BCR signaling is required for posttransplant lymphoproliferative disease in immunodeficient mice receiving human B cells.

Posttransplant lymphoproliferative disease (PTLD) is a major therapeutic challenge that has been difficult to study using human cells because of a lack of suitable models for mechanistic characterization. Here, we show that ex vivo-differentiated B cells isolated from a subset of healthy donors can elicit pathologies similar to PTLD when transferred into immunodeficient mice. The primary driver of PTLD-like pathologies were IgM-producing plasmablasts with Epstein-Barr virus (EBV) genomes that expressed genes commonly associated with EBV latency. We show that a small subset of EBV+ peripheral blood-derived B cells expressing self-reactive, nonmutated B cell receptors (BCRs) expand rapidly in culture in the absence of BCR stimulation. Furthermore, we found that in vitro and in vivo expansion of EBV+ plasmablasts required BCR signaling. Last, treatment of immunodeficient mice with the BCR pathway inhibitor, ibrutinib, delays onset of PTLD-like pathologies in vivo. These data have implications for the diagnosis and care of transplant recipients who are at risk of developing PTLD.

A chemically inducible IL-2 receptor signaling complex allows for effective in vitro and in vivo selection of engineered CD4+ T cells.

Engineered T cells represent an emerging therapeutic modality. However, complex engineering strategies can present a challenge for enriching and expanding therapeutic cells at clinical scale. In addition, lack of in vivo cytokine support can lead to poor engraftment of transferred T cells, including regulatory T cells (Treg). Here, we establish a cell-intrinsic selection system that leverages the dependency of primary T cells on IL-2 signaling. FRB-IL2RB and FKBP-IL2RG fusion proteins were identified permitting selective expansion of primary CD4+ T cells in rapamycin supplemented medium. This chemically inducible signaling complex (CISC) was subsequently incorporated into HDR donor templates designed to drive expression of the Treg master regulator FOXP3. Following editing of CD4+ T cells, CISC+ engineered Treg (CISC EngTreg) were selectively expanded using rapamycin and maintained Treg activity. Following transfer into immunodeficient mice treated with rapamycin, CISC EngTreg exhibited sustained engraftment in the absence of IL-2. Furthermore, in vivo CISC engagement increased the therapeutic activity of CISC EngTreg. Finally, an editing strategy targeting the TRAC locus permitted generation and selective enrichment of CISC+ functional CD19-CAR-T cells. Together, CISC provides a robust platform to achieve both in vitro enrichment and in vivo engraftment and activation, features likely beneficial across multiple gene-edited T cell applications.

Peripheral Vγ9Vδ2 T Cells Are a Novel Reservoir of Latent HIV Infection.

Eradication of HIV infection will require the identification of all cellular reservoirs that harbor latent infection. Despite low or lack of CD4 receptor expression on Vδ2 T cells, infection of these cells has previously been reported. We found that upregulation of the CD4 receptor may render primary Vδ2 cells target for HIV infection in vitro and we propose that HIV-induced immune activation may allow infection of γδ T cells in vivo. We assessed the presence of latent HIV infection by measurements of DNA and outgrowth assays within Vδ2 cells in 18 aviremic patients on long-standing antiretroviral therapy. In 14 patients we recovered latent but replication-competent HIV from highly purified Vδ2 cells demonstrating that peripheral Vδ2 T cells are a previously unrecognized reservoir in which latent HIV infection is unexpectedly frequent.

Gold nanoparticles to improve HIV drug delivery.

BACKGROUND: Antiretroviral therapy (ART) has improved lifespan and quality of life of patients infected with the HIV-1. However, ART has several potential limitations, including the development of drug resistance and suboptimal penetration to selected anatomic compartments. Improving the delivery of antiretroviral molecules could overcome several of the limitations of current ART. RESULTS & CONCLUSION: Two to ten nanometer diameter inorganic gold crystals serve as a base scaffold to combine molecules with an array of properties in its surface. We show entry into different cell types, antiviral activity of an HIV integrase inhibitor conjugated in a gold nanoparticle and penetration into the brain in vivo without toxicity. Herein, gold nanoparticles prove to be a promising tool to use in HIV therapy.

