Ts Cells Research Articles

Cyclophosphamide-modified murine peritoneal macrophages induce CD4+ T contrasuppressor cells that protect contact sensitivity T effector cells from suppression.

Cyclophosphamide (CY) is one of the most widely used alkylating agents in the treatment of various cancers and some autoimmune diseases. Numerous reports suggest that CY exerts immunoregulatory effects. Animal studies have shown CY affects contact sensitivity (CS) response by depleting CD4+CD25+ T regulatory cells and CD8+ T suppressor (Ts) cells. In a mouse model of CS, we previously showed that in vivo treatment with CY shapes the immunogenic/immunoregulatory balance of peritoneal macrophages. The aim of the current study is to verify if macrophages (Mf) from CY-treated mice are indeed able to induce immunoregulatory cells that could protect from suppression. Adoptive cell transfer of CS was used to examine immunomodulating properties of peritoneal Mf from CY-treated mice. Isolation of peritoneal Mf from animals that were (Mf-CY) or were not (Mf) treated with CY were cultured to identify cytokine repertoire. Further, we assessed spleen cell (SPLC) cytokine production following immunization with trinitrophenyl-conjugated Mf from donors treated (TNP-Mf-CY) or non-treated (TNP-Mf) with CY. In vitro experiments identified that Mf-CY produce more IL-6, TNF-α and TGF-β than naïve Mf. Further, immunization with peritoneal TNP-Mf-CY induces CD4+ T contrasuppressor cells (Tcs) cells that protect CS-effector cells from suppression. Higher IL-17A secretion was observed from TNP-Mf-CY-treated mouse SPLC compared to SPLC from TNP-Mf injected mice suggesting that this cytokine might be important in mediating contrasuppression in this model. Our results show that in vivo treatment with CY influences mouse peritoneal Mf to induce CD4+ Tcs cells that protect CS-effector cells from suppressive signals of Ts cells.

Characterization of high frequency of recombination strains selected by integrative suppression of F’lac in dnaA, gyrA and gyrB temperature sensitive mutants

Integration of F’lac plasmid into chromosome of both gyrA(Ts) and gyrB(Ts) cells phenotypically suppress the thermosensitive mutations of the DNA gyrase enzymes. As the comparative strains isolated from dnaA(Ts), these high frequency of recombination (Hfr) derivatives were able to transfer chromosomal markers to recipient strains, showed a growth rate of about 60 min, and developed filamentous forms when incubated at the temperature of 43°C. Conversely to dnaA(Ts) Hfr selected isolates, the great majority of Hfr derivative of gyrase mutants resulted resistant to acridine orange and rifampin. Time-kill experiments carried out at the non-permissive temperature also revealed that nalidixic acid has no antibacterial activity on these Hfr strains while derivatives of dnaA(Ts) mutant, as well as the control strain HfrH, were strongly inhibited by this drug. Therefore F plasmid induced duplication of chromosome in the mutants even if the DNA gyrase enzymes are not working. Of a certain interest is that these bacteria exhibit physiological perturbations that affect the main cellular functions, however, they do not appear essential for the survival of the strains.

Numerical and functional defects in CD8+ CD28- T-suppressor lymphocytes from patients with primary immune thrombocytopenia.

Primary immune thrombocytopenia (ITP) is an acquired autoimmune disorder, and loss of immune tolerance has been implicated in ITP pathogenesis. CD8+ CD28- suppressor (Ts) cells have an immunosuppression function and are involved in several autoimmune disorders. However, the role of Ts cells in ITP is currently not clear. Here, flow cytometry was used to detect the CD8+ CD28- CD127- proportion, which was decreased in active ITP patients compared with that of controls. Function analysis showed that immunosuppression of CD8+ CD28- Ts cells in ITP patients was impaired. Mechanistic studies have shown that CD8+ CD28- Ts cells from controls can downregulate CD80 and upregulate LILRB4 (ITL3) and LILRB2 (ILT4) expression on CD14+ monocytes, whereas these abilities were not found in Ts cells from ITP patients. Furthermore, Inducible T-cell costimulatory (ICOS) expression on the Ts cell surface after activation was decreased whereas programmed death 1 and interleukin 10 expression was not changed in ITP patients compared with those of controls. In summary, the down-regulated quantity and function of Ts cells in active patients indicated that a Ts defect was involved in ITP. Moreover, decreased ICOS expression and the loss of the ability to regulate co-stimulator expression on antigen-presenting cells partly explained the defective Ts-mediated suppression.

