Xenogeneic transplantation of human spermatogonia

In the last 3 years, several studies have shown that xenogeneic transplantation of rodent spermatogonia is feasible. The treatment of infertile patients with spermatogenic arrest using the injection of immature germ cells has yielded only poor results. We attempted to establish a complete spermatogenetic line in the testes of mutant aspermatogenic (W/Wv) and severe combined immunodeficient mice (SCID) transplanted with germ cells from azoospermic men. Spermatogenic cells were obtained from testicular biopsy specimens of men (average age of 34.3 +/- 9 years) undergoing infertility treatment because of obstructive and non-obstructive azoospermia. Testicular tissue was digested with collagenase to promote separation of individual spermatogenic cells. The germ cells were injected into mouse testicular seminiferous tubules using a microneedle (40 microm inner diameter) on a 10 ml syringe. To assess the penetration of the cell suspension into the tubules, trypan blue was used as an indicator. Mice were maintained for 50 to 150 days to allow time for germ cell colonisation and development prior to them being killed. Testes were then fixed for histological examination and approximately 100 cross-sectioned tubules were examined for human spermatogenic cells. A total of 26 testicular cell samples, 16 frozen and 10 fresh, were obtained from 24 men. The origin of the azoospermia was obstructive (OA) in 16 patients and non-obstructive (NOA) in 8 patients. The concentration of spermatogenic cells in the OA group was 6.6 x 10(6) cells/ml, and 1.3 x 10(6) cells/ml in the NOA group (p < 0.01). The different spermatogenic cell types were distributed equally in the OA samples, ranging from spermatogenia to fully developed spermatozoa, but in the NOA group the majority of cells were spermatogonia and spermatocytes. A total of 23 testes from 14 W/Wv mice and 24 testes from 12 SCID mice were injected successfully, as judged by the presence of spermatogenic cells in histological sections of testes removed immediately after the injection. However, sections from the remaining testes examined up to 150 days after injection showed tubules lined with Sertoli cells and xenogeneic germ cells were not found. The reason why the two strains of mouse used as recipients did not allow the implantation of human germ cells is probably due to interspecies specificity involving non-compatible cell adhesion molecules and/or immunological rejection.     

Cytochemical reactivity of T mu lymphocytes in human lymphatic tissue for dipeptidylaminopeptidase IV

Summary The enzyme dipeptidylaminopeptidase IV (DAP IV; EC 3.4.14.-) was recently shown cytochemically to be confined, in blood and bone marrow, to human T cells bearing, Fc receptors for IgM (T lymphocytes). This observation, confirmed by direct biochemical tests, stimulated us to study the histochemical distribution of DAP IV in normal human lymphatic tissue. In cryostat sections of lymph node, tonsil and thymus, DAP IV was detectable only in lymphocytes, Hassal's corpuscles and the endothelia of vessels. The distribution pattern of DAP IV-positive lymphocytes accorded well with results obtained with human T cell antisera. Compared to cytochemical reactions for other enzymes, such as acid esterase, DAP IV has the advantage that it does not stain monocytes, B lymphocytes or other mononuclear cells. Further, it does not depend on a particular type of staining pattern like, for example, the dot-like reaction product of acid esterase in T lymphocytes. Since the reaction for DAP IV remains more or less unchanged in month-old sections, it is easily adaptable to routine work and has the potentiality of being applied to the diagnosis of T cell lymphomas.     

Expression of fetal thymidine kinase in human cobalamin or folate deficient lymphocytes.

In extracts of peripheral blood lymphocytes of cobalamin or folate deficient patients thymidine kinase activity is increased three fold and exhibits properties of the fetal isoenzyme. Appropriate vitamin therapy results in reduction of this activity to normal levels and change from fetal to adult isoenzyme. The occurrence in cobalamin or folate deficiency of fetal thymidine kinase activity in non proliferating human lymphocytes is unique and may reflect events in the deficient marrow lymphoid progenitor cells.     

