Recruitment of effector lymphocytes by initiator lymphocytes. Recruited lymphocytes are immunospecific and include graft-versus-host-reactive lymphocytes.

We have shown previously that initiator T lymphocytes (ITL), sensitized in vitro against fibroblast antigens, recruit effector T cells in vivo. After injection into hind footpads of syngeneic recipients, sensitized ITL migrated to the draining popliteal lymph nodes (PLN) and activated a trapping mechanism by which circulating lymphocytes were recruited in the PLN. This paper reports experiments designed to test the immunospecificity of these recruited T lymphocytes (RTL). We found that immunospecific RTL were depleted from other lymphoid organs during recruitment in the PLN. However, immunospecific ITL were not depleted from spleens during PLN recruitment. Thus ITL and RTL are functionally distinguishable. We show that specific GVH reactive lymphocytes were also lost from spleens and distal lymph nodes during trapping of RTL in the PLN. Thus, the trapping phase of the recruitment response is immunospecific, as are the sensitization and effector phases. The trapped RTL are antigen-specific, and include the pool of GVH-reactive-lymphocytes committed to the same alloantigen. Thus, it appears that GVH-reactive cells respond to syngeneic ITL sensitized against allogeneic fibroblasts.     

Increased T lymphocytes and IgMEA-receptor lymphocytes in Hodgkin's disease spleens.

Splenic lymphocytes from 11 patients with Hodgkin's disease were compared to lymphocytes of six spleens from patients with nonlymphoproliferative diseases. T lymphocytes were increased in patients with histological involvement by Hodgkin's disease. Likewise, lymphocytes from spleens with histological involvement showed increased rosette formation with immunologlobulin M-coated sheep red blood cells (IgMEA). A similar increase in T lymphocytes and in IgMEA rosette formation was not observed with normal peripheral blood lymphocytes, control spleens, or with Hodgkin's disease spleens without evidence of histological involvement.     

Mitogen- and antigen-responsive milk lymphocytes.

Bovine and canine milk contained lymphocytes that responded to the nonspecific mitogens; phytohemagglutinin (PHA), concanavalin A (Con A), pokeweed mitogen (PWM) and gram negative bacterial lipopolysaccharide (LPS). It also was found that animals specifically sensitized with tuberculin or infected with infectious bovine rhinotracheitis virus (IBR) had antigen sensitive lymphocytes in their milk. In general, the responses of the milk lymphocytes from an individual animal were not identical to responses for the blood lymphocytes. Marked variation was observed in the daily responses of cells from milk samples from different quarters of the same animal, and between animals. The implications of antigen and mitogen responsive milk lymphocytes are discussed, in relation to their possible role in protective immunity.     

Recruitment of effector lymphocytes by initiator lymphocytes. Circulating lymphocytes are trapped in the reacting lymph node.

In previous studies, we have shown that initiator T lymphocytes (ITL) sensitized in vitro recruit effector T lymphocytes in vivo. When ITL were sensitized against fibroblast antigens in vitro and injected into footpads of syngeneic recipients, they induced enlargement of the draining popliteal lymph node (PLN) and the development there of specific effector lymphocytes of recipient origin. To study the basis of this lymph node response in recruitment, we injected 51Cr-labeled spleen cells i.v. into recipients of sensitized ITL and found that the labeled circulating lymphocytes were trapped in the reacting PLN. The trapping depended on surface properties of the labeled circulating lymphocytes, as revealed by various enzymatic treatments. The trapping process was radiosensitive, both on the part of the trapped lymphocytes and the lymph node-trapping mechanism. Thus, sensitized ITL injected into the hind footpads migrate to the PLN and induce the trapping of circulating recruitable lymphocytes, which either differentiate into or regulate the differentiation of effector T lymphocytes.     

Ontogeny of B lymphocytes. I. In vitro appearance of Ig-bearing lymphocytes

During the immediate postnatal period, a striking increase in the fraction and number of splenic lymphocytes which bear easily detectable surface immunoglobulin (Ig) occurs in BALB/c and CDF₁ mice. Thus, in mice < 8 hr of age, approximately 4% of splenic lymphoid cells bear surface Ig whereas 17% of splenic lymphocytes of mice 24 hr of age are positive for surface Ig. This increase in Ig-bearing lymphocytes can be obtained in vitro. Thus, cultures of spleen or liver cells from neonatal mice display a substantial increase in the percent and in the absolute number of cells with easily detectable surface Ig. The in vitro increase in Ig-bearing cells is largely inhibited by mitomycin C or puromycin treatment of neonatal cells. Interestingly, pretreatment of these cells with anti-κ antibody, with or without complement, inhibited the increase in Ig-bearing cells. These results indicate that a substantial portion of Ig-bearing lymphoid cells present at 24 hr of age derive from cells already present in the spleens and livers of neonatal mice. Many of these “precursor” cells appear to bear some surface Ig.     

Cytotoxic activity of lymphocytes. VIII. Lymphotoxin activity in cell-free extracts of activated lymphocytes.

