Th2 cell clonal expansion at diagnosis in human type 1 diabetes.

Soon after diagnosis with type 1 diabetes (T1D), many patients experience a period of partial remission. A longer partial remission is associated with a better response to treatment, but the mechanism is not known. The frequency of CD4+CD25+CD127hi (127-hi) cells, a cell subset with an anti-inflammatory Th2 bias, correlates positively with length of partial remission. The purpose of this study was to further characterize the nature of the Th2 bias in 127-hi cells. Single cell RNA sequencing paired with TCR sequencing of sorted 127-hi memory cells identifies clonally expanded Th2 clusters in 127-hi cells from T1D, but not from healthy donors. The Th2 clusters express GATA3, GATA3-AS1, PTGDR2, IL17RB, IL4R and IL9R. The existence of 127-hi Th2 cell clonal expansion in T1D suggests that disease factors may induce clonal expansion of 127-hi Th2 cells that prolong partial remission and delay disease progression.

CD4+CD25+CD127hi cell frequency predicts disease progression in type 1 diabetes.

Transient partial remission, a period of low insulin requirement experienced by most patients soon after diagnosis, has been associated with mechanisms of immune regulation. A better understanding of such natural mechanisms of immune regulation might identify new targets for immunotherapies that reverse type 1 diabetes (T1D). In this study, using Cox model multivariate analysis, we validated our previous findings that patients with the highest frequency of CD4+CD25+CD127hi (127-hi) cells at diagnosis experience the longest partial remission, and we showed that the 127-hi cell population is a mix of Th1- and Th2-type cells, with a significant bias toward antiinflammatory Th2-type cells. In addition, we extended these findings to show that patients with the highest frequency of 127-hi cells at diagnosis were significantly more likely to maintain ß cell function. Moreover, in patients treated with alefacept in the T1DAL clinical trial, the probability of responding favorably to the antiinflammatory drug was significantly higher in those with a higher frequency of 127-hi cells at diagnosis than those with a lower 127-hi cell frequency. These data are consistent with the hypothesis that 127-hi cells maintain an antiinflammatory environment that is permissive for partial remission, ß cell survival, and response to antiinflammatory immunotherapy.

Cancer-driven changes link T cell frequency to muscle strength in people with cancer: a pilot study.

BACKGROUND: Tumour growth can promote the loss of muscle mass and function. This is particularly disturbing because overall survival is significantly reduced in people with weaker and smaller skeletal muscle. The risk of cancer is also greater in people who are immune deficient. Muscle wasting in mice with cancer can be inhibited by infusion of CD4+ precursor T cells that restore balanced ratios of naïve, memory, and regulatory T cells. These data are consistent with the hypothesis that stronger anti-cancer T cell immunity leads to improved muscle mass and function. As a first step to testing this hypothesis, we determined whether levels of circulating T cell subsets correlate with levels of muscle strength in people with cancer. METHODS: The frequency of circulating CD4+ and CD8+ naïve, memory, and regulatory T cell subsets was quantified in 11 men with gastrointestinal cancer (aged 59.3 ± 10.1 years) and nine men without cancer (aged 60 ± 13 years), using flow cytometry. T cell marker expression was determined using real-time PCR and western blot analyses in whole blood and peripheral blood mononuclear cells. Handgrip strength, one-repetition maximum chest press, and knee extension tests were used to determine muscle strength. Performance was determined using a stair climb test. Body composition was determined using dual-energy X-ray absorptiometry scan. The Karnofsky and ECOG scales were used to assess functional impairment. Correlations between frequencies of cell subsets with strength, performance, and body composition were determined using regression analyses. RESULTS: Our data show significant correlations between (i) higher frequencies of CD8+ naïve (P = 0.02) and effector memory (P = 0.003) T cells and lower frequencies of CD8+ central memory T cells (P = 0.002) with stronger handgrip strength, (ii) lower frequency of regulatory cells with greater lean mass index (P = 0.04), (iii) lower frequency of CD8+ T cells that express CD95 with greater stair climb power (P = 0.003), (iv) higher frequency of T cells that co-express CD197 and CD45RA and greater one-repetition maximum knee extension strength (P = 0.008), and (iv) higher expression of CD4 in whole blood with greater functional impairment (P = 0.004) in people with cancer. CONCLUSIONS: We have identified significant correlations between levels of T cell populations and muscle strength, performance, and body composition in people with cancer. These data justify a follow-up study with a larger cohort to test the validity of the findings.

