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.

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.

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).

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.

A novel role for CD4+ T cells in the control of cachexia.

Cachexia is the dramatic weight loss and muscle atrophy seen in chronic disease states, including autoimmunity, cancer, and infection, and is often associated with lymphopenia. We have previously shown that CD4(+) T cells that express the lowest density of CD44 (CD4(+)CD44(v.low)) are significantly reduced in diabetic NOD mice that are cachexic compared with diabetic mice that are not cachexic. Using this model, and a model of cancer cachexia, we test the hypothesis that CD4(+)CD44(v.low) cells play an active role in protecting the host from cachexia. CD4(+)CD44(v.low) cells, but not CD4(+) cells depleted of CD44(v.low) cells, delay the onset of wasting when infused into either diabetic or prediabetic NOD recipients. However, no significant effect on the severity of diabetes was detected. In a model of cancer cachexia, they significantly reduce muscle atrophy, and inhibit muscle protein loss and DNA loss, even when given after the onset of cachexia. Protection from wasting and muscle atrophy by CD4(+)CD44(v.low) cells is associated with protection from lymphopenia. These data suggest, for the first time, a role for an immune cell subset in protection from cachexia, and further suggest that the mechanism of protection is independent of protection from autoimmunity.

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.

Cachexia in the non-obese diabetic mouse is associated with CD4+ T-cell lymphopenia.

One of the long-term consequences of Type I diabetes is weight loss with muscle atrophy, the hallmark phenotype of cachexia. A number of disorders that result in cachexia are associated with immune deficiency. However, whether immune deficiency is a cause or an effect of cachexia is not known. This study examines the non-obese diabetic mouse, the mouse model for spontaneous Type I diabetes, as a potential model to study lymphopenia in cachexia, and to determine whether lymphopenia plays a role in the development of cachexia. The muscle atrophy seen in patients with Type I diabetes involves active protein degradation by activation of the ubiquitin-proteasome pathway, indicating cachexia. Evidence of cachexia in the non-obese diabetic mouse was determined by measuring skeletal muscle atrophy, activation of the ubiquitin-proteasome pathway, and apoptosis, a state also described in some models of cachexia. CD4+ T-cell subset lymphopenia was measured in wasting and non-wasting diabetic mice. Our data show that the mechanism of wasting in diabetic mice involves muscle atrophy, a significant increase in ubiquitin conjugation, and upregulation of the ubiquitin ligases, muscle RING finger 1 (MuRF1) and muscle atrophy F box/atrogin-1 (MAFbx), indicating cachexia. Moreover, fragmentation of DNA isolated from atrophied muscle tissue indicates apoptosis. While CD4+ T-cell lymphopenia is evident in all diabetic mice, CD4+ T cells that express a very low density of CD44 were significantly lost in wasting, but not non-wasting, diabetic mice. These data suggest that CD4+ T-cell subsets are not equally susceptible to cachexia-associated lymphopenia in diabetic mice.

High-throughput sequencing of islet-infiltrating memory CD4+ T cells reveals a similar pattern of TCR Vß usage in prediabetic and diabetic NOD mice.

Autoreactive memory CD4(+) T cells play a critical role in the development of type 1 diabetes, but it is not yet known how the clonotypic composition and TCRß repertoire of the memory CD4(+) T cell compartment changes during the transition from prediabetes to diabetes. In this study, we used high-throughput sequencing to analyze the TCRß repertoire of sorted islet-infiltrating memory CD4(+)CD44(high) T cells in 10-week-old prediabetic and recently diabetic NOD mice. We show that most clonotypes of islet-infiltrating CD4(+)CD44(high) T cells were rare, but high-frequency clonotypes were significantly more common in diabetic than in prediabetic mice. Moreover, although the CD4(+)CD44(high) TCRß repertoires were highly diverse at both stages of disease development, dominant use of TRBV1 (Vß2), TRBV13-3 (Vß8.1), and TRBV19 (Vß6) was evident in both prediabetic and diabetic mice. Our findings strongly suggest that therapeutic targeting of cells specifically expressing the dominant TCRß might reduce pancreatic infiltration in prediabetic mice and attenuate the progression to diabetes.

T cell populations in the pancreatic lymph node naturally and consistently expand and contract in NOD mice as disease progresses.

Nonobese diabetic (NOD) mice develop spontaneous autoimmune Type 1 diabetes (T1D) that results from the destruction of insulin secreting ß cells by diabetogenic T cells. The activation of autoreactive T cells occurs in the pancreatic lymph nodes (PLN) from where effector T cells migrate to the pancreas. This study was designed to explore whether T cell populations in the NOD PLN expand in a predictable and reproducible way during disease progression. Complementary determining region (CDR) 3 length spectratype analysis of 19 TCR Vß families was used to identify the relative frequency of T populations in PLN of 4 and 10 week old NOD mice and mice at T1D onset. Significant and highly reproducible changes in specific T cell populations were detected in 14 of Vß families tested at all stages of disease. However, of these, the CDR3 spectratype of only four Vß families was significantly more perturbed at T1D onset than in 10 week old mice. Intriguingly, when diabetes was induced in 10 week old mice with cyclophosphamide (CYP) the same four Vß families, Vß5.1, Vß9, Vß10, and Vß15, were again significantly more perturbed than in the untreated non-diabetic age matched mice. Taken together the data show that while T cell responses in PLN of NOD mice are heterogeneous, they are ordered and consistent throughout disease development. The finding that within this heterogeneous response four Vß families are significantly more perturbed in diabetic mice, whether spontaneous or induced, strongly suggests their selection as part of the disease process.

