Chimerism, graft survival, and withdrawal of immunosuppressive drugs in HLA matched and mismatched patients after living donor kidney and hematopoietic cell transplantation.

Thirty-eight HLA matched and mismatched patients given combined living donor kidney and enriched CD34(+) hematopoietic cell transplants were enrolled in tolerance protocols using posttransplant conditioning with total lymphoid irradiation and anti-thymocyte globulin. Persistent chimerism for at least 6 months was associated with successful complete withdrawal of immunosuppressive drugs in 16 of 22 matched patients without rejection episodes or kidney disease recurrence with up to 5 years follow up thereafter. One patient is in the midst of withdrawal and five are on maintenance drugs. Persistent mixed chimerism was achieved in some haplotype matched patients for at least 12 months by increasing the dose of T cells and CD34(+) cells infused as compared to matched recipients in a dose escalation study. Success of drug withdrawal in chimeric mismatched patients remains to be determined. None of the 38 patients had kidney graft loss or graft versus host disease with up to 14 years of observation. In conclusion, complete immunosuppressive drug withdrawal could be achieved thus far with the tolerance induction regimen in HLA matched patients with uniform long-term graft survival in all patients.

Tolerance and withdrawal of immunosuppressive drugs in patients given kidney and hematopoietic cell transplants.

Sixteen patients conditioned with total lymphoid irradiation (TLI) and antithymocyte globulin (ATG) were given kidney transplants and an injection of CD34+ hematopoietic progenitor cells and T cells from HLA-matched donors in a tolerance induction protocol. Blood cell monitoring included changes in chimerism, balance of T-cell subsets and responses to donor alloantigens. Fifteen patients developed multilineage chimerism without graft-versus-host disease (GVHD), and eight with chimerism for at least 6 months were withdrawn from antirejection medications for 1-3 years (mean, 28 months) without subsequent rejection episodes. Four chimeric patients have just completed or are in the midst of drug withdrawal, and four patients were not withdrawn due to return of underlying disease or rejection episodes. Blood cells from all patients showed early high ratios of CD4+CD25+ regulatory T cells and NKT cells versus conventional naive CD4+ T cells, and those off drugs showed specific unresponsiveness to donor alloantigens. In conclusion, TLI and ATG promoted the development of persistent chimerism and tolerance in a cohort of patients given kidney transplants and hematopoietic donor cell infusions. All 16 patients had excellent graft function at the last observation point with or without maintenance drugs.

Alloantigen recognition is critical for CD8 T cell-mediated graft anti-tumor activity against murine BCL1 lymphoma after myeloablative bone marrow transplantation.

The goal of the current study was to determine whether whole bone marrow cells or splenic CD8(+) T cells from C57BL/6 (H-2(b)) donor mice, which are tolerant to BALB/c (H-2(d)) alloantigens, are capable of mediating graft anti-tumor activity against a BALB/c B-cell lymphoma after injection into irradiated BALB/c hosts. The experimental results show that high doses of splenic CD8(+) T cells mixed with T cell-depleted bone marrow cells from C57BL/6 non-tolerant (normal) donors eliminate the BCL(1) B-cell lymphoma cells and induce lethal graft-versus-host disease (GVHD). CD8(+) T cells from tolerant donors simultaneously lose both their ability to induce GVHD and their anti-tumor activity. Whole bone marrow cell transplants from normal donors eliminated BCL(1) tumor cells without inducing GVHD, and bone marrow cells from tolerant donors failed to eliminate the tumor cells. The infused BCL(1) tumor cells expressed an immunogenic tumor-specific idiotype antigen disparate from host alloantigens, indicating that recognition of the tumor-specific antigen alone was insufficient to elicit graft anti-tumor activity from unimmunized allotolerant donor splenic CD8(+) T cells or whole bone marrow cells. We conclude that CD8(+) T cells from unimmunized normal donor mice require alloantigen recognition to mediate their anti-tumor activity following allogeneic BMT.

Clonable progenitors committed to the T lymphocyte lineage in the mouse bone marrow; use of an extrathymic pathway.

