Thymocyte Cell Research Articles

Typical pneumonia among human immunodeficiency virus-infected patients in public hospitals in southern Ethiopia.

Typical pneumonia is a pressing issue in the treatment of human immunodeficiency virus (HIV) patients, especially in Sub-Saharan Africa, where it remains a significant menace. Addressing this problem is crucial in improving health outcomes and the reduction of the burden of diseases in this vulnerable category of patients. To determine the prevalence of community-acquired typical pneumonia among HIV patients in Public Hospitals in southern Ethiopia. A cross-sectional study was done among 386 HIV patients clinically suspected of typical pneumonia attending the anti-retroviral therapy (ART) clinics of two hospitals from March to September 2022. A pretested structured questionnaire was employed to collect the demographic, clinical, and behavioral data. Sputum samples were collected and inspected for bacteria following standard procedures, and antimicrobial susceptibility testing was performed employing the Kirby-Bauer disk diffusion method. Besides, extended-spectrum β-lactamase (ESβL) and carbapenemase-producing Gram-negative bacteria were inspected by the double disk synergy test and modified carbapenem inactivation method. Descriptive and inferential statistical analyses were also done. Overall, 39.1% (151/386) of sputum cultures (95% Confidence Interval: 32.4-44) were bacteriologically positive. A total of 151 bacteria were identified, comprising 72.8% (n = 110) of Gram-negative bacteria. The predominant isolate was Klebsiella pneumoniae (25.8%, n = 39), followed by Staphylococcus aureus (17.9%, n = 27); 59.6% (n = 90) of the entire isolates were multidrug-resistant (MDR). Forty percent (11/27) of S. aureus were methicillin-resistant S. aureus (MRSA), and 28.1% (n = 31) and 20.9% (n = 23) of Gram-negative bacteria were extended-spectrum beta-lactamases (ESBL) and carbapenemase producers, respectively. Occupational status, alcohol consumption, cluster of differentiation4 (CD4) Thymocyte cell count < 350, interruption of trimethoprim-sulfamethoxazole prophylaxis and antiretroviral treatment, and recent viral load ≥ 150 were found statistically significant. The higher rates of MDR, MRSA, ESBL, and carbapenem-resistant Enterobacterales (CRE) indicate that bacterial pneumonia is a vexing problem among HIV patients and therefore it is advisable to implement an antimicrobial stewardship program in the study area.

Surfing on the waves of the human γδ T cell ontogenic sea.

While γδ T cells are present virtually in all vertebrates, there is a remarkable lack of conservation of the TRG and TRD loci underlying the generation of the γδ T cell receptor (TCR), which is associated with the generation of species-specific γδ T cells. A prominent example is the human phosphoantigen-reactive Vγ9Vδ2 T cell subset that is absent in mice. Murine γδ thymocyte cells were among the first immune cells identified to follow a wave-based layered development during embryonic and early life, and since this initial observation, in-depth insight has been obtained in their thymic ontogeny. By contrast, less is known about the development of human γδ T cells, especially regarding the generation of γδ thymocyte waves. Here, after providing an overview of thymic γδ wave generation in several vertebrate classes, we review the evidence for γδ waves in the human fetal thymus, where single-cell technologies have allowed the breakdown of human γδ thymocytes into functional waves with important TCR associations. Finally, we discuss the possible mechanisms contributing to the generation of waves of γδ thymocytes and their possible significance in the periphery.

Cooperation between Mutant IL7R and TLX3 Generates Mixed Myeloid/Lymphoid Leukemia from Thymocyte Progenitors

