Unique ability of activated CD4⁺ T cells but not rested effectors to migrate to non-lymphoid sites in the absence of inflammation

Recent studies suggest that effector T cells generated by immune responses migrate to multiple non-lymphoid sites, even those without apparent expression of antigen or inflammation. To investigate the ability of distinct CD4+ T lymphocyte subsets to enter and persist in non-lymphoid, non-inflamed compartments, we examined the migration and persistence of naive, effector and rested effector CD4+ T cells generated in vitro following transfer to nonimmunized adoptive hosts. Th1 and Th2 effectors migrated to both lymphoid and non-lymphoid organs (peritoneum, fat pads and lung). In contrast, rested effectors and naive cells migrated only to lymphoid areas. Adhesion molecule expression, but not chemokine receptor expression, correlated with the ability to enter non-lymphoid sites. Donor cells persisted longer in lymphoid than in non-lymphoid sites. When hosts with naive and memory donor cells were challenged with antigen, effectors developed in situ, which also migrated to non-lymphoid sites. Memory cells showed an accelerated shift to non-lymphoid migration, in keeping with memory effector formation. These results suggest that only recently activated effector T cells can disperse to non-lymphoid sites in the absence of antigen and inflammation and as effectors return to rest, they lose this ability. These data also argue that memory cells in lymphoid sites are longer lived and not in equilibrium with those in non-lymphoid sites

Fibrin Facilitates Both Innate and T Cell-Mediated Defense against Yersinia pestis

The gram-negative bacterium Yersinia pestis causes plague, a rapidly progressing and often fatal disease. The formation of fibrin at sites of Y. pestis infection supports innate host defense against plague, perhaps by providing a non-diffusible spatial cue that promotes the accumulation of inflammatory cells expressing fibrin-binding integrins. This report demonstrates that fibrin is an essential component of T cell-mediated defense against plague but can be dispensable for antibody-mediated defense. Genetic or pharmacologic depletion of fibrin abrogated innate and T cell-mediated defense in mice challenged intranasally with Y. pestis. The fibrin-deficient mice displayed reduced survival, increased bacterial burden, and exacerbated hemorrhagic pathology. They also showed fewer neutrophils within infected lung tissue and reduced neutrophil viability at sites of liver infection. Depletion of neutrophils from wild type mice weakened T cell-mediated defense against plague. The data suggest that T cells combat plague in conjunction with neutrophils, which require help from fibrin in order to withstand Y. pestis encounters and effectively clear bacteria

Comparative Safety and Efficacy Profile of a Novel Oil in Water Vaccine Adjuvant Comprising Vitamins A and E and a Catechin in Protective Anti-Influenza Immunity

Non-replicating vaccines, such as those based on recombinant proteins, require adjuvants and delivery systems, which have thus far depended on mimicking pathogen danger signals and strong pro-inflammatory responses. In search of a safer and more efficacious alternative, we tested whether vaccinations with influenza recombinant hemagglutinin (HA) mixed with a novel vegetable oil in water emulsion adjuvant (Natural Immune-enhancing Delivery System, NIDS), based on the immune-enhancing synergy of vitamins A and E and a catechin, could protect against intra-nasal challenge with live influenza virus. Vaccinations of inbred Brag Albino strain c (BALB/c) mice, with HA mixed with NIDS compared to other adjuvants, i.e., a squalene oil in water emulsion (Sq. oil), and the Toll Like Receptor 3 (TLR3) agonist Poly (I:C), induced significantly lower select innate pro-inflammatory responses in serum, but induced significantly higher adaptive antibody and splenic T Helper 1 (TH1) or TH2, but not TH17, responses. Vaccinations with NIDS protected against infection, as measured by clinical scores, lung viral loads, and serum hemagglutination inhibition titers. The NIDS exhibited a strong dose sparing effect and the adjuvant action of NIDS was intact in the outbred CD1 mice. Importantly, vaccinations with the Sq. oil, but not NIDS, induced a significantly higher Serum Amyloid P component, an acute phase reactant secreted by hepatocytes, and total serum IgE. Thus, the NIDS may be used as a clinically safer and more efficacious vaccine adjuvant against influenza, and potentially other infectious diseases

TNFα and IFNγ but not perforin are critical for CD8 T cell-mediated protection against pulmonary Yersinia pestis infection.

Septic pneumonias resulting from bacterial infections of the lung are a leading cause of human death worldwide. Little is known about the capacity of CD8 T cell-mediated immunity to combat these infections and the types of effector functions that may be most effective. Pneumonic plague is an acutely lethal septic pneumonia caused by the Gram-negative bacterium Yersinia pestis. We recently identified a dominant and protective Y. pestis antigen, YopE69-77, recognized by CD8 T cells in C57BL/6 mice. Here, we use gene-deficient mice, Ab-mediated depletion, cell transfers, and bone marrow chimeric mice to investigate the effector functions of YopE69-77-specific CD8 T cells and their relative contributions during pulmonary Y. pestis infection. We demonstrate that YopE69-77-specific CD8 T cells exhibit perforin-dependent cytotoxicity in vivo; however, perforin is dispensable for YopE69-77-mediated protection. In contrast, YopE69-77-mediated protection is severely impaired when production of TNFα and IFNγ by CD8 T cells is simultaneously ablated. Interestingly, TNFα is absolutely required at the time of challenge infection and can be provided by either T cells or non-T cells, whereas IFNγ provided by T cells prior to challenge appears to facilitate the differentiation of optimally protective CD8 T cells. We conclude that cytokine production, not cytotoxicity, is essential for CD8 T cell-mediated control of pulmonary Y. pestis infection and we suggest that assays detecting Ag-specific TNFα production in addition to antibody titers may be useful correlates of vaccine efficacy against plague and other acutely lethal septic bacterial pneumonias

YopE_69–77-specific CD8 T cells lacking the capacity to produce TNFα and IFNγ fail to protect mice and control bacterial burden.

