Elevated CD4⁺ T-cell responses to relevant Ags in patients with mycobacterial- or fungal-associated IRIS events. CD4⁺ T-cell responses to TB, MAC, C neoformans, and H capsulatum were analyzed. For this purpose, patients were divided into 3 groups: those with IRIS events to mycobacterial- or fungal-associated OI and stimulated with the relevant Ag (mycobacterial/fungal-associated IRIS; red), those with IRIS events to other Ags, which can include mycobacterial/fungal Ags, but stimulated here with IRIS-irrelevant Ags (other IRIS; pink), and non-IRIS patients (non-IRIS; blue). Each data point represents stimulation with one Ag only. Shown are the response magnitudes by Ag: TB (A), MAC (B), C neoformans (C), and H capsulatum (D), as well as grouped for all 4 Ags (E). (A-D) Longitudinal data points are only connected for IRIS patients with IRIS manifestations associated with the given Ag. Cytokine pattern (F), T-cell subset distribution (G), and activation phenotype (H; tp3 only) of CD4⁺ T cells reactive to TB, MAC, C neoformans, or H capsulatum were also determined. Gray boxes indicate the first time point within 3 months of clinical manifestation of IRIS. Dashed lines separate pre-ART from on-ART samples. All groups were compared within time points with the mycobacterial/fungal-associated IRIS group (color-coded asterisks above bars or pies), as well as with corresponding pre-ART measurements within groups: red for IRIS; and blue asterisks, non-IRIS (indicated below graphs or pies). (G) The number of samples per pie are indicated (see “Data analysis”; these numbers also apply to data in panel H. i.d. indicates insufficient data. *P < .05; **P < .01; ***P < .001).