Mixed lymphocyte culture reactivity of fetal baboons: application for in utero bone marrow transplantation.

Bone marrow transplantation offers a potential cure for patients suffering from genetic diseases such as inborn errors of metabolism. The optimal time to transplant many of these affected individuals would be early in gestation. To date, little information is available on the cellular immune reactivity of fetal primate lymphocytes. Therefore, we tested peripheral blood lymphocytes obtained in utero from baboon fetuses (Papio sp.) for their ability to respond in mixed lymphocyte culture (MLC) against their mothers, against a pool of unrelated animals, and in the case of fetuses given unrelated bone marrow transplants in utero, against their specific bone marrow donors. The majority of fetuses as young as 80 gestational days (182-day normal gestation period) were capable of responding strongly to maternal and unrelated lymphocytes in MLC. Of six fetuses that were transplanted, three did not engraft as indicated by undetectable levels of the donor-specific type B allele of glucose phosphate isomerase in fetal blood samples 1 month post-transplant. The three fetuses that did engraft all lost their grafts before birth. These data demonstrate that fetal lymphocytes obtained in utero can be tested for MLC reactivity and suggest that MLC testing can be used to select appropriate donor-recipient combinations for in utero bone marrow transplantation.

Dithiothreitol-dependent antilisterial activity of lysates from normal macrophages exposed in vitro to culture fluids from spleen cells of BCG-immunized mice.

The effect of culture fluids from mouse lymphocytes on the antilisterial activity of normal macrophages in vitro was determined. Titers of lysates of macrophages incubated with or without culture fluids from lymphocytes from control mice had titers of 10 to 270 with or without a reducing agent, dithiothreitol. Lysates of macrophages after incubation with culture fluids of spleen lymphocytes from BCG-immunized mice had antilisterial titers with and without dithiothreitol of 197,000 and 810, respectively. This may indicate a dithiothreitol-dependent antilisterial system inherent in activated macrophages of cell-mediated immunity. Lysates of incubated spleen cells and their culture fluids also had slight antilisterial activity.

Subpopulations of splenic T cells regulating an anti-hapten antibody response. II. Distinct functions of, and sequential requirement for, helper and amplifier cells.

In adoptive transfer experiments, two classes of peripheral T lymphocytes, carrier-primed helper (TH) and carrier-primed amplifier (TA) cells, synergized in the induction of a primary anti-hapten IgG response by virgin B cells. Purified TH and TA had separate functions and were required at different times during the antigen-driven development of the response. TH were required early, and provided an initiating signal to B cells in the presence of the specific hapten-carrier conjugate. The differentiative nature of this signal was inferred from the threshold dose-response relationship and the insensitivity of the TH-directed event to the antimitotic agent vinblastine. TA were required 4 days later and provided an amplifying signal to B cells in the presence of the same hapten-carrier conjugate. The proliferative nature of this second signal was inferred from the exponential dose-response relationship and the exquisite sensitivity of the TA-directed event to vinblastine. Virgin B cells became susceptible to the TA signal only after having received the TH signal. TH and TA did not synergize, however, in true secondary responses since hapten-primed B cells depended only on the TH signal to generate large numbers of IgG antibody-forming cells.

Subpopulations of splenic T cells regulating an antihapten antibody response. I. Helper and amplifier cells.

There was a pronounced quantitative difference between the helper activities of B6C3F1 splenic T cells sensitized with unmodified vs modified antigens of SRBC. Modified SRBC induced the greater helper activity which was measured by the magnitude of an anti-TNP response (IgM and IgG) elicited in vivo by virgin B lymphocytes. Antigen modification was produced by conjugating SRBC with the hapten or simply by incubating SRBC in cacodylate buffer. There were restrictions with respect to both erythrocyte species and mouse strains for this differential priming to occur. The relatively poor performance of SRBC-primed T lymphocytes was apparently not due to suppressor T cells, but rather to activation of only one of two identified T cell subpopulations required for full helper activity. Unmodified SRBC activated a subpopulation of "helper" cells characterized as sensitive to elimination by ATS and long-lived after ATx, but failed to activate in B6C3F1 mice a second subpopulation of "amplifier" cells resistant to elimination by ATS and short-lived after ATx. In contrast, modified SRBC activated both helper and amplifier cells. Under appropriate conditions these subsets of T cells were strongly synergistic in promoting antihapten antibody formation especially of the IgG class. The involvement of two distinct types of T lymphocytes in the positive regulation of antibody responses raises interesting and novel questions concerning the sequence of events in the triggering of B cells and the subsequent development of the response.