Acute lymphoblastic leukemia (ALL) is the most common cancer in children. Despite current pediatric treatment protocols that have resulted in 5-year overall survival rates up to 90%, treatments have substantial side effects. Therefore, it is important to identify more specific therapeutic targets and develop more effective and less toxic drugs and drug combinations. We have shown that overexpression of TLX3 in association with mutant IL7R (mutIL7R) is sufficient to generate aggressive leukemia from transduced CD4-CD8- (DN) thymocyte progenitors. To better understand mutIL7R/TLX3-mouse leukemia, we combined single cell RNA sequencing (scRNAseq), Cellular Indexing of Transcriptomes and Epitopes by Sequencing (CITE-seq), DNA Next Generation Sequencing (NGS) and flow cytometry to explore the cooperation of mutIL7R and TLX3 in orchestrating leukemogenesis. We found that mutIL-7R+TLX3 induced murine mixed T/myeloid leukemia. Myeloid cell clones (CD11b+) expressed identical TCR rearrangements with T cell clones (CD8+), indicating that mutant IL-7R+TLX3 generates mixed leukemia from T cell progenitors. Moreover, the passaged leukemia lost the T-cell phenotype and acquired a pro-B-like phenotype, suggesting preferential expansion of pro-B cells (CD19+, B220+ IgM-), which were not detected in the founder leukemia presumably due to its low frequency. We next sought to determine whether the oncogene combination is sufficient to drive a similar immunophenotype profile in vitro. We showed that ectopic expression of mutIL7R and TLX3 in DN primary thymocytes pushed the cells to myeloid (CD11b+, Gr1+) and B-cell (B220) phenotype over a time course of 4, 8 and 20 days, while T-cell development was blocked, suggesting that TLX3 expression prevented differentiation into CD4+/CD8+ T-cells. To further characterize the mixed T/myeloid leukemia at the molecular level, we compared the leukemia expressed genes to a TLX-bound genes public dataset. We found that 42 TLX3 target genes are expressed in mutIL7R+TLX3 leukemia. Interaction network analysis suggested significant Myc stimulation. We then confirmed Myc upregulation at the protein level in a thymocyte cell line, indicating that mutIL7R alone induces Myc but requires TLX-3 cooperation to induce maximal Myc expression levels. Taken together, we generated a high-resolution dataset with detailed information about the transcriptional landscape and immunophenotype of leukemia generated by mutIL-7R+TLX3, a combination that is observed in patient samples, and suggests new therapeutic drug strategies. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Two Step Selection for Bias in β Chain V-J Pairing.

The β chain rearrangement in T cells is a two-step process where first Dβ and Jβ bind, and only then Vβ is joined to the complex. We here show that the frequency of human and mouse Vβ Jβ combinations deviates from the one expected based on each gene usage frequency. This bias is observed mainly in functional (F) rearrangements, but also slightly in non-functional (NF) rearrangements. Preferred Vβ Jβ combinations in F clones are shared between donors and samples, suggesting a common structural mechanism for these biases in addition to any host-specific antigen-induced peripheral selection. The sharing holds even in clones with J β 1 that share the same Dβ 1 gene. Vβ Jβ usage is correlated with the Molecular Weight and Isoelectric Point in F clones. The pairing is also observed in the Double Positive cells in mice thymocytes, suggesting that the selection leading to such a pairing occurs before thymic selection. These results suggest an additional structural checkpoint in the beta chain development prior to thymic selection during the T cell receptor expression. Understanding this structural selection is important for the distinction between normal and aberrant T cell development, and crucial for the design of engineered TCRs.

Potential role of inducible GPR3 expression under stimulated T cell conditions

G protein-coupled receptor 3 (GPR3) constitutively activates Gαs proteins without any ligands and is predominantly expressed in neurons. Since the expression and physiological role of GPR3 in immune cells is still unknown, we examined the possible role of GPR3 in T lymphocytes. The expression of GPR3 was upregulated 2 h after phorbol 12-myristate 13-acetate (PMA)/ionomycin stimulation and was sustained in Jurkat cells, a human T lymphocyte cell line. In addition, the expression of nuclear receptor 4 group A member 2 (NR4A2) was highly modulated by GPR3 expression. Additionally, GPR3 expression was linked with the transcriptional promoter activity of NR4A in Jurkat cells. In mouse CD4+ T cells, transient GPR3 expression was induced immediately after the antigen receptor stimulation. However, the expression of NR4A2 was not modulated in CD4+ T cells from GPR3-knockout mice after stimulation, and the population of Treg cells in thymocytes and splenocytes was not affected by GPR3 knockout. By contrast, spontaneous effector activation in both CD4+ T cells and CD8+ T cells was observed in GPR3-knockout mice. In summary, GPR3 is immediately induced by T cell stimulation and play an important role in the suppression of effector T cell activation.

