Miles, B., Miller, S. M., Folkvord, J. M., Kimball, A., Chamanian, M., Meditz, A. L., Arends, T., McCarter, M. D., Levy, D. N., Rakasz, E. G., Skinner, P. J., & Connick, E. (2015). Follicular regulatory T cells impair follicular T helper cells in HIV and SIV infection. Nature communications, 6, 8608.
Human and simian immunodeficiency viruses (HIV and SIV) exploit follicular lymphoid regions by establishing high levels of viral replication and dysregulating humoral immunity. Follicular regulatory T cells (TFR) are a recently characterized subset of lymphocytes that influence the germinal centre response through interactions with follicular helper T cells (TFH). Here, utilizing both human and rhesus macaque models, we show the impact of HIV and SIV infection on TFR number and function. We find that TFR proportionately and numerically expand during infection through mechanisms involving viral entry and replication, TGF-β signalling, low apoptosis rates and the presence of regulatory dendritic cells. Further, TFR exhibit elevated regulatory phenotypes and impair TFH functions during HIV infection. Thus, TFR contribute to inefficient germinal centre responses and inhibit HIV and SIV clearance.
Haas, M. K., Levy, D. N., Folkvord, J. M., & Connick, E. (2015). Distinct patterns of Bcl-2 expression occur in R5- and X4-tropic HIV-1-producing lymphoid tissue cells infected ex vivo. AIDS research and human retroviruses, 31(3), 298-304.
Most HIV-1 replication occurs in secondary lymphoid tissues in T cells within B cell follicles. Mechanisms underlying the accumulation of HIV-1-producing cells at these sites are not understood. Antiapoptotic proteins such as Bcl-2 could promote follicular CD4(+) T cell survival, contributing to sustained virus production. Tonsils obtained from subjects without known HIV infection were disaggregated and analyzed for Bcl-2 expression in follicular (CXCR5(+)) and extrafollicular (CXCR5(-)) CD3(+)CD4(+) cells by flow cytometry. Additional tonsil cells were cultured with phytohemagglutinin (PHA) and interleukin-2 (IL-2) for 2 days, infected with either CCR5(R5) or CXCR4-tropic (X4) GFP reporter viruses, and analyzed for Bcl-2 expression. In freshly disaggregated CD3(+)CD4(+) tonsil cells, mean florescence intensity (MFI) for Bcl-2 was higher in CXCR5(+) (median, 292) compared to CXCR5(-) cells (median, 194; p=0.001). Following in vitro stimulation with PHA and IL-2, Bcl-2 MFI was higher in both CXCR5(+) cells (median, 757; p=0.03) and CXCR5(-) cells (median, 884; p=0.002) in uninfected cultures compared to freshly isolated tonsil cells. Bcl-2 MFI was higher in GFP(+)CD3(+)CD8(-) R5-producing cells (median, 554) than in X4-producing cells (median, 393; p=0.02). Bcl-2 MFI was higher in R5-producing CXCR5(+) cells (median, 840) compared to all other subsets including R5-producing CXCR5(-) cells (median, 524; p=0.04), X4-producing CXCR5(+) cells (median, 401; p=0.02), and X4-producing CXCR5(-) cells (median, 332; p=0.008). Bcl-2 expression is elevated in R5 HIV-1-producing CXCR5(+) T cells in vitro, which may contribute to propagation of R5 virus in B cell follicles in vivo.
Moore, C. M., MaWhinney, S., Forster, J. E., Carlson, N. E., Allshouse, A., Wang, X., Routy, J. P., Conway, B., & Connick, E. (2017). Accounting for dropout reason in longitudinal studies with nonignorable dropout. Statistical methods in medical research, 26(4), 1854-1866.
Dropout is a common problem in longitudinal cohort studies and clinical trials, often raising concerns of nonignorable dropout. Selection, frailty, and mixture models have been proposed to account for potentially nonignorable missingness by relating the longitudinal outcome to time of dropout. In addition, many longitudinal studies encounter multiple types of missing data or reasons for dropout, such as loss to follow-up, disease progression, treatment modifications and death. When clinically distinct dropout reasons are present, it may be preferable to control for both dropout reason and time to gain additional clinical insights. This may be especially interesting when the dropout reason and dropout times differ by the primary exposure variable. We extend a semi-parametric varying-coefficient method for nonignorable dropout to accommodate dropout reason. We apply our method to untreated HIV-infected subjects recruited to the Acute Infection and Early Disease Research Program HIV cohort and compare longitudinal CD4+ T cell count in injection drug users to nonusers with two dropout reasons: anti-retroviral treatment initiation and loss to follow-up.
Connick, E., Webb, G. M., Li, S., Mwakalundwa, G., Folkvord, J. M., Greene, J. M., Reed, J. S., Stanton, J. J., Legasse, A. W., Hobbs, T., Martin, L. D., Park, B. S., Whitney, J. B., Jeng, E. K., Wong, H. C., Nixon, D. F., Jones, R. B., Skinner, P. J., & Sacha, J. B. (2018). The human IL-15 superagonist ALT-80 directs SIV-specific CD8+ T cells into B cells follicles. Blood Advances.
Miles, B., Miller, S. M., Folkvord, J. M., Levy, D. N., Rakasz, E. G., Skinner, P. J., & Connick, E. (2016). Follicular Regulatory CD8 T Cells Impair the Germinal Center Response in SIV and Ex Vivo HIV Infection. PLoS pathogens, 12(10), e1005924.
During chronic HIV infection, viral replication is concentrated in secondary lymphoid follicles. Cytotoxic CD8 T cells control HIV replication in extrafollicular regions, but not in the follicle. Here, we show CXCR5hiCD44hiCD8 T cells are a regulatory subset differing from conventional CD8 T cells, and constitute the majority of CD8 T cells in the follicle. This subset, CD8 follicular regulatory T cells (CD8 TFR), expand in chronic SIV infection, exhibit enhanced expression of Tim-3 and IL-10, and express less perforin compared to conventional CD8 T cells. CD8 TFR modestly limit HIV replication in follicular helper T cells (TFH), impair TFH IL-21 production via Tim-3, and inhibit IgG production by B cells during ex vivo HIV infection. CD8 TFR induce TFH apoptosis through HLA-E, but induce less apoptosis than conventional CD8 T cells. These data demonstrate that a unique regulatory CD8 population exists in follicles that impairs GC function in HIV infection.