Precise Quantitation of the Latent HIV-1 Reservoir: Implications for Eradication Strategies.

The quantitative viral outgrowth assay (QVOA) provides a precise minimal estimate of the reservoir of resting CD4(+) T-cell infection (resting cell infection [RCI]). However, the variability of RCI over time during antiretroviral therapy (ART), relevant to assess potential effects of latency-reversing agents or other interventions, has not been fully described. We performed QVOA on resting CD4(+) T cells obtained via leukapheresis from 37 human immunodeficiency virus (HIV)-infected patients receiving stable suppressive ART for a period of 6 years. Patients who started ART during acute (n = 17) or chronic (n = 20) HIV infection were studied once HIV RNA levels were <50 copies/mL for ≥ 6 months. Using random effects analysis of 160 RCI measurements, we found that RCI declined significantly over time (P < .001), with an estimated mean half-life of 3.6 years (95% confidence interval, 2.3-8.1 years), remarkably consistent with findings of prior studies. There was no evidence of more rapid decay in acute versus chronic HIV infection (P = .99) for patients suppressed ≥ 6 months. RCI was reliably estimated with longitudinal measurements generally showing < 2-fold variation from the previous measure. When QVOA is performed in this format, RCI decreases of >6-fold were rare. We suggest that a 6-fold decline is a relevant threshold to reliably identify effects of antilatency interventions on RCI.

Quantitation of replication-competent HIV-1 in populations of resting CD4+ T cells.

UNLABELLED: Central memory (TCM) CD4(+) T cells are the principal reservoir of latent HIV-1 infection that persists despite durable, successful antiretroviral therapy (ART). In a study that measured HIV DNA in 17 patients and replication-competent HIV in 4 patients, pools of resting and activated transitional memory (T(TM)) CD4(+) T cells were found to be a reservoir for HIV infection. As defective viruses account for the majority of integrated HIV DNA and do not reflect the actual frequency of latent, replication-competent proviral infection, we assessed the specific contribution of resting T(TM) cells to latent HIV infection. We measured the frequency of replication-competent HIV in purified resting memory cell subpopulations by a limiting-dilution, quantitative viral outgrowth assay (QVOA). HIV was routinely detected within the resting central memory compartment but was infrequently detected within the resting T(TM) compartment. These observations suggest that prolonged ART may limit persistent latent infection in the T(TM) compartment. Our results confirm the importance of latent infection within the TCM compartment and again focus attention on these cells as the most important latent viral reservoir. While proliferation may drive expansion of detectable viral genomes in cells, the frequency of replication-competent HIV must be carefully assessed. Latent infection appears to wane within the transitional memory compartment in patients who have sustained successful viral suppression via ART or were treated very early in infection. IMPORTANCE: Antiretroviral therapy (ART) has led to a significant decrease in morbidity and mortality among HIV-infected patients. However, HIV integrates into the genome of CD4(+) T cells, generating pools of long-lived cells that are reservoirs of latent HIV. Two main subsets of CD4(+) T cells, central memory and transitional memory cells, were reported to be major reservoirs of HIV infection. However, this study primarily measured the HIV DNA content, which also includes defective proviruses that would not be able to replicate and initiate new rounds of infection. By analyzing the replication-competent virus in both cell subsets, we showed that transitional memory cells may not be a durable reservoir in patients on successful ART.

HIV-1 expression within resting CD4+ T cells after multiple doses of vorinostat.

