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Department of Pathology and Center for Immunology, Howard Hughes Medical Institute, Washington University School of Medicine, St. Louis, MO 63110;
Department of Genetics, Howard Hughes Medical Institute, Harvard Medical School, Boston, MA 02115;
Whitehead Institute for Biomedical Research and Department of Biology, Massachusetts Institute of Technology, Cambridge, MA 02142; and
§
Roche Bioscience, Palo Alto, CA 94303
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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, lymphotoxin, and IL-2, whereas Th2 cells produce IL-4, IL-5,
and IL-10 (1). The development of Th1 and Th2 subsets is regulated by
several factors, including the cytokines IL-12 and IL-4 (2, 3, 4, 5). Other
factors that can influence the overall balance of Th1 and Th2
development include the nature or affinity of the antigenic peptide (6, 7) and genetic factors (2, 3, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22). Because Th1 and Th2 cells promote different effector activities, the type of T helper response may influence host susceptibility to a pathogen or disease. For example, genetic background influences development in humans of atopic immune responses (23). Th2-derived cytokines, particularly IL-4 and IL-5, have been implicated in the pathogenesis of asthma and allergy (24). Certain phenotypic markers of asthma and atopy, specifically total serum IgE (25, 26) and airway hyperreactivity (27), have been genetically linked to a region on the long arm of human chromosome 5 containing a cluster of cytokine genes (e.g., IL-4, IL-3, IL-5), and other genes (IFN-regulatory factor-1 (IRF1)5) that may influence Th1/Th2 development (28, 29).
The genetic influence on Th1 and Th2 responses is also seen in murine
experimental Leishmania major infection (10, 20, 21, 22). In
this model, elimination of infection requires macrophage activation by
IFN-. Although some early resistance occurs from innate immune
responses, ultimate control of disease requires development of
pathogen-specific Th1 cells. Various murine strains differ in the type
of Th response generated against L. major. BALB/c mice
produce a Th2 response to L. major and succumb to infection,
whereas other strains, including B10.D2 and C57BL/6, produce a Th1
response and are resistant (30, 31). The underlying basis for
differential Th1/Th2 responses between murine strains is not yet
completely understood. However, differences in both T cell and non-T
cell hemopoietic cells seem to be involved, contributing to the
polygenic control of disease susceptibility (32, 33).
We recently analyzed the genetic control of Th1/Th2 development in
vitro using 
ß TCR-transgenic CD4+ T cells (8, 34, 35, 36).
We crossed the DO11.10 ß TCR transgenes onto BALB/c and B10.D2
backgrounds to allow direct comparisons of Th phenotype development. We
found that under neutral conditions (i.e., cytokines are not directly
manipulated), BALB/c T cells develop a more highly Th2-polarized
phenotype compared with B10.D2 T cells (8). Further, we traced this
difference to the failure of BALB/c T cells, but not B10.D2 T cells, to
express IL-12 receptors (34, 36). Finally, we showed that differential
IL-12 responsiveness between BALB/c and F1(B10.D2 x BALB/c) T
cells is controlled by a single dominant genetic locus which we termed
T cell phenotype modifier-1 (Tpm1) (35). Using simple
sequence length polymorphism (SSLP) analysis (37, 38) of experimental
backcrosses between these strains, we mapped Tpm1 to a
region of mouse chromosome 11 containing a cluster of genes important
for T cell differentiation, including IL-4, IL-5, IL-3, and IRF1 (35).
This region is syntenic with the homologous gene cluster on human
chromosome 5 previously linked to several phenotypic markers of atopy
(25, 26, 27).
In this study, we generated and analyzed a cohort of first-generation backcross recombinants to generate a higher resolution linkage map around Tpm1. We chose four informative BC1 recombinants to generate BC2 animals and BALB/c recombinant inbred lines. This analysis significantly reduces the genetic boundaries of Tpm1 and now allows us to exclude certain previous candidates of Tpm1, including IRF1, from consideration. Cellular analysis of T cells demonstrates that Tpm1 exerts its effect on IL-12 receptor expression in a cell-autonomous manner. This analysis strongly suggests that Tpm1, despite its proximity of the IL-13/IL-4 gene cluster, is not an allelic variant of one of these Th2-inducing cytokines. Thus, Tpm1 appears to be a noncytokine gene near the IL-4 gene cluster that influences early IL-12 receptor expression.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Heterozygous DO11.10 ß TCR (DO-TCR)-transgenic mice were
maintained on the BALB/c background for >10 generations (39). BALB/c
homozygous DO-TCR+/+-transgenic mice were generated from
sibling crosses of BALB/c background heterozygous TCR-transgenic mice
and identified using Southern analysis and progeny testing (K.M.M.,
unpublished data). The DO-TCR was previously crossed onto the B10.D2
background (8, 36) and is maintained by crossing B10.D2 heterozygous
DO-TCR+/- males to B10.D2/nSnJ females.
