Fig 1.
Tfh cell subsets, including Tfr, are present in greater proportions in HIV+ spleens.
(A) Splenocytes were stained for T helper cell markers and analyzed by flow cytometry: according to the expression of CD45RA and CCR7, CD4 T cells were subdivided into CD45RA+CCR7+ naïve, CD45RA-CCR7- effector memory (TEM), CD45RA+CCR7- terminally differentiated effector memory (TEMRA) and CD45RA-CCR7+ central memory (TCM) T cells. Regulatory T (Treg) cells were identified as CD4+ CD45RA- Foxp3+ CD25+. HIV-ITP- n = 8, HIV+ITP- n = 4, HIV-ITP+ n = 3 and HIV+ITP+ n = 9. (B) Tfh cells were identified as CD3+ CD4+ CD45RA- CCR7- CXCR5+, GCTfh are the CD57+ subset of Tfh, and Tfr are Foxp3+ Tfh cells (S1 Fig). The frequency of Tfh, GCTfh and Tfr cells is represented as the percentage of total memory CD4+ T cells. HIV-ITP- n = 8, HIV+ITP- n = 4, HIV-ITP+ n = 3 and HIV+ITP+ n = 9. Frequency of Treg and Foxp3+ Tfh (Tfr) were identified for HIV-ITP- n = 4, HIV-ITP+ n = 3, HIV+ITP- n = 3 and HIV+ITP+ n = 4. Statistics were obtained using the non parametric Mann-Whitney test *p<0.05, **p<0.005, ***p<0.001.
Fig 2.
GC B cells accumulate whereas memory B cell compartment is reduced in HIV+ spleens.
(A) Using flow cytometry, B cell maturation was assessed according to the expression of CD38 and IgD markers by CD19+ cells (34). The gating strategy is presented in S1 Fig. In HIV chronically infected patients, B cell maturation is biased towards GC B cells and PC subsets as compared to HIV- donors and ITP+ patients. (B) The transitional B cell population was identified as CD19+CD38++IgD+CD27-IgM+ and plasma cells as CD19+CD38++IgD-. In the box-and-whiskers plot, box size represents the limits of data for the second and third quartiles, with medians shown as bars. Whiskers define the minimum and maximum of the data presented. HIV-ITP- n = 8, HIV-ITP+ n = 3, HIV+ITP- n = 5 and HIV+ITP+ n = 8. Statistics were obtained using the non parametric Mann-Whitney test *p<0.05, **p<0.005, ***p<0.001.
Fig 3.
HIV infection severely impacts transcription profile of splenic Tfh and GCTfh cells.
GCTfh and Tfh were sorted using flow cytometry based on the expression of CXCR5 and PD-1 associated or not with CD57 marker respectively (S2 Fig). Sorted cells were used to perform high throughput gene expression measurement with real time PCR in a microfluidic dynamic array. After cleansing and normalization, consistent data were used for statistical analysis. We focused on genes specifically implicated in T cell functions (S1 Table). (A) 2D Principal component analysis (PCA) plot representation of gene expression profile of Tfh (triangle) and GCTfh (diamond) cells from HIV- ITP- (n = 4, empty symbols) and HIV+ ITP+ (n = 5, filled symbols) spleens. The projection of the data on the first and second principal components efficiently discriminates HIV+ samples, suggesting that HIV-infection deeply impacts gene expression profile of Tfh and GCTfh cells. (B) mRNA expression in Tfh and GCTfh sorted from splenocytes of uninfected (n = 4) and HIV+ (n = 5) individuals. The data are expressed in 40-Ct where Ct represents the threshold cycle number and 40 is chosen because the PCR run stops after 40 cycles. This value is directly correlated with the initial amount of RNA and therefore allows a quantitative comparison for 18 genes encoding cytokines/chemokines and proteins implicated in Tfh differentiation, costimulation, immune regulation and signal transduction. Statistics were obtained using the non parametric Mann-Whitney test *p<0.05, **p<0.005, ***p<0.001.
Fig 4.
Activated HIV+ splenocytes fail to produce IL-4 and IL-10: Total splenocytes were stimulated with PHA for 2 days and culture supernatants were analyzed for IL-1ß, IL-6, Il-4 and IL-10 using enhanced sensitivity BD CBA flex set assay.
Horizontal bars represent mean and error bars show SEM. HIV-ITP- n = 4, HIV-ITP+ n = 3, HIV+ITP- n = 4 and HIV+ITP+ n = 5. Statistics were obtained using the non parametric Mann-Whitney test *p<0.05, **p<0.005, ***p<0.001.
Fig 5.
Splenic Tfh subset harbor high amount of HIV-1 DNA integration.
qRT-PCR quantification of HIV proviral DNA levels in GCTfh, Tfh, memory and naïve CD4+ T cell subsets isolated from splenocytes of five HIV+ ITP+ individuals as described in S1 Fig. Symbols represent individual samples: horizontal bars represent mean; and error bars show SEM. Statistics were obtained using the non parametric Mann-Whitney test *p<0.05, **p<0.005, ***p<0.001.