FACS analysis of multilineage human hematopoiesis in humanized Rag1^−/−γc^−/− mice.

<p>Single cell suspensions were made from spleen, bone marrow, lymph node and thymus of humanized Rag1^−/−γc^−/− mice and were stained with different antibodies to detect human hematopoietic cell sub-sets. Human anti-CD45 antibody was used to define human leukocytes. From this population human CD3+ T cells (A) as well as CD4+ and CD8+ T cell subsets were identified in the lymph node, spleen and thymus (B). Dendritic cells were identified by their lack of lineage staining (CD3, CD19, CD14, CD16, CD20 and CD56) and expression of HLA DR (C). Both myeloid (CD11c) and plasmacytoid (CD123) dendritic cells (D) were identified in lymphoid organs as were CD14 expressing monocytes (E) and two subsets of CD19 and CD20 expressing B cells (F).</p

Immunohistochemical staining of human hematopoietic cells in lymphoid organs.

<p>Tissue sections of lymphoid organs (spleen, lymph node, and thymus) from humanized Rag1^−/−γc^−/− mice were stained for the presence of human leukocytes. CD45⁺ leukocytes (A,B,C), CD68⁺ macrophages/dendritic cells (D,E,F), CD3⁺ T cells (G,H,I), CD4⁺ helper T cells (J,K,L), CD8⁺ cytotoxic T cells (M,N,O) and CD20⁺ B cells (P,Q,R) were detected in each of the organs assayed.</p

Human CD45 cell engraftment levels in humanized Rag1^−/−γc^−/− mice.

<p>Human CD34 cell reconstituted mice were bled at 12 weeks post-engraftment. RBCs were lysed and the white blood cell fraction was stained for human panleukocyte CD45 marker and FACS analyzed. The level of human cell engraftment for each mouse is depicted.</p

Comparison of Rag1^−/−γc^−/− versus Rag2^−/−γc^−/− mice for generation of humanized mice (Hu Mice).

<p>Comparison of Rag1^−/−γc^−/− versus Rag2^−/−γc^−/− mice for generation of humanized mice (Hu Mice).</p

Humanized Rag1^−/−γc^−/− mice are permissive to HIV-1 infection by vaginal route and show CD4 T cell decline.

<p>Mice were infected by vaginal route with R5 BaL HIV-1. Blood was collected weekly and viral RNA extracted from the plasma fraction. Viral RNA loads were determined by Q-RT-PCR as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0020169#s2" target="_blank">methods</a>. Levels of CD4 T cells were monitored on a weekly basis by FACS to determine their decline. Baseline values for each of the mice were established prior to infection as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0020169#s2" target="_blank">Methods</a>. A. RNA viral loads. B. CD4 T cell levels.</p

Human antibody production in humanized Rag1^−/−γc^−/− mice.

<p>Blood was drawn at 16 weeks post human CD34 cell engraftment and sera were analyzed by ELISA to detect different human immunoglobulin classes.</p

Supraphysiologic control over HIV-1 replication mediated by CD8 T cells expressing a re-engineered CD4-based chimeric antigen receptor

<div><p>HIV is adept at avoiding naturally generated T cell responses; therefore, there is a need to develop HIV-specific T cells with greater potency for use in HIV cure strategies. Starting with a CD4-based chimeric antigen receptor (CAR) that was previously used without toxicity in clinical trials, we optimized the vector backbone, promoter, HIV targeting moiety, and transmembrane and signaling domains to determine which components augmented the ability of T cells to control HIV replication. This re-engineered CAR was at least 50-fold more potent <i>in vitro</i> at controlling HIV replication than the original CD4 CAR, or a TCR-based approach, and substantially better than broadly neutralizing antibody-based CARs. A humanized mouse model of HIV infection demonstrated that T cells expressing optimized CARs were superior at expanding in response to antigen, protecting CD4 T cells from infection, and reducing viral loads compared to T cells expressing the original, clinical trial CAR. Moreover, in a humanized mouse model of HIV treatment, CD4 CAR T cells containing the 4-1BB costimulatory domain controlled HIV spread after ART removal better than analogous CAR T cells containing the CD28 costimulatory domain. Together, these data indicate that potent HIV-specific T cells can be generated using improved CAR design and that CAR T cells could be important components of an HIV cure strategy.</p></div

ICOS, CD27, and 4-1BB costimulation impair control of HIV replication <i>in vitro</i>.

