Thymus Of Normal Mice Research Articles

Deletion of self-reactive CCR7- thymocytes in the absence of MHC expression on thymic epithelial cells.

The selection of αβ T cells in the thymus is punctuated by checkpoints at which thymocytes differentiate or undergo apoptosis. Wave 1 deletion is defined as apoptosis within nascent αβ T-cell antigen receptor (TCR)-signalled thymocytes that lack CCR7 expression. The antigen-presenting cell (APC) types that mediate wave 1 deletion are unclear. To measure wave 1 deletion, we compared the frequencies of TCRβ + CD5 + Helios + CCR7- cells in nascent thymocyte cohorts in mice with normal or defective apoptosis. This thymocyte population is small in mice lacking major histocompatibility complex (MHC) expression. The scale of wave 1 deletion was increased by transgenic expression of the self-reactive Yae62 TCRβ chain, was almost halved when haemopoietic APCs lacked MHC expression and, surprisingly, was unchanged when epithelial cells lacked MHC expression. These findings demonstrate efficiency, and some redundancy, in the APC types that mediate wave 1 deletion in the normal mouse thymus.

Demonstration of Functional Heterogeneity of T lymphocytes and Identification of Their Two Major Subsets.

Demonstration of Functional Heterogeneity of T lymphocytes and Identification of Their Two Major Subsets.

Insights into the mechanisms of thymus involution and regeneration by modeling the glucocorticoid-induced perturbation of thymocyte populations dynamics

T-cells develop in the thymus and based on CD4 and CD8 expressions there are four main thymocyte populations in a normal mouse thymus. Currently, there are several mathematical models that describe the dynamics of thymocyte populations in a normal thymus, but only a few of them model the transient perturbation of their homeostasis. Our aim is to model the perturbation in the dynamics of each thymocyte population which is induced by the administration of a glucocorticoid, i.e. dexamethasone. The proposed approach relies on extending a four compartment thymus model based on differential equations by adding perturbation terms either globally (at the level of each equation) or locally (at the level of proliferation, death, and transfer rates). By fitting the perturbed model with experimental data on mice thymi collected before and after the administration of dexamethasone, it was possible to estimate the relevant parameters using a population-based stochastic search method. The fitted model is further used to conduct a quantitative analysis on the differentiated impact of dexamethasone on each T-cell population and on proliferation, death, and transfer processes. The obtained quantitative information on the perturbation could be used to explore and modify the flow of thymocytes between thymus compartments in order to elucidate the mechanisms of thymus involution and its subsequent regeneration. Since glucocorticoids are raised in many pathological situations, such a model could be useful in evaluating the impact of diseases on thymocyte dynamics in the thymus.

Analysis of illegitimate Notch1 and Bcl11b V(D)J deletion events during mouse development (P4400)

Abstract Previously, others identified hotspots for deletional illegitimate V(D)J rearrangements in normal mouse thymus involving cryptic RSSs in Notch1 and Bcl11b, onco/tumor suppressor genes involved in T cell development. These deletions are associated with radiation-induced and other T cell malignancies. We are conducting expanded frequency and junctional analyses of deletions during mouse development to examine and compare age- and gender-specific detection and occurrence of these events. Deletions in thymus increase in frequency between fetal and neonatal stages, are seen through at least 7 months of age, and are found in spleen. Deletions are more frequent at Bcl11b than Notch1 as previously observed, but both genes show clonality of deletion bearing cells. Junctional processing analysis indicates that nucleotide loss is more frequent and extensive at Bcl11b than Notch1, thus P nucleotide insertions are more abundant in Notch1 junctions. N nucleotide insertions increase between fetal and juvenile stages, with recessed palindromes (Pr nucleotides) corresponding mostly to single-base additions consistent with normal TdT activity. An in depth age and gender junctional processing analysis is in progress as are determinations of postnatal inducibility of deletions using inducible RAG transgenic mice. We present our results in the context of similar studies of V(D)J deletions at the nonimmune HPRT locus and normal V(D)J recombination at antigen receptor loci.

