Deterioration of cellular immunity during aging. The relationship between age-dependent impairment of delayed-type hypersensitivity reactivity, interleukin-2 production capacity, and frequency of Thy-1+,Lyt-2- cells in C57BL/Ka and CBA/Rij mice

The effect of aging on the delayed-type hypersensitivity (DTH) to sheep red blood cells (SRBC) in vivo and the interleukin-2 (IL-2) production capacity in vitro by spleen cells from young (17 weeks) and old (125 weeks) CBA/Rij and C57BL/Ka mice were investigated. For both CBA/Rij and C57BL/Ka mice an age-related decline in the DTH response to SRBC and the IL-2 production capacity was observed. Both parameters are mediated by Thy-1+,Lyt-2- spleen cells. For both mouse strains the proportion of Thy-1+,Lyt-2- spleen cells declined less strongly with aging than the DTH reactivity and the IL-2 production capacity. From this it was concluded that not only a quantitative but also a qualitative decrease of T-cell function occurs during senescence. It was also investigated whether the proportion of Thy-1+,Lyt-2- peripheral blood lymphocytes can be used as a predictive value with regard to the decline of DTH with aging of the corresponding mouse. This was indeed found to be the case in CBA/Rij mice, but not in C57BL mice.     

Isolation of monoclonal antibodies to antigen Lyt-3.2

A hybridoma producing monoclonal antibodies (McAb) NATF9.9 (F9) was obtained from fusion of murine myeloma X63 and splenocytes of AKR mice immunized with a single intravenous injection of 5 X 10(7) thymocytes of CBA mice. F9 McAb were cytotoxic for 80% thymocytes, 10% splenocytes, 20% lymph node cells, 85% cortical and 32% medullary thymocytes of CBA, C57BL/6, BALB/c, DBA/2 and SJL but not for the cells of C58 and AKR mice. F9 McAb reacted only with T cells and did not react with B cells and EL4 thymoma cells (Thy-1.2+, Lyt-1+2-3-). The proportion of F9+ cells accounts for about 40% among T lymphocytes of the lymph nodes and spleen as tested by flow-type cytometry. Lymph node cells treated with F9 McAb plus complement completely lost their reactivity with rat anti-Lyt-2 McAb and only partly (by 30%) with anti-Lyt-1 McAb. The reactivity pattern of F9 McAb attests to their specificity for Lyt-3.2 antigen.     

Genetic control of T-Cell subset representation in inbred mice

Lyt-2+ T cells constitute a significantly greater proportion of the total peripheral T-cell population in C57BL mice than in BALB/c and other mouse strains. The inheritance of this differential representation of Lyt-2- vs. Lyt-2+ T cells was studied by two-color immunofluorescence analysis of peripheral T cell subsets in BALB/c, C57BL, F1 and F2 generations, and in CXB recombinant inbred strains. It was shown that the C57BL phenotype (low Lyt-2-/Lyt-2+ ratio) is a dominant Mendelian character. Studies of subpopulations of thymocytes and of early thymus emigrants indicate that the representation of mature Lyt-2- and Lyt-2+ T cells is influenced by mechanisms of selection or differential turnover in the peripheral lymphoid organs, but that thymic and prethymic influences may also play a role.     

Suppression of alloimmune cytotoxic T lymphocyte (CTL) generation by depletion of NK cells and restoration by interferon and/or interleukin 2

