‘Medusa head ataxia’: the expanding spectrum of Purkinje cell antibodies in autoimmune cerebellar ataxia. Part 3: Anti-Yo/CDR2, anti-Nb/AP3B2, PCA-2, anti-Tr/DNER, other antibodies, diagnostic pitfalls, summary and outlook

Serological testing for anti-neural autoantibodies is important in patients presenting with idiopathic cerebellar ataxia, since these autoantibodies may indicate cancer, determine treatment and predict prognosis. While some of them target nuclear antigens present in all or most CNS neurons (e.g. anti-Hu, anti-Ri), others more specifically target antigens present in the cytoplasm or plasma membrane of Purkinje cells (PC). In this series of articles, we provide a detailed review of the clinical and paraclinical features, oncological, therapeutic and prognostic implications, pathogenetic relevance, and differential laboratory diagnosis of the 12 most common PC autoantibodies (often referred to as ‘Medusa head antibodies’ due to their characteristic somatodendritic binding pattern when tested by immunohistochemistry). To assist immunologists and neurologists in diagnosing these disorders, typical high-resolution immunohistochemical images of all 12 reactivities are presented, diagnostic pitfalls discussed and all currently available assays reviewed. Of note, most of these antibodies target antigens involved in the mGluR1/calcium pathway essential for PC function and survival. Many of the antigens also play a role in spinocerebellar ataxia. Part 1 focuses on anti-metabotropic glutamate receptor 1-, anti-Homer protein homolog 3-, anti-Sj/inositol 1,4,5-trisphosphate receptor- and anti-carbonic anhydrase-related protein VIII-associated autoimmune cerebellar ataxia (ACA); part 2 covers anti-protein kinase C gamma-, anti-glutamate receptor delta-2-, anti-Ca/RhoGTPase-activating protein 26- and anti-voltage-gated calcium channel-associated ACA; and part 3 reviews the current knowledge on anti-Tr/delta notch-like epidermal growth factor-related receptor-, anti-Nb/AP3B2-, anti-Yo/cerebellar degeneration-related protein 2- and Purkinje cell antibody 2-associated ACA, discusses differential diagnostic aspects and provides a summary and outlook

Tolerance to the Neuron-Specific Paraneoplastic HuD Antigen

Experiments dating back to the 1940's have led to the hypothesis that the brain is an immunologically privileged site, shielding its antigens from immune recognition. The paraneoplastic Hu syndrome provides a powerful paradigm for addressing this hypothesis; it is believed to develop because small cell lung cancers (SCLC) express the neuron-specific Hu protein. This leads to an Hu-specific tumor immune response that can develop into an autoimmune attack against neurons, presumably when immune privilege in the brain is breached. Interestingly, all SCLC express the onconeural HuD antigen, and clinically useful tumor immune responses can be detected in up to 20% of patients, yet the paraneoplastic neurologic syndrome is extremely rare. We found that HuD-specific CD8+ T cells are normally present in the mouse T cell repertoire, but are not expanded upon immunization, although they can be detected after in vitro expansion. In contrast, HuD-specific T cells could be directly activated in HuD null mice, without the need for in vitro expansion. Taken together, these results demonstrate robust tolerance to the neuronal HuD antigen in vivo, and suggest a re-evaluation of the current concept of immune privilege in the brain

The Onconeural Antigen cdr2 Is a Novel APC/C Target that Acts in Mitosis to Regulate C-Myc Target Genes in Mammalian Tumor Cells

Cdr2 is a tumor antigen expressed in a high percentage of breast and ovarian tumors and is the target of a naturally occurring tumor immune response in patients with paraneoplastic cerebellar degeneration, but little is known of its regulation or function in cancer cells. Here we find that cdr2 is cell cycle regulated in tumor cells with protein levels peaking in mitosis. As cells exit mitosis, cdr2 is ubiquitinated by the anaphase promoting complex/cyclosome (APC/C) and rapidly degraded by the proteasome. Previously we showed that cdr2 binds to the oncogene c-myc, and here we extend this observation to show that cdr2 and c-myc interact to synergistically regulate c-myc-dependent transcription during passage through mitosis. Loss of cdr2 leads to functional consequences for dividing cells, as they show aberrant mitotic spindle formation and impaired proliferation. Conversely, cdr2 overexpression is able to drive cell proliferation in tumors. Together, these data indicate that the onconeural antigen cdr2 acts during mitosis in cycling cells, at least in part through interactions with c-myc, to regulate a cascade of actions that may present new targeting opportunities in gynecologic cancer

Characterization of HuD p321-specific CD8+ T.