Experimental BALB/c DO-TCR+/- mice were generated from BALB/c DO-TCR+/+ males crossed to nontransgenic BALB/c females. Experimental F1 DO-TCR+/- mice were generated from BALB/c DO-TCR+/+ males crossed to nontransgenic B10.D2/nSnJ females. Experimental B10 DO-TCR+/- mice were generated from B10.D2 DO-TCR+/- males crossed to nontransgenic B10.D2/nSnJ females.
To produce first generation backcrossed (BC1) mice, F1 DO-TCR+/- (B10.D2 x BALB/c) males were crossed with female BALB/c mice. In this study, 580 BC1 mice were generated and tail DNA was analyzed. The genetic recombinations in 4 BC1 animals were identified as being potentially informative (animals 12, 145, 208, and 477). These were each crossed to homozygous DO-TCR+/+ mice to produce experimental animals with the crossover and to generate congenic lines for each crossover on the BALB/c background.
Female mice 4–6 wk old were purchased from Harlan Sprague-Dawley (Indianapolis, IN) (BALB/c), or The Jackson Laboratory (Bar Harbor, ME) (BALB/cByJ, B10.D2/nSnJ). Mice were housed in pathogen-clean facilities at Washington University Medical Center.
Tissue culture media and peptide
Cultures were maintained in Iscove’s modified Dulbecco’s Eagle medium supplemented with 10% FCS (HyClone, Logan, UT), 2 mM L-glutamine, 0.1 mM sodium pyruvate, 0.1 mM MEM nonessential amino acids, 100 U/ml penicillin, 100 µg/ml streptomycin, and 50 µM 2-ME. The antigenic peptide OVA 323–339 was synthesized as described (4) and purified by HPLC.
Transgenic T cell purification and culture
CD4+ T cells from DO-TCR-transgenic mice were
prepared from peripheral lymph nodes of 5–7-wk old mice as described
(8). T cells (1.25 x 105/well) were stimulated in
1-ml cultures with 0.3 µM OVA peptide presented by I-Ad
expressing BALB/c splenocytes (2000 rad, 2.5 x
106/well) and expanded threefold after 72 h into fresh
medium. On days 7 to 10, T cells were harvested, washed, and
restimulated at 1.25 x 105 cells/well with APC and
0.3 µM OVA peptide, with or without recombinant murine IL-12 (5 U/ml)
as indicated in the figure legends. To determine immediate production
of IFN- in response to IL-12, OVA was presented by the
I-Ad-expressing B cell hybridoma TA3 (10,000 rad, 2.5
x 105/well) (9), which does not produce IL-12 or IFN-
(34). Supernatants were collected after 48 h and IFN-
quantified by capture ELISA (2).
mAbs, cytokines, and reagents
Recombinant IL-12 was the gift of Dr. S. F. Wolf (Genetic
Institute, Cambridge, MA). The mAb 3E7 was the generous gift of Dr.
Kenneth Rock. Anti- CD25-FITC (IL-2R), and Cychrome-streptavidin
were obtained from PharMingen (San Diego, CA). The anti-clonotypic
mAb KJ1–26 has been described (40).
