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Functionally bivalent non-covalent Fab dimers (Bi-Fabs) specific for the TCR/CD3 complex promote CD3 signaling on T cells. While comparing functional responses to stimulation with Bi-Fab, F(ab')2 or mAb specific for the same CD3 epitope, we observed fratricide requiring anti-CD3 bridging of adjacent T cells. Surprisingly, anti-CD3 Bi-Fab ranked first in fratricide potency, followed by anti-CD3 F(ab')2 and anti-CD3 mAb. Low resolution structural studies revealed anti-CD3 Bi-Fabs and F(ab')2 adopt similar global shapes with CD3-binding sites oriented outward. However, under molecular dynamic simulations, anti-CD3 Bi-Fabs crosslinked CD3 more rigidly than F(ab')2. Furthermore, molecular modelling of Bi-Fab and F(ab')2 binding to CD3 predicted crosslinking of T cell antigen receptors located in opposing plasma membrane domains, a feature fitting with T cell fratricide observed. Thus, increasing rigidity of Fab-CD3 crosslinking between opposing effector-target pairs may result in stronger T cell effector function. These findings could guide improving clinical performance of bi-specific anti-CD3 drugs.

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American Indian/Alaska Native (AI/AN) persons in the US experience a disparity in chronic respiratory diseases compared to white persons. Using Behavioral Risk Factor Surveillance System (BRFSS) data, we previously showed that the AI/AN race/ethnicity variable was not associated with asthma and/or chronic obstructive pulmonary disease (COPD) in a BRFSS-defined subset of 11 states historically recognized as having a relatively high proportion of AI/AN residents. Here, we investigate the contributions of the AI/AN variable and other sociodemographic determinants to disease disparity in the remaining 39 US states and territories. Using BRFSS surveys from 2011 to 2019, we demonstrate that irrespective of race, the yearly adjusted prevalence for asthma and/or COPD was higher in the 39-state region than in the 11-state region. Logistic regression analysis revealed that the AI/AN race/ethnicity variable was positively associated with disease in the 39-state region after adjusting for sociodemographic covariates, unlike in the 11-state region. This shows that the distribution of disease prevalence and disparity for asthma and/or COPD is non-uniform in the US. Although AI/AN populations experience this disease disparity throughout the US, the AI/AN variable was only observed to contribute to this disparity in the 39-state region. It may be important to consider the geographical distribution of respiratory health determinants and factors uniquely impactful for AI/AN disease disparity when formulating disparity elimination policies.

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BACKGROUND: American Indian/Alaska Native (AI/AN) populations have been disproportionately affected by chronic respiratory diseases for reasons incompletely understood. Past research into disease disparity using population-based surveys mostly focused on state-specific factors. The present study investigates the independent contributions of AI/AN racial status and other socioeconomic/demographic variables to chronic respiratory disease disparity in an 11-state region with historically high AI/AN representation. Using data from the Behavioral Risk Factor Surveillance System (BRFSS) spanning years 2011-2018, this work provides an updated assessment of disease disparity and potential determinants of respiratory health in AI/AN populations. METHODS: This cross-sectional study used data from the BRFSS survey, 2011-2018. The study population included AI/AN and non-Hispanic white individuals resident in 11 states with increased proportion of AI/AN individuals. The yearly number of respondents averaged 75,029 (62878-87,350) which included approximately 5% AI/AN respondents (4.5-6.3%). We compared the yearly adjusted prevalence for chronic respiratory disease, where disease status was defined by self-reported history of having asthma and/or chronic obstructive pulmonary disease (COPD). Multivariable logistic regression was performed to determine if being AI/AN was independently associated with chronic respiratory disease. Covariates included demographic (age, sex), socioeconomic (marital status, education level, annual household income), and behavioral (smoking, weight morbidity) variables. RESULTS: The AI/AN population consistently displayed higher adjusted prevalence of chronic respiratory disease compared to the non-Hispanic white population. However, the AI/AN race/ethnicity characteristic was not independently associated with chronic respiratory disease (OR, 0.93; 95% CI, 0.79-1.10 in 2017). In contrast, indicators of low socioeconomic status such as annual household income of <$10,000 (OR, 2.02; 95% CI, 1.64-2.49 in 2017) and having less than high school education (OR, 1.37; 95% CI, 1.16-1.63 in 2017) were positively associated with disease. These trends persisted for all years analyzed. CONCLUSIONS: This study highlighted that AI/AN socioeconomic burdens are key determinants of chronic respiratory disease, in addition to well-established risk factors such as smoking and weight morbidity. Disease disparity experienced by the AI/AN population is therefore likely a symptom of disproportionate socioeconomic challenges they face. Further promotion of public health and social service efforts may be able to improve AI/AN health and decrease this disease disparity.

