The mechanism of rosette formation between Rh (D) positive erythrocytes and peripheral blood lymphocytes from Rh isoimmunized individuals. The role of surface micro-projections

Peripheral blood leucocytes were obtained from Group A Rh (D)-negative male volunteers re-injected with A Rh (D)-positive erythrocytes and the mechanism by which they form rosettes in vitro with A Rh (D)-positive erythrocytes was studied. It was found that there was a marked reduction in the number of rosettes formed at 4° as compared with 37°. Moreover, those rosettes formed at 4° comprised a central rosette-forming cell surrounded by one layer of erythrocytes whereas at 37° some rosette-forming cells (RFC) appeared to be surrounded by agglutinated erythrocytes. No anti-D antibody-forming cells were detected by the localized haemolysis in gel technique despite the presence of large numbers of RFC in the preparations tested. Pretreatment of leucocyte aliquots with anti-sera specific for human Fab at 4° was nearly as effective as pretreatment with anti-Fab at 37° in inhibiting rosette formation.

Examination of serial sections of rosettes with the electron microscope revealed that erythrocytes were often attached to micro-projections from the surface of central RFC. Cytochalasin B partially inhibited rosette formation at 37°. Those rosettes formed in the presence of cytochalasin B consisted of a central rosette-forming cell with few adherent erythrocytes and with the electron microscope it was seen that erythrocytes were rarely attached to central RFC by micro-projections. It is suggested that in this system the contractile activity of the surface of rosette-forming cells is important in rosette formation.

     

Kinetics of the proliferative response to antigen in vitro of rabbit lymph node cells taken at various times after immunization

The antigen stimulation of thymidine uptake by immune rabbit lymph node cells in vitro was studied. The kinetics of the response varied depending on the time between immunization and culture. Cultures set up early after immunization showed a peak response over day 1–2 of culture while those set up late after immunization showed a peak response over day 3–4.

Studies using the metabolic inhibitor BUDR suggested that this is due at least in part to a larger recruitment of cells into the response during the third day of culture when lymph node cells taken late after immunization were used.

Removal of antigen from cultures after brief exposure of cells at 4° reduced the magnitude but did not eliminate the proliferative response, suggesting that some antigen is specifically bound to the cells in the cold. Readdition of antigen restored normal reactivity.

Holding cells at 4° for 4 hours without antigen had no effect on their response to subsequent addition of antigen. However, if cells were held at 4° for 3 hours with antigen present a severe degree of depression of subsequent thymidine incorporation was observed in some but not all experiments. This depression of responsiveness was interpreted as an in vitro phenomenon comparable to immunologic tolerance.

     

The relationship of antibody-forming cells to rosette-forming cells

Rosette-forming cells (RFC) increased in numbers in the spleens of mice following injection of sheep red blood cells (SRBC). Very sensitive assay system for detecting plaque-forming cells (PFC) showed that RFC and PFC were present in approximately equal numbers at the height of the immune response. Thereafter PFC numbers declined much more rapidly than RFC.

Two techniques were used to study the contribution of PFC to RFCs (a) velocity sedimentation through foetal calf serum gradients and (b) transfer of individual RFC by micromanipulator into the PFC assay gel containing complement and rabbit antimouse IgG antiserum to identify what proportion of RFC were secreting antibody.

It was shown that, when rosettes were prepared at 4°, <10 per cent were formed by PFC at 4 days after immunization, and <2 per cent at 6 days. Rosettes prepared at 37° contained up to 16 per cent PFC.

It was concluded that PFC had either no cell bound antigen-binding receptors or that the receptors were not demonstrable at 4° by the particular rosette preparation used in the study.

Rosettes prepared late in the immune response were more resistant to mechanical agitation than those prepared early in the response.

     

Studies on actively allergized cells: IV. Light and electron microscopic studies on the morphology of rosette-forming cells with special reference to plasma cells

A proportion of the plasma cells in lymph nodes of allergized guinea-pigs and mice were found to possess membrane-bound receptors for antigen when tested for rosette-formation at 4° by the suspension-centrifugation technique. This was shown by staining rosetted cells with pyronin-methyl green. Rosette-forming cells of the guinea-pig were further examined by staining with fluorescein-conjugated antigen (rabbit Fab′) and by electron microscopy. By these techniques it was found that many plasma cells which contain antigen-specific intracellular antibodies do not form rosettes, and that cells of the plasmacytic series represented in the rosetted population are limited to plasmablasts and immature plasma cells. The lack of rosette-forming mature plasma cells suggests that a loss of receptors from the cell-membrane occurs as an accompaniment to maturation.     

