Method for concentrating marrow stem cells using the IBM 2991 washer. Necessary preparation before in vitro treatment of bone marrow by pharmacologic or immunologic means

The technique using the IBM 2991 blood cell processor is an effective technique for the concentration of mononuclear cells from large volumes of bone marrow. The marrow cells are layered on to Ficoll Metrizoate using the IBM processing set. The mononuclear cells and CFU-GM recoveries are in close relationship with the hematocrit of the cell suspension processed. Twenty two bone marrows have been collected and purified according to this protocol. The mononuclear cell recovery is an average of 78,3% (range: 44-92%) and the CFU-GM recovery is in average of 67,5% (range: 40-89%). At the end of the procedure the cell viability is satisfying (97,1% +/- 1,7 are trypan blue negatives). When it is necessary to remove from the bone marrow collected either malignant cells prior autologous bone marrow graft or T lymphocytes in an attempt to prevent GVHD in allogeneic BMT, the purity of marrow cell suspension become a fundamental parameter.     

Acute graft-versus-host disease after allogeneic bone marrow transplantation.

In 25 patients receiving allogeneic bone marrow transplants methotrexate was used to prevent acute graft-versus-host disease (GVHD). Acute GVHD, grades 2 to 4, developed in only 5 (20%) of the patients. The incidence of acute GVHD in other series of recipients of bone marrow transplants has ranged from 5% to 76%. A review of the literature suggests that this variation cannot be completely accounted for by age, type of disease treated by transplantation or type of GVHD prophylaxis. However, transfusion of allogeneic lymphocytes that have not been completely inactivated by irradiation (e.g., in platelet and granulocyte preparations) and inadequate isolation-decontamination procedures may increase the probability of GVHD following bone marrow transplantation.     

Allogeneic peripheral blood stem cell transplantation

Granulocyte colony stimulating factor (G-CSF)-mobilized peripheral blood stem cells (PBSC) are now widely used instead of bone marrow for autologous transplantation due to earlier hematopoietic recovery after transplant. The low toxicity of G-CSF has prompted phase I and II studies to evaluate PBSC for allogeneic transplantation; these studies have demonstrated that engraftment of neutrophils, red blood cells and platelets is faster with peripheral blood cells compared to marrow. In randomized studies comparing mobilized PBSC and marrow for allogeneic transplantation, most trials have confirmed significantly earlier engraftment with PBSC and similar risks of acute graft-vs.-host disease (GVHD). In some trials, an increase of 10-15% in grade II-IV GVHD has been noted with PBSC. All studies showed a trend towards more chronic GVHD with PBSC. Some randomized studies have shown improved survival and disease-free survival with the use of PBSC due to lowered transplant-related mortality and fewer relapses in recipients of PBSC as a result of improved immune reconstitution and a graft-vs.-leukemia (GVL) effect. This survival benefit is most apparent in patients with more advanced hematologic malignancies, but further studies are needed to define the relative benefits of PBSC for patients with less advanced disease. The GVL effect of PBSC is currently being exploited with the use of non-ablative allografts.     

Effects of T cell depletion in radiation bone marrow chimeras. I. Evidence for a donor cell population which increases allogeneic chimerism but which lacks the potential to produce GVHD

The opposing problems of graft-vs-host disease (GVHD) and failure of alloengraftment present major obstacles to the application of bone marrow transplantation (BMT) across complete MHC barriers. The addition of syngeneic T-cell-depleted (TCD) bone marrow (BM) to untreated fully allogeneic marrow inocula in lethally irradiated mice has been previously shown to provide protection from GVHD. We have used this model to study the effects of allogeneic T cells on levels of chimerism in recipients of mixed marrow inocula. The results indicate that T cells in allogeneic BM inocula eliminate both coadministered recipient-strain and radioresistant host hematopoietic elements to produce complete allogeneic chimerism without clinical GVHD. To determine the role of GVH reactivity in this phenomenon, we performed similar studies in an F1 into parent combination, in which the genetic potential for GVHD is lacking. The presence of T cells in F1 marrow inocula led to predominant repopulation with F1 lymphocytes in such chimeras, even when coadministered with TCD-recipient-strain BM. These results imply that the ability of allogeneic BM cells removed by T cell depletion to increase levels of allochimerism may be mediated by a population which is distinct from that which produces GVHD. These results may have implications for clinical BM transplantation.     

