Validation of autoradiography for recognition of antigen-binding lymphocytes in blood and lymphoid tissues: quantitation and specificity of binding

Antigen-binding lymphocytes were recognized by their reaction with radioiodine labelled antigens such as flagellin and haemocyanin. Counts varied according to the antigen and species studied. For flagellin, counts in human blood of antigen-binding lymphocytes (mean ± 1 SD per 1000 lymphocytes) were 19·0±3·0, and in foetal thymus 18·2±5·0 and spleen 3·5±0·5. Results depended on contact time of cells with antigen, concentration of antigen, autoradiographic exposure, presence of natural antibody and antibody levels after immunization. Antigen-binding lymphocytes in blood were not antibody-producing cells. The specificity of the antigen-binding reaction was shown by exposing lymphocytes to 0·5 μg of two antigenically distinct flagellins; there was a 67–100% increase in the counts in contrast to the 20–45% increase on doubling the dose (0·5 μg to 1 μg) of flagellin from Salmonella adelaide. Cytophilic antibody as the cause of antigen binding was excluded.

The binding of flagellin to lymphocytes was prevented by anti-human IgM and light chain antisera, but not anti-human IgG sera. The binding of labelled flagellin was prevented by unlabelled flagellin but 100 times more was needed for blood lymphocytes than thymocytes. It is inferred that thymocytes, T cells, have considerably fewer receptors than most β lymphocytes detectable in blood.

Using standardized conditions, radiolabelled antigen binding provides a reproducible, immunologically specific and flexible technique allowing study of the nature and role of antigen-binding cells and cell surface receptors.

     

Antigen-binding peripheral blood lymphocytes in guinea-pigs immunized with human thyroglobulin in BCG

Guinea-pigs were immunized with an emulsion of human thyroglobulin (Tg) and BCG vaccine in Freund's incomplete adjuvant. Peripheral blood lymphocytes binding Tg and purified protein derivative of mammalian tuberculin (PPD) labelled with ¹²⁵I were demonstrated with an autoradiographic technique. The percentage of these cells was compared with the size of the delayed hypersensitivity (DH) skin reactions to Tg and PPD, and Tg antibody titres at intervals up to 35 days after immunization. The individual responses to Tg showed the time course of primary immune reactions although they did not parallel each other closely. By contrast, a depression of cutaneous DH to PPD was observed in spite of the presence of substantial numbers of ¹²⁵I-PPD-binding peripheral blood lymphocytes. In non-immune guinea-pigs, approximately 2·8% of peripheral blood lymphocytes bound ¹²⁵I-PPD whereas <0·1% bound ¹²⁵I-Tg.

The significance of antigen-binding lymphocytes in the immune response is discussed, and it is suggested that the presence of a substantial pool of antigen-binding lymphocytes in unprimed may be of importance in the induction of DH.

     

The human secondary immune response to keyhole limpet haemocyanin

The human secondary immune response to the protein antigen Keyhole limpet haemocyanin (KLH) was studied in thirteen normal adults. For secondary immunization and for measurement of delayed hypersensitivity, 100 μg of KLH was given intradermally to each individual between 81 and 375 days after primary immunization. Immunity was assessed by delayed hypersensitivity skin testing and measurement of antibody formation and KLH stimulated in vitro lymphocyte blastogenesis. Twelve of the thirteen subjects had positive KLH delayed hypersensitivity reactions. Prior to secondary immunization all thirteen had circulating lymphocytes responsive in vitro to KLH and all had circulating haemagglutinating antibody. After secondary immunization there was no further increase in the mean KLH-stimulated blastogenesis while the KLH haemagglutinin titre rose due to increased 7S antibody formation. Primary immunization with small doses of KLH was as effective in inducing immunological memory and a subsequent secondary response as immunization with large doses of the antigen.     

Human peripheral lymphocytes bearing both B-cell complement receptors and T-cell characteristics for sheep erythrocytes detected by a mixed rosette method

Human peripheral lymphocyte preparations were tested with a mixed rosette method for the presence of lymphocytes bearing both the complement receptors characteristic of B lymphocytes, and the capacity of T lymphocytes to spontaneously bind with sheep red blood cells (SRBC). The large and oval shaped pigeon red blood cells (PRBC) which do not form T-cell rosettes were utilized as indicators for rosette formation with the complement receptor-bearing B cell. In every individual (an average of 2·06%) lymphocytes were observed which formed rosettes with both SRBC and PRBC indicators. These findings show that independent markers for both T and B cells may be present on the surface of the same lymphocytes.     

