Sara A. J. Thompson

Both CD4+CD25+ and CD4+CD25− Regulatory Cells Mediate Dominant Transplantation Tolerance

CD4+CD25+ T cells have been proposed as the principal regulators of both self-tolerance and transplantation tolerance. Although CD4+CD25+ T cells do have a suppressive role in transplantation tolerance, so do CD4+CD25− T cells, although 10-fold less potent. Abs to CTLA-4, CD25, IL-10, and IL-4 were unable to abrogate suppression mediated by tolerant spleen cells so excluding any of these molecules as critical agents of suppression. CD4+CD25+ T cells from naive mice can also prevent rejection despite the lack of any previous experience of donor alloantigens. However, this requires many more naive than tolerized cells to provide the same degree of suppression. This suggests that a capacity to regulate transplant rejection pre-exists in naive mice, and may be amplified in “tolerized” mice. Serial analysis of gene expression confirmed that cells sorted into CD4+CD25+ and CD4+CD25− populations were distinct in that they responded to TCR ligation with very different programs of gene expression. Further characterization of the differentially expressed genes may lead to the development of diagnostic tests to monitor the tolerant state.

IL-10 Gene Expression Is Controlled by the Transcription Factors Sp1 and Sp3

IL-10 is an 18-kDa cytokine with a key role in homeostatic control of inflammatory and immune responses. We have investigated how transcription of the IL-10 gene is regulated, so as to be able to understand the circumstances of IL-10 expression in both health and disease. In the mouse, IL-10 gene expression is regulated by a TATA-type promoter with a critical cis-acting element containing GGA repeats located at −89 to −77. Its complementary sequence is similar to the cis-acting elements (TCC repeats) in the promoters of genes encoding epidermal growth factor receptor and CD58. All these elements comprise a common CCTCCT sequence with less conserved C + T-rich sequences. Eliminating this CCTCCT sequence results in a marked reduction in promoter activity, suggesting a necessary role in IL-10 gene expression. Despite its dissimilarity to the G + C-rich Sp1 consensus sequence (GC box), Sp1 and Sp3 transcription factors could be shown to bind to this motif. The requirement for Sp1 and Sp3 in transcription of IL-10 was confirmed using Drosophila SL2 cells, which lack endogenous Sp factors. These results suggest that the transcription of IL-10 is positively regulated by both Sp1 and Sp3.

Lymphocyte homeostasis following therapeutic lymphocyte depletion in multiple sclerosis

Following lymphocyte depletion, homeostatic mechanisms drive the reconstitution of lymphocytes. We prospectively studied this process in 16 patients for 1 year after a single pulse of treatment with Campath‐1H, a humanised anti‐CD52 monoclonal antibody. We observed two phases of lymphocyte reconstitution. In the first 6 months after treatment the precursor frequency and proliferation index of the patients’ autologous mixed lymphocyte reaction increased; the depleted T cell pool was dominated by memory T cells, especially CD4+CD25high T cells, a putative regulatory phenotype; and there was a non‐significant rise in peripheral mononuclear cell FoxP3 mRNA expression and fall in constitutive cytokine mRNA expression. In the later phase, from 6‐to‐12 months after Campath‐1H, these changes reversed and there was a rise in ROG mRNA expression. However, total CD4+ numbers remained below 50% of pre‐treatment levels at 12 months, perhaps reflecting a failure in homeostasis. This was not due to an impaired IL‐7 response, as in rheumatoid arthritis, nor to a lack of IL‐7 receptors, which are found on fewer human CD4+CD25high than naive cells. We speculate that CCL21 and IL‐15 responses to lymphopaenia may be suboptimal in multiple sclerosis.

