John Iacomini

RAG-1-deficient mice have no mature B and T lymphocytes

The V(D)J recombination activation gene RAG-1 was isolated on the basis of its ability to activate V(D)J recombination on an artificial substrate in fibroblasts. This property and the expression pattern in tissues and cell lines indicate that RAG-1 either activates or catalyzes the V(D)J recombination reaction of immunoglobulin and T cell receptor genes. We here describe the introduction of a mutation in RAG-1 into the germline of mice via gene targeting in embryonic stem cells. RAG-1-deficient mice have small lymphoid organs that do not contain mature B and T lymphocytes. The arrest of B and T cell differentiation occurs at an early stage and correlates with the inability to perform V(D)J recombination. The immune system of the RAG-1 mutant mice can be described as that of nonleaky scid mice. Although RAG-1 expression has been reported in the central nervous system of the mouse, no obvious neuroanatomical or behavioral abnormalities have been found in the RAG-1-deficient mice.

Oocyte Generation in Adult Mammalian Ovaries by Putative Germ Cells in Bone Marrow and Peripheral Blood

It has been suggested that germline stem cells maintain oogenesis in postnatal mouse ovaries. Here we show that adult mouse ovaries rapidly generate hundreds of oocytes, despite a small premeiotic germ cell pool. In considering the possibility of an extragonadal source of germ cells, we show expression of germline markers in bone marrow (BM). Further, BM transplantation restores oocyte production in wild-type mice sterilized by chemotherapy, as well as in ataxia telangiectasia-mutated gene-deficient mice, which are otherwise incapable of making oocytes. Donor-derived oocytes are also observed in female mice following peripheral blood transplantation. Although the fertilizability and developmental competency of the BM and peripheral blood-derived oocytes remain to be established, their morphology, enclosure within follicles, and expression of germ-cell- and oocyte-specific markers collectively support that these cells are bona fide oocytes. These results identify BM as a potential source of germ cells that could sustain oocyte production in adulthood.

T cell receptor δ gene mutant mice: Independent generation of αβ T cells and programmed rearrangements of γδ TCR genes

Abstract T cells bearing T cell receptor (TCR) γ and δ chain heterodimers are first generated early in ontogeny. They form distinct subsets that differ in their TCR repertoires and tissue distribution. Disruption of the mouse TCR Cδ gene segment by a gene targeting method caused the complete loss of T cells bearing TCR γδ chains, but had little or no effect on the development of T cells bearing TCR αβ chains. The analyses of TCR γ and δ genes in the mutant mice suggest that intracellular mechanisms acting at the level of DNA rearrangement play key roles in the differential γ and δ gene rearrangements and in the generation of the highly restricted junctional sequences during fetal thymic development.

Identification of a microRNA signature of renal ischemia reperfusion injury

Renal ischemia reperfusion injury (IRI) is associated with significant morbidity and mortality. Given the importance of microRNAs (miRNAs) in regulating gene expression, we examined expression profiles of miRNAs following renal IRI. Global miRNA expression profiling on samples prepared from the kidneys of C57BL/6 mice that underwent unilateral warm ischemia revealed nine miRNAs (miR-21, miR-20a, miR-146a, miR-199a-3p, miR-214, miR-192, miR-187, miR-805, and miR-194) that are differentially expressed following IRI when compared with sham controls. These miRNAs were also differently expressed following IRI in immunodeficient RAG-2/common γ-chain double-knockout mice, suggesting that the changes in expression observed are not significantly influenced by lymphocyte infiltration and therefore define a lymphocyte-independent signature of renal IRI. In vitro studies revealed that miR-21 is expressed in proliferating tubular epithelial cells (TEC) and up-regulated by both cell-intrinsic and -extrinsic mechanisms resulting from ischemia and TGF-β signaling, respectively. In vitro, knockdown of miR-21 in TEC resulted in increased cell death, whereas overexpression prevented cell death. However, overexpression of miR-21 alone was not sufficient to prevent TEC death following ischemia. Our findings therefore define a molecular fingerprint of renal injury and suggest miR-21 may play a role in protecting TEC from death.