By using rabbit antiserum to a glycolipid, ganglio-n-tetraosylceramide (ASGM1), the accessory effect of natural killer (NK) cells on the generation of alloimmune CTL in mice was investigated. When normal C3H/He mice were immunized with C57BL/6 or BALB/c spleen cells, they generated alloimmune CTL with a surface marker phenotype of Thy-1+ Lyt-1-2+ ASGM1-, preceded by early augmentation of cytotoxic activity of NK cells with a Thy-1-Lyt-1-2-ASGM1+ phenotype. Administration of anti-ASGM1 (10 microliters) in mice resulted in a complete depletion of NK activity and ASGM1+ cells in the spleen even 1 day after injection, but no changes in the proportions of T (Thy-1+) cells and their Lyt-1 and Lyt-2 subsets as revealed by an immunofluorescence analyzer (FACS) and phagocytic cells. When these anti-ASGM1-treated mice were immunized with allogeneic cells, they showed neither augmented NK activity nor generation of alloimmune CTL, and spleen cells isolated from these anti-ASGM1-treated mice produced no CTL response to alloimmunization in vitro. Normal spleen cells treated with the antiserum and complement in vitro also showed a complete NK depletion without any deterioration of T cells and their Lyt-1 and Lyt-2 subsets, and when stimulated with allogeneic cells they generated no CTL. Spleen NK (ASGM1+) cells were purified by Percoll-gradient centrifugations followed by complement-dependent killing of T cells with the use of anti-Thy-1 monoclonal antibody, and were further purified by panning methods with anti-ASGM1, giving a preparation consisting of greater than 90% ASGM1+, Ly-5+ cells, and less than 0.5% of Thy-1+, Lyt-1+, and Lyt-2+ cells. These purified ASGM1+ Thy-1- cells alone generated no alloimmune CTL in response to alloantigens, suggesting that ASGM1+ NK cells contained no precursors of alloimmune CTL. When added into NK-depleted spleen cells, they restored the normal alloimmune CTL response of the spleen cells, indicating that ASGM1+ fractions contained cells to provide an accessory function for CTL generation. Lyt-1+ cells purified by panning methods did not restore the CTL response of NK-depleted spleen cells. These results indicate that ASGM1+ NK cells, but not Lyt-1+ helper T cells contaminating ASGM1+ fractions at undetectable levels, are responsible for the accessory function. When these purified ASGM1+ Thy-1- cells were stimulated with allogeneic cells, they produced IL 2 and IFN.(ABSTRACT TRUNCATED AT 400 WORDS)     

Asialo-GM1-positive T killer cells are generated in F1 mice injected with parental spleen cells

(C57BL/6 x DBA/2)F1 mice transplanted with parental C57BL/6 spleen cells become splenic chimeras, show donor antihost cytotoxic T cell activity, and lose their T cell-mediated, humoral, and natural immunity. Injection of anti-asialo-GM1 (ASGM1) into transplanted mice strongly suppresses splenic cytotoxic activity and causes a significant reduction of spleen cells expressing ASGM1, Thy-1, and Lyt-2. In vitro treatment of spleen cells from transplanted mice with antibody and complement shows that the cytotoxic effector cells are ASGM1+, Thy-1+, Lyt-2+, L3T4-, NK1.1-, and H-2d-, hence of donor origin. The cytotoxic effector cells are specific for H-2d targets and lack NK activity. In an attempt to explore whether in vivo elimination of the cytotoxic effector cells has any influence on splenic chimerism or humoral immunity, F1 mice injected with parental splenocytes were treated with anti-ASGM 1. Results show that this treatment eliminates a substantial proportion of cytotoxic effector cells but has no effect on splenic chimerism or restoration of humoral immunity. It therefore appears that cytotoxic effector cells are not primarily responsible for induction of chimerism or suppression of humoral immunity. In support of this injection of parental spleen cells with the nu/nu mutation induces killer cells in F1 mice but fails to induce splenic chimerism or immunosuppression. In contrast, injection of parental spleen cells with the bg/bg mutation generates both splenic chimerism and suppression of humoral immunity although their ability to generate cytotoxic effector cells in F1 hosts is seriously impaired and comparable to the cytotoxic potential of C57BL/6 nu/nu cells. It is concluded that the ASGM1 + cytotoxic T cells are not primarily responsible for splenic chimerism and suppression of humoral immunity and that the two effects are likely caused by parental cells with a different phenotype and function.     

