Phenotypic characterization of skin-infiltrating T cells in cutaneous T- cell lymphoma: comparison with benign cutaneous T-cell infiltrates

Using a panel of monoclonal antibodies, we have studied cell surface antigens of infiltrating mononuclear cells in skin biopsies from patients with cutaneous T-cell lymphoma (CTCL) and compared them with the T-cell surface phenotype seen in benign cutaneous T-cell infiltrations (e.g., contact dermatitis, delayed hypersensitivity skin tests, granuloma annulare) and in dermal infiltrates of lymphomatoid granulomatosis patients. We found that unlike circulating CTCL (Sezary) cells, CTCL cells infiltrating skin epidermis frequently expressed the T-cell antigen 3A1. Cutaneous infiltrates in 10 patients with mycosis fungoides (MF) and 1 patient with Sezary syndrome were OKT4 (inducer T cell), OKT8 (suppressor/cytotoxic T cell); 2 patients with MF were OKT4- , OKT8; and one MF patients's skin T cell were OKT4-, OKT8+. Similar to CTCL infiltrating cells, most of the benign skin T-cell infiltrates were usually 3A1+. OKT4+, and OKT8-. Our study shows the complex nature of T-cell antigen patterns in inflammatory and malignant skin T-cell infiltrations. We demonstrated that the CTCL the skin epidermal infiltrating T-cell phenotype is not invariate, and in many cases, is similar to the phenotype of clinically benign cutaneous T-cell infiltrations.     

Expression of three cell proliferation-associated antigens, DNA polymerase alpha, Ki-67 antigen and transferrin receptor in nodal and cutaneous T-cell lymphomas.

We studied the expression of three cell proliferation-associated antigens: DNA polymerase alpha, Ki-67 antigen, and transferrin receptor, in 35 T-cell lymphomas of nodal origin (T-NL) and 40 cutaneous T-cell lymphomas (CTCL). Immunohistochemical staining was carried out on frozen tissue sections of these specimens using three monoclonal antibodies, DAKO-PC, CL22-2-42B (DNA polymerase alpha), and OKT9. The proportion of cells positive for CL22-2-42B, DAKO-PC, or OKT9 among all tumor cells was correlated with four histologic subtypes: malignant lymphoma (ML), diffuse, small; mixed; large; and large cell immunoblastic in both T-NL and CTCL. A strong correlation was noted between positivity for CL22-2-42B and that for DAKO-PC or OKT9. On the other hand, no difference in the expression of these three antigens was noted between T-NL and CTCL in the high, intermediate or low grade-malignancy group. In CTCL as well as in T-NL, cells positive for CL22-2-42B, DAKO-PC or OKT9 were significantly more numerous in the high-grade group than the intermediate-grade group, and in the intermediate-grade group than the low-grade group. Furthermore, a significant correlation between survival period and the number of CL22-2-42B-positive cells was noted when the T cell malignancies, CTCL and T-NL were considered (t value = 2.015, p less than 0.05). Thus, the expression of DNA-polymerase alpha, Ki-67 antigen or OKT9 seems to well reflect the biological behavior and/or clinical prognosis of T-cell lymphoma.     

Analysis with OKT monoclonal antibodies of T-lymphocyte subsets present in blood and liver of patients with chronic active hepatitis

The relative distribution of T lymphocyte subsets, as defined by the monoclonal antibodies OKT, was determined by cytofluorimetric analysis in peripheral blood and in cells isolated from liver biopsies of 31 patients with chronic active hepatitis (CAH). The percentage of peripheral blood lymphocytes binding OKT8 (directed against cytotoxic/suppressor T cells) was found to be elevated in patients with HBsAg and HBeAg positive chronic active hepatitis. Patients with CAH who had seroconverted to anti-HBe, had an increased number of OKT3-positive cells in their blood, which was directed against a common T cell surface antigen, associated with a decreased number of OKT8 positive cells. Lymphocytes isolated from liver biopsies of patients with CAH presented a general increase of OKT8-positive cells associated with a decreased number of OKT4-positive (helper/inducer) T cells. It is likely that OKT8-positive cells found in liver biopsies represent cytotoxic T cells directed against either viral or liver cell determinants.     

