Circulating B cells in type 1 diabetics exhibit fewer maturation-associated phenotypes.

Although autoantibodies have been used for decades as diagnostic and prognostic markers in type 1 diabetes (T1D), further analysis of developmental abnormalities in B cells could reveal tolerance checkpoint defects that could improve individualized therapy. To evaluate B cell developmental progression in T1D, immunophenotyping was used to classify circulating B cells into transitional, mature naïve, mature activated, and resting memory subsets. Then each subset was analyzed for the expression of additional maturation-associated markers. While the frequencies of B cell subsets did not differ significantly between patients and controls, some T1D subjects exhibited reduced proportions of B cells that expressed transmembrane activator and CAML interactor (TACI) and Fas receptor (FasR). Furthermore, some T1D subjects had B cell subsets with lower frequencies of class switching. These results suggest circulating B cells exhibit variable maturation phenotypes in T1D. These phenotypic variations may correlate with differences in B cell selection in individual T1D patients.

Lenalidomide therapy in treatment-refractory cutaneous lupus erythematosus: histologic and circulating leukocyte profile and potential risk of a systemic lupus flare.

BACKGROUND: Lenalidomide is a thalidomide analogue that may serve as an adjunctive therapy for treatment-refractory cutaneous lupus erythematosus (CLE). OBJECTIVES: We evaluate the use of lenalidomide in CLE and describe the skin and circulating leukocyte profile of treatment-refractory patients before and after treatment. METHODS: Five subjects were treated with lenalidomide in an unblinded open-label study. Immunohistochemistry of skin was performed for T-cell markers, glycosaminoglycans, and CXCL10, an interferon-inducible chemokine, before and after treatment. Immunophenotyping and measurement of interferon-inducible genes from peripheral blood mononuclear cells was also performed before and after treatment. RESULTS: Four subjects demonstrated clinical improvement of their skin, however one of these responders subsequently developed symptoms of systemic lupus erythematosus. Small changes in rare circulating leukocyte subsets, plasmacytoid dendritic cells, and regulatory T cells were observed with treatment and may correlate with clinical response. Treatment was associated with increased circulating HLA-DR expression and decreased markers of interferon-mediated pathways, regardless of clinical response. LIMITATIONS: Our results are limited by small sample size and the measurement of rare populations of circulating cell subsets. CONCLUSIONS: Lenalidomide may have usefulness as therapy for severe, treatment-refractory CLE. However, our preliminary data suggest that lenalidomide may activate T cells and trigger systemic disease in some patients with CLE. We also saw a different histologic and circulating leukocyte phenotype in the nonresponding subject. Further characterization of the skin and circulating leukocyte profile of treatment-refractory patients will improve our understanding of CLE.

Editing and escape from editing in anti-DNA B cells.

Tolerance to dsDNA is achieved through editing of Ig receptors that react with dsDNA. Nevertheless, some B cells with anti-dsDNA receptors escape editing and migrate to the spleen. Certain anti-dsDNA B cells that are recovered as hybridomas from the spleens of anti-dsDNA H chain transgenic mice also bind an additional, Golgi-associated antigen. B cells that bind this antigen accumulate intracellular IgM. The intracellular accumulation of IgM is incomplete, because IgM clusters are observed at the cell surface. In the spleen, B cells that express the heavy and light chains encoding this IgM are surface IgM-bright and acquire the CD21-high/CD23-low phenotype of marginal zone B cells. Our data imply that expression of an Ig that binds dsDNA and an additional antigen expressed in the secretory compartment renders B cells resistant to central tolerance. In the periphery, these B cells may be sequestered in the splenic marginal zone.

SARS-CoV-2 seropositivity and seroconversion in patients undergoing active cancer-directed therapy.

Multiple studies have demonstrated the negative impact of cancer care delays during the COVID-19 pandemic, and transmission mitigation techniques are imperative for continued cancer care delivery. To gauge the effectiveness of these measures at the University of Pennsylvania, we conducted a longitudinal study of SARS-CoV-2 antibody seropositivity and seroconversion in patients presenting to infusion centers for cancer-directed therapy between 5/21/2020 and 10/8/2020. Participants completed questionnaires and had up to five serial blood collections. Of 124 enrolled patients, only two (1.6%) had detectable SARS-CoV-2 antibodies on initial blood draw, and no initially seronegative patients developed newly detectable antibodies on subsequent blood draw(s), corresponding to a seroconversion rate of 0% (95%CI 0.0-4.1%) over 14.8 person-years of follow up, with a median of 13 healthcare visits per patient. These results suggest that cancer patients receiving in-person care at a facility with aggressive mitigation efforts have an extremely low likelihood of COVID-19 infection.

SARS-CoV-2 Seropositivity and Seroconversion in Patients Undergoing Active Cancer-Directed Therapy.