Immune ignorance of a commensal bacterial antigen by endogenous antigen-specific CD4+ T cells in the gut. (49.14)

Abstract The immune system must discriminate between beneficial and harmful microbes in the gut, and failure to maintain a proper balance of immune responsiveness and immune tolerance to gut microbes can lead to intestinal pathology. In particular, inflammatory bowel diseases such as Crohn's Disease and ulcerative colitis are thought to be the result of aberrant immune responses targeting commensal bacteria normally present in the gut lumen. The early events that break steady-state tolerance and trigger an immune response directed to commensal bacterial antigens remain poorly understood. Using a peptide:MHC-II tetramer-based enrichment approach, we characterized endogenous CD4+ T cells specific for Cbir1, a flagellin antigen from commensal bacteria present in the gut of normal mice. Our data show that endogenous Cbir1-specific T cells develop in the thymus of normal mice at the double positive stage. These cells survive negative selection and populate the peripheral lymphoid organs without becoming activated. Following immunization with their cognate antigen, they undergo massive proliferation and upregulate activation markers. Despite the presence of Cbir1 in the gut lumen, Cbir1-specific T cells in the lamina propria do not differentiate into regulatory T cells, nor do they induce colitis in normal mice. These data suggest that immune ignorance, rather than regulatory T cell induction, is a major mechanism of immune tolerance to commensal bacteria flagellin in the gut.

Intrathymic injection of lentiviral vector curtails the immune response in the periphery of normal mice

Gene transfer in the thymus, based on HIV-derived lentiviral vectors, is a promising avenue for modulation of T cell selection and autoimmunity. However, the impact of intrathymic (IT) injections on an antigen-specific immune response elicited in the periphery of normal mice has not been investigated yet. Highly concentrated stocks of lentiviral vectors expressing the soluble form of hemaglutinin of the influenza virus (LvHA) were injected in the thymus of normal BALB/c mice. The CD4 and CD8-mediated immune responses to HA after peripheral immunization were measured by various parameters. We first show that a lentiviral vector expressing the luciferase was detected for at least 2 months after IT-injections. We then show that the LvHA vector could elicit a functional CD4- and CD8-T cell-mediated immune responses in the peripheral lymphoid organs of BALB/c mice. IT-injection of the LvHA vector significantly curbed this response: lower numbers of transferred HA-specific CD4(+) T cells were found in LvHA-injected compared to control animals. Furthermore, lower frequencies of HA-specific CD8(+) T cells, interferon γ-producing cells and cytotoxic cells were detected from 3 weeks to 3 months in LvHA-injected mice compared to controls. However, these reduced CD8-mediated responses were not increased after depletion of CD25(+) cells in vitro or in vivo. The results obtained in the present study show that injection of the LvHA lentiviral vector significantly curtailed the immune response to the same antigen in the periphery. Increased selection of HA-specific regulatory T cells and negative selection of HA-specific CD8(+) T cell precursors may explain the results. Our work establish the feasibility of IT-injections of lentiviral vectors to manipulate T cell tolerance in the thymus of normal mice, for basic and pre-clinical research.

Abstract 1904: Reversal of thymic involution and immunosuppression in tumor-bearing mice by IFN-γ

Abstract The thymus is a central lymphoid organ critical for the development and maintenance of an effective peripheral T cell repertoire. Most important, it provides a specialized environment for the selection of rearranged clones that will function appropriately in the adaptive immune response. The tumor-induced immunosuppression observed during murine mammary tumorigenesis has been associated with a profound thymus involution accompanied with a severe depletion of the most abundant subset of thymocytes; CD4+CD8+ double positive (DP) immature cells. Thymic involution has been observed in several model systems; including graft-vs-host disease, aging, and tumor development, however, the mechanisms involved in this phenomenon remain to be elucidated. Previous results from our laboratory have reported that the severe thymic atrophy and impaired T cell development seen in mammary tumor bearers are associated with an arrest in at least two steps of T cell differentiation, changes in the levels of crucial cytokines expressed in the thymus microenvironment, and a progressive increase in apoptosis during the tumor development mainly due to downregulation of important molecules that control programmed cell death. Moreover, we have shown that mice bearing the murine parental D1-DMBA-3 mammary tumor and tumors derived from the parental tumor, the DA-3, develop many changes in their cytokine production. For example, in mice bearing these tumors there is a decrease in the IFN-γ in the peripheral circulation and production of this crucial cytokine is greatly diminished in the T cells. In the present study we have used our DA-3 mammary tumor model to investigate whether the levels of IFN-γ have an important role in anti-tumor activity, in the pathogenesis of thymic atrophy as well as the tumor-induced immunosuppression in these mice. Interestingly, our results show that the overexpression of IFN-γ in DA-3 tumor cells results in a marked delay of tumor growth and a blockage in the tumor-associated thymus atrophy. Importantly, peripheral T cells from IFN-γ-transfected DA-3 tumor-bearing mice, show markedly high levels of this cytokine. Moreover, when thymuses from tumor-bearing mice that received IFN-γ were analyzed, thymic populations were very similar to those observed in the thymuses of normal mice. Collectively, our data suggest that the replacement of the faulty levels of IFN-γ in tumor bearers results in a delayed tumor growth and thymic involution leading to a decrease of tumor-induced immune suppression caused by the mammary tumor development. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1904.