IL-21 drives secondary autoimmunity in patients with multiple sclerosis, following therapeutic lymphocyte depletion with alemtuzumab (Campath-1H)

Phase II clinical trials revealed that the lymphocyte-depleting humanized monoclonal antibody alemtuzumab (Campath-1H) is highly effective in the treatment of early relapsing-remitting multiple sclerosis. However, 30% of patients develop autoimmunity months to years after pulsed exposure to alemtuzumab, usually targeting the thyroid gland and, more rarely, blood components. In this study, we show that autoimmunity arose in those patients with greater T cell apoptosis and cell cycling in response to alemtuzumab-induced lymphocyte depletion, a phenomenon that is driven by higher levels of IL-21. Before treatment, patients who went on to develop secondary autoimmunity had more than 2-fold greater levels of serum IL-21 than the nonautoimmune group. We suggest that serum IL-21 may, therefore, serve as a biomarker for the risk of developing autoimmunity months to years after alemtuzumab treatment. This has implications for counseling those patients with multiple sclerosis who are considering lymphocyte-depleting therapy with alemtuzumab. Finally, we demonstrate through genotyping that IL-21 expression is genetically predetermined. We propose that, by driving cycles of T cell expansion and apoptosis to excess, IL-21 increases the stochastic opportunities for T cells to encounter self antigen and, hence, for autoimmunity.

Regulatory T Cells Overexpress a Subset of Th2 Gene Transcripts

There is now compelling evidence for subpopulations of CD4+ T cells whose role is to prevent immune pathology in both autoimmunity and transplantation. We have cloned CD4+ T cells against a male transplantation Ag that, unlike Th1 or Th2 clones, suppresses the rejection of male skin grafts and are therefore considered examples of regulatory T cells. We have identified, using serial analysis of gene expression, transcripts that are overexpressed in regulatory T cells compared with Th1 and Th2 clones. Some of these transcripts are increased in tolerated rather than rejecting skin grafts and in addition are expressed by the natural regulatory CD4+CD25+ subpopulation of naive mice. These genes include prepro-enkephalin, GM2 ganglioside activator protein, glucocorticoid-induced TNFR superfamily member 18, and integrin αEβ7. They seem to represent a subset of transcripts shared with Th2 cells, suggesting that transplantation tolerance and normal immunoregulation may represent a unique form of Th2-like differentiation.

Posttranscriptional regulation of IL-10 gene expression through sequences in the 3′-untranslated region.

IL-10 is an 18-kDa immunoregulatory cytokine the transcription of which is controlled by the ubiquitously expressed transcription factors Sp1 and Sp3. Although many cell types express IL-10 mRNA, not all make detectable amounts of protein, and levels of protein expression vary enormously. We show here that much of this variation can be accounted for by posttranscriptional mechanisms. Multiple copies of potential mRNA destabilizing motifs AUUUA and related sequences can be found to the 3′-untranslated region (UTR) of IL-10 mRNA distributed through three potential regulatory regions. Evidence of RNA-destabilizing activities in all three regions was deduced from luciferase reporter assays. The half-life of RNA containing the 3′-UTR of IL-10 mRNA was quite short in both nonstimulated (t1/2 = 1 h), and PMA-stimulated EL-4 cell (t1/2 = 3 h). In contrast, the half-life of RNA lacking the 3′-UTR was much longer (t1/2 = >12 h) whether cells were stimulated or not. This suggests that many cells are poised to secrete IL-10 and will do so if they receive appropriate posttranscriptional signals.

Regulatory T cells and dendritic cells in transplantation tolerance: molecular markers and mechanisms.

Summary:  Transplantation tolerance can be induced in adult rodents using monoclonal antibodies against coreceptor or costimulation molecules on the surface of T cells. There are currently two well‐characterized populations of T cells, demonstrating regulatory capacity: the ‘natural’ CD4+CD25+ T cells and the interleukin (IL)‐10‐producing Tr1 cells. Although both types of regulatory T cells can induce transplantation tolerance under appropriate conditions, it is not clear whether either one plays any role in drug‐induced dominant tolerance, primarily due to a lack of clear‐cut molecular or functional markers. Similarly, although dendritic cells (DCs) can be pharmacologically manipulated to promote tolerance, the phenotype of such populations remains poorly defined. We have used serial analysis of gene expression (SAGE) with 29 different T‐cell and antigen‐presenting cell libraries to identify gene‐expression signatures associated with immune regulation. We found that independently derived, regulatory Tr1‐like clones were highly concordant in their patterns of gene expression but were quite distinct from CD4+CD25+ regulatory T cells from the spleen. DCs that were treated with the tolerance‐enhancing agents IL‐10 or vitamin D3 expressed a gene signature reflecting a functional specification in common with the most immature DCs derived from embryonic stem cells.