We searched for clonable committed T cell progenitors in the adult mouse bone marrow and isolated rare (approximately 0.05%) cells with the Thy-1hiCD2-CD16+CD44hiCD25-Lin- phenotype. In vivo experiments showed that these cells were progenitors committed only to reconstituting the T cell lineage of irradiated Ly5 congenic hosts. Reconstitution of the thymus was minimal compared with that of the bone marrow, spleen, and lymph nodes. At limiting dilutions, donor T cell reconstitution of the spleen frequently occurred without detectable donor cells in the thymus. Progenitors were capable of rapidly reconstituting athymic hosts. In conclusion, the clonable bone marrow progenitors were capable of T cell reconstitution predominantly by means of an extrathymic pathway.

Identification of an early T cell progenitor for a pathway of T cell maturation in the bone marrow.

We have identified a rare ( approximately 0.05-0.1%) population of cells (Thy-1(hi)CD16(+)CD44(hi)CD2(-)TCRalphabeta(-)B220(-)M ac-1(-)NK1. 1(-)) in the adult mouse bone marrow that generates CD4(+) and CD8(+) TCRalphabeta(+) T cells after tissue culture for 48 hr in the presence of Ly5 congenic marrow cells. The essential stages in the maturation of the progenitors were determined; the stages included an early transition from CD2(-)CD16(+)CD44(hi)TCRalphabeta(-) to CD2(+)CD16(int/-)CD44(int/-)TCRalphabeta(-) cells, and a later transition to CD4(+)CD8(+)TCRalphabeta(+) double-positive T cells that rapidly generate the CD4(+) and CD8(+) single-positive T cells. The maturation of the progenitors is almost completely arrested at the CD2(+)TCRalphabeta(-) stage by the presence of mature T cells at the initiation of cultures. This alternate pathway is supported by the marrow microenvironment; it recapitulates critical intermediary steps in intrathymic T cell maturation.

Heterogeneity of NK1.1+ T cells in the bone marrow: divergence from the thymus.

NK1.1+ T cells in the mouse thymus and bone marrow were compared because some marrow NK1.1+ T cells have been reported to be extrathymically derived. Almost all NK1.1+ T cells in the thymus were depleted in the CD1-/-, beta2m-/-, and Jalpha281-/- mice as compared with wild-type mice. CD8+NK1.1+ T cells were not clearly detected, even in the wild-type mice. In bone marrow from the wild-type mice, CD8+NK1.1+ T cells were easily detected, about twice as numerous as CD4+NK1.1+ T cells, and were similar in number to CD4-CD8-NK1.1+ T cells. All three marrow NK1.1+ T cell subsets were reduced about 4-fold in CD1-/- mice. No reduction was observed in CD8+NK1.1+ T cells in the bone marrow of Jalpha281-/- mice, but marrow CD8+NK1.1+ T cells were markedly depleted in beta2m-/- mice. All NK1.1+ T cell subsets in the marrow of wild-type mice produced high levels of IFN-gamma, IL-4, and IL-10. Although the numbers of marrow CD4-CD8-NK1.1+ T cells in beta2m-/- and Jalpha281-/- mice were similar to those in wild-type mice, these cells had a Th1-like pattern (high IFN-gamma, and low IL-4 and IL-10). In conclusion, the large majority of NK1.1+ T cells in the bone marrow are CD1 dependent. Marrow NK1.1+ T cells include CD8+, Valpha14-Jalpha281-, and beta2m-independent subsets that are not clearly detected in the thymus.

Bone marrow NK1.1(-) and NK1.1(+) T cells reciprocally regulate acute graft versus host disease.

Sorted CD4(+) and CD8(+) T cells from the peripheral blood or bone marrow of donor C57BL/6 (H-2(b)) mice were tested for their capacity to induce graft-versus-host disease (GVHD) by injecting the cells, along with stringently T cell-depleted donor marrow cells, into lethally irradiated BALB/c (H-2(d)) host mice. The peripheral blood T cells were at least 30 times more potent than the marrow T cells in inducing lethal GVHD. As NK1.1(+) T cells represented <1% of all T cells in the blood and approximately 30% of T cells in the marrow, the capacity of sorted marrow NK1.1(-) CD4(+) and CD8(+) T cells to induce GVHD was tested. The latter cells had markedly increased potency, and adding back marrow NK1.1(+) T cells suppressed GVHD. The marrow NK1.1(+) T cells secreted high levels of both interferon gamma (IFN-gamma) and interleukin 4 (IL-4), and the NK1.1(-) T cells secreted high levels of IFN-gamma with little IL-4. Marrow NK1.1(+) T cells obtained from IL-4(-/-) rather than wild-type C57BL/6 donors not only failed to prevent GVHD but actually increased its severity. Together, these results demonstrate that GVHD is reciprocally regulated by the NK1.1(-) and NK1.1(+) T cell subsets via their differential production of cytokines.