Interleukin 5 (IL-5) Treatment Prevents Chronic Allograft Rejection by Promoting IL-5 Receptor Expressing CD4 + CD25 + T Regulatory Ts2 Cells

Interleukin 5 (IL-5) Treatment Prevents Chronic Allograft Rejection by Promoting IL-5 Receptor Expressing CD4 + CD25 + T Regulatory Ts2 Cells

Human trophoblast stem cells: Real or not real?

Abnormal trophoblast differentiation is the root cause of many placenta-based pregnancy complications, including preeclampsia and fetal growth restriction. Human trophoblast differentiation is difficult to study due to the lack of a stem cell model. Such a multipotent “trophoblast stem” (TS) cell, with the ability to differentiate into all trophoblast subtypes, has been derived from mouse blastocysts, but attempts to derive similar human cells have failed. We consider here several possibilities for the TS cell niche in the human placenta. Aside from discussion of such a niche in the pre-implantation blastocyst, we discuss evidence for these TS cells residing in the post-implantation villous cytotrophoblast layer, or even in the non-trophoblast portions, of the human placenta. It is our hope that recognition of the niche would lead to successful derivation and in vitro establishment of such cells, which could then be disseminated widely to the placental biology community for advancing the field. Availability of self-renewing human TS cells, whose gene expression and environment could be manipulated, will provide a platform, not just for the study of pathophysiology of placental disease, but also for the discovery of diagnostic biomarkers and therapeutic targets for common pregnancy complications.

Identifying Tissue-Resident Memory T Cells in Graft-Versus-Host Disease

Tissue-resident memory T cells (TRM) are a newly described subset of transcriptionally-distinct memory CD4 and CD8 cells that persist in barrier and non-barrier tissues. They are non-circulating, able to facilitate the recruitment of circulating effector cells and elicit rapid recall responses. The majority of TRM cells can be identified by the expression of CD69 and αE integrin, CD103. However, CD69+CD103- TRM cells have also been described.In graft-vs-host disease (GVHD), alloreactive effector T cells enter GVHD target tissues and mediate tissue damage through direct and indirect mechanisms.The recruitment of effector T cells into tissues is in general not dependent on the tissue expressing the target antigen; and even if a target antigen is available in a tissue, there could be niches free of presented alloantigen. We therefore hypothesized that even in GVHD where alloantigen is ubiquitous, TRM may develop.We first explored TRM formation in a CD4 T cell receptor (TCR) transgenic (Tg) GVHD system wherein donor BALB/c RAG2-/- TS1 TCR Tg T cells target the S1 peptide derived from HA, which is expressed ubiquitously in BALB/c RAG2-/- HA104 mice. In this model, GVHD is induced by <1000 TS1 cells and is manifest by weight loss, death, and TS1-infiltrative pathology of the skin, liver, small bowel and colon. We harvested tissues from GVHD mice at days 21 and 28 post-transplant and quantitated TS1 cells with a TRM phenotype. At day 21, a fraction of TS1 cells expressed CD69+CD103+ (as % of total TS1 cells) in the epidermis (2.8% ± 1.2), dermis (11.3% ±7.9), colon (11.5% ±4.5) and small intestine (SI) intraepithelial (IEL) (33.4% ±5.8) and lamina propria (LP) (6.8% ±0.8). At day 28, CD69+CD103+ TS1 cells (% of total TS1) were present in the epidermis (13.6% ± 1.9), dermis (15.9% ± 7.7), colon (30.8% ± 6.7) and the SI IEL (69.1% ± 9.2) and SI LP (18.7 ±3.2). While there were no CD69+CD103+ TS1 in the spleen, bone marrow (BM) or liver (days 21 and 28), a small number (1.5% ±0.7, day 21; 1.7% ± 0.3, day 28) were found in the mesenteric lymph node (mLN). CD103- TRM have been described in the liver and secondary lymphoid organs. Consistent with this, CD69+CD103- TS1 cells (% of total TS1) were found in the liver (24.6% ±6.3, day21; 31.5% ±3.1, day 28), BM (23.1% ±3.1, day 21; 35.4% ±3.2, day 28), spleen (1.8% ±0.6, day 21; 9.1% ±0.7, day 28) and mLN (9.9% ±4.5, day 21; 23.5% ± 3.7, day 28). We are currently confirming the TRM identity of TS1 cells based on their transcriptional and migratory profiles and these data will be presented.Alloreactive TRM were also identified in the B6 (H-2b) into 129 (H-2b) MHC-matched, multiple minor histocompatibility antigen (miHA)-mismatched model in which GVHD is induced by a mix of CD4 and CD8 cells. A fraction of CD8 cells target the Kb-restricted miHA LTFNYRNL derived from H60, which can be tracked with tetramers (TetH60). At day 22 post-transplant, CD69+CD103+ CD4 cells (% of total CD4 T cells) were found in the epidermis (22.9% ±1.2), dermis (19.7% ±2.8), colon (9.4% ±3.1), SI IEL (26.5% ±2.6), SI LP (16.1% ± 3.7) and mLN (6.9% ±3.0). CD8+TetH60+ T cells (% of total CD8T cells) were detected in the epidermis (7.5% ± 3.5), dermis (10.6% ± 6.7), colon (6.9% ± 2.7), SI IEL (8.9% ±6.2), SI LP (10.2% ±3.8), mLN (3.0 ± 0.7) and spleen (9.0% ±2.9). A fraction of CD8+TetH60+ cells in the dermis (7.3% ±1.9), colon (18.1% ±12.2), SI IEL (50.1% ±4.3), SI LP (52.6% ±13.6), and mLN (6.3% ± 0.8) were CD69+CD103+, suggesting that alloreactive H60-directed CD8 T cells acquired a TRM phenotype.Using two different murine models, we found GVHD-inducing T cells with TRM phenotypes. Future experiments will confirm the TRM identity of these cells and will determine their importance in the maintenance of GVHD, perhaps by serving as a reservoir of cells that maintain GVHD locally. DisclosuresNo relevant conflicts of interest to declare.

