Apoptotic Endonuclease EndoG Induces Alternative Splicing of Telomerase TERT Catalytic Subunit,Caspase-2, DNase I,and BCL-x in Human,Murine, and Rat CD4<Superscript>+</Superscript> T Lymphocytes

Apoptotic endonuclease EndoG plays a key role in the alternative splicing of mRNA of human TERT telomerase catalytic subunit. The aim of this work was to test the ability of EndoG to induce alternative splicing of mRNA of other genes and in other organisms. To determine new mRNA splice-variants, EndoG overexpression was induced in human, mouse and rat CD4⁺-T-lymphocytes followed by sequencing of total RNA of these cells. Sequencing results showed that besides TERT, EndoG induced alternative splicing of deoxyribonuclease I (DNase I), caspase-2 (Casp-2) and BCL-x. The expression level of EndoG strongly correlated with mRNA splicing-variants of TERT, DNase I, Casp-2, and BCL-x in intact CD4⁺-T cells of healthy donors as well as different lines of mice and rats. EndoG overexpression induced down-regulation of fulllength mRNAs of TERT, DNase I, Casp-2, and BCL-x and up-regulation of their short-length mRNAs. Alternative splicing of studied mRNAs resulted in down-regulation of enzymatic activity of proteins in vitro and in vivo. The results of this work confirm the ability of endonuclease EndoG to induce alternative splicing of several mRNAs in human, mice and rats.     

Apoptotic endonuclease EndoG regulates alternative splicing of human telomerase catalytic subunit hTERT

Human telomerase catalytic subunit hTERT is subjected to alternative splicing results in loss of its function and leads to decrease of telomerase activity. However, very little is known about the mechanism of hTERT pre-mRNA alternative splicing. Apoptotic endonuclease EndoG is known to participate this process. The aim of this study was to determine the role of EndoG in regulation of hTERT alternative splicing. Increased expression of β-deletion splice variant was determined during EndoG overexpression in CaCo-2 cell line, after EndoG treatment of cell cytoplasm and nuclei as well as after nuclei incubation with EndoG digested cell RNA. hTERT alternative splicing was induced by 47-mer RNA oligonucleotide in naked nuclei and in cells after transfection. Identified long non-coding RNA, that is the precursor of 47-mer RNA oligonucleotide. Its size is 1754 nucleotides. Based on the results the following mechanism was proposed. hTERT pre-mRNA is transcribed from coding DNA strand while long non-coding RNA is transcribed from template strand of hTERT gene. EndoG digests long non-coding RNA and produces 47-mer RNA oligonucleotide complementary to hTERT pre-mRNA exon 8 and intron 8 junction place. Interaction of 47-mer RNA oligonucleotide and hTERT pre-mRNA causes alternative splicing.     

Identity of mysterious CD4<Superscript>+</Superscript>CD25<Superscript>-</Superscript>Foxp3<Superscript>+</Superscript>cells in systemic lupus erythematosus

Various abnormalities in CD4⁺CD25⁺ regulatory T cells (Tregs) in systemic lupus erythematosus (SLE) include increased Foxp3⁺ cells that are CD25 negative. Barring methodological technical factors, these cells could be atypical Tregs or activated non-Treg CD4⁺ cells that express Foxp3. Two groups have reached opposite conclusions that could possibly reflect the subjects studied. One group studied untreated new-onset SLE and suggested that these T cells were mostly CD25^-Foxp3⁺ non-Tregs. The other group studied patients with long-standing disease and suggested that these cells are mostly dysfunctional Tregs. A third group reported increased Foxp3⁺CD4⁺CD25^dim rather than CD25^- cells in active SLE and these were also non-Tregs. Thus, it is likely that not all Foxp3⁺ T cells in SLE have protective suppressive activity.     

CD4<Superscript>+</Superscript>CD25<Superscript>+</Superscript> immunoregulatory T cells may not be involved in controlling autoimmune arthritis

Accumulating evidence suggests that regulatory T cells play a crucial role in preventing autoimmunity. Recently, a naturally occurring CD4⁺CD25⁺ T-cell subset that is anergic and also suppressive has been shown to suppress autoimmunity in several animal models. We used proteoglycan-induced arthritis (PGIA) as a study model to investigate the role of the CD4⁺CD25⁺ regulatory T cells in autoimmune arthritis. There was no significant change in the percentage of CD4⁺CD25⁺ T cells during the immunization period when proteoglycan- or ovalbumin-immunized BALB/c and C57BL/6 mice were compared. An adoptive transfer study showed that the CD4⁺CD25⁺ T cells did not protect severe combined immunodeficient mice from arthritis when they were cotransferred with splenocytes from arthritic animals. Similarly, depletion of the CD4⁺CD25⁺ T cells did not enhance the onset of the disease or disease severity in severe combined immunodeficient mice. Moreover, CD28-deficient mice, which have very few CD4⁺CD25⁺ T cells, were highly resistant to PGIA. These findings indicate that the CD4⁺CD25⁺ regulatory T cells may not play a critical role in controlling PGIA.     

