Deep functional immunophenotyping predicts risk of cytomegalovirus reactivation after hematopoietic cell transplantation.

Cytomegalovirus (CMV) is the most common viral infection in hematopoietic cell transplantation (HCT) recipients. We performed deep phenotyping of CMV-specific T cells to predict CMV outcomes following allogeneic HCT. By using 13-color flow cytometry, we studied ex vivo CD8+ T-cell cytokine production in response to CMV-pp65 peptides in 3 clinically distinct subgroups of CMV-seropositive HCT patients: (1) Elite Controllers (n = 19): did not have evidence of CMV DNAemia on surveillance testing; (2) Spontaneous Controllers (n = 16): spontaneously resolved low-grade CMV DNAemia without antiviral therapy; and (3) Noncontrollers (NC; n = 21): experienced clinically significant CMV. Two CMV-specific CD8+ T-cell functional subsets were strongly associated with risk of CMV: (i) the nonprotective signature (NPS; IL-2-IFN-γ+TNF-α-MIP-1ß+), found at increased levels among NC; and (ii) the protective signature (PS; IL-2+IFN-γ+TNF-α+MIP-1ß+) found at low levels among NC. High levels of the NPS and low levels of PS were associated with an increased 100-day cumulative incidence of clinically significant CMV infection (35% vs 5%; P = .02; and 40% vs 12%; P = .05, respectively). The highest predictive value was observed when these signatures were combined into a composite biomarker consisting of low levels of the PS and high levels of the NPS (67% vs 10%; P < .001). After adjusting for steroid use or donor type, this composite biomarker remained associated with a fivefold increase in the risk of clinically significant CMV infection. CMV-specific CD8+ T-cell cytokine signatures with robust predictive value for risk of CMV reactivation should prove useful in guiding clinical decision making in HCT recipients.

Axitinib plus pembrolizumab in patients with advanced sarcomas including alveolar soft-part sarcoma: a single-centre, single-arm, phase 2 trial.

BACKGROUND: VEGF promotes an immunosuppressive microenvironment and contributes to immune checkpoint inhibitor resistance in cancer. We aimed to assess the activity of the VEGF receptor tyrosine-kinase inhibitor axitinib plus the anti-PD-1 immune checkpoint inhibitor pembrolizumab in patients with sarcoma. METHODS: This single-centre, single-arm, phase 2 trial was undertaken at a tertiary care academic medical centre in Miami, FL, USA, and participants were recruited from all over the USA and internationally. Patients were eligible if they were aged 16 years or older, and had histologically confirmed advanced or metastatic sarcomas, including alveolar soft-part sarcoma (ASPS); measurable disease with one site amenable to repeated biopsies; an ECOG performance status of 0-1; and progressive disease after previous treatment with at least one line of systemic therapy (unless no standard treatment existed or the patient declined therapy). The first five patients were enrolled in a lead-in cohort and were given axitinib 5 mg orally twice daily and pembrolizumab 200 mg intravenously for 30 min on day 8 and every 3 weeks for cycles of 6 weeks for up to 2 years. Thereafter, patients received escalating doses of axitinib (2-10 mg) plus flat dose pembrolizumab according to the schedule above. The primary endpoint was 3-month progression-free survival. All patients were evaluable for survival and safety analyses. This study is registered with ClinicalTrials.gov, number NCT02636725, and is closed to accrual. FINDINGS: Between April 19, 2016, and Feb 7, 2018, of 36 patients assessed for eligibility, 33 (92%) were enrolled and given study treatment (intention-to-treat population and safety population), 12 (36%) of whom had ASPS. With a median follow-up of 14·7 months (IQR 10·1-19·1), 3-month progression-free survival for all evaluable patients was 65·6% (95% CI 46·6-79·3). For patients with ASPS, 3-month progression-free survival was 72·7% (95% CI 37·1-90·3). The most common grade 3 or 4 treatment-related adverse events included hypertension (five [15%] of 33 patients), autoimmune toxicities (five [15%]), nausea or vomiting (two [6%]), and seizures (two [6%]). Serious treatment-related adverse events occurred in seven (21%) patients, including autoimmune colitis, transaminitis, pneumothorax, haemoptysis, seizures, and hypertriglyceridemia. There were no treatment-related deaths. INTERPRETATION: Axitinib plus pembrolizumab has manageable toxicity and preliminary activity in patients with advanced sarcomas, particularly patients with ASPS, warranting further investigation in randomised controlled trials. FUNDING: Merck, Pfizer, American Cancer Society, and Sylvester Comprehensive Cancer Center.