A peripheral CD4+ T cell precursor for naive, memory, and regulatory T cells.

Mechanisms that control the size of the T cell pool, the ratio between naive cells and memory cells, the number and frequency of regulatory T cells, and T cell receptor (TCR) diversity are necessary to maintain immune integrity and avoid disease. We have previously shown that a subset of naive CD4(+) T cells, defined by the expression on their surface of a very low density of CD44 (CD44(v.low) cells), can inhibit wasting and wasting-associated lymphopenia in mice with cancer. In this study, we further investigate the properties of CD44(v.low) cells and show that they are significantly more efficient than the remaining naive (CD44(low) or CD44(int)) and memory CD4(+) cell subsets in reconstituting the overall size of the CD4(+) T cell pool, creating a T cell pool with a diverse TCR repertoire, generating regulatory T cells that express forkhead box P3 (FoxP3), and promoting homeostatic equilibrium between naive, memory, and Foxp3(+) regulatory T cell numbers. T cell population reconstitution by CD44(v.low) cells is thymus independent. Compared with CD44(int) cells, a higher percentage of CD44(v.low) cells express B cell leukemia/lymphoma 2, interleukin-7 receptor, and CD5. The data support a key role for CD4(+) CD44(v.low) cells as peripheral precursors that maintain the integrity of the CD4(+) T cell pool.

CD8 blockade promotes antigen responsiveness to nontolerizing antigen in tolerant mice by inhibiting apoptosis of CD4+ T cells.

Using the DO11.10 CD4+ TCR-transgenic mouse system, we have recently shown that CD8 blockade promotes the expansion of Ag-specific regulatory CD4+ T cells in mice made tolerant to OVA with anti-CD4 mAb. We now show that CD8 blockade is also critical to promoting responses to nontolerizing Ag in anti-CD4 mAb-treated tolerant mice. Previously published work shows that treatment with anti-CD4 mAb without CD8 blockade induces Ag-specific tolerance. We now show that, in addition to inducing tolerance, anti-CD4 mAb treatment also significantly reduces responsiveness to irrelevant, nontolerizing Ag, and this unresponsiveness is associated with significant apoptosis of the CD4+ T cells. Anti-CD4 mAb-induced apoptosis is inhibited by cotreatment with anti-CD8 mAb and responsiveness to irrelevant Ag is restored, while Ag-specific tolerance is maintained. These data suggest that CD8 blockade promotes responsiveness to nontolerizing Ags in tolerant mice by inhibiting CD4+ T cell apoptosis.

IL-4 expression delays eosinophil-independent vasculopathy and fibrosis during allograft rejection in the mouse.

Transplant vasculopathy in the mouse is thought to be dependent on IL-4 and mediated by IL-5 and eosinophils, whereas in the rat and human systems, IL-4 is associated with the absence of transplant vasculopathy and down-regulation of a Th1-type response. In this study we tested the possibility that the apparent difference in the role of IL-4 in transplant vasculopathy is related to protocol differences rather than to the species being studied. Using a protocol that closely resembles that used in rat and human studies, we developed a model of transplant vasculopathy in the mouse that is associated with Th1-type cytokines and independent of IL-5 and eosinophil infiltration. In this model IL-4 promotes a significant delay in vasculopathy in the graft (P = 0.04) and a decrease in the incidence of allograft rejection (P = 0.02). The data suggest that the role of IL-4 in transplant vasculopathy can be controlled by the protocol used to treat the transplant recipient.

Increasing transplant mass results in long-term allograft survival and recovery from transplant vasculopathy.

Late allograft rejection due to transplant vasculopathy continues to be a major clinical problem. Increasing the ratio of donor transplant size to recipient weight has been shown to reduce the incidence of late allograft failure. Using a murine pancreas transplant model we have tested the hypothesis that increasing the donor transplant size in a recipient can promote long-term allograft survival by promoting recovery from transplant vasculopathy. Recipients of an allograft that showed extensive vasculopathy were transplanted with a second donor transplant. The effect of the second allograft on the vasculopathy present in the first graft was measured. Transplanting a second allograft reversed all signs of ongoing rejection, including transplant vasculopathy, resulting in long-term survival of the first graft. Vasculopathy was only reversed if the first and second grafts were from the same mouse strain, suggesting an antigen-specific mechanism. However, the recovery of the first graft was not associated with antigen-specific peripheral tolerance.