Predominance of epidermal CD8+ T lymphocytes in bullous cutaneous reactions caused by beta-lactam antibiotics

The phenotype and functional characteristics of skin-infiltrating lymphocytes in beta-lactam antibiotic-induced vesiculobullous exanthemas were studied in vivo and in vitro. Immunohistochemical analysis demonstrated that CD8+ T lymphocytes were the predominant epidermal T-cell subset in these reactions. Epidermal T lymphocytes were isolated and expanded for in vitro studies. Fluorescence-activated cell sorter analysis showed the majority of epidermal T cells to be CD3+, T-cell receptor alpha/beta+, CD4-, CD8+, and HLA-DR+, which correlated with the predominance of epidermal CD8+ T lymphocytes found in situ. Three CD8+ epidermal T-cell clones derived from cutaneous lesions proliferated in response to penicillin-pulsed autologous antigen-presenting cells but not allogeneic antigen-presenting cells, indicating that those clones were antigen and major histocompatibility complex specific. All T-cell clones produced significant amounts of interleukin-2, interferon-gamma, and granulocyte-macrophage colony-stimulating factor. Additionally, the T-cell clones displayed cytotoxicity against epidermal cells in lectin-mediated cytotoxicity and against B-cell lines in T-cell receptor-triggered cytotoxicity. These data demonstrate the presence of epidermal drug-specific CD8+ T cells in bullous drug reactions. Because these CD8+ T cells have a cytotoxic potential, they may contribute to the necrosis of keratinocytes associated with drug-induced blister formation.     

Prognosis of the skin UV-incidence increase in Poland

We studied the morphologic and immunohistochemical features of 10 peripheral T-cell lymphomas of a cytotoxic phenotype (CD3+/CD4-/CD8+), encountered among 98 peripheral T-cell lymphomas (PTCLs). Nine tumors were positive for both cytotoxic molecules, namely perforin (Pf) and granzyme B (GrB), and strong positivity was seen in the majority of the malignant cells. We also studied the expression of these molecules in 92 other cases of T-cell and natural killer (NK) cell neoplasms; 18 anaplastic large cell lymphomas (ALCLs); 63 CD4+ PTCLs; 10 CD56+ nasal lymphomas; and 1 NK-cell leukemia. Most of the CD4+ PTCLs (62 of 63) were negative for GrB, but all of the nasal lymphomas and the NK cell leukemia were positive for both Pf and GrB. Variable expression was seen among the 18 ALCLs. Within the 10 CD8+ PTCLs, 4 involved the skin, 3 of which were diagnosed as primary cutaneous lymphomas. Five patients died within 1 year of diagnosis. According to the Revised European-American Classification of Lymphoid Neoplasms, seven cases were categorized as "PTCL, unspecified," and three as "angioimmunoblastic T-cell lymphoma," "adult T-cell lymphoma/leukemia," or "small cell lymphoma," respectively. Three cases had characteristic morphologic features consisting of large lymphomatous cells with massive necrosis and nuclear fragmentation. Epstein-Barr virus mRNA was detected by in situ hybridization in three cases. Although the degree of apoptosis varied, apoptotic cells were detected in all cases by terminal deoxynucleotidyl transferase-mediated deoxyuridine 5-triphosphate-biotin nick end labeling. We conclude that CD8+ PTCLs are relatively rare, often involve extranodal sites, have an aggressive clinical course, and are often associated with Epstein-Barr virus. Compared with ALCLs, which have recently been considered as neoplasms of cytotoxic T-cells, we think that CD8+ PTCLs are more lineage-specific neoplasms of mature, cytotoxic, T lymphocytes.     

A tumor-specific Th2 clone initiating tumor rejection via primed CD8+ cytotoxic T-lymphocyte activation in mice