Impact of Cytomegalovirus Viral Load on Probability of Spontaneous Clearance and Response to Preemptive Therapy in Allogeneic Stem Cell Transplantation Recipients.

The optimal viral load threshold at which to initiate preemptive cytomegalovirus (CMV) therapy in hematopoietic cell transplantation (HCT) recipients remains to be defined. In an effort to address this question, we conducted a retrospective study of 174 allogeneic HCT recipients who underwent transplantation at a single center between August 2012 and April 2016. During this period, preemptive therapy was initiated at the discretion of the treating clinician. A total of 109 patients (63%) developed CMV viremia. The median time to reactivation was 17 days (interquartile range, IQR, 7-30 days) post-HCT. A peak viremia ≥150 IU/mL was strongly associated with a reduced probability of spontaneous clearance (relative risk, .16; 95% confidence interval, .1-.27), independent of established clinical risk factors, including CMV donor serostatus, exposure to antithymocyte globulin, and underlying lymphoid malignancy. The median time to clearance of viremia was significantly shorter in those who started therapy at CMV <350 IU/mL (19 days; IQR, 11-35 days) compared with those who started antiviral therapy at higher viremia thresholds (33 days; IQR, 21-42 days; P = .02). The occurrence of treatment-associated cytopenias was frequent but similar in patients who started preemptive therapy at CMV <350 IU/mL and those who started at CMV >350 IU/mL (44% versus 57%; P = .42). Unresolved CMV viremia by treatment day 35 was associated with increased risk of therapeutic failure (32% versus 0%; P = .001). Achieving eradication of CMV viremia by treatment day 35 was associated with a 74% reduction in 1-year nonrelapse mortality (NRM) (adjusted hazard ratio [HR], .26; 95% confidence interval [CI], .1-.8; P = .02), whereas therapeutic failure was associated with a significant increase in the probability of 1-year NRM (adjusted HR, 26; 95% CI, 8-87; P <.0001). We conclude that among allogeneic HCT patients, a peak CMV viremia ≥150 IU/mL is associated with a >80% reduction in the probability of spontaneous clearance independent of ATG administration, CMV donor serostatus, and lymphoid malignancy, and is a reasonable cutoff for preemptive therapy. Delaying initiation of therapy until a CMV value ≥350 IU/mL is associated with more protracted CMV viremia, and unresolved viremia by treatment day 35 is associated with a significant increase in NRM.

MEK inhibitors selectively suppress alloreactivity and graft-versus-host disease in a memory stage-dependent manner.

Immunosuppressive strategies currently used in hematopoietic stem cell transplantation reliably decrease graft-versus-host disease (GVHD) rates, but also impair pathogen-specific immunity. Experimental transplant studies indicate that GVHD-initiating alloreactive T cells reside primarily in naive and central memory T-cell compartments. In contrast, virus-specific T cells comprise a more differentiated memory population. After finding that the rat sarcoma/mitogen-activated protein kinase kinase/extracellular receptor kinase (RAS/MEK/ERK) pathway is preferentially activated in naive and central memory human T cells, we hypothesized that MEK inhibitors would preferentially inhibit alloreactive T cells, while sparing more differentiated virus-specific T cells. Confirming our hypothesis, we found that MEK inhibitors including selumetinib preferentially inhibited cytokine production and alloreactivity mediated by naive and central memory human CD4(+) and CD8(+) T cells while sparing more differentiated T cells specific for the human herpesviruses cytomegalovirus and Epstein-Barr virus. We then demonstrated that short-term posttransplant administration of selumetinib in a major histocompatibility complex major- and minor-mismatched murine model significantly delayed the onset of GVHD-associated mortality without compromising myeloid engraftment, demonstrating the in vivo potential of MEK inhibitors in the setting of hematopoietic stem cell transplantation. These findings demonstrate that targeting memory-dependent differences in T-cell signaling is a potent and selective approach to inhibition of alloreactivity.

eHealth mindfulness-based music therapy for patients undergoing allogeneic hematopoietic stem cell transplantation: A pilot randomized controlled trial protocol.