Generation of specific anti-melanoma reactivity by stimulation of human tumor-infiltrating lymphocytes with MAGE-1 synthetic peptide

The MAGE-1 gene encodes a tumor-specific antigen, MZ2-E, which is recognized by cloned, specific cytolytic T cells (CTL) derived from the peripheral blood of a patient with melanoma. We have produced a MAGE-1-specific CTL line derived from the tumor-infiltrating lymphocytes (TIL) of a melanoma patient by weekly restimulation with autologous EBV-B cells pulsed with the synthetic HLA-A1-restricted MAGE-1 epitope nonapeptide EADPTGHSY. The 1277. A TIL line grew in long-term culture in low-dose interleukin-2 (IL-2) and IL-4, and exhibited antigen-specific, MHC-class-I-restricted lysis of HLA-A1-bearing MAGE-1⁺ cell lines. Cytolysis of target cells pulsed with the synthetic MAGE-1 decapeptide KEADPTGHSY was superior to that of cells pulsed with the immunodominant nonapeptide. Single amino-acid or even side-chain substitutions in the immunodominant nonamer abrogated cytolysis. 1277. A TIL specifically secreted tumor necrosis factor after co-incubation with HLA-A1-expressing MAGE-1⁺ cell lines or fresh tumor. These data suggest that tumor-antigen-specific, MHC-restricted CTL may be grown from TIL in the presence of synthetic epitope peptides and expanded for adoptive immunotherapy in melanoma patients.     

Xenogeneic organ transplantation

Over the past few years, several major advances have occurred in the understanding of how the humoral and cellular immune system of humans recognizes and destroys transplant cells, tissues and organs derived from animal sources. Consequently, armed with this new knowledge, several laboratories have now developed novel immunoprotective technologies that may allow xenotransplantation to be clinically feasible.     

Efficient transfer of a tumor antigen-reactive TCR to human peripheral blood lymphocytes confers anti-tumor reactivity.

The tumor-associated-Ag MART-1 is expressed by most human melanomas. The genes encoding an alphabeta TCR from a MART-1-specific, HLA-A2-restricted, human T cell clone have been efficiently transferred and expressed in human PBL. These retrovirally transduced PBL cultures were MART-1 peptide reactive, and most cultures recognized HLA-A2+ melanoma lines. Limiting dilution clones were generated from three bulk transduced PBL cultures to investigate the function of individual clones within the transduced cultures. Twenty-nine of 29 CD8+ clones specifically secreted IFN-gamma in response to T2 cells pulsed with MART-1(27-35) peptide, and 23 of 29 specifically secreted IFN-gamma in response to HLA-A2+ melanoma lines. Additionally, 23 of 29 CD8+ clones lysed T2 cells pulsed with the MART-1(27-35) peptide and 15 of 29 lysed the HLA-A2+ melanoma line 888. CD4+ clones specifically secreted IFN-gamma in response to T2 cells pulsed with the MART-1(27-35) peptide. TCR gene transfer to patient PBL can produce CTL with anti-tumor reactivity in vitro and could potentially offer a treatment for patients with metastatic melanoma. This approach could also be applied to the treatment of other tumors and viral infections. Additionally, TCR gene transfer offers unique opportunities to study the fate of adoptively transferred T cells in vivo.     

Response of human fetal lymphocytes in xenogeneic mixed leukocyte culture: Phylogenetic and ontogenetic aspects

Cells from liver, thymus, and spleen of human fetuses at different stages of development were capable of a proliferation response against xenogeneic and allogeneic lymphocytes. The kinetics of fetal responses against rat lymphocytes were identical to those of fetal and adult responses against allogeneic cells. With all of the cell types studied, including adult lymphocytes, allogeneic responses were stronger than xenogeneic. Xenogeneic responses against lymphocytes from rat, mouse, or sheep were stronger than those against lymphocytes from rabbit, chicken, snake, or frog. These results are interpreted to indicate that recognition of foreign lymphocytes by human lymphocytes depends on the phylogenetic position of the species used as a source of stimulating cells. The degree of recognition decreases as the phylogenetic distance increases. Specific elimination of responding cells and restimulation with another cell population was used to study the specificity of proliferation responses against mouse and rat lymphocytes. Responses by prethymic liver cells from human fetuses were not due to the existence of specifically recognizing subpopulations. Thymus and spleen at 16 weeks' gestation contained specific subpopulations capable of differentiating between xenogeneic and allogeneic cells, as well as between xenogeneic cells with different intraspecies histocompatibility patterns. Generation of receptor diversity on T lymphocytes is discussed briefly in the light of these findings.     