In utero bone marrow transplantation of fetal baboons with mismatched adult baboon marrow.

In utero bone marrow transplantation to fetuses offers the potential advantage of ameliorating the effects of genetic disorders by transplanting allogeneic hematopoietic stem cells into recipients who are immunoincompetent and require no preparative regimen. Therefore, we undertook studies to examine the feasibility of in utero bone marrow transplantation of unrelated allogeneic adult bone marrow into fetal baboons. Thirty-one baboon fetuses were transplanted between the ages of 60 and 160 days gestation (normal gestation, 182 days) with unrelated allogeneic adult bone marrow containing a different isozyme of glucose-phosphate isomerase (GPI). Approximately one third of the 80-day fetuses demonstrated engraftment 1 month after transplantation. Three of three of the initial chimeras died in utero 45 to 80 days after transplantation and the remaining chimeras lost their graft. Furthermore, 80-day fetal baboons were able to recognize donor cells, maternal cells, and other adult baboon peripheral blood cells in a mixed lymphocyte culture (MLC) reaction but still could engraft with allogeneic bone marrow. In contrast all nonchimeric animals survived to term. These data suggest that fetal transplantation of primates is feasible using techniques employed in these studies and that transplantation of younger fetuses who are immunocompetent should be attempted.

CD34+ marrow cells, devoid of T and B lymphocytes, reconstitute stable lymphopoiesis and myelopoiesis in lethally irradiated allogeneic baboons.

CD34+ cells devoid of detectable mature and immature T and B lymphocytes, expressing the CD2, CD10, and CD20 antigens, were isolated from marrows of three pairs of sex-mismatched, mixed lymphocyte culture (MLC) nonreactive, sibling baboons. Reciprocal transplants were performed between members of each pair, using the sex chromosomes, identified by standard cytogenetic techniques, as markers of the transplanted cells. Five animals from these three pairs were transplanted with 0.6 to 2.1 x 10(6)/kg of isolated cryopreserved and/or fresh isolated cells that were greater than 95% to 97% CD34+. Before transplantation, animals were treated with either single (920 or 1,020 cGy) or split (700 cGy x 2) dose total body irradiation. All animals engrafted with donor cells, as demonstrated by cytogenetic analysis of bone marrow metaphase cells 4 weeks after transplantation, with days to white blood cell count (WBC) greater than 500 being 19 +/- 2, to WBC greater than 1,000 23 +/- 2, to absolute neutrophil count greater than 500 24 +/- 3, and to platelets greater than 20,000 30 +/- 7. Three animals died of infectious-related complications at 34, 42, and 109 days after transplantation with evidence of host and donor cells (mixed chimerism) in marrow. Two animals remain alive and healthy more than 545 and 455 days after transplantation with stable mixed chimerism in marrow and blood. For these two animals, cytogenetic analysis of granulocyte/macrophage and erythroid colonies derived from marrow precursors between weeks 25 and 42 posttransplant showed evidence of mixed chimerism. Cytogenetic studies of CD2+ T cells and CD20+ B cells isolated from blood of these two animals between weeks 21 and 51 posttransplant showed the presence of mixed chimerism in both lymphocyte populations. Thus, isolated allogeneic CD34+ marrow cells devoid of detectable mature and immature T and B lymphocytes can engraft and reconstitute stable long-term myelopoiesis and lymphopoiesis in lethally irradiated baboons. These results are consistent with the hypothesis that CD34+ marrow cells contain pluripotent hematopoietic stem cells capable of fully reconstituting lymphohematopoiesis in the transplanted host.