BACKGROUND: Allogeneic stem cell transplantation (allo-SCT) is the preferred therapy for patients with high-risk or relapsed hematologic malignancies, but may be complicated by psychological distress (e.g., depression, anxiety) and symptom burden (e.g., fatigue, pain). Mindfulness-based music therapy (MBMT), a relatively novel integrative medicine intervention that draws from mindfulness and music therapy principles, has shown promise in improving psychosocial outcomes and symptom burden in cancer patients. We outline an eHealth-based MBMT (eMBMT) intervention protocol examining: (1) feasibility, acceptability, and intended effects of eMBMT in improving HRQOL, symptom burden, and clinical markers of disease activity (e.g., infections), and (2) the extent to which eMBMT music therapy component-associated improvements in HRQOL, symptom burden, and disease activity are mediated by improvements in psychosocial and physiological (e.g., systemic inflammation, immune recovery) adaptation. METHODS: Participants (n = 60) with a hematologic malignancy undergoing allo-SCT will be randomized to receive eMBMT or an eHealth-based mindfulness meditation (eMM) intervention. eMBMT includes eight 60-min sessions facilitated by a music therapist focusing on mindfulness and music therapy. eMM includes eight 60-min self-led MM practices. RESULTS: Feasibility, acceptability, HRQOL, symptom burden, disease activity, and mediation effects of psychosocial and physiological adaptation will be assessed at baseline, pre-infusion, and post-engraftment with blood collection at baseline and post-engraftment. CONCLUSION: The current pilot RCT is the first eMBMT intervention to address the HRQOL and symptom burden of patients who are undergoing allo-SCT. Results will inform a fully powered RCT to establish preliminary efficacy of eMBMT on improvements in HRQOL, symptom burden, and disease activity.

Cell-Autonomous Cxcl1 Sustains Tolerogenic Circuitries and Stromal Inflammation via Neutrophil-Derived TNF in Pancreatic Cancer.

We have shown that KRAS-TP53 genomic coalteration is associated with immune-excluded microenvironments, chemoresistance, and poor survival in pancreatic ductal adenocarcinoma (PDAC) patients. By treating KRAS-TP53 cooperativity as a model for high-risk biology, we now identify cell-autonomous Cxcl1 as a key mediator of spatial T-cell restriction via interactions with CXCR2+ neutrophilic myeloid-derived suppressor cells in human PDAC using imaging mass cytometry. Silencing of cell-intrinsic Cxcl1 in LSL-KrasG12D/+;Trp53R172H/+;Pdx-1Cre/+(KPC) cells reprograms the trafficking and functional dynamics of neutrophils to overcome T-cell exclusion and controls tumor growth in a T cell-dependent manner. Mechanistically, neutrophil-derived TNF is a central regulator of this immunologic rewiring, instigating feed-forward Cxcl1 overproduction from tumor cells and cancer-associated fibroblasts (CAF), T-cell dysfunction, and inflammatory CAF polarization via transmembrane TNF-TNFR2 interactions. TNFR2 inhibition disrupts this circuitry and improves sensitivity to chemotherapy in vivo. Our results uncover cancer cell-neutrophil cross-talk in which context-dependent TNF signaling amplifies stromal inflammation and immune tolerance to promote therapeutic resistance in PDAC. SIGNIFICANCE: By decoding connections between high-risk tumor genotypes, cell-autonomous inflammatory programs, and myeloid-enriched/T cell-excluded contexts, we identify a novel role for neutrophil-derived TNF in sustaining immunosuppression and stromal inflammation in pancreatic tumor microenvironments. This work offers a conceptual framework by which targeting context-dependent TNF signaling may overcome hallmarks of chemoresistance in pancreatic cancer. This article is highlighted in the In This Issue feature, p. 1275.

Simultaneous measurement of ERα, HER2, and phosphoERK1/2 in breast cancer cell lines by flow cytometry.

