Seasonal influenza vaccine responsiveness is dependent on a specialised subset of CD4+ T-cells that are susceptible to infection with HIV. The role of CXCR5+ circulating T-follicular helper cells (cTFH) in this response is unclear in individuals with treated suppressed HIV infection. Investigations of potential biomarkers for HIV integration have identified rare CD4+ T-cells highly expressing the FC gamma receptor CD32, but with unknown function. We hypothesised that CD32 is upregulated on cTFH in response to seasonal influenza vaccine and used unsupervised computer algorithms to explore the cellular relationships arising.
16 men with treated, suppressed HIV infection and 14 healthy control subjects receiving quadrivalent influenza vaccine (QIV) during the 2017-18 Northern Hemisphere influenza season were studied. Peripheral blood mononuclear cells (PBMCs) were collected prior to and after vaccination. Thawed PBMCs were stained with a pre-optimised cocktail of flurochrome-conjugated antibodies, before acquisition on a BD Fortessa flow cytometer. The data were analysed using T-stochastic neighbour embedding analysis (t-SNE) and Spanning-tree progression analysis of density-normalized events in FlowJo v10.4.2 and FCS express v6plus.
cTFH more frequently expressed CD32 at Day 7 post QIV (p=0.0009) and returned to baseline at Day 28 (p<0.0001) with no difference in those with and without HIV infection. t-SNE identified three populations (P1, P2 and P3) of CD4+ T-cells that were defined by their expression of CXCR5 and CD32. P1 (CXCR5hiCD32hi) and P3 (CXCR5midCD32lo/mid) frequency was constant but P2 (CXCR5lo/midCD32lo/mid) was more frequent at Day 7 (p=0.0261) and expressed the cTFH activation markers programmed cell death 1 (PD-1) and inducible T-cell co-stimulator (ICOS). SPADE indicated a branched hierarchy of clustered nodes corresponding to P1, 2 and 3. Consistently, a central memory CXCR5mid node gave rise to a CXCR5hiCD32hi node that was unaffected by QIV and two vaccine-inducible activated ICOS+PD-1+CD38+CXCR3+CXCR5+CD32mid/hi nodes.
Circulating CXCR5+CD4+ T-cells fall into three major related populations. A parent population of cTFH-like cells gives rise to a vaccine-responsive cTFH population that upregulates CD32 and a vaccine-unresponsive population persistently expressing CD32 and CXCR5. These relationships were present irrespective of HIV infection in individuals receiving QIV and could be used to inform vaccine design.