Genotyping and generation of genetic linkage map
High m.w. genomic DNA was prepared from mouse tail biopsy and
SSLP mapping analysis performed using a standard procedure (38). BC1
tail DNA was examined by PCR for the markers D11 Mit235 and D11 Mit177
that flank the previously identified interval containing
Tpm1 (35). All BC1 DNA suggesting a meiotic recombination
event between these two markers (homozygous at D11 Mit235 and
heterozygous at D11 Mit177, or heterozygous at D11 Mit235 and
homozygous at D11 Mit177) were evaluated for genotype for all available
polymorphic PCR-based markers residing between D11 Mit235 and D11
Mit177. These included all available (37, 41) anonymous markers with
the prefix "D11 Mit" (Research Genetics, Huntsville, AL) as well as
polymorphic markers contained within the genes IL-13, IL-4, IL-5, and
granulocyte-macrophage (GM)-CSF (Table I
). The IL-4 gene is marked both by a
marker in the promoter (Table I) and by marker D11 Mit111, located in
the second intron. The map was manually constructed by the method of
minimization of double recombinants using the data from the 580 BC1
mice from this study combined with the data from 90 BC1 mice from our
previous study (35). The interval bounded by D11 Mit153 and D11 Mit164
is contained within the D11 Mit235-D11 Mit177 interval and displayed in
Fig. 1.
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For each selected BC mouse line, multiple independent
experiments were performed (see Table III). IFN- values, after log
transformation, were compared between BC, BALB/c, and F1
mice by analysis of variance, treating data from multiple experiments
within a mouse line as a random effect. Statistical calculations were
performed using the SAS (SAS Institute, Cary, NC) GLM procedure
for the analyses of variance and the NESTED procedure to compute
appropriate means and SEM.
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Five days after secondary antigenic activation, T cells were
harvested, washed, counted, and plated at 5.0 x
105/ml in a 48-well plate. The cultures were incubated with
40 U/ml IL-2 alone or with 40 U/ml IL-2 plus 5 U/ml IL-12 for 48
h. The cells were then harvested, washed, and stained first with
3E7-biotin (anti-Ly6A.2) and then with FITC-conjugated
anti-CD25 and Cychrome-streptavidin. The stained cells were then
analyzed via flow cytometry (Becton Dickinson, Mountain View,
CA). After gating for live cells using forward scatter
and side scatter, and then gating for Ly6A.2 expression, cells were
analyzed for CD25 expression (IL-2R).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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We previously mapped Tpm1 to an 10-cM interval on
chromosome 11, with strong linkage with the IL-13/IL4 genes (D11
Mit111; logarithm of odds score = 6.5) (35). To define the
interval containing Tpm1 at higher resolution, we used the
method of genomic exclusion mapping. First, we generated a more
detailed genetic map by analyzing the recombination in this interval of
670 BC1 mice from the interspecific cross (F1(B10.D2 x BALB/c) x
BALB/c) described in Materials and Methods. Fig. 1
shows the
recombination that occurred within this region for 4 of these 670 BC1
mice. The genetic positions of anonymous markers and polymorphic
markers from IL-4, IL-5, IL-13, and GM-CSF are indicated along the
vertical axis. An open or closed box shows the genotype of each mouse
at the indicated markers. One mouse (no. 477) had a meiotic
recombination that was in the middle of a dense cluster of polymorphic
markers which allowed one group of polymorphic markers, group 1 (87,
111, 240, 273, IL-4, IL-13), to be placed centromeric to another, group
2 (23, 86, 140, 310, IL-5, GM-CSF).
To determine the order of markers among groups 1 and 2, we examined
their physical locations within a YAC contig WC11.20 by determining
their presence or absence on individual YACS (average size in library,
820 kbp) (42) (Table II). The locations
of most anonymous D11 Mit markers within this contig were available
from the mouse genome mapping and sequencing initiative (37) and were
independently confirmed by us (Table II). The WC11.20 contig data
indicate that among the markers analyzed, IL4 (111) is the most
telomeric of group 1 and IL-5 is the most centromeric of group 2. Thus,
mouse 477 exhibits a meiotic recombination between IL-4 and IL-5. From
contig WC11.20, the placement of IRF1 was indistinguishable from IL-5
(Table II). We could not identify a polymorphic marker for IRF1 to
distinguish BALB/c from B10.D2 alleles and thus could not place IRF1
into group 1 or 2 by genetics. Moreover, the IRF1 cDNA from B10.D2 and
BALB/c had 100% sequence identity (T.M.M. and K.M.M., data not shown),
excluding a structural difference in IRF1 as a Tpm1
candidate. However, a previous study determined that IRF1 is telomeric
to IL-5 (43), so that the order of markers therefore (IL-5-IRF1-D11
Mit23). IL-13 and IL-4 are tightly linked (physical distance, <50 kbp
(44) and their order is not distinguished here. Together, the available
information allows ordering of markers in the interval D11 Mit153 to
D11 Mit164 as [153-314-87-240-273-141-IL-4
(111)/IL-13-IL-5-IRF1-23-86-140-310-207/241-312-64/272-131/274/313-164]
and agrees with the most recent release of sequence-tagged site
analysis data for this murine YAC library
(http://www.genome.wi.mit.edu/cgi-bin/mouse/index) for chromosome 11.
