Reduced skin homing by functional Treg in vitiligo

In human vitiligo, cutaneous depigmentation involves cytotoxic activity of autoreactive T cells. It was hypothesized that depigmentation can progress in the absence of regulatory T cells (Treg). The percentage of Treg among skin infiltrating T cells was evaluated by immunoenzymatic double staining for CD3 and FoxP3, revealing drastically reduced numbers of Treg in non-lesional, perilesional and lesional vitiligo skin. Assessment of the circulating Treg pool by FACS analysis of CD4, CD25, CD127 and FoxP3 expression, and mixed lymphocyte reactions in presence and absence of sorted Treg revealed no systemic drop in the abundance or activity of Treg in vitiligo patients. Expression of skin homing receptors CCR4, CCR5, CCR8 and CLA was comparable among circulating vitiligo and control Treg. Treg from either source were equally capable of migrating towards CCR4 ligand and skin homing chemokine CCL22, yet significantly reduced expression of CCL22 in vitiligo skin observed by immunohistochemistry may explain failure of circulating, functional Treg to home to the skin in vitiligo. The paucity of Treg in vitiligo skin is likely crucial for perpetual anti-melanocyte reactivity in progressive disease.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/78696/1/j.1755-148X.2010.00688.x.pd

Membrane-Bound IL-21 Promotes Sustained Ex Vivo Proliferation of Human Natural Killer Cells

NK cells have therapeutic potential for a wide variety of human malignancies. However, because NK cells expand poorly in vitro, have limited life spans in vivo, and represent a small fraction of peripheral white blood cells, obtaining sufficient cell numbers is the major obstacle for NK-cell immunotherapy. Genetically-engineered artificial antigen-presenting cells (aAPCs) expressing membrane-bound IL-15 (mbIL15) have been used to propagate clinical-grade NK cells for human trials of adoptive immunotherapy, but ex vivo proliferation has been limited by telomere shortening. We developed K562-based aAPCs with membrane-bound IL-21 (mbIL21) and assessed their ability to support human NK-cell proliferation. In contrast to mbIL15, mbIL21-expressing aAPCs promoted log-phase NK cell expansion without evidence of senescence for up to 6 weeks of culture. By day 21, parallel expansion of NK cells from 22 donors demonstrated a mean 47,967-fold expansion (median 31,747) when co-cultured with aAPCs expressing mbIL21 compared to 825-fold expansion (median 325) with mbIL15. Despite the significant increase in proliferation, mbIL21-expanded NK cells also showed a significant increase in telomere length compared to freshly obtained NK cells, suggesting a possible mechanism for their sustained proliferation. NK cells expanded with mbIL21 were similar in phenotype and cytotoxicity to those expanded with mbIL15, with retained donor KIR repertoires and high expression of NCRs, CD16, and NKG2D, but had superior cytokine secretion. The mbIL21-expanded NK cells showed increased transcription of the activating receptor CD160, but otherwise had remarkably similar mRNA expression profiles of the 96 genes assessed. mbIL21-expanded NK cells had significant cytotoxicity against all tumor cell lines tested, retained responsiveness to inhibitory KIR ligands, and demonstrated enhanced killing via antibody-dependent cell cytotoxicity. Thus, aAPCs expressing mbIL21 promote improved proliferation of human NK cells with longer telomeres and less senescence, supporting their clinical use in propagating NK cells for adoptive immunotherapy

Strain and Virulence Diversity in the Mouse Pathogen Chlamydia muridarum▿

The mouse chlamydial pathogen Chlamydia muridarum has been used as a model organism for the study of human Chlamydia trachomatis urogenital and respiratory tract infections. To date, two commonly used C. muridarum isolates have been used interchangeably and are essentially taken to be identical. Herein, we present data that indicate that this is not the case. The C. muridarum Weiss isolate and C. muridarum Nigg isolate varied significantly in their virulences in vivo and possessed different growth characteristics in vitro. Distinct differences were observed in intravaginal 50% infectious doses and in challenge infections, with the Weiss isolate displaying greater virulence. Respiratory infection by the intranasal route also indicated a greater virulence of the Weiss isolate. In vitro, morphometric analysis revealed that the Weiss isolate produced consistently smaller inclusions in human cervical adenocarcinoma cells (HeLa 229) and smaller plaques in monolayers of mouse fibroblasts (L929) than did the Nigg isolate. In addition, the Weiss isolate possessed significantly higher replicative yields in vitro than did the Nigg isolate. In plaque-purified isolates derived from our stocks of these two strains, total genomic sequencing identified several unique nonsynonymous single nucleotide polymorphisms and insertion/deletion mutations when our Weiss (n = 4) and Nigg (n = 5) isolates were compared with the published Nigg sequence. In addition, the two isolates shared 11 mutations compared to the published Nigg sequence. These results prove that there is genotypic and virulence diversity among C. muridarum isolates. These findings can be exploited to determine factors related to chlamydial virulence and immunity

