A Detailed Protocol for Characterizing the Murine C1498 Cell Line and its Associated Leukemia Mouse Model

The intravenous injection of C1498 cells into syngeneic or congenic mice has been performed since 1941. These injections result in the development of ...

INSERM, UMR-S-1172, Centre de Recherche Jean-Pierre Aubert (JPARC)

Université de Lille

Dendritic cells (DC) are potent inducers of immunity to foreign Ags, but also contribute to self-tolerance by induction of regulatory T cells or deletion/anergy of self-reactive T cells. In this study, we have studied the capacity of DC to activate naturally occurring CD4+CD25+ regulatory T cells as well as the ability of CD4+CD25+ T cells to suppress the DC-mediated activation of CD4+CD25- T cells. Mature bone marrow-derived dendritic cells, but not splenic DC, were able to induce the proliferation of CD4+CD25+ T cells in the presence of a polyclonal stimulus and in the absence of exogenous IL-2. The DC-induced proliferative response of the CD4+CD25+ T cells was partially dependent on IL-2 produced by a small number of contaminating CD25+ effector cells. Because bone marrow-derived dendritic cells induce proliferation of both CD4+CD25+ and CD4+CD25- T cells in vitro, it was impossible to assay the suppressive function of the CD4+CD25+ T cells using [3H]TdR uptake or CFSE dilution. We therefore measured IL-2 production in cocultures of CD4+CD25+ and CD4+CD25- T cells using the IL-2 secretion assay. Surprisingly, CD4+CD25+ T cells markedly suppressed IL-2 secretion by the CD4+CD25- T cells without inhibiting their proliferation. Collectively, these results suggest that Ag presentation by DC can induce the expansion of CD4+CD25+ T cells while simultaneously activating their ability to suppress cytokine secretion by effector T cells.

Bone marrow-derived dendritic cells reverse the anergic state of CD4+CD25+ T cells without reversing their suppressive function.

Several strategies are being designed to test the therapeutic potential of Ag-specific regulatory T cells to prevent or treat autoimmune diseases. In this study, we demonstrate that naive CD4+ Foxp3- T cells specific for a naturally expressed autoantigen (H+/K+ ATPase) can be converted to Foxp3+ T regulatory cells (Tregs) when stimulated in presence of TGFbeta. TGFbeta-induced Tregs (iTregs) have all the characteristics of naturally generated regulatory T cells in vitro, and more importantly, are effective at preventing organ-specific autoimmunity in a murine model of autoimmune gastritis. H+/K+ ATPase specific iTregs were able to inhibit the initial priming and proliferation of autoreactive T cells, and appear to do so by acting on H+/K+ ATPase presenting dendritic cells (DC). DC exposed to iTregs in vivo were reduced in their ability to stimulate proliferation and cytokine production by H+/K+ ATPase specific T cells. iTregs specifically reduced CD80 and CD86 expression on the surface of H+/K+ ATPase presenting DC in vitro. These studies reveal the therapeutic potential of Ag specific iTregs to prevent autoimmunity, and provide a mechanism by which this population of regulatory T cells, and perhaps others, mediate their suppressive effects in vivo.

Autoantigen-specific TGFbeta-induced Foxp3+ regulatory T cells prevent autoimmunity by inhibiting dendritic cells from activating autoreactive T cells.

CD4+CD25+forkhead box p3 (Foxp3)+ regulatory T cells (Treg) control peripheral tolerance. Although Treg are anergic when stimulated through the TCR, mature bone marrow-derived, but not splenic, dendritic cells (DC) can induce their proliferation after TCR stimulation in the absence of IL-2. One possibility is that the DC produce proinflammatory cytokines such as IL-1 or IL-6 that function as growth factors for Treg. We have analyzed the costimulatory effects of IL-1 on the expansion of Foxp3+ Treg in vitro. When CD4+CD25+ T cells were cultured in the presence of splenic DC and IL-1, marked expansion of the Foxp3+ T cells was observed. The effects of IL-1 were mediated on CD4+CD25+Foxp3(-) T cells present in the starting population rather than on the DC or on the CD4+CD25+Foxp3+ T cells. In contrast, stimulation of CD4+CD25+ T cells with plate-bound anti-CD3 and IL-1 in the absence of DC resulted in the outgrowth of a CD4+CD25+Foxp3(-) T cell population composed of NKT cells and non-NKT, IL-17-producing cells. Foxp3+ Treg purified from mice expressing the reporter gene enhanced GFP in the Foxp3 locus failed to proliferate when costimulated with IL-1. These findings have important implications for the design of protocols for the expansion of CD4+CD25+ T cells for cellular biotherapy.

Costimulatory effects of IL-1 on the expansion/differentiation of CD4+CD25+Foxp3+ and CD4+CD25+Foxp3- T cells.

Acute graft-versus-host disease (aGVHD), mediated by CD4(+) and CD8(+) effector T cells, is a life-threatening complication in hematopoietic stem cell transplantation. CD4(+)CD25(hi) regulatory T cells (T(reg)) have been shown to modulate tolerance to aGVHD in murine models. Based on these observations, we examined their role in the prevention of aGVHD in patients who underwent transplantation with peripheral blood-mobilized hematopoietic stem cells after administration of granulocyte colony-stimulating factor. The effects of the G-CSF on the phenotype, frequency, and function of CD4(+)CD25(hi) T cells were analyzed in grafts and after transplantation to determine whether these cells were regulatory T cells. CD4(+)CD25(hi) T cells could be detected at the same frequency before and after granulocyte colony-stimulating factor administration in the donors' peripheral blood. The isolation of these cells from the grafts or from the recipients' peripheral blood after transplantation revealed that they were suppressive to the same extent as T(reg) isolated from healthy volunteers. Their number and frequency were estimated in the grafts and the results indicated that protection against aGVHD was not dependent on the T(reg) amount transferred to the recipients. Similarly there was no correlation between the number of circulating CD4(+)CD25(hi) T cells in the recipients' peripheral blood during the early period after transplantation and the outcome of aGVHD.