An alternate pathway for T cell development supported by the bone marrow microenvironment: recapitulation of thymic maturation.

In the principal pathway of alpha/beta T cell maturation, T cell precursors from the bone marrow migrate to the thymus and proceed through several well-characterized developmental stages into mature CD4+ and CD8+ T cells. This study demonstrates an alternative pathway in which the bone marrow microenvironment also supports the differentiation of T cell precursors into CD4+ and CD8+ T cells. The marrow pathway recapitulates developmental stages of thymic maturation including a CD4+CD8+ intermediary cell and positive and negative selection, and is strongly inhibited by the presence of mature T cells. The contribution of the marrow pathway in vivo requires further study in mice with normal and deficient thymic or immune function.

Subsets of transgenic T cells that recognize CD1 induce or prevent murine lupus: role of cytokines.

T cells with T cell receptor (TCR) transgenes that recognized CD1 on syngeneic B cells stimulated B cells to secrete immunoglobulins in vitro. The CD4+, CD8+, or CD4-CD8- T cells from the spleen of the TCR transgenic BALB/c donors induced lupus with anti-double stranded DNA antibodies, proteinuria, and immune complex glomerulonephritis in irradiated BALB/c nude mice reconstituted with nude bone marrow. Injection of purified CD4-CD8- T cells from the marrow of transgenic donors prevented the induction of lupus by the transgenic T cells. Transgenic T cells that induced lupus secreted large amounts of interferon (IFN)-gamma and little interleukin (IL)-4, and those that prevented lupus secreted large amounts of IL-4 and little IFN-gamma or IL-10.

Granulocyte colony-stimulating factor reduces the capacity of blood mononuclear cells to induce graft-versus-host disease: impact on blood progenitor cell transplantation.

The feasibility of transplanting peripheral blood mononuclear cells (PBMC) from granulocyte colony-stimulating factor (G-CSF)-treated normal human donors to myeloablated allogeneic hosts has been demonstrated recently. The current work examined the ability of recombinant G-CSF to alter peripheral blood T-cell function and graft-versus-host disease (GVHD) in a murine model of allogeneic G-CSF-mobilized PBMC transplantation. Administration of recombinant G-CSF to C57BL/Ka mice markedly increased the capacity of PBMC to reconstitute lethally irradiated syngeneic hosts. T- and B-lineage lymphocytes were depleted about 10-fold in the bone marrow of the treated mice, and the T-cell yield in the blood was increased about fourfold. The ability of PBMC or purified CD4+ and CD8+ T cells to induce acute lethal GVHD in irradiated BALB/c mice was reduced after the administration of G-CSF. This was associated with decreased secretion of interferon gamma and interleukin-2 (IL-2) and an increased secretion of IL-4. The donor cell inoculum, which was most successful in the rescue of irradiated allogeneic hosts, was the low-density fraction of PBMC from G-CSF-treated mice. These low-density cells were enriched for CD4-CD8-NK1.1+ T cells and secreted about 10-fold more IL-4 than the unfractionated cells from the G-CSF-treated donors.

Different patterns of TCR transgene expression in single-positive and double-negative T cells. Evidence for separate pathways of T cell maturation.

Two lines of transgenic mice were established using the TCR alpha (V alpha 4.4-J alpha 24)- and beta (V beta 9-D beta 1.1-J beta 2.1)-chain genes from a cloned CD4-CD8-alpha beta + (double-negative; DN) T cell line from BALB/c mice. The TCR genes were expressed in CD4+CD8- and CD4-CD8+ (single-positive; SP) and double-positive (DP) T cells in the thymus, and in SP T cells in the peripheral lymphoid tissues, and marrow in one transgenic mouse line, and predominantly in DN T cells in the other. Bone marrow precursor cells from only the DN mouse line generated T cells expressing the V beta 9 transgene during tissue culture. V beta 9+ T cells were found in DN but not SP transgenic mice backcrossed to BALB/c nu/nu mice. The results suggest two separate pathways of T cell maturation, one which generates SP T cells in the thymus, and another which generates DN T cells in both the thymus and bone marrow.