We established a CD4+ T-cell clone specific for syngeneic methylcholanthrene-induced sarcoma, S1509a raised in an A/J mouse, involved in tumor regression. The phenotype of the T-cell clone was CD3+, TCR-beta+, CD4+, CD45RB+, LFA-1+, ICAM-1+, CD44+, and VLA-4+. The CD4+ T-cell clone specifically proliferated through antigen stimulation with attenuated S1509a in the presence of syngeneic accessory cells, and this antigen-induced proliferation was inhibited with anti-CD4 and anti-I-Ek monoclonal antibodies. The CD4+ T-cell clone designated YS1093 secreted interleukin (IL) 4, IL-5, and IL-6, but not IFN-gamma, tumor necrosis factor alpha, or IL-2, thus indicating that the clone belongs to the Th2 type. YS1093 cells and their culture supernatant after antigen stimulation augmented the primed cytotoxic T lymphocyte killing activity at the effector phase. YS1093 cells having Th2-type characteristics made the homologous growing tumor regress in the tumor-bearing syngeneic mice when YS1093 cells were transferred into the tumor-bearing mice i.v. The in vivo tumor regression initiated by YS1093 cell transfer essentially required the presence of CD8+ T cells in the tumor-bearing hosts, thus suggesting that some specific Th2 cells are positively involved in tumor regression by activating primed CD8+ cytotoxic T lymphocytes against the homologous tumor in situ.     

Oocyte donation: does a previous attempt affect a subsequent attempt?

Epidermal infiltration by neoplastic CD4+ T cells is a characteristic histologic feature of early stage mycosis fungoides, the most common type of cutaneous T cell lymphoma (CTCL). The mechanisms involved in epidermotropism are unknown. It has been suggested that the CXC chemokines IL-8 and interferon-gamma inducible protein 10 (IP-10) may play a role, but evidence that these chemokines are produced within the epidermis in epidermotropic CTCL is lacking. In this study skin biopsies from 17 CTCL patients, including 12 mycosis fungoides, four pleomorphic CTCL, and one CD8+ CTCL, were investigated for epidermal IL-8 and IP-10 mRNA expression by RNA in situ hybridization. In addition, the expression of monokine induced by gamma-interferon (Mig) mRNA, a CXC chemokine closely related to IP-10, was studied as well. The expression of IL-8 receptors A and B (CXCR1 and CXCR2, respectively) was investigated by immunohistochemistry. The results were correlated with the number and phenotype of epidermotropic T cells. Epidermal expression of IP-10 and Mig mRNA was detected in 10 of 11 and seven of 11 epidermotropic CTCL, respectively, but not in five nonepidermotropic CTCL biopsies or normal human skin. Epidermal IP-10 and Mig mRNA expression correlated with epidermal infiltration of CD4+ T cells, but not of CD8+ T cells. IL-8 mRNA was demonstrated in the epidermis of only two of 15 CTCL biopsies, and was associated, in both cases, with accumulation of neutrophils. Consistently, immunostaining of the (intraepidermal) T cells with antibodies against CXCR1 and CXCR2 was not observed. In conclusion, the results of this study indicate that IP-10, and to a lesser extent Mig, but not IL-8 is involved in the preferential infiltration of neoplastic CD4+ T cells in CTCL.     

Disclosure of concerns by hospice patients and their identification by nurses

Immunohistologic studies were performed to identify the phenotype and distribution of neoplastic lymphocytes in the spleens of BLV-negative animals examined by PCR and diagnosed as having sporadic bovine leukosis. Tumor cells from three cases of sporadic bovine leukosis were identified as of B-cell lineage. Tumor cells from three additional cattle were identified as CD3+ CD4- CD8+, CD3+ CD4- CD8-, and CD3+ CD4- WC1+, respectively. The last case was diagnosed as a gamma/delta T-cell lymphoma. Differences in morphology proliferative characteristics were recognized between B- and T-cell type lymphomas. The tumor cells in B-cell type lymphoma were characterized as follows: medium or large in size, round or polymorphic nucleus with rough chromatin with some tumor cells containing a convoluted nucleus. These tumor cells of B-cell type lymphoma were present in the red pulp and periarteriolar lymphoid sheath. Tumor cells of the T-cell type lymphoma were uniformly smaller than B-cell type and present around arteries or replaced red pulp of the spleen.     

