In vitro maturation of immature thymocytes into immunocompetent T cells in the absence of direct thymic influence.

Peanut lectin (PNL) binds to a majority of mouse thymocytes (Thc) in suspension. By using cell affinity chromatography on a column of anti-PNL antibody, Thc populations at least 96 percent pure in PNL + or - cells, as judged by immunofluorescence, were obtained. PNL(+) cells are rich in Thy 1 and poor in H(2) antigens, cortisone sensitive, unresponsive to phytohemagglutinin (PHA), and immunologically incompetent, as judged by mixed lymphocyte reaction, popliteal lymph node graft-versus-host assay, and by testing helper activity in a primary in vitro antibody response to sheep erythrocytes; the converse is true of PNL(-) cells. Thus, PNL(+) and (-) cells appear to correspond to cortical and medullary Thc, respectively, as previously suggested. In culture, PNL(+) Thc show poor viability and a weak proliferative response to concanavalin A (Con A), except when supernate (SUP) of 24 h Con A stimulated lymph node lymphocyte cultures, or irradiated lymph node cells, are added, in which cases a strong proliferative response to the mitogen is observed. A variety of control experiments showed that the proliferating cells did not result from preferential stimulation of a few contaminating PNL(-) Thc present in the PNL(+) Thc cultures. The blasts resulting from PNL(+) Thc proliferation display mitogen-induced cytotoxicity, and give rise to a population of medium-sized lymphocytes, mostly PNL(-), poor in Thy 1 and rich in H(2) antigens, PHA responsive, and immunologically competent in the above-mentioned assays. Fresh PNL(+) Thc responded in mixed lymphocyte reaction in the presence of SUP (lectin depleted) and since incubation in SUP alone did not confer reactivity on PNL(+) Thc, it appears therefore that (a) immature Thc possess alloantigen and mitogen-specific surface receptors but lack the capacity to respond by proliferation to receptor triggering without the help of extracellular factor(s) released by mature lymphoid cells stimulated by mitogens (b) cell division is associated with the acquisition of immunological responsiveness, characteristic of mature T lymphocytes. The implications of these findings for the ontogenesis of thymus-derived lymphocytes, and for the possible traffic of Thc within and from the thymus, are discussed.

Post-thymic T lymphocyte maturation during ontogenesis.

Peripheral T lymphocytes from newborn (4-6-d-old) mice, isolated from the spleen or lymph nodes, show phenotypic features of immature cortical thymocytes, such as high frequencies of proliferating cells and of peanut lectin-binding cells. These are features of peripheral T cells of recent thymic origin, as shown by in situ labeling of thymocytes and subsequent observation of the migrants to the spleen, which were mainly peanut lectin-binding cells. The function of newborn peripheral T cells was compared, on a per T cell basis, with that of thymocytes and of fully mature peripheral T cells of the adult, using preparations of newborn lymph node cells containing approximately 80% of T lymphocytes. They were strikingly (about 10-fold) less competent than adult T cells in their phytohemagglutinin responsiveness, their capacities to induce a graft vs. host reaction, to proliferate in the mixed lymphocyte reaction, and to help B lymphocytes in a humoral response in vivo and in vitro. In contrast, newborn T lymphocytes were comparable to those of adults in their capacity to generate cytotoxic T lymphocytes. No suppressive effect of newborn T lymphocytes could be demonstrated in several of these assays. These results argue for an asynchronous maturation of two T cell subsets during ontogeny and demonstrate that at least some T lymphocytes leave the thymus as immature T cells resembling cortical thymocytes and further mature at the periphery. Investigation of mice submitted to thymectomy of 5 d of age showed that these incompetent post-thymic T lymphocytes are capable of considerable expansion and maturation in the peripheral lymphoid organs in the absence of a thymic influence.

Functional polymorphism of each of the two HLA-DR beta chain loci demonstrated with antigen-specific DR3- and DRw52-restricted T cell clones.