Altered balance of immunoregulatory T lymphocyte subsets in autoimmune thyroid diseases

Three monoclonal antibodies recognizing cell surface antigens of total peripheral (OKT3), helper/inducer (OKT4) and suppressor/cytotoxic (OKT8) T lymphocytes were used by an indirect immunofluorescence technique to enumerate peripheral T lymphocytes in 25 patients with Graves' disease (including 4 euthyroid Graves' patients), 16 patients with Hashimoto's thyroiditis and 22 normal controls. Total lymphocyte count and percentages of overall T and helper/inducer T cells among peripheral lymphocytes in these conditions showed no significant difference from those of the controls. Percentage of suppressor/cytotoxic T cells, however, was decreased in Graves' disease patients with or without hyperthyroidism. The ratio of helper/inducer T cells to suppressor/cytotoxic T cells was increased in Graves' disease population and slightly increased in hypothyroid Hashimoto's thyroiditis patients. The ratio correlated with the mitogenic response of peripheral mononuclear cells to phytohaemagglutinin, but not with the serum levels of thyroid hormones nor with the titres of thyroid autoantibodies. These findings are in accordance with the results of previous functional studies and indicate possible defects in suppressor T lymphocytes in autoimmune thyroid disease.     

T lymphocyte subsets in patients with newly diagnosed Type 1 (insulin-dependent) diabetes: A prospective study

T lymphocyte subsets in peripheral blood from 11 newly diagnosed Type 1 (insulin-dependent) diabetic patients were studied prospectively at three time intervals: as soon as possible after diagnosis, 3 weeks and 5 months later. Lymphocytes were marked with monoclonal OKT antibodies and examined in a fluorescence-activated cell sorter. The percentage of T lymphocytes (OKT3) did not change significantly at the three study times. The percentage of helper/inducer T cells (OKT4) was high the first week after diagnosis, but decreased at the 5-month examination (p less than 0.05). The percentage of suppressor/cytotoxic T cells (OKT8) was low at diagnosis but increased at 3 weeks (p less than 0.02) and 5 months (p less than 0.01). The ratio OKT4/OKT8 lymphocytes was 2.28 at diagnosis, decreasing to 1.77 at 3 weeks and 1.87 at 5 months, compared with 1.46 for 16 age-matched control subjects. There was no significant change in the absolute number of lymphocytes. It is concluded that the distribution of T cell subsets was abnormal at the time of diagnosis, but changed towards normal within a few weeks, after which there was no significant change at 5 months. It is as yet unknown whether the high proportion of helper/inducer T cells and/or the low percentage of suppressor/cytotoxic T cells at diagnosis favour immune reactions involved in the pathogenesis of Type 1 diabetes.     

Tissue distribution of lymphocytes in rheumatic heart valves as defined by monoclonal anti-T cell antibodies

Fresh cardiac valvular tissues and atrial appendages removed from 106 Indian patients with rheumatic heart disease at the time of corrective cardiac surgery were examined to determine the characteristics of valvular interstitial lymphocytic infiltrates using conventional histologic staining along with indirect immunofluorescent techniques. Precise identification of the phenotypic profiles of inflammatory mononuclear cells was attempted using anti-IgG, anti-Ia, and monoclonal mouse hybridoma reagents identifying T cells (OKT3) as well as T cell subsets (OKT4 helper/inducer and OKT8 suppressor/cytotoxic cells). A similar group of 21 patients undergoing cardiac valvular resection in Albuquerque was studied. The mean age of Indian patients providing valve tissues was 27.7, whereas in those in Albuquerque, it was 52 years. Twenty-five percent of rheumatic heart valves in Indian patients showed significant interstitial lymphoid infiltrates, and one third of the rheumatic valves from patients in Albuquerque showed similar mononuclear cell collections. Lymphoid infiltrates contained a predominance of T cells (70 to 80 percent) and only occasional B cells. Most of the T cells were OKT4-positive, with only a minor representation of suppressor/cytotoxic OKT8-positive T cells. In many instances, OKT4-positive helper T cell collections were closely juxtaposed to fibroblasts and collagen fibrils. These findings suggest that the chronic rheumatic scarring process may involve helper/inducer T cells as an ancillary factor in the indolent contracture and fibrosis of deformed cardiac valvular structures. Attempts to demonstrate residual streptococcal antigens by indirect immunofluorescence using a wide panel of heterologous rabbit F(ab')2 reagents with specificity for group A streptococcal membranes, cell wall mucopeptide, or group A carbohydrate gave negative results.     