The activation of human epidermal growth factor receptor-2 (HER2) results in the activation of the mitogen-activated protein kinase (MAPK) cascade that may lead to the resistance to anti-estrogen therapy in estrogen receptor (ERα) expressing breast cancer by means of phosphorylation of ERα in the N-terminal region by phosphorylated extracellular signal-regulated kinase 1/2 (pERK1/2) and by means of decreasing ERα expression. Immunohistochemistry is the most widely used technique for the detection of ERα and HER2 in breast cancer specimens, however, is inadequate in its ability to assess the relationship between ERα, HER2, and MAPK cascade at the single cell level. To clear this major hurdle, we devised a novel flow cytometric method to quantify the expression of ERα, HER2, and the activation of MAPK cascade simultaneously in single cells. The method was validated by concurrent Western blotting in established cell lines: MDA-231 (ERα and HER2-negative), MCF-7 (ERα-positive, HER2-negative), MCF-7 cells overexpressing ERα after long-term incubation in estrogen-free medium, and HER2 transfected MCF7 cells. Using the flow cytometry method, we confirmed the previous finding that ERα expression is down-regulated upon epidermal growth factor mediated ERK1/2 phosphorylation in EGFR/MCF-7 cells. To our knowledge, this is the first such assay to incorporate simultaneous single cell measurement for all of these pathways, which may prove useful to determine the intratumoral heterogeneity in breast tumors or the receptor status in circulating tumor cells.

Human late memory CD8+ T cells have a distinct cytokine signature characterized by CC chemokine production without IL-2 production.

Late memory T cell skewing is observed in the setting of immune recovery after cord blood transplantation, and may be associated with inferior control of viral reactivation and cancers. Therefore, we sought to understand how late memory cells differ functionally from earlier stage memory T cells, and whether surface phenotypes associated with differentiation stages were predictably associated with functional signatures. Higher order cytokine flow cytometry allows characterization of human T cells based on complex phenotypic markers and their differential capacity to simultaneously secrete effector proteins, including cytokines and chemokines. We used 8-color, 10-parameter cytokine flow cytometry to characterize the functional activation of human late memory CD8(+) T cells defined by CD45RA and CD27 expression (CD27(-)CD45RA(+)). We assessed the 15 possible functional signatures of cells defined by production of IL-2, IFN-gamma, TNF-alpha, and MIP-1beta alone or in combination, following activation with Ags stimulating bypassing surface proteins (PMA:ionomycin) or through the TCR (e.g., viral Ags). Late memory CD8(+) T cells produced abundant amounts of CC chemokines (MIP-1beta, MIP-1alpha, and RANTES) but not IL-2. IL-2/IFN-gamma coproduction, characteristic of protective immune responses to viral infections, was absent in late memory CD8(+) T cells. These data demonstrate that functional cytokine signatures are predictably associated with CD8(+) maturation stages, and that the polarization of late memory CD8(+) T cells toward CC chemokine production and away from IL-2 production suggests a unique functional role for this subset.

Adoptive transfer of PR1 cytotoxic T lymphocytes associated with reduced leukemia burden in a mouse acute myeloid leukemia xenograft model.

BACKGROUND AIMS: Tumor antigen-specific cytotoxic T lymphocytes (CTL) have been used in the treatment of human cancer, including leukemia. Several studies have established PR1 peptide, an HLA-A2.1-restricted peptide derived from proteinase 3 (P3), as a human leukemia-associated antigen. PR1-specific CTL elicited in vitro from healthy donors have been shown to lyse P3-expressing AML cells from patients. We investigated whether PR1-CTL can be adoptively transferred into NOD/SCID mice to eliminate human leukemia cells. METHODS: PR1-CTL were generated in bulk culture from peripheral blood mononuclear cells (PBMC) stimulated with autologous dendritic cells. Human acute myeloid leukemia (AML) patient samples were injected and engrafted in murine bone marrow at 2 weeks post-transfer. RESULTS: Following adoptive transfer, bone marrow aspirate from mice that received AML alone had 72-88% blasts in a hypercellular marrow, whereas mice that received AML plus PR1-CTL co-infusion had normal hematopoietic elements and only 3-18% blasts in a hypocellular marrow. The PR1-CTL persisted in the bone marrow and liver and maintained a CD45RA⁻CD28+ effector phenotype. CONCLUSIONS: We found that adoptive transfer of PR1-CTL generated in vitro is associated with reduced AML cells in NOD/SCID mice. PR1-CTL can migrate to the sites of disease and maintain their capacity to kill the AML cells. The surface phenotype of PR1-CTL was consistent with their trafficking pattern in both vascular and end-organ tissues.