Intravascular tumor: a previously unreported finding of glucagonoma

The distinction between reactive and neoplastic cutaneous T-cell infiltrates is difficult and requires good clinicopathologic correlation. Many cases manifest changes that are at the borderline between the two. The polymerase chain reaction (PCR) has been reported to detect monoclonality in 52-90% of cutaneous T-cell lymphomas and may be of use in the diagnosis of histologically borderline lesions. We have investigated the use of PCR in a series of borderline lesions including borderline biopsy samples from patients who subsequently developed cutaneous lymphoma. PCR amplification of T-cell receptor (TCR)-gamma chain gene was performed on formalin-fixed, paraffin-embedded tissue from 27 cases of clinically and histologically typical mycosis fungoides (MF), 22 borderline biopsy samples from 10 patients who subsequently developed MF (pre-MF), 32 clinically suspicious, histologically borderline lesions, and 31 cases of chronic dermatitis. Monoclonality was demonstrated in 16 of 27 (59%) cases of MF, 10 of 22 (50%) pre-MF biopsy samples (six of 10 patients), and six of 32 (19%) borderline biopsy samples. The same size monoclonal band was detected in pre-MF biopsy samples from six of seven patients in which a band was demonstrated in the diagnostic MF biopsy. Sequencing confirmed that the MF biopsy sample and the pre-MF biopsy sample contained the same clone. The 31 dermatitis cases gave rise to polyclonal PCR products. Monoclonality can be demonstrated using PCR in 59% of MF cases, which is comparable with other T-cell lymphomas and in up to 50% of borderline biopsy samples in patients who later develop lymphoma. Detection of T-cell monoclonality by PCR is strong evidence of an established or evolving cutaneous T-cell lymphoma.     

Analysis of tumor-infiltrating lymphocytes in cutaneous squamous cell carcinoma

OBJECTIVE: To characterize tumor-infiltrating lymphocytes (TILs) within lesions of cutaneous squamous cell carcinoma (SCC) and related disorders. DESIGN: Case series with 1- and 2-color immunohistologic, molecular biological analysis of T-cell clonality and in vitro cytotoxicity assays. SETTING: Academic medical center. PATIENTS: Twenty-one patients, including 6 with actinic keratoses, 4 with SCC in situ, and 11 with invasive SCC. RESULTS: CD8+ TILs were present within lesions of cutaneous SCC and AK. These cells constituted a variable minority of the total T-cell infiltrate, and many expressed a phenotype consistent with major histocompatibility complex-restricted cytotoxic T lymphocytes: CD3+, TIA1+, CD16-, CD56-, CD57-. They also expressed HLA-DR, suggesting their activation in vivo. Virtually all T cells were T-cell receptor (TCR)-beta + delta, indicating that they expressed the TCR-alpha beta protein heterodimer. Molecular biological analysis of TCR-gamma gene rearrangements by the polymerase chain reaction and denaturing gradient gel electrophoresis technique indicated that the TILs were polyclonal. Functional studies suggested that TILs derived from SCC lesions were cytotoxic for autologous tumor cell targets. CONCLUSION: Tumor-infiltrating lymphocytes within cutaneous SCC lesions contain a subpopulation of polyclonal, major histocompatibility complex-restricted cytotoxic T lymphocytes expressing the TCR-alpha beta heterodimer.     

Long-term depletion of naive T cells in patients treated for Hodgkin's disease

We investigated the representation of T cells in patients who had been treated for Hodgkin's disease (HD). We found a marked depletion in both CD4 and CD8 naive T-cell counts that persists up to 30 years after completion of treatment. In contrast, CD4 and CD8 memory T-cell subsets recovered to normal or above normal levels by 5 years posttreatment. Thus, the previously-reported long-term deficit in total CD4 T-cell counts after treatment for HD is due to specific depletion of naive T cells. Similarly, total CD8 T-cell counts return to normal by 5 years only because CD8 memory T cells expand to higher than normal levels. These findings suggest that the treatment (mediastinal irradiation) results in a longterm dysregulation of T-cell subset homeostasis. The profound depletion of naive T cells may explain the altered T-cell function in treated patients, including the poor response to immunization after treatment for HD. Further, in some individuals, we identified expansions of unusual subsets expressing low levels of CD8. Eight-color fluorescence-activated cell sorting analyses showed that these cells largely express CD8alphaalpha homodimers and CD57, consistent with the phenotype of potentially extrathymically derived T cells. In addition, these cells, both CD4+ and CD4-, are probably cytotoxic lymphocytes, as they express high levels of intracellular perforin. In adults treated for HD, an increased activity of extrathymic T-cell differentiation may partially compensate for the loss of thymic-derived T cells.     