Monoclonal antibody analysis of human T lymphocyte subpopulations exhibiting autologous mixed lymphocyte reaction.

In autologous mixed lymphocyte reaction (MLR), T cells proliferate in response to the stimulation by autologous non-T cells. In the present study, human T cell subpopulations, defined by murine monoclonal antibodies OKT4, OKT8, or 9.3, were examined for their capacity to proliferate in autologous and allogeneic MLR. It was observed that the treatment of responder T cells with OKT4 or 9.3 antibody (both defining helper/inducer T cells) and complement (C') ablated their proliferative response in autologous MLR and markedly reduced their allogeneic MLR proliferative response. In contrast, treatment of T cells with OKT8 antibody (defining suppressor/cytotoxic T cells) and C' had no or minimal effect on their proliferative response in autologous MLR. Furthermore, OKT4, 9.3, 9.6 (PAN), or 7.2 anti-human Ia (framework specific) but OKT8 antibody, when present during the entire culture period, in the absence of C' inhibited in a dose-dependent manner the proliferative response in both autologous and allogeneic MLR. Inhibition of proliferation was also observed when the responder T cells were pretreated with OKT4 or 9.3 antibody, washed, and then stimulated with irradiated autologous or allogeneic non-T cells. Pretreatment of T cells with OKT8 or 7.2 anti-Ia antibody in the absence of C' did not influence their proliferative response in autologous MLR. Thus, T cells containing cells with helper/inducer activity defined by OKT4 or 9.3 antibody appear to be the major responder T-cell subpopulation in autologous MLR.     

Analysis of T cell subsets in the peripheral blood and synovial fluid of patients with rheumatoid arthritis by means of monoclonal antibodies.

In an attempt to define the immunoregulatory mechanisms operating in rheumatoid arthritis we have enumerated T cell subsets in the peripheral blood and synovial fluid of patients with this disease. The peripheral blood analysis revealed an elevation of the ratio of inducer T cells (OKT4 positive) to suppressor/cytotoxic T cells (OKT5 positive) in patients with clinically active rheumatoid arthritis when compared with normal persons. This was due to a reduction in the percentage of suppressor/cytotoxic T lymphocytes in these patients. The synovial fluid in rheumatoid arthritis differed from the peripheral blood in 2 respects. Firstly, synovial fluid was characterised by a lower helper: suppressor ratio due to an increased number of suppressor/cytotoxic cells, and, secondly, it contained an increased number of activated T cells bearing HLA DR antigens. The majority of these activated T cells belonged to the helper/inducer T cell subset.     

Ia+ T cells in synovial fluid and tissues of patients with rheumatoid arthritis

Markedly elevated levels of T cells expressing Ia antigens were found in the synovial membranes and synovial fluids of patients with rheumatoid arthritis. The primary increase in expression of the Ia antigens was on the OKT 8+ (suppressor/cytotoxic) T cell subset. In addition, the total percentage of OKT 8+ T cells in intraarticular sites was usually greater than levels in peripheral blood. Small numbers of OKT 4+ (helper/inducer) cells bearing Ia antigens were also identified. The characteristic increase in the Ia+ T cells in peripheral blood was not encountered in most patients treated with D-penicillamine.     