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Four mice (mice 12, 145, 477, and 208) exhibited meiotic
recombination very close to IL-4, the previous peak linkage for
Tpm1 (35). We previously found the penetrance of
Tpm1 to be 80–90%, with remaining impenetrance
attributable to undefined parameters specific to the in vitro assay of
individual recombinant animals (J.D.G. and M.L.G., unpublished data).
Thus, to minimize impenetrance from assay variability, we determined in
several replicate experiments the phenotypes from multiple groups of
4–6 BC2 mice derived from the BC1 mice 12, 145, 477, and 208 (Fig. 2). These BC2 mice were generated by
mating BC1 mice 12, 145, 477, or 208 to BALB/c DO-TCR+/+
transgenic mice.
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). T cells were harvested after 7
days, washed, and restimulated with Ag in the absence or presence of
IL-12 and IFN- production determined at 48 h. Fig. 2 shows
results from three representative experiments. As expected (35), F1 T
cells maintained IL-12 responsiveness, producing high levels of IFN-
in response to IL-12, whereas by contrast, BALB/c T cells lost IL-12
responsiveness. Among the four experimental mouse lines tested, T cells
from animals of line 208 (recombinants BC2.208) and line 12 (BC2.12)
maintained IL-12 responsiveness, whereas T cells from animals of line
145 (recombinants BC2.145) and line 477 (BC2.477) lost IL-12
responsiveness (Fig. 2).
To confirm these results and minimize the influence of incomplete
penetrance, several more independent experiments were performed using
additional BC2 cohorts (Fig. 3, Table III). Fig. 3 presents the pooled means
and SEM of IL-12-induced IFN- responses from each recombinant line,
and Table III presents the pooled statistical analysis for these
experiments. The four recombinant lines were compared with F1 and
BALB/c mice by analysis of variance (see Materials and
Methods). A recombinant line was considered similar to an F1 if
the (experimental vs F1) p value was larger than the
corresponding (experimental vs BALB/c) p value. Conversely,
a recombinant line was considered similar to the BALB/c line if the
(experimental vs BALB/c) p value was larger than the
corresponding (experimental vs F1) p value. By these
criteria, recombinant lines BC2.208 and BC2.12 are similar to F1, and
lines BC2.145 and BC2.477 are similar to BALB/c (Table III).
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). Thus, B10.D2 genetic
material derived from the D11 Mit153 to D11 Mit207/241 genetic interval
is sufficient to confer IL-12 responsiveness, placing Tpm1
within this interval. The pooled analysis also confirmed that T cells
from recombinant lines BC2.145 and BC2.477 are similar to BALB/c T
cells. Because line 145 has a meiotic recombination between D11 Mit314
and D11 Mit87 (Fig. 1) and line 477 harbors a meiotic recombination
between IL-4 and IL-5 (Fig. 1, Table II), Tpm1 must be
located in the 0.45-cM interval with centromeric boundary of D11 Mit314
and telomeric boundary of IL-5. This analysis excludes IL-5, GM-CSF,
and IRF1 as candidates for Tpm1 but does not rule out IL-4
or IL-13 or other genes in the region centromeric to IL-5. A single B10.D2 Tpm1 allele is sufficient to maintain IL-12 responsiveness in BALB/c mice
Our previous data with BC1 mice (35) and the data above with BC2
mice suggest that one B10.D2 Tpm1 allele is sufficient to
maintain IL-12 responsiveness in the BALB/c background. To directly
test this hypothesis, the BC1.208 founder mouse was mated for three
successive generations to the BALB/c background. In each generation,
the presence of the B10.D2/BALB/c recombination on chromosome 11 was
confirmed by SSLP analysis by PCR markers along the 153-164 interval.
