Immunologic and clinical effects of antibody blockade of cytotoxic T lymphocyte-associated antigen 4 in previously vaccinated cancer patients.

Cytotoxic T lymphocyte-associated antigen 4 (CTLA-4) functions as a negative regulator of endogenous and vaccine-induced antitumor immunity. The administration of fully human anti-CTLA-4 blocking monoclonal antibodies to advanced-cancer patients increases immune-mediated tumor destruction in some subjects. Nonetheless, patients that respond also frequently manifest serious inflammatory pathologies, raising the possibility that the therapeutic and toxic effects of CTLA-4 blockade might be linked. Here we show that periodic infusions of anti-CTLA-4 antibodies after vaccination with irradiated, autologous tumor cells engineered to secrete GM-CSF (GVAX) generate clinically meaningful antitumor immunity without grade 3 or 4 toxicity in a majority of metastatic melanoma patients. The application of this sequential immunotherapy to advanced ovarian carcinoma patients also revealed that tumor destruction and severe inflammatory pathology could be dissociated, although further refinements are required to increase clinical responses and to minimize toxicity in this population. The extent of therapy-induced tumor necrosis was linearly related to the natural logarithm of the ratio of intratumoral CD8(+) effector T cells to FoxP3(+) regulatory T cells (Tregs) in posttreatment biopsies. Together, these findings help clarify the immunologic and clinical effects of CTLA-4 antibody blockade in previously vaccinated patients and raise the possibility that selective targeting of antitumor Tregs may constitute a complementary strategy for combination therapy.

Stem cell marker upregulation in normal cutaneous vessels following pulsed-dye laser exposure and its abrogation by concurrent rapamycin administration: implications for treatment of port-wine stain birthmarks.

Port-wine stains (PWS) represent a group of vascular malformations that are usually accompanied by psychological distress for affected patients, often reflected in high treatment demand. Although the pulsed-dye laser (PDL) was established as standard therapy for PWS more than a decade ago, therapeutic outcome may be unsatisfactory. One of the main drawbacks to successful PDL therapy is PWS revascularization shortly after laser exposure. Therefore, inhibition of revascularization should improve therapeutic outcome of PDL therapy. In this study, we first evaluated the effects of various light energies on normal cutaneous vessels over a period of 14 days, particularly the proliferation and stem cell marker expression of dermal endothelial cells, which were found to be highest 8 days following laser exposure. We found that PDL exposure induced dose-dependent damage of dermal vessels up to energy densities of 6 J/cm(2), above which no increase in PDL-induced effects were observed with the energies employed in this study. In dermal endothelial cells of PDL-exposed skin, we found strong expression of the proliferation marker Ki-67 as well as the stem cell marker nestin but not other stem cell markers such as CD133 and CD166. The influence of rapamycin (RPM), used as an adjuvant to PDL exposure, was also investigated. RPM administration reduced Ki-67 and nestin expression in dermal endothelial cells and increased PDL-induced destruction of dermal vessels, indicating that the use of RPM after PDL exposure may be an interesting new approach for prolonging and improving PWS laser therapeutic outcome.

Focus on TILs: prognostic significance of tumor infiltrating lymphocytes in human melanoma.

Tumors contain variable numbers of lymphocytes, referred to as tumor infiltrating lymphocytes (TILs). In melanoma, the intensity of this lymphocytic infiltrate is believed to correlate with outcome, though there is some debate about the applicability of this finding for all melanomas. Much research has gone into classifying TILs with respect to antigen receptor structure and the antigen to which melanoma-specific T cells react. However, these studies for the most part did not immunophenotype TILs, and recent data has revealed that the composition of tumoral lymphocytes is not homogenous, but rather represents varying contributions from many lymphocytic subsets. Furthermore, the function of TILs is often compromised as a result of the accumulation of immunoregulatory cells and various tumor escape mechanisms. These recent insights stress the need to collect more data on the composition and function of TIL infiltrates before definitive conclusions about the prognostic significance of TILs can be drawn. Advances in immunology have also facilitated the development of immunotherapeutic strategies, examples of which will be discussed with a special emphasis on blocking antibodies against CTLA-4, which are prototypical immunotherapeutic agents. This flurry of novel "biological" therapies will undoubtedly complicate our already incomplete understanding of TIL immunobiology as each of these agents has the potential to uniquely distort the series of immunological events which normally occur in untreated melanoma. Therefore, considerable research is needed to better elucidate the function and prognostic significance of TILs in both untreated melanoma and tumors treated with "biological" therapy.