Host NKT cells can prevent graft-versus-host disease and permit graft antitumor activity after bone marrow transplantation

Allogeneic bone marrow transplantation is a curative treatment for leukemia and lymphoma, but graft-vs-host disease (GVHD) remains a major complication. Using a GVHD protective nonmyeloablative conditioning regimen of total lymphoid irradiation and antithymocyte serum (TLI/ATS) in mice that has been recently adapted to clinical studies, we show that regulatory host NKT cells prevent the expansion and tissue inflammation induced by donor T cells, but allow retention of the killing activity of donor T cells against the BCL1 B cell lymphoma. Whereas wild-type hosts given transplants from wild-type donors were protected against progressive tumor growth and lethal GVHD, NKT cell-deficient CD1d-/- and Jalpha-18-/- host mice given wild-type transplants cleared the tumor cells but died of GVHD. In contrast, wild-type hosts given transplants from CD8-/- or perforin-/- donors had progressive tumor growth without GVHD. Injection of host-type NKT cells into Jalpha-18-/- host mice conditioned with TLI/ATS markedly reduced the early expansion and colon injury induced by donor T cells. In conclusion, after TLI/ATS host conditioning and allogeneic bone marrow transplantation, host NKT cells can separate the proinflammatory and tumor cytolytic functions of donor T cells.     

Mechanism of protection from graft-versus-host disease mortality by IL-2. III. Early reductions in donor T cell subsets and expansion of a CD3+CD4-CD8- cell population.

Reducing the graft-vs-host disease (GVHD)-promoting capacity of allogeneic T cells while maintaining alloengraftment and graft-vs-leukemia effects remains an important but elusive goal in clinical bone marrow transplantation (BMT). We have recently demonstrated that a short course of high dose IL-2 administered at the time of BMT has a powerful protective effect against GVHD mortality in mice. This short course of IL-2 is able to protect mice from both acute and chronic GVHD without sacrificing alloengraftment or graft-vs-leukemia effects of allogeneic T cells. Because the early administration of IL-2 seems to be crucial for this effect, we have studied the early lymphoid repopulation events after lethal irradiation and allogeneic BMT. These studies show that there are consistent delays in splenic repopulation by allogeneic cells after BMT in IL-2-treated animals compared with their untreated cohorts. Even greater percent reductions were seen in donor splenic T cell populations in the first few days after BMT in IL-2-treated animals. Splenic cells with the CD3+CD4-CD8- phenotype were increased in IL-2 treated animals at days 3 and 4 after BMT. This phenotype resembles that of bone marrow-derived cells which have been previously shown to inhibit GVHD, suggesting a possible mechanism for the protective effect of IL-2.     

Donor APCs are required for maximal GVHD but not for GVL

Graft-versus-host disease (GVHD) is a major source of morbidity in allogenic stem cell transplantation. We previously showed that recipient antigen-presenting cells (APCs) are required for CD8-dependent GVHD in a mouse model across only minor histocompatibility antigens (minor H antigens). However, these studies did not address the function of donor-derived APCs after GVHD is initiated. Here we show that GVHD develops in recipients of donor major histocompatibility complex class I-deficient (MHC I(-)) bone marrow. Thus, after initial priming, CD8 cells caused GVHD without a further requirement for hematopoietic APCs, indicating that host APCs are necessary and sufficient for GHVD. Nonetheless, GVHD was less severe in recipients of MHC I(-) bone marrow. Therefore, once initiated, GVHD is intensified by donor-derived cells, most probably donor APCs cross-priming alloreactive CD8 cells. Nevertheless, donor APCs were not required for CD8-mediated graft-versus-leukemia (GVL) against a mouse model of chronic-phase chronic myelogenous leukemia. These studies identify donor APCs as a new target for treating GVHD, which may preserve GVL.     