In the last generation, the 208-line animal was crossed to BALB/c
homozygous DO-TCR+/+-transgenic mice to allow in vitro
analysis as before. At this stage, T cells from the backcross 4
generation animals (BC4.208) were analyzed for in vitro maintenance of
IL-12 responsiveness (Fig. 4). T cells
from BC4.208 mice maintained IL-12 responsiveness in a range similar to
that of F1 controls, whereas BALB/c T cells were significantly lower in
response (Fig. 4, Table III). Thus, even when 
95% of the genome is
of BALB/c origin (predicted of BC4 generation mice), a single B10.D2
allele at Tpm1 confers IL-12 responsiveness equivalent to
the level of an F1 animal.
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We previously found that B10.D2 CD4+ T cells could transfer the property of IL-12 responsiveness to BALB/c CD4+ T cells during coculture of cells from each type (34). Thus, when B10.D2 were added in excess at a 4:1 ratio to BALB/c cells, BALB/c T cells then maintained IL-12 responsiveness (34, 36). This ability of B10.D2 cells to transfer IL-12 responsiveness in coculture is consistent with the hypothesis that one genetic difference between B10.D2 and BALB/c T cells could be in the production of a cytokine such as IL-4.
In this study, we show that maintenance of IL-12 responsiveness in F1
CD4+ T cells is determined entirely by Tpm1, but
our analysis has thus far not excluded IL-4 as a Tpm1
candidate. We therefore performed experiments to determine whether the
maintenance of IL-12 responsiveness is transferable by F1
CD4+ T cells to BALB/c CD4+ T cells, as
expected if a secreted molecule such as IL-4 controls this phenotype.
To allow single-cell analysis in coculture experiments, we measured
IL-12 responsiveness by the ability of IL-12 to up-regulate the cell
surface expression of the IL-2R-chain (34, 36). T cells were
stimulated by Ag, washed, and cultured in the presence or absence of
IL-12, and IL-2R expression determined by FACS analysis. Staining of
IL-2R was unchanged in BALB/c CD4+ T cells cultured in
the absence or presence of IL-12 (Fig. 5A), confirming lack of IL-12
responsiveness. By contrast, both B10.D2 or F1 CD4+ T cells
maintained IL-12 responsiveness shown by the increased IL-2R surface
staining by cells treated with IL-12 (Fig. 5A).
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B).
In B10.D2/BALB/c cocultures, B10.D2 T cells maintained IL-12
responsiveness, whether they were numerically in the minority
(B10.D2:BALB/c = 1:5; Fig. 5B, top left) or
in the majority (B10.D2:BALB/c = 5:1; Fig. 5B, top
right). BALB/c T cells lost IL-12 responsiveness when mixed with a
minority of B10.D2 cells (B10.D2:BALB/c = 1:5; Fig. 5B, top
left) but showed a degree of IL-12 responsiveness when cultured
with a majority of B10.D2 cells (B10.D2:BALB/c = 5:1; Fig. 5B, top right). Thus, when the culture milieu was dominated
by B10.D2 T cells, BALB/c T cells maintained IL-12 responsiveness, as
we previously reported (34). This suggests that full B10.D2 T cells can
transfer IL-12 responsiveness to BALB/c T cells.
In contrast to B10.D2 T cells, F1 T cells did not exhibit the capacity
to transfer IL-12 responsiveness in coculture. Thus, in F1/BALB/c T
cell cocultures, F1 T cells maintained IL-12 responsiveness whether
they were numerically in the minority (F1:BALB/c = 1:5; Fig. 5B, bottom left) or in the majority (F1:BALB/c =
5:1; Fig. 5B, bottom right). BALB/c T cells lost IL-12
responsiveness when cocultured with a minority of F1 T cells
(F1:BALB/c = 1:5; Fig. 5B, bottom left). However,
unlike the B10.D2:BALB/c T cell coculture, BALB/c T cells lost IL-12
responsiveness even when cultured with a majority of F1 T cells
(F1:BALB/c = 5:1; Fig. 5B, bottom
right).