Surface immunoglobulins of rat immunocytes: quantitation and fate of cell-bound peroxidase-labeled antibody and Fab fragment

By using antibody-peroxidase conjugates, quantitative data were obtained concerning events following interactions between anti-Ig antibodies and surface Ig. About 5 × 10⁵ molecules of peroxidase-labeled anti-rat Ig antibodies were bound per positive lymph node cell, at conjugate excess, at 4 °C. Similar quantities of anti-Fcγ or anti-Fab were bound per positive cell, suggesting that cell surface IgG antigenic determinants are largely exposed. Experiments performed on thymocytes showed that the number of peroxidase molecules bound per 10⁶ cells was 12 to 16 times higher with lymph node lymphocytes than with thymocytes. After incubation of cells at 4 °C with peroxidase-labeled rabbit anti-rat Ig, the cells were washed and post incubated for various times at 37 °C. The fate of the peroxidase conjugates was followed by measuring enzymatic activity on the cell surface, in the cell, and in the incubation medium. During the first 15 min of incubation at 37 °C, peroxidase activity disappeared rapidly from the cell membrane, while enzymatic activity appeared and rapidly increased in the cell supernatant. After this time, membrane-bound peroxidase activity was found to be constant, while enzymatic activity of the supernatant increased slightly. After 1 h of incubation at 37 °C, about 20 % of the peroxidase activity had been eluted into the culture medium, 20 % remained on the cell surface, and about 35 % had been pinocytozed. Similar results were obtained with peroxidase-labeled sheep-Fab anti-rat Ig, except that more peroxidase activity was found in the supernatant and pinocytosis was less pronounced than with whole antibody. Immunoadsorbents were used to determine if cell-derived Ig was linked to the peroxidase liberated into the supernatant at 37 °C. Polyacrylamide beads to which were linked anti-rat Ig antibodies or Fab fragments fixed 40 % of the peroxidase activity. Molecular filtration of the supernatants showed that about 60 % of the peroxidase activity was eluted in the void volume of a Sephadex G-200 column, the remainder being eluted as free conjugate. Thus, part of the conjugate initially fixed on cells at 4 °C was eluted as cell surface Ig-anti-Ig complexes, while another part was apparently released uncomplexed.     

T cell rosette formation in primates, pigs, and guinea pigs. The influence of immunosuppresive agents

Out of 144 combinations of lymphocytes from 8 species and erythrocytes from 18, significant numbers of rosettes were found in only 14. Human and chimpanzee lymphocytes formed rosettes in the greatest number of combinations (5), pig lymphocytes with 3, and guinea pig lymphocytes with only one erythrocyte (rabbit). In all positive combinations tested, the percentage of rosette-forming cells was highest in the thymus, intermediate with lymph node and peripheral blood lymphocytes, and lowest in the spleen. Using the guinea pig rosette-forming cell as an experimental test for T lymphocytes, the effect of several immunosuppressive drugs on the percentage of lymphocytes that are T cells was measured. Cyclophosphamide injected into guinea pigs increased this percentage the most, cortisone and tilorone increased the percentage of rosette-forming cells slightly, and azathioprine and vinblastine caused no change. After in vitro incubation with the lymphocytes, cyclophosphamide, azathioprine, and tilorone increased the percentage of rosette-forming cells and vinblastine reduced this percentage.     

Physical Factors affecting Maximum Precipitation of the BSA-AntiBSA Fowl System

Studies were made ascertaining the effects of temperature and length of incubation period on the amount of precipitate formed in the BSA-antiBSA fowl serum system. The specific factors considered were centrifugal temperature, temperature of incubation and length of incubation. Reactions were analysed for the entire precipitin curve, using doubling dilutions of antigen, and also for the region of equivalence using intervals of 1 μg. nitrogen.

Reactions mixtures were incubated at 37° for 3 hours and then centrifuged at 22° or at 4° and the precipitate was analysed for total N precipitated. In addition, secondary incubation periods, following this initial incubation treatment of 3 hours at 37°, were made. The secondary periods were either 18, 66 or 118 hours at 4° or 18 hours at 37° and were followed by centrifugation at either 22° or 4°.