Overexpression of hTLX3 in Association with Mutant IL7Rα Is Sufficient to Generate T-ALL In Vivo and to Transform Thymocytes in Vitro

Mutations of the IL-7Rα chain occur in approximately 10% of pediatric T-cell acute lymphoblastic leukemia cases. While we have shown that mutant IL7Ra is sufficient to transform an immortalized thymocyte cell line, mutation of IL7Ra alone was insufficient to cause transformation of primary T cells, suggesting that additional genetic lesions may be present contributing to initiate leukemia. Studies addressing the combinations of mutant IL7Ra plus TLX3 overexpression indicates in vitro growth advantage, suggesting this gene as potential collaborative candidate. Furthermore, patients with mutated IL7R were more likely to have TLX3 or HOXA subgroup leukemia. We sought to determine whether combination of mutant hIL7Ra plus TLX3 overexpression is sufficient to generate T-cell leukemia in vivo. Double negative thymocytes were isolated from C57BL/6J mice and transduced with retroviral vectors containing mutant hIL7R plus hTLX3, or the genes alone. The combination mutant hIL7R wild type and hTLX3 was also tested. Transduced thymocytes were cultured on the OP9-DL4 bone marrow stromal cell line for 5-13 days and accessed for expression of transduced constructs and then injected into sublethally irradiated Rag-/- mice. Mice were euthanized at onset of clinical signs, and cells were immunophenotyped by flow cytometry. Thymocytes transduced with muthIL-7R-hTLX3 transformed to cytokine-independent growth and expanded over 30 days in the absence of all cytokines. Mice injected with muthIL7R-hTLX3 cells, but not the controls (wthIL7R-hTLX3or mutIL7R alone) developed leukemia approximately 3 weeks post injection, characterized by GFP expressing T-cells in blood, spleen, liver, lymph nodes and bone marrow. Furthermore, leukemic mice had increased white blood cell counts and presented with splenomegaly. Phenotypic analysis revealed a higher CD4-CD8- T cell population in the blood, bone marrow, liver and spleen compared in the mutant hIL7R + hTLX3 mice compared with mice injected with mutant IL7R alone indicating that the resulting leukemia from the combination mutant hIL7R plus hTLX3 shows early arrest in T-cell development. Taken together, these data show that oncogenic IL7R activation is sufficient for cooperation with hTLX3 in ex vivo thymocyte cell transformation, and that cells expressing the combination muthIL7R-hTLX3 is sufficient to trigger T-cell leukemia in vivo. Figure Disclosures No relevant conflicts of interest to declare.

Examination of Fas-Induced Apoptosis of Murine Thymocytes in Thymic Tissue Slices Reveals That Fas Is Dispensable for Negative Selection.

The death receptor Fas can induce cell death through the extrinsic pathway of apoptosis in a variety of cells, including developing thymocytes. Although Fas-induced cell death has been researched and modeled extensively, most of the studies have been done in vitro because of the lethality of Fas triggering in vivo. Thus, little is known about the time line of this type of cell death in vivo, specifically, how does the presence of macrophages and pro-survival cytokines affect apoptosis progression. In addition, although the sequence and timing of events during intrinsic pathway activation in thymocytes in situ have been described, no corresponding data for the extrinsic pathway are available. To address this gap in our knowledge, we established a novel system to study Fas-induced thymocyte cell death using tissue explants. We found that within 1 h of Fas ligation, caspase 3 was activated, within 2 h phosphatidylserine was externalized to serve as an “eat-me” signal, and at the same time, we observed signs of cell loss, likely due to efferocytosis. Both caspase 3 activation and phosphatidylserine exposure were critical for cell loss. Although Fas ligand (FasL) was delivered simultaneously to all cells, we observed significant variation in the entry into the cell death pathway. This model also allowed us to revisit the role of Fas in negative selection, and we ruled out an essential part for it in the deletion of autoreactive thymocytes. Our work provides a timeline for the apoptosis-associated events following Fas triggering in situ and confirms the lack of involvement of Fas in the negative selection of thymocytes.

Abstract B47: Overexpression of TLX3 or HOXA9 in association mutant IL7Rα are sufficient to generate T-ALL in vivo

