. Author manuscript; available in PMC: 2020 Jan 1.

Published in final edited form as: Bioessays. 2018 Dec 10;41(1):e1800128. doi: 10.1002/bies.201800128

Table 1.

Hypotheses conforming the proposed model of CD38-mediated indirect pathogenesis in HIV infection, and possible experimental approaches to test each.

Hypothesis	Possible experiments
2. 1 Catalytic activity where CD38 is present	Comparison of catalytic activity between CD38⁺ and CD38⁻ cells. Comparison of catalytic activity of CD4 T cells from HIV+ patients and healthy controls. Analysis of correlation of catalytic activity with CD38 expression. Possible methods: Degradation of NAD or an analog in culture media by colorimetry or HPLC-MS.
2.2 NAD depletion by CD38 activity	NAD measurement in lysates from CD4 T cells from patients and controls (colorimetry or HPLC-MS), and determination of its correlation with CD38 expression.
2. 3 cADPR- and ADPR production by CD38	ADPR and cADPR concentration determination in culture media and lysates of CD4 T cells from patients and healthy controls. Correlation with CD38 expression. Possible method: HPLC-MS
2. 4 Metabolic switch induced by NAD depletion	Determination of oxygen consumption rate (OCR, a measure of mitochondrial respiration) and extracellular acidification rate (ECAR, a measure of glycolytic activity) in CD4 T cells from HIV⁺ patients and controls. Purified cells with the same degree of differentiation should be used^*. Possible method: OCR/ECAR ratio by mitocondrial stress and glycolytic stress kinetic determinations with available instrumentation and kits.
2. 5 Increased cytoplasmic Ca²⁺	Comparison of basal Ca²⁺ levels in CD4 T cells from HIV⁺ patients and healthy controls^. Analysis of correlation with CD38 expression. Comparison of Ca²⁺ flux after TCR-mediated stimulation in CD4 T cells from HIV⁺ patients and healthy controls with the same degree of differentiation. ^ Analysis of correlation with CD38 expression. Effect of antagonists of cADPR (8-Br-cADPR) and of ADPR in cytoplasmic Ca²⁺ concentrations and functionality (see below) of CD4 T cells from HIV⁺ patients and healthy controls^*. Possible method: flow cytometry using a Ca²⁺ molecular probe.
2. 6. Mitochondrial damage	Mitochondrial morphology determination (e.g. mitocondrial fusion or fission and cristae integrity by confocal and electronic microspcopy)^. Mitochonrial biogenesis determination. Possible methods: microscopy and flow Cytometry with mitocondria-specific molecular probes^.
2.7 Altered survival, proliferation, cytokine production, and differentiation	Effect of pharmacological inhibition of CD38’s catalytic function in proliferation, survival, differentiation, and cytokine production after stimulation of CD4 T cells from patients and controls ^*. Determination of functional properties of CD4 T cell lines (for instance, Jurkat cells) expressing functional or non-catalytic variants of CD38. Possible method: CRISPR-Cas9 deletion of CD38 gen, followed by transfection with plasmids codifying for CD38 variants with or without catalytic function. Functional tests may be carried out by flow Cytometry using fluorochrome-conjugated monoclonal antibodies, cell division molecular probes, and viabiity molecular probes.

The use of CD4 T cells from patients and controls with a same degree of differentiation is proposed to rule out confounding effects of differentiation. Variables affected by differentiation include cytokine production, viability, proliferative capacity, differentiation capacity, and metabolic profiles.