Chimeric antigen receptor (CAR)–modified T cell therapy is effective in treating lymphomas, leukemias, and multiple myeloma in which the tumor cells express high amounts of target antigen. However, achieving durable remission for these hematological malignancies and extending CAR T cell therapy to patients with solid tumors will require receptors that can recognize and eliminate tumor cells with a low density of target antigen. Although CARs were designed to mimic T cell receptor (TCR) signaling, TCRs are at least 100-fold more sensitive to antigen. To design a CAR with improved antigen sensitivity, we directly compared TCR and CAR signaling in primary human T cells. Global phosphoproteomic analysis revealed that key T cell signaling proteins—such as CD3δ, CD3ε, and CD3γ, which comprise a portion of the T cell co-receptor, as well as the TCR adaptor protein LAT—were either not phosphorylated or were only weakly phosphorylated by CAR stimulation. Modifying a commonplace 4-1BB/CD3ζ CAR sequence to better engage CD3ε and LAT using embedded CD3ε or GRB2 domains resulted in enhanced T cell activation in vitro in settings of a low density of antigen, and improved efficacy in in vivo models of lymphoma, leukemia, and breast cancer. These CARs represent examples of alterations in receptor design that were guided by in-depth interrogation of T cell signaling.

Peptides bind Class II Major Histocompatibility Complex molecules (MHCII) through a thermodynamically non-additive process consequent to the flexibility of the reactants. Currently, how the specific outcome of this binding process effects the ensuing epitope selection needs resolution. Calorimetric assessment of binding thermodynamics for HA306-319 peptide variants to the human MHCII HLA-DR1 (DR1) and a mutant DR1 reveals that peptide/DR1 complexes can be formed with different enthalpic and entropic contributions. Complexes formed with a smaller entropic penalty feature circular dichroism spectra consistent with a non-compact form, and molecular dynamics simulation shows a more flexible structure. The opposite binding mode, compact and less flexible, is associated with greater entropic penalty. These structural variations are associated with rearrangements of residues known to be involved in DM binding, affinity of DM for the complex, and complex susceptibility to DM-mediated peptide exchange. Thus, the thermodynamic mechanism of peptide binding to DR1 correlates with the structural rigidity of the complex, and DM mediates peptide exchange by “sensing” flexible complexes in which the aforementioned residues are rearranged at a higher frequency than in more rigid ones.

BCG vaccination has been demonstrated to increase levels of activated CD4+ T cells, thus potentially influencing mother-to-child transmission of human immunodeficiency virus (HIV). To assess the risk of BCG vaccination in HIV infection, we randomly assigned newborn rhesus macaques to receive BCG vaccine or remain unvaccinated and then undergo oral simian immunodeficiency virus (SIV) challenges 3 weeks later. We observed elevated levels of activated peripheral CD4+ T cells (ie, HLA-DR+CD38+CCR5+ CD4+ T cells) by week 3 after vaccination. BCG was also associated with an altered immune gene expression profile, as well as with monocyte activation in both peripheral blood and the draining axillary lymph node, indicating significant BCG vaccine–induced immune activation. Despite these effects, BCG vaccination did not increase the rate of SIV oral transmission or disease progression. Our findings therefore identify patterns of T-cell and monocyte activation that occur after BCG vaccination but do not support the hypothesis that BCG vaccination is a risk factor for postnatal HIV transmission or increased pathogenesis in infants.

Therapy with CD19 directed chimeric antigen receptor (CAR) T cells has transformed the treatment of advanced B-cell malignancies. However, loss of or low antigen expression can enable tumor escape and limit the duration of responses achieved with CAR-T cell therapy. Engineering bispecific CAR-T cells that target two tumor antigens could overcome antigen negative escape. We found that CD79a and b, which are heterodimeric components of the B cell receptor, were expressed on 84.3% of lymphoma cases by immunohistochemistry, and that 87.3% of CD79ab positive tumors coexpressed CD19. We generated three bispecific permutations: tandem, bicistronic and pooled products of CD79a-CD19 or CD79b-CD19 CAR-T cells and showed that bispecific CAR-T cells prevented the outgrowth of antigen negative cells in a CD19-loss lymphoma xenograft model. However, the tandem and bicistronic CAR-T cells were less effective than monospecific CD19 or CD79a CAR-T cells for the treatment of tumors that only expressed CD19 or CD79, respectively. When compared to monospecific CAR-T cells, T cells expressing a tandem CAR exhibited reduced binding of each target antigen and T cells expressing a bicistronic CAR vector exhibited reduced phosphorylation of downstream CAR signaling molecules. Our study showed that despite added specificity, tandem and bicistronic CAR-T cells exhibit different defects that impair recognition of tumor cells expressing a single antigen. Our data provide support for targeting multiple B cell antigens to improve efficacy and identify areas for improvement of bispecific receptor designs.

We explored the use of filter paper soaked in whole blood for measuring carbon (C) and nitrogen (N) stable isotopes, often used in feeding ecology or diet studies, to better understand drivers of exposure to contaminants. Our results showed no statistically or biologically relevant differences in C and N stable isotope measures between our gold standard (whole blood with anticoagulant) and eluates from processed, blood-soaked filter paper. Our data supported the effectiveness of using filter paper for assessing C and N stable isotopes in blood to address feeding ecology and other uses. The ease of sampling and processing should allow blood-soaked filter paper to be used in sampling of live (e.g., captured, stranded) and lethally taken (e.g., hunter-killed) wild vertebrates.