Regulatory T cell profiles in patients with N-methyl-ᴅ-aspartate receptor-antibody encephalitis

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encephalitis. 2023;3(1):15-23

Publication date (electronic) : 2023 January 3

doi : https://doi.org/10.47936/encephalitis.2022.00052

Mildred A. Iro^,1,2, Christine S. Rollier^3,4, Sarosh R. Irani^5,6, Manish Sadarangani^7,8, Adam Al-Diwani^5,9, Andrew J. Pollard³, Elizabeth A. Clutterbuck³

¹Faculty of Medicine and Institute of Life Sciences, University of Southampton, United Kingdom

²Department of Paediatric Infectious Diseases, Southampton Children’s Hospital, University Hospital Southampton NHS Foundation Trust, Southampton, United Kingdom

³Oxford Vaccine Group, Department of Paediatrics, University of Oxford, and the NIHR Oxford Biomedical Research Centre, Oxford, United Kingdom

⁴School of Biosciences, Faculty of Health and Medical Sciences, University of Surrey, Guildford, United Kingdom

⁵Oxford Autoimmune Neurology Group, Nuffield Department of Clinical Neurosciences, University of Oxford, Oxford, United Kingdom

⁶Department of Neurology, John Radcliffe Hospital, Oxford University Hospitals NHS Foundation, Trust, Oxford, United Kingdom

⁷Vaccine Evaluation Center, BC Children’s Hospital Research Institute, Vancouver, BC, Canada

⁸Department of Pediatrics, University of British Columbia, Vancouver, BC, Canada

⁹University Department of Psychiatry, University of Oxford, Oxford, United Kingdom

Correspondence: Mildred A. Iro Faculty of Medicine and Institute of Life Sciences, University of Southampton, SO17 1BJ, United Kingdom E-mail: m.a.iro@soton.ac.uk

Received 2022 August 2; Revised 2022 September 24; Accepted 2022 September 30.

Abstract

Introduction

Autoimmune encephalitis (AIE) occurs due to a misdirected immune response against self-antigens expressed in the central nervous system (CNS). When the resulting autoantibodies target membrane surface antigens, they are considered potentially pathogenic. This is the case for N-methyl-ᴅ-aspartate receptor antibody encephalitis (NMDAR-Ab-E), a common form of autoimmune encephalitis characterized by psychosis, amnesia, seizures, and dyskinesias, where antibodies target the extracellular domain of the NR1 subunit of the NMDAR. While B cells are likely to be key to an autoantibody-mediated pathology, T cell functions and imbalances between several limbs of an acquired immune system are likely to have both pathogenic and therapeutic roles [1]. For example, in the NMDAR-Ab-E–associated ovarian teratomas, dense infiltrations of both T and B cells are observed [2].

Patients with NMDAR-Ab-E show variable responses to treatment and ~20% of patients suffer disease relapses after first-line immunotherapies [3], indicating the need to identify alternative treatment approaches. Th17 cells play a pathogenic role in the development of autoimmune diseases through production of proinflammatory cytokines that cause tissue damage. Excessive activation of the Th17 pathway has been implicated in the immunopathology of NMDAR-Ab-E [3,4]. Notably, an increase in cerebrospinal fluid (CSF) concentration of the Th17 inflammatory cytokines interleukin (IL) 17 and IL6 was observed in NMDAR-Ab-E patients [3]. Additionally, Zeng et al. [3] found an increased number of Th17 CD4 T cells in the CSF of NMDAR-Ab-E patients. Thus, a common paradigm for intervention with the greatest presumptive benefit in AIE, and indeed NMDAR-Ab-E, should center on early attenuation of the underlying CNS inflammation.

Regulatory T cells (Tregs) are T cells that suppress inflammatory and autoimmune responses promoting immune tolerance, a function proven to be critical for the control of several autoimmune diseases. Treg deficiencies can lead to microglial activation, inflammation, and neuronal injury. Poorly functioning Tregs and/or reduced Treg numbers have been observed in patients with autoimmune diseases including multiple sclerosis and vasculitides [5,6]. Restoring immune homeostasis and tolerance through activation or adoptive transfer of Tregs has become an attractive approach for the treatment of several autoimmune diseases [7,8]. A better understanding of the role of Tregs in NMDAR-Ab-E could shed more light on the potential utility of Treg-directed treatment strategies or indeed provide evidence to support repurposing of already existing treatments to improve outcomes for affected patients.