Abstract Background: Mutations of the IL7Rα chain occur in approximately 10% of pediatric T-cell acute lymphoblastic leukemia cases. While we have shown that mutant IL7Rα is sufficient to transform an immortalized thymocyte cell line, mutation of IL7Rα alone was insufficient to cause transformation of primary T cells, suggesting that additional genetic lesions may be present, contributing to initiate leukemia. Studies addressing the combinations of mutant IL7Rα plus TLX3 or HOXA9 overexpression indicate in vitro grow advantage, suggesting these two genes as potential collaborative candidates. Furthermore, patients with mutated IL7Rα were more likely to have TLX3 or HOXA subgroup leukemia. Objective: We sought to determine whether combination of mutant hIL7Rα plus TLX3 or HOXA9 overexpression is sufficient to generate T-cell leukemia in vivo. Methods: Double-negative thymocytes were isolated from C57BL/6J mice and transduced with retroviral vectors containing mutant hIL7Rα plus TLX3 or HOXA9. The same combinations were tested for the hIL7Rα wild-type. Transduced thymocytes were cultured on the OP9-DL4 bone marrow stromal cell line for 7-13 days and accessed for driver oncogenes expression and then injected into sublethally irradiated Rag-/- mice. Mice were euthanized at onset of clinical signs, and cells were immunophenotyped by flow cytometry. Results: Thymocytes transduced with muthIL7Rα-TLX3 showed overexpression of c-Myc and Pim-1. Nonetheless, there was no difference in the protein level expression for these two proteins in either thymocytes transduced with muthIL-7Rα-HOXA9 or in the control wthIL7Rα-TLX3. Mice injected with either muthIL7Ra-TLX3 or muthIL7Rα-HOXA9 cells, but not the controls (wthIL7Rα-TLX3 or wthIL7Rα-HOXA9), developed leukemia approximately 14 days post injection, characterized by GFP-expressing T-cells in blood, spleen, liver, lymph nodes, and bone marrow. Conclusion: Thymocytes transduced with muthIL7Rα-TLX3 showed c-Myc and Pim-1 upregulation. Cells expressing the combination muthIL7Rα-TLX3 or muthIL7Rα-HOXA9 were sufficient to trigger T-cell leukemia in vivo. Citation Format: Gisele O.L. Rodrigues, Julie Hixon, Hila Winer, Wenqing Li, Scott Durum. Overexpression of TLX3 or HOXA9 in association mutant IL7Rα are sufficient to generate T-ALL in vivo [abstract]. In: Proceedings of the AACR Special Conference on the Advances in Pediatric Cancer Research; 2019 Sep 17-20; Montreal, QC, Canada. Philadelphia (PA): AACR; Cancer Res 2020;80(14 Suppl):Abstract nr B47.

Cofilin1-driven actin dynamics controls migration of thymocytes and is essential for positive selection in the thymus.

Actin dynamics is essential for T-cell development. We show here that cofilin1 is the key molecule for controlling actin filament turnover in this process. Mice with specific depletion of cofilin1 in thymocytes showed increased steady-state levels of actin filaments, and associated alterations in the pattern of thymocyte migration and adhesion. Our data suggest that cofilin1 is controlling oscillatory F-actin changes, a parameter that influences the migration pattern in a 3-D environment. In a collagen matrix, cofilin1 controls the speed and resting intervals of migrating thymocytes. Cofilin1 was not involved in thymocyte proliferation, cell survival, apoptosis or surface receptor trafficking. However, in cofilin1 mutant mice, impaired adhesion and migration resulted in a specific block of thymocyte differentiation from CD4/CD8 double-positive thymocytes towards CD4 and CD8 single-positive cells. Our data suggest that tuning of the dwelling time of thymocytes in the thymic niches is tightly controlled by cofilin1 and essential for positive selection during T-cell differentiation. We describe a novel role of cofilin1 in the physiological context of migration-dependent cell differentiation.

Treatment with Lead Chloride During Pregnancy and the Postnatal Period Alters Cell Proliferation and Immune Function in Swiss Albino Mice.

In the current study, we investigated the effect of lead chloride (PbCl2) administration (50 and 100 ppm) on organ and body weight as well as its bioaccumulation during pregnancy and the postnatal period in mice. We showed that lead has no effect on the body weight of mice. However, spleen weight is affected by the two doses of PbCl2 while liver and kidney weights are altered only by the 100-ppm dose. Inductively coupled plasma atomic emission spectrometry (ICP-AES) analysis showed that lead accumulates in the blood, spleen, and thymus. Both doses of PbCl2 significantly reduced splenocyte and thymocyte cell counts after stimulation with lipopolysaccharide (LPS) and phytohemagglutinin A (PHA), respectively. On the other hand, we showed that the levels of Th1 cytokines (interleukin-2 (IL-2), interferon gamma (IFN-γ)), and tumor necrosis factor alpha (TNF-α) were reduced in the serum of mice treated with PbCl2 in a dose-dependent manner, as measured by ELISA. The levels of interleukin-4 (IL-4) and interleukin-10 (IL-10) were very low in untreated mice and were also reduced by treatment with PbCl2. The levels of IL-2, IFN-γ, IL-4, IL-10, and TNF-α secretion differentially decreased in LPS-stimulated splenocytes in lead-treated mice. Using PHA-stimulated thymocytes, we observed a reduction in the levels of IL-2, IL-4, IL-10, and TNF-α in the PbCl2-treated groups. However, IFN-γ concentration in the supernatant of these cells was not decreased when mice were treated with 50 ppm of lead.