<p><b>(A)</b> Gag staining on day 9 of co-culture for CD8 negative T cells. <b>(B)</b> Summary data for a single experiment performed in triplicate, gated on the CD8 negative T cells. Error bars indicate SEM. Significance was detected using a 1-way ANOVA test, stratifying based on the E:T ratio (p values: ns >0.05, *<0.05, **<0.01, ***<0.0001). Data is representative of three independent experiments. <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006613#ppat.1006613.s018" target="_blank">S18 Fig</a> shows each of the 3 independent experiments.</p

Lentiviral backbone augments CAR expression and control over HIV replication.

<p><b>(A-D)</b> Primary human CD8 T cells were activated with αCD3/αCD28 coated beads and were either left <b>(A)</b> nontransduced (NTD), <b>(B)</b> transduced with the original MMLV-based CD4 CAR, or <b>(C)</b> transduced with the same CAR placed in a HIV-based lentiviral vector, both driven by the PGK promoter. After eight days T cells were stained for CD4 and CD8 by flow cytometry. Median fluorescence intensity (MFI) is indicated on each graph. (<b>D)</b> Overlying histograms of the data shown in <b>(A-C). (E)</b> Eight days post activation, qPCR was performed and the number of integrated vector copies per cell was calculated. (<b>F</b>) Schematic of experimental design to study the control over HIV replication by T cells expressing HIV-specific CARs. Briefly, following activation with αCD3/αCD28 coated beads, CD4 T cells were infected with HIV Bal, and 24 hours later the indicated CD8 T cells were mixed at the indicated effector to target (E:T) ratios. After 7 days of co-culture, the expression of surface CD4, CD8, and intracellular Gag was measured by flow cytometry. <b>(G)</b> Intracellular Gag staining on CD8 negative cells, and <b>(H)</b> Intracellular Gag staining on CD8 positive cells. <b>(I)</b> Summary data for a single experiment, performed in triplicate, gating on the CD8 negative cells. Error bars indicate standard error of the mean (SEM). Significance was detected using a 1-way ANOVA test, stratifying based on the E:T ratio (p values: ns >0.05, *<0.05, **<0.01, ***<0.0001). This data is representative of three independent experiments. <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006613#ppat.1006613.s014" target="_blank">S14 Fig</a> shows each of the 3 independent experiments. (<b>J)</b> Measurement of levels of intracellular Gag in CD8 negative T cells over the time course of an experiment. Each graph represents a different E:T ratio. Error bars indicate SEM (n = 3).</p

CD4 CARs respond to Env⁺ cells and not MHC class II⁺ cells.

<p><b>(A)</b> Primary human CD8 T cells were activated with either left NTD or transduced with the indicated CD4 CARs. Two weeks post activation, the CD8 T cells were co-cultured for 6 hours at a 1:1 ratio with unmodified K562 cells, K562 cells expressing high levels of HLA-DR, or K562 cells expressing HIV-1 YU2 GP160. Intracellular IFNγ and MIP-1β expression is shown on the left, and intracellular IL-2 expression and CD107a surface mobilization is shown on the right. <b>(B)</b> A co-culture assay was designed to demonstrate that CD4 CAR⁺ CD8 T cells do not kill MHC class II-expressing target cells. Briefly, NTD or CD4 28z CAR transduced CD8 T cells from <b>(A)</b> were co-cultured with K562 cells expressing both HLA-A2 and GFP as well as K562 expressing both HLA-DR*0401 and mCherry at a 1:1:1 ratio. Flow cytometry measuring GFP and mCherry expression was performed immediately after mixing (0 hr) and after 3 days of co-culture (72 hr). <b>C)</b> Summary data for a single experiment performed in triplicate, measuring the ratio of HLA-A2/GFP-expressing cells to HLA-DR*0401/mCherry-expressing cells after 24, 48, and 72 hours of culture. Error bars indicate SEM. Data is representative of three independent experiments.</p