in either preintegration or postintegration phases of infection . As preintegration latency does not persist during long term ART, we sought to identify postintegration Parietin latency in infected resting CD4 T cells in BLT mice. We first demonstrated the presence of resting CD4 T cells in naive BLT mice. For this analysis, we isolated mononuclear cells from peripheral blood, bone marrow, spleen, lymph nodes, liver, and lung according to published protocols . All mononuclear cells from individual mice were combined and resting human CD4 T cells were negatively selected by magnetic separation essentially using the same approach used to isolate resting cells from human leukapheresis product . Briefly, cells were ubated with antibodies specific for mouse CD45 and TER119 and for human CD8, CD14, CD16, CD19, CD56, glycophorin A, CD41, HLA DR, CD25 , and CD31 and CD105 .
Antibody bound cells were removed using magnetic selection and the flowthrough fraction containing the purified resting cells was collected. This negative selection approach resulted in a 97% pure resting CD4 T cell population as defined by expression of CD4, CCR7, and CD27 and lack of expression Bcl-2 Signaling Pathway of CD8, CD25, HLA DR, and CD11b . After establishing that resting CD4 T cells are present in BLT mice and that they can be isolated with the same procedure used to obtain similar cells from human peripheral blood, we determined whether a latent reservoir of HIV infected cells is present in infected BLT mice on ART. The general approach used to accomplish this objective is described in Fig. 3A.
Administration of ART to infected BLT mice resulted in an average 3.2 log reduction in plasma viral load . Mononuclear cells from peripheral blood, lymph nodes, human thymic organoid, spleen, bone marrow, lungs and liver were collected and all cells from each individual mouse were pooled for resting cell isolation. Resting CD4 T cell enrichment was performed on each pool paraffin of cells, as described above . The average yield of resting cells per BLT mouse was 3.0 106 . To limit the potential contribution of nonintegrated HIV DNA to the outgrowth assay results, the enriched population of resting cells was cultured in the presence of 15 nM efavirenz and 1 Mraltegravir for 2 days prior to any stimulation for viral outgrowth . The lack of ongoing virion production in resting cells maintained in efavirenz and raltegravir was confirmed prior to coculture and stimulation of viral outgrowth as previously published .
The frequency of resting cell infection was determined using a maximum likelihood method, and the results were expressed as infectious units per million resting CD4 T cells . The coculture assay results yielded an average of 9.9 IUPM resting CD4 T cells per mouse . These results demonstrate that HIV infection of humanized mice results in the establishment of a population of resting cells latently infected with HIV that can be readily isolated and induced to express HIV ex vivo, recapitulating key aspects of the human condition. Because of the relatively short frame of time necessary to observe ART efficacy in this model, preclinical evaluation of successful HIV eradication interventions can be rapidly In summary, we have established a novel application of the humanized BLT mouse model for the study.