Cell-free extracts of human lymphocytes activated by PHA contain a cytotoxin that kills mouse L cells. Such cells elaborate two different kinds of toxins into the culture supernatant fluids, called α- and β-lymphotoxin (-LT), which differ in size, stability, and antigenicity. The amount of intracellular toxin is 1 to 24% of that found in supernatants of different tonsil donors. Equal amounts of intracellular toxin appear in both microsomal fraction (100,000g pellet) and soluble supernatant fractions of the cell-free extracts (CFE). The toxin can be solubilized from the membrane by digestion with papain or extraction with a nonionic detergent, but not by repeated sonication. The molecular weight of both the microsomal and soluble cellular cytotoxin is 45,000 ± 5000. The intracellular toxin differs from the extracellular toxins secreted by the same cells in two major characteristics: one, although its size approximates that of supernatant β-LT (and is smaller than the 76,000 M_r α-LT), antibody-inhibiting α-LT but not β-LT inhibits both the microsomal and soluble CFE-LT. Two, the intracellular LT does not display the charge heterogeneity so characteristic of supernatant α-LT. Supernatant α-LT and CFE-LT are similar in their patterns of inactivation by heating to 80 °C and treatments with sodium dodecylsulfate (SDS), guanidine, proteases, and heavy metal ions, and are similarly unaffected by treatment with 8 M urea, N-ethylmaleimide, and sodium periodate. These results suggest that the single polypeptide intracellular LT is the precursor of the more complex secreted α-LT molecule.     

Cytotoxic activity of lymphocytes. VI. Heterogeneity of cytotoxins in supernatants of mitogen-activated lymphocytes.

Two different lymphotoxins synthesized by human blood lymphocytes stimulated with phytohemagglutin (PHA) are described. The two toxins are called alpha-LT and beta-LT relative to their elution order on gel chromatography. Their m.w. are 75,000 and 45,000 daltons, respectively. Both toxins appear as early as 7 hr after the addition of PHA, with the amount of beta-toxin exceeding that of alpha-LT initially. Both toxins are differentiated from a third toxin (adherent cell toxin, ACT) made by plastic-adherent cells without requiring mitogen-stimulation. Depletion of macrophages or B cells does not affect the synthesis of either lymphotoxin. Monospecific antisera were elicited to alpha-LT. Antisera elicited to beta-LT also neutralized alpha-LT but to a significantly lesser degree. Alpha-LT appears to be the lymphotoxin described by most other workers. Beta-LT is unstable at 37 degrees C which may explain why the low m.w. lymphotoxin has not been described previously.     

D cyclins in lymphocytes.

BACKGROUND: D cyclins are essential for the progression of cells through the G1 phase of the cell cycle. There are three distinct D cyclins. Cyclin D1 has been shown to be expressed by many different types of cells but not by lymphocytes. Cyclins D2 and D3 have been found in lymphocytes. METHODS: We used high-resolution enzymatic amplification staining technology in conjunction with flow cytometry and confocal microscopy and with immunoblotting to reassess the expression of the D cyclins in human lymphocytes. RESULTS: Using high-resolution technology for flow cytometry, we found all three D cyclins in quiescent human peripheral blood lymphocytes. Cyclin D1 was expressed in quiescent and activated cells at levels commensurate with those of actively proliferating tumor cell lines. Cyclin D1 was functional inasmuch as it was complexed with CDK4. In the quiescent cells, cyclin D1 was expressed in the cytoplasm but, after activation, was found in the nucleus. CONCLUSIONS: These findings demonstrate that lymphocytes express cyclin D1 and necessitate a reappraisal of the hypothesis that the D cyclins subsume redundant activities with tissue-specific expression.     

Chemotaxis of rat lymphocytes.

Rat lymphocytes obtained from spleens, lymph nodes, and thymus glands showed migratory responses to a variety of factors including fluids from mixed lymphocyte culture fluids from concanavalin A-stimulated cells, fluids from phagocytizing macrophages, and to anti-rat IgG. Migratory responses to the last factor were bimodal over a dose range of anti-Ig; at high concentrations of anti-Ig, the response appeared to be nonspecific, whereas, at low concentrations, the responses seemed to be chemotactic in character. When lymphocytes from spleens, lymph nodes, and thymic glands were compared, qualitative and quantitative differences on the responses were evident with use of the three attractants. When spleen lymphocytes were separated into T cell- and B cell-enriched fractions, T cells responded to the culture fluids from mixed lymphocyte cultures, whereas B cells seemed to respond poorly, if at all. Only B cells responded to anti-Ig. These findings may explain, at least in part, the accumulation of lymphoid cells at sites of inflammatory stimuli.     

Activation of rat B lymphocytes. II. Functional and structural changes in "aged" rat B lymphocytes

Anti-mu, anti-gamma, and anti-delta antibodies induce proliferation of splenic B lymphocytes from young Lewis rats, measured by 3H-TdR uptake. In contrast, splenic B cells of aged Lewis rats respond poorly or not at all to these reagents. T lymphocytes or interleukin 2 (IL-2) of young or aged rats augment the uptake of 3H-TdR in cultures of "young" B cells responding to anti-Ig reagents or LPS and DxS, but have no significant effect on the responses of "old" B cells. Analysis of spleen cells of young and aged rats in a fluorescence-activated cell sorter indicates the density of mu, gamma, and delta isotypes is reduced in "old" B cells, and that B cells of aged rats are significantly larger than those of young rats. These results delineate anatomic and structural changes in B lymphocytes of aged rats.