Donor-But Not Recipient-Derived Cells Produce Collagen-1 in Chronically Rejected Cardiac Allografts

Fibrosis is a prominent feature of chronic allograft rejection, caused by an excessive production of matrix proteins, including collagen-1. Several cell types produce collagen-1, including mesenchymal fibroblasts and cells of hematopoietic origin. Here, we sought to determine whether tissue-resident donor-derived cells or allograft-infiltrating recipient-derived cells are responsible for allograft fibrosis, and whether hematopoietic cells contribute to collagen production. A fully MHC-mismatched mouse heterotopic heart transplantation model was used, with transient depletion of CD4+ T cells to prevent acute rejection. Collagen-1 was selectively knocked out in recipients or donors. In addition, collagen-1 was specifically deleted in hematopoietic cells. Tissue-resident macrophages were depleted using anti-CSF1R antibody. Allograft fibrosis and inflammation were quantified 20 days post-transplantation. Selective collagen-1 knock-out in recipients or donors showed that tissue-resident cells from donor hearts, but not infiltrating recipient-derived cells, are responsible for production of collagen-1 in allografts. Cell-type-specific knock-out experiments showed that hematopoietic tissue-resident cells in donor hearts substantially contributed to graft fibrosis. Tissue resident macrophages, however, were not responsible for collagen-production, as their deletion worsened allograft fibrosis. Donor-derived cells including those of hematopoietic origin determine allograft fibrosis, making them attractive targets for organ preconditioning to improve long-term transplantation outcomes

Cross‐presentation of dead‐cell‐associated antigens by DNGR‐1⁺ dendritic cells contributes to chronic allograft rejection in mice

The purpose of this study was to elucidate whether DC NK lectin group receptor-1 (DNGR-1)-dependent cross-presentation of dead-cell-associated antigens occurs after transplantation and contributes to CD8(+)T cell responses, chronic allograft rejection (CAR), and fibrosis. BALB/c or C57BL/6 hearts were heterotopically transplanted into WT, Clec9a(-/-), or Batf3(-/-)recipient C57BL/6 mice. Allografts were analyzed for cell infiltration, CD8(+)T cell activation, fibrogenesis, and CAR using immunohistochemistry, Western blot, qRT(2)-PCR, and flow cytometry. Allografts displayed infiltration by recipient DNGR-1(+)DCs, signs of CAR, and fibrosis. Allografts in Clec9a(-/-)recipients showed reduced CAR (p < 0.0001), fibrosis (P= 0.0137), CD8(+)cell infiltration (P < 0.0001), and effector cytokine levels compared to WT recipients. Batf3-deficiency greatly reduced DNGR-1(+)DC-infiltration, CAR (P < 0.0001), and fibrosis (P= 0.0382). CD8 cells infiltrating allografts of cytochrome C treated recipients, showed reduced production of CD8 effector cytokines (P < 0.05). Further, alloreactive CD8(+)T cell response in indirect pathway IFN-gamma ELISPOT was reduced in Clec9a(-/-)recipient mice (P= 0.0283). Blockade of DNGR-1 by antibody, similar to genetic elimination of the receptor, reduced CAR (P= 0.0003), fibrosis (P= 0.0273), infiltration of CD8(+)cells (p= 0.0006), and effector cytokine levels. DNGR-1-dependent alloantigen cross-presentation by DNGR-1(+)DCs induces alloreactive CD8(+)cells that induce CAR and fibrosis. Antibody against DNGR-1 can block this process and prevent CAR and fibrosis

Severe T cell hyporeactivity in ventilated COVID-19 patients correlates with prolonged virus persistence and poor outcomes

Coronavirus disease 2019 (COVID-19) can lead to pneumonia and hyperinflammation. Here we show a sensitive method to measure polyclonal T cell activation by downstream effects on responder cells like basophils, plasmacytoid dendritic cells, monocytes and neutrophils in whole blood. We report a clear T cell hyporeactivity in hospitalized COVID-19 patients that is pronounced in ventilated patients, associated with prolonged virus persistence and reversible with clinical recovery. COVID-19-induced T cell hyporeactivity is T cell extrinsic and caused by plasma components, independent of occasional immunosuppressive medication of the patients. Monocytes respond stronger in males than females and IL-2 partially restores T cell activation. Downstream markers of T cell hyporeactivity are also visible in fresh blood samples of ventilated patients. Based on our data we developed a score to predict fatal outcomes and identify patients that may benefit from strategies to overcome T cell hyporeactivity.Coronavirus disease 2019 (COVID-19) can lead to pneumonia and hyperinflammation. Here we show a sensitive method to measure polyclonal T cell activation by downstream effects on responder cells like basophils, plasmacytoid dendritic cells, monocytes and neutrophils in whole blood. We report a clear T cell hyporeactivity in hospitalized COVID-19 patients that is pronounced in ventilated patients, associated with prolonged virus persistence and reversible with clinical recovery. COVID-19-induced T cell hyporeactivity is T cell extrinsic and caused by plasma components, independent of occasional immunosuppressive medication of the patients. Monocytes respond stronger in males than females and IL-2 partially restores T cell activation. Downstream markers of T cell hyporeactivity are also visible in fresh blood samples of ventilated patients. Based on our data we developed a score to predict fatal outcomes and identify patients that may benefit from strategies to overcome T cell hyporeactivity