<p>TCRβδ-deficient (TCRbdKO) mice were lethally irradiated and reconstituted with 75% TCRβδKO bone marrow cells and 25% of either WT, TNFαKO, IFNγKO, PKO, or TNFαIFNγ DKO bone marrow cells. Six weeks later they were immunized with CT mixed with YopE_69–77 or OVA_257–264 and then challenged intranasally with 20 MLD <i>Y. pestis</i> strain D27. (A) Survival. In comparison with OVA_257–264-immunized mice reconstituted with WT T cells (n = 20), the YopE_69–77-immunized chimeric mice reconstituted with WT (n = 25), TNFαKO (n = 17), IFNγKO (n = 19), PKO (n = 8) or TNFαIFNγ DKO (n = 33) T cells all showed significant protection. (B) The percentage of CD8+ T cells that stained positive for MHC class I tetramer K^bYopE_69–77 in PBL on the day before challenge. Solid bar depicts the mean. All groups of chimeric mice that were immunized with YopE_69–77 had significantly increased frequency of K^bYopE_69–77+CD8+ T cells in compared with the chimeric mice immunized with OVA_257–264 (p<0.001). YopE_69–77-immunized chimeric mice reconstituted with TNFαKO T cells had significantly higher frequency of K^bYopE_69–77+CD8+ T cells in compared with YopE_69–77-immunized chimeric mice reconstituted with WT T cells (p<0.01). Data for (A) and (B) are pooled from 6 independent experiments. (C and D) Bacterial burden in lung (C) and liver (D) tissues was measured at day 4 after challenge (Kruskal-Wallis test). Data are pooled from 3 independent experiments. Solid bar depicts median; broken line depicts the limit of detection.</p

TNFα and IFNγ are critical for T cell-mediated protection against <i>Y. pestis</i>.

<p>(A and B) Wild-type (WT), TNFα-deficient (TNFaKO), IFNγ-deficient (IFNgKO) C57BL/6 mice were immunized intranasally with CT adjuvant alone or CT mixed with YopE_69–77 peptide and then challenged intranasally with 20 MLD <i>Y. pestis</i> strain D27. In comparison with YopE_69–77–immunized WT mice (n = 15–25), YopE_69–77–immunized TNFα-deficient (n = 22) and IFNγ-deficient (n = 18) mice exhibited significantly reduced survival. Data were pooled from 3–5 independent experiments. (C) Wild-type C57BL/6 mice were immunized intranasally with CT alone or CT mixed with YopE_69–77 peptide and then challenged intranasally with 20 MLD <i>Y. pestis</i> strain D27. One day before the challenge, the YopE_69–77-immunized mice received neutralizing mAb specific for TNFα (anti-TNF), IFNγ (anti-IFN), or an isotype-matched mAb (Ctrl Ig). In comparison with CT-immunized mice (n = 20), YopE_69–77-immunized mice treated with isotype-matched mAb (n = 19) or IFNγ-neutralizing mAbs (n = 10) but not TNFα-neutralizing mAb (n = 20) were protected against <i>Y. pestis</i> challenge. Data were pooled from 4 independent experiments.</p

Selective depletion of TNFα from either macrophages/neutrophils or T cells does not impact the protection conferred by YopE_69–77 immunization.

<p>Mice with (A) monocyte/neutrophil-specific (MN-TNF KO) or (B) T cell-specific (T-TNF KO) deletion of TNFα were immunized with CT adjuvant alone or CT mixed with YopE_69–77 peptide and challenged intranasally with 20 MLD <i>Y. pestis</i> strain D27. Littermate TNF-floxed (TNF flox/flox) mice were used as controls. In comparison with CT-immunized mice (n = 10–14), YopE_69–77-immunized MN-TNF KO mice (n = 20) and T-TNF KO mice (n = 19) were protected against <i>Y. pestis</i> challenge (p<0.0001) with no significant difference from YopE_69–77-immunized TNF flox/flox mice (n = 12–13 mice/group). Data were pooled from 3 (A) and 5 (B) independent experiments.</p

Immunization with YopE_69–77 peptide protects mice against <i>Y. pestis</i>.

<p>Wild-type C57BL/6 mice were immunized intranasally with CT adjuvant alone (CT) or CT mixed with YopE_69–77 peptide (YopE) and then challenged intranasally with (A) 20 MLD (2×10⁵ CFU) or (B) 200 MLD (2×10⁶ CFU) <i>Y. pestis</i> strain D27 or (C) 10 MLD (1×10⁴ CFU) <i>Y. pestis</i> strain CO92. In comparison with CT–immunized mice (n = 10–40), YopE_69–77–immunized mice (n = 15–39) exhibited significantly increased survival. Data were pooled from 2–5 independent experiments.</p