The role of alpha beta+ T cells and homeostatic T cell proliferation in Y-chromosome-associated murine lupus

Male BXSB mice develop an early life, severe lupus-like disease largely attributed to an undefined Y-chromosome-associated autoimmunity accelerator, termed YAA: Although the exact disease pathogenesis is uncertain, indirect evidence suggests that T cells play an important role in the male BXSB disease. We have developed TCR alpha-chain gene-deleted BXSB mice to directly examine the role of alphabeta+ T cells and the mode by which Yaa promotes disease in this strain. All disease parameters, including hypergammaglobulinemia, autoantibody production, glomerulonephritis, and the unique monocytosis of BXSB males, were severely reduced or absent in the alphabeta+ T cell-deficient mice. Adoptively transferred CD4+ T cells of either male or female BXSB origin showed equal homeostatic proliferation in alphabeta+ T cell-deficient male recipients. Moreover, deficient male mice eventually developed equally severe lupus-like disease after adoptive transfer and homeostatic expansion of T cells from wild-type BXSB males or females. The results directly demonstrate that the Yaa-mediated disease requires alphabeta+ T cells that are not, in themselves, abnormal in either composition or properties, but are engaged by a Yaa-encoded abnormality in a non-T cell component. In addition, homeostatic anti-self proliferation of mature T cells derived from a small number of precursors can induce systemic autoimmunity in an appropriate background.     

Human T cells constitutively express IL-15 that promotes ex vivo T cell homeostatic proliferation through autocrine/juxtacrine loops

Homeostatic proliferation of T cells in vivo is responsible for the maintainance of the T cell pool, and IL-15 is a pivotal cytokine implicated in this process. Known cell sources providing physiologically active IL-15 are monocytes/macrophages, dendritic cells, and stromal cells. T lymphocyte expression of functionally active IL-15 and its possible role in T cell biology have not been investigated. In this study, we demonstrate that human T cells constitutively express IL-15 that acts through autocrine/juxtacrine loops to promote ex vivo homeostatic T cell proliferation.     

Protein energy malnutrition impairs homeostatic proliferation of memory CD8 T cells

Nutrition is a critical but poorly understood determinant of immunity. There is abundant epidemiological evidence linking protein malnutrition to impaired vaccine efficacy and increased susceptibility to infections; yet, the role of dietary protein in immune memory homeostasis remains poorly understood. In this study, we show that protein-energy malnutrition induced in mice by low-protein (LP) feeding has a detrimental impact on CD8 memory. Relative to adequate protein (AP)-fed controls, LP feeding in lymphocytic choriomeningitis virus (LCMV)-immune mice resulted in a 2-fold decrease in LCMV-specific CD8 memory T cells. Adoptive transfer of memory cells, labeled with a division tracking dye, from AP mice into naive LP or AP mice demonstrated that protein-energy malnutrition caused profound defects in homeostatic proliferation. Remarkably, this defect occurred despite the lymphopenic environment in LP hosts. Whereas Ag-specific memory cells in LP and AP hosts were phenotypically similar, memory cells in LP hosts were markedly less responsive to polyinosinic-polycytidylic acid-induced acute proliferative signals. Furthermore, upon recall, memory cells in LP hosts displayed reduced proliferation and protection from challenge with LCMV-clone 13, resulting in impaired viral clearance in the liver. The findings show a metabolic requirement of dietary protein in sustaining functional CD8 memory and suggest that interventions to optimize dietary protein intake may improve vaccine efficacy in malnourished individuals.     

Il-12 enhances CD8 T cell homeostatic expansion

The size of the T lymphocyte pool is maintained by regulation of T cell production, proliferation, and survival. Under the pressure of a T lymphopenic environment, mature naive T cells begin to proliferate in the absence of Ag, a process called homeostatic expansion. Homeostatic expansion involves TCR recognition of self peptide/MHC ligands, but less is known about the soluble factors that regulate this process. Here we show that IL-12 dramatically enhanced the homeostatic proliferation of CD8 T cells. In contrast, IL-2 had no beneficial effect on homeostatic expansion and, in fact, inhibited T cell expansion induced by IL-12. Using gene-targeted mice, we showed that IL-12 acted directly on the T cells to enhance homeostatic expansion, but that IL-12 cannot override the requirement for TCR interaction with self peptide/MHC ligands in homeostatic expansion. These data indicate that inflammatory cytokines may modulate T cell homeostasis after lymphopenia and have implications for regulation of the T cell repertoire and autoimmunity.     