HLA-DR3- and HLA-DRw52-associated functional polymorphism was investigated with selected tetanus toxoid (TT)-specific T cell clones. We have shown earlier that HLA-DR antigens are encoded by two distinct loci, DR beta I and DR beta III. The alloantigenic determinant(s) defined by the serological HLA-DR3 specificity map to the former, while the supratypic HLA-DRw52 determinants map to DR beta III. Furthermore, we have recently recognized by DNA sequencing three alleles of HLA-DRw52 at locus DR beta III, referred to as 52 a, b, and c. Our objective was to correlate the pattern of T cell restriction with the gene products of individual DR beta chain loci and with the three newly described alleles of locus DR beta III. Among the selected T cell clones, 5 reacted exclusively when TT was presented by HLA-DR3+ APCs (TT-DR3-APC). In contrast, two T cell clones were stimulated by TT-DRw52-APC. More specifically, these two T cell clones (Clones 10 and 16) were stimulated by different subsets of TT-DRw52-APC. Clone 16 responded to some DR3 and TT-DRw6-APC, while clone 10 was stimulated by other TT-DR3 and TT-DRw6, and all TT-DR5-APC. This same pattern of DRw52 restriction was found in panel, as well as in family studies. Because this suggested a correlation with the pattern of DRw52 polymorphism observed earlier by DNA sequencing and oligonucleotide hybridization, the APC used in these experiments were typed for the 52 a, b, and c alleles of locus DR beta III by allele-specific oligonucleotide probes. This distribution overlapped exactly with the stimulation pattern defined by the T cell clones. Clone 16 responded to TT-52a-APC, clone 10 to TT-52b-APC, and both clones to a TT-52c-APC. The response of the T cell clones was inhibited differentially by mAbs to DR. Raising TT concentration, or increasing HLA-class II expression with INF-gamma both affected the magnitude of response of the TT-specific clones but did not modify their specificities. These results demonstrate that a restriction specificity can be attributed to the DR beta III locus and illustrate the functional relevance of the polymorphism observed at this locus. This is of special interest in view of the striking difference in the pattern of structural diversity among alleles of DR beta I and DR beta III.

Correlation of structure with T cell responses of the three members of the HLA-DRw52 allelic series.

A third allele at the DRB3 locus, DRw52c, represents an intermediate sequence between DRw52a and DRw52b and may have arisen by a gene conversion-like event. The recognition of cells bearing these molecules by a number of alloreactive and antigen-specific DR-restricted T cell clones was analyzed. On the basis of a theoretical model of HLA class II structure, distinct amino acid clusters have been identified as motifs controlling TCR recognition. These are located both in the cleft and in the alpha-helical edge of the MHC class II recognition platform. Motifs shared between two alleles may restrict public T cell clones.

"Contractile interstitial cells" in pulmonary alveolar septa: a possible regulator of ventilation-perfusion ratio? Ultrastructural, immunofluorescence, and in vitro studies.

In the lungs of healthy rats, humans, lambs, and monkeys, about 50% of the alveolar interstitial cells-resembling fibroblasts-contain bundles of fibrils measuring 30-80 A in diameter. Immunofluorescence studies on frozen sections of rat lung demonstrate that many interstitial cells bind sera containing antiactin antibodies. On account of these two sets of findings and our additional in vitro studies suggesting alveolar tissue contraction due to hypoxia or epinephrine, we postulate that the alveolar septa contain contractile cells different from that of smooth muscle. For these cells we propose the name of "contractile interstitial cells." Such cells lie within the thick portion of the air-blood barrier and around the pre- or postcapillary vessels. Hence it is possible that they play a role in the autoregulation of ventilation/perfusion (V/Q) ratio, particularly in hypoxic pulmonary hypertension. These findings, demonstrating a contractile system other than bronchial and arterial smooth muscle, suggest that the alveolus should no more be conceived as a passive "organ."

Quantity and nature of residual bone marrow T cells after treatment of the marrow with Campath-1.

Precise characterization of T-cell depletion in marrows used for transplantation is necessary for the evaluation of their potential contribution to engraftment and to graft-versus-host disease. A limiting dilution culture method that allows the determination of small numbers of bone marrow T cells is described. It can detect less than one T cell in 10(4) cells. Bone marrow T-cell depletion with the rat monoclonal antibody Campath-1 and fresh human complement results in a median decrease of 1.7 log (range, 1.6-2.1 log) of marrow T cells. A 2.7 to 3.3-log reduction is obtained when peripheral blood T cells are treated. A second incubation with fresh complement removes additional T cells from peripheral blood and from marrow samples, indicating the importance of bystander marrow cells to complement activity. The assay system described also allows the phenotypic study of responding cells growing in culture. These studies demonstrate that, after treatment with Campath-1 plus complement, no T-subset imbalance occurs. Furthermore, the T cells in these cultures are derived from mature T cells contained in the samples.

Rapid purification of peanut agglutinin by sialic acid-less fetuin-sepharose column.

A simple purification of peanut agglutinin (PNL) with a sialic acid-less fetuin-Sepharose column is described. Immunofluorescence studies show PNL to be a suitable surface marker of potential use in the study of subpopulations of thymocytes and T cells.

Alloreactive T cell responses between HLA-identical siblings. Detection of anti-minor histocompatibility T cell clones induced in vivo.

Cytotoxic T cell (CTL) clones were isolated from the peripheral blood of a patient 13 days following marrow transplantation from her HLA-identical brother. Nineteen of the clones were specifically reactive with host--but not donor--cells, but one clone was reactive with donor but not host cells. Family studies using the 19 clones showed reactivity patterns consistent with three non-HLA, minor histocompatibility antigens (minor-HA). The frequency of reactivity on a large panel of unrelated cells indicated a relatively limited degree of polymorphism. Each of the clones was restricted in its activity by a class I epitope common to HLA-B7, B27, and B40. These data demonstrate that by clonal analysis it is possible to identify in vivo antidonor and antihost alloreactive CTL clones directed against HLA determinants following marrow transplantation.