Thus, both B10.D2 and F1 T cells maintained IL-12 responsiveness in these cocultures, even when cocultured with a majority of BALB/c T cells. This suggests that B10 and F1 T cells possess a cell-autonomous mechanism different from BALB/c T cells that allows for differential IL-12 responsiveness. However, only B10.D2 T cells, and not F1 T cells, could transfer the IL-12 responsiveness to BALB/c T cells, and then only when added at a high cellular ratio. The simplest explanation for these results is that IL-12 responsiveness in F1 T cells is not mediated by allelic expression of a secreted factor. Thus, it would appear that Tpm1 does not encode a cytokine such as IL-4. Rather, Tpm1 seems to encode for a cell-autonomous mechanism in B10 and F1 T cells, which allows for maintenance of IL-12 responsiveness.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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In this study, we refine the genetic bounds of Tpm1 and demonstrate that it is sufficient to influence in vitro Th cell phenotype development. In our previous study, we identified the 95% confidence interval of Tpm1, with peak logarithm of odds score of 6.5, centered on the IL-4 cytokine gene cluster. Through isolation of recombinants in this region and analysis of their in vitro phenotype, we narrowed Tpm1 to a 0.45-cM region again centered around the IL-4 cytokine gene cluster. Interestingly, this region has been associated with genetic linkages in several pathologic states, including atopy, pathogen susceptibility, and autoimmune diseases.
The syntenic human genomic region 5q31.1 contains also contains the IL-13/IL-4/IL-5 gene cluster and is linked to high serum IgE levels and airway hyperresponsiveness (25, 26, 27). Atopy depends on environmental and genetic factors and is not inherited as a simple trait (23, 48). Common to various atopic conditions is elevated expression of Th2 cytokines (49, 50). IL-4 induces IgE isotype switching, sensitizes mast cells for Ag-mediated degranulation, and is a mast cell growth factor. IL-5 induces eosinophil growth, influx, and degranulation. BALB/c mice exhibit greater expression of Th2 markers than the B10.D2-related C57BL/6 strain. BALB/c mice produce higher serum IgE (51, 52) and show greater Ag-induced airway hyperreactivity (53).
Genetic effects on Th1/Th2 balance also participate in resistance to pathogens such as L. major (54). IL-12 treatment of susceptible BALB/c mice concurrent with L. major infection generates a curative Th1-type CD4+ response (12, 55). Interestingly, IL-12 administration 1 wk after infection fails to induce Th1 responses or a cure in BALB/c mice (12), suggesting a temporal limit for response to IL-12. In BALB/c or C57BL/6 mice, IL-12 mRNA is undetectable until 1 wk after experimental L. major infection (56). By comparison, other pathogens such as Listeria monocytogenes induce IL-12 very rapidly (56). In addition, BALB/c T cells induced to develop toward Th2 cells in vitro lose expression of functional IL-12 receptors as early as 3 days after primary activation (57). B10.D2 and BALB/c T cells developing under neutral conditions differentially maintain responsiveness to IL-12 (34) and differentially maintain functional IL-12 receptors (36). Thus, the failure of BALB/c T cells to induce IL-12 receptors under neutral conditions may prevent their subsequent responses to the delayed IL-12 induced by L. major, perhaps contributing their susceptibility to this pathogen.
In studies of 7 recombinant inbred strains (C57BL/6 x BALB/c), a locus conferring susceptibility to L. major was mapped to a large region of mouse chromosome 11 (58). More recent in vivo mapping studies of L. major susceptibility using serial backcrossing have identified several other loci on chromosomes 6, 7, 10, 11, 15, and 16, important in controlling disease outcome (33). Another study reported other loci, on chromosomes 9, and 17, to influence in vivo L. major susceptibility. These studies confirm the polygenic control of L. major susceptibility and do not claim to have identified any single all-important locus. In the study by Beebe et al. (33), no one locus in isolation promoted pathogen resistance. However, a congenic mouse homozygous for the centromeric 40 cM of the B10.D2 chromosome 11 in the BALB/c background demonstrated a sex-influenced ability to control L. major infection (33). This 40-cM centromeric region of chromosome 11 contains one centromeric locus identified in that study as well as the more telomeric portion of chromosome 11 containing Tpm1.