Evaluation of the data showed centrifugation at 22° gave higher amounts of precipitate than at 4° in all cases and the differences were statistically significant in most of the instances. In eighteen of the twenty sera tested, precipitation was at a maximum after 3 hours' incubation at 37° and warm centrifugation in contrast to additional incubation periods and/or cold centrifugation.

     

Effect of incubation temperature on mitogen responses of lymphocytes from adult peripheral blood and from cord blood

Lymphocytes from normal adults, when cultured with phytohaemagglutinin (PHA) or Concanavalin A (Con A) at 39°C, showed an enhancement and earlier onset of ³H-thymidine incorporation compared with cultures incubated at 37°C. In contrast, the peak responses of cord blood lymphocytes incubated at either 35°C or 39°C did not differ significantly from those incubated at 37°C. Cultures of adult lymphocytes showed an exponential rise and fall in ³H-thymidine incorporation, which was much more rapid at 39°C than at 37°C. However, the kinetics of thymidine incorporation into mitogen-stimulated cord blood lymphocytes incubated at 39°C were similar to those at 37°C. The following results suggested that temperature acted predominantly on the proliferative phase of the transformation response. Firstly, by inhibiting binding of Con A using methyl-α-D-mannopyranoside, it was found that activation of adult lymphocytes took place within the same time period at both 39°C and 37°C. Secondly, cultures incubated at 37°C for 3 days, and labelled for 4 hr at either 37°C or 39°C showed no significant difference in ³H-thymidine uptake, whereas cultures incubated at 39°C for 3 days, and labelled for 4 hr at 37°C showed significantly higher responses than those both incubated and labelled at 37°C. Thirdly, the major increase in thymidine uptake occurred after incubation at 39°C for the second and third days of culture. These findings were consistent with a shortening of the cell cycle at the higher temperature. Thus, the failure of cord blood lymphocytes to show increased thymidine uptake after incubation at 39°C apparently reflects an insensitivity to temperature of certain of the metabolic pathways involved in cell replication in the neonate.     

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.     

Sensitivity of SARS‐CoV‐2 to different temperatures

This study was designed to investigate the sensitivity of SARS‐CoV‐2 to different temperatures, to provide basic data and a scientific basis for the control of COVID‐19 epidemic. The virus was dispersed in 1 mL basal DMEM medium at a final concentration of 10^3.2 TCID₅₀/mL and then incubated at 4, 22, 30, 35, 37, 38, 39 and 40°C for up to 5 days. The infectivity of residual virus was titrated using the Vero E6 cell line. The results showed that the virus remained viable for 5 days at 4°C, and for 1 day only at 22 and 30°C. We found that the infectivity of the virus was completely lost after less than 12 hours at 37, 38 and 39°C, while at 40°C, the inactivation time of the virus was rapidly reduced to 6 hours. We show that SARS‐CoV‐2 is sensitive to heat, is more stable at lower temperatures than higher temperature, remains viable for longer at lower temperatures, and loses viability rapidly at higher temperatures.     

The kinetics and morphology of the rosette-forming cell response in the popliteal lymph nodes of rats

A modified ICA technique was used to study the kinetics and morphology of the RFC response in rat popliteal lymph nodes after an inoculation of SRBC into the hind footpad. The primary response was followed over a 10-day period. RFC were classified as either macrophages, haemocytoblasts, plasmacytes or lymphocytes.RFC present in the popliteal lymph nodes of uninoculated rats were identified as macrophages and lymphocytes. After inoculation the number of RFC rose rapidly to reach a peak at 5–6 days. It was shown that after incubation at 37° certain RFC from inoculated rats had several layers of adherent SRBC and it was suggested that this was an indication of an active secretion of haemagglutinin. 3–4 days after innoculation large mature haemocytoblasts were actively secreting haemagglutinin whilst from the 5th to the 10th day plasmacytes were the RFC involved in the haemagglutinin production. It is suggested that the large haemagglutinin producing haemocytoblasts arise without mitosis via a process of cell transformation and that RF plasmacytes arise via lymphocyte activation into small haemocytoblasts, mitotic division and eventual maturation into immature plasmacytes. RF lymphocytes were thought not to be involved to any extent in haemagglutinin production.     