<p>(a) 5×10⁶ HuD p321-specific <i>in vitro</i> stimulated CD8+ T cells were adoptively transferred into Rag^−/− mice (n = 2) with 2×10⁶ C57BL/6 DC pulsed with p321. Mice also received PTx and IL-2. Eight days post transfer, mice were injected with CFSE-labeled syngeneic splenocytes pulsed with HuD p321 (CFSE^hi) or βgal p96 (CFSE^lo). A naïve control mouse without transferred CD8+ T cells was injected with CFSE-labeled splenocytes. 6 hours after target injection, splenocytes were analyzed by FACS for <i>in vivo</i> target cell lysis. A representative mouse is shown. Data is representative of two experiments. (b) C57BL/6 mice (n = 2) were used as recipients of adoptively transferred HuD p321-specific CD8+ T cells as in (a). A representative mouse is shown. Data is representative of two experiments. (c) Primary kidney cells from C57BL/6 mice (D^b+/K^b+) or transgenic Bm1 mice (D^b+/K^b−) were irradiated and pulsed with HuD p321 or βgal p96 and used as stimulators in an IFNγ ELIPOST assay (5×10⁴/well) with 3× restimulated HuD p321-specific or βgal p96-specific CD8+ T cells (10⁴/well). The assay was performed in triplicate. Means are plotted and error bars represent standard deviations of the mean. Data is representative of two experiments. (d) C57BL/6 mice were immunized with either AdVHuD or influenza virus or left untreated (2 mice per group). 15 days after immunization, CD8+ T cells were isolated from the spleen and stained directly <i>ex vivo</i> with anti-CD8+ antibody and PE-labeled tetramer. A portion of splenocytes from each mouse was stimulated <i>in vitro</i> with cognate peptide for 7 days. Naïve mice were stimulated with HuD p321. CD8+ T cells from <i>in vitro</i> stimulation cultures were stained with anti-CD8+ antibody and PE-labeled tetramer. Plots are gated on CD8+ T cells. Data is representative of two experiments.</p

Comparison of HuA p321-specific CD8+ T cells and HuD p321-specific CD8+ T cells.

<p>(a) Sequences of HuD p321 and HuA p321 (b) RMA/S cells were incubated with serial dilutions of peptide and stained for D^b MHC I. HuD p321 and HuA p321 were assayed. The A2.1 epitope of influenza (M1) was used as a negative control. The D^b epitope of influenza (NP) was used as a positive control. (c) C57BL/6 mice were immunized with individual peptides (NP, HuA p321, or HuD p321) in TiterMax adjuvant (2 mice per group). 7 days later, draining lymph node CD8+ T cells were plated in an IFNγ ELISPOT assay (2×10⁵/well) with peptide pulsed EL4 cells (5×10⁴/well). The assay was performed in triplicate. Means are plotted and error bars represent standard deviations of the mean. Data is representative of four experiments.</p

C57BL/6 mice are tolerized to HuD.

<p>(a) C57BL/6 mice were immunized with AdVHuD or AdVβgal+PTx (2 mice per group). 13 days later, CD8+ T cells were isolated from the spleen and plated in an IFNγ ELISPOT assay (2×10⁵/well) with EL4 pulsed with 10 uM peptide (5×10⁴/well). The assay was performed in triplicate. Means are plotted and error bars represent standard deviations of the mean. Data is representative of four experiments. (b) C57BL/6 mice were immunized with AdVHuD−/+PTx (2 mice per group). 13 days later, splenocytes were stimulated <i>in vitro</i> with 0.5 uM HuD p321. On day 7, CD8+ T cells were plated in an IFNγ ELISPOT assay (10⁴/well) with DC pulsed with 10 uM peptide (7×10³/well). The assay was performed in triplicate. Means are plotted and error bars represent standard deviations of the mean Data is representative of two experiments. (c) Individual HuD^+/+ or HuD^−/− mice were immunized with AdVHuD+PTx and used in an IFNγ ELISPOT assay as described in (a). The assay was performed in triplicate. Means are plotted and error bars represent standard deviations of the mean. Data is representative of four experiments. (d) Half of the spleens from mice immunized in (c) were stimulated <i>in vitro</i> with HuD p321. After 7 days, CD8+ T cells were isolate from stimulation cultures and plated in an IFNγ ELISPOT (10⁴/well) with peptide pulsed EL4 cells (5×10⁴/well).</p