Human CD4+ CD25+ CD127hi cells and the Th1/Th2 phenotype.

CD4+ T cells that co-express CD25 and CD127 (CD25+CD127+) make up around 20% of all circulating CD4+ memory T cells in healthy people. The clinical significance of these cells is that in children with type 1 diabetes their relative frequency at diagnosis is significantly and directly correlated with rate of disease progression. The purpose of this study was to further characterize the CD25+CD127hi cells. We show that they are a mix of Th1 and Th2 cells however, they have a significantly higher relative frequency of pre-committed and committed Th2 cells, and secrete significantly higher levels of Th2-type cytokines than CD25- memory T cells. Further, these cells are neither exhausted nor senescent and proliferate to the same extent as CD25- memory cells. Thus, CD25+CD127hi cells are a highly active subset of memory T cells that might play a role in controlling inflammation via anti-inflammatory Th2-type deviation.

Memory T Cells in Type 1 Diabetes: the Devil is in the Detail.

PURPOSE OF REVIEW: Therapies that target beta-cell antigen-specific T cells subsets have not been as successful in patients with type 1 diabetes as in mice. This might be explained by complexities in the repertoire of beta-cell antigen-specific T cells and the variety of T cell subsets involved in type 1 diabetes development in human. RECENT FINDINGS: T cells that infiltrate islets of people with type 1 diabetes (i) react towards known islet cell antigens but also unknown antigens, (ii) differ from one patient to another, and (iii) are also present in the circulation, but not in the islets, of healthy people. Moreover, several circulating memory T cell subsets not recognized as relevant in mouse are significantly associated with clinical outcome. A more detailed understanding of the specificity, phenotype, and function of T cells that are associated with defined clinical outcomes might identify new pathways for therapeutic intervention.

Data on correlations between T cell subset frequencies and length of partial remission in type 1 diabetes.

Partial remission in patients newly diagnosed with type 1 diabetes is a period of good glucose control that can last from several weeks to over a year. The clinical significance of the remission period is that patients might be more responsive to immunotherapy if treated within this period. This article provides clinical data that indicates the level of glucose control and insulin-secreting ß-cell function of each patient in the study at baseline (within 3 months of diagnosis), and at 3, 6, 9, 12, 18 and 24 months post-baseline. The relative frequency of immune cell subsets in the PBMC of each patient and the association between the frequency of immune cell subsets measured and length of remission is also shown. These data support the findings reported in the accompanying publication, "A pilot study showing associations between frequency of CD4+ memory cell subsets at diagnosis and duration of partial remission in type 1 diabetes" (Moya et al., 2016) [1], where a full interpretation, including biological relevance of the study can be found.

Short-term pyrrolidine dithiocarbamate administration attenuates cachexia-induced alterations to muscle and liver in ApcMin/+ mice.

Cancer cachexia is a complex wasting condition characterized by chronic inflammation, disrupted energy metabolism, and severe muscle wasting. While evidence in pre-clinical cancer cachexia models have determined that different systemic inflammatory inhibitors can attenuate several characteristics of cachexia, there is a limited understanding of their effects after cachexia has developed, and whether short-term administration is sufficient to reverse cachexia-induced signaling in distinctive target tissues. Pyrrolidine dithiocarbamate (PDTC) is a thiol compound having anti-inflammatory and antioxidant properties which can inhibit STAT3 and nuclear factor κB (NF-κB) signaling in mice. This study examined the effect of short-term PDTC administration to ApcMin/+ mice on cachexia-induced disruption of skeletal muscle protein turnover and liver metabolic function. At 16 weeks of age ApcMin/+ mice initiating cachexia (7% BW loss) were administered PDTC (10mg/kg bw/d) for 2 weeks. Control ApcMin/+ mice continued to lose body weight during the treatment period, while mice receiving PDTC had no further body weight decrease. PDTC had no effect on either intestinal tumor burden or circulating IL-6. In muscle, PDTC rescued signaling disrupting protein turnover regulation. PDTC suppressed the cachexia induction of STAT3, increased mTORC1 signaling and protein synthesis, and suppressed the induction of Atrogin-1 protein expression. Related to cachectic liver metabolic function, PDTC treatment attenuated glycogen and lipid content depletion independent to the activation of STAT3 and mTORC1 signaling. Overall, these results demonstrate short-term PDTC treatment to cachectic mice attenuated cancer-induced disruptions to muscle and liver signaling, and these changes were independent to altered tumor burden and circulating IL-6.