NKT Cells in Mice Originate from Cytoplasmic CD3-Positive, CD4\u2212CD8\u2212 Double-Negative Thymocytes that Express CD44 and IL-7R\u03b1

Although natural killer T cells (NKT cells) are thought to be generated from CD4+CD8+ (DP) thymocytes, the developmental origin of CD4−CD8− (DN) NKT cells has remained unclear. In this study, we found the level of NK1.1 expression was highest in DN cells, followed by CD4 and CD8 (SP) and DP cells. The level of NK1.1 expression was highest in CD44+CD25− (DN1) cells, after that CD44+CD25+ (DN2), finally, CD44−CD25− (DN3) and CD44− CD25+ (DN4) cells. Unexpectedly, cytoplasmic CD3 was not only expressed in SP and DP thymocytes but also in most DN thymocytes at various stages. The mean fluorescence of cytoplasmic and surface CD3 in DN cells was significantly lower than in mature (SP) T and NKT cells in the thymus and spleen. Interestingly, there were more NKT cells in DN-cytoplasmic CD3 expression cells was higher than in DN-surface CD3 expression cells. There were more CD3-NKT cells in DN1 thymocytes than in TCR-β-NKT cells. NKT cells expressed higher levels of IL-7Rα which was correlated with CD44 expression in the thymus. Our data suggest that T cells and NKT cells follow similar patterns of expression with respect to cytoplasmic and surface CD3. Cytoplasmic CD3 could be used as a marker for early stage T cells. Both cytoplasmic CD3 and surface CD3 were expressed in mature T cells and immature T cells, including the immature cytoplasmic CD3+ surface CD3− and surface CD3+TCR-β− cells in DN1-NKT thymocytes. CD44 could be used as an additional marker of NKT cells which may originate from cytoplasmic CD3-positive DN thymocytes that express CD44 and IL-7Rα in mice.

Peroxisome Proliferator-Activated Receptor–δ Supports the Metabolic Requirements of Cell Growth in TCRβ-Selected Thymocytes and Peripheral CD4+ T Cells

During T cell development, progenitor thymocytes undergo a large proliferative burst immediately following successful TCRβ rearrangement, and defects in genes that regulate this proliferation have a profound effect on thymus cellularity and output. Although the signaling pathways that initiate cell cycling and nutrient uptake after TCRβ selection are understood, less is known about the transcriptional programs that regulate the metabolic machinery to promote biomass accumulation during this process. In this article, we report that mice with whole body deficiency in the nuclear receptor peroxisome proliferator-activated receptor-δ (PPARδmut) exhibit a reduction in spleen and thymus cellularity, with a decrease in thymocyte cell number starting at the double-negative 4 stage of thymocyte development. Although in vivo DNA synthesis was normal in PPARδmut thymocytes, studies in the OP9-delta-like 4 in vitro system of differentiation revealed that PPARδmut double-negative 3 cells underwent fewer cell divisions. Naive CD4+ T cells from PPARδmut mice also exhibited reduced proliferation upon TCR and CD28 stimulation in vitro. Growth defects in PPAR-δ-deficient thymocytes and peripheral CD4+ T cells correlated with decreases in extracellular acidification rate, mitochondrial reserve, and expression of a host of genes involved in glycolysis, oxidative phosphorylation, and lipogenesis. By contrast, mice with T cell-restricted deficiency of Ppard starting at the double-positive stage of thymocyte development, although exhibiting defective CD4+ T cell growth, possessed a normal T cell compartment, pointing to developmental defects as a cause of peripheral T cell lymphopenia in PPARδmut mice. These findings implicate PPAR-δ as a regulator of the metabolic program during thymocyte and T cell growth.