The separation and reactivity in vitro of a subpopulation of human lymphocytes which bind histamine. Correlation of histamine reactivity with cellular maturation.

Lymphocytes reactive with histamine were fractionated on beads of Sepharose-albumin to which histamine was attached (SAH). Histamine reactive cells were present in blood, tonsil, and thymus. Using membrane and functional criteria, both T and B cells were shown to contain histamine-binding cells, while precursor cells did not. In T cell populations, lectin induced proliferation, cell-mediated lymphotoxicity, and secretion of mediators (lymphocytes mitogenic factor and immunoglobulin secretion inducing activity) were restricted to histamine reactive lymphocyte. However, antigen induced proliferation and mixed lymphocyte culture response mainly present in histamine-binding cells, also occurred in nonbinding populations. Reactivity with sheep erythrocytes was present in both reactive and non-reactive cells. In B cell populations, reactivity with antisera to IgM occurred in both binding and non-binding cells, while reactivity with antisera to IgG as well as T-induced IgG secretion were confined to histamine-binding cells. Membrane reactivity to erythrocyte coated with the first four components of complement was characteristic of histamine-unreactive cells. In T and B populations, histamine unreactive cells responded to lymphocyte mitogenic factor. These facts, together with data obtained from longterm lymphoid lines showing differentiation from nonbinding to binding cells after cell division, lead to the concept that generation of detectable histamine sites was a differentiative lymphocyte process. The generation of histamine-binding cells in precursor cell cultures supports this hypothesis. A possible role of histamine as a physiological lymphocyte regulating agent is suggested by the inhibition by soluble histamine of proliferative, cytotoxic, and secretory responses of histamine-binding T cells.     

Natural cytotoxic reactivity of human lymphocytes against a myeloid cell line: characterization of effector cells.

Using a series of techniques to identify and deplete various peripheral blood lymphocyte subpopulations, we studied the cytotoxic reactivity of normal individuals against the myeloid cell line K-562 in a 4-hr 51chromium-release assay. Depletion of lymphocytes bearing complement receptors had a variable, usually negligible effect on cytotoxicity. In contrast, depletion of lymphocytes bearing Fc receptors abrogated target cell lysis. Separation of lymphocytes with high-affinity binding of sheep red blood cells (SRBC) evidenced by rosette formation at 29 degrees C yielded a population of rosette-forming cells containing few cytotoxic cells, whereas separation of total E-RFC under optimal rosetting conditions produced a rosette fraction containing a major proportion of the effector cells. These data indicate that the cytotoxic lymphocyte in this system is Fc receptor positive, largely complement receptor negative, and may possess low density or low affinity receptors for SRBC.     

Differential reactivity of human lymphocytes allosensitized in vitro in hormonally defined medium or medium supplemented with plasma

We compared the in vitro allogeneic response of human lymphocytes cultured in hormonally defined medium (HDM) with that of those cultured in plasma-containing medium (PCM). Differences occurred only in secondary MLR or PLT and in cell-mediated cytotoxicity in terms of specificity, which was either greater or modified when HDM was used.     

Xenogeneic mixed leucocyte response

Xenogeneic mixed leucocyte cultures composed of human, chimpanzee, baboon, goat, sheep, pig, and dog cells were set up with a variety of plasma culture supplements. The characteristics of the human leucocyte response to xenogeneic cells was similar to its response to allogeneic cells. Peak response to xenogeneic stimulation occurred on the same culture day as the peak response to allogeneic stimulation. Similar numbers of xenogeneic and allogeneic cells produced peak stimulation of cells from any one individual. There was, however, a wide variation in the response of human lymphocytes to both allogeneic and xenogeneic cells. A factor in the plasma supplement specific for responding or stimulating cells inhibited the mixed leucocyte response in some combinations and could be removed by absorption techniques.     