Thymus-homing peripheral dendritic cells constitute two of the three major subsets of dendritic cells in the steady-state thymus

Many dendritic cells (DCs) in the normal mouse thymus are generated intrathymically from common T cell/DC progenitors. However, our previous work suggested that at least 50% of thymic DCs originate independently of these progenitors. We now formally demonstrate by parabiotic, adoptive transfer, and developmental studies that two of the three major subsets of thymic DCs originate extrathymically and continually migrate to the thymus, where they occupy a finite number of microenvironmental niches. The thymus-homing DCs consisted of immature plasmacytoid DCs (pDCs) and the signal regulatory protein α–positive (Sirpα+) CD11b+ CD8α− subset of conventional DCs (cDCs), both of which could take up and transport circulating antigen to the thymus. The cDCs of intrathymic origin were mostly Sirpα− CD11b− CD8αhi cells. Upon arrival in the thymus, the migrant pDCs enlarged and up-regulated CD11c, major histocompatibility complex II (MHC II), and CD8α, but maintained their plasmacytoid morphology. In contrast, the migrant cDCs proliferated extensively, up-regulated CD11c, MHC II, and CD86, and expressed dendritic processes. The possible functional implications of these findings are discussed.

Keratinocyte Growth Factor Induces Expansion of Murine Peripheral CD4+Foxp3+ Regulatory T Cells and Increases Their Thymic Output

Keratinocyte growth factor (KGF) has been shown to reduce the incidence and severity of graft-versus-host disease by prevention of epithelial damage and by modulating alloreactivity. Since regulatory T cells (Treg) play a crucial role in immune modulation, we evaluated the effects of exogenous KGF on peripheral CD4(+)Foxp3(+) Treg and the generation of Treg in the thymus of normal mice. A 3-day course of KGF induced a rapid selective increase in the number of highly suppressive CD4(+)Foxp3(+) Treg. Blood Treg numbers remained elevated for >2 mo, but the frequency normalized after 2 wk due to a concomitant increase in CD4(+)Foxp3(-) T cells. Analysis of single joint TCR excision circles frequency and Ki-67 expression in peripheral blood Treg showed that the early selective increase of Treg was predominantly accounted for by peripheral expansion. Thymectomy before KGF administration did not affect the early selective increase of Treg but abrogated the late increase in CD4(+) T cell numbers, thereby showing its dependence on thymic output. Collectively, these results show that KGF induces an increase in blood CD4(+)Foxp3(+) Treg numbers via two independent mechanisms. First by selective peripheral expansion of Treg and thereafter by enhanced thymic output of newly developed Treg.

Homeostatic maintenance of natural Foxp3+ CD25+ CD4+ regulatory T cells by interleukin (IL)-2 and induction of autoimmune disease by IL-2 neutralization

Interleukin (IL)-2 plays a crucial role in the maintenance of natural immunologic self-tolerance. Neutralization of circulating IL-2 by anti–IL-2 monoclonal antibody for a limited period elicits autoimmune gastritis in BALB/c mice. Similar treatment of diabetes-prone nonobese diabetic mice triggers early onset of diabetes and produces a wide spectrum of T cell–mediated autoimmune diseases, including gastritis, thyroiditis, sialadenitis, and notably, severe neuropathy. Such treatment selectively reduces the number of Foxp3-expressing CD25+ CD4+ T cells, but not CD25− CD4+ T cells, in the thymus and periphery of normal and thymectomized mice. IL-2 neutralization inhibits physiological proliferation of peripheral CD25+ CD4+ T cells that are presumably responding to normal self-antigens, whereas it is unable to inhibit their lymphopenia-induced homeostatic expansion in a T cell–deficient environment. In normal naive mice, CD25low CD4+ nonregulatory T cells actively transcribe the IL-2 gene and secrete IL-2 protein in the physiological state. IL-2 is thus indispensable for the peripheral maintenance of natural CD25+ CD4+ regulatory T cells (T reg cells). The principal physiological source of IL-2 for the maintenance of T reg cells appears to be other T cells, especially CD25low CD4+ activated T cells, which include self-reactive T cells. Furthermore, impairment of this negative feedback loop via IL-2 can be a cause and a predisposing factor for autoimmune disease.