A pilot study showing associations between frequency of CD4(+) memory cell subsets at diagnosis and duration of partial remission in type 1 diabetes.

In some patients with type 1 diabetes the dose of insulin required to achieve euglycemia is substantially reduced soon after diagnosis. This partial remission is associated with ß-cell function and good glucose control. The purpose of this study was to assess whether frequencies of CD4(+) T cell subsets in children newly diagnosed with type 1 diabetes are associated with length of partial remission. We found that the frequency of CD4(+) memory cells, activated Treg cells and CD25(+) cells that express a high density of the IL-7 receptor, CD127 (CD127(hi)) are strongly associated with length of partial remission. Prediction of length of remission via Cox regression is significantly enhanced when CD25(+) CD127(hi) cell frequency is combined with either Insulin Dependent Adjusted A1c (IDAA1c), or glycosylated hemoglobin (HbA1c), or C-peptide levels at diagnosis. CD25(+) CD127(hi) cells do not express Foxp3, LAG-3 and CD49b, indicating that they are neither Treg nor Tr1 cells.

CD4(+) CD44(v.low) cells are unique peripheral precursors that are distinct from recent thymic emigrants and stem cell-like memory cells.

CD4(+) CD44(v.low) cells are peripheral precursor T cells that inhibit lymphopenia by generating a large CD4(+) T cell pool containing balanced numbers of naïve, memory, and regulatory Foxp3(+) cells with a diverse TCR repertoire. Recent thymic emigrants (RTE) and stem cell-like memory T cells (T(SCM)) can also replenish a T cell pool. In this study we formally test whether CD44(v.low) cells are the same population as RTE and T(SCM). Our data show that, in contrast to RTE, CD44(v.low) cells express high levels of CD45RB and low levels of CD24. Moreover, CD44(v.low) cells isolated from mice devoid of RTE retain their capacity to repopulate lymphopenic mice with naïve and memory cells and Foxp3(+) Tregs. In addition, CD44(v.low) cells do not express IL-2Rß, Sca-1, and CXCR3, the phenotypic hallmarks of T(SCM). Overall, these data demonstrate that CD44(v.low) cells are neither RTE nor T(SCM).

Liver inflammation and metabolic signaling in ApcMin/+ mice: the role of cachexia progression.

The ApcMin/+ mouse exhibits an intestinal tumor associated loss of muscle and fat that is accompanied by chronic inflammation, insulin resistance and hyperlipidemia. Since the liver governs systemic energy demands through regulation of glucose and lipid metabolism, it is likely that the liver is a pathological target of cachexia progression in the ApcMin/+ mouse. The purpose of this study was to determine if cancer and the progression of cachexia affected liver endoplasmic reticulum (ER)-stress, inflammation, metabolism, and protein synthesis signaling. The effect of cancer (without cachexia) was examined in wild-type and weight-stable ApcMin/+ mice. Cachexia progression was examined in weight-stable, pre-cachectic, and severely-cachectic ApcMin/+ mice. Livers were analyzed for morphology, glycogen content, ER-stress, inflammation, and metabolic changes. Cancer induced hepatic expression of ER-stress markers BiP (binding immunoglobulin protein), IRE-1α (endoplasmic reticulum to nucleus signaling 1), and inflammatory intermediate STAT-3 (signal transducer and activator of transcription 3). While gluconeogenic enzyme phosphoenolpyruvate carboxykinase (PEPCK) mRNA expression was suppressed by cancer, glycogen content or protein synthesis signaling remained unaffected. Cachexia progression depleted liver glycogen content and increased mRNA expression of glycolytic enzyme PFK (phosphofrucktokinase) and gluconeogenic enzyme PEPCK. Cachexia progression further increased pSTAT-3 but suppressed p-65 and JNK (c-Jun NH2-terminal kinase) activation. Interestingly, progression of cachexia suppressed upstream ER-stress markers BiP and IRE-1α, while inducing its downstream target CHOP (DNA-damage inducible transcript 3). Cachectic mice exhibited a dysregulation of protein synthesis signaling, with an induction of p-mTOR (mechanistic target of rapamycin), despite a suppression of Akt (thymoma viral proto-oncogene 1) and S6 (ribosomal protein S6) phosphorylation. Thus, cancer induced ER-stress markers in the liver, however cachexia progression further deteriorated liver ER-stress, disrupted protein synthesis regulation and caused a differential inflammatory response related to STAT-3 and NF-κB (Nuclear factor-κB) signaling.