Identifying the Clonal Origins of Gvhd-Causing T Cells

In graft-versus-host disease (GVHD) donor αβ T cells in the allograft recognize host tissues as non-self and cause multi-organ damage through direct and indirect mechanisms. This multi-organ disease is mediated by T cells acquiring diverse phenotypes, including expression of homing receptors, adhesion molecules, cytokines and other effector molecules. How this diversity is generated at a clonal level is not understood. In the one extreme, each type of effector could be derived from a single T cell expressing a unique antigen receptor that may even target a unique antigen. Alternatively, a single T cell targeting a single antigen may be able to differentiate into a variety of effectors (Fig. 1). A second related question is whether GVHD is maintained within affected organs. That is, what is the importance of the continued recruitment of new blood-derived alloreactive effectors in maintaining GVHD.To answer these questions, we needed 1) a system in which GVHD-inducing T cells could be unambiguously tracked, as in polyclonal systems it is difficult if not impossible to know which T cells are disease causing; and 2) a way to distinguish GVHD-inducing T cells at the clonal level. To address the first point, we used a CD4 T cell receptor (TCR) transgenic (Tg) model of GVHD wherein GVHD is induced by the transfer of <1000 BALB/c RAG2-/- TS1 TCR Tg T cells, which recognize the S1 peptide derived from HA, into BALB/c RAG2-/- HA104 Tg recipients that ubiquitously express HA. GVHD is manifest by weight loss, death, and TS1-infiltrative pathology of the skin, liver, small intestine and colon. In order to determine the clonal contributions, we crossed TS1 Tg mice to combinations of CD45.1, CD45.2, Thy1.1, Thy1.2 and GFP backgrounds to create a matrix of up to 9 distinguishable TS1 cells.Using this model, we first characterized the variety of TS1 effector phenotypes being generated across tissues at day 14 and 21 post-transplant and will be presenting data detailing these TS1 phenotypes, including the expression of CCR9, α4β7 integrin, and cutaneous lymphocyte antigen (CLA).To confirm that different matrix cells perform similarly, we assessed the development of total TS1 effector cells in mice that had received an equal number of 5 distinct TS1 cell combinations. We found that within individual mice (n=5), there is comparable distribution of GVHD-causing TS1 effector cells (% of total TS1 cells) from all 5 TS1cell matrices across tissues 14-21 days post-transplant (Table 1).We next tested our ability to detect total effector cells derived from transferring low numbers of TS1 cells (5-500 cells) of a given matrix combination. We found that within individual mice (n=9), the transfer of 5 and 10 TS1 clones of a given matrix results in disparate distribution of TS1 effectors (% of total TS1 cells) across tissues 14-21 days post-transplant. As an example for one mouse at day 14, the fraction of TS1effectors derived from 10 TS1clones of one matrix was 0.8% (spleen), 1.9% (mLN), 1.4% (BM), 0.6% (colon), 4.4% (SI IEL), 1.1% (SI LP), 1.2% (skin) and 1.5% (liver). In that same mouse, the fraction of TS1effectors derived from 5 TS1clones of a different matrix was 0.7% (spleen), 0.9% (mLN), 0.4% (BM), 1.1% (colon), 1.1% (SI IEL), 0.6% (SI LP), 0.6% (skin) and 2.7% (liver). We made similar observations in mice at day 21, where the percentage of TS1 effectors derived from 5 TS1 clones in one mouse was 2.5% (spleen), 2.0% (BM), 2.8% (mLN), 0.2% (SI IEL), 2.3% (SI LP), 1.1% (skin) and 1.6% (liver). When focusing on specific TS1 phenotypes derived from 5 and 10 TS1clones, we also found unequal distributions of these cells across different tissues. For example, the distribution of CD69+CD103+ TS1 cells (% of total TS1 cells) derived from 5 TS1clones was 2.9% (colon), 1.5% (SI IEL) and 1.9% (SI LP) in one mouse at day 14. Similarly, TS1 cells expressing α4β7 integrin also varied in distribution across tissues, with one mouse having 2.2% (spleen), 0.5% (mLN) and 0% (BM) α4β7+ TS1 cells. These data suggest that tissue GVHD effectors are not simply in equilibrium with blood, consistent with some local GVHD maintenance. Furthermore, the presence of 5 TS1 clones in more than one effector type suggests that single cells can differentiate into multiple effectors. We are now directly addressing these possibilities by analyzing progeny derived from many sorted single cells. [Display omitted] [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Loss of mutL homolog-1 (MLH1) expression promotes acquisition of oncogenic and inhibitor-resistant point mutations in tyrosine kinases.

