Asymmetric division and stem cell renewal without a permanent niche: lessons from lymphocytes.

Numerous tissues in long-lived organisms are composed of short-lived cells. The continual regeneration of some barrier surfaces, for example, relies on adult stem cells that have the capacity to divide and produce one daughter cell destined for terminal differentiation and function and another daughter cell that renews the stem cell fate. The immune system of higher animals possesses a cellular component called lymphocytes, which face a similar need for regeneration. A lymphocyte that is recruited during an infection must give rise to cellular progeny that undergo terminal differentiation to eliminate an invading microbe, yet retain progeny that replace the recruited cell in order to maintain immunity to reinfection. Emerging evidence suggests that specifying the divergent cell fates necessary for immunity relies on the ability of the lymphocyte to exploit an evolutionarily conserved strategy for making kindred cells different--asymmetric cell division. Although the lymphocyte does not possess constitutive polarity, it appears to use a facultative interaction with another cell to nucleate unequal segregation of fate determinants relative to its plane of division. Herein, we propose that other mobile and nonadherent cells, such as blood and cancer stem cells, might exploit provisional interactions with their niche or microenvironment to achieve diversity among their daughter cells.

Cell cycle controlling the silencing and functioning of mammalian activators.

Naïve CD4(+) helper T (T(H)) cells respond to stimulation by terminally differentiating into two mature classes, T(H)1 cells, which express interferon gamma (IFN-gamma), and T(H)2 cells, which express interleukin 4 (IL-4). The transcriptional activators T-bet and Gata-3 mediate commitment to the T(H)1 and T(H)2 fates, respectively, including chromatin remodeling of signature genes. The cytokine IL-12 fosters growth of committed T(H)1 cells, while IL-4 fosters growth of committed T(H)2 cells. IL-12 and IL-4 also play critical roles in commitment by promoting transcriptional silencing of Gata-3 and T-bet, respectively. We now show that both T-bet and Gata-3 are induced in a cell cycle-independent manner in bipotent progenitor cells. In contrast, both lineage-restricted gene induction by the activator proteins and heritable silencing of the transcription of each activator, the hallmarks of terminal differentiation, are cell cycle dependent. We found that cells that cannot cycle remain uncommitted and bipotent in response to the most polarizing signals for maturation. These results provide mechanistic insight into a mammalian model of terminal differentiation by illustrating that cell cycle-coupled epigenetic effects, as originally described in yeast, may represent an evolutionarily conserved strategy for organizing signaling and cell fate.

Induction of cytotoxic T lymphocyte antigen 4 (CTLA-4) restricts clonal expansion of helper T cells.

Cytotoxic T lymphocyte antigen (CTLA)-4 plays an essential role in immunologic homeostasis. How this negative regulator of T cell activation executes its functions has remained controversial. We now provide evidence that CTLA-4 mediates a cell-intrinsic counterbalance to restrict the clonal expansion of proliferating CD4(+) T cells. The regulation of CTLA-4 expression and function ensures that, after approximately 3 cell divisions of expansion, most progeny will succumb to either proliferative arrest or death over the ensuing three cell divisions. The quantitative precision of the counterbalance hinges on the graded, time-independent induction of CTLA-4 expression during the first three cell divisions. In contrast to the limits imposed on unpolarized cells, T helper type 1 (Th1) and Th2 effector progeny may be rescued from proliferative arrest by interleukin (IL)-12 and IL-4 signaling, respectively, allowing appropriately stimulated progeny to proceed to the stage of tissue homing. These results suggest that the cell-autonomous regulation of CTLA-4 induction may be a central checkpoint of clonal expansion of CD4(+) T cells, allowing temporally and spatially restricted growth of progeny to be dictated by the nature of the threat posed to the host.

Role of T-bet in commitment of TH1 cells before IL-12-dependent selection.

How cytokines control differentiation of helper T (TH) cells is controversial. We show that T-bet, without apparent assistance from interleukin 12 (IL-12)/STAT4, specifies TH1 effector fate by targeting chromatin remodeling to individual interferon-gamma (IFN-gamma) alleles and by inducing IL-12 receptor beta2 expression. Subsequently, it appears that IL-12/STAT4 serves two essential functions in the development of TH1 cells: as growth signal, inducing survival and cell division; and as trans-activator, prolonging IFN-gamma synthesis through a genetic interaction with the coactivator, CREB-binding protein. These results suggest that a cytokine does not simply induce TH fate choice but instead may act as an essential secondary stimulus that mediates selective survival of a lineage.

Helper T cell differentiation, inside and out.