PURPOSE: Multiple studies have demonstrated the negative impact of cancer care delays during the COVID-19 pandemic, and transmission mitigation techniques are imperative for continued cancer care delivery. We aimed to gauge the effectiveness of these measures at the University of Pennsylvania. METHODS: We conducted a longitudinal study of SARS-CoV-2 antibody seropositivity and seroconversion in patients presenting to infusion centers for cancer-directed therapy between May 21, 2020, and October 8, 2020. Participants completed questionnaires and had up to five serial blood collections. RESULTS: Of 124 enrolled patients, only two (1.6%) had detectable SARS-CoV-2 antibodies on initial blood draw, and no initially seronegative patients developed newly detectable antibodies on subsequent blood draw(s), corresponding to a seroconversion rate of 0% (95% CI, 0.0 TO 4.1%) over 14.8 person-years of follow up, with a median of 13 health care visits per patient. CONCLUSION: These results suggest that patients with cancer receiving in-person care at a facility with aggressive mitigation efforts have an extremely low likelihood of COVID-19 infection.

Genomic landscape of metastatic breast cancer identifies preferentially dysregulated pathways and targets.

Nearly all breast cancer deaths result from metastatic disease. Despite this, the genomic events that drive metastatic recurrence are poorly understood. We performed whole-exome and shallow whole-genome sequencing to identify genes and pathways preferentially mutated or copy-number altered in metastases compared with the paired primary tumors from which they arose. Seven genes were preferentially mutated in metastases - MYLK, PEAK1, SLC2A4RG, EVC2, XIRP2, PALB2, and ESR1 - 5 of which are not significantly mutated in any type of human primary cancer. Four regions were preferentially copy-number altered: loss of STK11 and CDKN2A/B, as well as gain of PTK6 and the membrane-bound progesterone receptor, PAQR8. PAQR8 gain was mutually exclusive with mutations in the nuclear estrogen and progesterone receptors, suggesting a role in treatment resistance. Several pathways were preferentially mutated or altered in metastases, including mTOR, CDK/RB, cAMP/PKA, WNT, HKMT, and focal adhesion. Immunohistochemical analyses revealed that metastases preferentially inactivate pRB, upregulate the mTORC1 and WNT signaling pathways, and exhibit nuclear localization of activated PKA. Our findings identify multiple therapeutic targets in metastatic recurrence that are not significantly mutated in primary cancers, implicate membrane progesterone signaling and nuclear PKA in metastatic recurrence, and provide genomic bases for the efficacy of mTORC1, CDK4/6, and PARP inhibitors in metastatic breast cancer.

Circulating lymphocyte subsets in normal adults are variable and can be clustered into subgroups.

BACKGROUND: Flow cytometry is used to monitor lymphocyte subsets in both the clinical and research settings. An understanding of the degree of inter- and intrasubject variability of these populations is critical for data interpretation. METHODS: Peripheral blood lymphocytes of 18 healthy adults were analyzed on two separate occasions using a multicolor flow cytometric panel with B, T, and NK cell markers. Variability was calculated using the coefficient of variation and compared between and within individuals using agglomerative clustering. RESULTS: Each subject appears to have B and T cell subset profiles that are stable over the two time points, but differ from the profiles of other subjects. Thus, the range of measurements for a particular B or T cell subset is larger between subjects and narrower for an individual. In addition, the level of variability correlates inversely with the size of the lymphocyte subset. When lymphocyte profiles are analyzed by agglomerative clustering, replicate samples from the same individual tend to cluster. When single samples from different individuals are analyzed, individuals appear to cluster into different subgroups. CONCLUSIONS: Variability of lymphocyte subsets is usually greater between individuals than within a single individual and each person appears to have a characteristic profile of lymphocyte subsets. These results underscore the importance of obtaining a baseline value for each subject when investigating the impact of a treatment on lymphocyte subsets over time. These results also highlight the potential utility of cluster analysis as a tool for immune subset profiling and biomarker discovery. © 2011 International Clinical Cytometry Society.

RS rearrangement frequency as a marker of receptor editing in lupus and type 1 diabetes.

Continued antibody gene rearrangement, termed receptor editing, is an important mechanism of central B cell tolerance that may be defective in some autoimmune individuals. We describe a quantitative assay for recombining sequence (RS) rearrangement that we use to estimate levels of antibody light chain receptor editing in various B cell populations. RS rearrangement is a recombination of a noncoding gene segment in the kappa antibody light chain locus. RS rearrangement levels are highest in the most highly edited B cells, and are inappropriately low in autoimmune mouse models of systemic lupus erythematosus (SLE) and type 1 diabetes (T1D), including those without overt disease. Low RS rearrangement levels are also observed in human subjects with SLE or T1D.