Inadequate T-cell control of Kaposi sarcoma-associated herpesvirus (KSHV) infection predisposes to development of Kaposi sarcoma (KS), but little is known about the T-cell response to KSHV. Postulating that KS tumors contain abundant KSHV-specific T-cells, we performed transcriptional profiling and T-cell receptor (TCR) repertoire analysis of tumor biopsies from 144 Ugandan adults with KS. We show that CD8+ T-cells and M2-polarized macrophages dominate the tumor micro-environment (TME). The TCR repertoire of KS tumor infiltrating lymphocytes (TIL) is shared across non-contiguous tumors and persists across time. Clusters of T-cells with predicted shared specificity for uncharacterized antigens, potentially encoded by KSHV, comprise ~25% of KS TIL, and are shared across tumors from different time points and individuals. Single-cell RNA-sequencing of blood identifies a non-proliferating effector memory phenotype and captured the TCRs in 14,698 putative KSHV-specific T-cells. These results suggest that a polyspecific KSHV-specific T-cell response inhibited by M2 macrophages exists within the KS TME, and provide a foundation for studies to define its specificity at a large scale.

Allogeneic antibodies against minor histocompatibility antigens encoded on the Y chromosome (HY-Abs) develop after hematopoietic cell transplant (HCT) of male recipients with female donors (F→M). However, the temporal association between HY-Ab development and chronic graft-versus-host disease (cGVHD) has yet to be elucidated. We studied 136 adult F→M HCT patients, with plasma prospectively collected through 3 years posttransplant, and measured immunoglobulin G against 6 H-Y antigens. Multiple HY-Abs were frequently detected beginning at 3 months posttransplant: 78 (57%) of F→M patients were seropositive for at least 1 of the 6 HY-Abs, and 3-month seropositivity for each HY-Ab was associated with a persistent seropositive response throughout the posttransplant follow-up period (P < .001 in each). There were no associations between pretransplant features and 3-month overall HY-Ab development. Detection of multiple HY-Abs at 3 months (represented by HY score) was significantly associated with an increased risk of cGVHD (P < .0001) and nonrelapse mortality (P < .01). Compared to clinical factors alone, the addition of HY score to clinical factors improved the predictive potential of cGVHD (P < .01). Monitoring HY-Ab development thus stratifies cGVHD risk in F→M HCT patients and may support preemptive prophylaxis therapy for cGVHD beginning at 3 months posttransplant.

Pretargeted radioimmunotherapy (PRIT) is a multi-step method of selectively delivering high doses of radiotherapy to tumor cells while minimizing exposure to surrounding tissues. Yttrium-90 (90Y) and lutetium-177 (177Lu) are two of the most promising beta-particle emitting radionuclides used for radioimmunotherapy, which despite having similar chemistries differ distinctly in terms of radiophysical features. These differences may have important consequences for the absorbed dose to tumors and normal organs. Whereas 90Y has been successfully applied in a number of preclinical and clinical radioimmunotherapy settings, there have been few published pretargeting studies with 177Lu. We therefore compared the therapeutic potential of targeting either 90Y or 177Lu to human B-cell lymphoma xenografts in mice.

With age, the immune system becomes less effective, causing increased susceptibility to infection. Chronic CMV infection further impairs immune function and is associated with increased mortality in the elderly. CMV exposure elicits massive CD8+ T cell clonal expansions and diminishes the cytotoxic T cell response to subsequent infections, leading to the hypothesis that to maintain homeostasis, T cell clones are expelled from the repertoire, reducing T cell repertoire diversity and diminishing the ability to combat new infections. However, in humans, the impact of CMV infection on the structure and diversity of the underlying T cell repertoire remains uncharacterized. Using TCR β-chain immunosequencing, we observed that the proportion of the peripheral blood T cell repertoire composed of the most numerous 0.1% of clones is larger in the CMV seropositive and gradually increases with age. We found that the T cell repertoire in the elderly grows to accommodate CMV-driven clonal expansions while preserving its underlying diversity and clonal structure. Our observations suggest that the maintenance of large CMV-reactive T cell clones throughout life does not compromise the underlying repertoire. Alternatively, we propose that the diminished immunity in elderly individuals with CMV is due to alterations in cellular function rather than a reduction in CD8+ T cell repertoire diversity.

Immunotherapy is an effective treatment for a subset of cancer patients, and expanding the benefits of immunotherapy to all cancer patients will require predictive biomarkers of response and immune-related adverse events (irAEs). To support correlative studies in immunotherapy clinical trials, we are developing highly validated assays for quantifying immunomodulatory proteins in human biospecimens.

Radioimmunotherapy (RIT) for treatment of hematologic malignancies has primarily employed monoclonal antibodies (Ab) labeled with 131I or 90Y which have limitations, and alternative radionuclides are needed to facilitate wider adoption of RIT. We therefore compared the relative therapeutic efficacy and toxicity of anti-CD45 RIT employing 90Y and 177Lu in a syngeneic, disseminated murine myeloid leukemia (B6SJLF1/J) model. Biodistribution studies showed that both 90Y- and 177Lu-anti-murine CD45 Ab conjugates (DOTA-30F11) targeted hematologic tissues, as at 24 hours 48.8 ± 21.2 and 156 ± 14.6% injected dose per gram of tissue (% ID/g) of 90Y-DOTA-30F11 and 54.2 ± 9.5 and 199 ± 11.7% ID/g of 177Lu-DOTA-30F11 accumulated in bone marrow (BM) and spleen, respectively. However, 90Y-DOTA-30F11 RIT demonstrated a dose-dependent survival benefit: 60% of mice treated with 300 µCi 90Y-DOTA-30F11 lived over 180 days after therapy, and mice treated with 100 µCi 90Y-DOTA-30F11 had a median survival 66 days. 90Y-anti-CD45 RIT was associated with transient, mild myelotoxicity without hepatic or renal toxicity. Conversely, 177Lu- anti-CD45 RIT yielded no long-term survivors. Thus, 90Y was more effective than 177Lu for anti-CD45 RIT of AML in this murine leukemia model.