KHSV(-) EBV(-) post-transplant effusion lymphoma with plasmablastic features: variant of primary effusion lymphoma?

Primary effusion lymphoma (PEL) is a rare type of B-cell non-Hodgkin lymphoma (NHL), which predominantly occurs in HIV-infected individuals, and is pathogenetically linked with Kaposi sarcoma (KS)-associated herpes virus/human herpes virus-8 (KSHV/HHV-8) infection with or without evidence of Epstein-Barr virus (EBV) co-infection. Although uncommon, PELs have been reported in immunocompetent patients and recipients of solid organ allografts. Rare cases of KSHV(-) EBV(+) post-transplant effusion lymphomas resembling PEL have also been described, as have KSHV(-) EBV(-) effusion lymphomas, the latter including those arising in individuals with chronic liver disease. We report a unique KSHV(-) EBV(-) post-transplant effusion lymphoma associated with serum paraproteins, occurring in an HIV(-) individual, which had cytologic features and phenotype similar to PEL, and displayed a complex karyotype including isochromosome 12p and translocation t(8;22), resulting in rearrangement of c-MYC.

CTLA-4 blockade with ipilimumab induces significant clinical benefit in a female with melanoma metastases to the CNS.

BACKGROUND: A 63-year-old female presented to her primary physician with numbness and weakness in her left leg, which progressed over several days to involve her entire lower extremities. MRI of the spine and brain revealed multiple metastases. The patient received ipilimumab and after 3 months experienced intermittent confusion and focal seizures. INVESTIGATIONS: Electroencephalogram and MRI scans of the spine and brain, followed by surgical removal of a left frontal cortical brain metastasis and subsequent histological and pathological analyses. DIAGNOSIS: Metastatic melanoma from an unknown primary tumor. MANAGEMENT: The patient was treated with ipilimumab on a compassionate-use program and dexamethasone, celecoxib, and levetiracetam to treat the symptoms and seizures. Postoperative stereotactic radiosurgery was initiated.

A novel T cell receptor transgenic animal model of seborrheic dermatitis-like skin disease.

We have characterized a novel animal model of the common inflammatory skin disease seborrheic dermatitis (SD) that involves the expression of the self-specific 2C transgenic T cell receptor on the DBA/2 genetic background. Opportunistic fungal pathogens are present in the primary histological lesions and severe disease can be mitigated by the administration of fluconazole, demonstrating a role for infection in disease pathogenesis. Spontaneous disease convalescence occurs at 70-90 d of age and is preceded by an expansion of CD4+ T cells that partially restores the T cell lymphopenia that occurs in these animals. The adoptive transfer of syngeneic CD4+ T cells into pre-diseased DBA/2 2C mice completely abrogates the development of cutaneous disease. The pattern of disease inheritance in DBA/2 backcrosses suggests that one, or a closely linked group of genes, may control disease penetrance. Bone marrow reconstitution experiments demonstrated that the DBA/2 susceptibility factor(s) governing disease penetrance is likely non-hematopoietic since bone marrow from disease-resistant 2C mice can adoptively transfer the full disease phenotype to non-transgenic DBA/2 animals. This model implicates fungal organisms and CD4+ T cell lymphopenia in the development of a SD-like condition and, as such, may mimic the development of SD in acquired immunodeficiency syndrome.