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Adaptive immunity is mediated by antigen receptors that can induce weak or strong immune responses depending on the nature of the antigen that is bound. In T lymphocytes, antigen recognition triggers signal transduction by clustering T cell receptor (TCR)/CD3 multiprotein complexes. In addition, it hypothesized that biophysical changes induced in TCR/CD3 that accompany receptor engagement may contribute to signal intensity. Nonclustering monovalent TCR/CD3 engagement is functionally inert despite the fact that it may induce changes in conformational arrangement or in the flexibility of receptor subunits. We report that the intrinsically inert monovalent engagement of TCR/CD3 can specifically enhance physiologic T cell responses to weak antigens in vitro and in vivo without stimulating antigen-unengaged T cells and without interrupting T cell responses to strong antigens, an effect that we term as "co-potentiation." We identified Mono-7D6-Fab, which biophysically altered TCR/CD3 when bound and functionally enhanced immune reactivity to several weak antigens in vitro, including a gp100-derived peptide associated with melanoma. In vivo, Mono-7D6-Fab induced T cell antigen-dependent therapeutic responses against melanoma lung metastases, an effect that synergized with other anti-melanoma immunotherapies to significantly improve outcome and survival. We conclude that Mono-7D6-Fab directly co-potentiated TCR/CD3 engagement by weak antigens and that such concept can be translated into an immunotherapeutic design. The co-potentiation principle may be applicable to other receptors that could be regulated by otherwise inert compounds whose latent potency is only invoked in concert with specific physiologic ligands.

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Generated by proteolytic cleavage of immunoglobulin, Fab fragments possess great promise as blocking reagents, able to bind receptors or other targets without inducing cross-linking. However, aggregation of Fab preparations is a common occurrence, which generates intrinsic stimulatory capacity and thwarts signal blockade strategies. Using a panel of biochemical approaches, including size exclusion chromatography, SDS-PAGE, mass spectrometry, and cell stimulation followed by flow cytometry, we have measured the oligomerization and acquisition of stimulatory capacity that occurs in four monoclonal IgG Fabs specific for TCR/CD3. Unexpectedly, we observed that all Fabs spontaneously formed complexes that were precisely bivalent, and these bivalent complexes possessed most of the stimulatory activity of each Fab preparation. Fabs composing bivalent complexes were more susceptible to proteolysis than monovalent Fabs, indicating a difference in conformation between the Fabs involved in these two different states of valency. Because osmolytes represent a class of compounds that stabilize protein folding and conformation, we sought to determine the extent to which the amino acid osmolyte l-proline might impact bivalent Fab complexation. We found that l-proline (i) inhibited the adoption of the conformation associated with bivalent complexation, (ii) preserved Fab monovalency, (iii) reversed the conformation of preformed bivalent Fabs to that of monovalent Fabs, and (iv) separated a significant percentage of preformed bivalent complexes into monovalent species. Thus, Fab fragments can adopt a conformation that is compatible with folding or packing of a bivalent complex in a process that can be inhibited by osmolytes.

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