T cells in patients with narcolepsy target self-antigens of hypocretin neurons

Welcome to our Monthly Journal Club! Each month I post a paper or two that I have read and find interesting. I use this as a forum for open discussion about the paper in question. Anyone can participate in the journal club, and provide comments/critiques on the paper. This month’s paper is “T cells in patients with narcolepsy target self-antigens of hypocretin neurons” by Frederica Sallusto and colleagues at ETH-Zurich. I will provide a brief overview of the techniques/approaches used to make it more understandable to potential non-expert readers. If I am not familiar with something, I’ll simply say so.

Hypocretin (green) neurons in the mouse lateral hypothalamus co-stained with anti-cFos (red). Latorre et al., 2018 demonstrate that autoreactive CD4+ T-cells in patients with narcolepsy specifically target epitopes present on these neurons, leading to their destruction (Credit: JCB).

Narcolepsy is a relatively rare neurological disease caused by the selective destruction of hypocretin (hcrt; also known as orexin) neurons in the lateral hypothalamus. Destruction of these neurons causes patients to experience excessive daytime sleepiness, cataplexy (spontaneous loss of muscle tone), dream-like hallucinations, and sleep paralysis. There is a strong genetic association between development of narcolepsy and specific genotypes of the antigen presentation complex HLA-DQB1 (HLA-DQB1*06:02), additional evidence of immune dysfunction, and increased incidence of the disease following influenza vaccination. These findings suggest a role for the immune system in the etiology of narcolepsy. Indeed, the hunt for autoreactive T-cells targeting hypocretin or hypocretin receptors has been on for over a decade. The present paper fills this gap by empirically showing that patients with sporadic narcolepsy have CD4+ T-cells that target hypocretin, essentially proving an autoimmune etiology for this disease.

This has been a hard problem to crack as five epitopes from the hcrt precursors were predicted to bind HLA-DQB1*06:02, however a recent study failed to find evidence of auto-reactive CD4+ T-cell targeting of hcrt precursors (Kornum et al., 2017). Additionally, a study claiming to find CD4+ T-cell mediated autoimmunity targeting hcrt and cross-reactivity to an epitope present on the 2009 H1N1 influenza virus was retracted when the authors failed to replicate their own findings (de la Herran-Arita et al., 2014). Others have demonstrated (in mouse models) that H1N1 infection can lead to narcolepsy-like symptoms in mice (Tesoriero et al, 2015), and reprogramming of T-cells to target hypocretin neurons leads to their destruction and the development of a narcolepsy phenotype (Bernard-Valnet et al., 2016).

Specific CD4+ autoreactive T-cell clones targeting hypocretin peptides in patients with narcolepsy (Latorre et al., 2018).

Sallusto and colleagues began by obtaining peripheral blood samples from 16 patients with narcolepsy (and who had the HLA-DQB1*06:02 allele) and complementary healthy controls (also containing the putative autoimmune allele). As autoreactive T-cells are extremely rare to begin with, they began by sorting memory T cells (CD45RA− CD4+) to high purity (>98%), through labeling them with carboxyfluorescein succinimidyl ester (CFSE). These cells were then ‘pulsed’ with antigen presenting cells (APCs; monocytes) containing different amino acid sequences of the protein precursor (pre-prohypocretin) to Hcrt-1 and Hcrt-2. This method only showed 1 patient with an ‘activated’ response of the T cells to stimulation with APCs containing peptides of pre-prohypocretin. Dissatisfied with this result, the authors tried a more sensitive approach through generated ‘T cell libraries’.

Hcrt-specific autoreactive T cells detected using the T cell library method. Each dot represents a single T cell, with proliferation measured in response to peptide stimulation reported in counts per minute (c.p.m) after incubation with [3H]-thymidine to label proliferating cells. ‘Positive’ T cell responses were considered > 2,000 c.p.m as the background (unstimulated) proliferation rate was ~1,500 c.p.m. Note the strong proliferative response of T cells to hcrt peptide fragments in narcoleptics (P#) versus controls (C#). (NT1 = narcolepsy type 1, NT2 = narcolepsy type 2) (Latorre et al., 2018).

When autoreactive T cells from narcoleptics were pulsed with B cells containing a ‘hypocretin peptide pool’, nearly all (all but one) patient samples showed a strong response to hcrt peptides, indicating that they indeed recognized and responded to this amino acid sequence. In contrast, there were only 3 (out of 12) proliferating responses in control patient samples. Regardless, the magnitude of the response was much higher in narcoleptics than in controls. This approach let them determine that the frequency of the hcrt-reactive T cells was very small (~21.4 cells per 10,000,000 CD4+ T cells). Interestingly, these autoreactive T cells were also found in patients lacking the HLA-DQB1*06:02 allele, and those without hcrt deficiency.

Epitope mapping of hcrt and TRIB2-specific autoreactive T cells. Each patient sample contains T-cell populations that react to different regions along the hypocretin and TRIB2 amino acid sequences. This is driven by antigen presentation through HLA-DR/DQ/DP, as blockade of these interactions prevent T cell expansion upon stimulation (Latorre et al., 2018).