Several models in which the Th1/Th2 balance can influence autoimmunity
have recently shown a genetic linkage to this region of mouse
chromosome 11. T cell-dependent destruction of the islet ß cell in
the nonobese diabetic mouse shows linkage with several genetic
loci. This disease is mediated by Th1, but not by Th2, cells
(59). One nonobese diabetic locus, idd4, maps to a large
region of chromosome 11 (60). In a transgenic autoimmune diabetes model
(61), the B10.D2 background conferred susceptibility to diabetes and a
Th1 profile, while the BALB/c background conferred disease resistance
and a Th2 profile. Finally, in murine experimental allergic
encephalomyelitis, a Th1-dependent autoimmune process of the central
nervous system mediates tissue pathology (62, 63). Here again, a recent
study, using backcrosses between high and low responder inbred mouse
strains, identified the middle portion of chromosome 11 (peak
2 at IL-4) as a potent modifier of
experimental autoimmune encephalomyelitis disease severity (64).
Therefore, in the context of distinct genetic and environmental
settings, Tpm1 could influence several immunologically
mediated pathological states, including pathogen susceptibility, atopy,
and autoimmunity.
This region of mouse chromosome 11 contains genes for several cytokines
that can influence Th1/Th2 development. IL-4 is a potential candidate
since it promotes Th2 development from naive T cells (2, 5) with loss
of IL-12 responsiveness (57). Thus, an overproduction of IL-4 by BALB/c
allele could explain loss of IL-12 receptors and increased Th2
development observed in BALB/c mice. If Tpm1 were the IL-4
gene, one must propose that the BALB/c allele is more active than the
B10.D2 allele. This hypothesis predicts that F1 mice, by harboring one
BALB/c IL-4 allele, should also overproduce IL-4 relative to B10.D2
mice and that the Tpm1 phenotype should act in an extrinsic,
rather than cell-autonomous, manner. However, we observe that F1 mice
show a B10-like phenotype and retain IL-12 responsiveness. Further,
experiments with cocultured B10.D2, F1 and BALB/c T cells show that
Tpm1 acts independently of the extracellular environment
(Fig. 5). Thus, the behavior of Tpm1 is not consistent with
its being due to allelic differences of the IL-4 gene.
These data suggest an alternative model in which Tpm1 plays
a role in an intracellular process that is involved in IL-12R
expression. For example, Tpm1 could regulate the sensitivity
of T cells to an extracellular factor that can influence IL-12R
expression, such as IL-4 or IFN-. We previously thought that IRF1
(65) was a reasonable candidate, since it could have mediated the known
induction of IL-12R expression by IFN-. However, our current genetic
exclusion mapping has placed IRF1 outside the region containing
Tpm1. Tpm1 could directly influence a pathway controlling
the transcription of the IL-12R ß2 subunit. The current
chromosomal region does contain other candidates for Tpm1
that could generate intracellular differences between BALB/c and B10.D2
CD4+ T cells. These include T cell factor-1 (66) and
IL-2-inducible T cell kinase (67), which each encode signaling
molecules or transcription factors expressed in T cells that could
influence IL-12R expression.
Finally, these experiments imply the existence of genetic loci other
than Tpm1 that influence the maintenance of IL-12
responsiveness. The coculture experiments indicate that B10.D2 and
F1(B10.D2 x BALB/c) T cells differ in phenotype. Both B10.D2 and
F1 T cells maintain IL-12 responsiveness. However, B10.D2 T cells, but
not F1 T cells, transfer the ability to maintain IL-12 responsiveness
to BALB/c T cells. Thus, cell-autonomous maintenance of IL-12
responsiveness segregates genetically from the ability to transfer that
property. This observation allows us to hypothesize the existence of a
"transferability" locus or loci. Indeed, initial coculture
experiments (data not shown) using BC1 (F1x B10.D2) T cells and BALB/c
T cells suggest the presence of a "transferability" locus
(Tpm2) on murine chromosome 15 (Table IV).
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Acknowledgments |
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Footnotes |
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2 These authors contributed equally to this work.
3 Current address: Department of Pathology, Dartmouth Medical School-DHMC, Lebanon, NH 03756.
4 Address correspondence and reprint requests to Dr. Kenneth M. Murphy, Department of Pathology, Washington University School of Medicine, 660 South Euclid Avenue, St. Louis, MO 63110. E-mail address:
5 Abbreviations used in this paper: IRF1, IFN-regulatory factor-1; DO-TCR, DO11.10 ß TCR; Tpm1, T cell phenotype modifier-1; SSLP, simple sequence length polymorphism; GM, granulocyte-macrophage.
Received for publication June 3, 1998. Accepted for publication October 15, 1998.
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