Immunologic regulation of experimental cutaneous leishmaniasis. V. Characterization of effector and specific suppressor T cells

Resistant CBA mice infected with Leishmania tropica promastigotes develop concomitant and convalescent immunity against reinfection. This can be adoptively transferred by splenic and lymph node T cells with a threshold dosage of 1 to 2.5 x 10(7). The effector cells are of Thy-1+, Lyt-1+2- phenotype. The same immune cell population also adoptively transfers specific DTH to L. tropica, which is restricted by the major histocompatibility complex. On the other hand, highly susceptible BALB/c mice infected with L. tropica develop antigen-specific suppressor T (Ts) cells (previously shown to inhibit the induction and expression of DTH), which are capable, on transference, of reversing the healing of lesions induced by prior sublethal irradiation of BALB/c mice. As few as 10(6) of these T cells are effective in abrogating the potent prophylactic effect of 550 rad. The Ts cells are of Thy-1+, Lyt-1+2-, and I-J- phenotype. Sublethally irradiated and infected BALB/c mice produce antibody responses quantitatively and isotypically similar during the critical first 40 days after infection whether or not they are injected with 10(7) Ts cells (nonhealing vs healing). Thus, impairment of DTH and curative immune responses in BALB/c mice cannot be attributed to a helper function of these Thy-1+, Lyt-1+2- cells for the formation of suppressive antibody.     

The role of suppressor T cells in BCNU-mediated rejection of a syngeneic tumor

The present investigation was initiated to determine the mechanism by which 1,3-bis(2-chloro-ethyl)-1-nitrosourea (BCNU) treatment of tumor-bearing mice results in a high percentage of surviving mice which are resistant to subsequent homologous tumor challenge. Spleen cells from C57BL/6 mice bearing the syngeneic LSA ascites tumor failed to demonstrate significant tumor-specific cytotoxic T lymphocyte (CTL) activity when stimulated in vitro with irradiated tumor cells. This lack of CTL activity correlated with the presence and high activity of two types of CTL-regulatory suppressor T cells (Ts), tumor-specific Thy-1+, Lyt-1-2+ and tumor-nonspecific Thy-1+, Lyt-1+2+ cells, as demonstrated by a double-positive selection technique. In contrast, spleen cells from BCNU-treated tumor-bearing mice generated high tumor-specific CTL activity when stimulated in vitro with irradiated tumor cells. This CTL activity correlated with the lack of demonstrable tumor-specific Ts and greatly diminished tumor-nonspecific Ts activity. The tumor-specific helper activity of Thy-1+, Lyt-1+,2- cells was found to be similar in both BCNU-treated and untreated tumor-bearing mice. BCNU-treated mice that survived a primary LSA tumor challenge (referred to as BCNU-cured mice) resisted subsequent challenge with the homologous (LSA) but not with a heterologous syngeneic tumor (EL-4). However, rejection of a secondary challenge with LSA tumor by BCNU-cured mice was inhibited by adoptive transfer of spleen cells from either normal mice or mice bearing LSA tumors. Furthermore, LSA tumor cells that failed to evoke tumor-specific CTL activity in normal mice could induce high CTL activity in BCNU-cured mice. The present study suggests that, in addition to its direct tumoricidal activity, BCNU inhibits the induction of tumor-specific Ts, thereby explaining why a high percentage of mice survive a primary syngeneic tumor challenge after treatment with BCNU, and also resist subsequent rechallenge with the homologous tumor.     

Feedback suppression of the immune response in vivo. III. Lyt-1+ B cells are suppressor-inducer cells

We previously demonstrated that injection of a high dose (4 X 10(9] of sheep erythrocytes (SRBC) into C57BL/6 mice results in the generation of splenic B cells (plastic nonadherent, Thy-1- and Ig+) which, when transferred to normal syngeneic recipients, subsequently induce antigen-specific suppressor T cells to suppress the recipient's plaque-forming cell (PFC) responses to SRBC. In the present study we characterized the suppressor-inducer B cells phenotypically. Cytotoxic treatment of the donor's immune spleen cells with anti-Lyt-1 antibody plus complement (C'), but not with anti-Lyt-2 antibody plus C', relieved the suppression of PFC responses in recipients. The FcRr+ population separated by EA-rosette formation showed enriched suppressor-inducing activity, whereas the FcRr- population showed no activity. Our findings, taken together with the previous ones, suggest that suppressor-inducer cells are Thy-1-, Lyt-1+, Lyt-2-, FcRr+, and Ig+.     

