The highest Spearman rank correlations were noted for the spleen ( 0.70, 0.01), followed by the axillary and submandibular LNs ( 0.52, 0.05), suggesting that the majority of the radiotracer uptake in these organs is the result of specific binding of the radiotracer to CD4+ cells. were noted between radiotracer uptake in spleen, tonsil and axillary lymph nodes (LNs), and peripheral blood (PB) CD4+ T cell counts. Minimal uptake was noted in the gastrointestinal tract. These data highlighted the limitations of using the PB CD4+ T cell counts to infer the level of whole-body immunocompetence and challenged the prevalent notion that this gut is the major reservoir of CD4+ T cells in the body. It has been suggested that this increases in the numbers of CD4+ T cells in the PB of HIV-1Cinfected subjects after initiation of combination antiretroviral therapy (cART) result from a combination of 2 phenomena: decreases in immune activation leading to an increase in distribution of cells from lymphoid tissues (LTs) to PB, and increases in the total number of CD4 cells in the body due to a decrease in cell death (2). Determination of the relative contributions of these 2 components requires simultaneous and repeated measurements of both PB and total LTs. This is now Emicerfont possible through the use of PB sampling and noninvasive, total body imaging with CD4-specific probes. Initial longitudinal imaging studies to examine the effects of cART were compromised due to the immunogenicity of the probe (1). Accordingly, the probes were modified from intact antibodies Emicerfont into F(ab)2 fragments that were then coupled with the emitter 99mTechnetium (99mTc) without loss in binding affinity to CD4 receptors. Utilizing F(ab)2 fragments of the humanized CDR-OKT4A/huIgG4 and of the rhesus recombinant CD4R1-OKT4A/rhIgG1 mAbs, hereafter called F(ab)2-OKT4A and F(ab)2-CD4R1, respectively, longitudinal studies became possible, as recently exhibited by our group in the settings of hematopoietic stem cell transplantation following total body irradiation in NHPs (3). These altered radiotracers produced images with lower background compared with the intact mAb Emicerfont and considerably less immunogenicity than the intact antibodies. The current work examines longitudinal changes in the CD4 pool in SIV-infected NHPs following the initiation or interruption of cART. Results The in vitro and ex lover vivo specificity assessments and the in vivo stability and biodistribution studies were performed for both the 99mTc-F(ab)2-OKT4A, and the 99mTc-F(ab)2-CD4R1 radiotracers, as previously explained (1, 3). These preliminary studies showed high immunoreactivity and optimal stability of both probes, as reported in Supplemental Physique 1 (supplemental material available online with this short article; https://doi.org/10.1172/jci.insight.97880DS1). Associations between Emicerfont LTs and PB CD4 pools in SIV-infected NHPs. A summary of the imaging experiments and RPTOR characterizations of the monkeys imaged are shown in Supplemental Table 1. Eleven NHPs, spanning a range of CD4+ T cell counts from 28C1,324, received 6.04 1.27 mCi of 99mTc-F(ab)2-OKT4A, and 19 NHPs, spanning a range of CD4+ T cell counts from 7C1,718 cells/l, received 6.12 1.07 mCi of 99mTc-F(ab)2-CD4R1 i.v. after fasting for at least 8 hours. No association was found between the mean fluorescence intensity (MFI) of CD4 cells in the PB and the PB CD4+ T cell count (not shown). Therefore, any differences in probe binding likely reflected differences in total numbers of cells rather than differences in the number of antibody molecules bound per cell. Whole-body SPECT images were acquired from the top of the skull to the inguinal region for all animals at 4C6 hours after radiotracer injection (p.r.i.) and, for any subgroup of animals, also at 19 hours p.r.i. The semi-quantitative analyses of the 3D-SPECT images are here summarized for all the images acquired at 4C6 hours p.r.i. Whole-body region of interest (ROI) analyses revealed no significant correlations between PB CD4+ T cell count and hepatic uptake (OKT4A: = C0.04, = 0.46, = 11; CD4R1: = C0.21, = 0.20, = 19). In contrast, strong correlations were found between the radiotracer retention in LTs and PB CD4+ T cell count (Table 1 and Physique 1). The highest Spearman rank correlations were noted for the spleen ( 0.70, 0.01), followed by the axillary and submandibular LNs ( 0.52, 0.05), suggesting that the majority of the radiotracer uptake in these organs is the result of specific binding of the radiotracer to CD4+ cells. Moreover, nonstatistically significant associations were observed between radiotracer uptake in kidney, heart, thymus, and bone marrow (proximal and central humerus) and PB CD4+ T cell count, suggesting that the majority of the radiotracer uptake in those organs was the result of nonspecific uptake. This pattern of correlations, or lack thereof, was also observed with the SUVmax and, overall, were found to be amazingly much like those reported for.