Adoptive T cell therapy represents an attractive modality for the treatment of patients with cancer. Peripheral blood mononuclear cells have been used as a source of antigen specific T cells but the very low frequency of T cells recognizing commonly expressed antigens such as NY-ESO-1 limit the applicability of this approach to other solid tumors. To overcome this, we tested a strategy combining IL-21 modulation during in vitro stimulation with first-in-class use of tetramer-guided cell sorting to generate NY-ESO-1 specific cytotoxic T lymphocytes (CTL).

Recent studies have suggested that human RNA helicase, DDX3X, is important for DNA repair, but little is known about the nuclear activity of this protein. In vitro analysis of nuclear DDX3X interactions and localization with DNA damage pointed to a direct role for DDX3X in the DNA damage response. We aimed to investigate whether DDX3X plays a direct role in the DNA damage response in live cells. In order to track nuclear DDX3X, we generated a nuclear-export deficient DDX3X mutant construct and performed microirradiation in live cells. We found that DDX3X accumulates at sites of microirradiation shortly after DNA damage induction. We further found DDX3X recruitment to be mediated by its intrinsically disordered domains, similar to other RNA binding proteins that are recruited to sites of DNA damage. Inhibition of liquid-liquid phase separation also reduced DDX3X recruitment. CRISPR/Cas9-mediated knockout of PARP1 ablated DDX3X recruitment, which was restored upon transgenic expression of wild-type PARP1 but not catalytically inactive PARP1, suggesting that DDX3X recruitment is PARP1-dependent.

The single-arm DAWN trial (NCT01779791) of ibrutinib monotherapy in patients with relapsed/refractory follicular lymphoma (FL) showed an overall response rate (ORR) of 20.9% and a median response duration of 19.4 months. This biomarker analysis of the DAWN dataset sought to determine genetic classifiers for prediction of response to ibrutinib treatment.

Long-term antiretroviral therapy (ART) in people living with HIV (PLHIV) is associated with sustained increases in CD4+ T-cell count, but its effect on the peripheral blood T-cell repertoire has not been comprehensively evaluated. In this study, we performed serial profiling of the composition and diversity of the T-cell receptor β-chain (TRB) repertoire in 30 adults with HIV infection before and after the initiation of ART to define its long-term impact on the TRB repertoire. Serially acquired blood samples from 30 adults with HIV infection collected over a mean of 6 years (range, 1-12) years, with 1-4 samples collected before and 2-8 samples collected after the initiation of ART, were available for analysis. TRB repertoires were characterized via high-throughput sequencing of the TRB variable region performed on genomic DNA extracted from unsorted peripheral blood mononuclear cells. Additional laboratory and clinical metadata including serial measurements of HIV viral load and CD4 + T-cell count were available for all individuals in the cohort. A previously published control group of 189 TRB repertoires from peripheral blood samples of adult bone marrow transplant donors was evaluated for comparison. ART initiation in PLHIV was associated with a sustained reduction in viral load and a significant increase in TRB repertoire diversity. However, repertoire diversity in PLHIV remained significantly lower than in the control group even after long-term ART. The composition of TRB repertoires of PLHIV after ART also remained perturbed compared to the control cohort, as evidenced by large persistent private clonal expansions, reduced efficiency in the generation of TRB CDR3 amino acid sequences, and a narrower range of CDR3 lengths. Network analysis revealed an antigen-experienced structure in the TRB repertoire of PLHIV both before and after ART initiation that was quite distinct from the structure of control repertoires, with a slight shift toward a more naïve structure observed after ART initiation. Though we observe significant improvement in TRB repertoire diversity with durable viral suppression in PLHIV on long-term ART, the composition and structure of these repertoires remain significantly perturbed compared to the control cohort of adult bone marrow transplant donors.

The role of the immune microenvironment in maintaining disease remission in patients with multiple myeloma (MM) is not well understood. In this study, we comprehensively profile the immune system in patients with newly diagnosed MM receiving continuous lenalidomide maintenance therapy with the aim of discovering correlates of long-term treatment response. Leveraging single-cell RNA sequencing and T cell receptor β sequencing of the peripheral blood and CyTOF mass cytometry of the bone marrow, we longitudinally characterize the immune landscape in 23 patients before and one year after lenalidomide exposure. We compare patients achieving sustained minimal residual disease (MRD) negativity to patients who never achieved or were unable to maintain MRD negativity. We observe that the composition of the immune microenvironment in both the blood and the marrow varied substantially according to both MRD negative status and history of autologous stem cell transplant, supporting the hypothesis that the immune microenvironment influences the depth and duration of treatment response.