Cutting edge: differential self-peptide/MHC requirement for maintaining CD8 T cell function versus homeostatic proliferation

Memory T cells do not require self-peptide/MHC (spMHC) complexes to survive long term in vivo. However, memory CD4 T cells lose the ability to reject skin grafts when transiently placed in an environment in which these low-level TCR stimulations are absent. Whether or not spMHC alters the ability of CD8 T cells to respond to stimulation in vivo remains unknown. Here, we show that memory CD8 T cells retain the ability to respond to dendritic cell-mediated stimulation after adoptive transfer into either TAP(-/-) (MHC class I-deficient) or wild-type mice. Surprisingly, naive CD8 T cells, which fail to undergo homeostatic proliferation and erode in number in the absence of MHC class I, also retain the ability to respond to dendritic cell-mediated antigenic stimulation for at least 1 wk after transfer into TAP(-/-) mice. These findings suggest a differential requirement for spMHC signals for maintenance of CD8 T cell function and homeostatic proliferation.     

Cutting edge: homeostatic proliferation of peripheral T lymphocytes is regulated by clonal competition

Homeostatic proliferation functions to maintain peripheral T cell numbers and is regulated by cytokines. In this study, we provide evidence that T cell homeostasis is also regulated by clonal competition. Naive polyclonal T cells divided when transferred to TCR transgenic hosts, as did monoclonal T cells when transferred to TCR transgenic hosts of differing clonotype. However, T cells did not divide in hosts of identical clono-type. Transgenic T cell proliferation was inhibited in irradiated hosts of the same clonotype, while cotransferred nontransgenic T cells proliferated extensively. These results show that clonal competition is a component of homeostasis that may contribute to selection of the peripheral T cell repertoire.     

Activation-driven T cell death. II. Quantitative differences alone distinguish stimuli triggering nontransformed T cell proliferation or death.

Clonal deletion is the major mechanism by which T cell tolerance is achieved in vivo. The process of activation-driven cell death, originally characterized with T cell hybridomas, likely represents the mechanism of clonal deletion because it shares a number of properties with the in vivo process, especially the ability to be triggered in an Ag-specific manner, the cell-autonomous nature of the response, and its sensitivity to the drug cyclosporin A. We now have extended our analysis of activation-driven cell death to clonal populations of nontransformed T cells. Activation-driven cell death can be induced in nontransformed T lymphocytes by combinations of mitogenic stimuli. In particular, two mitogenic stimuli at high dose, one a lymphokine and the other delivered via the TCR or another activation structure, are required to induce activation-driven cell death. Activation-driven cell death is an active cell suicide process with attributes typical of physiological cell death, including early nuclear disintegration and a requirement for macromolecular synthesis, and is distinct from death by factor deprivation. Susceptibility to the induction of cell death by antigenic or activating stimulation is a common aspect of most T cells and is consistent with observations that clonal deletion can occur throughout T cell ontogeny. Most importantly, the alternative cellular responses of cell death and cell proliferation in nontransformed T cells appear to be triggered solely as a function of quantitative differences in the doses of identical stimuli. This can be viewed as a dose-dependent switch that determines cell fate. Developmental regulation of this switch may explain the processes of positive and negative selection during T cell ontogeny and also provide a mechanistic rationale for a strategy of selective anti-tumor therapy.     

