Daniel E. Lowther

Sodium chloride inhibits the suppressive function of FOXP3+ regulatory T cells

FOXP3+ Tregs are central for the maintenance of self-tolerance and can be defective in autoimmunity. In multiple sclerosis and type-1 diabetes, dysfunctional self-tolerance is partially mediated by a population of IFNγ-secreting Tregs. It was previously reported that increased NaCl concentrations promote the induction of proinflammatory Th17 cells and that high-salt diets exacerbate experimental models of autoimmunity. Here, we have shown that increasing NaCl, either in vitro or in murine models via diet, markedly impairs Treg function. NaCl increased IFNγ secretion in Tregs, and reducing IFNγ - either by neutralization with anti-IFNγ antibodies or shRNA-mediated knockdown - restored suppressive activity in Tregs. The heightened IFNγ secretion and loss of Treg function were mediated by the serum/glucocorticoid-regulated kinase (SGK1). A high-salt diet also impaired human Treg function and was associated with the induction of IFNγ-secreting Tregs in a xenogeneic graft-versus-host disease model and in adoptive transfer models of experimental colitis. Our results demonstrate a putative role for an environmental factor that promotes autoimmunity by inducing proinflammatory responses in CD4 effector cells and Treg pathways.

Regulatory T cells in the central nervous system

Summary:  Regulatory T cells (Tregs) are critical to the human immune system, providing appropriately scaled immune responses and mediating peripheral tolerance. A central role for forkhead box protein 3 (FoxP3)+ Tregs has been shown in the pathogenesis of mechanistically diverse central nervous system (CNS) diseases from autoimmune diseases such as multiple sclerosis to glioblastomas. Understanding how tumors induce Treg function to escape immune surveillance in marked contrast to autoimmune diseases, where there is loss of Treg function, will provide valuable lessons regarding Treg biology and potential therapeutic targets for CNS diseases.

Th1 not Th17 cells drive spontaneous MS-like disease despite a functional regulatory T cell response

Multiple sclerosis is considered a disease of complex autoimmune etiology, yet there remains a lack of consensus as to specific immune effector mechanisms. Recent analyses of experimental autoimmune encephalomyelitis, the common mouse model of multiple sclerosis, have investigated the relative contribution of Th1 and Th17 CD4 T cell subsets to initial autoimmune central nervous system (CNS) damage. However, inherent in these studies are biases influenced by the adjuvant and toxin needed to break self-tolerance. We investigated spontaneous CNS disease in a clinically relevant, humanized, T cell receptor transgenic mouse model. Mice develop spontaneous, ascending paralysis, allowing unbiased characterization of T cell immunity in an HLA-DR15-restricted T cell repertoire. Analysis of naturally progressing disease shows that IFNγ+ cells dominate disease initiation with IL-17+ cells apparent in affected tissue only once disease is established. Tregs accumulate in the CNS but are ultimately ineffective at halting disease progression. However, ablation of Tregs causes profound acceleration of disease, with uncontrolled infiltration of lymphocytes into the CNS. This synchronous, severe disease allows characterization of the responses that are deregulated in exacerbated disease: the correlation is with increased CNS CD4 and CD8 IFNγ responses. Recovery of the ablated Treg population halts ongoing disease progression and Tregs extracted from the central nervous system at peak disease are functionally competent to regulate myelin specific T cell responses. Thus, in a clinically relevant mouse model of MS, initial disease is IFNγ driven and the enhanced central nervous system responses unleashed through Treg ablation comprise IFNγ cytokine production by CD4 and CD8 cells, but not IL-17 responses.

The immune cell infiltrate populating meningiomas is composed of mature, antigen-experienced T and B cells

BACKGROUND Meningiomas often harbor an immune cell infiltrate that can include substantial numbers of T and B cells. However, their phenotype and characteristics remain undefined. To gain a deeper understanding of the T and B cell repertoire in this tumor, we characterized the immune infiltrate of 28 resected meningiomas representing all grades. METHODS Immunohistochemistry was used to grossly characterize and enumerate infiltrating lymphocytes. A molecular analysis of the immunoglobulin variable region of tumor-infiltrating B cells was used to characterize their antigen experience. Flow cytometry of fresh tissue homogenate and paired peripheral blood lymphocytes was used to identify T cell phenotypes and characterize the T cell repertoire. RESULTS A conspicuous B and T cell infiltrate, primarily clustered in perivascular spaces, was present in the microenvironment of most tumors examined. Characterization of 294 tumor-infiltrating B cells revealed clear evidence of antigen experience, in that the cardinal features of an antigen-driven B cell response were present. Meningiomas harbored populations of antigen-experienced CD4+ and CD8+ memory/effector T cells, regulatory T cells, and T cells expressing the immune checkpoint molecules PD-1 and Tim-3, indicative of exhaustion. All of these phenotypes were considerably enriched relative to their frequency in the circulation. The T cell repertoire in the tumor microenvironment included populations that were not reflected in paired peripheral blood. CONCLUSION The tumor microenvironment of meningiomas often includes postgerminal center B cell populations. These tumors invariably include a selected, antigen-experienced, effector T cell population enriched by those that express markers of an exhausted phenotype.