Human autoimmunity after lymphocyte depletion is caused by homeostatic T-cell proliferation

Significance This paper identifies the mechanism by which patients with multiple sclerosis develop secondary autoimmunity after treatment with the lymphocyte-depleting humanized monoclonal antibody alemtuzumab (Campath-1H). In identifying this mechanism, it shows that T-cell homeostatic proliferation can lead to autoimmunity in humans. Alemtuzumab is one of the most effective treatments of multiple sclerosis tested to date; it is currently licensed in the European Union and under consideration by the Food and Drug Administration. Understanding what drives its most significant side effect is of clear clinical importance. The association between lymphopenia and autoimmunity is recognized, but the underlying mechanisms are poorly understood and have not been studied systematically in humans. People with multiple sclerosis treated with the lymphocyte-depleting monoclonal antibody alemtuzumab offer a unique opportunity to study this phenomenon; one in three people develops clinical autoimmunity, and one in three people develops asymptomatic autoantibodies after treatment. Here, we show that T-cell recovery after alemtuzumab is driven by homeostatic proliferation, leading to the generation of chronically activated (CD28−CD57+), highly proliferative (Ki67+), oligoclonal, memory-like CD4 and CD8 T cells (CCR7−CD45RA− or CCR7−CD45RA+) capable of producing proinflammatory cytokines. Individuals who develop autoimmunity after treatment are no more lymphopenic than their nonautoimmune counterparts, but they show reduced thymopoiesis and generate a more restricted T-cell repertoire. Taken together, these findings demonstrate that homeostatic proliferation drives lymphopenia-associated autoimmunity in humans.

Dehydroepiandrosterone replacement in patients with Addison's disease has a bimodal effect on regulatory (CD4+CD25hi and CD4+FoxP3+) T cells

Oral replacement of the near‐total deficiency of dehydroepiandrosterone (DHEA) in patients with Addisons disease (adrenal insufficiency) enhances mood and well‐being and reduces fatigue. We studied the immunological effects of 12 wk of oral DHEA treatment in ten patients with Addisons disease receiving their normal mineralo‐ and glucocorticoid hormone replacement. We found that baseline circulating regulatory T cells were reduced in Addisons disease patients compared to controls, a hitherto unrecognised defect in this disorder. Oral DHEA treatment had a bimodal effect on naturally occurring regulatory (CD4+CD25hiFoxP3+) T cells and lymphocyte FoxP3 expression. Oral DHEA replacement restored normal levels of regulatory T cells and led to increased FoxP3 expression. These effects were probably responsible for a suppression of constitutive cytokine expression following DHEA withdrawal. In contrast, oral DHEA treatment led to reduced FoxP3 expression induced by TCR engagement and so augmented the cytokine response, but without a bias towards the Th1 or Th2 phenotype. NK and NKT cell numbers fell during DHEA treatment, and homeostatic lymphocyte proliferation was increased. We conclude that DHEA replacement in Addisons disease has significant immunomodulatory properties and propose that it has a greater impact on the human immune system than would be expected from its classification as a dietary supplement.

A Novel Strategy To Reduce the Immunogenicity of Biological Therapies

Biological therapies, even humanized mAbs, may induce antiglobulin responses that impair efficacy. We tested a novel strategy to induce tolerance to a therapeutic mAb. Twenty patients with relapsing–remitting multiple sclerosis received an initial cycle of alemtuzumab (Campath-1H), up to 120 mg over 5 d, preceded by 500 mg SM3. This Ab differs from alemtuzumab by a single point mutation and is designed not to bind to cells. Twelve months later, they received a second cycle of alemtuzumab, up to 72 mg over 3 d. One month after that, 4 of 19 (21%) patients had detectable serum anti-alemtuzumab Abs compared with 145 of 197 (74%) patients who received two cycles of alemtuzumab without SM3 in the phase 2 CAMMS223 trial (p < 0.001). The efficacy and safety profile of alemtuzumab was unaffected by SM3 pretreatment. Long-lasting “high-zone” tolerance to a biological therapy may be induced by pretreatment with a high i.v. dose of a drug variant, altered to reduce target-binding.