The objective of this study was to evaluate the proportion of Tregs in peripheral blood of affected patients compared with healthy controls. Given the reciprocal relationship between Th17 cells and Tregs [9] and data supporting activation of the Th17 pathway in NMDAR-Ab-E [3,4], we hypothesized that the frequency of Tregs in peripheral blood would be lower in NMDAR-Ab-E patients than in healthy controls, suggesting a loss of immune tolerance in affected patients.

Methods

Definitions

Tregs were identified by flow cytometry based on the expression of CD25 and FoxP3 on T (CD3+ CD4+) cells. The Treg population was then classified into different subpopulations based on their expression of additional cell surface and intracellular markers, as described elsewhere [10] (Gating strategies in Figure 1A and B) into: (i) naïve Tregs (CD3+ CD4+ CD25+ FoxP3+ CD45RA+), (ii) memory Tregs (CD3+ CD4+ CD25+ FoxP3+ CD45RA–), (iii) effector memory (EM) Tregs (CD3+ CD4+ CD25+ FoxP3+ CD45RA– CCR7–), and (iv) central memory (CM) Tregs (CD3+ CD4+ CD25+ FoxP3+ CD45RA– CCR7+). The expression of the activation marker CTLA on Tregs and the homing marker CCR6 were also evaluated.

Figure 1.

Gating strategy

(A) Gating strategy for fluorescence minus one control for intracellular markers. (B) Gating strategy used for identification of regulatory T cells (Tregs) and the different subpopulations. Subsequent gating is done on the population highlighted either in the circle of rectangle in the preceding figure. CTLA, cytotoxic T-lymphocyte associated protein 4; FMO, fluorescence minus one control; SSC, side scatter; FSC, forward scatter; PECF, 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-(carboxyfluorescein); APC, allophycocyanin; CCR, chemokine receptor. Tregs, CD3+ CD4+ CD25+ FoxP3+; memory Tregs, CD3+ CD4+ CD25+ FoxpP3+ CD45RA–; effector memory (EM) Tregs, CD3+ CD4+ CD25+ FoxP3+ CD45RA– CCR7–; central memory (CM) Tregs, CD3+ CD4+ CD25+ FoxP3+ CD45RA– CCR7+.

Results

Study participants

Eight participants were included in this study: four patients with NMDAR-Ab-E and four age- and sex-matched healthy controls (Table 1). Patient samples were obtained either in the acute (n = 3) or convalescent (n = 1) phase of the disease. Clinical characteristics of the NMDAR-Ab-E patient cohort are provided in Tables 1 and 2. Three of the four NMDAR-Ab-E patients had received at least one immunomodulatory treatment (intravenous methylprednisolone, n=3; oral prednisolone, n=2; and plasma exchange, n=1), prior to sampling (Table 1). All patients were female. The mean age (±standard deviation) of the study population was 23.50 years (±5.447) and 28.00 years (±2.944) for the NMDAR-Ab-E and healthy control groups, respectively.

Table 1

Characteristics of enrolled participants

Table 2

Cerebrospinal fluid (CSF) parameters of enrolled participants

Comparison of peripheral lymphocyte counts between NMDAR-Ab-E patients and healthy controls

Firstly, we compared the frequency of total lymphocytes between NMDAR-Ab-E patients and healthy controls and found no significant differences (p = 0.0571) (Figure 2A and Table 3).

Figure 2.

Comparison of different cell populations between NMDAR-Ab-E patients and healthy controls

(A) Lymphocyte comparison between NMDAR-Ab-E patients and healthy controls. (B) Comparison of proportion of CD3+ CD4+ T cells between NMDAR-Ab-E patients and healthy controls. (C) Comparison of proportion of Tregs between NMDAR-Ab-E patients and healthy controls. (D) Comparison of proportion of naïve Tregs between NMDAR-Ab-E patients and healthy controls. (E) Comparison of proportion of Tregs expressing CCR6 between NMDAR-Ab-E patients and healthy controls. (F) Comparison of proportion of effector memory Tregs between NMDAR-Ab-E patients and healthy controls. (G) Comparison of proportion of effector memory Tregs expressing CCR6 between NMDAR-Ab-E patients and healthy controls. (H) Comparison of proportion of central memory Tregs between NMDAR-Ab-E patients and healthy controls.

NMDAR-Ab-E, N-methyl-ᴅ-aspartate receptor-antibody encephalitis; PBMC, peripheral blood mononuclear cell; CCR, chemokine receptor; Tregs, regulatory T cells.

Table 3

Comparison of proportions of the different populations between patients with NMDAR-Ab-E and healthy controls

Discussion

Our data show differences in the frequency of naïve Tregs, EM Tregs, and CCR6+ Tregs between NMDAR-Ab-E patients and healthy controls, without differences in the proportion of total Tregs.