Repopulation of the mouse thymus after sublethal fission neutron irradiation. I. Sequential appearance of thymocyte subpopulations

The T cell composition of the thymus of sublethal fission neutron-irradiated CBA/H mice was analyzed with cytofluorometry and immunohistology, using monoclonal antibodies directed to the cell surface antigens Thy-1, T-200, MT-4, Lyt-1, Lyt-2, and MEL-14. The results of this investigation show that whole body irradiation with 2.5 Gy fission neutrons results in a severe reduction and degeneration of the cortex, whereas the medulla is affected to a lesser extent. Irradiation selects, within 24 hr, for a population of dull Thy-1+, bright T-200+, bright Lyt-1+ cells localized in the medulla. Phenotype analysis of the regeneration of the thymus, which starts at about 5 days after irradiation, reveals the sequential appearance of: 1) "null" cells, i.e., lymphoblasts negative for all tested antigens, mainly in the subcapsular area but also in the medulla; 2) Thy-1+ "only" and T-200+ "only" cells in the subcapsular area; 3) Thy-1+, T-200+ cells; and 4) Thy-1+, T-200+, MT-4+, Lyt+ cells in the cortex. In addition, an increased MEL-14 expression is observed in correlation with the expression of Thy-1 and T-200 determinants during the regeneration of the thymus. From day 10 on up to at least 150 days after irradiation, no differences can be observed in the thymus of irradiated and age-matched sham-irradiated control mice, as measured by the expression and distribution of Thy-1, T-200, MT-4, Lyt-1, Lyt-2, and MEL-14 antigens. The observed sequence in phenotype shift in the regeneration of the thymus after irradiation is discussed in view of recently published data on the differentiation of the T cell system.     

Qualitative and quantitative heterogeneity in Pgp-1 expression among murine thymocytes

A proportion of Pgp-1+ cells in the thymus have been shown to have progenitor activity. In adult AKR/Cum mice the total Pgp-1+ population in the thymus differs from that of the bulk of thymocytes and is antigenically heterogeneous when examined by flow cytometry. Pgp-1+ thymocytes are enriched for several minor cell populations compared to total thymocytes: B2A2-, interleukin-2-receptor+ (IL-2R+), and Lyt-2-, L3T4-. However, these subsets are still a minor proportion of the Pgp-1+ cells, the majority being Lyt-2+ and/or L3T4+ and B2A2+. Pgp-1+ thymocytes also differ from the bulk of thymocytes in having lower amounts of Thy-1 and in showing a higher proportion of single positive (Lyt-2+, L3T4- or Lyt-2-, L3T4+) cells. Populations of adult thymocytes that are enriched in progenitor cells can be isolated by cytotoxic depletion using either anti-Thy-1 antibody (Thy-1 depletion) or anti-Lyt-2 and anti-L3T4 antibody (Lyt-2, L3T4 depletion). Pgp-1+ cells in progenitor cell-enriched populations are also phenotypically heterogeneous. Pgp-1+ cells in both populations may be IL-2R+ or IL-2R- and B2A2+ or B2A2-. The population of Pgp-1+ cells in progenitor cell-enriched populations in the adult differs from that of the fetus at 14 days of gestation in that in the 14-day fetus, most Pgp-1+ cells are IL-2R+. By Day 15 of gestation, distinct populations of Pgp-1+, IL-2R-; Pgp-1+, IL-2R+; and Pgp-1-, IL-2R+ cells are observed. In the 15-day fetus, as in the adult, many Pgp-1+ thymocytes express low to moderate levels of Thy-1. The total percentage of Pgp-1+ cells in the thymus varies among different mouse strains, ranging from 4 to 35% in the thymus of young adult mice. Pgp 1.1 strains contain more detectably Pgp-1+ thymocytes than Pgp 1.2 strains; however, there is variability in the proportion of Pgp-1+ cells, even among Pgp 1.2 strains. In contrast to AKR/Cum mice, the Pgp-1+ thymocyte population in BALB/c mice, which contain a high proportion of Pgp-1+ thymocytes, closely resembles the total thymocyte population.     