Therapy for pneumonitis and sialadenitis by accumulation of CCR2-expressing CD4<Superscript>+</Superscript>CD25<Superscript>+</Superscript>regulatory T cells in MRL/lpr mice

Adoptive transfer of CD4⁺CD25⁺ regulatory T cells has been shown to have therapeutic effects in animal models of autoimmune diseases. Chemokines play an important role in the development of autoimmune diseases in animal models and humans. The present study was performed to investigate whether the progression of organ-specific autoimmune diseases could be reduced more markedly by accumulating chemokine receptor-expressing CD4⁺CD25⁺ regulatory T cells efficiently in target organs in MRL/MpJ-lpr/lpr (MRL/lpr) mice. CD4⁺CD25⁺Foxp3⁺ T cells (Treg cells) and CD4⁺CD25⁺Foxp3⁺ CCR2-transfected T cells (CCR2-Treg cells) were transferred via retro-orbital injection into 12-week-old MRL/lpr mice at the early stage of pneumonitis and sialadenitis, and the pathological changes were evaluated. Expression of monocyte chemoattractant protein-1 (MCP-1)/CCL2 was observed in the lung and submandibular gland of the mice and increased age-dependently. The level of CCR2 expression and MCP-1 chemotactic activity of CCR2-Treg cells were much higher than those of Treg cells. MRL/lpr mice to which CCR2-Treg cells had been transferred showed significantly reduced progression of pneumonitis and sialadenitis in comparison with MRL/lpr mice that had received Treg cells. This was due to more pronounced migration of CCR2-Treg cells and their localization for a longer time in MCP-1-expressing lung and submandibular gland, resulting in stronger suppressive activity. We prepared chemokine receptor-expressing Treg cells and demonstrated their ability to ameliorate disease progression by accumulating in target organs. This method may provide a new therapeutic approach for organ-specific autoimmune diseases in which the target antigens remain undefined.     

Tacrolimus differentially regulates the proliferation of conventional and regulatory CD4<Superscript>+</Superscript> T cells

Tacrolimus is a widely used T cell targeted immunosuppressive drug, known as a calcineurin inhibitor. However, the exact pharmacological effects of tacrolimus on CD4⁺ T cells have yet to be elucidated. This study investigated the effects of tacrolimus on CD4⁺ T cell subsets. Mouse or human CD4⁺ T cells were cultured with immobilized anti-CD3/CD28 antibodies in the presence of tacrolimus. The cell division of CD4⁺ T cells was analyzed using a flow cytometer according to the expression of Foxp3. The gene expression patterns of tacrolimus-exposed T cells were examined by quantitative PCR. In the case of conventional CD4⁺ T cells (Tconv cells), tacrolimus inhibited T cell receptor stimulation-induced cell division. In contrast, the cell division of regulatory CD4⁺ T cells (Treg cells) was even promoted in the presence of tacrolimus, especially in humans. Tacrolimus did not promote conversion of Tconv to Treg cells in mice. Furthermore, tacrolimus modified the expression levels of Foxp3-regulated T cell receptor signal related-genes, PTPN22 and Itk, in human Treg cells. Immunosuppressive effect of tacrolimus may be attributed to the relatively enhanced proliferation of Treg cells in association with altered gene expression levels of TCR signaling molecules.     

CD4<Superscript>+</Superscript> T-cell recognition of human 5T4 oncofoetal antigen: implications for initial depletion of CD25<Superscript>+</Superscript> T cells

Background The human 5T4 (h5T4) oncofoetal antigen is expressed by a wide variety of human carcinomas including colorectal, ovarian, gastric and renal, but rarely on normal tissues. Its restricted expression on tumour tissues as well as its association with tumour progression and bad prognosis has driven the development of a MVA-based vaccine (TroVax) which has been tested in several early phase clinical trials and these studies have led to the start of a phase III trial in renal cell carcinoma patients. We have recently shown that CD8⁺ T cells recognizing h5T4 can be generated in the absence of CD4⁺ T cells from peripheral blood lymphocytes of human healthy individuals. Results We report the existence and expansion of human CD4⁺ T cells against h5T4 by stimulation with autologous monocyte-derived dendritic cells infected with a replication defective adenovirus encoding the h5T4 cDNA (Ad-h5T4). The h5T4-specific T-cell responses in normal individuals are enhanced by initial depletion of CD25⁺ cells (putative T regulatory cells) prior to the in vitro stimulation. We have identified a novel h5T4-derived 15-mer peptide recognized by CD4⁺ T cells in HLA-DR4 positive healthy individuals. Interestingly, CD4⁺ T cells spontaneously recognizing a different 5T4 epitope restricted by HLA-DR were identified in tumour-infiltrating lymphocytes isolated from a regressing renal cell carcinoma lung metastasis. Conclusion Our data show that CD4⁺ T cells recognizing h5T4 can be expanded and detected in healthy individuals and a renal cell carcinoma patient. Such h5T4-specific CD4⁺ T cells boosted or induced by vaccination could act to modulate both cell or antibody mediated anti-tumour responses. This work was supported by Cancer Research UK.     