Haploidentical Natural Killer Cells Infused before Allogeneic Stem Cell Transplantation for Myeloid Malignancies: A Phase I Trial

Allogeneic stem cell transplantation is an effective treatment for high-risk myeloid malignancies, but relapse remains the major post-transplantation cause of treatment failure. Alloreactive natural killer (NK) cells mediate a potent antileukemic effect and may also enhance engraftment and reduce graft-versus-host disease (GVHD). Haploidentical transplantations provide a setting in which NK cell alloreactivity can be manipulated, but they are associated with high rates of GVHD. We performed a phase I study infusing escalating doses of NK cells from an HLA haploidentical-related donor-selected for alloreactivity when possible-as a component of the preparative regimen for allotransplantation from a separate HLA-identical donor. The goal of infusing third-party alloreactive NK cells was to augment the antileukemic effect of the transplantation without worsening GVHD and, thus, improve the overall outcome of hematopoietic transplantation. Twenty-one patients with high-risk acute myeloid leukemia (AML), myelodysplastic syndrome (MDS), or chronic myelogenous leukemia refractory or beyond first remission received a preparative regimen with busulfan and fludarabine followed by infusion of apheresis-derived, antibody-selected, and IL-2-activated NK cells. Doses were initially based on total nucleated cell (TNC) content and later based on CD56(+) cells to reduce variability. CD56(+) content ranged from .02 to 8.32 × 10(6)/kg. IL-2, .5 × 10(6) units/m(2) subcutaneously was administered daily for 5 days in the final cohort (n = 10). CD3(+) cells in the NK cell product were required to be < 10(5)/kg. Median relapse-free, overall, and GVHD-free/relapse-free survival for all patients enrolled was 102, 233, and 89 days, respectively. Five patients are alive, 5 patients died of transplantation-related causes, and 11 patients died of relapse. Despite the small sample size, survival was highly associated with CD56(+) cells delivered (P = .022) and development of ≥ grade 3 GVHD (P = .006). There were nonsignificant trends toward higher survival rates in those receiving NK cells from KIR ligand-mismatched donors and KIR-B haplotype donors. There was no association with disease type, remission at time of transplantation, or KIR content. GVHD was not associated with TNC, CD56(+), or CD3(+) cells infused in the NK cell product or the stem cell product. This trial demonstrates a lack of major toxicity attributable to third-party NK cell infusions delivered in combination with an HLA-compatible allogeneic transplantation. The infusion of haploidentical alloreactive NK cells was well tolerated and did not interfere with engraftment or increase the rate of GVHD after allogeneic hematopoietic transplantation. Durable complete remissions occurred in 5 patients at high risk for disease recurrence. This approach is being further developed in a phase I/II trial with ex vivo-expanded NK cells to increase the NK cell dose with the objective of reducing relapse and improving the outcome of allogeneic hematopoietic transplantation for AML/MDS

Therapeutic implications of autoimmune vitiligo T cells

Vitiligo is an autoimmune disease presenting with progressive loss of skin pigmentation. The disease strikes 1% of the world population, generally during teenage years. The progressive loss of melanocytes from depigmenting vitiligo skin is accompanied by cellular infiltrates containing both CD4+ and CD8+ T lymphocytes. Infiltrating cytotoxic T cells with high affinity T cell receptors have likely escaped clonal deletion in the thymus, allowing such T cells to enter the circulation. Through the expression of CLA, these T cells home to the skin where they express type 1-cytokine profiles and mediate melanocyte apoptosis via the granzyme/perforin pathway. T cells found juxtapositionally apposed to remaining melanocytes can be isolated from the skin. Vitiligo T cells have demonstrated reactivity to antigens previously recognized as target antigens for T cells infiltrating melanoma tumors. In a comparison to existing melanoma-derived T cells, vitiligo T cells displayed superior reactivity towards melanoma cells. It is thought that genes encoding the TCRs expressed by vitiligo skin infiltrating T cells can be cloned and expressed in melanoma T cells, thereby generating a pool of circulating T cells with high affinity for their targets that can re-direct the immune response towards the tumo

NK cell expansion and purity after repeated weekly stimulation with aAPCs expressing membrane-bound cytokines.

