Anti-PD-1 chimeric antigen receptor T cells efficiently target SIV-infected CD4+ T cells in germinal centers.

Programmed cell death protein 1 (PD-1) is an immune checkpoint marker commonly expressed on memory T cells and enriched in latently HIV-infected CD4+ T cells. We engineered an anti-PD-1 chimeric antigen receptor (CAR) to assess the impact of PD-1 depletion on viral reservoirs and rebound dynamics in SIVmac239-infected rhesus macaques (RMs). Adoptive transfer of anti-PD-1 CAR T cells was done in 2 SIV-naive and 4 SIV-infected RMs on antiretroviral therapy (ART). In 3 of 6 RMs, anti-PD-1 CAR T cells expanded and persisted for up to 100 days concomitant with the depletion of PD-1+ memory T cells in blood and tissues, including lymph node CD4+ follicular helper T (TFH) cells. Loss of TFH cells was associated with depletion of detectable SIV RNA from the germinal center (GC). However, following CAR T infusion and ART interruption, there was a marked increase in SIV replication in extrafollicular portions of lymph nodes, a 2-log higher plasma viremia relative to controls, and accelerated disease progression associated with the depletion of CD8+ memory T cells. These data indicate anti-PD-1 CAR T cells depleted PD-1+ T cells, including GC TFH cells, and eradicated SIV from this immunological sanctuary.

CD20 CAR T cells safely and reversibly ablate B cell follicles in a non-human primate model of HIV persistence.

Chimeric antigen receptor (CAR) T cell therapies have demonstrated immense clinical success for B cell and plasma cell malignancies. We tested their impact on the viral reservoir in a macaque model of HIV persistence, comparing the functions of CD20 CAR T cells between animals infected with simian/human immunodeficiency virus (SHIV) and uninfected controls. We focused on the potential of this approach to disrupt B cell follicles (BCFs), exposing infected cells for immune clearance. In SHIV-infected animals, CAR T cells were highly functional, with rapid expansion and trafficking to tissue-associated viral sanctuaries, including BCFs and gut-associated lymphoid tissue (GALT). CD20 CAR T cells potently ablated BCFs and depleted lymph-node-associated follicular helper T (TFH) cells, with complete restoration of BCF architecture and TFH cells following CAR T cell contraction. BCF ablation decreased the splenic SHIV reservoir but was insufficient for effective reductions in systemic viral reservoirs. Although associated with moderate hematologic toxicity, CD20 CAR T cells were well tolerated in SHIV-infected and control animals, supporting the feasibility of this therapy in people living with HIV with underlying B cell malignancies. Our findings highlight the unique ability of CD20 CAR T cells to safely and reversibly unmask TFH cells within BCF sanctuaries, informing future combinatorial HIV cure strategies designed to augment antiviral efficacy.

Oral desensitization therapy for peanut allergy induces dynamic changes in peanut-specific immune responses.

BACKGROUND: The PALISADE study, an international, phase 3 trial of peanut oral immunotherapy (POIT) with AR101, resulted in desensitization in children and adolescents who were highly allergic to peanut. An improved understanding of the immune mechanism induced in response to food allergen immunotherapy would enable more informed and effective therapeutic strategies. Our main purpose was to examine the immunological changes in blood samples from a subset of peanut-allergic individuals undergoing oral desensitization immunotherapy with AR101. METHODS: Blood samples obtained as part of enrollment screening and at multiple time points during PALISADE study were used to assess basophil and CD4+ T-cell reactivity to peanut. RESULTS: The absence of clinical reactivity to the entry double-blinded placebo-controlled peanut challenge (DBPCFC) was accompanied by a significantly lower basophil sensitivity and T-cell reactivity to peanut compared with DBPCFC reactors. At baseline, peanut-reactive TH2A cells were observed in many but not all peanut-allergic patients and their level in peripheral blood correlates with T-cell reactivity to peanut and with serum peanut-specific IgE and IgG4 levels. POIT reshaped circulating peanut-reactive T-cell responses in a subset-dependent manner. Changes in basophil and T-cell responses to peanut closely paralleled clinical benefits to AR101 therapy and resemble responses in those with lower clinical sensitivity to peanut. However, no difference in peanut-reactive Treg cell frequency was observed between groups. CONCLUSION: Oral desensitization therapy with AR101 leads to decreased basophil sensitivity to peanut and reshapes peanut-reactive T effector cell responses supporting its potential as an immunomodulatory therapy.

Immune inactivation of anti-simian immunodeficiency virus chimeric antigen receptor T cells in rhesus macaques.