Flow cytometric determination of Src phosphorylation in pediatric patients treated with dasatinib.

Tyrosine kinase inhibitors, such as imatinib, have dramatically improved the outcomes for patients with selected cancers. For imatinib, western blotting of phospho-CrkL was an insensitive, indirect, and descriptive method to determine drug efficacy. Greater use of targeted therapies should involve more quantitative evaluation of the target's dose-inhibition. The Src/Abl kinase inhibitor dasatinib has recently been approved for use in Ph+ leukemias after failure with imatinib. Src family kinases (SFK) also play a critical role in nonhematologic cancers. We have developed a flow cytometric assay to measure SFK autophosphorylation levels in blood mononuclear cells and observed a direct correlation between its inhibition and patient dosage. This method provides a sensitive, quick, and quantitative tool to assess drug efficacy.

Correction to: Angiosarcoma patients treated with immune checkpoint inhibitors: a case series of seven patients from a single institution.

Following publication of the original article [1], the authors have reported that the following sentence "While of the same IgG1 class as ipilimumab, preclinical data suggests this molecule may have enhanced activity against T regulatory cells".

Angiosarcoma patients treated with immune checkpoint inhibitors: a case series of seven patients from a single institution.

BACKGROUND: Angiosarcoma is an uncommon endothelial malignancy and a highly aggressive soft tissue sarcoma. Due to its infiltrative nature, successful management of localized angiosarcoma is often challenging. Systemic chemotherapy is used in the metastatic setting and occasionally in patients with high-risk localized disease in neoadjuvant or adjuvant settings. However, responses tend to be short-lived and most patients succumb to metastatic disease. Novel therapies are needed for patients with angiosarcomas. METHODS: We performed a retrospective analysis of patients with locally advanced or metastatic angiosarcoma, who were treated with checkpoint inhibitors at our institution. We collected their clinical information and outcome measurements. In one patient with achieved complete response, we analyzed circulating and infiltrating T cells within peripheral blood and tumor tissue. RESULTS: We have treated seven angiosarcoma (AS) patients with checkpoint inhibitors either in the context of clinical trials or off label [Pembrolizumab + Axitinib (NCT02636725; n = 1), AGEN1884, a CTLA-4 inhibitor (NCT02694822; n = 2), Pembrolizumab (n = 4)]. Five patients had cutaneous angiosarcoma, one primary breast angiosarcoma and one radiation-associated breast angiosarcoma. At 12 weeks, 5/7 patients (71%) had partial response of their lesions either on imaging and/or clinical exam and two (29%) had progressive disease. 6/7 patients are alive to date and, thus far, 3/7 patients (43%) have progressed (median 3.4 months)- one achieved partial response after pembrolizumab was switched to ongoing Nivolumab/Ipilimumab, one died of progressive disease at 31 weeks (primary breast angiosarcoma) and one was placed on pazopanib. One patient had a complete response (CR) following extended treatment with monotherapy AGEN1884. No patient experienced any ≥ grade 2 toxicities. CONCLUSIONS: This case series underscores the value of targeted immunotherapy in treating angiosarcoma. It also identifies genetic heterogeneity of cutaneous angiosarcomas and discusses specific genetic findings that may explain reported benefits from immunotherapy.

A novel strategy for rapid and efficient isolation of human tumor-specific CD4(+) and CD8(+) T-cell clones.