Study of T cell subsets in patients with cutaneous leishmaniasis

Summary The proportion of T lymphocyte populations was assessed using monoclonal antibodies (OKT 3, OKT 4, OKT 8) and spontaneous E-rosette tests with SRBC (for total and active TE cells) in 36 patients with cutaneous leishmaniasis subdivided according to the clinical variety. The T cell profile was normal in patients with simple cutaneous leishmaniasis of early ulcerative type whereas active TE cells and OKT 4 positive cells were reduced in some cases of late ulcerative type. A significant reduction of active TE cells and of OKT 4 positive cells (helper/inducerphenotype) and increase of OKT 8 positive cells (suppressor/ cytotoxic phenotype) was observed in peripheral blood of all patients with persistent active lesions and leishmaniasis recidivans. In patients with highly ulcerated persistent lesions a low proportion of active TE cells was also demonstrated in cellular extracts from dermal tissue. The results support the data obtained in experimental leishmaniasis that lack of helper/inducer cells and generation of suppressor T cells may be responsible for the development of chronic leishmaniasis.     

Regulation of human peripheral blood erythroid burst-forming unit growth by T lymphocytes and T lymphocyte subpopulations defined by OKT4 and OKT8 monoclonal antibodies

To reexamine the influence that T lymphocytes have on the regulation of human peripheral blood burst-forming unit (BFU-E) proliferation in the absence of a statistically significant number of monocytes, very low numbers (3 to 10 X 10(3)/mL) of a null cell fraction highly enriched for BFU-E were cultured alone and in the presence of 5 X 10(5) sheep erythrocyte-purified, autologous T lymphocytes in a methylcellulose culture system containing erythropoietin. T lymphocytes consistently enhanced the growth of BFU-E from the null cell fraction, as reflected in both their number and size. Irradiation of T lymphocytes prior to coculture with null cells markedly reduced this enhancement, strongly suggesting that T lymphocytes synthesize erythroid burst-promoting factors (BPA). To determine whether there were functional differences between the two major T lymphocyte populations as defined by OKT4 (T helper/inducer) and OKT8 (T suppressor/cytotoxic) murine monoclonal antibodies to stimulate the growth of BFU-E, both T cell subpopulations were isolated by negative (panning) or positive (fluorescence-activated cell sorting) selection and cocultured with null cells. No statistically significant differences emerged between unseparated, OKT4+ and OKT8+ T lymphocytes in their ability to stimulate the growth of BFU-E. Thus, these studies provide further evidence that T lymphocytes are a major population of BPA-producing cells and further that OKT4+ and OKT8+ T lymphocytes equally elaborate these factors.     

Peripheral blood T lymphocyte subpopulations defined by monoclonal antibodies in rheumatoid arthritis: distribution in patients untreated and treated by oral gold therapy

The distribution of T lymphocyte subsets was determined in peripheral blood (PB) of two groups of patients with rheumatoid arthritis by using monoclonal antibodies (OKT). In untreated patients the percentage of OKT4+ cells (helper/inducer) was found to be significantly increased as compared to healthy controls. In patients receiving oral gold therapy a similar increase in OKT4+ cells was confirmed; furthermore, these patients showed a significant decrease in OKT8+ cell population (cytotoxic/suppressor) compared to untreated patients and to normal controls. A small numerical superimposition of values of OKT4+ and OKT8+ lymphocytes was observed in untreated but not in treated patients.     

Disturbances of T lymphocyte subsets preceding refractory anemia with excess blasts in transformation in a child

A 5-year-old girl with refractory anaemia with excess of blasts in transformation (RAEBT) terminating in acute nonlymphocytic leukemia is reported. The patient's serum had an inhibitory effect on normal hemopoiesis. T cell subsets defined by monoclonal antibodies revealed a decrease in helper/inducer T cell population (OKT4-positive cells) and inversion of the T4/T8 ratio. It is suggested that imbalance in immunoregulatory T cells, which has a crucial role in the control of hemopoiesis, may be a primary event in the pathogenesis of myelodysplastic syndrome which eventually develops into an overt leukemia.     