Extrathymic maturation of alpha beta T cells from hemopoietic stem cells.

The object of the study was to determine whether alpha beta T cells can develop from hemopoietic stem cells in the absence of the thymus. C57BL/6 (Ly-5.1 and Thy-1.2) mice were thymectomized or sham-thymectomized at 4 wk of age, and received lethal whole body irradiation 2 wk later. These mice were reconstituted with an i.v. injection of 500 highly purified hemopoietic stem cells (Mac-1-, B220-, TER-119-, CD3-, CD4-, CD8-, Thy 1low, SCA-1+) obtained from the bone marrow of C57BL/6 (Ly-5.2 and Thy-1.1) donors. A similar percentage of Ly-5.2+ alpha beta T cells (donor) was found in the marrow of thymectomized recipients, sham-thymectomized recipients, and normal donor mice at least 3 mo after stem cell transplantation. The percentage of Ly-5.2+ alpha beta T cells in the spleens of sham-thymectomized and normal donor mice was similar. The percentage in the spleens of thymectomized recipients was reduced by about 50%, and approximately one-half of the latter T cells expressed the CD4-CD8- alpha beta+ phenotype. A purified population of Ly-5.2+ alpha beta- cells obtained from the marrow of thymectomized recipients was incubated in vitro for 48 h without exogenous growth factors. After the incubation procedure a proportion of the marrow cells acquired alpha beta TCR surface receptors. The results show that alpha beta T cells can develop from hemopoietic stem cells in the absence of the thymus.

Enrichment of bone marrow and blood progenitor (CD34+) cells by density gradients with sufficient yields for transplantation.

We have evaluated the use of iso-osmolar Percoll density gradients to enrich CD34+ hematopoietic progenitor cells and to reduce T cells for purposes of bone marrow or mobilized peripheral blood cell transplantation (BMT or PBCT). Samples from 12 normal BM donors and 11 patients undergoing mobilization of PB cells using chemotherapy and G-CSF were placed over a five-step density gradient from 40 to 50% Percoll. In BM, low-density fractions 1 to 3 (40 to 45% Percoll) accounted for 3% of starting nucleated cells with a 10- to 20-fold enrichment of hematopoietic progenitors (CD34+ cells) and a 20-fold reduction of CD4+ and CD8+ T cells. In PB, fractions 1 to 3 accounted for 20 to 30% of the starting nucleated cells with a five-fold enrichment of hematopoietic progenitors. Based on these values, such populations have been used for clinical transplantation using a single apheresis. The reduced cell numbers in the low-density fractions can facilitate tumor purging, and the reduced T cell numbers present in the marrow may ameliorate graft-vs.-host disease.

The role of purified CD8+ T cells in graft-versus-leukemia activity and engraftment after allogeneic bone marrow transplantation.

The ability of highly purified CD8+ T cells to mediate GVL activity and facilitate engraftment of allogeneic bone marrow cells was studied in the C57BL/Ka-->BALB/c mouse strain combination. Splenic CD8+ T cells were enriched by depletion of CD4+ T cells by "planning" or purified by positive selection by cell sorting. Although C57BL/Ka bone marrow cells reconstitute lethally irradiated BALB/c mice without severe GVHD, the addition of at least 1.0 x 10(6) donor spleen cells induced uniform acute lethal GVHD. Equivalent doses of spleen cells depleted of CD4+ T cells failed to induce lethal GVHD. Allogeneic bone marrow cells alone failed to mediate antitumor activity against the BCL1 B cell leukemia/lymphoma as compared with syngeneic bone marrow and spleen cell injections. Despite the inability to induce severe GVHD, CD4+ T cell-depleted allogeneic spleen cells prevented the progressive growth of the BCL1 tumor, and eliminated BCL1 idiotype-positive tumor cells in the blood. In order to determine whether CD8+ T cells can prevent tumor growth in the absence of other spleen cell subsets, such as NK cells, that are present in the CD4- populations, highly purified CD8+ T cells were obtained by positive selection using flow cytometry. The latter cells prevented the progressive growth of the tumor, and markedly reduced the level of tumor cells in the blood. Sorted CD8+ T cells facilitated the engraftment of allogeneic marrow cells in sublethally irradiated hosts. Thus, addition of highly purified CD8+ T cells to marrow cells provides GVL activity and facilitates engraftment without inducing severe GVHD in most recipients.