Acute Graft-Versus-Host Disease of the Kidney in Allogeneic Rat Bone Marrow Transplantation

Allogeneic hematopoietic cell or bone marrow transplantation (BMT) causes graft-versus-host-disease (GVHD). However, the involvement of the kidney in acute GVHD is not well-understood. Acute GVHD was induced in Lewis rats (RT1^l) by transplantation of Dark Agouti (DA) rat (RT1^a) bone marrow cells (6.0×10⁷ cells) without immunosuppression after lethal irradiation (10 Gy). We examined the impact of acute GVHD on the kidney in allogeneic BMT rats and compared them with those in Lewis-to-Lewis syngeneic BMT control and non-BMT control rats. In syngeneic BMT and non-BMT control rats, acute GVHD did not develop by day 28. In allogeneic BMT rats, severe acute GVHD developed at 21–28 days after BMT in the skin, intestine, and liver with decreased body weight (>20%), skin rush, diarrhea, and liver dysfunction. In the kidney, infiltration of donor-type leukocytes was by day 28. Mild inflammation characterized by infiltration of CD3+ T-cells, including CD8+ T-cells and CD4+ T-cells, and CD68+ macrophages to the interstitium around the small arteries was noted. During moderate to severe inflammation, these infiltrating cells expanded into the peritubular interstitium with peritubular capillaritis, tubulitis, acute glomerulitis, and endarteritis. Renal dysfunction also developed, and the serum blood urea nitrogen (33.9±4.7 mg/dL) and urinary N-acetyl-β-D-glucosaminidase (NAG: 31.5±15.5 U/L) levels increased. No immunoglobulin and complement deposition was detected in the kidney. In conclusion, the kidney was a primary target organ of acute GVHD after BMT. Acute GVHD of the kidney was characterized by increased levels of urinary NAG and cell-mediated injury to the renal microvasculature and renal tubules.     

Effect of graft-versus-host disease (GVHD) on host hematopoietic progenitor cells is mediated by Fas-Fas ligand interactions but this does not explain the effect of GVHD on donor cells.

The acute graft-versus-host disease (GVHD) generated in BDF1 mice by the injection of spleen cells from the C57BL/6 parental strain induces a direct cell-mediated attack on host lymphohematopoietic populations, resulting in the reconstitution of the host with donor hematopoietic stem cells. We examined the effect of GVHD on the donor and host hematopoiesis in parental-induced acute GVHD. The bone marrow was hypoplastic and the number of hematopoietic progenitor cells significantly decreased at 4 weeks after GVHD induction. However, extramedullary splenic hematopoiesis was present and the number of hematopoietic progenitor cells in the spleen significantly increased at this time. Fas expression on the host spleen cells and bone marrow cells significantly increased during weeks 2 to 8 of GVHD. Host cell incubation with anti-Fas Ab induced apoptosis, and the number of hematopoietic progenitor cells decreased during these weeks. A significant correlation between the augmented Fas expression on host bone marrow cells and the decreased number of host bone marrow cells by acute GVHD was observed. Furthermore, the injection of Fas ligand (FasL)-deficient B6/gld spleen cells failed to affect host bone marrow cells. Although Fas expression on repopulating donor cells also increased, Fas-induced apoptosis by the repopulating donor cells was not remarkable until 12 weeks, when more than 90% of the cells were donor cells. The number of hematopoietic progenitor cells in the bone marrow and the spleen by the repopulating donor cells, however, decreased over an extended time during acute GVHD. This suggests that Fas-FasL interactions may regulate suppression of host hematopoietic cells but not of donor hematopoietic cells. Hematopoietic dysfunctions caused by the reconstituted donor cells are independent to Fas-FasL interactions and persisted for a long time during parental-induced acute GVHD.     