Natural Killer Dendritic Cells Have Both Antigen Presenting and Lytic Function and in Response to CpG Produce IFN-γ via Autocrine IL-12

We have isolated rare cells bearing the NK cell surface marker NK1.1, as well as the dendritic cell (DC) marker CD11c, from the spleen, liver, lymph nodes, and thymus of normal mice. These cells possess both NK cell and DC function because they can lyse tumor cells and subsequently present Ags to naive Ag-specific T cells. Interestingly, in response to IL-4 plus either IL-2 or CpG, NKDC produce more IFN-gamma than do DC, or even NK cells. We determined that CpG, but not IL-2, induces NKDC to secrete IFN-gamma via the autocrine effects of IL-12. In vivo, CpG dramatically increases the number of NKDC. Furthermore, NKDC induce greater Ag-specific T cell activation than do DC after adoptive transfer. Their unique ability to lyse tumor cells, present Ags, and secrete inflammatory cytokines suggests that NKDC may play a crucial role in linking innate and adaptive immunity.

Scotin, a novel p53-inducible proapoptotic protein located in the ER and the nuclear membrane.

p53 is a transcription factor that induces growth arrest or apoptosis in response to cellular stress. To identify new p53-inducible proapoptotic genes, we compared, by differential display, the expression of genes in spleen or thymus of normal and p53 nullizygote mice after γ-irradiation of whole animals. We report the identification and characterization of human and mouse Scotin homologues, a novel gene directly transactivated by p53. The Scotin protein is localized to the ER and the nuclear membrane. Scotin can induce apoptosis in a caspase-dependent manner. Inhibition of endogenous Scotin expression increases resistance to p53-dependent apoptosis induced by DNA damage, suggesting that Scotin plays a role in p53-dependent apoptosis. The discovery of Scotin brings to light a role of the ER in p53-dependent apoptosis.

The ontogeny of B cells in the thymus of normal, CD3 epsilon knockout (KO), RAG-2 KO and IL-7 transgenic mice.

The ontogeny of thymic B cells was determined by three-color flow cytometry and the presence or absence of B cell progenitors confirmed by cell culture experiments. In the thymus of young normal mice, CD117(+), B220(low) pro- and pre-B cells are present but disappear with age. B220(low), CD5(+), B-1 B cells are present in the thymus of older animals following the appearance of similar cells in the peritoneal cavity and blood. In CD3 epsilon gene-deleted mice, the phenotypic progression and number of thymic B cells remains unaltered, showing that blocking T cell development does not automatically result in an increase of thymic B lymphopoiesis. Pro-B cells in RAG-2 knockout mice are found in the fetal and neonatal blood, spleen and thymus, but with increasing age are only found in the bone marrow. B lymphopoiesis in adult IL-7 transgenic mice is dramatically altered with CD117(+) pro- and pre-B cells present in spleen, lymph node and blood. In the thymus of adult IL-7 transgenic mice, the fraction of CD117(+) thymic B cells is significantly increased. These results show that in the steady state, the phenotype of thymic B cells is critically dependent on both mouse age and the phenotype of circulating B cells.

Thymus function in drug-induced lupus.

Autoimmunity develops when a lupus-inducing drug is introduced into the thymus of normal mice, but the relevance of this model to the human disorder is unclear in part because it is widely assumed that the thymus is non-functional in the adult. We compared thymus function in 10 patients with symptomatic procainamide-induced lupus to that in 13 asymptomatic patients who only developed drug-induced autoantibodies. T cell output from the thymus was quantified using a competitive polymerase chain reaction that detects T cell receptor DNA excision circles in peripheral blood lymphocytes. Despite the advanced age of the patient population under study, newly generated T cells were detected in all subjects. Although there was no overall quantitative difference between the symptomatic and asymptomatic patients, we found a positive correlation between the level of T cell receptor excision circles in peripheral lymphocytes and serum IgG anti-chromatin antibody activity in patients with drug-induced lupus. The association between autoantibodies and nascent peripheral T cells supports the requirement for T cells in autoantibody production. Our observations are consistent with findings in mice in which autoreactive T cells derived from drug-induced abnormalities in T cell development in the thymus.