Apoptosis of CD4<Superscript>+</Superscript>CD25<Superscript>high</Superscript> T cells in response to Sirolimus requires activation of T cell receptor and is modulated by IL-2

Targeted molecular therapies inhibit proliferation and survival of cancer cells but may also affect immune cells. We have evaluated the effects of Sirolimus and Sorafenib on proliferation and survival of lymphoid cell subsets. Both drugs were cytotoxic to CD4⁺CD25^high T cells, and were growth inhibitory for CD4⁺ and CD8⁺ T cells. Cytotoxicity depended on CD3/CD28 stimulation and was detectable within 12 h, with 80–90% of CD4⁺CD25^high cells killed by 72 h. Cell death was due to apoptosis, based on Annexin V and 7AAD staining. Addition of IL-2 prevented the apoptotic response to Sirolimus, potentially accounting for reports that Sirolimus can enhance proliferation of CD4⁺CD25^high cells. These results predict that Sirolimus or Sorafenib would reduce CD4⁺CD25^high cells if administered prior to antigenic stimulation in an immunotherapy protocol. However, administration of IL-2 protects CD4⁺CD25^high T cells from cytotoxic effects of Sirolimus, a response that may be considered in design of therapeutic protocols.     

Priming of naive CD8+ T cells in the presence of IL-12 selectively enhances the survival of CD8<Superscript>+</Superscript>CD62L<Superscript>hi</Superscript> cells and results in superior anti-tumor activity in a tolerogenic murine model

During the antigen-dependant activation process several subsets CD8⁺ T cells appear with different phenotypic and functional characteristics. Recent studies indicate that the state of T cell differentiation radically affects their ability to effectively respond to tumor challenge, with early effector CD8⁺ T (CD62L^high) cells having better anti-tumor activity. Thus strategies aimed at optimizing the generation of such subpopulations could significantly enhance the effectiveness of adoptive cell therapy (ACT) for cancer. In this study, we show that priming of naïve CD8⁺ T cells in the presence of IL-12 selectively rescued early CD8⁺ CD62L^hi from activation induced cell death and resulted in the increased accumulation of this subset of CD8⁺ T cells. Furthermore, we demonstrated that IL-12 directly modulated the expression of CD62L on activated CD8⁺ T cells. When used for ACT, naïve CD8⁺ T cells primed in vitro in the presence of IL-12 showed superior anti-tumor activity toward B16 melanoma. Importantly, using the Pmel-1 model, priming pmel-1 cells in vitro with IL-12 reduced the state of functional tolerance associated with the non-mutated “self” tumor antigen gp100, as demonstrated by significant tumor responses in the absence of vaccination. Together, our results suggest that in vitro conditioning of naïve CD8⁺ T cells with IL-12 prior to ACT could significantly enhance their anti-tumor activity.     

Dynamic Changes of CD4<Superscript>+</Superscript>CD25<Superscript>+</Superscript> Regulatory T Cells in NOD.H-2<Superscript>h4</Superscript> Mice with Iodine-Induced Autoimmune Thyroiditis

Iodine is an essential trace element for thyroid hormone synthesis and metabolism, either low or high intake may lead to thyroid disease, but the pathogenetic mechanisms by which iodine interacts with the thyroid autoimmune are poorly understood. We investigated the dynamic changes of CD4⁺CD25⁺ regulatory T cells in NOD.H-2^h4 mice with iodine-induced autoimmune thyroiditis (AIT), and explore potential immune mechanism of AIT induced by iodine. NOD.H-2^h4 mice were randomly divided into two groups, and received plain water or water containing 0.005% sodium iodide. Eight weeks after iodine provision, the incidences of thyroiditis, relative weights of thyroids, and serum thyroglobulin antibody titers in the iodine-supplied groups were significantly increased compared to the control groups (p < 0.05). The AIT mice had fewer CD4⁺CD25⁺Foxp3⁺ T cells and reduced Foxp3 mRNA expression in splenocytes compared with the controls (p < 0.01), and maintained relatively low levels during the development of thyroiditis. The changes described above aggravated gradually with the extension of iodine treatment. These data suggest that CD4⁺CD25⁺ regulatory T cells may be involved in the pathogenesis and development of AIT induced by iodine.     

The expansion of CD4<Superscript>+</Superscript>CD28<Superscript>-</Superscript> T cells in patients with rheumatoid arthritis

Clonal expansion of CD4⁺CD28^- T cells is a characteristic finding in patients with rheumatoid arthritis (RA). Expanded CD4⁺ clonotypes are present in the peripheral blood, infiltrate into the joints, and persist for years. CD4⁺CD28^- T cells are oligoclonal lymphocytes that are rare in healthy individuals but are found in high percentages in patients with chronic inflammatory diseases. The size of the peripheral blood CD4⁺CD28^- T-cell compartment was determined in 42 patients with RA and 24 healthy subjects by two-color FACS analysis. The frequency of CD4⁺CD28^- T cells was significantly higher in RA patients than in healthy subjects. Additionally, the number of these cells was significantly higher in patients with extra-articular manifestations and advanced joint destruction than in patients with limited joint manifestations. The results suggest that the frequency of CD4⁺CD28^- T cells may be a marker correlating with extra-articular manifestations and joint involvement.