Human Innate Mycobacterium tuberculosis–Reactive αβTCR+ Thymocytes

The control of Mycobacterium tuberculosis (Mtb) infection is heavily dependent on the adaptive Th1 cellular immune response. Paradoxically, optimal priming of the Th1 response requires activation of priming dendritic cells with Th1 cytokine IFN-γ. At present, the innate cellular mechanisms required for the generation of an optimal Th1 T cell response remain poorly characterized. We hypothesized that innate Mtb-reactive T cells provide an early source of IFN-γ to fully activate Mtb-exposed dendritic cells. Here, we report the identification of a novel population of Mtb-reactive CD4− αβTCR+ innate thymocytes. These cells are present at high frequencies, respond to Mtb-infected cells by producing IFN-γ directly ex vivo, and display characteristics of effector memory T cells. This novel innate population of Mtb-reactive T cells will drive further investigation into the role of these cells in the containment of Mtb following infectious exposure. Furthermore, this is the first demonstration of a human innate pathogen-specific αβTCR+ T cell and is likely to inspire further investigation into innate T cells recognizing other important human pathogens

Human Neonatal Dendritic Cells Are Competent in MHC Class I Antigen Processing and Presentation

Neonates are clearly more susceptible to severe disease following infection with a variety of pathogens than are adults. However, the causes for this are unclear and are often attributed to immunological immaturity. While several aspects of immunity differ between adults and neonates, the capacity of dendritic cells in neonates to process and present antigen to CD8+ T cells remains to be addressed. We used human CD8+ T cell clones to compare the ability of neonatal and adult monocyte-derived dendritic cells to present or process and present antigen using the MHC class I pathway. Specifically, we assessed the ability of dendritic cells to present antigenic peptide, present an HLA-E–restricted antigen, process and present an MHC class I-restricted antigen through the classical MHC class I pathway, and cross present cell-associated antigen via MHC class I. We found no defect in neonatal dendritic cells to perform any of these processing and presentation functions and conclude that the MHC class I antigen processing and presentation pathway is functional in neonatal dendritic cells and hence may not account for the diminished control of pathogens

DC from adult and neonatal blood are similar in their ability to process and directly present the HLA-A2 restricted antigen HCMV pp65 epitope NVLPMVATV following recombinant vaccinia infection.

<p>HLA-A2⁺ DC from adults (<i>n = </i>11) and neonates (<i>n = </i>7) were infected with vvpp65 (moi = 1.5) overnight. Infected and uninfected DC were titrated over a range (10,000 to 1,100 cells/well) and incubated overnight with pp65-specific CD8⁺ T cell clone D2-1-D2 (10,000 cells/well). IFN-γ production was detected by ELISPOT. HLA-A2⁻ DC from both adults and neonates did not induce any IFN-γ production by CD8⁺ T cell clone D2-1-D2. There was no statistically significant difference between adult and neonatal DC in processing and directly presenting antigen following recombinant vaccinia infection (p = 0.6489).</p

DC from adults and neonates are equivalent in their ability to cross present cell-associated antigen.

<div><p><b>4A</b>. To confirm inactivation of vaccinia virus, HLA-A2⁺LCL (30,000 cells/well) were co-incubated for 24 hrs with vvpp65-infected HLA-A2⁻ LCL (60,000 cells/well) prior to (left well) or after (right well) heat-inactivation (30 minutes at 56C) and UV-treatment (200mJ²). Direct presentation was then detected by IFN-γ ELISPOT after an overnight incubation with CD8⁺ T cell clones D2 1-D2 (10,000/well), specific for the HLA-A2-restricted antigen HCMV pp65.</p><p><b>4B.</b> Representative ELISPOT wells of the cross presentation assay are shown. DC (30,000/well) from five individual adult (top row) or neonatal donors (bottom row) were incubated overnight with cell-associated antigen, namely, vvpp65-infected LCL (60,000/well) that were heat (30 minutes at 56C) and UV-treated (200 mJ²). CD8⁺ T cell clones D2 1-D2, specific for the pp65 antigen, were added (10,000 cells/well) and IFN-γ was detected the following day by ELISPOT. Only HLA-A2⁺ DC cross presented the pp65 antigen (right 4 columns) and HLA-A2⁻ DC (left column) never cross presented the antigen. HLA-A2⁺ DC incubated with uninfected HLA-A2⁻ LCL never elicited a response by CD8⁺ T cell clone D2-1-D2 (not shown). In addition, vvpp65-infected HLA-A2⁻ LCL alone in the absence of HLA-A2+ DC never elicited a response by CD8⁺ T cell clone D2-1-D2 (not shown).</p><p><b>4C</b>. Combined data from 3 separate cross presentation experiments using adult (<i>n = </i>18) and neonatal (<i>n = </i>14) HLA-A2⁺ DC. No significant differences were observed (p = 0.8587).</p></div

DC from adults and neonates are comparable in their ability to present the <i>M. tuberculosis</i> antigen pronase-digested cell wall to HLA-E-restricted clone D160-1-23.

<p>DC from adult (<i>n = </i>5) and neonatal (<i>n = </i>6) blood were incubated overnight with or without the pronase-treated cell wall fraction from <i>M.tuberculosis</i>. The DC were diluted over a range of concentrations (90,000 to 1100 cells/well), and incubated with the cognate CD8⁺ HLA-E-restricted clone D160-1-23 (10,000 cells/well) overnight. IFN-γ was detected by ELISPOT. There was no statistically significant difference between adult and neonatal DC in presentation of pronase-digested cell wall to HLA-E-restricted CD8⁺ T cells (p = 0.7943).</p