Early Cellular Events in Systemic Autoimmunity Driven by Chromatin-Reactive T Cells

In vivo exposure of the thymus of normal mice to procainamide–hydroxylamine, a lupus-inducing drug, causes development of chromatin-reactive T cells. Autoantibodies subsequently appear, but their origin and significance are unknown. The current studies were undertaken to determine the specificities of B cells that respond to chromatin-reactive T cells at the initiation of this autoimmune process. Three days after adoptive transfer of 6 × 106 chromatin-reactive T cells, B cells with the capacity to secrete IgM anti-chromatin antibodies were detected in 1/106 splenocytes, and these became 10- to 50-fold more numerous if either the donor T cells or the recipient had defective Fas due to the lpr allele. Five days later these mice developed IgG anti-chromatin-secreting B cells at a precursor frequency of 3–6 × 10−5. B cells with dDNA-binding activity isolated from mice primed in vivo to a complex of methylated pigeon cytochrome c and dDNA could stimulate naive, cytochrome c-reactive T cells in vitro, demonstrating that B cells can internalize dDNA-bound proteins through their dDNA immunoblobulin receptor and can functionally present a T cell epitope. However, no capacity of chromatin for binding anti-dDNA antibodies was detected, and IgM dDNA-specific B cells did not expand when challenged with chromatin-reactive T cells in vivo. The rapid and robust expansion of anti-chromatin-secreting B cells indicates that the normal immune repertoire includes nontolerant autoreactive B cells that respond to strong T cell drive and are readily manifested if Fas-mediated activation-induced cell death is inhibited.

The Importation of Hematogenous Precursors by the Thymus Is a Gated Phenomenon in Normal Adult Mice

Hematogenous precursors repopulate the thymus of normal adult mice, but it is not known whether this process is continuous or intermittent. Here, two approaches were used to demonstrate that the importation of prothymocytes in adult life is a gated phenomenon. In the first, age-dependent receptivity to thymic chimerism was studied in nonirradiated Ly 5 congenic mice by quantitative intrathymic and intravenous bone marrow (BM) adoptive transfer assays. In the second, the kinetics of importation of blood-borne prothymocytes was determined by timed separation of parabiotic mice. The results showed that >60% of 3-18-wk-old mice developed thymic chimerism after intrathymic injection of BM cells, and that the levels of chimerism (range, 5-90% donor-origin cells) varied cyclically (periodicity, 3 to 5 wk). In contrast, only 11-14% of intravenously injected recipients became chimeric, and chimerism occurred intermittently (receptive period approximately 1 wk; refractory period approximately 3 wk). In the intravenously injected mice, chimerism occurred simultaneously in both thymic lobes; gate opening occurred only after most intrathymic niches for prothymocytes had emptied; and the ensuing wave of thymocytopoiesis encompassed two periods of gating. These kinetics were confirmed in parabiotic mice, and in cohorts of mice in whom gating was synchronized by an initial intrathymic injection of BM cells. In addition, a protocol was developed by which sequential intravenous injections of BM cells over a 3 to 4 wk period routinely induces thymic chimerism in the apparent absence of stem cell chimerism. Hence, the results not only provide a new paradigm for the regulation of prothymocyte importation during adult life, but may also have applied implications for the selective induction of thymocytopoiesis in nonmyeloablated hosts.

A Nondeletional Mechanism for Central T-Cell Tolerance

To be positively selected, immature thymocytes must receive signaling through their T-cell receptor (TCR), and engagement of relatively low-affinity self-peptides permits further T-cell maturation. However, mature T cells no longer overtly respond to such low-affinity antigens, indicating that T cells acquire a higher threshold for activation during thymopoiesis. We wondered whether partial interference in positive selection could produce T cells that respond to the selecting self-peptide. This possibility was tested by injecting procainamide-hydroxylamine (PAHA), a lupus-inducing drug, into the thymus of adult normal mice. Three weeks after the second injection, IgG antichromatin antibodies appeared in the circulation and remained for several months. The murine antichromatin antibodies reacted with the (H2A-H2B)-DNA subnucleosome complex, the predominant specificity in patients with procainamide-induced lupus. In thymus organ and reaggregate cultures, PAHA had no effect on negative selection of T cells with high affinity for a co-present antigen, but acted on CD4+ CD8+ immature T cells as they underwent positive selection. TCR transgenic T cells specific to cytochrome c peptide 88-104 acquired the capacity to respond to the low-affinity analogue at position 99 (lys-->ala) if PAHA was present during their development. PAHA also blocked the capacity of a T-cell line to become anergic after anti-CD3 treatment, suggesting that PAHA prevents the production of negative regulators that accumulate in response to partial signaling through the TCR. These results are consistent with the view that T cells acquire self-tolerance during positive selection, and disruption of this process can result in autoreactive T cells and systemic autoimmunity.

