Diversity of the CD8(+) T-Cell Response to Herpes Simplex Virus Type 2 Proteins among Persons with Genital Herpes

Cytolytic T cells play a major role in controlling herpes simplex virus type 2 (HSV-2) infections in humans. In an effort to more thoroughly evaluate the response to HSV-2 directly, ex vivo, we developed an enzyme-linked immunospot (ELISPOT) assay that utilized pools of overlapping synthetic peptides presented by autologous dendritic cells to purified CD8(+) T cells. Donor response rates to individual open reading frames (ORFs) ranged from fewer than 5% responding to as many as 70% responding, with the greatest frequency of responses (by ORF) being directed against UL39, UL25, UL27, ICP0, UL46, and UL47 in descending order of frequency. HSV-2-seropositive subjects responded to as few as 3 or as many as 46 of the 48 ORFs tested, with a median of 11 ORFs recognized. HLA-B*07 expression correlated with stronger responses overall that were directed primarily against UL49 and UL46. Cumulative precursor frequencies in the blood ranged from 500 to almost 6,000 HSV-2 spot-forming units/10(6) CD8(+) T cells. The magnitude and breadth of the response in the infected population were greater than previously appreciated. Whether this variability in the CD8(+) T-cell response within individuals is associated with the frequency of viral reactivation warrants further study

Tracking the fate and origin of clinically relevant adoptively transferred CD8 +

Adoptively transferred tumor-specific cells can mediate tumor regression in cancers refractory to conventional therapy. Autologous polyclonal tumor-specific cytotoxic T cells (CTL) generated from peripheral blood and infused into patients with metastatic melanoma show enhanced persistence, compared to equivalent numbers of more extensively expanded monoclonal CTL, and are associated with complete remissions (CR) in select patients. We applied high-throughput T cell receptor Vβ sequencing (HTTCS) to identify individual clonotypes within CTL products, track them in vivo post-infusion and then deduce the pre-adoptive transfer (endogenous) frequencies of cells ultimately responsible for tumor regression. The summed in vivo post-transfer frequencies of the top 25 HTTCS-defined clonotypes originally detected in the infused CTL population were comparable to enumeration by binding of antigen peptide-HLA multimers, revealing quantitative HTTCS is a reliable, multimer-independent alternative. Surprisingly, the polyclonal CTL products were composed predominantly of clonotypes that were of very low frequency (VLF) in the endogenous samples, often below the limit of HTTCS detection (0.001%). In patients who achieved durable CRs, the composition of transferred CTLs was dominated (57–90%) by cells derived from a single VLF clonotype. Thus, HTTCS now reveals that tumor-specific CTL enabling long-term tumor control originate from endogenous VLF populations that exhibit proliferative/survival advantages. Along with results indicating that naïve cell populations are most likely to contain cells that exist at VLF within the repertoire, our results provide a strong rationale for favoring T cells arising from VLF populations and with early-differentiation phenotypes when selecting subset populations for adoptive transfer

T cell receptor gene therapy targeting WT1 prevents acute myeloid leukemia relapse post-transplant.

To access publisher's full text version of this article click on the hyperlink belowRelapse after allogeneic hematopoietic cell transplantation (HCT) is the leading cause of death in patients with acute myeloid leukemia (AML) entering HCT with poor-risk features1-3. When HCT does produce prolonged relapse-free survival, it commonly reflects graft-versus-leukemia effects mediated by donor T cells reactive with antigens on leukemic cells4. As graft T cells have not been selected for leukemia specificity and frequently recognize proteins expressed by many normal host tissues, graft-versus-leukemia effects are often accompanied by morbidity and mortality from graft-versus-host disease5. Thus, AML relapse risk might be more effectively reduced with T cells expressing receptors (TCRs) that target selected AML antigens6. We therefore isolated a high-affinity Wilms' Tumor Antigen 1-specific TCR (TCRC4) from HLA-A2+ normal donor repertoires, inserted TCRC4 into Epstein-Bar virus-specific donor CD8+ T cells (TTCR-C4) to minimize graft-versus-host disease risk and enhance transferred T cell survival7,8, and infused these cells prophylactically post-HCT into 12 patients ( NCT01640301 ). Relapse-free survival was 100% at a median of 44 months following infusion, while a concurrent comparative group of 88 patients with similar risk AML had 54% relapse-free survival (P = 0.002). TTCR-C4 maintained TCRC4 expression, persisted long-term and were polyfunctional. This strategy appears promising for preventing AML recurrence in individuals at increased risk of post-HCT relapse.Juno Therapeutics Immunotherapy Integrated Research Center at the Fred Hutchinson Cancer Research Center Damon Runyon Guillot Family ZachAttacksLeukemia Foundatio

Interleukin-21 Enhances Rituximab Activity in a Cynomolgus Monkey Model of B Cell Depletion and in Mouse B Cell Lymphoma Models

<div><p>Rituximab, a monoclonal antibody targeting CD20 on B cells, is currently used to treat many subtypes of B cell lymphomas. However, treatment is not curative and response rates are variable. Recombinant interleukin-21 (rIL-21) is a cytokine that enhances immune effector function and affects both primary and transformed B cell differentiation. We hypothesized that the combination of rIL-21 plus rituximab would be a more efficacious treatment for B cell malignancies than rituximab alone. We cultured human and cynomolgus monkey NK cells with rIL-21 and found that their activity was increased and proteins associated with antibody dependent cytotoxicity were up-regulated. Studies in cynomolgus monkeys modeled the effects of rIL-21 on rituximab activity against CD20 B cells. In these studies, rIL-21 activated innate immune effectors, increased ADCC and mobilized B cells into peripheral blood. When rIL-21 was combined with rituximab, deeper and more durable B cell depletion was observed. In another series of experiments, IL-21 was shown to have direct antiproliferative activity against a subset of human lymphoma cell lines, and combination of murine IL-21 with rituximab yielded significant survival benefits over either agent alone in xenogeneic mouse tumor models of disseminated lymphoma. Therefore, our results do suggest that the therapeutic efficacy of rituximab may be improved when used in combination with rIL-21.</p></div

Targeting an alternate Wilms' tumor antigen 1 peptide bypasses immunoproteasome dependency.

