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The peripheral blood represents only a small fraction of the total number of lymphocytes in the body. To develop a more thorough understanding of T cell dynamics, including the effects of SIV/SHIV/HIV infection on immune cell depletion and immune reconstitution following combination antiretroviral therapy (cART), one needs to utilize approaches that allow direct visualization of lymphoid tissues. In the present study, noninvasive in vivo imaging of the CD4+ T cell pool has revealed that the timing of the CD4+ T cell pool reconstitution following initiation of ART in SIV-infected nonhuman primates (NHPs) appears seemingly stochastic among clusters of lymph nodes within the same host. At 4 weeks following initiation or interruption of cART, the changes observed in peripheral blood (PB) are primarily related to changes in the whole-body CD4 pool rather than changes in lymphocyte trafficking. Lymph node CD4 pools in long-term antiretroviral-treated and plasma viral load-suppressed hosts appear suboptimally reconstituted compared with healthy controls, while splenic CD4 pools appear similar between the 2 groups.

Ionizing irradiation is used routinely to induce myeloablation and immunosuppression. However, it has not been possible to evaluate the extent of ablation without invasive biopsy. For lymphoid recovery, peripheral blood (PB) lymphocytes (PBLs) have been used for analysis, but they represent

It has not been possible to evaluate immune recovery in tissues without invasive biopsy. Investigators have had to rely on peripheral blood lymphocytes (PBLs) for analysis. PBLs, however, represent only a very small percentage of cells in lymphoid tissues (LTs). Using a combination of SPECTanda radiotracer, rhesus IgG1 anti-CD4R1 Tc-99m(Fab')2,we sequentially imaged CD4+ cell recovery in rhesus macaques following varying doses of total body irradiation (TBI) and reinfusion of vector transduced, autologous CD34+ cells. Seven rhesus were used in this study. Two received a dose of 3Gy on two sequential days (3Gyx2days) of TBI (6Gy total), three received 4Gyx2days TBI, and two 5Gyx2days TBI. CD34+ cells were immunoselected from a leukapheresis product following G-CSF and SCF mobilization over 5 days. On the last day of TBI a mean (SD) of 5.9(1.7)x106/kg autologous CD34+ cells were reinfused after being transduced once (moi=50) with a SIV/HIV chimeric lentiviral vector expressing EGFP. SPECT/CT images were taken prior to transplant (Figure 1) and at approximately 6, 30, 90, 150, 260 and 380 days post-TBI. Development of immunogenicity to the radiotracer was monitored using size exclusion HPLC and an in vitro cell binding assay. Following 3Gyx2days TBI, both animals rejected their CD34+ cell graft despite achieving a PBL nadir of Unlike the 3Gyx2days TBI animals, no immunogenicity to the radiotracer was detected in the 4Gyx2days TBI rhesus monkeys until at least one year post transplant. After the 6 or 7th exposure, all three animals developed an immune response to the radiotracer. This suggests immune recovery remained incomplete in the animals receiving a dose of 4Gyx2days TBI or higher for at least one year following transplant. No immune response has been observed at 5Gyx2 at d.245. Our results present for the first time a non-invasive method in real time to evaluate CD4+ cell immune recovery in LTs following hematopoietic stem cell transplantation using SPECT/CT imaging. Despite myeloablation of circulating leukocytes following TBI, we did not observe total depletion of CD4+ cells in LTs such as the spleen. This is problematic if one is concerned about residual disease or endogenous contributions to recovery. The methodologies presented here have direct applications to the evaluation of immunosuppressive therapies and other immunosuppressive disorders, such as those associated with viral infections. Figure 1 Figure 1. Baseline SPECT image depicting lymphoid organs. Figure 2 Figure 2. SPECT images comparing 3Gyx2 (ZI12), 4Gyx2 (ZG21, ZH32, ZG41), and 5Gyx2 (ZG70 and ZI64) d.6 post-TBI and an animal chronically infected with SHIVDH12R(G43). Despite low circulating levels of CD4+ cells in all animals, residual CD4+ cells remain within LT such as the spleen following TBI, and more complete depletion associated with chronic SHIV infection. Figure 3 Figure 3. Control and d.6 post-TBI anti-CD3 IHC of spleen and axillary LN. Disclosures No relevant conflicts of interest to declare.