The somatostatin immunoregulatory circuit present at sites of chronic inflammation.

Somatostatin is part of an immunoregulatory circuit that helps limit interferon-gamma (IFNgamma) production at sites of chronic inflammation. In murine schistosomiasis. parasite eggs induce focal, chronic granulomatous inflammation in the liver and intestines. These granulomas produce somatostatin 1-14 and express somatostatin receptor subtype number 2 (SSTR2), which is the exclusive somatostatin receptor present in this inflammation. Granuloma and splenic macrophages as well as macrophage cell lines make somatostatin. There appears to be no other inflammatory cell source of the peptide. Various inflammatory mediators induce this expression, whereas substance P inhibits somatostatin production. Somatostatin can suppress IFN-gamma secretion from T cells via interaction with the SSTR2 receptor expressed on these cells. Other cells within the granuloma also display SSTR2. The effect of somatostatin on these other cell types remains unknown. The thymus of normal mice has a complete somatostatin regulatory circuit. The thymic epithelial and dendritic cells make somatostatin. Like the granulomas of murine schistosomiasis, the thymus expresses only SSTR2. Somatostatin likely has an important role in thymic T cell education and selection.

Cloning of DLM-1, a novel gene that is up-regulated in activated macrophages, using RNA differential display.

Tumors interact with their environment, reprogramming host cells to induce responses such as angiogenesis, inflammation, immunity and immune suppression. To understand these processes, it is important to identify and isolate new genes whose expression is induced in host tissues in response to tumors. Ascites tumors offer an attractive model for isolating such genes, because responding host peritoneal lining tissues can be cleanly separated from tumor cells growing in suspension within the peritoneal cavity. We here report the cloning by differential display of a novel gene, DLM-1, that is highly up-regulated in the peritoneal lining tissue of mice bearing MOT ascites tumors. Mouse peritoneal macrophages, stimulated by IFN-gamma or LPS, also expressed significant amounts of DLM-1. Up-regulation of DLM-1 became evident by 4h after stimulation with IFN-gamma and was not blocked by cycloheximide, suggesting the presence of IFN responding elements in its transcription regulation region. DLM-1 RNA was detected at significant levels in normal mouse lung, intestinal epithelium, liver and thymus by Northern blot analysis. In situ hybridization of MOT and HT-29 mouse subcutaneous transplanted solid tumors revealed strong DLM-1 expression in the host reactive stromal cells, but not the tumor cells. Sequence analysis of the full-length cDNA clone revealed that it encodes a protein of approx. M(r) 44330 with multiple potential protein kinase C and casein kinase II phosphorylation sites. Our data suggest that DLM-1 plays a role in such important processes as host response in neoplasia.

Structure and function of the murine chemokine receptor CXCR3

The gene encoding the murine homologue of human CXCR3 exists in a single copy consisting of two exons with an intron interrupting the coding sequence between nucleotides 10 and 11. The deduced amino acid sequence is 86 % identical to the predicted human sequence. Murine CXCR3 mRNA is detectable in bone marrow cells cultured in the presence of IL-2 but not unstimulated cells. It is also detectable at low abundance in normal mouse spleen, lymph node, mammary gland, and thymus. Transfection of murine CXCR3 in murine pre-B lymphocyte line (CXCR3 / L1.2) conferred binding of the ligands IP10, ITAC and Mig with KD's of 1.35 ± 0.56, 1.41 ± 0.20, and 11.65 ± 0.90 nM, respectively. Lower affinity binding was observed for several beta or CC chemokines (eotaxin, MCP-3, MIP3α and SLC / 6Ckine / Exodus 2). ITAC, IP10 and Mig induced chemotaxis with an order of potency ITAC > IP10 = Mig. The chemokines also increased intracellular calcium concentration and were variably desensitized to repeated agonist stimulation. The hierarchy for cross- desensitization was ITAC > Mig > IP10. Thus, while Mig, ITAC and IP10 all act on the same receptor for binding and agonist stimulation, they may interact with different receptor conformational isoforms to produce divergent responses.