Critical role of TLR9 in acute graft-versus-host disease

Graft-vs-host disease (GVHD) is a major complication after allogeneic bone marrow transplantation. Different studies have demonstrated that intestinal bacterial breakdown products and loss of gastrointestinal tract integrity, both induced by conditioning regiments, are critical in the pathogenesis of acute GVHD. Using C57BL/6 knockout mice, we evaluated the role of TLR4 and TLR9, which recognize bacterial LPS and DNA, respectively, in the GVHD associated with allogeneic bone marrow transplantation. When myeloablative-irradiated TLR9 knockout (TLR9(-/-)) mice were used as graft recipients, survival and clinical score of acute GVHD were improved as compared with the wild-type recipient mice (18/30 vs 1/31 mice still alive at day 70 in a total of four experiments); while no differences were observed using recipient TLR4 knockout (TLR4(-/-)) mice. The reduced mortality and morbidity in TLR9(-/-) mice related with reduced stimulatory activity of TLR9(-/-) spleen APCs after conditioning and reduced proliferation of allogeneic donor T cells. Experiments using TLR9(+/+) into TLR9(-/-) and TLR9(-/-) into TLR9(+/+) chimeric mice as recipients indicated a critical role for nonhematopoietic TLR9(+/+) cells interacting with bacterial breakdown products released in myeloablated mice. Altogether these data reveal a novel important role of TLR9 in GVHD, a finding that might provide tools to reduce this complication of allogeneic transplantation.     

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.     

Alterations of glycosphingolipid-based blood group antigen expression on erythrocytes and in plasma studied on consecutive samples after a blood group O to A bone marrow transplantation.

A blood group A1Le(a-b+) individual with chronic myeloid leukaemia had received a bone marrow graft from an HLA-identical OLe(a+b-) donor. Twelve months after bone marrow transplantation (BMT), the red blood cells of the patient became agglutinable with anti-A blood group reagents. To elucidate whether the blood group A antigen expression was of plasma or of bone marrow origin, total non-acid glycosphingolipid fractions were prepared from red blood cells and plasma collected 17 months after BMT, and from plasma collected 13, 15 and 19 weeks after BMT. The glycolipid fractions were analysed by thin-layer chromatography and immunostained with monoclonal A-antibodies, and permethylated and permethylated-reduced derivatives of selected plasma samples were analysed by mass spectrometry. The results strongly indicate the presence of host bone marrow-produced blood group A red blood cells. Furthermore, the presence of a blood group H active pentaglycosylceramide type 1 (H-5-1) (Table I), characteristic for an OLe(a-b-) secretor, was seen in plasma 3-4 weeks before clinical chronic graft versus host disease (GVHD). After treatment of chronic GVHD, this expression disappeared. The blood group ALeb (A-7-1) antigen produced by the recipient seems to be present and to increase with time in all plasma samples. This also seems to be the case for the Leb and A-6-1 antigens.     

Development of immunity in human severe primary T cell deficiency following haploidentical bone marrow stem cell transplantation

Recent advances in the prevention of graft-vs-host disease (GVHD) have allowed the use of haploidentical bone marrow cells for correction of lethal genetic defects of the immune system. Sequential analyses of blood lymphocyte phenotypes and functions were done before and after transplantation of haploidentical marrow stem cells into 17 infants with severe primary T cell deficiencies. The marrow was depleted of post-thymic T cells and most other mature marrow cells by soy lectin agglutination and sheep erythrocyte rosetting. The studies were performed to define the time course and extent of appearance of immune function, and to identify factors leading to resistance to engraftment. No pretransplant immunosuppression was used. T cell function was detected between 34 and 287 days after transplantation, but a sharp rise usually occurred between 84 and 115 days, and normal function was reached between 113 and 210 days. Fifteen of the patients are alive from 6 to 41 mo post-transplantation, 12 have improved or have normal T lymphocyte function, and nine have proven T cell chimerism. Increased immunoglobulins of several isotypes have been noted in 11 patients and specific antibodies in seven patients, although B cell chimerism has been detected in only one patient. B cell function required 2 to 2.5 yr for normalization. No GVHD occurred in 14 patients, and the other three had only transient mild skin rashes. Two patients died of viral infections. Failure to engraft was correlated with some pre-transplant lymphocyte responses to mitogens and allogeneic cells (three cases), but not with the presence of pre-transplant natural killer cell function (five cases) nor with the presence of purine salvage pathway enzyme deficiencies (four cases). The latter, however, was associated with poor lymphoid function in two patients. These studies indicate that the thymic microenvironment of most infants with severe combined immunodeficiency disease is capable of differentiating donor stem cells to mature and functioning T lymphocytes which can cooperate with apparently normal host B cells for antibody production.     