To investigate further, the researchers examined whether these patients also had autoreactive T cells targeting another protein highly expressed in hypocretin-neurons, called tribbles homologue 2 (TRIB2). Indeed, patient T cells showed a highly proliferative response to stimulation with pieces of this protein as well. The authors further confirmed ‘killer’ CD8+ T cell responses to hypocretin peptides from patient samples, but not controls. They further characterized these cells by examining what proteins they secrete and what mRNAs they transcribe in response to hcrt stimulation.

By analyzing the T-cell receptor β -chain variable region (TRBV) in each T cell clone, the authors demonstrated that these autoreactive T cells are not homogenous, and target multiple epitopes along hcrt-1 and hcrt-2. Finally, and importantly the authors failed to find evidence for ‘molecular mimicry’ between hcrt and influenza antigens, as T-cells from patients with narcolepsy failed to proliferate in response to an influenza vaccine containing A/California/7/2009 H1N1 strains or to one containing CA09 H1 haemagglutinin. Therefore, the association between vaccination and the development of narcolepsy still remains a mystery.

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Modulation of anti-tumor immunity by the brain's reward system

Welcome to our Monthly Journal Club! Each month I post a paper or two that I have read and find interesting. I use this as a forum for open discussion about the paper in question. Anyone can participate in the journal club, and provide comments/critiques on the paper. This month’s paper is “Modulation of anti-tumor immunity by the brain’s reward system” by Asya Rolls and colleagues at the Technion - Israel Institute of Technology. I will provide a brief overview of the techniques/approaches used to make it more understandable to potential non-expert readers. If I am not familiar with something, I’ll simply say so.

Figure 1: Using DREADDs to selectively manipulate VTA-Dopamine neurons in the context of cancer.

Discussion

In their paper, Rolls and colleagues used viral vectors encoding Cre-dependent Designer Receptors Exclusively Activated by Designer Drugs (DREADDs) to investigate how the brain alters peripheral cancer growth. By injecting these viral constructs into the ventral tegmental area of tyrosine hydroxylase-Cre mice, specific DREADD expression within only VTA-dopamine neurons (TH+) was accomplished. Gq-coupled DREADDs allow for activation of these neurons by systemic injections of the inert* molecule clozapine-N-oxide (CNO).

This is a good approach for several reasons. (1) it allows for cell-type specific control, (2) the kinetics of CNO are well known, allowing for a good amount of temporal precision, (3) it allows for non-invasive (IP injection) control of the brain, without damaging or destroying the cells. Despite this, there are caveats, such as receptor desensitization from repeated administrations of CNO, and the daily stress of IP injections. These findings will have to be replicated using disparate techniques, like optogenetics, or a more ‘natural’ way to activate these cells, via a ‘CNO drinking’ protocol, or even social/sexual interaction which are known to activate VTA-DA neurons.

After achieving cell-type specific DREADD expression, the authors gave mice subcutaneous tumors (LLC or B16 cancer cells), and then gave them daily injections of CNO. Mice that were ‘VTA-activated’ had smaller tumors than control mice that did not express the DREADD in the VTA (as seen in Figure 1). To examine how this signal from the brain might reach the tumor, the authors ablated the sympathetic nervous system using 6-hydroxydopamine (6-OHDA), a neurotoxin which destroys adrenergic nerve terminals (distinguishing feature of the sympathetic nervous system). Mice that were SNS-ablated (or received a beta-adrenergic receptor antagonist) failed to show an effect of VTA-DA activation on tumor growth. They further showed that VTA activation altered norepinephrine concentrations specifically in the bone marrow, an important immune compartment. This strongly supports the hypothesis that VTA-DA neurons alter tumor growth via SNS innervation of the bone marrow.

Narrowing in on the bone marrow, they showed that VTA activation reduces the number of myeloid derived suppressor cells (MDSCs), which can promote tumor growth via immune suppression and the promotion of angiogenesis. MDSCs contained beta-2 adrenergic receptors, which made them sensitive to VTA-DA activation (through the SNS). Finally, adoptively transferring MDSCs from VTA-activated mice to control mice recapitulated the anti-tumor effect of VTA-activation. This suggests that modulation of the immune system via a discrete population of neurons within the brain acts (at least in part) to suppress tumor growth.

Figure 4: ‘VTA-activated’ myeloid derived suppressor cells (MDSCs) are necessary and sufficient to suppress tumor growth. Adoptive transfer of ‘activated’ MDSCs suppressed tumor growth in mice that had not been ‘VTA-activated’.

These findings are in line with early research showing rats with a hyperreactive dopaminergic system have reduced tumor growth, metastasis, and angiogenesis compared to control rats (Teunis et al., 2002). These results need to be confirmed through alternative methodology and cancer models, but this paper represents an exciting new target for peripheral cancer suppression (through modulation of the brain). Indeed, the authors acknowledged this possibility in their earlier paper showing that VTA-DA activation alters both adaptive and innate immunity, suggesting that this may (at least in part) be responsible for the ‘placebo effect’ (Ben-Shaanan et al., 2016).

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*Note: recent research has demonstrated that CNO’s action is likely through it’s metabolism to the bioactive molecule clozapine (Gomez et al., 2017).

Banner Image: VTA-dopamine neurons expressing a sgRNA against BMAL1 (Credit: JCB).