Multilevel human secondary lymphoid immune system compartmentalization revealed by complementary imaging approaches.

Secondary human lymphoid tissue immune reactions take place in a highly coordinated environment with compartmentalization representing a fundamental feature of this organization. In situ profiling methodologies are indispensable for the understanding of this compartmentalization. Here, we propose a complementary experimental approach aiming to reveal different aspects of this process. The analysis of human tonsils, using a combination of single cell phenotypic analysis based on flow cytometry and multiplex imaging and mass spectrometry-based methodologies, revealed a compartmentalized organization at the cellular and molecular levels. More specifically, the skewed distribution of highly specialized immune cell subsets and relevant soluble mediators was accompanied by a compartmentalized localization of several lipids across different anatomical areas of the tonsillar tissue. The performance of such combinatorial experimental approaches could lead to the identification of novel in situ interactions and molecular targets for the in vivo manipulation of lymphoid organ, particularly the germinal center, immune reactions.

Trispecific antibody targeting HIV-1 and T cells activates and eliminates latently-infected cells in HIV/SHIV infections.

Agents that can simultaneously activate latent HIV, increase immune activation and enhance the killing of latently-infected cells represent promising approaches for HIV cure. Here, we develop and evaluate a trispecific antibody (Ab), N6/αCD3-αCD28, that targets three independent proteins: (1) the HIV envelope via the broadly reactive CD4-binding site Ab, N6; (2) the T cell antigen CD3; and (3) the co-stimulatory molecule CD28. We find that the trispecific significantly increases antigen-specific T-cell activation and cytokine release in both CD4+ and CD8+ T cells. Co-culturing CD4+ with autologous CD8+ T cells from ART-suppressed HIV+ donors with N6/αCD3-αCD28, results in activation of latently-infected cells and their elimination by activated CD8+ T cells. This trispecific antibody mediates CD4+ and CD8+ T-cell activation in non-human primates and is well tolerated in vivo. This HIV-directed antibody therefore merits further development as a potential intervention for the eradication of latent HIV infection.

Potent anti-viral activity of a trispecific HIV neutralizing antibody in SHIV-infected monkeys.

Broadly neutralizing antibodies (bNAbs) represent an alternative to drug therapy for the treatment of HIV-1 infection. Immunotherapy with single bNAbs often leads to emergence of escape variants, suggesting a potential benefit of combination bNAb therapy. Here, a trispecific bNAb reduces viremia 100- to 1000-fold in viremic SHIV-infected macaques. After treatment discontinuation, viremia rebounds transiently and returns to low levels, through CD8-mediated immune control. These viruses remain sensitive to the trispecific antibody, despite loss of sensitivity to one of the parental bNAbs. Similarly, the trispecific bNAb suppresses the emergence of resistance in viruses derived from HIV-1-infected subjects, in contrast to parental bNAbs. Trispecific HIV-1 neutralizing antibodies, therefore, mediate potent antiviral activity in vivo and may minimize the potential for immune escape.

Concordance of immunological events between intrarectal and intravenous SHIVAD8-EO infection when assessed by Fiebig-equivalent staging.

Primary HIV-1 infection can be classified into six Fiebig stages based on virological and serological laboratory testing, whereas simian-HIV (SHIV) infection in nonhuman primates (NHPs) is defined in time post-infection, making it difficult to extrapolate NHP experiments to the clinics. We identified and extensively characterized the Fiebig-equivalent stages in NHPs challenged intrarectally or intravenously with SHIVAD8-EO. During the first month post-challenge, intrarectally challenged monkeys were up to 1 week delayed in progression through stages. However, regardless of the challenge route, stages I-II predominated before, and stages V-VI predominated after, peak viremia. Decrease in lymph node (LN) CD4+ T cell frequency and rise in CD8+ T cells occurred at stage V. LN virus-specific CD8+ T cell responses, dominated by degranulation and TNF, were first detected at stage V and increased at stage VI. A similar late elevation in follicular CXCR5+ CD8+ T cells occurred, consistent with higher plasma CXCL13 levels at these stages. LN SHIVAD8-EO RNA+ cells were present at stage II, but appeared to decline at stage VI when virions accumulated in follicles. Fiebig-equivalent staging of SHIVAD8-EO infection revealed concordance of immunological events between intrarectal and intravenous infection despite different infection progressions, and can inform comparisons of NHP studies with clinical data.

Immunogenicity of Protein Therapeutics: A Lymph Node Perspective.

The continuous development of molecular biology and protein engineering technologies enables the expansion of the breadth and complexity of protein therapeutics for in vivo administration. However, the immunogenicity and associated in vivo development of antibodies against therapeutics are a major restriction factor for their usage. The B cell follicular and particularly germinal center areas in secondary lymphoid organs are the anatomical sites where the development of antibody responses against pathogens and immunogens takes place. A growing body of data has revealed the importance of the orchestrated function of highly differentiated adaptive immunity cells, including follicular helper CD4 T cells and germinal center B cells, for the optimal generation of these antibody responses. Understanding the cellular and molecular mechanisms mediating the antibody responses against therapeutics could lead to novel strategies to reduce their immunogenicity and increase their efficacy.

Intestinal Mucosal Barrier Function Restoration in Mice by Maize Diet Containing Enriched Flavan-4-Ols.

Inflammatory bowel disease (IBD), a chronic intestinal inflammatory condition, awaits safe and effective preventive strategies. Naturally occurring flavonoid compounds are promising therapeutic candidates against IBD due to their great antioxidant potential and ability to reduce inflammation and improve immune signaling mediators in the gut. In this study, we utilized two maize near-isogenic lines flavan-4-ols-containing P1-rr (F+) and flavan-4-ols-lacking p1-ww (F-) to investigate the anti-inflammatory property of flavan-4-ols against carboxymethylcellulose (CMC)-induced low-grade colonic inflammation. C57BL/6 mice were exposed to either 1% CMC (w/v) or water for a total of 15 weeks. After week six, mice on CMC treatment were divided into four groups. One group continued on the control diet. The second and third groups were supplemented with F+ at 15% or 25% (w/w). The fourth group received diet supplemented with F- at 15%. Here we report that mice consuming F+(15) and F+(25) alleviated CMC-induced increase in epididymal fat-pad, colon histology score, pro-inflammatory cytokine interleukin 6 expression and intestinal permeability compared to mice fed with control diet and F-(15). F+(15) and F+(25) significantly enhanced mucus thickness in CMC exposed mice (p < 0.05). These data collectively demonstrated the protective effect of flavan-4-ol against colonic inflammation by restoring intestinal barrier function and provide a rationale to breed for flavan-4-ols enriched cultivars for better dietary benefits.