A role of cellular prion protein in programming T-cell cytokine responses in disease

The cellular prion protein (PrPC) is widely expressed in neural and non‐neural tissues, but its function is unknown. Elucidation of the part played by PrPC in adaptive immunity has been a particular conundrum: increased expression of cell surface PrPChas been documented during T‐cell activation, yet the functional significance of this activation remains unclear, with conflicting data on the effects of Prnp gene knockout on various parameters of T‐cell immunity. We show here that Prnp mRNA is highly inducible within 8‐24 h of T‐cell activation, with surface protein levels rising from 24 h. When measured in parallel with CD69 and CD25, PrPC is a late activation antigen. Consistent with its up‐regulation being a late activation event, PrP deletion did not alter T‐cell‐antigen presenting cell conjugate formation. Most important, activated PrP0/0T cells demonstrated much reduced induction of several T helper (Th) 1, Th2, and Th17 cytokines, whereas others, such as TNF‐α and IL‐9, were unaffected. These changes were investigated in the context of an autoimmune model and a bacterial challenge model. In experimental autoimmune encephalomyelitis, PrP‐knockout mice showed enhanced disease in the face of reduced IL‐17 responses. In a streptococcal sepsis model, this constrained cytokine program was associated with poorer local control of infection, although with reduced bacteremia. The findings indicate that PrPC is a potentially important molecule influencing T‐cell activation and effector function.—Ingram, R.J., Isaacs, J.D., Kaur, G., Lowther, D.E., Reynolds, C.J., Boyton,R. J., Collinge, J., Jackson, G.S., Altmann, D.M. A role of cellular prion protein in programming T‐cell cytokine responses in disease. FASEB J. 23, 1672–1684 (2009)

The nature of innate and adaptive interleukin‐17A responses in sham or bacterial inoculation

Streptococcus pyogenes is the causative agent of numerous diseases ranging from benign infections (pharyngitis and impetigo) to severe infections associated with high mortality (necrotizing fasciitis and bacterial sepsis). As with other bacterial infections, there is considerable interest in characterizing the contribution of interleukin‐17A (IL‐17A) responses to protective immunity. We here show significant il17a up‐regulation by quantitative real‐time PCR in secondary lymphoid organs, correlating with increased protein levels in the serum within a short time of S. pyogenes infection. However, our data offer an important caveat to studies of IL‐17A responsiveness following antigen inoculation, because enhanced levels of IL‐17A were also detected in the serum of sham‐infected mice, indicating that inoculation trauma alone can stimulate the production of this cytokine. This highlights the potency and speed of innate IL‐17A immune responses after inoculation and the importance of proper and appropriate controls in comparative analysis of immune responses observed during microbial infection.

Functional differences between PD-1+ and PD-1- CD4+ effector T cells in healthy donors and patients with glioblastoma multiforme

Immune checkpoint inhibitors targeting programmed cell death protein 1 (PD-1) have been highly successful in the treatment of cancer. While PD-1 expression has been widely investigated, its role in CD4+ effector T cells in the setting of health and cancer remains unclear, particularly in the setting of glioblastoma multiforme (GBM), the most aggressive and common form of brain cancer. We examined the functional and molecular features of PD-1+CD4+CD25—CD127+Foxp3—effector cells in healthy subjects and in patients with GBM. In healthy subjects, we found that PD-1+CD4+ effector cells are dysfunctional: they do not proliferate but can secrete large quantities of IFNγ. Strikingly, blocking antibodies against PD-1 did not rescue proliferation. RNA-sequencing revealed features of exhaustion in PD-1+ CD4 effectors. In the context of GBM, tumors were enriched in PD-1+ CD4+ effectors that were similarly dysfunctional and unable to proliferate. Furthermore, we found enrichment of PD-1+TIM-3+ CD4+ effectors in tumors, suggesting that co-blockade of PD-1 and TIM-3 in GBM may be therapeutically beneficial. RNA-sequencing of blood and tumors from GBM patients revealed distinct differences between CD4+ effectors from both compartments with enrichment in multiple gene sets from tumor infiltrating PD-1—CD4+ effectors cells. Enrichment of these gene sets in tumor suggests a more metabolically active cell state with signaling through other co-receptors. PD-1 expression on CD4 cells identifies a dysfunctional subset refractory to rescue with PD-1 blocking antibodies, suggesting that the influence of immune checkpoint inhibitors may involve recovery of function in the PD-1—CD4+ T cell compartment. Additionally, co-blockade of PD-1 and TIM-3 in GBM may be therapeutically beneficial.