Formation of mouse erythrocyte rosettes by human lymphocytes. A B-cell marker

26·5±8·6% of human peripheral lymphocytes form rosettes with mouse erythrocytes. There is a significant correlation between the numbers of mouse erythrocyte rosette-forming (MERF) lymphocytes and immunoglobulin-bearing cells. By density gradient centrifugation the MERF cells can be separated from the other lymphocytes. Studies on the isolated MERF cells indicate that every MERF lymphocyte is an Ig-bearing cell, but they do not possess sheep erythrocyte-binding receptors and cannot be stimulated with phytohaemagglutinin. Accordingly, the MERF lymphocytes are regarded as B cells.     

The relationship between the morphology of rosette-forming cells and their mode of rosette formation

Rats were injected in the footpad with sheep erythrocytes. The proportion of each morphological type of rosette-forming cell in the popliteal lymph node was measured, in vitro, performing the immunocyto-adherence technique at 4° and 37°. Among the different morphological types of rosette-forming cell, which appear after immunization, few plasmacytes form rosettes at 4°.

The number of rosettes formed at 37° was increased by the addition of anti-rat immunoglobulin (Ig) to aliquots of lymph node preparations from rats 7 days after immunization. This increase was accounted for by an increase in the number of rosette-forming plasmacytes and rosette-forming cells intermediate between small blasts and plasmacytes, and was thought to be due to the detection of cells secreting non-haemagglutinating antibody. The enhancing effect of anti-rat Ig at 37° contrasted with its inhibitory effect at 4°. The presence of 10^-3 M puromycin only slightly reduced the number of rosettes as compared with untreated control aliquots, but it prevented or reduced the enhancing effect of anti-rat Ig. The addition of anti-rat Ig to aliquots of lymph node preparations from rats 4 days after immunization reduced the number of rosettes formed as compared with control aliquots. It is considered that plasmacytes lack a surface associated antigen-binding receptor and depend for their rosette-forming ability on the secretion of antibody.

     

The determination of numbers of T and B lymphocytes in the blood of children and adults by the direct immunofluorescence technique

Lymphocytes in the peripheral blood of sixty-five children ranging in age from newborns to 14-year-olds and twenty-one adults were studied by the direct immunofluorescence technique for B- and T-membrane determinants, with a GaHu–Fab fluorescein isothiocyanate (FITC) conjugate as a B-cell marker and a tetramethyl rhodamine isothocyanate (TRITC) labelled horse anti-human T-cell conjugate (ATC) as a T-cell marker. The ATC was prepared from a commercial horse anti-human thymocyte IgG fraction and made specific for human T lymphocytes by means of extensive absorption with all kinds of human blood cells. Lymphocytes were also tested for E-rosette formation with sheep red blood cells (SRBC). In adults, an average of 79% of peripheral blood lymphocytes reacted with the ATC, 14% with the anti-Fab conjugate, 2% with both conjugates and 5% with neither of the conjugates. An average of 76% of peripheral blood lymphocytes (PBL) formed E rosettes. Relative numbers of fluorescent B and T lymphocytes in blood from children showed no significant differences as compared to adults. The percentage of E rosettes in cord blood was lower than in any of the other age groups studied, but only in comparison with the 3–12-month-old age group was the difference significant (P<0·05). In the 3–6-month-old age group, the percentage of fluorescent T lymphocytes was highest and the percentage of fluorescent B lymphocytes lowest, but a significant difference was observed only for the B-lymphocyte percentage compared with the first month of age (P<0·02). Up to 2 years of age, absolute values for total blood lymphocytes, fluorescent T and B lymphocytes and E rosettes were significantly higher (P<0·001) than in the older age groups and adults. After the second year of life, those values were the same as in adults.     

Circulating lymphocyte subpopulations in Hashimoto thyroiditis

Peripheral blood and T and B lymphocytes and [¹²⁵I]thyroglobulin-binding lymphocytes were investigated in twenty-two euthyroid Hashimoto thyroiditis patients and in twenty-two age- and sex-matched normal subjects. Although the total lymphocyte count in Hashimoto patients (mean±SEM = 1226±187/mm³) was lower than in normal subjects (1603±156/mm³) this difference was not statistically significant. There was, however, a statistically significant reduction in the proportion of circulating T lymphocytes in the Hashimoto patients (mean±SEM = 57·4±2·5%) as assessed by the sheep red-cell rosette method when compared with the normal controls (mean±SEM = 66·7±1·8%). The proportion of B lymphocytes in the peripheral blood as assessed by indirect immunofluorescence, was not significantly different being 21·6±2·1% in the Hashimoto patients and 20·2±1·1% in normal subjects.