Cellular and molecular requirements for rejection of B16 melanoma in the setting of regulatory T cell depletion and homeostatic proliferation

We have recently demonstrated that adoptive transfer of regulatory T cell-depleted polyclonal T cells into lymphopenic mice leads to rejection of B16 melanoma, which generated an opportunity to study host requirements for tumor rejection when it effectively occurred. CD8(+) T cell priming and tumor rejection required tumor Ag cross-presentation, as evidenced by tumor outgrowth in Kb(-/-) bone marrow chimeric or B71/2(-/-) mice. CD4(+) T cells were additionally required for optimal tumor control, although not through classical CD4 "help," as the frequency of primed CD8(+) T cells was similar in the absence of CD4(+) T cells, and tumor rejection did not depend upon CD40-CD40L interactions or on IL-2 production by CD4(+) T cells. Rather, CD4(+) T cells appeared to act at the effector phase of tumor rejection and responded to B16-derived Ags in vitro. At the effector phase, IFN-γ production by transferred T cells, but not host cells, was necessary. IFN-γ acted either on host or tumor cells and was associated with reduced tumor vascularity. Finally, tumor rejection occurred after transfer of TNF-α, perforin, or FasL-deficient T cells. However, perforin/FasL double-knockout T cells failed to reject, arguing that the killing of B16 melanoma cells could occur either via the cytotoxic granule or Fas pathways. Collectively, these results support a model in which host tumor Ag cross-presentation primes adoptively transferred T cells, which remain functional in the setting of homeostatic proliferation and regulatory T cell depletion, and which promote tumor rejection via IFN-γ and lysis via cytotoxic granules and/or FasL.     

Quantitative analysis of topoisomerase IIalpha to rapidly evaluate cell proliferation in brain tumors

Immunohistochemical cell proliferation analyses have come into wide use for evaluation of tumor malignancy. Topoisomerase IIalpha (topo IIalpha), an essential nuclear enzyme, has been known to have cell cycle coupled expression. We here show the usefulness of quantitative analysis of topo IIalpha mRNA to rapidly evaluate cell proliferation in brain tumors. A protocol to quantify topo IIalpha mRNA was developed with a real-time RT-PCR. It took only 3 h to quantify from a specimen. A total of 28 brain tumors were analyzed, and the level of topo IIalpha mRNA was significantly correlated with its immuno-staining index (p<0.0001, r=0.9077). Furthermore, it sharply detected that topo IIalpha mRNA decreased in growth-inhibited glioma cell. These results support that topo IIalpha mRNA may be a good and rapid indicator to evaluate cell proliferate potential in brain tumors.     

Bone marrow is a preferred site for homeostatic proliferation of memory CD8 T cells

Proliferative renewal of memory CD8 T cells is essential for maintaining long-term immunity. In this study, we examined the contributions that various tissue microenvironments make toward the homeostatic proliferation of Ag-specific memory CD8 T cells. We found that dividing memory T cells were present in both lymphoid and nonlymphoid tissues. However, the bone marrow was the preferred site for proliferation and contained a major pool of the most actively dividing memory CD8 T cells. Adoptive transfer studies indicated that memory cells migrated through the bone marrow and divided there preferentially. These results show that the bone marrow is not only the source of stem cells for generating naive T cells but also provides the necessary signals for the self-renewal of memory T cells.     

Quantitative analysis of the role of receptor recycling in T cell polarization

Activation of T cells of the immune system involves recognition of the antigen by the T cell receptor and subsequent internalization and recycling of this receptor. We present a numerical model for this process that accounts for the polarity of the intracellular traffic determined by the polarization of the microtubule-organizing center to the immunological synapse. Unexpectedly, the model explains the observed accumulation of receptors at the immunological synapse mainly as dynamic maintenance of the receptor density there, while the surface receptors everywhere else are depleted, even though the internalization occurs primarily at the synapse. In the case of an unsuccessful polarization of the microtubule-organizing center, which alters the polarity of the receptor trafficking, the model explains the absence of receptor accumulation as a dynamic downregulation at the synapse. The experiment shows that in this case the interaction of the T cell with its target is aborted. Disruption of recycling leads in the experiment to accumulation of the incompletely polarized cells. We propose that receptor recycling is a mechanism whereby the cell can sense its internal structure and detect polarity errors, analogous to checkpoint signaling mechanisms that ensure fidelity of cell division.     