Designing effective antileukemic immunotherapy will require understanding mechanisms underlying tumor control or resistance. Here, we report a mechanism of escape from immunologic targeting in an acute myeloid leukemia (AML) patient, who relapsed 1 year after immunotherapy with engineered T cells expressing a human leukocyte antigen A*02 (HLA-A2)-restricted T cell receptor (TCR) specific for a Wilms' tumor antigen 1 epitope, WT1 (T). Resistance occurred despite persistence of functional therapeutic T cells and continuous expression of WT1 and HLA-A2 by the patient's AML cells. Analysis of the recurrent AML revealed expression of the standard proteasome, but limited expression of the immunoproteasome, specifically the beta subunit 1i (β1i), which is required for presentation of WT1. An analysis of a second patient treated with T demonstrated specific loss of AML cells coexpressing β1i and WT1. To determine whether the WT1 protein continued to be processed and presented in the absence of immunoproteasome processing, we identified and tested a TCR targeting an alternative, HLA-A2-restricted WT1 epitope that was generated by immunoproteasome-deficient cells, including WT1-expressing solid tumor lines. T cells expressing this TCR (T) killed the first patients' relapsed AML resistant to WT1 targeting, as well as other primary AML, in vitro. T controlled solid tumor lines lacking immunoproteasome subunits both in vitro and in an NSG mouse model. As proteasome composition can vary in AML, defining and preferentially targeting these proteasome-independent epitopes may maximize therapeutic efficacy and potentially circumvent AML immune evasion by proteasome-related immunoediting

rIL21 induced STAT1 signaling and growth inhibition for IM-9 but not Raji lymphoma cells.

<p>Growth curves for (A) IM-9 and (B) Raji cells when cultured with 50 ng/ml of rIL-21 after 3 days pre-treatment under the same conditions. Insets show phospho-STAT1 signaling IM-9 and Raji cells after 15 min culture with 10 ng/ml IL-21. Lower panels show survival curves for SCID mice (6–10/group) injected i.v. with 10⁶ cells of the (C) IM-9, (D) Raji, or (E) H-S Sultan lymphoma lines. Human rIL-21 or control treatment (PBS) was administered by 28-day mini-osmotic pump implanted one day after tumor cell injection.</p

Murine IL-21 plus rituximab prolongs the survival of SCID mice bearing disseminated lymphoma tumor cells.

<p>(<b>A, B</b>) SCID mice (n = 10/group) were injected i.v. with 10⁶ lymphoma cells and then treated with rituximab alone (20 µg on days 3, 7, 11, 15, 19), mIL-21 alone (100 µg days 1–5), or mIL-21 and rituximab. Significant prolongation of survival was observed in the rituximab plus mIL-21 group when compared to rituximab alone (<i>p</i> = 0.0006) in the HS-Sultan model (A). Mice in the Raji model (B) were treated as above, except that mIL-21 was given on days 3–7 and rituximab was given on days 5, 9, 13, 17, and 21. Mice in the combination group survived longer than those treated with rituximab alone (<i>p</i> = 0.0079). (<b>C, D, E</b>) Test of effector cell function with Raji lymphoma models established in SCID or NOD/SCID mice as described above. (C) NOD/SCID mice with impaired NK cells compared to SCID mice. Equivalent survival times were observed for NOD/SCID and SCID mice given rituximab plus rIL-21 (p = 0.012) or rituximab alone. (D) SCID mice injected i.p. with 50 µg of anti-Gr-1 antibody on day -1, 4, 9 and 14 to deplete granulocytes or with PBS (control). Survival was significantly decreased for depleted mice given rituximab plus rIL-21 (p = 0.012) or rituximab alone (p = 0.001) compared with non-depleted mice. (E) SCID mice injected i.v. with liposomes containing clodronate to deplete macrophages or PBS (control). Survival was significantly decreased for depleted mice given rituximab plus rIL-21 treatment (p = 0.0115) or rituximab alone (p = 0.0011) compared with non-depleted mice.</p

Activation of innate effector cells in cynomolgus monkeys treated with rIL-21.

<p>(A) ADCC activity in PBMC preparations in animals treated with vehicle control, weekly rituximab (0.05 mg/kg), rIL-21 (0.5 mg/kg for 3 days), or a combination of rituximab (0.05 mg/kg) and rIL-21 (0.5 mg/kg for 3 days). Gray bars indicate rIL-21 dosing days; rituximab was co-administered before the first rIL-21 dose in each cycle (days 1, 8, and 20). (B) Percentage of peripheral blood NK cells and cytotoxic T lymphocytes (CTL) staining positive for perforin. Gray bars in C indicate dosing days, error bars show standard error of the mean (SEM). (A, B) N = 3 animals per group.</p