Subcutaneous panniculitis-like T-cell lymphoma: further evidence for a distinct neoplasm originating from large granular lymphocytes of T/NK phenotype

We report the case of a 20 year-old caucasian woman who presented a primary subcutaneous panniculitis-like T-cell lymphoma (SPTCL) as an invasive tumor of the chest wall. Herein, the neoplastic cells were found to express a CD3+CD8+ phenotype but also displayed variably the natural killer (NK)-associated antigens CD56 and CD57 as well as granzyme B. On cytological examination, these cells showed a large granular lymphocyte (LGL)-like morphology with presence of azurophilic granules in their cytoplasm. Electron dense and membrane bound granules like those found in cytotoxic T lymphocytes (CTL) were also demonstrated by electron microscopy. Neither rearrangement of the T-cell receptor subunits nor Epstein-Barr virus (EBV) genome was observed at the molecular level. The LGL-like features of the neoplastic cells found in this case and the presence of NK-associated antigens provide additional support to the cytotoxic derivation of most SPTCL.     

Improving tumour targeting and decreasing normal tissue uptake by optimizing the stoichiometry of a two-step biotinylated-antibody/streptavidin-based targeting strategy: studies in a nude mouse xenograft model

Peripheral T-cell lymphomas (PTCLs) are often diagnosed after demonstration of T-lineage-related antigen expression on neoplastic lymphocytes by paraffin immunoperoxidase (PIP). However, complete T-cell subset analysis for helper, suppressor/cytotoxic, alphabeta, and gammadelta phenotypes has not been examined by PIP. Therefore, PIP was performed for CD4, CD8, T-cell intracellular antigen (TIA)-1, and betaF1 expression in 31 PTCLs previously studied for CD4 and CD8 by flow cytometry. The CD4 and CD8 results from both methods were compared. All betaF1- PTCLs were studied for T-cell receptor (TCR)gammadelta by PIP. PIP showed 71% correlation with the 21 PTCLs that had distinct CD4+ CD8- or CD4- CD8+ phenotypes by flow cytometry, with 64% and 90% sensitivity for CD4 and CD8 expression, respectively. Tumor cells in four of six PTCLs that had no clear CD4 or CD8 predominance or coexpression of these antigens by flow cytometry were shown to be CD4+ CD8- or CD4- CD8+ by PIP. Twelve (39%) PTCLs demonstrated a cytotoxic (TIA-1+) phenotype by PIP, including eight CD4- CD8+, one CD4+ CD8- and three CD4- CD8- cases. Of 30 immunoreactive PTCLs, 26 (87%) were alphabeta (betaF1+) by PIP. Both large cell cases among four betaF1- PTCLs were TCRgammadelta+ by PIP, including one gammadelta+ case confirmed by flow cytometry. We conclude that CD4 and CD8 T-cell subsets can be assigned for most PTCLs by PIP, with CD4 showing moderate and CD8 showing strong correlation with flow cytometric results. PIP can also define CD4 or CD8 expression on tumor cells in the PTCLs in which flow cytometry produces inconclusive results. Cytotoxic PTCLs can be identified easily with TIA-1, which can also distinguish cytotoxic from "suppressor" CD8+ PTCLs. Most PTCLs are derived from alphabeta T-cells, however some large cell gammadelta PTCLs may be identified by PIP.     

In situ characterization of gingival mononuclear cells in rapidly progressive periodontitis

Rapidly progressive periodontitis (RPP) has been suggested as a distinct clinical entity within the spectrum of early onset periodontitis. Immunological mechanisms have been considered in the pathogenesis of RPP. This study was designed to evaluate the distribution and phenotypic properties of the lymphocyte populations within the affected gingival tissue of patients with RPP. Biopsies were obtained from 16 patients between 22 and 33 years of age. The tissue samples were processed for both histopathological and immunohistochemical examinations. Gingival tissue T lymphocytes (CD3+), helper T cells (CD4+), suppressor-cytotoxic T cells (CD8+), and cells positive for HLA-DR antigen were identified using monoclonal antibodies with an immunoperoxidase technique. Intracytoplasmic immunoglobulin-containing cells were also stained immunohistochemically with polyclonal antibodies. CD3+ cells were mainly located beneath the pocket epithelium. CD4+ and CD8+ cells were evenly distributed within this T-cell infiltrate with a CD4+/CD8+ ratio of 1:12. Numerous HLA-DR+ cells were also observed in the lymphocytic infiltrates. The majority of mononuclear cells located throughout the stroma were IgG+ plasma cells. Our results indicate that RPP patients present an IgG-bearing plasma cell dominated lesion with equal participation of both T-cell subpopulations. These findings suggest that activation and proliferation of B-cells play an important role in the pathogenesis of periodontal diseases.     