BACKGROUND: A single dose of the histone deacetylase inhibitor vorinostat (VOR) up-regulates HIV RNA expression within resting CD4(+) T cells of treated, aviremic human immunodeficiency virus (HIV)-positive participants. The ability of multiple exposures to VOR to repeatedly disrupt latency has not been directly measured, to our knowledge. METHODS: Five participants in whom resting CD4(+) T-cell-associated HIV RNA (rc-RNA) increased after a single dose of VOR agreed to receive daily VOR Monday through Wednesday for 8 weekly cycles. VOR serum levels, peripheral blood mononuclear cell histone acetylation, plasma HIV RNA single-copy assays, rc-RNA, total cellular HIV DNA, and quantitative viral outgrowth assays from resting CD4(+) T cells were assayed. RESULTS: VOR was well tolerated, with exposures within expected parameters. However, rc-RNA measured after dose 11 (second dose of cycle 4) or dose 22 (second dose of cycle 8) increased significantly in only 3 of the 5 participants, and the magnitude of the rc-RNA increase was much reduced compared with that after a single dose. Changes in histone acetylation were blunted. Results of quantitative viral outgrowth and other assays were unchanged. CONCLUSIONS: Although HIV latency is disrupted by an initial VOR dose, the effect of subsequent doses in this protocol was much reduced. We hypothesize that the global effect of VOR results in a refractory period of ≥ 24 hours. The optimal schedule for VOR administration is still to be defined.

Latent HIV-1 infection of resting CD4⁺ T cells in the humanized Rag2⁻/⁻ γc⁻/⁻ mouse.

Persistent human immunodeficiency virus type 1 (HIV-1) infection of resting CD4⁺ T cells, unaffected by antiretroviral therapy (ART), provides a long-lived reservoir of HIV infection. Therapies that target this viral reservoir are needed to eradicate HIV-1 infection. A small-animal model that recapitulates HIV-1 latency in resting CD4⁺ T cells may accelerate drug discovery and allow the rational design of nonhuman primate (NHP) or human studies. We report that in humanized Rag2⁻/⁻ γ(c)⁻/⁻ (hu-Rag2⁻/⁻ γ(c)⁻/⁻) mice, as in humans, resting CD4⁺ T cell infection (RCI) can be quantitated in pooled samples of circulating cells and tissue reservoirs (e.g., lymph node, spleen, bone marrow) following HIV-1 infection with the CCR5-tropic JR-CSF strain and suppression of viremia by ART. Replication-competent virus was recovered from pooled resting CD4⁺ T cells in 7 of 16 mice, with a median frequency of 8 (range, 2 to 12) infected cells per million T cells, demonstrating that HIV-1 infection can persist despite ART in the resting CD4⁺ T cell reservoir of hu-Rag2⁻/⁻ γ(c)⁻/⁻ mice. This model will allow rapid preliminary assessments of novel eradication approaches and combinatorial strategies that may be challenging to perform in the NHP model or in humans, as well as a rigorous analysis of the effect of these interventions in specific anatomical compartments.

The effect of an environmental enrichment device on individually caged rabbits in a safety assessment facility.

The primary enclosure of a laboratory animal's environment should encourage species-typical behavior and enhancement of the animal's well-being, as indicated by the Guide. Enrichment devices have been documented to decrease the incidence of stereotypical behaviors and increase overall activity of rabbits. An 8-week study was performed to evaluate the effect of an environmental enrichment device, stainless-steel rabbit rattles on spring clips, on individually housed rabbits in a Safety Assessment facility. We used 48 New Zealand White rabbits; the devices were placed on cages of 32 study rabbits, and 16 control rabbits had no devices. Food consumption measurements and observations of device manipulations (taken during a predetermined peak interaction 1-h timeframe) were collected 5 days per week. All rabbits were bled for evaluation of hematologic parameters for the stress triad (neutrophilia, lymphopenia, and eosinopenia) and weighed weekly. No significant differences were found between study and control rabbits when body weights, food consumption, and hematologic parameters were analyzed. Our study supports previous findings that interaction with enrichment devices decreases over time, thus indicating the need for frequent rotation of different enrichment devices. In addition, no adverse effects of the analyzed parameters were found, indicating that stainless-steel rabbit rattles on spring clips are suitable devices for safety assessment studies, in which the introduction of new variables is often unacceptable.