Evaluation of T cell subsets with monoclonal antibodies in synovial fluid in rheumatoid arthritis

Monoclonal antibodies that react with all T cells (OKT3), the inducer-helper T cell subset (OKT4), and the suppressor-cytotoxic T cell subset (OKT8) have been used to evaluate T cell and T cell subpopulations in synovial fluid (SF) from patients with rheumatoid arthritis (RA). We found that the immunoregulatory ratio of SF lymphocytes ranged from normal values to extremely low values indicating normal to excess numbers of OKT8+ cells. The rheumatoid synovium in patients with RA is characterized by the presence of lymphoid aggregates in which HLA-DR+ interdigitating cells form close contacts with a large number of OKT4+ T cells, while the OKT8+ T cells are sparse. It may be that the localizing mechanisms for OKT8+ suppressor cells in lymphoid aggregates are deficient in RA and the cells are thus permitted to pass into synovial exudates, resulting in an increase of OKT8+ cells in the SF.     

Abnormalities of peripheral blood T lymphocyte subsets in polymyalgia rheumatica

The T lymphocyte subsets were studied by monoclonal antibodies in the blood of eight patients with polymyalgia rheumatica. The percentages of circulating T cells (OKT3+) were lower when compared with normal controls (p less than 0.02), as were the proportions of suppressor/cytotoxic (OKT8+) T cells (p less than 0.001), whereas the proportions of helper/inducer (OKT4+) T lymphocytes were not changed. Consequently, the OKT4:OKT8 ratio was higher in patients with polymyalgia rheumatica than in controls (p less than 0.001). On the contrary, circulating B cells were not altered. The decrease in peripheral blood OKT3+ and OKT8+ lymphocyte subsets suggests that there is an impairment of cell-mediated immunity in polymyalgia rheumatica.     

Decreased level of suppressor/cytotoxic T cells (OKT8+) in polymyalgia rheumatica and temporal arteritis: relation to disease activity

Separated peripheral blood mononuclear cells were analyzed by fluorescent microscopy with monoclonal antisera for T cells (OKT3+), helper/inducer T cells (OKT4+) and suppressor/cytotoxic T cells (OKT8+). Thirty-seven patients with polymyalgia rheumatica (PMR), 13 of whom had positive biopsies for arteritis, were studied and compared with 25 age and sex matched normal subjects. The percentages of OKT3+ and OKT4+ T cells were similar in the PMR group and controls, but percentage of OKT8+ T cells was significantly reduced in patients (14.8 +/- 6.8) compared with controls (22.1 +/- 6.3). Values of OKT8+ T cells were extremely low in untreated patients with active, acute disease (7.8 +/- 4.4%) and significantly lower than in prednisone treated patients with inactive disease (17.3 +/- 5.9). These findings indicate that low values of circulating OKT8+ T cells is a feature of PMR and is related to disease activity.     

Cells in lymph draining normal human skin-monoclonal antibody analysis

The immune cells which migrate into the human skin from the blood and subsequently leave it via lymph vessels play an important role in immune processes. We made use of the monoclonal antibodies, characterizing cell populations which migrate into the normal skin and which having traversed the tissue, could be recovered from the afferent lymph vessels. The percentage of OKM1+ cells (monocytes/macrophages, null cells) in lymph was low (8.9 +/- 1.6%) when compared to that of blood (16.5 +/- 4.6%) (p less than 0.05). The OKM1 antibody labeled only 40% of the large macrophage-like lymph cells. The percentage of OKT3+ (T cells) in lymph was higher (75.4 +/- 4.0%) than in blood (54.0 +/- 4.5%) (p less than 0.05) as was that of the OKT4+ (inducer/helper) subset (41.5 +/- 9.5 and 33.3 +/- 4.8%, p less than 0.05), while cells of the OKT8+ (suppressor/cytotoxic) subset were found to be less numerous in lymph than in blood. (18.4 +/- 6.2% and 20.3 +/- 4.9%, p less than 0.05). The OKI a1+ cell population consisted of large veiled macrophage-like cells and only very few small cells. Around 60% of the large mononuclear cells present in lymph reacted with OKT6 antibody specific for cortical thymocytes. The finding of high proportions of T cells, cells bearing la-like antigens, and a high inducer/suppressor ratio in normal prenodal lymph reflects the intensity of "physiological" immune processes in the normal skin.     