Genomic instability drives cancer progression by promoting genetic abnormalities that allow for the multi-step clonal selection of cells with growth advantages. We previously reported that the IL-9-dependent TS1 cell line sequentially acquired activating substitutions in JAK1 and JAK3 upon successive selections for growth factor independent and JAK inhibitor-resistant cells, suggestive of a defect in mutation avoidance mechanisms. In the first part of this paper, we discovered that the gene encoding mutL homolog-1 (MLH1), a key component of the DNA mismatch repair system, is silenced by promoter methylation in TS1 cells. By means of stable ectopic expression and RNA interference methods, we showed that the high frequencies of growth factor-independent and inhibitor-resistant cells with activating JAK mutations can be attributed to the absence of MLH1 expression. In the second part of this paper, we confirm the clinical relevance of our findings by showing that chronic myeloid leukemia relapses upon ABL-targeted therapy correlated with a lower expression of MLH1 messenger RNA. Interestingly, the mutational profile observed in our TS1 model, characterized by a strong predominance of T:A>C:G transitions, was identical to the one described in the literature for primitive cells derived from chronic myeloid leukemia patients. Taken together, our observations demonstrate for the first time a causal relationship between MLH1-deficiency and incidence of oncogenic point mutations in tyrosine kinases driving cell transformation and acquired resistance to kinase-targeted cancer therapies.

Effects of Anti-CD45RB Monoclonal Antibody for T Lymphocyte Subsets in Mice Heart Transplantation Model.

Anti-CD45RB monoclonal antibody (anti-CD45RBmAb), as a new immune tolerance inducer, may inhibit T cell proliferation and induce immune tolerance through competitive combination with CD45RB on the T cell surface, which blocks the conduction of activation signals. However, how anti-CD45RBmAb plays its role on T lymphocyte subsets during immunosuppression remains unclear. In this work, we investigate the effects of anti-CD45RBmAb on CD3(+) T lymphocyte both invitro and in allogeneic heart transplant model invivo. Interestingly, anti-CD45RBmAb could inhibit the proliferation of T cells, promote the transformation of T lymphocyte to Treg and Th2 cells, suppress the transformation to Th17 and Th1 cells, increase the number of Ts cells, decrease the number of Tm cells and thus play a role in immune inhibition and induction of immune tolerance.