[¹²⁵I]thyroglobulin-binding lymphocytes, as assessed by autoradiography were present in the circulation of nineteen Hashimoto patients with a mean frequency of 8·37±1·15/10⁴ lymphocytes and in thirteen normal subjects with a mean of 8·84±0·93/10⁴ lymphocytes. There was no difference in the degree of [¹²⁵I]thyroglobulin binding between the two groups as determined by grain count analysis. There was no apparent correlation between age or thyroglobulin antibody titres and the frequency of [¹²⁵I]thyroglobulin-binding lymphocytes. Thyroglobulin-binding lymphocytes were increased 100-fold in a Hashimoto thyroid biopsy in comparison to the patient's peripheral blood.

     

Membrane-associated immunoglobulins of human lymphocytes in immunologic disorders

Membrane-associated immunoglobulins of peripheral blood lymphocytes were studied by indirect immunofluorescence for γ, α, μ, κ and λ chains in healthy subjects and patients with immunologic disease.

In healthy subjects, heavy chains were found on 30·7% of lymphocytes (γ 15·3%, α 7·2% and μ 8·2%) and light chains on 32·8% of cells (κ 20·4% and λ 12·4%). Patients with humoral immune deficiencies had fewer immunoglobulin-bearing cells; sarcoidosis or thymectomy patients had normal or decreased immunoglobulin-bearing lymphocytes; cells with light chains were fewer than those with heavy chains on their lymphocytes. In some cases, normal levels of serum immunoglobulins were found in the absence of the corresponding immunoglobulin-bearing cells, and in others normal immunoglobulin-bearing lymphocytes were present in the absence of the corresponding serum immunoglobulins.

These data suggest that (1) immunoglobulin-bearing lymphocytes in blood do not reflect the condition of immunoglobulin-synthesizing cells in peripheral lymphoid tissues, and (2) in certain immunologic disorders, either some B-lymphocytes do not synthesize immunoglobulins, or immunoglobulins are in such a situation that the whole molecule or part of the molecule is not visualized by current methods.

     

Colony formation by subpopulations of human T lymphocytes. I. Effects of phytohaemagglutinin and lymphocytosis-promoting factor from Bordetella pertussis

A method is described for the growth in semi-solid agar medium of human lymphocyte colonies in response to stimulation by lymphocytosis-promoting factor (LPF) derived from Bordetella pertussis.

Colony formation was dependent on (a) a liquid pre-culture step with LPF prior to agar seeding, (b) presence of LPF in the agar medium, (c) a cell density in the agar culture of more than 5000 cells per ml.

Optimal colony formation was obtained with 30 μl LPF preparation in the liquid pre-culture step and 20 μl LPF preparation in the agar medium. Moreover, colony development improved after addition of 5 × 10⁻⁵M 2-mercaptoethanol and 0·6% human AB serum. The frequency of LPF-induced T-lymphocyte colonies in ten normal adult donors was 5450±1800 per 10⁶ mononuclear blood cells. In comparison, the frequency of phytohaemagglutinin (PHA)-induced colonies was 20,000±2500 per 10⁶ mononuclear cells plated directly in the agar medium. Lymphocytes seeded in agar medium with LPF started to divide within 2–3 days of culture and formed colonies of 30 to 200 cells on day 7 of culture. Cultures became moribund at day 8. The colony cells were negative for surface immunoglobulin and approximately 75% formed rosettes with sheep red blood cells (SRBC). No synergistic effect between LPF and PHA on colony formation was observed when PHA and LPF were used in the first and second step of culture respectively. Similarly, addition of LPF did not influence the growth of PHA-induced colonies.

When mononuclear cells were depleted of monocytes prior to agar culture addition of supernate factor(s) from cultures of adherent blood mononuclear cells (AC-CM) was necessary to ensure optimal development of colonies.

Mononuclear cells were separated by various rosette-depletion techniques. Colony-forming cells were recovered in the sheep red blood cell-rosetting fraction (E-RFC) of human blood lymphocytes indicating that these precursor cells are themselves T lymphocytes. E-RFCs were separated into Fc(γ)-receptor-positive and negative subpopulations. The frequency of colony-forming cells in the Fc(γ)-receptor-negative T-lymphocyte population was two to ten times higher than that of the Fc(γ)-positive T-cell population. Co-culture of equal numbers of Fc-positive and Fc-negative T lymphocytes reduced the frequency of T colonies by 20 to 95%.