CD4+CD25+ regulatory T cells preserve graft-versus-tumor activity while inhibiting graft-versus-host disease after bone marrow transplantation

Mature donor T cells cause graft-versus-host disease (GVHD), but they are also the main mediators of the beneficial graft-versus-tumor (GVT) activity of allogeneic bone marrow transplantation. Suppression of GVHD with maintenance of GVT activity is a desirable outcome for clinical transplantation. We have previously shown that donor-derived CD4+CD25+ regulatory T cells inhibit lethal GVHD after allogeneic bone marrow transplantation across major histocompatibility complex (MHC) class I and II barriers in mice. Here we demonstrate that in host mice with leukemia and lymphoma, CD4+CD25+ regulatory T cells suppress the early expansion of alloreactive donor T cells, their interleukin-2-receptor (IL-2R) alpha-chain expression and their capacity to induce GVHD without abrogating their GVT effector function, mediated primarily by the perforin lysis pathway. Thus, CD4+CD25+ T cells are potent regulatory cells that can separate GVHD from GVT activity mediated by conventional donor T cells.     

The p55 TNF-alpha receptor plays a critical role in T cell alloreactivity

TNF-alpha is known to be an important mediator of tissue damage during allograft rejection and graft-vs-host disease (GVHD), but its role in supporting T cell responses to allogeneic Ags is unclear. We have studied this question by comparing normal mice with those lacking the p55 (p55 TNFR-/-) or p75 (p75 TNFR-/-) TNF-alpha receptors as donors in well-defined bone marrow transplant (BMT) models. Recipients of p55 TNFR-/- cells had significantly reduced mortality and morbidity from GVHD compared with the other two sources of T cells. In vitro, T cells lacking the p55 (but not the p75) TNF-alpha receptor exhibited decreased proliferation and production of Th1 cytokines in MLC. This defect was only partially restored by exogenous IL-2 and affected both CD4+ and CD8+ populations. CD8+ p55 TNFR-/- proliferation was impaired independently of IL-2 whereas CTL effector function was impaired in an IL-2-dependent fashion. Inhibition of TNF-alpha with TNFR:Fc in primary MLC also impaired the proliferation and Th1 differentiation of wild-type T cells. BMT mixing experiments demonstrated that the reduced ability of p55 TNFR-/- donor cells to induce GVHD was due to the absence of the p55 TNFR on T cells rather than bone marrow cells. These data highlight the importance of TNF-alpha in alloreactive T cell responses and suggest that inhibition of the T cell p55 TNF-alpha receptor may provide an additional useful therapeutic maneuver to inhibit alloreactive T cell responses following bone marrow and solid organ transplantation.     

Bone marrow transplantation for the treatment of leukaemia-results of the Munich Cooperative Group

This report summarizes the results of marrow transplantation from HLA-identical siblings and syngeneic twins for treatment of acute myelogenous leukaemia, chronic myelogenous leukaemia, acute lymphoblastic and undifferentiated leukaemia from 1975 until December 1986. Three conditioning regimens and treatment of the marrow graft in vitro with absorbed antithymocyte globulin or the monoclonal antibody "Campath 1" for prophylaxis of graft-versus-host disease (GVHD) have been studied and analyzed retrospectively. The regimen of total body irradiation in large fractions of 4 Gy and of cyclosphosphamide (200 mg/kg) has achieved the most favorable results. Inactivation of T-cells by treatment of the marrow "in vitro" has decreased the severity of GVHD without improving survival. The antileukaemic effect of the graft may be important for control of the disease and may be improved by better immunosuppression of the recipient.     