Similar rates of production of T and B lymphocytes in the bone marrow.

The rate of renewal of T lymphocytes in the bone marrow of euthymic C57BL/Ka and athymic nu/nu BALB/c mice was estimated by in vivo labeling with bromodeoxyuridine. T lymphocytes accounted for 16-18% of marrow cells in euthymic mice as judged by immunofluorescent staining with monoclonal antibodies for Thy-1, CD3, and alpha/beta T cell antigen receptor markers. About 70% of marrow cells expressed receptors (Mac-1, Gr-1, B220) for myeloid, macrophage, and B lineage cells. Approximately 13% of cells in the athymic bone marrow expressed alpha/beta T cell receptors. Sorted marrow T cells proliferated in response to stimulation with anti-alpha/beta antibodies in vitro and showed functional rearrangements of V beta and J beta genes. Sorted non-T cells did not respond to stimulation in vitro, and all V beta and J beta gene rearrangements identified were nonfunctional. In vivo labeling studies indicated that approximately 17 x 10(6) bone marrow T cells are renewed daily in euthymic mice and approximately 14 x 10(6) are renewed in athymic mice. Approximately 11 x 10(6) mature B cells (immunoglobulin M+) are renewed daily in the bone marrow of the latter mice. To determine whether marrow precursors can give rise to T cells directly, marrow cells from euthymic and athymic mice were depleted of T cells by cell sorting and incubated in vitro for 48 h in the absence of exogenous growth factors or thymic stromal cells. Examination of the cells after culture showed that 10-12% stained brightly for alpha/beta T cell receptors. Although functional rearrangements of V beta and J beta genes were not detected before culture, the majority of rearrangements were functional after culture. The emergence of the bright alpha/beta T cells in culture was dependent on depletion T cells from the marrow cells before culture. The results suggest that most marrow T cells are generated in the marrow itself.

Homogeneous antigen receptor beta-chain genes in cloned CD4- CD8- alpha beta T suppressor cells.

The rearrangements of beta-chain genes of the T cell Ag receptor were examined in 12 CD4- CD8- alpha beta + T cell lines derived from the spleen or thymus of neonatal or adult BALB/c mice. Eleven of the lines were cloned and established from six independent cloning procedures from different mice. Five cloned lines used V beta 9, four cloned lines used V beta 15, and two cloned lines used V beta 7. Nucleotide sequencing of the beta-chain genes showed that clones that used a given V beta were identically rearranged even when they were derived from independent cloning procedures. In the case of V beta 7 and V beta 15 all nucleotides in the V-D-J joining region were in the germ line configuration without N region additions. Rearrangements of the V beta 7, V beta 9, and V beta 15 genes were functional. Each V beta 15 clone also had a homogeneous rearrangement of the V beta 13 gene, which was nonfunctional. The predicted amino acid sequence of the joining regions of the V beta 7, V beta 9, and V beta 15 rearrangements showed homology in four of seven amino acids in the peptide contact region.

T-cell subsets and suppressor cells in human bone marrow.

To characterize immune suppressive and hematopoietic features of enriched subsets of human marrow cells, we separated these cells on Percoll density gradients. CD4+ and CD8+ T cells (CD3+) were enriched in the high-density marrow cell fractions and reduced in low-density fractions. CD4-CD8- (CD3+) T cells expressing the alpha beta T-cell antigen receptor were at least 10 times less numerous than the CD4+ and CD8+ T cells in all fractions. Purified populations of the CD4-CD8- alpha beta + T cells obtained by flow cytometry suppressed the mixed leukocyte reaction (MLR). Another population of suppressor cells that expressed neither T-cell (CD3) nor natural killer cell (CD16) surface markers was also identified. The latter cells had the phenotypic and functional characteristics of "natural suppressor" cells. Suppressor cell activity was enriched in the low-density fractions along with hematopoietic progenitors (colony-forming unit-granulocyte-macrophage and burst-forming unit-erythroid). The progenitor and suppressor cell activities were depleted in high-density fractions. The latter fractions made vigorous responses in the MLR. The low-density fractions, which accounted for less than 10% of the input marrow cells, suppressed the MLR and did not respond. Further evaluation of the low-density fractions may be of value in allogeneic bone marrow transplantation due to the reduction of CD4+ and CD8+ T cells and the enrichment of hematopoietic progenitors as well as immune suppressor cells that may inhibit graft-versus-host disease.