MHC class I-positive dendritic cells (DC) control CD8 T cell homeostasis in vivo: T cell lymphopenia as a prerequisite for DC-mediated homeostatic proliferation of naive CD8 T cells

The sizes of peripheral T cell pools are regulated by competition for environmental signals within a given ecological T cell niche. Cytokines and MHC molecules have been identified as resources for which naive T cells compete to proliferate homeostatically in lymphopenic hosts to fill up their respective compartments. However, it still remains unclear to what extent CD4 and CD8 T cells intercompete for these resources and which role dendritic cells (DC) play in this scenario. Using transgenic mice in which only DC express MHC class I, we demonstrate that this type of APC is sufficient to trigger complete homeostatic proliferation of CD8 T cells in vivo. However, normal numbers of endogenous naive CD4 T cells, but not CD25(+)CD4(+) T regulatory cells, efficiently suppress this expansion in vivo. These findings identify DC as a major resource and a possible target for homeostatic competition between naive CD4 and CD8 T cells.     

Homeostatic T cell proliferation as a barrier to T cell tolerance

The maintenance of T cell numbers in the periphery is mediated by distinct homeostatic mechanisms that ensure the proper representation of na?ve and memory T cells. Homeostatic proliferation refers to the process by which T cells in lymphopenic hosts divide in the absence of cognate antigen to reconstitute the peripheral lymphoid compartment. During this process T cells acquire effector-memory like properties, including the ability to respond to low doses of antigen in the absence of CD28 costimulation. Furthermore, this capacity is retained long after proliferation has ceased. Accumulating data implicates homeostatic proliferation in autoimmune diseases and transplant rejection, and suggests that it may represent a barrier to tolerance in protocols that use T cell depletion. Implementing combination therapies that aim to promote the development and expansion of regulatory T cell populations while specifically targeting alloresponsive T cells may be the soundest approach to attaining allograft tolerance in the aftermath of T cell depletion and homeostatic proliferation.     

PSGL-1 regulates the migration and proliferation of CD8(+) T cells under homeostatic conditions

P-selectin glycoprotein ligand-1 (PSGL-1), a heavily glycosylated sialomucin expressed on most leukocytes, has dual function as a selectin ligand for leukocyte rolling on vascular selectins expressed in inflammation and as a facilitator of resting T cell homing into lymphoid organs. In this article, we document disturbances in T cell homeostasis present in PSGL-1(null) mice. Naive CD4(+) and CD8(+) T cell frequencies were profoundly reduced in blood, whereas T cell numbers in lymph nodes and spleen were at or near normal levels. Although PSGL-1(null) T cells were less efficient at entering lymph nodes, they also remained in lymph nodes longer than PSGL-1(+/+) T cells, suggesting that PSGL-1 supports T cell egress. In addition, PSGL-1(null) CD8(+) T cell proliferation was observed under steady-state conditions and PSGL-1(null) CD8(+) T cells were found to be hyperresponsive to homeostatic cytokines IL-2, IL-4, and IL-15. Despite these disturbances in T cell homeostasis, PSGL-1(null) mice exhibited a normal acute response (day 8) to lymphocytic choriomeningitis virus infection but generated an increased frequency of memory T cells (day 40). Our observations demonstrate a novel pleiotropic influence of PSGL-1 deficiency on several aspects of T cell homeostasis that would not have been anticipated based on the mild phenotype of PSGL-1(null) mice. These potentially offsetting effects presumably account for the near-normal cellularity seen in lymph nodes of PSGL-1(null) mice.     

Quantitative analysis of cell proliferation and differentiation in the cortex of the postnatal mouse cerebellum

The generation cycle of germinative cells (external matrix cells) in the external granular layer of the cerebellar cortex of the 10-to 11-day-old mouse was studied by radioautography following repeated injections of H³-thymidine. The generation time is 19 hr, presynthetic time 8.5 hr, DNA-synthetic time 8 hr, postsynthetic time 2 hr, and mitotic time 0.5 hr. These proliferating cells occupy the outer half of the external granular layer and make up the external matrix layer. Neuroblasts are differentiated from the external matrix cell, migrate out from the layer and accumulate in the inner half of the external granular layer to form the external mantle layer. The transit time of the neuroblasts in the external mantle layer is 28 hr. Thereafter, they migrate farther into the molecular layer and the internal granular layer. By means of long-term cumulative labeling, the rate of daily production of neuroblasts from the external matrix cell is studied in quantitative terms. It becomes clear that the entire population of the inner granule neurons arises postnatally in the external granular layer between 1 and 18 days of age and that 95% of them is produced between postnatal days 4 and 15. Finally, the fate of the cells in the external granular layer at its terminal stage was studied by marking the cells with H³-thymidine during 15–16 days of life and following their subsequent migration and developmental changes up to 21 days of life. Comparison of radioautographs taken before and after the migration disclosed that the external matrix cells give rise to a small number of neuroglia cells. This finding revealed their multipotential nature.     

