T cell repopulation from functionally restricted splenic progenitors: 10,000-fold expansion documented by using limiting dilution analyses

Mice depleted of T cells by thymectomy, lethal irradiation, and reconstitution with Thy-1-depleted syngeneic bone marrow were given graded doses of splenic T cells to see whether post-thymic cells had the ability to regenerate immune function in these hosts. Using limiting dilution methods to estimate the number of antigen- and mitogen-responsive cells in recipients 12 to 20 wk after reconstitution, we found that new helper and cytotoxic precursor cells were produced, but attained levels only 10 to 20% of normal. Because these repopulated mice were able to produce nearly normal levels of helper and cytotoxic activity in conventional, high density cultures, despite their relative paucity of precursors, we infer that their normal function in conventional assays may reflect a balanced deficiency of effector and regulatory cell types. Surface phenotyping of the progenitor cells responsible for repopulation showed that Lyt-2- cells were required for helper cell regeneration and that Lyt-2+ cells acted as progenitors only for the cytotoxic lineage, contrary to earlier speculation that the splenic Lyt-1+ 2+ (Ly-123) pool included cells antecedent to both effector lineages. Comparison of the number of injected progenitors needed to produce repopulation with the number of new precursor cells eventually produced suggests that the relevant progenitors are able to undergo 10,000-fold expansion in 12 to 20 wk. Numerical expansion in the periphery from thymic-processed cells could well be a major source of new lymphocytes in adult mice.     

Phenotypic heterogeneity of antisyngeneic tumor killer cells (ASTK) generated in allogeneic mixed lymphocyte reactions

By using monoclonal antibodies to Thy-1, Lyt-2, and Qa-5 differentiation antigens, we demonstrated a heterogeneity of cytotoxic cells developed in allogeneic mixed lymphocyte responses that lyse tumor cells syngeneic with the responder cells. There are minimally two Thy-1+ populations, one of which is Lyt-2+ and the other Lyt-2-. There is probably also a Thy-1- population. Most of the Lyt-2- tumor killer cells are Qa-5+, and most of the Lyt-2+ tumor killer cells are Qa-5-.     

TNF regulates thymocyte production by apoptosis and proliferation of the triple negative (CD3-CD4-CD8-) subset

TNF is a proinflammatory cytokine with opposing death/no-death effects in vivo and in vitro. Our studies showed that TNF regulates mouse thymocyte production, inducing both apoptosis and proliferation of the most immature CD3(-)CD4(-)CD8(-) triple negative (TN) subset within a broad range of dosages (10(1)-10(5) pg/ml) in the presence of IL-7. TNF apoptosis affected only the TN3 (CD44(-)CD25(+)) and TN4 (CD44(-)CD25(-)) subsets that expressed both TNFR-p55 and -p75. Although each TNFR alone could mediate TNF apoptosis, maximal apoptosis was seen in C57BL/6J wild type, which expressed both TNFRs. TNF also induced proliferation of TN3 cells at higher doses (10(4)-10(5) pg/ml) mediated only by TNFR-p75. Both anti-TNFR-p55 and -TNFR-p75 mAb inhibited apoptosis but only anti-p75 inhibited proliferation. TNF also regulated TN proliferation to IL-7 because TNFR knockout (KO), TNF KO, and TNF/lymphotoxin alpha and beta triple KO mice showed 2- to 3-fold increased responses not seen in C57BL/6J wild type. In vivo, TNFR KO mice showed thymic hypertrophy with a 60% increase in total thymocytes, with no effect on the CD4/CD8 subsets. We conclude that TNF maintains homeostatic control of total thymocyte production by negative selection of TN3 and TN4 prothymocytes and down-regulation of their proliferation to endogenous IL-7.     