Different subsets of T cells in the adult mouse bone marrow and spleen induce or suppress acute graft-versus-host disease.

Fractionation of normal adult mouse spleen and bone marrow cells (C57BL/Ka) was performed by discontinuous Percoll density gradients. The fractionated low density (1.050-1.060 g/ml) C57BL/Ka spleen cells completely suppressed acute lethal graft vs host disease (GVHD) when coinjected with unfractionated C57BL/Ka spleen cells into sublethally irradiated (400 rad) BALB/c mice. In dose response experiments, as few as 0.5 x 10(6) low density cells from the spleen fractions suppressed acute GVHD induced by 2.5 x 10(6) unfractionated allogeneic spleen cells. Although the low density spleen fractions inhibited acute GVHD, the high density (1.075-1.090 g/ml) spleen fractions induced acute GVHD in sublethally irradiated BALB/c recipients. Fractionation of C57BL/Ka bone marrow cells showed that none of the high or low density fractions or unfractionated cells induced lethal GVHD. When these fractions were tested for their capacity to suppress GVHD by coinjection with C57BL/Ka unfractionated spleen cells, all fractions protected the BALB/c recipients. Unfractionated bone marrow cells showed modest protection. Evaluation of the dose response characteristics of the suppressive activity of the low and middle density (1.060-1.068 g/ml) bone marrow cell fraction showed that reproducible protection could be achieved at a 5:1 ratio of inducing to suppressing cells. The low density fractions of both bone marrow and spleen cells had a marked depletion of typical TCR(+)-alpha beta CD4+ or CD8+ T cells, and a predominant population of TCR(+)-alpha beta CD4- CD8- T cells. Purified populations of the latter cells suppressed GVHD. Recipients given unfractionated C57BL/Ka spleen cells and protected with low-density bone marrow or spleen cells were chimeras.

Treatment of BCL1 leukemia by transplantation of low density fractions of allogeneic bone marrow and spleen cells.

C57BL/Ka bone marrow and spleen cells fractionated by density gradients were transplanted to lethally irradiated (800 rad) BALB/c recipients. Unfractionated bone marrow and spleen cell mixtures (1:1), or high density fractions of these cells induced acute lethal graft-vs-host disease (GVHD). In contrast, low and middle density fractions of bone marrow and spleen cell mixtures reconstituted the irradiated hosts, and the majority survived for at least 100 days. The latter cells contained sufficient hemopoietic cells for reconstitution, but were deficient in inducing GVHD. Examination of the T cell subsets in the low density fractions showed a reduction of typical alpha beta TCR+ CD4+ or CD8+ cells and little change in the proportion of alpha beta TCR+ CD4- CD8- cells. BALB/c mice injected with the BCL1 B cell leukemia/lymphoma were lethally irradiated and transplanted with unfractionated BALB/c or C57BL/Ka bone marrow and spleen mixtures or low density fractions of C57BL/Ka mixtures. All control unirradiated BALB/c mice given the BCL1 tumor cells died by day 55. Almost all BALB/c mice given the BCL1 tumor cells, irradiation, and injected with a syngeneic marrow and spleen mixture died by day 95. All of the latter recipients tested showed evidence of tumor relapse. Almost all BALB/c mice given BCL1 cells, irradiation, and a C57BL/Ka unfractionated marrow and spleen mixture died by day 40. The survival of BALB/c mice given BCL1 cells, irradiation, and a low density fraction of the C57BL/Ka mixture was markedly prolonged as compared to those recipients given unfractionated allogeneic or syngeneic mixtures. None of the low density fraction recipients tested showed evidence of tumor relapse. Similar results were obtained with leukemic C57BL/Ka x BALB/c F1 hybrid mice. Thus, the low density fraction fails to induce acute lethal GVHD, but retains graft-vs-leukemia activity.