Significance of clonal TCR gene rearrangement in acquired pure red cell aplastic anemia

To evaluate the significance of T-cell antigen receptor (TCR) gene rearrangement among patients with acquired pure red cell aplastic anemia (A-PRCA). For 16 patients with A-PRCA, an immunosuppressive regimen based on cyclosporin A (CsA) was applied. Rearrangement of the TCR gene was detected by PCR, and T lymphocyte subsets in peripheral blood specimens was detected with flow cytometry. Five patients had presented with TCR clonal rearrangement and were positive for both TCR γ and TCR δ. The blood of 13 patients have returned to normal with the treatment, which included 3 cases with bone marrow returning to normal. In 7 cases, the red cell hyperplasia of bone marrow is still down, but has increased with the treatment. Three patients were close to cure, 7 showed remission, 3 were improved, but 3 were ineffective. The rate of effective treatment in those with TCR rearrangement (2/5) was significantly lower than that those without (11/11, chi-square=8.123, P < 0.05). Compared with those without the TCR gene rearrangement, the Th cells and proportion of Th/Ts were significantly lower, while the Ts cell (CD3+CD8+) were significantly higher in those with the rearrangement (P < 0.05). TCR gene rearrangement may play a role in the pathogenesis of A-PRCA. CsA is effective for the treatment of A-PRCA, but patients presenting clonal TCR gene rearrangement may response poorly to the treatment.

Declined Natural Killer Cells Emerging in Women with Unexplained Recurrent Spontaneous Abortion and Further Reducing after Medical Therapy.

As focus grows on reproduction, the issue of Recurrent Spontaneous Abortion (RSA), especially for unexplained reasons (URSA), is grabbing more and more attention in gynecological immunology. We investigated the changes of peripheral lymphocyte subsets focusing on whether they had some relationship with development of URSA. The percentage and absolute count of lymphocyte subsets (T cells, Th cells, Ts cells, B cells, NK cells) were simultaneously evaluated by flow cytometry in URSA patients (n = 48) and healthy controls (HC, n = 22). Significantly lower percentage and absolute counts of NKT cells and NK cells were observed in URSA compared to the HC. After medical therapy, an obviously increase was shown in the percentage of both T cells and B cells, whereas it presented a reduction in the percentage of NK cells. The flow cytometry test in T, B, NK cells is a method available to identify URSA patients from healthy women and to provide reference guides for clinical therapy.

Study on pure red cell aplastic lymphocyte subsets characteristics and the effect of cyclosporin A

Objective To detect and analyse acquired pure red cell aplasia (PRCA) T lymphocyte subsets distribution and to assess its condition and cyclosporine immune function of T lymphocytes subsets. Methods Flow cytometry was applicated to detect peripheral blood T lymphocyte subsets of acquired PRCA patients before and after 3 months of treatment with cyclosporine-based immunosuppressive regimen and normal controls. Results Among 22 patients, 17 cases of blood returning normal (three cases of bone marrow returning normal), the total effective rate was 95.5 % (3 cases of cure, 14 cases of remission, 4 cases of improvement, 1 case of ineffectiveness). Th (CD3+ CD4+) cells and Th/Ts ratios in acquired PRCA patient group were lower than those in the normal control group, while Ts (CD3+ CD8+) cells was higher than that in the normal control group, the differences were statistically significant (P< 0.05). After 3 months of treatment, Th cells and Th/Ts ratio were higher than those before, and Ts cells were decreased compared with the previous (P< 0.05). Conclusion Disorders of T lymphocyte subsets in acquired PRCA patients lead to immune dysfunction, however, cyclosporine can improve T lymphocyte subsets in patients with imbalance, which is an effective way to treat the disease with significant curative effect and mild adverse reactions. Key words: Acquired pure red cell aplastic; T lymphocytes; Cyclosporine

The proportional changes of B regulatory cells and suppressor T cells in peripheral blood of patients with Hashimotos' thyroiditis

Objective To explore the proportional changes of regulatory B lymphocyte cells(Bregs)and suppressor T lymphocyte cell(Ts)in the peripheral blood of patients with Hashimotos' thyroiditis(HT). Method Sixty-one cases of HT and 38 healthy subjects with matched age and gender were recruited. Flow cytometry technology was used to detect the proportional changes of CD19+ CD24+ CD27+ B cells, CD19+ CD24hiCD38hiB cells, CD19+ IL-10+ B cells, and CD8+ CD28-T cells. Result Compared with control group, CD19+ CD24+ CD27+ B cells in HT group increased significantly(P<0.05). With the increase of thyroid peroxidase antibody, CD19+ CD24hiCD38hiB cells were decreased significantly. With the increase of antithyrogloblin antibody(TGAb), CD8+ CD28-T cells were decreased markedly(P<0.05). TT3 was positively related with CD19+ CD24hiCD38hiB cells while TT4 had a negative correlation with CD8+ CD28-T cells and positive correlation with CD19+ CD24+ CD27+ B cells. Logistic regression analysis showed that TGAb and FT4 were independent risk factors for decreased CD8+ CD28-T cells in lymphocyte percentage(P<0.05). TSH was an independent risk factor for the decreased proportion of CD19+ CD24hiCD38hiB cell. FT4 was the independent risk factors for increased CD19+ CD24+ CD27+ B cells(P<0.05). Conclusion The percentage of Ts cells in patients with HT was lowered, while the different phenotypic Bregs cells showed various changes. Thyroid autoantibodies and thyroid function were closely associated with the immunological changes in HT patients. (Chin J Endocrinol Metab, 2015, 31: 427-433) Key words: Hashimotos' thyroiditis; Regulatory B lymphocyte cells; Suppressor T lymphocyte cell

Macrophages play an essential role in antigen-specific immune suppression mediated by T CD8⁺ cell-derived exosomes.