CD8-deficient mice exhibit augmented mucosal immune responses and intact adjuvant effects to cholera toxin

We used normal, CD4 and CD8 gene-targeted mice to investigate the role of CD4+ and CD8+ T cells in the regulation of gut mucosal immune responses following oral immunizations with cholera toxin (CT) adjuvant. Phenotypic analysis of mucosa-associated tissues revealed normal CD3+ T-cell frequencies in CD4-/- and CD8-/- mice such that in CD4-/- mice the CD8+ and double-negative (DN) T cells were increased. In CD8-/- mice the CD4+ T cells were increased, with the exception that in the intraepithelial compartment the CD3+ T cells were predominantly DN gamma delta T-cell receptor (TCR)+ T cells. All mice, normal and deficient, failed to respond to oral immunization with the antigen, keyhole limpet haemocyanin (KLH), alone. In the presence of CT adjuvant, however, CD8-/- mice consistently exhibited three- to fivefold stronger gut mucosal responses compared to normal C57B1/6 mice. By contrast, no difference was observed for systemic responses between CD8-/- and normal mice. Thus the up-regulation selectively affected mucosal responses, suggesting that, contrary to the CD8-/- mouse gut, the normal gut mucosa may host CD8+ T cells that exert a local suppressive effect on T- and B-cell responses. The magnitude of the enhancing effect of CT was comparable in CD8-/- and normal mice, clearly demonstrating that the adjuvant mechanism of CT does not require CD8+ T cells. On the other hand, the adjuvant effect of CT required CD4+ T cells, because no or poor anti-KLH responses were observed in CD4-/- mice. In both normal and CD8-/- mice CT adjuvant promoted KLH-specific CD4+ T-cell printing without any selective effect on the differentiation towards a T-helper type-1 (Th1) or Th2 dominance. Furthermore, CT adjuvant increased the frequency of CD4+ T cells expressing a memory phenotype, i.e. CD44high, LECAM-1low and CD45RBlow. We have shown, using gene-targeted mice, that CD8+ T cells are not required for the adjuvant effect of CT, and that CD8+ T cells may exert local mucosal down-regulation of intestinal immune responses.     

CD8+CD103+ T cells analogous to intestinal intraepithelial lymphocytes infiltrate the pancreas in chronic pancreatitis

OBJECTIVE: Chronic pancreatitis is a painful chronic inflammatory disease of the exocrine pancreas that is associated with the replacement of functional parenchyma by extended fibrosis and with a massive infiltration of T lymphocytes. However, to date further characterization of infiltrating T cells in chronic pancreatitis has not been undertaken. METHODS: Using the novel method of multiepitope imaging with fluorochrome-tagged specific monoclonal antibodies, which allows the simultaneous localization and characterization of T cells in tissues, we analyzed the distribution and phenotypes of T cells infiltrating the pancreas in chronic pancreatitis. RESULTS: The mean CD4:CD8 ratio in 10 cases of chronic pancreatitis was 2.4:1. In order of decreasing frequency, the following markers were observed: CD45RO, CD18, TCRgammadelta, and CD103. The lymphocytes, especially of the CD4+ subset, were found mainly in the fibrous stroma, but T cells were also observed periductally. A T-cell subset bearing the phenotype CD8+CD103+, analogous to intestinal intraepithelial lymphocytes, was found intracalating between the cells of the ductal epithelium. CONCLUSIONS: Phenotyping of the T lymphocytes in chronic pancreatitis supports the concept of the involvement of cell-mediated cytotoxicity in the pathogenesis of this disease. In addition, intraepithelial lymphocytes were found interspersed between the ductal epithelial cells, pointing to a role of this T-cell subset as a first-line defense against deleterious epithelial events in chronic pancreatitis.     

