Eight adult Soay rams (four control and four cranially sympathectomized by removal of the superior cervical ganglia) were exposed to alternating 16-week periods of short days of 8 h light: 16 h darkness (8L : 16D) and long days (16L : 8D) for more than 3 years, and the changes in the diameter of the testes were recorded. Once during short days and once during long days blood samples were collected hourly for 28 h, and the concentrations of melatonin, prolactin, FSH, LH, testosterone, tri-iodothyronine (T3), thyroxine (T4) and cortisol were measured by radioimmunoassay. In the control rams the testes were reduced in size during long days compared to short days, and the blood concentrations (24-h mean values) of FSH, LH and testosterone were decreased. The levels of prolactin were much increased, while there was no significant change in the mean levels of melatonin, T3, T4 and cortisol. During both photoperiods there was considerable hour-to-hour variation in all eight hormones indicative of episodic secretion, as well as a consistent variation related to the time of day which was most pronounced for melatonin and T3. There was a clear difference in the daily profile of plasma melatonin levels between short and long days. In the superior cervical ganglionectomized rams there were no significant changes in the size of the testes or in the hormone titres between short and long days. Compared to the controls the plasma levels of LH, FSH, testosterone and prolactin were in the intermediate range. Some consistent diurnal variation was evident in the levels of all the hormones measured, with a pattern similar to the controls for a few of the hormones (e.g. T3) but quite different for others (e.g. melatonin).

Six rams of an ancient breed of domesticated sheep (SOAY) were subjected to an artificial light régime of alternating periods of long days (16 h light: 8 h darkness) and short days (8 h light: 16 h darkness) which induced seasonal development and regression of the testes during a period of 36 weeks. Over 2000 blood samples were taken, and the changes in plasma levels of FSH, LH and testosterone were related to the cycle of testicular activity. During long days plasma levels of gonadotrophins became very low and the testes regressed to about 20% of their maximum size; there was a corresponding reduction in plasma testosterone levels. When the rams were returned to short days reproductive development was again stimulated after 2-3 weeks with a progressive increase in plasma FSH and LH levels and consequent hypertrophy of the testes. It took about 16 weeks of short days for testicular activity to become maximal. Blood samples collected at hourly intervals for 24 h on ten occasions during the study revealed transitory peaks in plasma FSH and LH levels indicative of episodic release. Changes in gonadotrophin secretion were modulated primarily by alterations in the frequency of episodic releas; less than 1 spike per 24 h during long days increased to a maximum of 10 spikes/24 h under short daylengths. The peaks of FSH release were of smaller amplitude than those of LH, although during periods of frequent episodic release basal levels of fsh were increased to a greater extent than those of LH. A circadian rhythm was observed in the plasma levels of FSH, LH and testosterone, which was related to increased gonadotrophin release during the dark phase of the 24 h cycle; changes in blood haematocrit were also observed. The circadian changes appeared to be correlated with the activity cycle of the animals which in turn was dictated by daylight. A possible interrelationship between the circadian cycle and the seasonal cycle is discussed.

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Safe use of immune checkpoint blockade in patients with cancer and autoimmune disorders requires a better understanding of the pathophysiology of immunologic activation. We describe the immune correlates of reactivation of granulomatosis with polyangiitis (GPA)—an antineutrophil cytoplasmic antibody (ANCA)‐associated vasculitis—in a patient with metastatic urothelial carcinoma treated with pembrolizumab. After PD‐1 blockade, an inflammatory pulmonary nodule demonstrated a granulomatous, CD4+ T‐cell infiltrate, correlating with increased CD4+ and CD8+ naïve memory cells in the peripheral blood without changes in other immune checkpoint receptors. Placed within the context of the existing literature on GPA and disease control, our findings suggest a key role for PD‐1 in GPA self‐tolerance and that selective strategies for immunotherapy may be needed in patients with certain autoimmune disorders. We further summarize the current literature regarding reactivation of autoimmune disorders in patients undergoing immune checkpoint blockade, as well as potential immunosuppressive strategies to minimize the risks of further vasculitic reactivation upon rechallenge with anti‐PD‐1 blockade. Key Points Nonspecific imaging findings in patients with cancer and rheumatological disorders may require biopsy to distinguish underlying pathology. Patients with rheumatologic disorders have increased risk of reactivation with PD‐(L)1 immune checkpoint blockade, requiring assessment of disease status before starting treatment. Further study is needed to evaluate the efficacy of treatment regimens in preventing and controlling disease reactivation.

As checkpoint inhibitor immunotherapies gain traction among cancer researchers and clinicians, the need grows for assays that can definitively phenotype patient immune cells. Herein, we present an 8-color flow cytometry panel for lineage and immune checkpoint markers and validate it using healthy human donor peripheral blood mononuclear cells (PBMCs). Flow cytometry data was generated on a BD LSR Fortessa and supported by Luminex multiplex soluble immunoassay. Our data showed significant variation between donors at both baseline and different stages of activation, as well as a trend in increasing expression of checkpoint markers on stimulated CD4 + and CD8 + T-cells with time. Soluble immune checkpoint quantification assays revealed that LAG-3, TIM-3, CTLA-4, and PD-1 soluble isoforms are upregulated after stimulation. This 8-color flow cytometry panel, supported here by soluble immunoassay, can be used to identify and evaluate immune checkpoints on T-lymphocytes in cryopreserved human PBMC samples. This panel is ideal for characterizing checkpoint expression in clinical samples for which cryopreservation is necessary.