The characteristics of Staphylococcus aureus peptidoglycan in the spectrum of the reactivity of human lymphocytes to bacterial peptidoglycans]

The sensitivity of lymphocytes of healthy persons to S. aureus peptidoglycan as compared with that to the polyclonal stimulator zymosan C3b and peptidoglycans of other bacteria (Streptococcus faecalis, Escherichia coli, Bacterium bifidum) was analyzed with a test system permitting the determination of specific reactivity to peptidoglycans. The analysis showed that at the peak of luminol-dependent chemiluminescence (25-30 minutes) individual reactivity to S. aureus peptidoglycan varied within wide limits (the coefficient of lymphocyte stimulation was 1.4-9.6, 3.5 +/- 0.6), exceeding sensitivity to other bacteria, as well as the values obtained in the negative control. The conclusion of the wide spread of sensitization to S. aureus peptidoglycan and the possibility of using this preparation for the study of cell-mediated immunity reactions was made.     

Histocompatibility requirements of rat lymphocytes for fetal antigen recognition

The mitogenic response in vitro of DA rat splenic lymphocytes to concanavalin A has been found to be greatly enhanced (up to 800-fold) if the in vitro environment in which the cells are cultured is modified by the addition of nonmitogen-responsive, mitomycin C-treated “filler” cells. The results of the experiments suggest that filler cells may act as “spacer” cells and that the phenomenon is a physical effect in which the additional cells act as non-immunological cushions that modulate suppressive factors limiting cell responsiveness in vitro. Cell viability of the spacer cells was not necessary and the enhanced responses that follow the addition of spacer cells could be duplicated by formalin-fixed cells or even non-biologically active material such as Sephadex or Bio-Gel. Soluble factors released from spacer cell preparations also resulted in a modest increase in mitogenic responsiveness. The experiments further define the conditions for the culture of rodent lymphocytes and underscore the need for controls that eliminate nonbiological effects as explanatory mechanisms where cell collaboration is putatively involved in the generation of cell-mediated immune responses.     

Cell-mediated lympholysis by human maternal and neonatal lymphocytes: mother's reactivity against neonatal cells and vice versa.

Maternal reactivity in cell-mediated lympholysis (CML) against cells from her own child is, in average, half of the maternal reactivity against unrelated adult cells. This finding remains the same when cells from a newborn or from an older child are used, suggesting that the reduced maternal reactivity is based rather on the one haplotype identity between the mother and child than on the occurrence of specific maternal tolerance. Consistently, CML-capacity of the child, directed against cells of own mother, is half of the control values, again independently of the child's age.     

Conversion of human lymphocytes to antitumor immune reactivity by xenogeneic and allogeneic imune RNA detected by leukocyte-adherence inhibition tests

Summary Using leukocyte adherence inhibition (LAI) tests, we studied the activity of xenogeneic immune RNA (I-RNA) extracted from the spleen and lymph nodes of sheep after immunization with human breast carcinoma tissue or keyhole limpet hemocyanin (KLH) in inducing lymphocytes from normal healthy donors to mediate immune responses in vitro. Mononuclear cells isolated from venous blood of normal donors, depleted of monocytes and, in some experiments, separated into T cells and non-T cells, were incubated with and without anti-breast carcinoma I-RNA or anti-KLH I-RNA for 20 min at 37° C. Then, lymphocyte adherence was determined by a Coulter counter method in the presence of 3 M KCl extracts of breast carcinoma tissues, control tissue, or KLH. Following incubation with anti-breast carcinoma I-RNA, the adherence of lymphocytes from normal donors was found to be inhibited only in the presence of breast carcinoma extracts. Following incubation with anti-KLH I-RNA, lymphocyte adherence was inhibited only in the presence of KLH. The principal effector cells involved appeared to be T lymphocytes. I-RNA treatment with RNase, but not with DNase or pronase, completely abrogated the LAI responses. In a blind study utilizing coded samples of xenogeneic and allogeneic I-RNA of unknown origin, samples containing activity against breast cancer extracts were identified correctly by LAI. Abbreviations used: I-RNA, immune RNA; LAI, leukocyte adherence inhibition; KLH, Keyhole limpet hemocyanin; PBS, phosphate buffered saline; RNase, ribonuclease; DNase, deoxyribonuclease