Effects of in vitro heat shock on immune cells in diet-induced obese mice

Obesity has been associated with impaired immune responses and inflammation. The mechanisms underlying these immune disturbances in obesity are not yet clarified. This study investigated the effects of in vitro heat shock (HS) on immune cells from the point of view of thymocyte apoptosis and T-cell mitogen-stimulated splenocyte cytokine production as well as the heat shock protein 70 (HSP70) protein levels in diet-induced obese mice to explore a possible association between the disturbance of T cell immunity and HS response in obesity. Obese mice had increased apoptotic and necrotic thymocytes populations and increased splenocyte cytokine production of both proinflammatory and anti-inflammatory cytokines compared with lean mice. The in vitro HS at 42°C decreased the rate of live cells in thymocytes, and the degree of the decrease was larger in obese mice compared with lean mice. The in vitro HS increased the intracellular and extracellular HSP70 protein levels in thymocytes and splenocytes, while the effects of obesity on the HSP70 protein levels were not obvious. The in vitro HS prior to T cell mitogen stimulation decreased IFN-γ and IL-10 production by mitogen-stimulated splenocytes. This change in cytokine production due to HS was not affected by obesity. The obvious alteration of the HSP70 protein levels and association between cytokine production and the HS response in obesity were not found in this obesity model; however, our results indicate an association between the viability of thymocytes and an altered HS response in obesity and provide evidence that the increase in thymocyte apoptosis and acceleration of thymus involution in obesity could be, in part, due to the alteration of the HS response.

Cytotoxic T Lymphocyte–associated Protein 4 Acts on Local Draining Lymph Nodes in the Allogeneic Abdominal Skin Flap of a Rat Model to Extend Its Survival

BackgroundEarly acute allograft rejection is the result of immune cells mainly composed of thymocyte (T) cells, which are activated, mature, and differentiate in lymphoid tissue. MethodsCytotoxic T lymphocyte–associated protein 4 immunoglobulin (CTLA4-Ig) competitively suppresses the cluster of differentiation 28/CTLA4-B7 costimulatory signaling pathway and alters the ratio of T helper 1 to T helper 2, leading to the inhibition of T cell activation. ResultsWe used lentivirus-packed CTLA4-Ig to infect the local draining lymph nodes of the allograft skin flap in a rat model, which effectively extended the survival of the allograft. Meanwhile, it also reduced the dose of immunosuppressant necessary in the early stages of transplantation, which lowered the toxicity and side effects of the drug. ConclusionsOur findings provide new insights into transplantation immunology, facilitate the exploration of a safe and effective immunosuppressive therapy, and promote the wide clinical application of composite tissue allotransplantation.

The effect of dichloroacetate on male rat thymus and on thymocyte cell cycle

The study aim was to investigate the effect of dichloroacetate (DCA) on thymus and the thymocyte cycle in rats. Wistar male gonad-intact and castrated rats (4-5 weeks) were investigated in the following groups: (1) control; (2) treated with DCA; and (3) treated with the DCA and sodium valproate (NaVP) combination. Rats were treated for 4 weeks with DCA 200 mg/kg/day alone and 300 mg/kg/day of NaVP plus 200 mg/kg/day of DCA (every second week, beginning with NaVP). After the experiment, the thymus was weighted, and the thymus lobe was taken for thymocyte flow cytometry. In gonad-intact rats, the thymus weight of the control was higher than in rats treated with DCA (P <0.001) or with the NaVP-DCA combination (P <0.04); a comparison of thymus weight between DCA- and NaVP-DCA-treated groups revealed a higher thymus weight loss in the DCA-treated group (P <0.03). Flow cytometry shows that DCA treatment increased the percentage of cells in the G2-M phase (P <0.03) and reduced in G1-G0 (P <0.02). The DCA treatment effect was determined only in gonad-intact but not in castrated rats. The authors discuss the possible DCA and NaVP interaction mechanisms.

Identifying the Spatial Relationships of Thymic Stromal and Thymocyte Subsets by Immunofluorescence Analysis.

