Minimally invasive monitoring of CD4 T cells at multiple mucosal tissues after intranasal vaccination in rhesus macaques

Abstract

Studies in nonhuman primates (NHP) for prospective immune cell monitoring subsequent to infection and/or vaccination usually rely on periodic sampling of the blood samples with only occasional collections of biopsies from mucosal tissues because of safety concerns and practical constraints. Here we present evidence in support of cytobrush sampling of oral, rectal, and genital mucosal tissues as a minimally invasive approach for the phenotypic analyses of different T cells subsets de novo as well as prospectively after intranasal immunization in rhesus macaques. Significant percentages of viable lymphocytes were obtained consistently from both naïve and chronically SIV-infected rhesus macaques. The percentages of CD3+ T cells in the blood were significantly higher compared to those in the mucosal tissues analyzed in the naïve animals, while in the SIV+ animals the CD3+ T cells were significantly elevated in the rectal tissues, relative to all other sites analyzed. In the naïve, but not SIV+ macaques, the rectal and vaginal mucosal tissues, compared to oral mucosa and blood, showed higher diversity and percentages of CD4+ T cells expressing the HIV entry co-receptor CCR5 and mucosal specific adhesion (CD103) as well as activation (HLA-DR) and proliferation (Ki67) markers. Sequential daily cytobrush sampling from the oral, rectal, and genital mucosal tissues was performed in SIV+ animals from an ongoing study where they were administered intranasal immunization with adenoviral vectored vaccines incorporating the green fluorescent protein (GFP) reporter gene. We detected a transient increase in GFP+ CD4 T cells in only oral mucosa suggesting limited mucosal trafficking. In general, CD4+ and CD8+ T cells expressing Ki67 transiently increased in all mucosal tissues, but those expressing the CCR5, HLA-DR, and CD103 markers exhibited minor changes. We propose the minimally invasive cytobrush sampling as a practical approach for effective and prospective immune monitoring of the oral-genital mucosal tissues in NHP.

Determination of cell expansion and surface molecule expression on anti‐CD3/28 expanded CD4+ T cells

“…Our study shows that the expanded cells had a preferential increase in α4β7 expression to 82% of CD4 + T cells. In contrary, the level of another gut mucosal‐specific adhesion molecule, CD103, was low at the end of the culture (10% of CD4 + T cells) even though it was fivefold higher expressed in the expanded cells. It is then worth proposing that these cells feasibly migrate to the site of depletion and improve immune response at the gut‐associated lymphoid tissues.…”

Kinetics of Intratumoral Immune Cell Activation During Chemoradiation for Cervical Cancer

“…To analyze lymphocytes present in the tumor by using cytobrush, we followed a similar previously published method. (10, 12) Briefly, a sterile disposable cytobrush Plus GT (Cooper Surgical, Trumbull, CT) was used to collect cells from the cervix by rotating the brush 360° with 3 to 4 full rotations against the tumor. After collection of the sample, the brush is placed in complete RPMI 1640 media, placed on ice, and processed within 3 hours of sample collection.…”

“…(9) We recently reported the utility of this methodology for monitoring CD4 T cells at multiple mucosal tissues after intranasal vaccination in rhesus macaques. (10) By using this minimally invasive method, we characterized the immune infiltrate with multispectral flow cytometry at baseline and at weeks 1, 3, and 5 of CRT. Because T-cell infiltration has been associated with better prognosis in patients with cervical cancer and myeloid cells are known to modulate this infiltration, we focused our analysis on these populations.…”