Mononuclear cells were separated by passage through Ig-anti-Ig-coated columns. Attempts to grow LPF-stimulated colonies from passaged lymphocytes failed, suggesting that the colony-forming cells or cells necessary for colony formation were trapped in the column. In contrast, PHA-induced colony formation was enriched in cells which had passed through Ig-anti-Ig-coated columns.

     

Cytotoxic lymphocytes in Hashimoto thyroiditis: an in vitro assay system using 51Cr-labelled chicken red blood cells coated with thyroglobulin

An in vitro method is described to detect lymphocytes in patients with Hashimoto thyroiditis that are cytotoxic to thyroglobulin-coated chicken red blood cells. Using this technique, the cytotoxic index of lymphocytes from patients with Hashimoto thyroiditis was 25·46±3·81 (SEM), which is significantly different from that obtained with lymphocytes from control subjects, 6·28±0·80.     

Characterization of the cellular immune defect in lepromatous leprosy: a specific lack of circulating mycobacterium leprae-reactive lymphocytes

The blastogenic response of leucocyte cultures from patients with tuberculoid and lepromatous leprosy has been studied. The leucocytes from the two groups were studied simultaneously and cultivated in the same pool of normal human serum. While the leucocytes from twenty-eight tuberculoid patients responded quite strongly to Mycobacterium leprae after 7 days of culture (average lymphocyte transformation 11·1%), there was a complete lack of response in similar cultures from twenty-seven lepromatous patients (average 0·1% transformed cells). These results were confirmed by studies on cellular incorporation of ³H-thymidine in the cultures from four tuberculoid and four lepromatous patients.

This lack of response was quite specific as leucocytes from several lepromatous patients responded to BCG. Furthermore, four patients with both lepromatous leprosy and tuberculosis responded as strongly to BCG and PPD as tuberculous patients without leprosy. In the mixed leucocyte reaction, between two lepromatous or two tuberculoid patients respectively, the lepromatous cells responded well (average 15·0%) and comparably to tuberculoid cells (average 12·1%).

The blastogenic response of purified lymphocytes to M. leprae revealed a similar pattern, i.e. the tuberculoid cells responded well, while again there was a lack of response in the lepromatous group.

It is concluded that the lepromatous patients lack circulating lymphocytes responding to M. leprae, indicating that their immunological defect as observed in the present study has features in common with immunological tolerance.

     

B- and T-lymphocyte markers on transformed lymphocytes from mitogen-stimulated cultures of normal and CLL lymphocytes and on tonsil blasts

Mitogen-transformed human peripheral blood lymphocytes and tonsil blasts were examined by rosette formation to detect the presence of membrane-bound immunoglobulin (Ig) and surface receptors for fixed IgG and fixed C₃. In addition, the capacity of these cells to rosette with sheep erythrocytes was evaluated as a reaction characteristic of T lymphocytes. In order for clear morphological recognition of the rosetting transformed lymphocytes and the rosetting tonsil blasts a cytocentrifuge technique was developed and used in conjunction with autoradiography and/or with Romanowsky stains. Using these techniques and the culture methods described in this paper phytohaemagglutinin, pokeweed mitogen, streptococcal filtrates and purified protein derivative stimulated predominantly T cells in the peripheral blood of man. A minority of the transformed cells in these mitogen-stimulated cultures (<24%) did rosette with B lymphocyte markers and presumably represent a B-cell response. No significant differences were found between the T- or B-cell specificity of the mitogens investigated. Lymphoid preparations from tonsils excised from normal donors with recurrent tonsillitis were found to contain 6–15% lymphoblasts and the large majority of these cells formed rosettes with the B-cell markers, less than 20% of these lymphoblasts formed spontaneous sheep erythrocyte rosettes. Using a mixed rosetting technique a small proportion (<5%) of PHA-transformed cells and tonsil lymphoblasts were found to have combined sheep Fc or combined sheep C₃ receptors. The investigation of B- and T-lymphocyte surface markers on mitogentransformed lymphocytes was extended to neoplastic lymphocyte populations and it was found that the majority of transformed cells (> 70%) present in chronic lymphocytic leukaemia cultures stimulated with PHA after 6 days incubation were transformed T lymphocytes.     

Rosette formation with goat erythrocytes. A marker for human T lymphocytes.