We observed that patients with NMDAR-Ab-E have a lower circulating pool of naïve Tregs than healthy controls. Naïve Tregs differentiate into activated Tregs following antigenic exposure and are required to control immune responses through continuous replenishment of the activated Treg pool [11]. The lower frequency of naïve Tregs in NMDAR-Ab-E patients could indicate impaired Treg homeostasis due to an influence of Treg thymic development, or an increase in peripheral cell turnover and subsequent conversion to memory Tregs. The latter would seem more plausible due to the higher proportion of memory Tregs observed in NMDAR-Ab-E patients compared with healthy controls (data not shown).

One cross-sectional observational study of 29 patients with autoimmune encephalitis showed a nonsignificantly lower proportion of Tregs in peripheral blood of patients with synaptic antibodies (NMDAR-Ab-E, n = 4) compared with those with paraneoplastic antibodies [12]; no healthy controls were included in the study making it impossible to ascertain to what extent these differences are directly reflective of the underlying diagnosis. Although our data show that NMDAR-Ab-E patients have similar levels of circulating Tregs as healthy controls do, this finding may not necessarily reflect the Treg numbers in the CNS.

The results of this study indicate that NMDAR-Ab-E patients have a higher proportion of circulating EM Tregs than healthy controls. Contrary to their CM counterparts, EM Tregs represent more recently activated memory Tregs with increased suppressive function. Most of the NMDAR-Ab-E patients were in the active state of the disease; therefore, the higher proportion of EM Tregs in the patient cohort would suggest recent antigen reexposure with a significant shift towards an EM phenotype.

Though there was no difference in the overall proportion of Tregs between NMDAR-Ab-E patients and healthy controls, there was a trend towards a lower proportion of circulating CCR6+ Tregs in NMDAR-Ab-E patients. Also, we observed that the frequency of EM Tregs expressing CCR6 was lower in NMDAR-Ab-E patients than in healthy controls. Following antigenic stimulation, activated naïve T cells acquire specific homing receptors which allow them to efficiently migrate into peripheral tissues and sites of inflammation. Treg-mediated suppressive function involves Treg migration to lymphoid organs and to sites of inflammation where they act directly to control potentially destructive immune responses in inflamed tissues and prevent organ-specific autoimmune diseases [13]. CCR6 promotes Treg migration to inflamed sites through its physiological ligand macrophage inflammatory protein-3 alpha/CC chemokine ligand 20 (CCL20). CCR6+ memory Tregs have been shown to exhibit superior suppressive activity [14] and experimental studies indicate that CD4+ Tregs require CCR6 to downregulate the inflammatory process associated with experimental autoimmune encephalomyelitis [15]. In a study of 60 patients with NMDAR-Ab-E, significantly increased levels of CCR6 and CCL20 were observed in the CSF of NMDAR-Ab-E patients compared with patients with noninflammatory neurological disorders [3]. This finding is supported by earlier studies showing accumulation of CCR6+ Tregs and increased levels of CCL20 in the CNS during acute inflammation [16,17]. Consequently, CCR6-CCL20 axis has been suggested as a novel therapeutic target for NMDAR-Ab-E [3]. Our observation of lower frequencies of CCR6+ EM Tregs and a trend to lower frequencies of CCR6+ Tregs in NMDAR-Ab-E patients could indicate a genuine deficiency of CCR6 in these patients and this could have therapeutic implications. A more likely possibility is that this finding could reflect artificially low circulating levels of these Treg populations due to their recruitment to the CNS. Notably, Zeng et al. [3] demonstrated a significant increase in CCR6 expression by Th17 cells in the CSF but not peripheral blood of NMDAR-Ab-E patients and showed that addition of CCL20 significantly promoted the chemotactic ability of Th17 cells. Nonetheless, the exact relevance and implications of our findings are unclear and warrant further investigation.

The main limitations of this study are the potential confounding effects of steroid and plasma exchange therapy, and the small sample size. Due to the logistical challenges of CSF sampling particularly from healthy controls and the low numbers of lymphocytes found in the CSF in encephalitis, we focused on investigating the Treg population in peripheral blood. Findings in peripheral blood may not reflect activity in the CNS but it is likely the autoimmunization in these patients begins in the periphery [1].

Our preliminary data suggest that Tregs may have a role in the pathogenesis of NMDAR-Ab-E. It is not clear whether these findings are part of the disease pathogenesis in NMDAR-Ab-E implying that patients might benefit from modifying the Treg response, or just a response to the disease with no negative pathological consequence. Further studies in a larger cohort of patients using paired blood and CSF samples are necessary to confirm these observations and to elucidate the functional relevance of these findings.

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