expression outside
of the immune system indicates that these re-
ceptors may have broader functions. Thus, in
primates and rodents, TIM-1 is expressed on
renal tubular cells; in primates, an alternatively
spliced form is expressed on liver cells, where it
has been usurped as a receptor for hepatitis A
(1–3, 16). The functions of TIM receptors on
these nonhematopoietic cells are unknown.
Ferritin is a spherical protein complex that
stores up to 4,000 iron atoms as an oxidized
mineral core (17). It is a heteropolymer that is
T.T. Chen and L. Li contributed equally to this work.
M.R. Daws’ present address is Department of Anatomy,
University of Oslo, Oslo, Norway NO317.
The online version of this article contains supplemental material.
William E. Seaman:
Abbreviations used: GC,
germinal center; IHC, immuno-
histochemistry; RT, room
temperature; TIM, T cell
and mucin domain; TREM,
triggering receptor expressed
on myeloid cells.
TIM-2 IS A B CELL RECEPTOR FOR H-FERRITIN | Chen et al.
formed by 24 subunits of H- and L-ferritin; their ratios vary
in different tissues and in response to iron, growth factors,
inflammation, or malignancy (18). Ferritin primarily is ex-
pressed intracellularly, where it regulates iron mineralization
and sequestration, and thereby buffers reactive oxygen spe-
cies. This effect of H-ferritin is essential for the antiapoptotic
effect of NF-
B, and the transcription of H-ferritin is up-
regulated by NF-
B (19). However, ferritin also circulates,
and earlier evidence suggests that H-ferritin acts as an im-
mune regulator, through binding to subsets of lymphocytes
and myeloid cells. Thus, ferritin inhibits T cell proliferation
in response to mitogens, it impairs the maturation of B cells
in vitro (20, 21), and it has immunosuppressive effects in
vivo (22). Additionally, H-ferritin, but not L-ferritin, shows
saturable binding to subsets of human T and B cells (23–26).
Despite the evidence for H-ferritin receptors on the cell sur-
face, none had been identified.
We demonstrate that TIM-2 is expressed at low levels on
all splenic B cells and is expressed at higher levels on germi-
nal center (GC) B cells. Outside the hematopoietic system,
TIM-2 is expressed in liver, especially in bile duct epithelial
cells, and in renal tubule cells. We further demonstrate that
TIM-2 serves as a selective receptor for H-ferritin, but not
for L-ferritin, and that binding of H-ferritin to TIM-2 leads
to the endocytosis of extracellular H-ferritin. The expression
of a surface receptor for H-ferritin is consistent with a role
for H-ferritin in modulating cell function—beyond its role
in storing iron—and the endocytic function of TIM-2 pro-
vides a new pathway for altering levels of H-ferritin inde-
pendent of gene expression.
TIM-2 is expressed on all splenic B cells, with high levels on
GC B cells
An expressed sequence tag for TIM-2 was isolated from the
database based on its partial homology to the Ig domain of
triggering receptor expressed on myeloid cells
(27). By expression of this cDNA, we prepared a mAb
against the extracellular domain of TIM-2. To define the
levels of TIM-2 on lymphocyte cell subsets, mice were not
immunized or were immunized with T-dependent antigens;
staining with anti–TIM-2 was assessed on subsets that were
defined by their surface phenotype. Studies from unimmu-
nized or immunized mice revealed that although TIM-2 was
expressed on follicular B cells, it was expressed at
higher levels on GC B cells (range 1.6–3.5) (Fig. 1 A). In
contrast, TIM-2 was not detected on T cells (CD4
CD8) (Fig. 1 A).
The expression of TIM-2 in splenic B cells, and its pref-
erential expression in GC B cells was confirmed by quantita-
tive RT-PCR, using SYBR Green. For these studies,
splenic B cell subsets and T cells were isolated by fluores-
cence-activated cell sorting and used to prepare total RNA.
Transcripts for TIM-2 were
B cells than in follicular B cells, whereas levels of TIM-2
transcripts in the marginal zone B cells were between the
10-fold more abundant in GC
two (Fig. 1 B). Little or no transcripts for TIM-2 were de-
tected in splenic T cells.
The preferential expression of TIM-2 on GC B cells
also was evident by immunohistochemistry (IHC). For
these studies, we raised a rabbit antiserum against a peptide
from the cytoplasmic domain of TIM-2. On fixed sections
from spleen, this antiserum demonstrated clusters of TIM-2
cells in the center of lymphoid follicles, although scat-
tered cells were seen elsewhere, including the red pulp (Fig.