We demonstrate the use of goat erythrocytes in a rosette procedure for the classification of human lymphocytes. The population is almost perfectly overlapping with the lymphocytes which form rosettes with sheep red blood cells. 70·2 ± 7·5% of peripheral lymphocytes form rosettes with goat erythrocytes and less than 1% of these cells have surface immunoglobulins. Enrichment of goat rosette-forming cells results in a population with an increased percentage of both goat and sheep rosettes. This population retains activity to the T-cell mitogens Con A and PHA, while the cells depleted of goat rosettes have greatly diminished responses to these same mitogens. Tonsil and spleen lymphocytes form 50·2 ± 6·8% and 24% of goat rosettes respectively, while peripheral blood lymphocytes from patients with CLL rarely form goat rosettes. Cell lines maintained in vitro rosetted with goat cells in a parallel fashion to sheep cells. Thus T-cell lines, such as Molt-3, which form rosettes with SRBC also rosette with GRBC, while sheep rosette-negative lines, i.e. Molt-4, are negative for both erythrocytes. B-lymphoid cell lines were negative, as were several lymphoma cell lines. There was a slight variation in the binding of goat cells, depending on the source of the goat. Thus, as in sheep rosettes, some animals were better sources than others, although all the animals tested formed rosettes.Human lymphocytes are capable of binding goat red cells. The cells which bind to the erythrocytes seem identical to those binding sheep red blood cells, and should be considered as a T-cell population. Preliminary inhibition data suggests that the receptor on T cells is the very same structure for both erythrocytes.     

T cell subset alterations in idiopathic glomerulonephritis

Peripheral blood lymphocytes from 15 healthy controls and 59 patients with idiopathic glomerulonephritis were studied to determine whether an imbalance exists among human T cell subsets in these diseases. Twenty of the patients studied had a minimal change nephropathy (10 with nephrotic syndrome and 10 in sustained remission); 27 had a membranous glomerulonephritis (12 with nephrotic syndrome, six with isolated proteinuria and nine in complete remission); 12 patients had an IgA glomerulonephritis with heamaturia and mild proteinuria. Monoclonal antibodies directed at human T lymphocyte subsets termed OKT3, OKT4 and OKT8 were used in an indirect immunofluorescence assay in all cases. Patients with minimal change nephropathy, with or without nephrotic syndrome and patients with IgA glomerulonephritis showed mean values of OKT3⁺ cells (total peripheral T cells), helper OKT4⁺ cells, suppressor OKT8⁺ cells and OKT4⁺/OKT8⁺ cell ratio, in the normal range. Only the group of patients with membranous glomerulonephritis and nephrotic syndrome presented a mean OKT4⁺/OKT8⁺ ratio greater than the normal group (percentages: 2·43±0·3 vs 1·6±0·1 s.e.m.; P<0·02). This increased ratio was due to a reduction in the OKT8⁺ cell subset compared to the healthy subjects (percentages: 27·6±2·9 vs 36·8±1·4 s.e.m.; P<0·01). Our data shows that the functional lymphocyte disorders previously described in minimal change nephropathy and IgA glomerulonephritis are not due to a numerical imbalance of lymphocyte subsets. Such an imbalance of lymphocyte subsets was specifically observed in membranous glomerulonephritis with nephrotic syndrome. The true significance of this finding has to be clarified by longitudinal studies and functional tests.     

The functions of immune T and B rosette-forming cells

From 7 to 35 days after CBA mice were primed with SRBC their spleens were removed and anti-SRBC rosettes were formed. The rosettes were purified from other spleen cells by velocity sedimentation at 4° and rosette-enriched, rosette-depleted and various control populations were injected into lethally irradiated CBA recipients. These were challenged with SRBC and their spleens analysed for direct (IgM) and enhanced (IgG) PFC 7 days later. Removal of RFC depleted the primed spleen cells of their capacity adoptively to transfer an immune response. This depletion was antigen-specific. Purified rosettes alone prepared 7–8 days after priming transferred significant (relative to controls) immune reactivity to the irradiated recipients. Both B and T RFC were present at this stage and the response was dependent upon cell collaboration between these two populations. Later in the primary response (35 days) purified rosettes transferred negligible immune reactivity. But these RFC (90 per cent B) collaborated with rosette-depleted cells to restore full reactivity. B memory lymphocytes (AFCP) form rosettes from 7 to 35 days after immunization but T memory cells only do so for a limited stage during the peak of the primary response. The majority of T memory cells probably never form rosettes in this system. It is suggested that most T RFC may be cells mediating delayed hypersensitivity or are `passive' rosettes.     