Murine contact sensitivity (CS) reaction could be antigen-specifically regulated by T CD8(+) suppressor (Ts) lymphocytes releasing microRNA-150 in antibody light-chain-coated exosomes that were formerly suggested to suppress CS through action on macrophages (Mφ). The present studies investigated the role of Mφ in Ts cell-exosome-mediated antigen-specific suppression as well as modulation of Mφ antigen-presenting function in humoral and cellular immunity by suppressive exosomes. Mice depleted of Mφ by clodronate liposomes could not be tolerized and did not produce suppressive exosomes. Moreover, isolated T effector lymphocytes transferring CS were suppressed by exosomes only in the presence of Mφ, demonstrating the substantial role of Mφ in the generation and action of Ts cell regulatory exosomes. Further, significant decrease of number of splenic B cells producing trinitrophenyl (TNP) -specific antibodies with the alteration of the ratio of serum titres of IgM to IgG was observed in recipients of exosome-treated, antigen-pulsed Mφ and the significant suppression of CS was demonstrated in recipients of exosome-treated, TNP-conjugated Mφ. Additionally, exosome-pulsed, TNP-conjugated Mφ mediated suppression of CS in mice pre-treated with a low-dose of cyclophosphamide, suggesting de novo induction of T regulatory (Treg) lymphocytes. Treg cell involvement in the effector phase of the studied suppression mechanism was proved by unsuccessful tolerization of DEREG mice depleted of Treg lymphocytes. Furthermore, the inhibition of proliferation of CS effector cells cultured with exosome-treated Mφ in a transmembrane manner was observed. Our results demonstrated the essential role of Mφ in antigen-specific immune suppression mediated by Ts cell-derived exosomes and realized by induction of Treg lymphocytes and inhibition of T effector cell proliferation.

Induction of antigen specific CD4+CD25+Foxp3+T regulatory cells from naïve natural thymic derived T regulatory cells

CD4+CD25+FOXP3+T regulatory cells (Treg) play a major role in prevention of induction and control of immune responses, and contribute to induction of immune tolerance. Natural or thymic Treg (tTreg) have non-antigen specific suppressor action. Tolerance to a specific antigen is also mediated by CD4+CD25+FOXP3+Treg, but the source of these cells is disputed. Many suggest that they are derived from effector lineage CD4+CD25−FOXP3−T cells and are induced Treg (iTreg). Our work shows that tTreg with specific TCR for the antigen can be activated to more potent antigen specific Treg. We have demonstrated that initial activation of tTreg with antigen and IL-2 induces antigen specific Treg that express receptors for the late Th1 cytokines IFN-γ and IL-12. These antigen specific Treg suppress effector lineage T cells at much lower ratios than tTreg, and we call these Ts1 cells as they are activated by Th1 cytokines and express receptors for Th1 cytokines. Further activation of Ts1 cells with specific antigen and late Th1 cytokines such as IL-12 induces very potent Th1-like Treg, that express t-bet, the transcription factor for Th1 cells, as well as the Th1 cytokine IFN-γ. Similar Th1-like Treg can be induced in IL-2 activated tTreg, by IFN-γ or IL-27. tTreg activated by antigen in the presence of IL-4 induces antigen specific Treg that express the IL-5 receptor, and these Ts2 cells can be induced to Th2-like Treg by IL-5 and antigen. tTreg can be activated to antigen specific Tregs that induce tolerance and have therapeutic potential.

Aberrant hedgehog signaling is responsible for the highly invasive behavior of a subpopulation of hepatoma cells.