Phenotypic variation in clonal Abelson virus lymphoma cells

Two clonal A-MuLV lymphoma cell lines have the capacity to generate phenotypic variants when grown in vivo as ascites tumors. Variant lines differed from parental lymphoma cells in their expression of enzymatic or cell surface differentiation markers. Parental lines expressed the B220 and Lyb-2 glycoproteins characteristic of pre-B cells and bound B220-specific monoclonal antibodies such as 14.8. The parental cells expressed low levels of TdT activity but did not synthesize detectable mu-heavy chain, a cellular phenotype that may correspond to lymphoid progenitor cells. Three classes of phenotypic variants were recovered from the Thy-1- parental lines: 1) 14.8+, Lyt-1+, Thy-1- cells; 2) 14.8 +/-, Lyt-1+, Thy-1+ cells, and 3) 14.8-, Lyt-1+, Thy-1+ cells. Cell cloning experiments indicated that Thy-1+ variant cells can be recovered within 14 days of in vivo inoculation as a minor proportion (1/10(6] of the tumor cell population and subsequently become the predominant tumor cell population. These clonal tumor lines provide a model for the study of cellular and molecular alterations that occur during neoplastic differentiation and progression in the lymphoid system.     

Immunohistology of lymphoid and non-lymphoid cells in the thymus in relation to T lymphocyte differentiation

The present paper reports the distribution of lymphoid and non-lymphoid cell types in the thymus of mice. To this purpose, we employed scanning electron microscopy and immunohistology. For immunohistology we used the immunoperoxidase method and incubated frozen sections of the thymus with 1) monoclonal antibodies detecting cell-surface-differentiation antigens on lymphoid cells, such as Thy-1, T-200, Lyt-1, Lyt-2, and MEL-14; 2) monoclonal antibodies detecting the major histocompatibility (MHC) antigens, H-2K, I-A, I-E, and H-2D; and 3) monoclonal antibodies directed against cell-surface antigens associated with cells of the mononuclear phagocyte system, such as Mac-1, Mac-2, and Mac-3. The results of this study indicate that subsets of T lymphocytes are not randomly distributed throughout the thymic parenchyma; rather they are localized in discrete domains. Two major and four minor subpopulations of thymocytes can be detected in frozen sections of the thymus: 1) the majority of cortical thymocytes are strongly Thy-1+ (positive), strongly T-200+, variable in Lyt-1 expression, and strongly Lyt-2+; 2) the majority of medullary thymocytes are weakly Thy-1+, strongly T-200+, strongly Lyt-1+, and Lyt-2- (negative); 3) a minority of medullary cells are weakly Thy-1+, T-200+, strongly Lyt-1+, and strongly Lyt-2+; 4) a small subpopulation of subcapsular lymphoblasts is Thy-1+, T-200+, and negative for the expression of Lyt-1 and Lyt-2 antigens; 5) a small subpopulation of subcapsular lymphoblasts is only Thy-1+ but T-200- and Lyt-; and 6) a small subpopulation of subcapsular lymphoblasts is negative for all antisera tested. Surprisingly, a few individual cells in the thymic cortex, but not in the medulla, react with antibodies directed to MEL-14, a receptor involved in the homing of lymphocytes in peripheral lymphoid organs. MHC antigens (I-A, I-E, H-2K) are mainly expressed on stromal cells in the thymus, as well as on medullary thymocytes. H-2D is also expressed at a low density on cortical thymocytes. In general, anti-MHC antibodies reveal epithelial-reticular cells in the thymic cortex, in a fine dendritic staining pattern. In the medulla, the labeling pattern is more confluent and most probably associated with bone-marrow-derived interdigitating reticular cells and medullary thymocytes. We discuss the distribution of the various lymphoid and non-lymphoid subpopulations within the thymic parenchyma in relation to recently published data on the differentiation of T lymphocytes.     

