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Hemopoietic Spleen Colony Studies

III. Hemopoietic Nature of Spleen Colonies Induced by Lymph Node or Thymus Cells, with or Without Phytohemagglutinin¹

From the Division of Experimental Biology, Baylor University College of Medicine, Houston, Texas

Abstract

1. Suspensions of lymph node or thymus cells from normal mice, administered intravenously to lethally irradiated (820 to 950 r) isogenic mice in doses of 1 to 60 x 10⁶ (viable) cells, were found to have very few spleen-colony-forming units (CFU). These were detectable only if high cell doses were administered to sufficiently large numbers of irradiated mice.

2. Phytohemagglutinin (PHA) treatment of recipients appeared to increase slightly the frequency of spleen colonies in mice injected with thymus cells, and sometimes in mice injected with lymph node cells. Unpublished data indicate that this effect may be attributed, at least in part, to PHA stimulation of endogenous spleen colony formation.

3. PHA treatment of donor mice slightly increased the low frequency of spleen colonies induced by lymph node cells, but not of colonies induced by thymus cells.

4. PHA treatment of both donor and recipient had no additional effect on the numbers of spleen colonies formed by transfusion of thymus or lymph node cells.

5. Histologically, cytologically and cytochemically the colonies formed by injection of thymus and lymph node cells were indistinguishable from the several types of hemopoietic spleen colonies induced in much larger numbers by bone marrow cell transfusions, with the following exception. They occurred against a background of significant diffuse lymphocytic repopulation of the spleen and partial repopulation of the lymphoid follicles caused by the large doses of lymphoid cells necessary to induce macroscopic spleen colonies, and contained lymphocytes of that background population. No macroscopic lymphopoietic colonies were found.

6. Erythroid spleen colonies induced by transfusion of thymus or lymph node cells (the only type of spleen colony that might be misdiagnosed as "lymphoid colonies" on routine histologic examination) were found to take up ⁵⁹Fe, to stain for hemoglobin and to be repressed by polycythemia. This repression was prevented by administered erythropoietin. These findings are identical to those obtained with erythropoietic colonies induced by bone marrow cells.

7. On retransplantation, the spleen colonies induced by lymph node cells were shown to give the same spectrum of "clean" hemopoietic secondary spleen colonies as those induced by spleen colonies of bone marrow origin. There was no background of, or admixture with, diffuse lymphocytic repopulation of the spleen.

8. Significant numbers of hemolytic antibody-forming cells were found in the spleen colonies induced by lymph node cells from PHA-treated donors when the irradiated recipients had also received injections of sheep red blood cells (SRBC). However, the concentration of antibody-forming cells in the colonies was only about one-fourth that in the intercolonial tissue. Spleen colonies induced by bone marrow injection did not contain antibody-forming cells, but comparable numbers of antibody-forming cells were produced in all of the hemopoietic colonies induced by bone marrow cells, and in the intercolonial tissue, by injecting lymph node cells from normal mice along with the bone marrow cells. Irradiated mice injected with 5 to 50 x 10⁶ lymph node cells from normal donor mice, and with SRBC, had few or no spleen colonies, but sheep hemolytic antibody-forming cells appeared in their spleens in direct proportion to the number of lymph node cells administered.

9. It is concluded that the spleen colonies induced by thymus or lymph node cells, with or without PHA-treatment of the donor or irradiated recipient, are not "lymphoid clones." Rather, they are hemopoietic spleen colonies similar to those induced by bone marrow cells, with the exception that they are contaminated by the large numbers of transfused lymph node cells necessary to induce a few hemopoietic colonies. As such, they are unsuitable for clonal lymphoid studies for which they have been used and recommended.

Footnotes

This investigation was supported by United States Public Health Service Grants T1 CA 05021, CA 03367 and K6 CA 14219 from the National Cancer Institute.