Immunofluorescence analysis of thymic tissue sections is an indispensable technique for visualizing spatial relationships among thymocyte and stromal cell subsets. The thymus is organized into distinct microenvironmental zones in which particular thymic epithelial cell (TEC) subsets support specific stages of thymocyte maturation. Conversely, thymocytes and lymphoid tissue inducer cells support functional maturation of TECs. The composition and organization of TECs change during ontogeny to generate a maximally functional organ in the young adult. Deterioration of thymic architecture and stromal organization occurs with age as the thymus undergoes involution. Such changes can be monitored by immunofluorescent staining of thymic sections obtained at different ages throughout the life-span. Here we describe methods to generate frozen or paraffin-embedded thymic tissue sections for multicolor immunofluorescence staining using antibodies to surface and/or cytoplasmic antigens.

The JAK Inhibitor Ruxolitinib Is Effective in Treating T Cell Acute Lymphoblastic Leukemia with Gain of Function Mutations in IL-7R Alpha

Acute lymphoblastic leukemia (ALL) results from transformation of immature B or T cells, and is the most common pediatric cancer. Though the current cure rate of ALL is 80-90%, it is important to understand the underlying biology of ALL in order to develop refined therapies for patients who fail to respond to conventional chemotherapy as well as to reduce its toxicity. The IL-7 receptor (IL-7R) signaling pathway is necessary for the proliferation and survival of T cells. Together with collaborators, we have shown that 9% of patients with T cell acute lymphoblastic leukemia (T-ALL) have gain of function mutations in IL-7R alpha. These mutations promote homodimerization of IL-7R alpha subunits, resulting in constitutive activation of this pathway via Janus Kinase 1 (JAK1). As the JAK-STAT pathway is downstream of the IL-7 receptor, we hypothesized that JAK inhibitors could be used to treat T-ALL patients with IL-7R mutations. To demonstrate this, we first established a cell line model of T-ALL driven by constitutive IL-7R signaling. The model cells were established by transforming the D1 thymocyte cell line with a mutated IL-7R alpha derived from a patient sequence. These cells termed "D1_hIL7R_P1" are also GFP+, which allows us to monitor the proliferation of the cells in vivo.I have showed that D1_hIL7R_P1 cells delivered intravenously result in an aggressive leukemia with morbidity within 18-21 days. Ruxolitinib, a JAK1 inhibitor, inhibits survival, proliferation, and STAT5 activation of D1_hIL7R_P1 cells in vitro. To treat this leukemia in vivo, I administered ruxolitinib for 5 days at a dose of 150 mg/kg to mice starting 8 days after D1_hIL7R_P1 engraftment. Tissues were then harvested for analysis of GFP+ cells as a measure of leukemic burden via flow cytometry. Ruxolitinib reduced leukemic cells from 15% in the blood (as demonstrated in untreated mice) to 5%, from 30% to 10% in the spleen, and 40% to 20% in the lungs. With these promising results, I have acquired T-ALL patient samples that can be xenografted into NSG mice. TALL#5 cell line successfully engrafts in 30 days and can be detected in the bone marrow, spleen, and peripheral blood by flow cytometry. Additionally, TALL#5 expresses human IL-7R alpha and is sensitive to ruxolitinib treatment in vitro. I have also identified two T-ALL cell lines, DND41 and KOPTK1, which express high levels of IL-7R alpha. These cell lines are also potential candidates for studying the effects of ruxolitinib on T-ALL in NSG mice. TALL#5, DND41, and KOPTK1 will be engrafted into mice, and after leukemia is established, mice will be treated with ruxolitinib. If successful, this will give us insight into the effectiveness of JAK inhibitors in treating ALL, and which patients can be recruited for future clinical trials. DisclosuresOff Label Use: Ruxolitinib is a JAK1/2 inhibitor that is FDA approved for myelofibrosis. My experiments involve pre-clinical studies looking at the efficacy of ruxolitinib in leukemic mice..

Functional requirement of tyrosine residue 429 within CD5 cytoplasmic domain for regulation of T cell activation and survival

CD5 has been mainly described as a negative regulator of TCR and BCR signaling and recent evidence has shown an important role for this receptor in delivering pro-survival signals. However, the molecular mechanisms underlying these processes remain unresolved. TCR crosslinking leads to phosphorylation of three tyrosine residues within the cytoplasmic tail of CD5 (Y429, Y441 and Y463) leading to the recruitment of signaling molecules like PI3K, c-Cbl and RasGAP; nevertheless, the role of these residues in T cell survival has not yet been assessed. In this study, we show that alanine-scanning mutagenesis of such tyrosine residues, either singly or in combination, leads to an increased thymocyte cell death with or without α-CD3 stimulation. Remarkably, the T-cell death observed with each individual tyrosine mutant was Caspase 3-independent. Furthermore, Y429 mutation resulted in a hyper-phosphorylation of ERK suggesting that this tyrosine residue regulates cell survival through down modulation of TCR signaling. Mutation of Y441 or Y463 did not induce hyper-responsiveness to TCR activation, indicating that they promoted T-cell survival by a TCR signal-independent pathway. Our results show that three tyrosine-based domains within CD5 cytoplasmic tail promote T-cell survival through non-overlapping mechanisms. This study also reveals that Y429 domain of CD5, previously described as a “pseudo ITAM”, is functionally an ITIM domain in T cells.