Analysis of T cell subsets in cancer patients treated with interferon

T cell subsets were analyzed in 33 patients with advanced cancer who were treated with either of two interferon preparations: a partially purified human leukocyte interferon (HulFN-alpha (Le] and a highly purified recombinant interferon (lFLrA). Included in the lFLrA-treated group were eight patients with immunodeficiency and Kaposi's sarcoma. The OKT4+/OKT8+ ratio was used to define the balance between helper/inducer and suppressor/cytotoxic T cell subsets. With both interferon preparations, the mean OKT4+/OKT8+ ratio decreased 24 hours after the first interferon dose. Within the HulFN-alpha (Le) group, the decrease in ratio was related to an increase in OKT8+ cells; in the lFLrA group, it was accompanied by a small decrease in the proportion of OKT4+ cells that was greater than the decrease in OKT8+ cells. Patients treated with lFLrA were followed for the first three weeks of therapy. Most patients treated with lFLrA at all dose levels, ranging from 1 X 10(6) to 54 X 10(6) units per day, had a decrease in OKT4+/OKT8+ ratio on Day 1. No substantial change in the ratio was observed on Days 7, 14, and 22. Patients with immunodeficiency and Kaposi's sarcoma had responses similar to those of patients with other cancers treated with lFLrA. In conclusion, although both HulFN-alpha (Le) and lFLrA induce immediate decreases in the OKT4+/OKT8+ ratio, the T cell subset(s) primarily responsible for the decrease varies with the source of interferon.     

Cytochemistry of normal lymphocyte subsets defined by monoclonal antibodies and immunocolloidal gold

The cytochemical reactivities of 3 acid hydrolases, alpha-naphthyl acetate esterase (ANAE), acid phosphatase and beta-glucuronidase were investigated in normal peripheral blood lymphocyte subsets defined by monoclonal antibodies OKT3, 4, 8 and FMC4 (anti-Ia). A combined monoclonal antibody-immunocolloidal gold/cytochemical staining procedure was used to determine enzyme activities and distributions of reaction product in each subset. Cytochemical profiles for each lymphocyte subset were defined. The majority (greater than 85%) of T cells (OKT3+) were positive for all 3 enzymes whereas a minority (less than 40%) of B cells (FMC4+) displayed reactivity. The cytochemical profiles of T helper/inducer (OKT4+) and T suppressor/cytotoxic (OKT8+) cells were not significantly different and corresponded to that observed for OKT3+ cells; thus none of these enzymes can be used to distinguish normal lymphocyte subsets cytochemically. ANAE reactions were further analysed, in the respective subsets, on the basis of dot-like or scattered/diffuse reactivity. The ratios of cells displaying dot-like: scattered/diffuse reactivity, in the respective subsets, were OKT3+, 5.4:1; OKT4+, 8.1:1; OKT8+, 2.4:1; FMC4+, 0.4:1. The cytochemical profiles and ANAE reactivities of T cell subsets identified by monoclonal antibodies differ from those displayed by T cell subsets defined by Fc receptors and confirms that there is little correlation between subsets defined by these two methods.     

Subpopulations of T lymphocytes in Kenyan patients with visceral leishmaniasis

Populations of peripheral blood T lymphocytes from patients with Kenyan visceral leishmaniasis were studied using specifically defined antisera (monoclonal antibodies, Ortho-mune OKT3, OKT4, OKT6, and OKT8). The levels of total T lymphocytes and circulating thymocytes were within the same range as those of clinically normal individuals. However, the proportions of the helper/inducer T cells were lower in untreated patients than in the controls (18.9% vs. 39.7%) while the levels of suppressor/cytotoxic T cells were higher than in the controls (40.5% vs. 27.8%). After successful antileishmania treatment these levels showed a gradual return towards normal over a period of one year. It was concluded that immunosuppression observed is due to the levels of peripheral blood helper/inducer and suppressor/cytotoxic T lymphocytes.