The past year has seen remarkable progress in the field of helper T cell differentiation, including discovery of a novel transcription factor as well as a novel cytokine receptor of the Th1 lineage. The year has also brought new perspectives on the genetic and epigenetic control of gene expression. It is likely that mechanisms of immunity will continue to provide insight into the general problem of cellular decision-making.

Bone-marrow chimeras reveal hemopoietic and nonhemopoietic control of resistance to experimental Lyme arthritis.

Both genetic resistance and susceptibility to development of experimental Lyme arthritis are mediated by the innate immune response. To determine whether this process is mainly controlled by hemopoietic or nonhemopoietic cells, we created bone marrow (BM) chimeric mice between arthritis-resistant DBA/2J (DBA) and arthritis-susceptible C3H/HeJ (C3H) mice and infected them with Borrelia burgdorferi. Both sets of BM chimeric mice, C3H donors into DBA recipients (C-->D) and DBA donors into C3H recipients (D-->C), as well as DBA sham chimeric mice (D-->D) were resistant to the development of experimental Lyme arthritis as measured by ankle swelling and arthritis severity scores. Only the C3H sham chimeric mice (C-->C) developed severe arthritis. These results indicate that independent and nonoverlapping mechanisms exist in hemopoietic and nonhemopoietic cellular compartments that can provide protection against arthritic pathology.

Cytokines and T cells in host defense.

Soluble and cell-bound ligands profoundly influence the differentiative fate of lymphocytes during an immune response. Recent advances have been made in understanding the role of cytokine signals and costimulatory signals in the regulation of T cell responses associated with resistance or susceptibility to infection. There has also been recent progress in defining the requirements for the generation and maintenance of immunologic memory, a critical component of adaptive immunity.

Structural analysis of CTLA-4 function in vivo.

CTLA-4-mediated inhibition of T cell activation may be accomplished by competition for ligands and/or by signals mediated through the intracellular domain. Studies have implicated Tyr201 in the cytoplasmic domain of CTLA-4 in regulating CTLA-4 signal transduction and intracellular trafficking. To investigate the mechanism of CTLA-4 function in vivo, transgenes encoding wild-type CTLA-4 (FL), a mutant lacking the cytoplasmic domain of CTLA-4 (DeltaCTLA-4 tail), or a CTLA-4 Tyr201 mutant (Y201V) were introduced into CTLA-4-deficient mice. CTLA-4-/- mice display an autoimmune lymphoproliferative disorder resulting in tissue destruction and early death. When either the FL or the Y201V transgene was bred into CTLA-4-/- animals, a complete rescue from lymphoproliferation and autoimmunity was observed. In contrast, CTLA-4-/- mice expressing the DeltaCTLA-4 tail transgene were long lived with no evidence of multiorgan lymphocytic infiltration, but exhibited lymphadenopathy and accumulated large numbers of activated T cells. Furthermore, these animals displayed a Th2-biased phenotype which conferred susceptibility to Leishmania infection. These results indicate that the inhibitory effect of CTLA-4 is mediated in part through the ability of the extracellular domain to compete for ligands. The cytoplasmic domain of CTLA-4, however, is required for complete inhibitory function of the receptor and for regulation of Th cell differentiation in vivo.

Stat6 regulation of in vivo IL-4 responses.

Although in vitro development of a Th2 response from naive CD4+ T cells is Stat6 dependent, mice immunized with a goat Ab to mouse IgD have been reported to produce a normal primary IL-4 response in Stat6-deficient mice. Experiments have now been performed with mice immunized with more conventional Ags or inoculated with nematode parasites to account for this apparent discrepancy. The ability of an immunogen to induce a primary in vivo IL-4 response in Stat6-deficient mice was found to vary directly with its ability to induce a strong type 2 cytokine-biased response in normal mice. Even immunogens, however, that induce strong primary IL-4 responses in Stat6-deficient mice induce poor memory IL-4 responses in these mice. Consistent with this, Stat6-deficient CD4+ T cells make relatively normal IL-4 responses when stimulated in vitro for 3 days with anti-CD3 and anti-CD28, but poor IL-4 responses if they are later restimulated with anti-CD3. Thus, Stat6 signaling enhances primary IL-4 responses that are made as part of a type 0 cytokine response (mixed type 1 and type 2) and is required for normal development or survival of Th2 memory cells.

Genes outside the major histocompatibility complex control resistance and susceptibility to experimental Lyme arthritis.

