Transgender women (TW) are at increased risk for both human immunodeficiency virus (HIV) and cardiovascular disease (CVD). Antiretroviral therapy-treated HIV has been associated with a two-fold increased risk of CVD, potentially due to dysregulated Toll-like receptor (TLR)-induced immune activation. Use of estrogens in feminizing hormone therapy (FHT) may enhance inflammatory responses and the risk of cardiovascular mortality in TW. Despite this, the immunomodulatory effects of estrogen use in TW with HIV have been inadequately explored.
As an in vitro model for FHT, cryopreserved PBMCs (cryoPBMCs) from HIV negative (HIV-), HIV+ ART-suppressed (HIV+SP), and HIV+ ART-unsuppressed (HIV+USP) cisgender men were cultured overnight in the presence of 17-β estradiol or 17-α ethinylestradiol with and without the TLR4 agonist LPS or the TLR8 agonist ssPolyU. Monocyte activation (CD69, HLA-DR, CD38) was assessed by flow cytometry. Cytokine levels (IL-6, TNF-α, IL-1β, and IL-10) were measured in cell culture supernatants by Legendplex. Levels of phosphorylated TLR signaling molecules (JNK, MAPK p38) were assessed by Phosflow. Plasma levels of immune activation biomarkers (LPS-binding protein, monocyte activation markers sCD14 and sCD163, and inflammatory molecules IL-6 and TNF-α receptor I) were measured by ELISA.
PBMCs from people with HIV (PWH) produced greater levels of inflammatory cytokines following exposure to LPS or ssPolyU compared to levels from cells of HIV- individuals. While estrogen exposure alone induced mild changes in immune activation, LPS-induced TLR4 activation was elevated with estrogen in cisgender men (CM) with HIV, increasing monocyte activation and inflammatory cytokine production (IL-6, TNF-α). Interestingly, testosterone inhibited LPS-induced cytokine production in CM regardless of HIV status. Plasma markers of immune activation and microbial translocation (e.g., sCD14, sCD163, LPS-binding protein) were generally higher in PWH compared to HIV- CM, and these markers were positively associated with in vitro responsiveness to estrogen and LPS in CM with HIV.
Our in vitro data suggest that estrogen exposure may enhance innate immune activation in PWH. Further examination is needed to fully understand the complex interactions of FHT, HIV, and CVD in TW, and determine optimal FHT regimens or supplementary treatments aimed at reducing excess immune activation.
Copyright © 2022 Kettelhut, Bowman, Gabriel, Hand, Liyanage, Kulkarni, Avila-Soto, Lake and Funderburg.

Numerous observations indicate that red blood cells (RBCs) affect T-cell activation and proliferation. We have studied effects of packed RBCs (PRBCs) on T-cell receptor (TCR) signaling and the molecular mechanisms whereby (P)RBCs modulate T-cell activation. In line with previous reports, PRBCs attenuated the expression of T-cell activation markers CD25 and CD69 upon costimulation via CD3/CD28. In addition, T-cell proliferation and cytokine expression were markedly reduced when T-cells were stimulated in the presence of PRBCs. Inhibitory activity of PRBCs required direct cell-cell contact and intact PRBCs. The production of activation-induced cellular reactive oxygen species, which act as second messengers in T-cells, was completely abrogated to levels of unstimulated T-cells in the presence of PRBCs. Phosphorylation of the TCR-related zeta chain and thus proximal TCR signal transduction was unaffected by PRBCs, ruling out mechanisms based on secreted factors and steric interaction restrictions. In large part, downstream signaling events requiring reactive oxygen species for full functionality were affected, as confirmed by an untargeted MS-based phosphoproteomics approach. PRBCs inhibited T-cell activation more efficiently than treatment with 1 mM of the antioxidant N-acetyl cysteine. Taken together, our data imply that inflammation-related radical reactions are modulated by PRBCs. These immunomodulating effects may be responsible for clinical observations associated with transfusion of PRBCs.
Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.

Sequential allergen desensitization provides temporary tolerance for allergic patients. We adapted a clinical protocol to desensitize human blood basophils ex vivo and investigated the mechanism and allergen specificity.
We included 28 adult, grass allergic subjects. The optimal, activating allergen concentration was determined by measuring activated CD63(+) CD193(+) SS(Low) basophils in a basophil activation test with 8 log-dilutions of grass allergen. Basophils in whole blood were desensitized by incubation with twofold to 2.5-fold increasing allergen doses in 10 steps starting at 1 : 1000 of the optimal dose. Involvement of p38 mitogen-activated protein kinase (MAPK) was assessed after 3 min of allergen stimulation (n = 7). Allergen specificity was investigated by desensitizing cells from multi-allergic subjects with grass allergen and challenging with optimal doses of grass, birch, recombinant house dust mite (rDer p2) allergen or anti-IgE (n = 10).
Desensitization reduced the fraction of blood basophils responding to challenge with an optimal allergen dose from a median (IQR) 81.0% (66.3-88.8) to 35.4% (19.8-47.1, P < 0.0001). CD63 MFI expression was reduced from 68 248 (29 336-92 001) to 30 496 (14 046-46 179, P < 0.0001). Basophils from multi-allergic subjects were desensitized with grass allergen. Challenge with grass allergen resulted in 39.6% activation (15.8-58.3). An unrelated challenge (birch, rDer p2 or anti-IgE) resulted in 53.4% activation (30.8-66.8, P = 0.16 compared with grass). Desensitization reduced p38 MAPK phosphorylation from a median 48.1% (15.6-92.8) to 26.1% (7.4-71.2, P = 0.047) and correlated with decrease in CD63 upregulation (n = 7, r > 0.79, P < 0.05).
Desensitization attenuated basophil response rapidly and non-specifically at a stage before p38 MAPK phosphorylation.
© 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.