Phenotypic heterogeneity of antisyngeneic tumor killer cells (ASTK) generated in allogeneic mixed lymphocyte reactions

By using monoclonal antibodies to Thy-1, Lyt-2, and Qa-5 differentiation antigens, we demonstrated a heterogeneity of cytotoxic cells developed in allogeneic mixed lymphocyte responses that lyse tumor cells syngeneic with the responder cells. There are minimally two Thy-1+ populations, one of which is Lyt-2+ and the other Lyt-2-. There is probably also a Thy-1- population. Most of the Lyt-2- tumor killer cells are Qa-5+, and most of the Lyt-2+ tumor killer cells are Qa-5-.     

Induction of alloreactive cytotoxic T cells by acute virus infection of mice

Alloreactive cytotoxic T lymphocytes (CTL) distinct from virus-specific CTL and activated natural killer (NK) cells were generated during acute lymphocytic choriomeningitis virus (LCMV) infection of C57BL/6J mice. The alloreactive CTL shared similar antigenic markers (Thy-1.2+, Lyt-2.2+, and asialo GM1-) with the virus-specific CTL that appeared at the same time 7 days postinfection, but had different target specificities. These alloreactive CTL lysed allogeneic but not syngeneic or xenogeneic targets. These were distinct from activated NK cells, which lysed all target cell types, peaked 3 days postinfection, and had a phenotype of asialo GM1+, Thy-1 +/-, Lyt-2.2-. Cold target competition studies indicated that there were several subsets of alloreactive T cells with distinct specificities, and that these alloreactive T cells were not subsets of the virus-specific T cells. Similar types of alloreactive CTL were induced at much lower levels in C3H/St mice. This may indicate that the generation of this "aberrant" T cell activity is under genetic control. Hence, the LCMV infection of C57BL/6J mice induces several cytotoxic effector populations including alloreactive CTL, activated NK cells, and virus-specific CTL. Virus infections therefore have the ability not only to polyclonally stimulate B cells, as previously described, but also to stimulate CTL.     

L3T4+ T cells regulate Abelson virus-induced lymphomagenesis

To evaluate the role of T cells in regulation of lymphomagenesis, experiments were performed using Abelson murine leukemia virus (AMuLV). In vitro transformation of bone marrow target cells by this B lymphotropic retrovirus was inhibited by peripheral lymph node cells from naive mice. The inhibitory activity depended on Thy-1+ L3T4+ cells but did not require Lyt-2+ cells. In vivo depletion of L3T4+ T cells with a mAb (GK1.5) altered the course of AMuLV-induced lymphoma. L3T4 depletion of naturally resistant C57BL/6 mice resulted in dramatic susceptibility to lymphoma induction. Lymphoma cells from anti-L3T4-treated C57BL/6 mice infected with AMuLV displayed the B lineage transformation marker P1606C3. These studies reveal an important immunologic component of Abelson disease resistance involving L3T4+ T cells.     

In vivo dynamics of pulmonary lymphoid cell subpopulations generated against pulmonary metastasis: evaluation by bronchoalveolar lavage fluid.

The dynamics of lymphoid cell subpopulations in bronchoalveolar lavage fluid (BALF) and the systemic lymphoid organs of mice after intravenous injection of B16 melanoma cells were examined with a fluorescence-activated cell sorter. The lymphoid cell subpopulations of BALF and mediastinal lymph nodes showed significant changes in numbers and proportions, while those of other lymphoid organs including inguinal lymph nodes, thymus and spleen, showed little change. In week 1, the cells with a Thy-1.2+, Lyt-1+, L3T4-, Lyt-2- phenotype and asialo-Gm1+ cells in BALF significantly increased and L3T4+ cells slightly increased in number. By week 3, the numbers of Lyt-2+ cells in BALF markedly increased in number (by about 90 times) compared with controls. The number of Thy-1.2+ cells in mediastinal lymph nodes also increased significantly by week 3. Mice that had been pretreated with an immunosuppressive dose of cyclophosphamide were also inoculated intravenously with B16 melanoma cells. In these mice, a significantly increased number of pleural tumors developed and the number of Thy-1.2+ cells in BALF was markedly reduced from week 1 to 3. The results indicate that L3T4 and Lyt-2 double negative T-cells and natural killer (NK) cells may be generated and/or mobilized to the lung in an early phase of experimental metastasis of B16 melanoma cells and that at a later stage, when multiple metastases develop, T-cells with a Lyt-2+ phenotype markedly increase, probably as an expression of a host reaction against proliferating metastatic tumor cells.     