<p><i>A</i>, Mean expansion of CD3^neg/CD16-or-56^pos NK cells from 22 donors expanded for 21 days using aAPCs bearing mbIL15 or mbIL21. NK cells from five donors were also expanded with aAPCs expressing both mbIL15 and mbIL21. <i>B</i>, Mean expansion of NK cells from 4 donors expanded for 42 days using aAPCs bearing mbIL15 or mbIL21. <i>C</i>, Percent of CD3^pos T/NKT cells and CD3^neg/CD16-or-56^pos NK cells in the starting PBMC product (day 0) and at the end of 21 days of expansion on Clone 9.mbIL21 from 20 donors. <i>D</i>, Mean expansion of CD3^neg/CD16-or-56^pos NK cells on Clone 9.mbIL21 from unfractionated PBMC (n = 19), unfractionated PBMC followed by NK purification at day 14 of expansion (n = 3), or from NK cells purified from PBMC at day 0 prior to expansion (n = 13). Mean +/− SD is shown for each plot. All p values indicated are for <i>t-</i>test of fold expansion at the end of the expansion period.</p

Phenotype of NK cells expanded on aAPCs bearing membrane-bound cytokines.

<p>NK cells purified from PBMC were stimulated weekly with either Clone 4 (mbIL15) or Clone 9.mbIL21 for 3 weeks. Expression of NK cell receptors was determined by flow cytometry. A) Representative dot plots of NK cells expanded from the same donor on Clone 4 (mbIL15) or Clone 9.mbIL21. B) Component subpopulations of fresh NK cells or NK cells expanded on Clone 4 (mbIL15) or Clone 9.mbIL21 from 4 donors as determined by flow cytometry. Mean +/− SD is shown. P values are for 2-way repeated-measures ANOVA comparing against mbIL21-expanded NK cells with Bonferroni correction (all significant P values are indicated).</p

Schema for NK cell manufacturing with aAPCs.

<p>Artificial antigen-presenting cells (aAPCs) were produced by genetic modification of K562 to express costimulatory molecules and membrane-bound cytokines. To expand NK cells <i>ex</i> vivo, unfractionated PBMC are stimulated weekly with irradiated PBMC, inducing rapid proliferation of NK cells and in some cases non-specific expansion of T cells. Contaminating T cells may be depleted, and the remaining purified NK cells may be stimulated weekly by the aAPCs as needed to obtain sufficient numbers. Expanded NK cells may be used directly or cryopreserved for future use.</p

Gene expression in NK cells stimulated with mbIL15 or mbIL21 as assessed using the nCounter platform.

<p>Paired aliquots of purified NK cells from 4 donors were stimulated for one week with either Clone 4 (mbIL15) or Clone 9.mbIL21. Total RNA was purified and equal quantities hybridized for detection of expression of 96 genes. Gene expression was normalized to LDH (mean 6,076 copies detected), and the remaining data for each gene plotted as mean +/− SEM for each expansion condition. Genes with detection below background (set at ≤10 detected copies) were excluded as not biologically significant (grey box). The ten highest-expressed genes in addition to LDH are labeled, as are genes that are differentially expressed by >2 fold (red) or <0.5 fold (blue) in mbIL21-expanded cells. Differentially expressed genes were replotted (inset) for comparison, and two-tailed <i>t-</i>test applied.</p

Direct cytotoxicity and cytokine secretion of fresh NK cells, mbIL15-expanded, and mbIL21-expanded NK cells.

<p>NK cells were purified from four normal donor buffy coats and assessed directly, and then retested after being expanded for 21 days with Clone 9.mbIL21 and Clone 4 (mbIL15) (with CD3 depletion on day 14). NK cells were assessed for cytotoxicity against 721.221 (A) or cytokine secretion in response to K562 (B). NK cells from another four donors were purified and cryopreserved, and expanded with Clone 9.mbIL21 and then cryopreserved. Fresh (circles) and expanded (squares) NK cells were then thawed and activated in parallel for 24 h in 50 IU/mL IL-2, and then tested for cytotoxicity against HLA^null 721.221 targets (C), C*0702-transduced (Group C1) 721.221 targets (D), or B*5801-transduced (Group Bw4) 721.221 targets (E). All data points shown are mean +/− SEM of the means of four donors tested in triplicate.</p