Hepatoma exhibits a series of heterogeneous subpopulations in its cell surface markers, tumorigenicity, invasion and metastatic capability. We previously demonstrated that the CD133(-)/EpCAM(-) hepatoma subpopulation was more metastatic than its counterpart; however, the controlling mechanisms are unexplored. The present study aimed to delineate the significance of aberrant hedgehog (Hh) signaling in the mediation of metastases. Fluorescence-activated cell sorting-enriched CD133(-)/EpCAM(-) (double negative, DN), Huh-7 cells underwent a transwell selection for metastatic cells (transwell-selected, TS). The TS cells displayed much greater metastatic activity as evidenced by an increased invasion rate, extremely upregulated expression of matrix metalloproteinase (MMP)-1/2/9 genes compared with DN and double-positive (DP) subpopulations. In contrast to DP cells, TS cells lost E-cadherin and were all vimentin-positive as shown by immunocytochemistry. There was a transitional increase in Gli-1/2 gene expression levels from DP, DN to TS subpopulations, which was consistent with elevated Gli-1/2 or Twist-1 protein levels in the nuclear fraction. Furthermore, truncated Gli-1 (tGli-1), which transactivates molecules involved in metastasis, was detected in the highly invasive Huh-7 cell subpopulation, but not in less metastatic hepatoma cells or normal hepatocytes. The enhanced metastatic features with increased expression of MMPs as well as the presence of twist and snail genes in TS Huh-7 cells were reversed by LDE225, a potent Smoothened antagonist. In conclusion, the highly metastatic capability of a unique TS subpopulation was highly attributed to significant epithelial-mesenchymal transition, enhanced Hh activity and aberrant occurrence of a tGli-1 variant, which appears to be responsible for the highly invasive behavior.

Evaluation of humoral and cellular immune responses to a DNA vaccine encoding chicken type II collagen for rheumatoid arthritis in normal rats

A major challenge in the development of effective therapies for rheumatoid arthritis (RA) is finding a method for the specific inhibition of the inflammatory disease processes without the induction of generalized immunosuppression. Of note, the development of therapeutic DNA vaccines and boosters that may restore immunological tolerance remains a high priority. pcDNA-CCOL2A1 is a therapeutic DNA vaccine encoding chicken type II collagen(CCII). This vaccine was developed by our laboratory and has been shown to exhibit efficacy comparable to that of the current “gold standard” treatment, methotrexate (MTX). Here, we used enzyme-linked immunosorbent assays with anti-CII IgG antibodies, quantified the expression levels of Th1, Th2, and Th3 cytokines, and performed flow cytometric analyses of different T-cell subsets, including Th1, Th2, Th17, Tc, Ts, Treg, and CD4+CD29+T cells to systemically evaluate humoral and cellular immune responses to pcDNA-CCOL2A1 vaccine in normal rats. Similar to our observations at maximum dosage of 3 mg/kg, vaccination of normal rats with 300 μg/kg pcDNA-CCOL2A1 vaccine did not induce the production of anti-CII IgG. Furthermore, no significant changes were observed in the expression levels of pro-inflammatory cytokines interleukin (IL)-1α, IL-5, IL-6, IL-12(IL-23p40), monocyte chemotactic protein (MCP)-1, macrophage inflammatory protein (MIP)-1α, regulated on activation in normal T-cell expressed and secreted (RANTES), receptor activator for nuclear factor-κB ligand (RANKL), and granulocyte colony-stimulating factor (G-CSF) or anti-inflammatory cytokines IL-4 and IL-10 in vaccinated normal rats relative to that in controls(P > 0.05). However, transforming growth factor (TGF)-β levels were significantly increased on days 10 and 14, while interferon (IFN)-γ and tumor necrosis factor (TNF)-α levels were significantly decreased on days 28 and 35 after vaccination(P < 0.05). Similarly, there were no significant differences in the percentages of Tc, Ts, Th1/Th2, and Th17 cells between the 2 groups(P > 0.05), with the exception of Treg cells, which were significantly reduced on days 14 and 21 after vaccination (P < 0.05), and CD4+CD29+T cells, which were significantly increased on days 7 and 14 after vaccination(P < 0.05).Taken together, these results suggested that pcDNA-CCOL2A1 vaccine did not markedly affect the balance of immune system components in vaccinated normal rats, indicating that this DNA vaccine may have clinical applications in the treatment of RA.