Adoptive therapy with antigen-specific T cells is a promising approach for the treatment of infectious diseases and cancer. However, cloning of antigen-specific T cells by the traditional approach of limiting dilution is a time-consuming, laborious, and inefficient process. Here, we describe a novel flow cytometric strategy for rapid isolation of human tumor antigen-specific T-cell clones by using T-cell receptor (TCR) Vbeta antibodies in combination with carboxyfluorescein succinimidyl ester (CFSE)-based proliferation assay. The CFSE dilution following antigen stimulation identified proliferating antigen-specific T cells, and the TCRVbeta antibodies allowed distinguishing T cells at the clonal level from a heterogeneous T-cell population. This method of TCRVbeta/CFSE dilution was used for the isolation of four different human lymphoma and melanoma-specific CD4(+) and CD8(+) T-cell clones reactive against defined and undefined tumor antigens. Isolated tumor-specific T-cell clones could be expanded to large numbers ex vivo while maintaining phenotype, function, and tumor antigen specificity. The method was simple, efficient, and reproducible, and may have potential application for the development of adoptive immunotherapeutic strategies.

Chronic myelogenous leukemia shapes host immunity by selective deletion of high-avidity leukemia-specific T cells.

We have shown that cytotoxic T lymphocytes specific for PR1, an HLA-A2-restricted nonopeptide derived from proteinase 3, kill leukemia cells and may contribute to the elimination of chronic myelogenous leukemia (CML) after treatment with IFN or allogeneic bone marrow transplant. Some patients with persistent disease also have circulating PR1-specific T cells, however, suggesting the likelihood of immune tolerance. Here we show that both high- and low-avidity PR1-specific T cells from the peripheral blood of healthy donors can be identified and selectively expanded in vitro. Although high-avidity PR1-specific T cells killed CML more effectively than low-avidity T cells, only high-avidity T cells underwent apoptosis when stimulated with high PR1 peptide concentration or when exposed to leukemia that overexpressed proteinase 3. No high-avidity PR1-specific T cells could be identified or expanded from newly diagnosed leukemia patients, whereas low-avidity T cells were readily expanded. Circulating high-avidity PR1-specific T cells were identified in IFN-sensitive patients in cytogenetic remission, however. These results provide evidence that CML shapes the host immune response and that leukemia outgrowth may result in part from leukemia-induced selective deletion of high-avidity PR1-specific T cells.

The evolving art of hematopoietic stem cell transplantation: translational research in post-transplant immune reconstitution and immunosuppression.

Allogeneic hematopoietic stem cell transplantation (SCT) offers the best chance for cure and/or long-term survival for a broad range of diseases, including many high-risk hematologic malignancies, bone marrow failure states and subsets of inherited metabolic diseases and hemoglobinopathies. Clinical advances in allogeneic SCT have resulted in dramatically improved clinical outcomes over the past two decades, resulting in a significant expansion of transplant utilization to many recipients who would previously have been excluded from consideration, including elderly recipients and individuals lacking matched sibling or unrelated donors. Despite these advances, significant clinical challenges remain, including delayed immune reconstitution and the frequent occurrence of acute and chronic graft-versus-host disease, especially in the unrelated donor transplant setting. Translational laboratory efforts, facilitated by technical advances in our ability to measure thymopoiesis and functional T cell subsets in humans, have resulted in an improved understanding of immune recovery and have provided novel insights that may lead to more rational and selective immunosuppression.

Understanding the biology of ex vivo-expanded CD8 T cells for adoptive cell therapy: role of CD62L.

CD62L governs the circulation of CD8(+) T cells between lymph nodes and peripheral tissues, whereby the expression of CD62L by CD8(+) T cells promotes their recirculation through lymph nodes. As such, CD62L participates in the fate of adoptively transferred CD8(+) T cells and may control their effectiveness for cancer immunotherapy, including settings in which host preconditioning results in the acute lymphopenia-induced proliferation of the transferred cells. Indeed, previous studies correlated CD62L expression by donor CD8(+) cells with the success rate of adoptive cell therapy (ACT). Here, we analyzed the functions and fate of ex vivo-activated, tumor-specific CD62L(-/-) CD8(+) T cells in a mouse melanoma model for ACT. Unexpectedly, we observed that CD62L(-/-) CD8(+) T cells were functionally indistinguishable from CD62L(+/+) CD8(+) T cells, i.e., both greatly expanded in cyclophosphamide preconditioned animals, controlled subcutaneously and hematogenously spreading tumors, and generated anti-tumor-specific CD8(+) T cell memory. Moreover, even in hosts with rudimentary secondary lymphoid organs (LT(-/-) animals), CD8(+) T cells with and without CD62L expanded equivalently to those adoptively transferred into wild-type animals. These results put into question the utility of CD62L as a predictive biomarker for the efficacy of ex vivo-expanded T cells after ACT in lymphopenic conditions and also offer new insights into the homing, engraftment, and memory generation of adoptively transferred ex vivo-activated CD8(+) T cells.