Abstract 2287: A model system to treat T-cell acute lymphoblastic leukemia with JAK inhibitors

Abstract The IL-7 Receptor signaling pathway is necessary for the proliferation and survival of T cells. Together with collaborators, we have shown that 9% of patients with T cell acute lymphoblastic leukemia (T-ALL) have gain of function mutations in IL-7R exon 6. These mutations promote homodimerization of IL-7Rα subunits, resulting in constitutive activation of this pathway via JAK1. Approximately 20% of T-ALL patients fail to respond to conventional chemotherapy, so there is a need for additional options for these patients. We hypothesized that JAK inhibitors could be used to treat T-ALL patients, particularly those with IL-7R mutations. We established a cell line model of T-ALL driven by constitutive IL-7R signaling which grew as a leukemia in RAG1-/- mice. The model cells were established by transforming the D1 thymocyte cell line with a mutated IL-7Rα derived from a patient sequence. These cells termed D1_hIL7R_P1 are also GFP+ which allows us to monitor the proliferation of the cells in vivo. D1_hIL7R_P1 cells delivered intravenously result in an aggressive leukemia with morbidity within 18-21 days. Currently we are studying the chemotherapeutic effects of two JAK inhibitors, Ruxolitinib and Tofacitinib on T-ALL. The maximum tolerated doses for Tofacitinib and Ruxolitinib are 50 mg/kg and 150 mg/kg, respectively. Ruxolitinib appears to be the most effective, showing a reduction of leukemogenesis detected in blood and tissues after just three days of oral treatment. Additionally, we have obtained samples derived from pediatric T-ALL patients. Once growth kinetics in NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ mice is established, we will proceed to study the efficacy of the JAK inhibitors with human leukemia cells. Citation Format: Emilee Senkevitch, Julie Hixon, Wenqing Li, Scott Durum. A model system to treat T-cell acute lymphoblastic leukemia with JAK inhibitors. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2287. doi:10.1158/1538-7445.AM2015-2287

High dietary intake of vitamin C suppresses age-related thymic atrophy and contributes to the maintenance of immune cells in vitamin C-deficient senescence marker protein-30 knockout mice.

Vitamin C (VC) is an essential nutrient for humans and certain other animals. It has antioxidant properties and has been reported to ameliorate oxidative damage to lipids, DNA and proteins. However, the effects of VC on immune function are poorly understood, especially the influence of long-term high-dose VC intake on the number and function of immune cells. In the present study, to evaluate the immune effects of VC, VC-deficient senescence marker protein-30 knockout (SMP30KO) mice were fed a diet containing the recommended level of VC (20 mg/kg per d; 0·02 % VC) or a high level of VC (200 mg/kg per d; 0·2 % VC) for 1 year. The plasma VC concentration of the 0·02 % group was the same as that of age-matched C57BL/6 mice after 1 year of feeding; however, plasma VC concentration and thymus weight were significantly higher in the 0·2 % VC group than in the 0·02 % VC group. The total counts of leucocytes, lymphocytes, granulocytes and monocytes in the peripheral blood, as well as the number of splenocytes and thymocytes, were all significantly higher in the 0·2 % VC group than in the 0·02 % VC group. In addition, the number of naive T cells in peripheral blood lymphocytes, the number of memory T-cell populations in splenocytes, and the number of cluster of differentiation (CD)4⁺CD8⁺ or CD4⁺CD8⁻ or CD4⁻CD8⁺ T cells in thymocytes were all markedly higher in the 0·2 % VC group than in the 0·02 % VC group after 1 year of dietary treatment. These results suggest that a long-term high-dose intake of VC is effective in the maintenance of immune cells, partly through the suppression of age-related thymic involution in VC-deficient SMP30KO mice.