Effect of factors inhibiting HLA-DR antibodies before transplantation on kidney graft survival.

Blood transfusions administered before renal allografts are known to enhance graft survival. Among alternative hypotheses proposed to explain this effect, one of the most attractive is the possible induction of antiidiotypic antibodies directed against the specific antigen-binding site of donor-specific antibodies. In order to determine if such blocking antibodies are generated after blood transfusions, serial serum samples obtained before transplantation from 44 kidney recipients were analyzed for the development of HLA-DR alloantisera inhibitory activity by a microcytotoxicity inhibition assay. A significant correlation was found between the presence of inhibitory factors before transplantation and prolonged graft survival. However a clear relation between the development of inhibitory factors and the administration of transfusions could not be established. In addition the sera of 36 patients were studied for the presence of circulating immune complexes (CIC) before grafting. The presence of CIC was clearly associated with that of inhibitory factors, and with a prolonged graft survival. Thus these studies provide support for the development of blocking (possibly antiidiotypic) antibodies to anti-MHC in human renal graft recipients.

Change in functional phenotype of cloned human alloreactive cytolytic T cells.

Alloreactive T cell clones primed in vivo were tested for the expression of T cell differentiation antigens CD2, CD3, CD4, and CD8. Each of 29 different clones were found to express CD2 and CD3, but were variable in their expression of CD4 (7 positive clones) and CD8 (15 positive clones). Six clones were positive for both CD4 and CD8. One of the 29 clones expressed neither CD4 or CD8. Over a period of 12-18 weeks of culture, these clones began to lose their alloreactivity but acquired NK-like activity. By changing the concentration of TCGF, the "allo" and "NK-like" lytic activities could be modulated. After 18 weeks of culture, these clones lost their alloreactive specificity, but not their NK activity. The expression of surface markers was unchanged. CD2 and CD3 molecules were determined to play a role in both the alloreactive and NK activity of these clones.

An HLA-DRB alpha-helix motif shared by DR11 and DR8 alleles is implicated in the pluriallelic restriction of peptide-specific T-cell lines.

The T-cell recognition of HLA-DR-peptide complexes is generally restricted by the polymorphism of the DRB molecules but pluriallelic restriction has been described. The molecular basis of restriction and promiscuity of such peptide-specific responses is poorly understood. We isolated a panel of T-cell lines specific for the tetanus toxin peptide p2 (TT830-843) exhibiting pluriallelic restriction by DR11 and DR8 alleles. Fine restriction specificity of the T-cell lines was examined in functional assays against DR oligotyped APCs expressing different variants of DR11 and DR8 alleles. Our results show that (a) polymorphisms between serologically related alleles are relevant in terms of restriction of the peptide-specific T-cell response; in some instances, a single amino acid substitution can determine the restriction of a T-cell line; (b) different patterns of restriction are not the result of specific differences in DR-p2 binding as p2 peptide binds to all DR11 and DR8 alleles tested (DRB1* 1101, -1102, -1103, -1104, 110X, -0801, -0802, -0803, and -0806); and (c) pluriallelic restriction of the peptide-specific T-cell response correlates with the presence of a DRB1 alpha-helix motif (67-71-86) shared by some DR11 and DR8 alleles. Possible implications of pluriallelic restriction of peptide-specific T-cell response in autoimmune disorders associated with DR11 and DR8 are discussed.

Marrow transplantation for leukemia following fractionated total body irradiation. A comparative trial of methotrexate and cyclosporine.

Fifty-six patients, 30-47 yr of age, with leukemia in relapse received allogeneic marrow transplants from HLA-identical siblings. All patients were treated with cyclophosphamide (120 mg/kg) and 7 daily fractions of 2.25 Gy of total body irradiation (TBI) for seven consecutive days. Nine patients (16%) are currently alive and free of disease 324-845 days from transplantation. The actuarial relapse and survival rates at 2 yr were 56% and 9.5% respectively. These data were not remarkably different from those in previous studies using 10 Gy of TBI administered as a single dose. Thirty patients were randomized to receive methotrexate (MTX) and 26 to receive cyclosporine (CSP) as postgrafting prophylaxis for acute graft-versus-host disease (GVHD). The probability of developing significant acute GVHD by day 100 post-transplant was 71% for patients in the MTX group and 45% for patients in the CSP group (p less than 0.05). The probability of relapse was 37% for patients in the MTX group and 70% for patients in the CSP group (p less than 0.05). Transplant-related deaths were more frequent in the MTX group and leukemic deaths were more frequent in the CSP group although this may have been related to an uneven distribution of high-risk patients. Long-term disease-free survival was comparable. Patients in the MTX group had more severe mucositis, more alveolar pneumonias and possibly more deaths due to complications of acute and chronic GVHD. Patients in the CSP group had a higher incidence of hypertension, neurological complications and renal dysfunction.