Cooperating JAK1 and JAK3 mutants increase resistance to JAK inhibitors

AbstractThe acquisition of growth signal self-sufficiency is 1 of the hallmarks of cancer. We previously reported that the murine interleukin-9–dependent TS1 cell line gives rise to growth factor–independent clones with constitutive activation of the Janus kinase (JAK)- signal transducer and activator of transcription (STAT) pathway. Here, we show that this transforming event results from activating mutations either in JAK1, JAK3, or in both kinases. Transient and stable expression of JAK1 and/or JAK3 mutants showed that each mutant induces STAT activation and that their coexpression further increases this activation. The proliferation of growth factor–independent TS1 clones can be efficiently blocked by JAK inhibitors such as ruxolitinib or CMP6 in short-term assays. However, resistant clones occur upon long-term culture in the presence of inhibitors. Surprisingly, resistance to CMP6 was not caused by the acquisition of secondary mutations in the adenosine triphosphate–binding pocket of the JAK mutant. Indeed, cells that originally showed a JAK1-activating mutation became resistant to inhibitors by acquiring another activating mutation in JAK3, whereas cells that originally showed a JAK3-activating mutation became resistant to inhibitors by acquiring another activating mutation in JAK1. These observations underline the cooperation between JAK1 and JAK3 mutants in T-cell transformation and represent a new mechanism of acquisition of resistance against JAK inhibitors.

Abstract 2005: Aberrant hedgehog signaling is responsible for the highly invasive behavior of a subpopulation of hepatoma cells

Abstract Background: Hepatoma consists of heterogeneous subpopulations in terms of their cell surface markers, tumorigenicity, invasion and metastatic capability. In our previous study, we indicated that the CD133-/EpCAM- hepatoma subpopulation was more metastatic than its counterpart; however the controlling mechanisms are unexplored (J Hepatol 2011;55:838-845). The present study aims to delineate the significance of aberrant hedgehog signaling in the development of metastases. Methods: Huh-7 cells were FACS-enriched into CD133+/EpCAM+ (double positive, DP) and CD133-/EpCAM- (double negative, DN) subpopulations. The double negative cells further underwent Transwell-selection for metastatic cells (Transwell-selected, TS). The metastatic rate, matrix metalloproteinase (MMP) gene expression, epithelial mesenchymal transition (EMT) markers, and hedgehog signaling activities, as well as truncated Gli1 were determined in these subpopulations. Results: TS cells displayed much greater metastatic activity as evidenced by an increased Transwell invasion rate (105 vs. 68.7 and 58.0%, p&amp;lt;0.01), extremely elevated expression of MMP1, 2 and 9 genes (36-3000-fold, p&amp;lt;0.05-0.001) compared to DN and DP populations. There was a nearly 2-fold increase in TGF-β1 gene expression in TS cells compared to DN and DP subpopulations. TS cells lost E-cadherin and were all vimentin-positive as shown by immunocytochemistry in contrast to DP cells. There was a transitional increase in Gli1 gene expression levels from DP, DN to TS subpopulations, which is consistent with elevated Gli-2 and Twist1 levels in the nuclear fraction as detected by Western blot analysis and quantitative RT-PCR. Furthermore, truncated Gli1, which has been indicative of mediating expression of the molecules involved in metastasis, was detected in highly invasive hepatoma cell subpopulations and was the highest in the TS subpopulation; whereas, it did not appear in primary human hepatocytes. Flow cytometric analysis verified that these TS cells maintained a negative cell surface marker profile in their sub-passages. Conclusions: The highly metastatic capability of unique Transwell-selected cells is associated with negative expression of CD133 and EpCAM surface molecules, significant EMT, enhanced hedgehog activity and aberrant expression of truncated Gli1. The appearance of truncated Gli1 is probably responsible for highly invasive behavior of the transwell-selected double-negative subpopulation. Further exploration of molecular mechanisms of abnormal hedgehog signaling in modulating metastases will establish molecular targets for the therapeutic intervention of hepatoma metastases. Citation Format: Yahan Fan, Jia Ding, Jian Wu. Aberrant hedgehog signaling is responsible for the highly invasive behavior of a subpopulation of hepatoma cells. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2005. doi:10.1158/1538-7445.AM2014-2005

Thermotolerance in Saccharomyces cerevisiae is linked to resistance to anhydrobiosis

We have demonstrated that a thermotolerant yeast strain (Saccharomyces cerevisiae TS1) is much more resistant to dehydration–rehydration treatments than a mesophilic strain of S. cerevisiae. Yeast resistance to dehydration–rehydration was found to be similar in cells from exponential and stationary growth phases. Under controlled rehydration conditions involving gradual rehydration in water vapour, yeast cell viability was maintained at 90–95%. When S. cerevisiae TS1 cells were pre-grown at 37°C and then dried, controlled rehydration lead to restoration of plasma membrane integrity, indicating important differences in cell envelope architechture of mesophilic and thermotolerant yeast strains. Comparison of such strains provides new insight into anhydrobiosis in eukaryotic cells.