Diminished human immunodeficiency virus type 1 DNA yield from dried blood spots after storage in a humid incubator at 37 degrees C compared to -20 degrees C

Collecting whole blood on filter paper simplifies the processing, transport, and storage of specimens used for the diagnosis of human immunodeficiency virus type 1 (HIV-1) and other tests. Specimens may be collected in tropical or rural areas with minimal facilities for handling specimens. To compare simulated tropical conditions with freezer storage, we examined the stability of HIV-1 DNA in dried blood spots (DBS) stored in humid heat and at −20°C. DBS were created by spotting 50-μl aliquots of whole blood on 903 filter paper. DNA was extracted from DBS at baseline and after 2, 6, or 12 months of storage at −20°C or at 37°C with ~85% humidity. The DNA was tested undiluted or diluted using the Amplicor HIV-1 DNA PCR (Roche), version 1.5. Each reaction was scored positive, negative, or indeterminate based on optical density. Results were compared between storage conditions and over time. A total of 1,832 reactions from 916 DBS were analyzed, including 100 DBS at baseline, 418 stored at −20°C, and 398 stored at 37°C. A chi-square test showed fewer positive reactions for DBS stored at 37°C (55%) than for those stored at −20°C (78%) (P < 0.0001). Samples stored at −20°C showed little change in the probability of detection of HIV-1 DNA over time; the odds ratio (OR) was 0.93 after storage for 1 year. Samples stored at 37°C demonstrated a significant change in detection at 1 year (OR, 0.29). We conclude that exposure of DBS to 37°C and high humidity impaired the recovery of HIV-1 DNA from DBS, whereas DNA recovery was preserved when DBS were stored frozen.     

Restoration of anti-tetanus toxoid responses in patients initiating highly active antiretroviral therapy with or without a boost immunization: an INITIO substudy

INITIO is an open-labelled randomized trial evaluating first-line therapeutic strategies for human immunodeficiency virus-1 (HIV-1) infection. In an immunology substudy a tetanus toxoid booster (TTB) immunization was planned for 24 weeks after initiation of highly active antiretroviral therapy (HAART). All patients had received tetanus toxoid immunization in childhood. Generation of proliferative responses to tetanus toxoid was compared in two groups of patients, those receiving a protease inhibitor (PI)-sparing regimen (n = 21) and those receiving a PI-containing (n = 54) regimen. Fifty-two participants received a TTB immunization [PI-sparing (n = 15), PI-containing (n = 37)] and 23 participants did not [PI-sparing (n = 6) or PI-containing (n = 17)]. Cellular responses to tetanus antigen were monitored by lymphoproliferation at time of immunization and every 24 weeks to week 156. Proportions with a positive response (defined as stimulation index ≥ 3 and Δ counts per minute ≥ 3000) were compared at weeks 96 and 156. All analyses were intent-to-treat. Fifty-two participants had a TTB immunization at median 25 weeks; 23 patients did not. At weeks 96 and 156 there was no evidence of a difference in tetanus-specific responses, between those with or without TTB immunization (P = 0·2, P = 0·4). There was no difference in the proportion with response between those with PI-sparing or PI-containing regimens at both time-points (P = 0·8, P = 0·7). The proliferative response to tetanus toxoid was unaffected by initial HAART regimen. Anti-tetanus responses appear to reconstitute eventually in most patients over 156 weeks when treated successfully with HAART, irrespective of whether or not a TTB immunization has been administered.     

Combined treatment with a CXCL12 analogue and antibiotics improves survival and neutrophil recruitment and function in murine sepsis

Previous studies demonstrated that the CXCL12 peptide analogue CTCE-0214 (CTCE) has beneficial effects in experimental sepsis induced by caecal ligation and puncture (CLP). We examined the hypothesis that CTCE recruits neutrophils (polymorphonuclear leucocytes; PMN) to the site of infection, enhances PMN function and improves survival of mice in CLP-induced sepsis with antibiotic treatment. Mice with sepsis (n = 15) were administered imipenem (25 mg/kg) and CTCE (10 mg/kg) subcutaneously versus vehicle control at designated intervals post-CLP. CTCE treatment increased PMN recruitment in CLP-induced sepsis, as evidenced by increased PMN in blood, by 2·4 ± 0·6 fold at 18 hr, 2·9 ± 0·6 fold at 24 hr, and in peritoneal fluid by 2·0 ± 0·2 fold at 24 hr versus vehicle control. CTCE treatment reduced bacterial invasion in blood [colony-forming units (CFU) decreased 77 ± 11%], peritoneal fluid (CFU decreased 78 ± 9%) and lung (CFU decreased 79 ± 8% versus CLP vehicle). The improved PMN recruitment and bacterial clearance correlated with reduced mortality with CTCE treatment (20% versus 67% vehicle controls). In vitro studies support the notion that CTCE augments PMN function by enhancing phagocytic activity (1·25 ± 0·02 fold), increasing intracellular production of reactive oxygen species (32 ± 4%) and improving bacterial killing (CFU decreased 27 ± 3%). These composite findings support the hypothesis that specific CXCL12 analogues with ancillary antibiotic treatment are beneficial in experimental sepsis, in part, by augmenting PMN recruitment and function.     