CCR7 signaling inhibits T cell proliferation

CCR7 and its ligands, CCL19 and CCL21, are responsible for directing the migration of T cells and dendritic cells into lymph nodes, where these cells play an important role in the initiation of the immune response. Recently, we have shown that systemic application of CCL19-IgG is able to inhibit the colocalization of T cells and dendritic cells within secondary lymphoid organs, resulting in pronounced immunosuppression with reduced allograft rejection after organ transplantation. In this study, we demonstrate that the application of sustained high concentrations of either soluble or immobilized CCL19 and CCL21 elicits an inhibitory program in T cells. We show that these ligands specifically interfere with cell proliferation and IL-2 secretion of CCR7(+) cells. This could be demonstrated for human and murine T cells and was valid for both CD4(+) and CD8(+) T cells. In contrast, CCL19 had no inhibitory effect on T cells from CCR7 knockout mice, but CCR7(-/-) T cells showed a proliferative response upon TCR-stimulation similar to that of CCL19-treated wild-type cells. Furthermore, the inhibition of proliferation is associated with delayed degradation of the cyclin-dependent kinase (CDK) inhibitor p27(Kip1) and the down-regulation of CDK1. This shows that CCR7 signaling is linked to cell cycle control and that sustained engagement of CCR7, either by high concentrations of soluble ligands or by high density of immobilized ligands, is capable of inducing cell cycle arrest in TCR-stimulated cells. Thus, CCR7, a chemokine receptor that has been demonstrated to play an essential role during activation of the immune response, is also competent to directly inhibit T cell proliferation.     

Recent immune status determines the source of antigens that drive homeostatic T cell expansion

Homeostatic proliferation of naive T cells transferred to T cell-deficient syngeneic mice is driven by low-affinity self-MHC/peptide ligands and the cytokine IL-7. In addition to homeostatic proliferation, a subset of naive T cells undergoes massive proliferation in chronically immunodeficient hosts, but not in irradiated normal hosts. Such rapid T cell proliferation occurs largely independent of homeostatic factors, because it was apparent in the absence of IL-7 and in T cell-sufficient hosts devoid of functional T cell immunity. Strikingly, immunodeficient mice raised under germfree conditions supported only slow homeostatic proliferation, but not the marked T cell proliferation observed in conventionally raised immunodeficient mice. Thus, polyclonal naive T cell expansion in T cell-deficient hosts can be driven predominantly by either self-Ags or foreign Ags depending on the host's previous state of T cell immunocompetency.     

Distinct but complementary roles of Fas ligand and Bim in homeostatic T cell apoptosis

Vascular smooth muscle cell (VSMC) phenotypic modulation and proliferation are critical cellular events in the development of a variety of proliferative vascular diseases. However, the molecular mechanisms involved in cellular events are still unclear. MicroRNAs (miRNAs) represent a novel class of small, non-coding RNAs that negatively regulate gene expression via degradation or translational inhibition of their target mRNAs. In a previous study, we identified that miR-145 is the most abundant miRNA in normal arteries and VSMCs. However, the roles of miR-145 in VSMC biology and vascular disease are unknown. In our recent Circulation Research article, we found that the expression of miR-145 is significantly downregulated in dedifferentiated VSMCs and in balloon-injured arteries. Moreover, both in vitro and in vivo studies demonstrated that miR-145 is a critical modulator of VSMC phenotype and proliferation. This review article summarizes the current research progress regarding the roles of miR-145 in VSMC biology and discusses the potential therapeutic opportunities surrounding this miRNA in vascular disease.