CD4+ T cell depletion, immune activation and increased production of regulatory T cells in the thymus of HIV-infected individuals

Abstract

Mechanisms by which HIV affects the thymus are multiple and only partially known, and the role of thymic dysfunction in HIV/AIDS immunopathogenesis remains poorly understood. To evaluate the effects of HIV infection on intra-thymic precursors of T cells in HIV-infected adults, we conducted a detailed immunophenotypic study of thymic tissue isolated from 7 HIV-infected and 10 HIV-negative adults who were to undergo heart surgery. We found that thymuses of HIV-infected individuals were characterized by a relative depletion of CD4+ single positive T cells and a corresponding enrichment of CD8+ single positive T cells. In addition, thymocytes derived from HIV-infected subjects showed increased levels of activated and proliferating cells. Our analysis also revealed a decreased expression of interleukin-7 receptor in early thymocytes from HIV-infected individuals, along with an increase in this same expression in mature double- and single-positive cells. Frequency of regulatory T cells (CD25+FoxP3+) was significantly increased in HIV-infected thymuses, particularly in priorly-committed CD4 single positive cells. Our data suggest that HIV infection is associated with a complex set of changes in the immunophenotype of thymocytes, including a reduction of intrathymic CD4+ T cell precursors, increased expression of activation markers, changes in the expression pattern of IL-7R and enrichment of T regulatory cells generation.

Figure 1. Flow cytometry analysis of thymocytes.

Thymocytes were characterized by their forward (FSC) and side scattering (SSC) properties(A). Thymocytes were gated by their CD3+ expression and SSC characteristics (B) in CD3 ^High(gate F), CD3 ^low (gate G), CD3 ^neg (gate H). Dot plots CD3 ^High DP and SP (C) CD3 ^low DP (D) and CD3 ^neg TN and ISP (E) thymocytes are showed. DP = double positive; TN = triple negative; SP = single positive; ISP:immature single positive (CD4+).

Figure 2. Flow cytometry evaluation of thymopoietic stages in HIV-infected (grey triangles) and uninfected controls (white circles).

All thymopoietic stages were detected in both HIV+ and HIV- thymic tissues, but these stages were skewed in HIV-infected compared to uninfected thymuses. Thus, whereas the percentage of immature triple negative cells (CD3-CD4-CD8-, TN) and of immature single positive CD4+ cells (ISP4+) was comparable in HIV+ and in HIV- patients, an increase of CD3+CD4-CD8+ single positive (SP8+) cells (46.0% versus 22.5%) as well as a reduction of double-positive (DP) (CD3brightCD4+CD8+: 23.0% versus 31.05%) and CD3+CD4+CD8- single positive (SP4+) cells (13.9% versus 37.7%) were observed in HIV+ compared to HIV- subjects.

Figure 3. Expression of CD69, CD27 and Ki67 on thymocytes in HIV-infected (grey triangles) and uninfected controls (white circles).

HIV-infected subjects showed significantly higher percentages of CD69(a) expression in single positive CD8+ cells (HIV+ 7.9%; HIV- 0.6%; p = 0.02) compared to HIV-uninfected controls. A similar trend toward higher levels of CD27 (b) expression was seen in HIV-infected patients compared to HIV-negative subjects. This was observed in triple negative cells (HIV+ 6.0%; HIV- 0.625%, p = 0.05), in CD3+ double positive cells (HIV+ 4.7%; HIV- 1.25%), and in single positive cells (SP4+ HIV+ 10.2%, HIV- 1.0%; SP8+ HIV+11.0%, HIV-1.6%, p = 0.03). Significantly higher levels of Ki67 thymocytes (c) were detected in triple negative cells (HIV+ 39.5%, HIV- 17.2%, p = 0.04) and in double positive and CD8+ single positive cells of HIV-infected patients compared to uninfected controls (CD3+DP HIV+ 36.35%, HIV-11.45%, p = 0.04; SP4+ HIV+ 5.65%, HIV- 0.45%, p = 0.04; SP8+ HIV+ 15.9%, HIV- 1.15%, p = 0.02).

Figure 4. Expression of CD25 and FoxP3 on CD4+ SP and CD3+DP thymocytes in HIV-infected (grey triangles) and uninfected controls (white circles).

The expression of CD25 and FoxP3 on CD4+ SP cells was significantly increased in thymuses of HIV-infected individuals compared to uninfected controls (HIV+ 4.3%, HIV- 1.3%, p = 0.05), whereas the expression of CD25 and FoxP3 on CD4+CD8+ DP cells was comparable in the two groups of subjects (HIV+ 0.22%, HIV- 0.19%).