Deposition of IgM and complement at the dermoepidermal junction in acute and chronic cutaneous graft-vs-host disease in man.

The presence of cutaneous immunoglobulin and complement was investigated in 88 patients with and without graft-vs-host disease (GVHD) after transplantation of bone marrow from HLA identical siblings for the treatment of acute leukemia or aplastic anemia. For comparison, skin biopsies from the patients obtained before transplantation, from 58 healthy individuals (mostly marrow donors) and from four syngeneic marrow recipients were studied. A direct immunfluorescent staining technique was used. Dermo-epidermal IgM deposits were found in 11% of healthy individuals and patients before grafting but were present in 86% of patients with chronic and 39% of patients with acute GVHD. Patients with allogeneic grafts who never had GVHD or who had recovered from it and patients with syngeneic grafts showed findings not different from those in healthy individuals. Findings similar to those with IgM, although less striking, were made for C3, i.e., patients who had chronic or acute GVHD had a high incidence and intensity of C3 deposits at the dermo-epidermal junction. This observation raises the possibility that humoral immunity is involved in the development of GVHD.     

Sex chromatin analysis of lymphocytes invading host organs in transfusion-associated graft-versus-host disease

We report a method of sex chromatin analysis of lymphocytes separated from host organs in transfusion-associated graft-versus-host disease (GVHD), which enabled the demonstration of invasion by donor lymphocytes. The lymphocytes examined were separated from deparaffinized tissue blocks of skin, spleen and bone marrow from two female patients with transfusion-associated GVHD by incubation in 0.5% pepsin. The tissues had been removed at autopsy and fixed in formalin. Sex chromatin analysis was performed by fluorescence microscopy on separated lymphocytes stained with 0.005% quinacrine dihydrochloride. By this means Y chromatin-positive (i.e. male) lymphocytes were demonstrated in the skin, spleen and bone marrow of both female patients.     

Development of tolerogenic strategies in the clinic

The study of tolerance in the clinic can be divided into three areas: (i) focused evaluation of existing tolerant transplant recipients as to their mechanism of tolerance; (ii) prospective tolerance trials, such as combined bone marrow and kidney transplantation as well as T cell depletion followed by subsequent weaning of immunosuppression; and (iii) immunologic assays to assess the likelihood of rejection or tolerance. Frankly, a very small number of patients have been transplanted with the intention of removing all immunosuppressive therapy, but several clinical trials with this aim are currently in progress, largely sponsored by the Immune Tolerance Network, a joint venture between the National Institutes of Health and the Juvenile Diabetes Research Foundation. Similarly, a reliable assay to assess tolerance has not yet been developed but a variety of approaches towards assessing rejection, and in some cases tolerance, are being developed. It would be accurate to state that many of the experimental and preclinical approaches to the induction of tolerance have resulted in better immunosuppression for human transplantation, but reliable tolerance strategies in humans have not yet been achieved. Combined bone marrow and kidney transplantation may be considered as one exception to this, but such a strategy is not generally applicable to the vast majority of solid organ transplant recipients. This review will summarize efforts to date, particularly focusing on kidney transplantation.     

Sex chromatin analysis of lymphocytes invading host organs in transfusion-associated graft-versus-host disease

We report a method of sex chromatin analysis of lymphocytes separated from host organs in transfusion-associated graft-versus-host disease (GVHD), which enabled the demonstration of invasion by donor lymphocytes. The lymphocytes examined were separated from deparaffinized tissue blocks of skin, spleen and bone marrow from two female patients with transfusion-associated GVHD by incubation in 0.5% pepsin. The tissues had been removed at autopsy and fixed in formalin. Sex chromatin analysis was performed by fluorescence microscopy on separated lymphocytes stained with 0.005% quinacrine dihydrochloride. By this means Y chromatin-positive (i.e. male) lymphocytes were demonstrated in the skin, spleen and bone marrow of both female patients.