Carbohydrate differentiation antigens of murine T cells: expression on intestinal lymphocytes and intestinal epithelium

Carbohydrate differentiation antigens (CT antigens) which previously had been shown to be associated with cytotoxic T cells were found at high levels on intestinal intraepithelial lymphocytes (IEL) and on the intestinal epithelium. Histological examination of intestinal sections demonstrated that the CT1 MAb defined epitopes on IEL and on epithelial cells located in the base of the villi crypts. The CT2 MAb reacted with IEL but also bound to the majority of cells in the intestinal epithelium. When isolated intestinal cell populations were analyzed by flow cytometry, two major size classes of cells were evident. The smaller cells, corresponding to lymphocytes, were primarily Lyt-2+, with a high proportion expressing CT antigens. Another differentiation antigen defined by the MAb J11d was absent from IEL, indicating that those IEL of T cell origin are likely to be mature because thymocytes, but not peripheral T cells, express the J11d antigen. Two-color fluorescence analysis indicated that the CT determinants were present on the Thy-1+, Lyt-2+, and the Thy-1-, Lyt-2+ subsets of IEL. However, the small percentage of L3T4+ IEL were CT-, further supporting our previous demonstration of a correlation between CT expression and Lyt-2 expression. Interesting phenotypic characteristics of IEL other than CT antigen expression were also detected. IEL did not express the MEL-14 lymphocyte homing receptor, and the cell surface level of LFA-1 was significantly lower than that of other peripheral lymphocytes. It was also shown that a small percentage of IEL express a T cell receptor allotypic marker, indicating that at least some of the cells are mature in terms of T cell receptor gene rearrangements. The large intestinal cells, although CT+, were not hematopoietic in origin because they were T200- and were shown by using chimeric mice not to be bone marrow-derived. In contrast to previously reported results, the cytotoxic activity of IEL was negligible with detectable lysis against NK-sensitive cells and other tumor cells, being observed in only one of seven experiments. Thus, the expression of the CT determinants was not indicative of cytotoxic ability, as previously suggested. The presence of specific carbohydrate residues on the cell surface of a subset of lymphocytes in an anatomically distinct immune compartment suggests that a unique differentiation pathway is followed by these cells.     

Immunoregulation in experimental disseminated histoplasmosis: flow microfluorometry (FMF) studies of the Thy and Lyt phenotypes of T lymphocytes from infected mice

Previous studies have shown that mice infected i.v. with 6 X 10(5) yeast phase Histoplasma capsulatum (Hc) develop suppressed immune responses during weeks 1 to 4 of infection but that by weeks 8 to 12 of infection these responses return to normal. In this study total and differential cell counts showed that as early as the third day of infection there was a marked reduction in the number of lymphocytes recovered from the peripheral blood, bone marrow, and thymus of infected animals. Concomitantly, there was an increase in the number of splenic lymphocytes. By day 28 both the total and differential cell counts were similar in both infected and normal animals. Flow microfluorometric (FMF) studies comparing the Thy-1.2, Lyt-1, Lyt-2, and surface immunoglobulin (slg) phenotypes of lymphocytes from normal and infected mice were performed. Between days 5 and 7 the thymocytes from infected mice displayed a higher relative fluorescence intensity (RFI) of the Thy-1.2 marker than normal thymocytes, whereas at day 10, the RFI was less than that of normal thymic lymphocytes. Between days 7 and 10 of infection the RFI of the Lyt-2 marker was less on thymocytes from Hc-infected mice; however, there was no change in the Lyt-1 marker. Examination of these lymphocyte markers in blood, spleen, and mesenteric lymph nodes showed that there were decreases in the RFI of both the Thy-1.2 and Lyt-2 between days 5 and 10 of infection. No changes were observed in the Lyt-1 or slg markers. By day 28 there were no differences between the normal and infected mice with respect to any surface marker in any of the organs studied. In other experiments, the effect of adrenalectomy before infection on these surface markers was studied. Absolute numbers of Thy-1.2+, Lyt-1+, and Lyt-2+ cells were significantly increased in the spleen and significantly decreased in the thymus and peripheral blood of infected mice relative to normal controls. These studies suggest that there is a migration of cells from the thymus, blood, and bone marrow to the spleens of mice with disseminated Hc infection.     