2 A, middle panel). No staining was seen with control anti-
serum (Fig. 2 A, left panel); staining by the anti–TIM-2 an-
tiserum was blocked in the presence of the immunizing
peptide, which demonstrated that binding was antigen-spe-
cific (Fig. 2 A, right panel). Immunofluorescent studies
demonstrated that most, if not all, of the TIM-2
cytometric analysis of TIM-2 expression on mouse spleen cells. Spleens
were isolated 8 d after immunization with SRBCs. Each curve is normalized,
so that the peaks are of equal height. Staining of GC cells is more than
twice that of follicular B cells (Fol. B cells), although all B cells stain with
anti–TIM-2, compared with an isotype control mAb (dotted line; the control
shown is for GC B cells, but equivalent staining was seen with control
staining of follicular B cells, not depicted). TIM-2 was not detected on
CD4? or CD8? T cells. The numbers indicate geometric mean fluorescence
intensity (MFI) for TIM-2 staining of each cell subset. (B) Quantitative RT-PCR
analysis of TIM-2 expression. Total RNA was prepared from isolated subsets
of spleen cells (sorted by FACS) or from isolated tissues, and was analyzed
by quantitative RT-PCR. Expression of mRNA is shown as a ratio of TIM-2
mRNA/mRNA of two housekeeping genes, hypoxanthine-guanine phos-
phoribosyltransferase (HPRT) and GAPDH. Fol, follicular; MZ, marginal zone.
TIM-2 is expressed preferentially on GC B cells. (A) Flow
JEM VOL. 202, October 3, 2005
ide-mediated induction of ferritin synthesis in J774 macrophages by in-
flammatory cytokines: role of selective iron regulatory protein-2
downregulation. Blood. 91:1059–1066.
45. Kim, S., and P. Ponka. 2000. Effects of interferon-gamma and li-
popolysaccharide on macrophage iron metabolism are mediated by ni-
tric oxide-induced degradation of iron regulatory protein 2. J. Biol.
46. Wu, H., D.A. Windmiller, L. Wang, and J.M. Backer. 2003. YXXM
motifs in the PDGFbeta receptor serve dual roles as PI 3-kinase bind-
ing motifs and tyrosine-based endocytic sorting signals. J Biol Chem.
278:40425–40428 [erratum published in 278:45040].
47. Zuyderhoudt, F.M., P. Vos, G.G. Jorning, and J. Van Gool. 1985. Fer-
ritin in liver, plasma and bile of the iron-loaded rat. Biochim. Biophys.
48. Cleton, M.I., J.W. Sindram, L.H. Rademakers, F.M. Zuyderhoudt,
W.C. De Bruijn, and J.J. Marx. 1986. Ultrastructural evidence for the
presence of ferritin-iron in the biliary system of patients with iron
overload. Hepatology. 6:30–35.
49. Yang, D.C., F. Wang, R.L. Elliott, and J.F. Head. 2001. Expression of
transferrin receptor and ferritin H-chain mRNA are associated with
clinical and histopathological prognostic indicators in breast cancer.
Anticancer Res. 21:541–549.
50. Tripathi, P.K., and S.K. Chatterjee. 1996. Elevated expression of ferritin
H-chain mRNA in metastatic ovarian tumor. Cancer Invest. 14:518–526.
51. Gray, C.P., P. Arosio, and P. Hersey. 2003. Association of increased lev-
els of heavy-chain ferritin with increased CD4? CD25? regulatory
T-cell levels in patients with melanoma. Clin. Cancer Res. 9:2551–2559.
52. Gray, C.P., P. Arosio, and P. Hersey. 2002. Heavy chain ferritin acti-
vates regulatory T cells by induction of changes in dendritic cells.
53. Wu, C.G., M. Groenink, A. Bosma, P.H. Reitsma, S.J. van Deventer,
and R.A. Chamuleau. 1997. Rat ferritin-H: cDNA cloning, differen-
tial expression and localization during hepatocarcinogenesis. Carcino-
54. Chakravarti, S., C.A. Sabatos, S. Xiao, Z. Illes, E.K. Cha, R.A. Sobel,
X.X. Zheng, T.B. Strom, and V.K. Kuchroo. 2005. Tim-2 regulates T
helper type 2 responses and autoimmunity. J. Exp. Med. 202:437–444.
55. Allen, C.D., K.M. Ansel, C. Low, R. Lesley, H. Tamamura, N. Fujii,
and J.G. Cyster. 2004. Germinal center dark and light zone organiza-
tion is mediated by CXCR4 and CXCR5. Nat. Immunol. 5:943–952.