Detection of Corynebacterium kroppenstedtii in Granulomatous Lobular Mastitis Using Real-Time Polymerase Chain Reaction and Sanger Sequencing on Formalin-Fixed, Paraffin-Embedded Tissues.

CONTEXT.­: Associations between granulomatous lobular mastitis (GLM) and Corynebacterium kroppenstedtii have been reported since 2002, but large-scale studies to assess the actual prevalence of this bacterium in GLM have not been performed. OBJECTIVE.­: To assess the prevalence of C kroppenstedtii in GLM using real-time polymerase chain reaction and Sanger sequencing. DESIGN.­: We analyzed formalin-fixed, paraffin-embedded tissues from 67 cases of GLM by sequential DNA amplification and sequencing to assess the rate of C kroppenstedtii detection in GLM. A retrospective analysis including patient demographics, history of pregnancy and lactation, clinical signs and symptoms, radiographic findings, histologic pattern, Gram stain results, and microbial cultures was performed on 67 cases of GLM. In addition, 10 cases of nongranulomatous breast abscess were included as controls. RESULTS.­: C kroppenstedtii 16S rRNA SYBR real-time polymerase chain reaction was positive on formalin-fixed, paraffin-embedded tissues from 46 of 67 (68.7%) GLM cases, while all control cases were negative. Among the positive cases, the majority showed features of cystic neutrophilic granulomatous mastitis. CONCLUSIONS.­: C kroppenstedtii was highly prevalent in GLM cases and was not found to be associated with nongranulomatous breast abscess in our study (P < .001).

CD122-Selective IL2 Complexes Reduce Immunosuppression, Promote Treg Fragility, and Sensitize Tumor Response to PD-L1 Blockade.

The IL2 receptor (IL2R) is an attractive cancer immunotherapy target that controls immunosuppressive T regulatory cells (Treg) and antitumor T cells. Here we used IL2Rß-selective IL2/anti-IL2 complexes (IL2c) to stimulate effector T cells preferentially in the orthotopic mouse ID8agg ovarian cancer model. Despite strong tumor rejection, IL2c unexpectedly lowered the tumor microenvironmental CD8+/Treg ratio. IL2c reduced tumor microenvironmental Treg suppression and induced a fragile Treg phenotype, helping explain improved efficacy despite numerically increased Tregs without affecting Treg in draining lymph nodes. IL2c also reduced Treg-mediated, high-affinity IL2R signaling needed for optimal Treg functions, a likely mechanism for reduced Treg suppression. Effector T-cell IL2R signaling was simultaneously improved, suggesting that IL2c inhibits Treg functions without hindering effector T cells, a limitation of most Treg depletion agents. Anti-PD-L1 antibody did not treat ID8agg, but adding IL2c generated complete tumor regressions and protective immune memory not achieved by either monotherapy. Similar anti-PD-L1 augmentation of IL2c and degradation of Treg functions were seen in subcutaneous B16 melanoma. Thus, IL2c is a multifunctional immunotherapy agent that stimulates immunity, reduces immunosuppression in a site-specific manner, and combines with other immunotherapies to treat distinct tumors in distinct anatomic compartments. SIGNIFICANCE: These findings present CD122-targeted IL2 complexes as an advancement in cancer immunotherapy, as they reduce Treg immunosuppression, improve anticancer immunity, and boost PD-L1 immune checkpoint blockade efficacy in distinct tumors and anatomic locations.

Age effects of distinct immune checkpoint blockade treatments in a mouse melanoma model.

Cancer immunotherapy has shown remarkable recent progress. Immune checkpoint blocking antibodies have become the most successful anti-cancer agent class ever developed, with six distinct agents approved since 2011 for a wide variety of cancers. Although age is the biggest risk factor for cancer (aside from selected early-onset pediatric cancers), these agents were tested pre-clinically in young hosts, and there is remarkably little published on the effects of host age on treatment outcomes in pre-clinical studies or human clinical trials. The three principal immune checkpoints against which blocking antibodies have been FDA-approved for human use are CTLA-4, PD-1 and PD-L1. We used a mouse model of transplantable, orthotopic B16 melanoma to test age effects of treatments with anti-CTLA-4, anti-PD-1 and anti-PD-L1 antibodies. All three agents were highly effective in treating young tumor-bearing hosts as expected. Anti-PD-L1 as a single agent had no effect on tumor growth in aged hosts, anti-CTLA-4 had detectable, modest effects and anti-PD-1 was essentially as effective in aged as in young hosts, the first single agent we have identified not to lose efficacy with age in this model. Other important differences in young versus aged hosts included lack of anti-CTLA-4-mediated depletion of intratumor regulatory T cells in aged hosts and poorer ability of all three agents to activate T cells in aged versus young hosts. Anti-CTLA-4 efficacy appeared to improve when combined with anti-PD-L1. Regulatory T cell depletion with FDA-approved denileukin diftitox did not improve treatment by any single agent. Aged mice tolerated treatments as well as young mice without obvious toxicities at equivalent doses.

Regulation of caspase-9 activity by differential binding to the apoptosome complex.

Proteolytic processing of procaspase-9 is required for its activation, but processing in itself appears to be insufficient for its activity. We studied caspase activation in a cell-free system and found that incubation of cytosol from rat kidney proximal tubule cells with Cytochrome c (Cyt c) and dATP results in rapid autocatalytic processing of procaspase-9 from ~50 kD to ~38 kD size fragment. Moreover, Cyt c concentration influences the production of alternatively processed forms of caspase-9. At lower Cyt c concentration (0.01-0.05 mg/ml), two fragments of caspase-9 of the size 38 and 40 kD are produced. In contrast, at higher concentrations of Cyt c (>0.1 mg/ml) only 38 kD fragment will prevail. However, our failure to capture processed caspase-9 by affinity labeling or co-elution with Apaf-1 suggested that caspase-9 undergoes a conformational change during its enzymatic action on effector caspases, resulting in its release from the apoptosome complex and inactivation. In support of this hypothesis, catalytic inhibitors of caspase-9 prevented its release from the apoptosome complex without affecting its auto-processing and allowed successful capture of active caspase-9 (38 kD) and its complex by affinity labeling. These observations suggest that complex allosteric interactions with the apoptosome complex influence caspase-9 activity and function by controlling not only the induction of its enzymatic activity, but also its rapid termination.