A novel role of CD4 Th17 cells in mediating cardiac allograft rejection and vasculopathy

T-bet plays a crucial role in Th1 development. We investigated the role of T-bet in the development of allograft rejection in an established MHC class II–mismatched (bm12 into B6) model of chronic allograft vasculopathy (CAV). Intriguingly, and in contrast to IFN-γ−/− mice that are protected from CAV, T-bet−/− recipients develop markedly accelerated allograft rejection accompanied by early severe vascular inflammation and vasculopathy, and infiltration by predominantly IL-17–producing CD4 T cells. Concurrently, T-bet−/− mice exhibit a T helper type 1 (Th1)–deficient environment characterized by profound IFN-γ deficiency, a Th2 switch characterized by increased production of interleukin (IL) 4, IL-5, IL-10, and IL-13 cytokines, as well as increased production of the proinflammatory cytokines IL-6, IL-12p40, and IL-17. Neutralization of IL-17 inhibits accelerated allograft rejection and vasculopathy in T-bet−/− mice. Interestingly, CD4 but not CD8 T cell deficiency in T-bet−/− mice affords dramatic protection from vasculopathy and facilitates long-term graft acceptance. This is the first study establishing that in the absence of Th1-mediated alloimmune responses, CD4 Th17 cells mediate an aggressive proinflammatory response culminating in severe accelerated allograft rejection and vasculopathy. These results have important implications for the development of novel therapies to target this intractable problem in clinical solid organ transplantation.

Prevention of type 1 diabetes by gene therapy

The autoimmune disease type 1 diabetes in humans and NOD mice is determined by multiple genetic factors, among the strongest of which is the inheritance of diabetes-permissive MHC class II alleles associated with susceptibility to disease. Here we examined whether expression of MHC class II alleles associated with resistance to disease could be used to prevent the occurrence of diabetes. Expression of diabetes-resistant MHC class II I-Abeta chain molecules in NOD mice following retroviral transduction of autologous bone marrow hematopoietic stem cells prevented the development of autoreactive T cells by intrathymic deletion and protected the mice from the development of insulitis and diabetes. These data suggest that type 1 diabetes could be prevented in individuals expressing MHC alleles associated with susceptibility to disease by restoration of protective MHC class II expression through genetic engineering of hematopoietic stem cells.

Costimulation-dependent expression of microRNA-214 increases the ability of T cells to proliferate by targeting Pten.

T cell activation requires signaling through the TCR and costimulatory molecules, such as CD28. MicroRNAs (miRNAs) are small noncoding RNAs that regulate gene expression posttranscriptionally and are also known to be involved in lymphocyte development and function. In this paper, we set out to examine potential roles of miRNAs in T cell activation, using genome-wide expression profiling to identify miRNAs differentially regulated following T cell activation. One of the miRNAs upregulated after T cell activation, miR-214, was predicted to be capable of targeting Pten based on bioinformatics and reports suggesting that it targets Pten in ovarian tumor cells. Upregulation of miR-214 in T cells inversely correlated with levels of phosphatase and tensin homolog deleted on chromosome 10. In vivo, transcripts containing the 3′ untranslated region of Pten, including the miR-214 target sequence, were negatively regulated after T cell activation, and forced expression of miR-214 in T cells led to increased proliferation after stimulation. Blocking CD28 signaling in vivo prevented miR-214 upregulation in alloreactive T cells. Stimulation of T cells through the TCR alone was not sufficient to result in upregulation of miR-214. Thus, costimulation-dependent upregulation of miR-214 promotes T cell activation by targeting the negative regulator Pten. Thus, the requirement for T cell costimulation is, in part, related to its ability to regulate expression of miRNAs that control T cell activation.