Background Historically lymphocyte (Lc) recovery following hematopoietic transplantation, immunosuppressive therapies, and infectious diseases has relied quite heavily on peripheral blood (PB) analysis. The PB, however, comprises less than 2% of total Lcs in the body, hence small changes in trafficking rates (e.g., lymphoid tissue (LT) homing) could have profound effects on PB Lc count. In this study we explored the use of a radiotracer rhesus IgG1 anti-CD4R1 Tc99mFab'2 and single photon emission computed tomography (SPECT/CT) to sequentially image CD4+ Lc recovery in the whole-body following total body irradiation (TBI) and autologous CD34+ cell transplant. Methods Three specific pathogen free rhesus macaques were used in this study. Each received 4Gy TBI on two sequential days (8Gy total). CD34+ cells were immunoselected from a leukapheresis product following cytokine mobilization over 5 days using G-CSF and Stem Cell Factor. 4-9x106/kg CD34+ cells were reinfused on the last day of irradiation after being transduced in culture once with a SIV/HIV chimeric lentiviral vector expressing EGFP at a multiplicity of infection of 50. SPECT/CT images were taken prior to transplant as a baseline; following mobilization with AMD3100 at least two months prior to transplant; and at approximately 6, 30, 90, 150 and 260 days post-TBI/transplant. Development of immunogenicity to the radiotracer was monitored using size exclusion high performance liquid chromatography (HPLC) and an in vitro cell binding assay. Results Dynamic SPECT acquisition for two hours following mobilization with 1mg/kg AMD3100 SQ did not reveal measurable changes in the whole-body CD4 pool, despite an ∼ 2-fold maximum increase in CD4+ PB cell counts. This is consistent with a small percentage of CD4+ cells being released from tissues to PB without significantly affecting total Lc counts in the whole-body. Following 4Gyx2 TBI, all 3 animals experienced dramatic depletion of PB CD4 counts (a 90-98% decrease from baseline) (Table 1). Variability in tissue CD4 depletion was observed between and within hosts, with more dramatic decreases observed in axillary lymph nodes (73-92%) and only mild decreases in the spleen (36-40%). Similarly, recovery of the CD4 pools in LTs following transplant varied between and within hosts and appeared overall slower than the dynamics of repopulation in the PB, suggesting changes in trafficking rates between LTs and PB has a role in transplant recovery. By month 9 post-TBI, one animal (ZG41) had full recovery of the PB CD4+ Lc count and the CD4 pool in axillary LNs quantified by the SPECT/CT, but minimal recovery of CD4 pools were observed in the spleen. In another animal (ZG21), full recovery of the PB CD4+ cell count was observed by month 5 concomitant with full recovery of the spleen CD4 pool, despite still greater than 70% depletion of CD4 pool in axillary LNs. In the last animal (ZH32), by month 3 the CD4+ Lc count in PB was fully recovered, however, minimal CD4 pool recoveries were observed in LT through month 9 post-TBI (Figure 1). EGFP expressing Lcs first appeared in the circulation on average(SD) 69(48) days following transplant and were CD8+ and CD20+. In all animals, CD4+ EGFP+ Lcs were not observed until after month 5, suggesting that endogenous recovery preceded graft recovery in the PB. Of importance is that no immunogenicity to the radiotracer was detected despite sequential imaging up to month 5 in one animal and up to month 9 in the other two animals. This is in contrast to animals that were imaged several years post-transplant. In these animals immune complex formation to the radiotracer was observed by HPLC after a single exposure to the rhesus IgG1 anti-CD4R1 Tc99mFab'2. This suggests either tolerance was induced in animals exposed to the radiotracer shortly following transplant or that immune recovery remains incomplete after 150 days in these animals. Conclusions These results demonstrate for the first time that immune reconstitution of lymphoid tissues can be effectively monitored following transplant of autologous CD34+ cells and TBI, that the reconstitution appears to be discordant between PB and LT, as well as identifying a potential approach to decreasing immunogenicity to complex antigens through lymphocyte mobilization during antigen exposure. Disclosures: No relevant conflicts of interest to declare.