Specific neonatally induced tolerance to Mls locus determinants

Neonatal injection of CBA/HT6T6 (H-2k, Mlsb) mice with adult, Mls-incompatible (CBA/J [H-2k, Mlsd] X CBA/HT6T6)F1 spleen cells results in the abrogation of cell proliferation and interleukin 2 (IL 2) production in bulk mixed lymphocyte cultures, when spleen cells from the inoculated mice are tested at 6 to 8 wk of age with stimulator cells expressing the Mlsd of the tolerizing inoculum. In limiting dilution assays, this tolerant state was manifested in a 25- to 550-fold (280-fold average) decrease in the frequency of precursors of Mlsd-responsive IL 2-producing T cells. Tolerance was specific in that the frequencies of precursors of IL 2-producing cells responding to Con A, allogeneic H-2d, and self-Ia were not affected. The observed low frequency of Mls-responsive cells was due neither to extensive chimerism resulting in the dilution of Mlsd-responsive cells by the nonresponsive F1 cells of the inoculum, nor to the action of suppressor cells. These findings indicate that neonatal injection of Mls-incompatible spleen cells produces a state of specific tolerance by a clonal deletion or inactivation mechanism. This specific tolerance supports the view that 1) the Mls locus encodes or regulates the expression of defined alloantigenic determinants and 2) Mls-incompatible responder mice have specific receptors for Mls determinants on clonally distributed IL 2-producing responder T cells.     

Chromatographic Separation and Antigenic Analysis of Proteins of the Oncornaviruses IV. Biochemical Typing of Murine Viral Proteins

Tryptic peptide maps were prepared for four purified structural proteins derived from several murine leukemia viruses (MuLV's). Analyses of these peptide maps reveal that the p30 proteins of Rauscher, Moloney, and Gross MuLV's are very similar to each other, as are the p10's obtained from these three viruses. In contrast, the peptide maps of the individual p15's and p12's from the same viruses establish that each of these polypeptides is highly strain specific. For all four polypeptides studied, unique peptides appear in the Rauscher MuLV and Moloney MuLV tryptic profiles that are not present in the corresponding Gross MuLV profile. By this method of analysis it was possible to distinguish the p30's of N-tropic and B-tropic MuLV's derived from the same BALB/c mouse.     

Improved Isolation of Anaerobic Bacteria From the Gingival Crevice Area of Man

A roll tube technique (Hungate method) was employed in an attempt to cultivate a maximal portion of the organisms in the gingival crevice area of man. This technique achieves an anaerobic state by flushing the local environment with oxygen-free gas. Once collected, the crevicular debris was immediately placed into sterile oxygen-free test tubes which were flushed out by the oxygen-free gas. In this manner, the samples were weighed, dispersed, diluted, and cultured in roll tubes and plates. The medium for control (Brewer Jar technique) and Hungate techniques was Heart Infusion Agar fortified with 10% defibrinated horse blood. When the Hungate technique was used, the recovery of viable bacteria, as a percentage of the direct microscopic count, was significantly greater than plates incubated aerobically or utilizing the Brewer Anaerobic technique. Cultural counts by using the Hungate method averaged 41.3% for six samples when 90% nitrogen and 10% hydrogen were used, 70.4% for eight samples when 85% nitrogen, 10% hydrogen, and 5% carbon dioxide were used, and 63.4% for eight samples when 100% carbon dioxide was the gaseous atmosphere. At no time were cultural counts, by using anaerobic plates (Brewer Jar), more than 24% of the direct microscopic count. This suggests that exclusion of oxygen and the presence of carbon dioxide maximized recovery of gingival crevice bacteria.     

L3T4+, B2A2+ thymocytes from infant mice produce IL 2 after interaction with accessory cells expressing self class II antigens

The presence of a thymocyte population in infant (3 to 10 day old), but not adult mice, that produces IL 2 after self class II MHC antigen stimulation is described. The responding thymocyte expresses the antigenic phenotype: Thy-1+, Ly-1+, Ly-2-, L3T4+, B2A2+. Cell-cell mixing experiments and limiting dilution analysis were consistent with a loss of the self-Ia-reactive IL 2-producing thymocyte in the adult, with no evidence for active suppression found. Although thymocytes from adult animals failed to generate IL 2 after self class II stimulation, a similar frequency of IL 2-producing cells was demonstrated in both infant and adult after allo stimulation. These findings, taken with the recent demonstration of IL 2 receptors on neonatal thymocyte subpopulations, demonstrate a mechanism for IL 2 production in the thymus that may play a role in intrathymic differentiation of T cells.