Chimeric antigen receptor (CAR) T cell therapies are being investigated as potential HIV cures and designed to target HIV reservoirs. Monoclonal antibodies (mAbs) targeting the simian immunodeficiency virus (SIV) envelope allowed us to investigate the potency of single-chain variable fragment (scFv)-based anti-SIV CAR T cells. In vitro, CAR T cells expressing the scFv to both the variable loop 1 (V1) or V3 of the SIV envelope were highly potent at eliminating SIV-infected T cells. However, in preclinical studies, in vivo infusion of these CAR T cells in rhesus macaques (RMs) resulted in lack of expansion and no detectable in vivo antiviral activity. Injection of envelope-expressing antigen-presenting cells (APCs) 1 week post-CAR T cell infusion also failed to stimulate CAR T cell expansion in vivo. To investigate this in vitro versus in vivo discrepancy, we examined host immune responses directed at CAR T cells. A humoral immune response against the CAR scFv was detected post-infusion of the anti-SIV CAR T cells; anti-SIV IgG antibodies present in plasma of SIV-infected animals were associated with inhibited CAR T cell effector functions. These data indicate that lack of in vivo expansion and efficacy of CAR T cells might be due to antibodies blocking the interaction between the CAR scFv and its epitope.

Envelope-Specific Adaptive Immunity following Transplantation of Hematopoietic Stem Cells Modified with VSV-G Lentivirus.

Current approaches for hematopoietic stem cell gene therapy typically involve lentiviral gene transfer in tandem with a conditioning regimen to aid stem cell engraftment. Although many pseudotyped envelopes have the capacity to be immunogenic due to their viral origins, thus far immune responses against the most common envelope, vesicular stomatitis virus glycoprotein G (VSV-G), have not been reported in hematopoietic stem cell gene therapy trials. Herein, we report on two Fanconi anemia patients who underwent autologous transplantation of a lineage-depleted, gene-modified hematopoietic stem cell product without conditioning. We observed the induction of robust VSV-G-specific immunity, consistent with low/undetectable gene marking in both patients. Upon further interrogation, adaptive immune mechanisms directed against VSV-G were detected following transplantation in both patients, including increased VSV-G-specific T cell responses, anti-VSV-G immunoglobulin G (IgG), and cytotoxic responses that can specifically kill VSV-G-expressing target cell lines. A proportion of healthy controls also displayed preexisting VSV-G-specific CD4+ and CD8+ T cell responses, as well as VSV-G-specific IgG. Taken together, these data show that VSV-G-pseudotyped lentiviral vectors have the ability to elicit interfering adaptive immune responses in the context of certain hematopoietic stem cell transplantation settings.

CAR T-cell therapy for cancer and HIV through novel approaches to HIV-associated haematological malignancies.

People living with HIV are a global population with increased cancer risk but their access to modern immunotherapies for cancer treatment has been limited by socioeconomic factors and inadequate research to support safety and efficacy in this population. These immunotherapies include immune checkpoint inhibitors and advances in cellular immunotherapy, particularly chimeric antigen receptor (CAR) T-cell therapy. Despite the field of cancer immunotherapy rapidly expanding with ongoing clinical trials, people with HIV are often excluded from such trials. In 2019, post-approval evaluation of anti-CD19 CAR T-cell therapy in people with HIV and aggressive B-cell lymphoma showed the feasibility of CAR T-cell therapy for cancer in this excluded group. Along with expanded treatment options for people with HIV is the ability to assess the effects of immunotherapy on the latent HIV reservoir, with certain immunotherapies showing the ability to alleviate this burden. This Series paper addresses the increased cancer burden in people with HIV, the increasing evidence for the safety and efficacy of immunotherapies in the context of HIV and cancer, and opportunities for novel applications of CAR-T therapy for the treatment of both haematological malignancies and HIV.

Robust expansion of HIV CAR T cells following antigen boosting in ART-suppressed nonhuman primates.

Chimeric antigen receptor (CAR) T cells targeting CD19+ hematologic malignancies have rapidly emerged as a promising, novel therapy. In contrast, results from the few CAR T-cell studies for infectious diseases such as HIV-1 have been less convincing. These challenges are likely due to the low level of antigen present in antiretroviral therapy (ART)-suppressed patients in contrast to those with hematologic malignancies. Using our well-established nonhuman primate model of ART-suppressed HIV-1 infection, we tested strategies to overcome these limitations and challenges. We first optimized CAR T-cell production to maintain central memory subsets, consistent with current clinical paradigms. We hypothesized that additional exogenous antigen might be required in an ART-suppressed setting to aid expansion and persistence of CAR T cells. Thus, we studied 4 simian/HIV-infected, ART-suppressed rhesus macaques infused with virus-specific CD4CAR T cells, followed by supplemental infusion of cell-associated HIV-1 envelope (Env). Env boosting led to significant and unprecedented expansion of virus-specific CAR+ T cells in vivo; after ART treatment interruption, viral rebound was significantly delayed compared with controls (P = .014). In 2 animals with declining CAR T cells, rhesusized anti-programmed cell death protein 1 (PD-1) antibody was administered to reverse PD-1-dependent immune exhaustion. Immune checkpoint blockade triggered expansion of exhausted CAR T cells and concordantly lowered viral loads to undetectable levels. These results show that supplemental cell-associated antigen enables robust expansion of CAR T cells in an antigen-sparse environment. To our knowledge, this is the first study to show expansion of virus-specific CAR T cells in infected, suppressed hosts, and delay/control of viral recrudescence.