The mechanism controlling the development of experimental Lyme arthritis remains poorly understood. Mice with the H-2k haplotype have generally been thought to be more susceptible to Lyme arthritis development than those with the H-2d haplotype. In the present study the role of genes within the major histocompatibility complex (MHC) in determining resistance or susceptibility to the development of experimental Lyme arthritis was investigated. We found that C3H congenic mice were equally susceptible, and DBA congenic mice equally resistant, to arthritis development regardless of their H-2 haplotype (H-2k or H-2d). These results indicate that genes outside the murine MHC are the major determinants of both resistance and susceptibility to the development of experimental Lyme arthritis.

Robust B cell immunity but impaired T cell proliferation in the absence of CD134 (OX40).

CD134 (OX40) is a member of the TNF receptor family that is expressed on activated T lymphocytes. T cells from mice that lack expression of CD134 made strong responses to a range of challenges, but they showed impaired proliferation in response to direct stimulation through the TCR with monoclonal anti-CD3epsilon Ab. CD134-deficient mice controlled infection with Leishmania major, Nippostrongylus brasiliensis, and Theiler's murine encephalomyelitis virus, and they made overtly normal Ab responses to a variety of antigens. Thus, CD134 is not essential for many T cell responses in vivo, nor is it required for the provision of help to B cells. Nonetheless, a subtle role in the regulation of T cell reactivity is suggested by the effect of CD134 deficiency on in vitro T cell responses.

Experimental lyme arthritis in the absence of interleukin-4 or gamma interferon.

Genetic resistance and susceptibility to experimental Lyme arthritis have been linked with the production of interleukin-4 (IL-4) or gamma interferon (IFN-gamma), respectively. To determine the absolute requirement for these cytokines in disease outcome, we compared arthritis development in wild-type, IL-4-deficient (IL-4 degrees ), and IFN-gamma-deficient (IFN-gamma degrees ) mice. While susceptible C3H mice developed swelling of ankle joints during the second week of infection, this swelling was exacerbated in C3H IFN-gamma degrees mice. Their arthritis severity scores at day 21, however, were similar. Resolution of arthritis was also similar between C3H and C3H IFN-gamma degrees mice. Arthritis-resistant DBA mice did not develop ankle swelling during the experimental period. There were no differences in ankle swelling or arthritis severity scores between control DBA mice and DBA IL-4 degrees mice at any of the time points tested. While the presence of spirochetes in various tissues was similar among all strains at day 21, DBA IL-4 degrees mice had a higher presence of spirochetes in blood, heart, and spleen than the DBA, C3H, and C3H IFN-gamma degrees mice did at day 60. DBA IL-4 degrees mice also had impaired ability to produce Borrelia-specific antibody responses, especially immunoglobulin G1. Thus, while IFN-gamma and IL-4 are not absolutely required for arthritis susceptibility or resistance, the production of IL-4 does appear to play an important role in Borrelia-specific antibody production and spirochete clearance.

Development of lyme arthritis in mice deficient in inducible nitric oxide synthase.

Nitric oxide (NO) is a powerful antimicrobial agent and an important regulatory molecule of the innate immune response. To determine if NO has a role in experimental Lyme disease, arthritis-resistant DBA/2J and arthritis-susceptible C3H/HeJ mice were bred to be genetically deficient for inducible NO synthase (iNOS). Following footpad injection of Borrelia burgdorferi, arthritis was similar between iNOS-deficient and control animals regardless of their genetic background. Histologic examination and arthritis severity scores of ankles revealed no differences in arthritis development between iNOS-deficient and control animals. Despite being deficient in a key antimicrobial agent, iNOS-deficient mice had tissue levels of B. burgdorferi similar to those in control mice. Thus, NO does not have a critical role in susceptibility to Lyme arthritis through tissue damage via an overexuberant inflammatory response, nor is it required in resistance through the clearance of spirochetes from tissues.

Genetic control of experimental lyme arthritis in the absence of specific immunity.

Host genetics play an important role in determining resistance or susceptibility to experimental Lyme arthritis. While specific immunity appears to regulate disease resolution, innate immunity appears to regulate disease severity. Intradermal infection with Borrelia burgdorferi yields severe arthritis in C3H/He (C3H) mice but only minimal arthritis in BALB/c mice. Intradermal infection of immunodeficient C3H SCID mice also results in severe arthritis, but arthritis of only moderate severity in BALB/c SCID mice. In the present study, we examined immunodeficient recombinase-activating gene-knockout (RAG-1(-/-)) (RAG-) mice from resistant C57BL/6 (B6) and DBA/2 (DBA) mouse strains. B. burgdorferi-infected B6 RAG- and DBA RAG- mice had little or no ankle swelling, a low occurrence of inflammatory infiltrates in tibiotarsal joints, and low arthritis severity scores in comparison to RAG+ and RAG- BALB/c or C3H mice. Few differences in spirochete DNA levels in ankles of resistant and susceptible RAG- mice were seen. These data suggest that resistance to arthritis development following B. burgdorferi infection is not necessarily dependent on an acquired immune response and can occur despite the presence of high spirochete burden. Thus, genes expressed outside the specific immune response can be central regulators of experimental arthritis.