Modulation of Fc receptors on human peripheral blood lymphocytes by antisera against β2 microglobulin, Ia or immunoglobulin

The association between Fc receptors and other surface molecules was examined by EA-rosette (EAR) inhibition experiments. Twenty to 30% EAR were detected in suspensions of peripheral blood lymphocytes from normal individuals. Anti-β₂ microglobulin (βMi) sera fully suppressed EAR whereas anti-Ig, anti-Ia sera and heat aggregated IgG inhibited only 50–60% EAR. Thus almost half of the detected EAR were apparently not surface Ig positive B cells. Rabbit and monkey anti-βMi sera suppressed EAR effectively whereas rat and chicken antisera, despite strong βMi binding capacity, inhibited EAR poorly. The latter result was ascribed on the basis of immunofluorescent analysis to inadequate capping of βMi. Incubation of PBL with whole antisera suppressed EAR to a similar degree at either 0° or 37°, whereas F(ab′)₂ fragments were inhibitory only at 37°. Taken together the results suggest that Fc receptors can be inhibited by antibodies with specificity against any cell surface antigen. The blocking mechanism may be due to steric hindrance by the Fc part of antibody molecules and/or F(ab′)₂ fragment mediated co-capping.     

The mechanism of action of soluble lymphocytic mediators: I. A pulse exposure test for the measurement of macrophage migration inhibitory factor

Culture supernatants containing macrophage migration inhibitory factor (MIF) were obtained by incubating lymphocytes of guinea-pigs, immunized with Freund's complete adjuvant (FCA), with tuberculin PPD in vitro. Exposure of normal peritoneal macrophages to MIF-containing supernatants for 2 hours at 37° (pulse exposure), followed by suspension in culture medium and transfer to capillaries, resulted in inhibition of migration in vitro for the next 24 hours. No inhibition was seen when macrophages were incubated with MIF at 4°. On the other hand when exposure to MIF at 4° was followed by incubation of the cells for 2 hours at 37° in culture medium, in the absence of MIF, inhibition of migration was obtained. These results indicate that: (a) macrophages possess a specific receptor able to bind MIF at either 4° or 37°, and (b) inhibition of migration by receptor bound MIF requires a temperature-dependent active process, the nature of which remains unknown.

Passage of lymphocytes through columns of glass beads resulted in a population of cells with intact or heightened MIF-forming ability, as assessed by both conventional and pulse exposure techniques.

     

The velocity of lymph flow in the canine thoracic duct

1. The velocity and pattern of movement of lymph in the thoracic duct of anaesthetized and conscious dogs has been studied by observing the movement of droplets of ultrafluid lipiodol in the duct.

2. The velocity of flow when anaesthetized varied from 0·1-2·0 cm/sec, to 5·0 cm/sec when conscious.

3. The pattern of flow was affected by respiration and the cardiac cycle. Most movement occurred at the end of inspiration.

4. The duct of five autopsy preparations was perfused with saline to assess the volume and velocity of flow produced by the level of pressure gradients previously observed in the duct. These studies show that the small gradients (2-5 mmHg) observed during life are more than sufficient to produce the normal volume and velocity of flow measured. The mean resistance of the duct was 0·5 mmHg/ml. min.