Metastatic transitional cell carcinoma of the urinary bladder presenting as a mandibular gingival swelling.

Oral cavity metastases mostly originate from the breasts, lungs, or kidneys. Transitional cell carcinoma (TCC), the most frequent malignant tumor of the urinary bladder, rarely metastasizes to the jaws. To the best of our knowledge, only 8 cases of bladder carcinoma have been reported in the English literature to metastasize to the jawbones. A new case of mandibular metastasis of urinary bladder TCC with extension to the gingiva is presented in a 64-year-old white man. The patient was referred for a periodontal infection of the upper right first molar. The clinical examination also showed a gingival swelling located in the lower left premolar region with a hypoasthesia of the left side of the lower lip. The gingival mass was biopsied, and the microscopy showed a mandibular metastatic TCC of the urinary bladder extending to the gingiva. Periodontists should be aware that, although gingival metastases are rare, when they occur they may mimic other local benign pathological conditions.

Cell cultures and detection of minimal residual disease: significance and limitations.

This introductory article puts into perspective the usefulness of cell cultures in the area of detection of minimal residual disease. We stress the role of single cell cultures in selective media as a means to increase the number of residual tumor cells, thereby facilitating their detection by other methods. Prior treatment may affect the growth potential of cells for a very long time. Quantitation of the event to be detected is thus highly dependent on the characteristics of the cells which are also influenced by their growth environment. This environment may be optimized by the judicious use of growth factors in defined media.

Administration of recombinant interleukin 2 to mice enhances production of hemopoietic and natural killer cells.

Seven days of continuous perfusion of mice with human recombinant interleukin 2 (rIL 2) (approximately 3 X 10(4) U/day) increased the percentage of large mononuclear leukocytes (LML) among bone marrow, spleen, lymph node cells and liver interstitial cells (LIC). An increase in the lymphokine-activated killer (LAK) activity was evident in these organs. The greatest increase in the number of LML and in the LAK activity was observed among the liver interstitial cells (about 500-fold increase). The LML were nonphagocytic, Thy-1+, sIg-, Ly 2+, L3T4- and asialo Gm1+. Perfusion of athymic nude mice, or of thymectomized, irradiated radiation chimera, showed that the Thy-1+, LAK+ LML were the thymus and T lymphocyte-independent progeny of Thy-1- marrow precursors. The LML had no T cell function in a graft-vs.-host reactivity assay, neither did they have an inhibitory effect on T lymphocyte function in vivo. rIL 2 perfusion did not significantly affect the medullary hemopoiesis but did strongly enhance the extramedullary hemopoiesis, particularly within the interstice of the liver: the number of erythroid and myeloid cell was increased as well as the number of colony-forming units per spleen and colony-forming units per culture for various lineages (20-50-fold increment). These results show that in vivo, rIL 2 has a global enhancing effect on hemopoiesis together with a more selective influence on the production of LML.

[Quantitative determination of human bone marrow T cells following in vitro depletion by Campath-1 monoclonal antibody]. / Détermination quantitative des cellules T humaines médullaires après déplétion in vitro par l'anticorps monoclonal Campath-1.

Bone marrow T cell depletion is an effective graft vs host disease prophylaxis in allogeneic bone marrow transplantation. Standard methods of identifying T cells are not sensitive enough to determine small numbers of residual T cells following T cell depletion. A rapid and sensitive method, using limiting dilution culture technology and radionucleotide labelling and allowing the detection of 1/10(4)-1/10(5) residual T cells, has been developed. Its usefulness in the evaluation of the number of T cells after monoclonal antibody (Campath-1) + autologous complement marrow treatment is demonstrated.

[In vivo detection of alloreactive T cells of host origin after rejection of HLA-identical allogeneic bone marrow transplant]. / Détection in vivo de cellules T alloréactives d'origine de l'hôte après rejet de greffe de moelle osseuse allogénique HLA-identique.

Rejection and graft vs host disease after HLA-identical allogeneic bone marrow transplantation are probably due to in-vivo T-cell reactivity against non-MHC antigens. Peripheral blood lymphocytes from a patient transplanted for aplastic anemia have been obtained during graft failure and tested for their alloreactivity towards unrelated and marrow donor lymphocytes. Their caryotype was determined. Our results demonstrate (1) alloreactive T cells of host origin and (2) the presence of donor-specific host cells in vivo, providing evidence for rejection following in vivo sensitization to non-MHC antigens.