Targeting Tim-1 to overcome resistance to transplantation tolerance mediated by CD8 T17 cells

The ability to induce durable transplantation tolerance predictably and consistently in the clinic is a highly desired but elusive goal. Progress is hampered by lack of appropriate experimental models in which to study resistance to transplantation tolerance. Here, we demonstrate that T helper 1-associated T box 21 transcription factor (Tbet) KO recipients exhibit allograft tolerance resistance specifically mediated by IL-17-producing CD8 T (T17) cells. Neutralization of IL-17 facilitates long-term cardiac allograft survival with combined T cell co-stimulation (CD28-CD80/86 and CD154-CD40) blockade in Tbet KO recipients. We have used this T17-biased Tbet KO model of allograft tolerance resistance to study the impact of targeting a T cell-co-stimulatory pathway, and demonstrate that targeting T cell Ig and mucin domain-1 (Tim-1) with anti-Tim-1 overcomes this resistance by specifically inhibiting the pathogenic IL-17-producing CD8 T17 cells. These data indicate that in the absence of Th1 immunity, CD8 T17 alloreactivity constitutes a barrier to transplantation tolerance. Targeting TIM-1 provides an approach to overcome resistance to tolerance in clinical transplantation.

The Role of T Cell Help in the Production of Antibodies Specific for Galα1–3Gal

The majority of xenoreactive natural Abs in humans recognize the carbohydrate Ag present on pig tissue, Galα1–3Galβ1–4GlcNAc-R (αGal), synthesized by the enzyme UDP galactose:β-d-galactosyl-1,4-N-acetyl-d-glucosaminide α(1–3)galactosyltransferase or αGT. Using αGT knockout mice (GT0 mice), which like humans produce serum Abs that bind αGal, we examined the role of T cells in production of Abs specific for αGal. GT0 mice were crossed with TCR-β knockout mice (TCR-β0) to generate double-knockout mice (GT0/TCR-β0). While GT0/TCR-β+ mice exhibited an age-dependent increase in the serum titer of natural Abs specific for αGal, a similar increase was not observed in GT0/TCR-β0 mice, and the titer of αGal-specific Abs in double knockouts was significantly lower than in age-matched GT0/TCR-β+ mice. Immunization with pig cells resulted in a significant increase in the serum titer of αGal-specific Abs in GT0/TCR-β+ mice, but had no effect on the level of αGal-specific serum Abs in GT0/TCR-β0 mice. Treatment of GT0/TCR-β+ mice with anti-CD40L Abs before immunization with pig cells prevented sensitization to αGal. Our data suggest that the majority of αGal-specific Abs are T cell dependent and that production of αGal-specific Abs after sensitization can be prevented by blocking costimulatory pathways.

Critical role of proinflammatory cytokine IL-6 in allograft rejection and tolerance.

The proinflammatory cytokine IL‐6 plays an important role in controlling T‐cell differentiation, especially the development of Th17 and regulatory T cells. To determine the function of IL‐6 in regulating allograft rejection and tolerance, BALB/c cardiac grafts were transplanted into wild‐type or IL‐6‐deficient C57BL/6 mice. We observed that production of IL‐6 and IFN‐γ was upregulated during allograft rejection in untreated wild‐type mice. In IL‐6‐deficient mice, IFN‐γ production was greater than that observed in wild‐type controls, suggesting that IL‐6 production affects Th1/Th2 balance during allograft rejection. CD28‐B7 blockade by CTLA4‐Ig inhibited IFN‐γ production in C57BL/6 recipients, but had no effect on the production of IL‐6. Although wild‐type C57BL/6 recipients treated with CTLA4‐Ig rejected fully MHC‐mismatched BALB/c heart transplants, treatment of IL‐6‐deficient mice with CTLA4‐Ig resulted in graft acceptance. Allograft acceptance appeared to result from the combined effect of costimulatory molecule blockade and IL‐6‐deficiency, which limited the differentiation of effector cells and promoted the migration of regulatory T cells into the grafts. These data suggest that the blockade of IL‐6, or its signaling pathway, when combined with strategies that inhibit Th1 responses, has a synergistic effect on the promotion of allograft acceptance. Thus, targeting the effects of IL‐6 production may represent an important part of costimulation blockade‐based strategies to promote allograft acceptance and tolerance.