     

Studies on the nature of EA binding by lymphocytes from rheumatoid arthritis patients

Investigation of the nature of the increased erythrocyte-antibody (EA) binding activity of peripheral blood lymphocytes (PBL) from rheumatoid arthritis (RA) patients reported in the preceding paper has revealed that IgG is the active class of antibody in this rosette formation. Some IgM binding also occurs. SRBC sensitized with F(ab)₂ preparations of IgG do not give rosette formation even at high concentrations. EA binding is inhibited by prior incubation of lymphocytes with heat-aggregated human IgG but antigen-antibody complexes did not give significant inhibition.The majority of these rosettes were found to be stable at 4°C and room temperature but labile at 37°C.Enzyme studies with pronase, trypsin, neuraminidase and treatment with sodium azide gave results strongly supporting the conclusion that the increased binding observed is increased Fc-receptor activity. This activity appears not to be a result of Fc binding by cell-bound rheumatoid factor.A range of titres of antibody and of IgG was used to sensitize erythrocytes to form EA and the enhanced EA-rosette formation by PBL from RA patients occurred throughout the range of concentrations of sensitizing antibody. Significantly more EA were bound by individual lymphocytes from RA patients than control subjects. This data suggest that the Fc receptors on RA lymphocytes are more avid for EA than receptors on lymphocytes from healthy people.     

General methods for the study of cells and serum during the immune response: the response to dinitrophenyl in mice

Two new methods for antibody assay are reported: one for antibody in solution, the other for individual cells releasing antibody. They depend on the use of an immunoabsorbent-bound antigen to absorb the antibody, and radioactively-labelled anti-immunoglobulin antibody (anti-Ig) for its detection. In the first method immunoabsorbent was added to the solution containing antibody, washed free of serum components other than the bound antibody, treated with [¹³¹I]anti-Ig, and the uptake of radioactivity assessed by γ-ray counting. This method was standardized for anti-DNP antibody by comparison with results of equilibrium dialysis. It was shown to be independent of affinity, down to K₀ = 10⁶ 1/M. It would detect 0·1 ng antibody. For the study of lymphoid cells releasing antibody the cells and immunoabsorbent were dispersed in agarose gel on microscope slides, incubated at 37°C in the presence of [¹²⁵I]anti-Ig, washed, and autoradiographed. Antibody released by a cell could then be visualized as a circular area (`spot') of silver grains. When sheep red blood cells were used as the antigen, the number of spots approximated to the number of lytic plaques obtained by the addition of complement. The methods were shown to be generally useful for a number of antigens. Both methods were used in a study of the course of the primary immune response to dinitrophenyl in mice. Cells releasing anti-DNP antibody were detected from 2 days after immunization and rose to a peak number at 9 days of 2 × 10⁵ in the lymph nodes local to the site of injection.     

Lymphocytotoxins in infectious mononucleosis: a non-cytotoxic effect on their cytotoxicity in vitro

The lymphocytotoxic activity (LCA) of sera from patients with infectious mononucleosis (IM) was tested against lymphocytes under various experimental conditions. Firstly, lymphocytes from 11 healthy donors were preincubated with pools of normal human sera (NHS) or IM sera at 37°C and then tested for (a) reactivity with the same IM sera in a standard lymphocytotoxin assay at 15°C; (b) rosetting with various sheep erythrocyte (E) preparations (E, EA and EAC) and (c) stimulation by non-specific activators (phytohaemagglutinin, pokeweed mitogen and concanavalin A). These experiments showed that preincubation of normal cells with IM sera caused significant reduction in subsequent lymphocyte killing at 15°C (P<0·01) compared to unincubated cells or those preincubated with pooled NHS. There was no change in the binding of E, EA and EAC or mitogen stimulation following incubation. Culture of preincubated lymphocytes in lymphocytotoxin-free medium for 24 hr did not restore LCA at 15°C. Secondly, a pool of normal lymphocytes was incorporated into media containing either 2,4-dinitrophenol or sodium azide and tested for LCA against 11 acute IM sera and two NHS at both 15 and 37°C. No significant change in cell killing was observed at 15°C in the presence of these inhibitors, but there was a significant return of LCA at 37°C. Finally, normal lymphocytes and cells from two patients with IM were cultured at 37°C in lymphocytotoxin-free medium to determine the role of down-regulation of lymphocyte surface receptors in reducing autolymphocytotoxicity during the acute phase of the illness. There was no change in cell killing by IM sera after culture for 24 hr. These experiments show that lymphocytotoxic sera from patients with infectious mononucleosis interact with normal lymphocytes at 37°C without causing cell killing. This interaction caused a change in surface-binding characteristics that was not reversed by culture in ligand-free medium for 24 hr. Studies using metabolic inhibitors suggested that the failed lymphocytotoxicity at 37°C resulted, at least in part, from lymphocyte metabolism, although this did not inhibit the reaction between cytotoxic material and the lymphocyte surface.