Strain-Transcending Fc-Dependent Killing of Plasmodium falciparum by Merozoite Surface Protein 2 Allele-Specific Human Antibodies▿ †

It is widely accepted that antibody responses against the human parasitic pathogen Plasmodium falciparum protect the host from the rigors of severe malaria and death. However, there is a continuing need for the development of in vitro correlate assays of immune protection. To this end, the capacity of human monoclonal and polyclonal antibodies in eliciting phagocytosis and parasite growth inhibition via Fcγ receptor-dependent mechanisms was explored. In examining the extent to which sequence diversity in merozoite surface protein 2 (MSP2) results in the evasion of antibody responses, an unexpectedly high level of heterologous function was measured for allele-specific human antibodies. The dependence on Fcγ receptors for opsonic phagocytosis and monocyte-mediated antibody-dependent parasite inhibition was demonstrated by the mutation of the Fc domain of monoclonal antibodies against both MSP2 and a novel vaccine candidate, peptide 27 from the gene PFF0165c. The described flow cytometry-based functional assays are expected to be useful for assessing immunity in naturally infected and vaccinated individuals and for prioritizing among blood-stage antigens for inclusion in blood-stage vaccines

Specificity of activation of C7 clone and protection<b>.</b>

<p>Mice were first injected (iv) or not with 10³ live wild-type (<i>wt</i>) ANKA <i>Pb</i>SPZ 10 h before they received or not 7×10⁵ naïve-BALB/c hepatocytes and C7 clone as indicated above. Infected hepatocytes were isolated from BALB/c mice injected with 10⁶ ANKA <i>wt Pb</i>SPZ 2 h earlier; and naïve BALB/c hepatocytes were isolated from naïve BALB/c mice. Mice were considered protected if they remained parasite negative 2 weeks after infection.</p

BALB/c mice injected with iSPZ-loaded hepatocytes are protected against SPZ challenge.

<p>BALB/c mice were injected (IS transfer) with 7×10⁵<i>Pb</i>iSPZ-infected or naïve BALB/c hepatocytes. Infected hepatocytes were obtained from BALB/c mice immunized with 10⁶ ANKA <i>wt</i> iSPZ 2 h earlier. Recipient mice were then challenged with two different doses (2×10³ and 5×10³, respectively, A and B) of live <i>Pb</i>SPZ one week later. Mice were considered protected if they remained parasite negative 2 weeks after challenge.</p

Relative comparative <i>wt</i> and <i>spect (</i>−<i>)</i> parasite DNA load in spleen and liver of immunized mice.

<p>To compare relative load of <i>wt</i> and <i>spect (</i>−<i>)</i> parasite DNA in liver and spleen, BALB/c (3 mice per group) were immunized iv on tail with 1×10⁵ iSPZ (<i>wt</i> or <i>spect (</i>−<i>)</i>) in 500 µl of RPMI. Two hours later, a real-time PCR was performed following extraction of respective parasite RNA. Every sample was done in duplicate. To avoid any contamination by eventual parasite from blood, the liver and spleen were perfused with PBS before performing the RT-PCR. Figures <b>a</b> and <b>b</b> represent <i>wt</i> and <i>spect (</i>−<i>)</i> parasite DNA load, respectively, in the liver and spleen 2 h after iSPZ injection (iv). Naïve mice (receiving only 500 µl iv of DMEM) were used as a control.</p

Infected hepatocytes present a <i>Pb</i>CSP-specific epitope to primed CD8+ T-cells and protect mice against SPZ challenge.

<p>Each recipient <i>TAP−/−</i> mouse (H-2K^b) received 7×10⁵<i>Pb</i>SPZ-infected BALB/c (H-2k^d) hepatocytes by IS transfer as described in Materials and Methods. Before IS injection, infected hepatocytes were isolated from BALB/c mice that were injected (iv) with 10⁶ANKA <i>wt Pb</i>SPZ 2 h earlier. C7 and S14 (20 million cells per mouse, iv) were injected into the corresponding group 4 h after IS transfer. Mice were protected if they remained parasite negative 2 weeks after infected hepatocyte transfer.</p

Frequency of <i>Pb</i>CSP-specific CD8+ T-cells in different organs of BALB/c mice immunized with <i>wt</i> or <i>spect (</i>−<i>)</i> iSPZ.

<p>BALB/c mice (4 mice per group) were immunized (iv) with <i>wt</i> or <i>spect (</i>−<i>)</i> ANKA <i>Pb</i>iSPZ (one dose of 1×10⁵ iSPZ). Seven (7) days later, PBL, liver and spleen cells of mice were isolated to determine frequency of <i>Pb</i>CSP-specific CD8+ T-cells by flow cytometry (FACScan). The CD8+ T-cells were double stained with PE-conjugated <i>Pb</i>CSP epitope tetramer and FITC-conjugated anti-mouse CD8b antibody. The naive group received neither <i>wt</i> nor <i>spect (</i>−<i>)</i> iSPZ. p-value compares statistically significant mean of CD8+ T-cell frequency between <i>wt</i> and <i>spect (</i>−<i>)</i> iSPZ-immunized groups.</p