CD4 T cells (Thelper, TH) play a key role in antiviral immunity. The magnitude of HIV-specific TH responses generated by infection or vaccination is highly variable. Previous studies suggest that antiviral TH responses can be generated prior to pathogen exposure by cross-reactivity with other microorganisms, and may shape TH responses upon infection or immunization. However, the functionality of such pre-existing HIV-specific TH in HIV-negative subjects is unknown.
We investigated HIV-specific TH responses in HIV-uninfected donors (UD, n=18) and compared them to those of HIV-infected subjects (HI, n=53). We measured TH proliferative responses to HIV antigen (Ag) peptide pools using CFSE assays and grew HIV-specific TH cell lines (CL). We determined ex vivo responses firstly by ICS and secondly by co-upregulation of activation-induced markers (AIM) after Ag stimulation: i) CD69 and CD40L; or ii) CD25 and Ox40.
We identified a high prevalence of HIV-specific proliferative TH responses in UD; 33% had a robust CFSE response to one or more HIV Ags (Gag, Env, Nef or Pol; net >1% and >2x No Ag background). Gag was less immunodominant in UD than HI: the strongest response was against Gag in 33% of UD vs. 68% of HI (p=0.013, Fisher exact test). While ICS for Th0/1 cytokines (IFNγ, IL2, TNF) and CD40L failed to identify HIV-specific TH in PBMCs directly ex vivo, the same ICS assay on Gag- and gp41-specific CL derived from UD (n=12) showed that these responses were functional and dominated by TNF and CD40L, but produced little IFNγ. In contrast to ICS, AIM assays detected HIV-specific TH from UD without pre-expansion: net HIV-specific TH frequencies (cutoff: >2x No Ag value) ranged from 0.15 to 0.79% for CD69/CD40L and 0.3 to 1.60% for CD25/Ox40. Compared to total TH, HIV-specific Ox40+CD25+ TH were enriched in central memory (median 62% vs. 36%) and T follicular helper cells (median 20% vs. 14%), and preferentially expressed CXCR3 (median 46% vs. 35%). There was a direct correlation between the magnitudes of HIV-specific TH measured by the AIM and CFSE assays (n=20; CD69/CD40L: p=0.01, r=0.55; Ox40/CD25: p=0.01, r=0.56; Spearman).
These results demonstrate that HIV-specific TH cells exist in a substantial proportion of UD, can target diverse HIV Ags and are detectable by functional assays including CFSE and AIM. These cross-reactive CD4 T cells could impact the development of virus-specific TH responses upon acute HIV infection and influence vaccine-induced immunity.