Vitamin D receptor upregulation in alloreactive human T cells.

Vitamin D deficiency is adversely associated with diseases characterized by inflammation. The combination of the high incidence of vitamin D deficiency in patients undergoing allogeneic stem cell transplants (SCT) and the potential role of vitamin D deficiency in influencing graft-versus-host disease led us to further characterize the expression of VDR on alloreactive T cells. We hypothesized that vitamin D receptor expression may directly regulate alloreactive T cell responses. To overcome existing limitations in measuring VDR in bulk cellular populations, we developed a flow cytometric assay to measure cytoplasmic VDR in human T cells. Upon stimulation, VDR was expressed extremely early and exhibited sustained upregulation with chronic stimulation. VDR expression was also coupled to cytokine production, proliferation, and ERK1/2 phosphorylation. In addition, VDR exhibited a maturation stage-specific pattern of expression, with greatest expression on cells known to mediate GVHD, naïve and early memory T cells. Alloreactive T cells upregulated VDR, whereas the nonreactive T cells did not. Finally, repletion of vitamin D in vitro was sufficient to significantly reduce alloreactive T cell responses. These data suggest that vitamin D effects on T cells may be important in reducing graft versus host disease (GVHD) in the allogeneic stem cell transplant setting.

The role of antigen cross-presentation from leukemia blasts on immunity to the leukemia-associated antigen PR1.

Cross-presentation is an important mechanism by which exogenous tumor antigens are presented to elicit immunity. Because neutrophil elastase (NE) and proteinase-3 (P3) expression is increased in myeloid leukemia, we investigated whether NE and P3 are cross-presented by dendritic cells (DC) and B cells, and whether the NE and P3 source determines immune outcomes. We show that NE and P3 are elevated in leukemia patient serum and that levels correlate with remission status. We demonstrate cellular uptake of NE and P3 into lysosomes, ubiquitination, and proteasome processing for cross-presentation. Using anti-PR1/human leukocyte antigen-A2 monoclonal antibody, we provide direct evidence that B-cells cross-present soluble and leukemia-associated NE and P3, whereas DCs cross-present only leukemia-associated NE and P3. Cross-presentation occurred at early time points but was not associated with DC or B-cell activation, suggesting that NE and P3 cross-presentation may favor tolerance. Furthermore, we show aberrant subcellular localization of NE and P3 in leukemia blasts to compartments that share common elements of the classic major histocompatibility class I antigen-presenting pathway, which may facilitate cross-presentation. Our data demonstrate distinct mechanisms for cross-presentation of soluble and cell-associated NE and P3, which may be valuable in understanding immunity to PR1 in leukemia.

Functional impairment of central memory CD4 T cells is a potential early prognostic marker for changing viral load in SHIV-infected rhesus macaques.

In HIV infection there is a paucity of literature about the degree of immune dysfunction to potentially correlate and/or predict disease progression relative to CD4(+) T cells count or viral load. We assessed functional characteristics of memory T cells subsets as potential prognostic markers for changing viral loads and/or disease progression using the SHIV-infected rhesus macaque model. Relative to long-term non-progressors with low/undetectable viral loads, those with chronic plasma viremia, but clinically healthy, exhibited significantly lower numbers and functional impairment of CD4(+) T cells, but not CD8(+) T cells, in terms of IL-2 production by central memory subset in response to PMA and ionomycine (PMA+I) stimulation. Highly viremic animals showed impaired cytokine-production by all T cells subsets. These results suggest that functional impairment of CD4(+) T cells in general, and of central memory subset in particular, may be a potential indicator/predictor of chronic infection with immune dysfunction, which could be assayed relatively easily using non-specific PMA+I stimulation.