Eosinophilic/T-cell chorionic vasculitis and chronic villitis involve regulatory T cells and often occur together.

Eosinophilic/T-cell chorionic vasculitis (ETCV) is characterized by mixed T-cell, eosinophilic, and histiocytic infiltrates within the chorionic vessel wall. We sought to better characterize this lesion with respect to other pathologic correlates and the T-cell populations involved. Epidemiologic data and other pathologic diagnoses, including concurrent chronic villitis (CV), were tabulated for each case of ETCV diagnosed at our institution over a 6-year period. CD3, CD25, FOXP3, and dual FOXP3-CD3 immunostains were used to identify regulatory T-cell populations in ETCV and CV. Cells positive for CD3, FOXP3, and CD25 were quantitated by manual counts of ×40 fields at the sites of ETCV and CV, and FOXP3∶CD3 and CD25∶CD3 ratios were calculated. Digital analysis of ETCV and CV using the dual FOXP3-CD3 immunostain was also performed on select cases. Of 31 ETCV cases, 10 (32%) were accompanied by CV and 13 (42%) by a thrombus in the vessel affected by ETCV. The mean Treg cell marker∶CD3 ratios in ETCV ranged from 0.18 to 0.26 by manual count and digital analysis, but the counts did not statistically differ by method. The mean Treg cell marker∶CD3 ratios in CV ranged from 0.37 to 0.39 by manual count and 0.19 by digital analysis, but these counts also did not statistically differ by method. Chronic villitis was seen in one-third of ETCV cases. FOXP3+ and CD25+ regulatory T cells represent a significant subpopulation of T cells in ETCV and CV, suggesting that they may play a role in these entities.

Immunohistochemical analysis reveals an influx of regulatory T cells and focal trophoblastic STAT-1 phosphorylation in chronic villitis of unknown etiology.

Maternal T cells and fetal macrophages constitute the primary infiltrate of chronic villitis of unknown etiology (CVUE), but the role of CD25(+)/FOXP3(+) regulatory T (Treg) cells in CVUE has not been examined. Moreover, little is known about the expression of immune markers, such as the major histocompatibility complex (MHC) class II antigen, human leukocyte antigen-DR (HLA-DR), in trophoblasts in this disease. We, therefore, examined CVUE placentas for the presence of Treg cells and aberrant activation of HLA-DR in trophoblasts. Sequential formalin-fixed, paraffin-embedded tissue sections from 8 CVUE placentas and 10 control placentas were stained by immunohistochemistry with antibodies for CD3, CD4, CD8, CD20, CD25, FOXP3, CD56, CD68, HLA-DR, STAT-1, and phosphorylated STAT-1 [P-(Y701)-STAT-1]. T cells and histiocytes were confirmed as the inflammatory infiltrate in CVUE. In areas of CVUE, histiocytes strongly expressed HLA-DR and nuclear P-(Y701)-STAT-1, and the relative numbers of CD25(+)/FOXP3(+) Treg cells were increased, compared with control placentas. In 5 of 8 CVUE cases, there was patchy nuclear expression of P-(Y701)-STAT-1 in syncytiotrophoblast most extensively involved by villitis, but no other marker examined was detected in the trophoblast cell layer. We confirmed the influx of T cells and histiocytes in CVUE. Our results are the 1st, to our knowledge, to identify increased numbers of Treg cells in CVUE vs noninflamed placentas. However, we were unable to verify HLA-DR expression in trophoblasts of placentas with CVUE, suggesting that this does not contribute to the influx of T cells. Our observation that P-(Y701)-STAT-1 expression in a syncytiotrophoblast is restricted to regions of inflammation suggests that the JAK-STAT-1 pathway is aberrantly activated in these cells.

Alpha-CTLA-4 mAb-associated panenteritis: a histologic and immunohistochemical analysis.