Sex differences in the relationship of IL-6 signaling to cancer cachexia progression.

A devastating aspect of cancer cachexia is severe loss of muscle and fat mass. Though cachexia occurs in both sexes, it is not well-defined in the female. The Apc(Min/+) mouse is genetically predisposed to develop intestinal tumors; circulating IL-6 is a critical regulator of cancer cachexia in the male Apc(Min/+) mouse. The purpose of this study was to examine the relationship between IL-6 signaling and cachexia progression in the female Apc(Min/+) mouse. Male and female Apc(Min/+) mice were examined during the initiation and progression of cachexia. Another group of females had IL-6 overexpressed between 12 and 14 weeks or 15-18 weeks of age to determine whether IL-6 could induce cachexia. Cachectic female Apc(Min/+) mice lost body weight, muscle mass, and fat mass; increased muscle IL-6 mRNA expression was associated with these changes, but circulating IL-6 levels were not. Circulating IL-6 levels did not correlate with downstream signaling in muscle in the female. Muscle IL-6r mRNA expression and SOCS3 mRNA expression as well as muscle IL-6r protein and STAT3 phosphorylation increased with severe cachexia in both sexes. Muscle SOCS3 protein increased in cachectic females but decreased in cachectic males. IL-6 overexpression did not affect cachexia progression in female Apc(Min/+) mice. Our results indicate that female Apc(Min/+) mice undergo cachexia progression that is at least initially IL-6-independent. Future studies in the female will need to determine mechanisms underlying regulation of IL-6 response and cachexia induction.

Role of interleukin-6 in cachexia: therapeutic implications.

PURPOSE OF REVIEW: Interleukin-6 (IL-6) has emerged as a cytokine involved in cachexia progression with some cancers. This review will present the recent breakthroughs in animal models and humans related to targeting IL-6 as a cancer cachexia therapy. RECENT FINDINGS: IL-6 can target adipose, skeletal muscle, gut, and liver tissue, which can all affect cachectic patient recovery. IL-6 trans-signaling through the soluble IL-6R has the potential to amplify IL-6 signaling in the cachectic patient. In the skeletal muscle, chronic IL-6 exposure induces proteasome and autophagy protein degradation pathways that lead to wasting. IL-6 is also indirectly associated with AMP-activated kinase (AMPK) and nuclear factor kappa B (NF-κB) activation. Several mouse cancer models have clearly demonstrated that blocking IL-6 and associated signaling can attenuate cachexia progression. Additionally, pharmaceuticals targeting IL-6 and associated signaling can relieve some cachectic symptoms in cancer patients. Research with cachectic mice has demonstrated that exercise and nutraceutical administration can interact with chronic IL-6 signaling during cachexia progression. SUMMARY: IL-6 remains a promising therapeutic strategy for attenuating cachexia progression with many types of cancer. However, improvement of this treatment will require a better understanding of the indirect and direct effects of IL-6 as well as its tissue-specific actions in the cancer patient.

Quercetin supplementation attenuates the progression of cancer cachexia in ApcMin/+ mice.