Renal Cell Carcinoma (RCC) Tumors Display Large Expansion of Double Positive (DP) CD4+CD8+ T Cells With Expression of Exhaustion Markers.

Checkpoint inhibitors target the inhibitory receptors expressed by tumor-infiltrating T cells in order to reinvigorate an anti-tumor immune response. Therefore, understanding T cell composition and phenotype in human tumors is crucial. We analyzed by flow cytometry tumor-infiltrating lymphocytes (TILs) from two independent cohorts of patients with different cancer types, including RCC, lung, and colon cancer. In healthy donors, peripheral T cells are usually either CD4+ or CD8+ with a small percentage of CD4+ CD8+ DP cells (<5%). Compared to several other cancer types, including lung, and colorectal cancers, TILs from about a third of RCC patients showed an increased proportion of DP CD4+CD8+ T cells (>5%, reaching 30-50% of T cells in some patients). These DP T cells have an effector memory phenotype and express CD38, 4-1BB, and HLA-DR, suggesting antigen-driven expansion. In fact, TCR sequencing analysis revealed a high degree of clonality in DP T cells. Additionally, there were high levels of PD-1 and TIM-3 expression on DP T cells, which correlated with higher expression of PD-1 and TIM-3 in conventional single positive CD8 T cells from the same patients. These results suggest that DP T cells could be dysfunctional tumor-specific T cells with the potential to be reactivated by checkpoint inhibitors.

Human Immune Monitoring Techniques during Food Allergen Immunotherapy.

PURPOSE OF REVIEW: Encouraging results from recent food allergen immunotherapy clinical trials indicate that the immune system plays an essential role in peripheral tolerance to food allergen. Thus, the monitoring of changes in immune responses and their possible correlation with clinical outcome in allergic patients receiving immunotherapies could theoretically serve as surrogate markers and be harnessed as rationale for food allergen immunotherapy development. RECENT FINDINGS: A shift towards antigen specificity in recent assays has provided a solid foundation for the elucidation of cellular mechanisms involved in food allergen immunotherapy as well as the tracking of allergen-specific immune cells. In this review, we overview the current challenges and technologies used in immune monitoring during immunotherapy in allergic patients with a focus on cell-mediated immunity. We also discuss critical steps involved in some of the cellular immune assays utilized in clinical trials.

Efficient generation of monoclonal antibodies against peptide in the context of MHCII using magnetic enrichment.

Monoclonal antibodies specific for foreign antigens, auto-antigens, allogeneic antigens and tumour neo-antigens in the context of major histocompatibility complex II (MHCII) are highly desirable as novel immunotherapeutics. However, there is no standard protocol for the efficient generation of monoclonal antibodies that recognize peptide in the context of MHCII, and only a limited number of such reagents exist. In this report, we describe an approach for the generation and screening of monoclonal antibodies specific for peptide bound to MHCII. This approach exploits the use of recombinant peptide:MHC monomers as immunogens, and subsequently relies on multimers to pre-screen and magnetically enrich the responding antigen-specific B cells before fusion and validation, thus saving significant time and reagents. Using this method, we have generated two antibodies enabling us to interrogate antigen presentation and T-cell activation. This methodology sets the standard to generate monoclonal antibodies against the peptide-MHCII complexes.

Shaping T cell - B cell collaboration in the response to human immunodeficiency virus type 1 envelope glycoprotein gp120 by peptide priming.

Prime-boost vaccination regimes have shown promise for obtaining protective immunity to HIV. Poorly understood mechanisms of cellular immunity could be responsible for improved humoral responses. Although CD4+ T-cell help promotes B-cell development, the relationship of CD4+ T-cell specificity to antibody specificity has not been systematically investigated. Here, protein and peptide-specific immune responses to HIV-1 gp120 were characterized in groups of ten mucosally immunized BALB/c mice. Protein and peptide reactivity of serum antibody was tested for correlation with cytokine secretion by splenocytes restimulated with individual gp120 peptides. Antibody titer for gp120 correlated poorly with the peptide-stimulated T-cell response. In contrast, titers for conformational epitopes, measured as crossreactivity or CD4-blocking, correlated with average interleukin-2 and interleukin-5 production in response to gp120 peptides. Antibodies specific for conformational epitopes and individual gp120 peptides typically correlated with T-cell responses to several peptides. In order to modify the specificity of immune responses, animals were primed with a gp120 peptide prior to immunization with protein. Priming induced distinct peptide-specific correlations of antibodies and T-cells. The majority of correlated antibodies were specific for the primed peptides or other peptides nearby in the gp120 sequence. These studies suggest that the dominant B-cell subsets recruit the dominant T-cell subsets and that T-B collaborations can be shaped by epitope-specific priming.