Monoclonal antibodies (mAbs) against the cytotoxic T lymphocyte antigen-4 (CTLA-4) molecule are used as an adjuvant to experimental tumor immunization protocols in the treatment of malignant melanomas and ovarian cancers. Aside from noted early therapeutic successes, a spectrum of adverse effects, including severe gastroenteritis, has been reported. We report herein our observations of 5 patients who developed severe gastrointestinal toxicity affecting the gastric, small intestinal, and colonic mucosa. The endoscopic findings were variable, ranging from normal to diffusely erythematous and ulcerated mucosa. The constant histologic findings included a lymphoplasmacytic expansion of the lamina propria with increase in intraepithelial lymphocytes. Increased epithelial apoptosis was also a distinctive feature. Cryptitis and glandular inflammation were observed in the colon, ileum, and stomach, whereas villous blunting was present in the ileal and duodenal mucosa. Immunohistochemical analysis revealed a marked increase of all T-cell subsets (CD3+, CD4+, and CD8+) and of CD4CD25 regulatory T cells. We conclude that the panenteritis associated with injection of alpha-CTLA-4 mAbs demonstrates histology resembling autoimmune enteropathy. Furthermore, although the pathogenesis of immune dysregulation after the infusion of alpha-CTLA-4 mAbs remains unclear, we suspect that the increased number of regulatory T cells in the gastrointestinal mucosa may play a role in the pathogenicity.

Functional deficiencies of granulocyte-macrophage colony stimulating factor and interleukin-3 contribute to insulitis and destruction of beta cells.

The pathogenesis of type 1 diabetes (T1D) involves the immune-mediated destruction of insulin-producing beta cells in the pancreatic islets of Langerhans. Genetic analysis of families with a high incidence of T1D and nonobese diabetic (NOD) mice, a prototypical model of the disorder, uncovered multiple susceptibility loci, although most of the underlying immune defects remain to be delineated. Here we report that aged mice doubly deficient in granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin-3 (IL-3) manifest insulitis, destruction of insulin-producing beta cells, and compromised glucose homeostasis. Macrophages from mutant mice produce increased levels of p40 after LPS stimulation, whereas concurrent ablation of interferon-gamma (IFN-gamma) ameliorates the disease. The administration of antibodies that block cytotoxic T lymphocyte associated antigen-4 (CTLA-4) to young mutant mice precipitates the onset of insulitis and hyperglycemia. These results, together with previous reports of impaired hematopoietic responses to GM-CSF and IL-3 in patients with T1D and in NOD mice, indicate that functional deficiencies of these cytokines contribute to diabetes.

A comparison of thyroxine- and polyamine-mediated enhancement of rat facial nerve regeneration.

Thyroid hormones and spermidine, a motor neuron trophic polyamine (PA), have been shown to enhance peripheral motor nerve regeneration; however, the mechanism by which these treatment modalities exert their effect is unknown. Similarities in treatment outcome suggest that these molecules may be working via a common mechanism. Such an explanation is plausible since thyroid hormone is a potent inducer of ornithine decarboxylase (ODC), which is the rate-limiting enzyme involved in polyamine synthesis. This study was designed to morphologically evaluate the effects of exogenous thyroxine and spermidine on the regeneration of the rat facial nerve. Myelinated fiber density, axonal size, and degree of myelination were assayed by light and electron microscopy 21 days following facial nerve crush. Strikingly, the two treatment modalities had identical effects on all parameters tested. Each significantly enhanced the density of myelinated axons in regenerating nerves relative to the vehicle control. In addition, relative to the control treatment, both thyroxine and spermidine significantly increased the cross-sectional area of regenerating axons (P < 0.05). Interestingly, neither of the drug treatments had any effect on remyelination at the position where this parameter was analyzed. The concurrent administration of both thyroxine and spermidine did not synergistically enhance motor neuron regeneration. These data support the hypothesis that thyroxine and spermidine enhance neural regeneration by a common mechanism.