Although there are currently no approved treatments for cancer cachexia, there is an intensified interest in developing therapies because of the high mortality index associated with muscle wasting diseases. Successful treatment of the cachectic patient focuses on improving or maintaining body weight and musculoskeletal function. Nutraceutical compounds, including the natural phytochemical quercetin, are being examined as potential treatments because of their anti-inflammatory, antioxidant, and anticarcinogenic properties. The purpose of this study was to determine the effect of quercetin supplementation on the progression of cachexia in the adenomatous polyposis coli (Apc)(Min/+) mouse model of colorectal cancer. At 15 wk of age, C57BL/6 and male Apc(Min/+) mice were supplemented with 25 mg/kg of quercetin or vehicle solution mix of Tang juice and water (V) daily for 3 wk. Body weight, strength, neuromuscular performance, and fatigue were assessed before and after quercetin or V interventions. Indicators of metabolic dysfunction and inflammatory signaling were also assessed. During the treatment period, the relative decrease in body weight in the Apc(Min/+) mice gavaged with V (Apc(Min/+)V; -14% ± 2.3) was higher than in control mice gavaged with V (+0.6% ± 1.0), control mice gavaged with quercetin (-2% ± 1.0), and Apc(Min/+) mice gavaged with quercetin (Apc(Min/+)Q; -9% ± 1.3). At 18 wk of age, the loss of grip strength and muscle mass shown in Apc(Min/+)V mice was significantly attenuated (P < 0.05) in Apc(Min/+)Q mice. Furthermore, Apc(Min/+)V mice had an induction of plasma interleukin-6 and muscle signal transducer and activator of transcription 3 phosphorylation, which were significantly (P < 0.05) mitigated in Apc(Min/+)Q mice, despite having a similar tumor burden. Quercetin treatment did not improve treadmill run-time-to-fatigue, hyperglycemia, or hyperlipidemia in cachectic Apc(Min/+) mice. Overall, quercetin supplementation positively affected several aspects of cachexia progression in mice and warrants further exploration as a potential anticachectic therapeutic.

Skeletal muscle glycoprotein 130's role in Lewis lung carcinoma-induced cachexia.

Chronic inflammation is associated with cachexia-induced skeletal muscle mass loss in cancer. Levels of IL-6 cytokine family members are increased during cancer-related cachexia and induce intracellular signaling through glycoprotein130 (gp130). Although muscle STAT3 and circulating IL-6 are implicated in cancer-induced muscle wasting, there is limited understanding of muscle gp130's role in this process. Therefore, we investigated the role of skeletal muscle gp130 (skm-gp130) in cancer-induced alterations in the regulation of muscle protein turnover. Lewis lung carcinoma (LLC) cells were injected into 8-wk-old skm-gp130-knockout (KO) mice or wild-type mice. Skeletal muscle loss was attenuated by 16% in gp130-KO mice, which coincided with attenuated LLC-induced phosphorylation of muscle STAT3, p38, and FOXO3. gp130 KO did not restore mTOR inhibition or alter AMP-activated protein kinase (AMPK) expression. The induction of atrogin expression and p38 phosphorylation in C2C12 myotubes exposed to LLC-treated medium was attenuated by gp130 inhibition, but mTOR inhibition was not restored. STAT signaling inhibition in LLC-treated myotubes did not attenuate the induction of p38 or AMPK phosphorylation. We concluded that, during LLC-induced cachexia, skm-gp130 regulates muscle mass signaling through STAT3 and p38 for the activation of FOXO3 and atrogin, but does not directly regulate the suppression of mTOR.

Characterization of the male ApcMin/+ mouse as a hypogonadism model related to cancer cachexia.

Cancer cachexia, the unintentional loss of lean body mass, is associated with decreased quality of life and poor patient survival. Hypogonadism, involving a reduction in circulating testosterone, is associated with the cachectic condition. At this time there is a very limited understanding of the role of hypogonadism in cancer cachexia progression. This gap in our knowledge is related to a lack of functional hypogonadal models associated with cancer cachexia. The Apc(Min/+) mouse is an established colorectal cancer model that develops an IL-6 dependent cachexia which is physiologically related to human disease due to the gradual progression of tumor development and cachexia. The purpose of this study was to assess the utility of the Apc(Min/+) mouse for the examination of hypogonadism during cancer cachexia and to investigate if IL-6 has a role in this process. We report that Apc(Min/+) mice that are weight stable have comparable testosterone levels and gonad size compared to wild type mice. Cachectic Apc(Min/+) mice exhibit a reduction in circulating testosterone and gonad size, which has a significant association with the degree of muscle mass and functional strength loss. Circulating testosterone levels were also significantly associated with the suppression of myofibrillar protein synthesis. Skeletal muscle and testes androgen receptor expression were decreased with severe cachexia. Although testes STAT3 phosphorylation increased with severe cachexia, systemic IL-6 over-expression for 2 weeks was not sufficient to reduce either testes weight or circulating testosterone. Inhibition of systemic IL-6 signaling by an IL-6 receptor antibody to Apc(Min/+) mice that had already initiated weight loss was sufficient to attenuate a reduction in testes size and circulating testosterone. In summary, the Apc(Min/+) mouse becomes hypogonadal with the progression of cachexia severity and elevated circulating IL-6 levels may have a role in the development of hypogonadism during cancer cachexia.