The response of human leucocyte cultures to stimulation by tuberculin and phytohaemagglutinin measured by the uptake of radioactive thymidine

A method is described for assessing the lymphocyte transformation of human leucocyte cultures induced by phytohaemagglutinin (PHA) and tuberculin purified protein derivative (PPD) by the measurement of the uptake of [¹⁴C]thymidine. The thymidine uptake of the cultures has been expressed as percentage of the total activity added. The mean basal uptake of twenty-two unstimulated cultures was 0·96%. The mean uptake of twenty-four PHA stimulated cultures was 19·5%, whilst the mean uptake of eleven PPD stimulated cultures from seven subjects, who were strongly positive on skin testing to tuberculin, was 20·1%. A rise of thymidine uptake was detected in five out of six tuberculin negative subjects (mean uptake 3·8%). Hydrocortisone hemisuccinate in final concentrations of 5 and 50 μg/ml usually reduced the thymidine uptake of unstimulated and PHA and PPD stimulated cultures, though the total cell count was reduced in only four out of ten cultures. An increase of thymidine from 1 to 64 μg/culture resulted in twoto three-fold increase in the uptake of thymidine; further increase of thymidine added to the cultures to 256 μg/culture resulted in a decrease of thymidine uptake.     

Use of cryopreserved cells in quality control of human lymphocyte assays: analysis of variation and limits of reproducibility in long-term replicate studies

Cryopreserved cells obtained by leucopheresis of normal donors were found to be useful as quality control reagents in long-term studies of human lymphocytes. Enough cells were obtained from one donor on a single day to test aliquots in parallel with patient samples for periods of up to 10 months. The results of E-rosette assays of two such cryopreserved panels, expressed as the mean percentage of lymphoid cells±s.d., were 63·7±8·4 (panel 1) and 52·3±7·8 (panel 2). The results of EAC-rosette assays on these cells were 6·2±1·7 and 11·3±6·0. These data were comparable to the values of 69·3±2·3 for E-rosettes and 6·5±4·9 for EAC-rosettes obtained using fresh cells from normal individuals. The long-term variation in rosette assays was also comparable to that in replicate samples counted on a single day. Detailed analysis of 155 serial tests showed that much of the variation was inherent in the microscope counting procedures and sampling errors. The cryopreserved cells were also used in lymphocyte stimulation assays with the mitogens concanavalin A, phytohaemagglutinin P and pokeweed. In mitogen assays, the principal sources of variation were found to be initial cell viability and incubation conditions. The major limitation of cryopreserved cells was that they did not control for the variation encountered in the gradient procedures used to separate lymphocytes from blood. Nevertheless, cryopreserved cells have been shown to be valuable for defining the limits of reproducibility in these assays and their consistent use has increased our confidence in the interpretation of day to day results on patient samples.     

Rosetting of human red blood cells to thymocytes and thymus-derived cells

Human thymocytes bind with human red blood cells (HRBC) and neuraminidase-treated HRBC (nHRBC) forming rosettes. Peripheral blood lymphocytes and tonsillar lymphocytes bind to nHRBC but not HRBC. The conditions which lead to nHRBC rosette formation are similar to those required for rosette formation with sheep red blood cells (SRBC). The rosetting of human lymphocytes to nHRBC does not require serum but is prevented by pretreatment of the lymphocytes with sodium azide, proteolytic enzymes, 50°C for 7 min, or anti-T cell (but not anti-B cell) serum. Peripheral lymphocytes from patients with chronic lymphatic leukaemia do not rosette with either HRBC or nHRBC. Therefore, the nHRBC rosetting lymphocyte is a living T cell. It is proposed that this in vitro test may simulate an in vivo mechanism for immune surveillance.     

Lymphocyte-dependent antibody-mediated cytotoxicity in Hashimoto thyroiditis

In the presence of normal human lymphocytes, decomplemented sera from twentynine out of thirty-nine patients with Hashimoto thyroiditis caused significant lysis of thyroglobulin-coated chicken red blood cells, as estimated by the release of ⁵¹Cr; the mean% specific ⁵¹Cr release being 14·1 ± 1·9 (SEM). Serum from twenty-one control subjects studied concurrently caused no significant lysis of thyroglobulin-coated chicken red blood cells; the mean% specific ⁵¹Cr release being −1·6±0·7 (SEM).