Polarized helper-T-cell responses against Leishmania major in the absence of B cells.

B-cell-to-T-cell signaling can shape helper T (Th) cell responses. During infection with Leishmania major, Th response is critical in determining the outcome of disease. Resistance depends on the generation of a protective Th1 response, while susceptibility is mediated by the generation of a Th2 response. In this study, we determined whether B cells are required for the development of polarized Th1 and Th2 responses during infection with L. major. Mice lacking B cells due to disruption of the immunoglobulin M locus (microMT) were infected with L. major, and disease progression and Th cell development were assessed. On the genetically resistant C57BL background, both wild-type and microMT mice controlled the infection and mounted a Th1 response. On the genetically susceptible BALB/c background, both wild-type and microMT mice were susceptible to infection and generated Th2 responses. Thus, during L. major infection, neither direct antigen presentation or costimulation by B cells nor antibody-mediated effector functions are essential for the development of polarized Th responses.

Activation of natural killer cells in arthritis-susceptible but not arthritis-resistant mouse strains following Borrelia burgdorferi infection.

Infection of susceptible mouse strains with Borrelia burgdorferi, the agent of Lyme disease, results in the development of arthritis. Components of the innate immune system may be important mediators of this pathology. To investigate the potential role of NK cells in development of experimental Lyme arthritis, we examined their activation in vivo in both resistant and susceptible mouse strains. Following inoculation of B. burgdorferi into the footpad, lymph node NK cells from susceptible C3H/HeJ (C3H) mice produced more gamma interferon than NK cells from resistant DBA/2J mice. Lymph node cells from susceptible C3H and AKR mice also had increased ability to lyse YAC-1 target cells 2 days following infection. Antibody depletion of NK cells from susceptible mice, however, did not alter the development of arthritis following B. burgdorferi challenge. In addition, NK cell depletion had little effect on spirochete burden. Thus, there is a marked activation of NK cells in susceptible mouse strains following infection. Although NK cells are not absolutely required for arthritis, events occurring prior to NK cell activation might be important in mediating pathology in experimental Lyme disease.

Impaired negative selection in CD28-deficient mice.

T cell antigen receptors (TCR) expressed on developing T cells can react with self-peptides presented by proteins encoded by the major histocompatibility complex (MHC). Depending on the relative strength of these interactions, thymocytes are either negatively selected as potentially autoreactive and deleted or positively selected to become mature T cells. Developmental selection may also be regulated by signals in addition to those mediated through the TCR. In peripheral T cells, the CD28 receptor plays an important role in enhancing the survival and expansion of T cells activated by TCR engagement. Therefore, we have investigated the role of CD28 in regulating the selection of thymocytes using CD28-deficient mice. Surprisingly, we found a 50% increase in cell number in the thymi of CD28-deficient compared to wildtype mice, suggesting that CD28 might play a role in negative selection. Negative selection of double-positive thymocytes was found to be significantly reduced in response to either antigen or antibody crosslinking of the TCR complex in CD28-deficient animals. This was not due to a generalized defect in thymocyte survival as thymocytes from CD28-deficient and wildtype mice displayed similar sensitivity to apoptosis initiated by either gamma-irradiation or dexamethasone. In contrast to its role in T cell activation and survival in the peripheral immune system, the CD28 receptor appears to participate in the intracellular signaling events that result in negative selection in the thymus.

Helper T cell differentiation is controlled by the cell cycle.

Helper T (Th) cell differentiation is highly regulated by cytokines but initiated by mitogens. By examining gene expression in discrete generations of dividing cells, we have delineated the relationship between proliferation and differentiation. Initial expression of IL-2 is cell cycle-independent, whereas effector cytokine expression is cell cycle-dependent. IFNgamma expression increases in frequency with successive cell cycles, while IL-4 expression requires three cell divisions. Cell cycle progression and cytokine signaling act in concert to relieve epigenetic repression and can be supplanted by agents that hyperacetylate histones and demethylate DNA. Terminally differentiated cells exhibit stable epigenetic modification and cell cycle-independent gene expression. These data reveal a novel mechanism governing Th cell fate that initially integrates proliferative and differentiative signals and subsequently maintains stability of the differentiated state.