The T-cell receptor repertoires expressed by CD4+ and CD4- large granular lymphocytes derived from the same patients suggest the persistent action of an immune-mediated selection process

The lymphoproliferative syndrome with large granular lymphocytes (LGL) is an heterogeneous disorder of unknown etiology. The analysis of T-cell receptor (TCR) genes rearrangements has shown that, in most cases, the disease is associated with clonal proliferation of CD8+CD57+ LGL. However, the putative neoplastic nature of these expansions remains questionable because clonal proliferations of CD8+ cells have recently been found also in physiologic conditions. To obtain more precise information on the mechanisms responsible for LGL expansions, we decided to compare the molecular characteristics of TCRBV chains expressed by LGL with different phenotype and function, but derived from the same patients. To this end, we characterized, at the molecular level, the TCR repertoires of fractionated T-cell populations of two unusual patients with concurrent expansions of CD4+CD57+ and CD4-CD57+ LGL. Our results show that the dominant TCRBV chains expressed by the different CD4+ and CD4- LGL populations were strictly oligoclonal. However, the molecular characteristics of the dominant V-D-J rearrangements also imply that the selection of these clones was not due to a neoplastic event. Rather, our data suggest that these particular LGL proliferations can be ascribed to a chronic T-cell-mediated immune response that involves recognition by the engaged TCR of antigens that are not necessarily presented to immune system in the classical major histocompatibility complex-restricted pathway.     

Immunity to porcine rubulavirus infection in adult swine

The immune response against the porcine rubulavirus was analyzed in experimentally infected adult pigs. High titers of virus neutralizing and hemagglutinating inhibitory antibodies were identified in infected animals. The antibody specificity was directed towards HN, M, and NP rubula virion proteins; immunodominance of HN proteins was demonstrated. Peripheral blood mononuclear cells from infected, but not from non-infected pigs proliferated in vitro in response to virus antigenic stimuli, showing a bell-shaped plot with the highest peak at 5 weeks post-infection. Virus-induced lymphoblasts expressed CD4+ CD8+ phenotype, whereas lectin-induced lymphoblasts were mainly identified as CD4+ CD8- cells. Phenotype analysis of freshly prepared PBMC revealed increased number of both monocytes (PoM1+) and total T lymphocytes (CD2+) early during infection, with reduced values of B lymphocytes at 4 weeks post-infection. Decrease in CD4+ CD8- blood cells was observed at 3 weeks post-infection, whereas both CD4- CD8+ and CD4+ CD8+ cells increased 1 and 4 weeks post-infection, respectively. This work discusses the relevance of CD4+ CD8+ T cells in the control of porcine rubulavirus infection.     

Genealogy reconstruction from short tandem repeat genotypes in an Amazonian population

We report seven cases of particular cutaneous tumors selected from the register of the French Study Group on Cutaneous Lymphomas. The patients (three men, four women) were aged 37-86 years. They initially presented with cutaneous nodules or papules. Three cases presented with regional lymph nodes. Stagings were negative, except for one patient with bone marrow involvement. Histological features were relevant with pleomorphic medium T-cell lymphoma, but these cells exhibited a distinguishing phenotype. They were positive for CD4, CD56, and also CD45, CD43, and HLA-DR. All other T-cell and B-cell markers were negative. The myelomonocytic markers (CD13, CD14, CD15, CD33, CD117, myeloperoxidase, and lysozyme) were negative excepted CD68, which was clearly positive in four cases and weakly in two cases. Others natural killer cell markers (CD16, CD57, TiA1, granzyme B), TdT, and CD34 were negative. Polymerase chain reaction studies did not detect any B or T clonal rearrangement. The cytogenetic studies, performed in five cases, showed a del(5q) in two cases. All patients were treated successfully by polychemotherapy, but relapsed quickly in the skin, between 4 and 28 months. Five patients developed bone marrow involvement, with leukemia in three cases, and they died in 5-27 months. One patient died at 17 months with skin progression. The seventh patient is alive at 33 months, with cutaneous progression. The origin of these cells is unclear. Despite expression of CD4 or CD56, we failed to demonstrate a T-cell, natural killer cell origin. However, CD4 and CD56 are not specific for T or natural killer lineages. Although these two markers are also known to be expressed by monocytic cells, classic myeloid antigens were negative. These seven cases, together with other rare similar cases already reported, seem to represent a distinct entity likely developed from hematological precursor cells.