Regulatory CD4+CD25hi T cells conserve their function and phenotype after granulocyte colony-stimulating factor treatment in human hematopoietic stem cell transplantation.

We examined TCR:MHC/peptide interactions and in vivo epitope availability to explore the Th1- or Th2-like phenotype of autoimmune disease in two TCR Tg mouse models of autoimmune gastritis (AIG). The TCR of strains A23 and A51 recognize distinct IA(d)-restricted peptides from the gastric parietal cell H/K-ATPase. Both peptides form extremely stable MHC/peptide (MHC/p) complexes. All A23 animals develop a Th1-like aggressive, inflammatory AIG early in life, while A51 mice develop indolent Th2-like AIG at 6-8 wk with incomplete penetrance. A51 T cells were more sensitive than A23 to low doses of soluble antigen and to MHC/p complexes. Staining with IA(d)/peptide tetramers was only detectable on previously activated T cells from A51. Thus, despite inducing a milder AIG, the A51 TCR displays a higher avidity for its cognate IA(d)/peptide. Nonetheless, in vivo proliferation of adoptively transferred A51 CFSE-labeled T cells in the gastric lymph node was relatively poor compared with A23 T cells. Also, DC from WT gastric lymph node, presenting processed antigen available in vivo, stimulated proliferation of A23 T cells better than A51. Thus, the autoimmune potential of these TCR in their respective Tg lines is strongly influenced by the availability of the peptide epitope, rather than by differential avidity for their respective MHC/p complexes.

Availability of autoantigenic epitopes controls phenotype, severity, and penetrance in TCR Tg autoimmune gastritis.

In this study, we explored the potential of human naive CD4(+) T cells to acquire regulatory properties upon stimulation. We demonstrated that, in vitro, pre-activated naive CD4(+)CD25(-)CD45RA(+) T cells could become anergic and suppressive CD4(+)CD25(+) T cells upon lower intensity TCR stimulation. These CD4(+)CD25(+) T cells generated in vitro potently suppress the proliferation of allogenic CD4(+)CD25(-) T cells independently of cytokines and in a contact-dependent manner. Our data indicate that expression of Foxp3 is not necessary to induce the suppressive T cell activity. We demonstrate that these CD4(+)CD25(+) T cells are unresponsive upon re-stimulation and that their suppressive activity is transient. However, we showed that the anergy and the suppressive function could be reversed by increasing the stimulus and their level of activation. We concluded from our data that these anergy and suppressive activities are related to a fine tuning of TCR activation threshold.

Modulation of the TCR stimulation strength can render human activated CD4+ T cells suppressive.

This unit presents two methods for preparing dendritic cells (DCs), a highly specialized type of antigen-presenting cell (APC). The first method involves the isolation of DCs from mouse spleen, resulting in a cell population that is highly enriched in accessory cell and APC function. A support protocol for collagenase digestion of splenocyte suspensions is described to increase the yield of dendritic cells. The second method involves generating large numbers of DCs from mouse bone marrow progenitor cells. In that technique, bone marrow cells are cultured in the presence of granulocyte/macrophage colony-stimulating factor (GM-CSF) to yield 5-10 x 10(6) cells, 60% of which express DC surface markers (e.g., B-7-2/CD86). Additional techniques for isolating DCs from mouse spleens or other mouse tissues, as well as from human tissues, are also discussed.

Isolation of dendritic cells.

Recent studies on acute myelogenous leukemia (AML) patients have revealed the existence of T-cell immunodeficiencies, characterized by peripheral T lymphocytes that are unable to interact with blasts, reduced thymic emigrants and oligoclonal restricted repertoires. These observations suggest that there is a profound thymic dysregulation, which is difficult to study in AML patients. Using the C1498 AML mouse model, we demonstrated that leukemia development was associated with thymus atrophy, which was defined by abnormal organ weight and reduced cellularity. In addition, we observed a dramatic loss of peripheral CD4(+) and CD8(+) T-cell numbers with increased frequencies of CD4(+) FoxP3(+) regulatory and activated/memory T cells. Investigating the mechanisms leading to this atrophy, we observed a significant accumulation of the monocyte chemoattractant protein 1 (MCP-1/CCL2) in thymi of leukemic mice. Treatment of AML-bearing animals with a blocking anti-CCL2 antibody revealed a lower tumor burden, augmented antileukemic T-cell responses, and improved survival rate compared to nontreated mice. These results were not observed when neutralization of CCL2 was performed in thymectomized mice. Altogether, we show that the CCL2 protein participates in thymic atrophy in AML mice, and this could have important implications for future immunotherapeutic strategies.

Carine Brinster

Institut pour la Recherche sur le Cancer de Lille (IRCL)

INSERM, UMR-S-1172, Centre de Recherche Jean-Pierre Aubert (JPARC)

A Detailed Protocol for Characterizing the Murine C1498 Cell Line and its Associated Leukemia Mouse Model

Carine Brinster

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INSERM, UMR-S-1172, Centre de Recherche Jean-Pierre Aubert (JPARC)

A Detailed Protocol for Characterizing the Murine C1498 Cell Line and its Associated Leukemia Mouse Model

Institut pour la Recherche sur le Cancer de Lille (IRCL)