Thy-1+ epidermal cells proliferate in response to concanavalin A and interleukin 2

Mouse epidermal cells (EC) are composed of at least two phenotypically discrete populations of cells that in epidermal sheets have a dendritic morphology: Ia+ Langerhans cells (LC) and dendritic, bone marrow-derived, Ia- cells that express Thy-1 antigen (Thy-1+ dEC). Thy-1+ dEC lack other typical T cell markers such as L3T4, Lyt-1, and Lyt-2; however they do express Ly-5 and asialo GM1 in common with NK cells and certain other leukocytes. To investigate the functional capabilities of Thy-1+ dEC in vitro, cell suspensions prepared from trypsin-disaggregated sheets of mouse body wall epidermis were first enriched to 8 to 20% Ia+ and 20 to 40% Thy-1+ cells by centrifugation over Isolymph and then were cultured for 2 to 10 days with Concanavalin A (Con A) and/or partially purified rat IL 2. Con A-induced proliferation of EC was readily seen, with the maximal response occurring at a Con A concentration of 2.5 micrograms/ml on day 5 of culture. Con A responses were significantly enhanced by the continuous presence of 1 microgram/ml indomethacin. Responses both in the presence and absence of Con A were significantly enhanced by the addition of 5 to 10 U/ml of partially purified rat IL 2; proliferation in cultures stimulated by both Con A and IL 2 continued to increase throughout the 10-day culture period. Culture of fluorescence-activated cell sorter (FACS)-separated EC suspensions revealed that Thy-1-depleted EC and irradiated Thy-1+ EC failed to proliferate in response to Con A and IL 2, whereas unirradiated purified Thy-1+ EC gave enhanced Con A- and IL 2-induced responses compared with the unseparated population. Finally, to distinguish between the proliferation of small numbers of mature peripheral T cells and that of Thy-1+ dEC, antibody and complement-depletion studies were conducted with an unusual monoclonal anti-Thy-1 reagent, 20-10-5S, and with the anti-T cell reagents, anti-L3T4 and anti-Lyt-2. Thy-1+ dEC, but not LC, express the 20-10-5S determinant; furthermore, in CBA (Thy-1.2) mice 20-10-5S reacts with Thy-1+ dEC, thymocytes, and peripheral T cells, whereas in AKR/J (Thy-1.1) mice, it reacts only with Thy-1+ dEC and thymocytes and not with peripheral T cells. Pretreatment of AKR/J EC with 20-10-5S and complement abolished the capacity of such cells to respond to Con A and to IL 2.(ABSTRACT TRUNCATED AT 400 WORDS)     

Age-related changes in the capacity of bone marrow cells to differentiate in thymic organ cultures

The capacity of the bone marrow to give rise to T cells in advanced age was studied in vitro by reconstituting fetal thymic lobes from 14-day C57BL/Ka (Thy-1.1) mice with bone marrow cells from old (24-month) or young (3-month) C57BL/6 (Thy-1.2) mice. The use of these congenic strains enabled distinguishing between donor and host contribution to the developing T cells. We found that bone marrow cells from aged mice maintained their capacity to reconstitute fetal thymic explants and to differentiate into various T-cell subsets as assessed by distinct T-cell-specific surface markers (Thy-1, Lyt-1, Lyt-2, and L3T4) and functions (concanavalin A-induced proliferative and cytotoxic responses). However, when mixtures of old and young bone marrow cells reconstituted fetal thymic explants, the cells of old mice were less efficient than those of young in their capacity to give rise to T cells. These results indicate that bone marrow cells from aged mice can reconstitute the thymus and differentiate into T cells; however, their reconstituting capacity is inferior to that of bone marrow cells from young mice.