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F W Fitch

Publications and source records attributed to F W Fitch.

At least 91 records · Page 5Linked to original sources
PubMed1986

Interleukin-2 differentially regulates IL-2 receptors on murine cloned cytolytic and helper T cells.

The effects of interleukin-2 (IL-2) on the expression of IL-2 receptors by cloned cytolytic and helper T lymphocytes were studied using three anti-IL-2 receptor antibodies. IL-2 enhanced the expression of IL-2 receptors on all clones tested. A steep dose-response curve was observed with no measurable effect seen below 2 units/ml and maximal IL-2 receptor expression with greater than 5 units/ml. IL-2 receptor expression peaked 24-48 hr after the addition of IL-2. The subsequent decrease in IL-2 receptor expression correlated with a decrease in the levels of IL-2 remaining in the culture supernatants of cytolytic T lymphocyte cells. Removing residual IL-2 from cultures resulted in the rapid return of IL-2 receptor expression to unstimulated levels. The daily addition of low levels of IL-2 to cultures resulted in the prolonged expression of high levels of IL-2 receptors by non-IL-2-producing cloned cytolytic T cells. Cloned helper T cells which make IL-2 showed the initial increase in IL-2 receptor levels, but the daily addition of IL-2 did not prolong IL-2 receptor expression in these cells. These data suggest that IL-2 receptors on those cells which do not make IL-2 are regulated differently from receptors on cells which themselves make IL-2.

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PubMed1985

Cell-surface modification with an iodinatible imidoester to enhance radiolabeling.

A procedure to enhance incorporation of radioactive iodine into cell-surface proteins is described. The effects of chemical modification of cells with the iodinatible haptens N-succinimidyl-3-(4-hydroxyphenyl) propionate (Bolton-Hunter reagent) and methyl-p-hydroxybenzimidate HC1 (Wood reagent) are compared. The Wood reagent, that does not alter the charge of the modified amino group, is superior in retention of cell viability and function. Cell-surface modification with the Wood reagent prior to radiolabeling is particularly useful for detecting antigens not readily iodinated using standard techniques.

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PubMed1985

I-region restriction of alloantigen recognition: alloantigen expressed on the surface of a hybridoma cell must be presented in the context of self class II major histocompatibility complex determinants for recognition by a dual-reactive T-cell clone.

A helper-T-lymphocyte clone, designated A10, proliferated in response to both hen egg ovalbumin (OVA) presented in the context of self I-Ak and to the alloantigen I-As. The alloantigen source could be provided by irradiated H-2s spleen cells and also by paraformaldehyde-fixed H-2s spleen cells. However, for fixed allogeneic spleen cells to stimulate proliferation of the cloned cells, it was necessary to add irradiated syngeneic I-Ak-bearing spleen cells, as fixed H-2s spleen cells added, by themselves, to A10 cells were nonstimulatory. We have extended these findings by generating a monoclonal hybridoma cell which expressed the I-As allodeterminant. Similar to our results with fixed allogeneic spleen cells, this source of alloantigen could stimulate A10 cells to proliferate only if irradiated syngeneic spleen cells were added to the cultures. These proliferative responses were effectively inhibited by anti-I-Ak monoclonal antibody (mAb) and by anti-I-As mAb. Furthermore, the response of A10 cells to the alloantigen-bearing hybridoma cells were also inhibited by the anti-L3T4 mAb GK1.5. Collectively, these data indicate that, in some situations, alloreactivity may be mediated by self class II major histocompatibility complex restriction of alloantigen-driven proliferation.

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PubMed1984

Comparative characterization of monoclonal antibodies to carbonic anhydrase.

Monoclonal antibodies (Mabs) were generated to avian carbonic anhydrase-C and characterized; their reactivity with human, murine, bovine, chicken and fish erythrocyte carbonic anhydrase-C, and with human carbonic anhydrase-B was investigated by ELISA and electroblot techniques. Reactivity of the Mabs with native and SDS-denatured carbonic anhydrase was compared. Mabs that recognize antigenic determinants shared by all the carbonic anhydrases examined were identified. The results demonstrate the potential usefulness of these particular probes for investigating various aspects of function, evolution, development and regulation of this important, but not well understood group of enzymes.

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PubMed1984

Alloreactive T cell clones.

T cell clones are useful models for studying lymphocyte function both at the level of the individual cell and in interacting systems. Murine cytolytic and non- cytolyic T cell clones have been obtained with relative ease, and the particular procedure used to derive and maintain T cell clones may influence profoundly the characteristics of the resulting cells. The method of choice depends on the specific question to be asked. Although some clones have characteristics that would have been expected on the basis of results observed with bulk cell populations, other clones have rather unexpected properties. Although most T cell clones appear to be either cytolytic or non-cytolytic, this distinction is not always absolute. A high proportion of both cytolytic and non-cytolytic T cell clones have dual reactivity. This is true for cells which by other criteria appear to be true clones. The frequency of such cells is high enough to suggest that most if not all T cells may have reactivity for more than one antigenic determinant or that antigenic determinants recognized by T cells are shared widely and unexpectedly. It is not clear whether one or two different antigen receptors account for such dual reactivity. The nature of the T cell receptor for antigen remains obscure. T cell clones, because of their homogeneous nature, should make it easier to answer these important immunological questions. Although it remains to be determined how many distinct molecules account for the numerous biological activities found in the culture supernatants from antigen-stimulated T cell clones, it is clear that these factors influence several different types of cells that are involved directly and indirectly in immune responses. IL-2 stimulates both cytolytic and non-cytolytic T cells to proliferate. BCSF causes polyclonal activation of B cells, and there may be other factors which influence B cell responses to antigenic stimulation. IL-3 apparently stimulates maturation of immature T cells. CSF stimulates production of macrophages from precursor cells found in the bone marrow. Supernatants also stimulate expression of Ia antigens by macrophages, and antigen-presenting cells have been found to bear Ia antigens. Interferon augments natural killer cell activity. Thus, regardless of how many molecules are involved in these effects, activated non-cytolytic T cells appear to be involved in a variety of ways in the modulation of immune responses.(ABSTRACT TRUNCATED AT 400 WORDS)

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PubMed1984

Contributions of T cell clones to an understanding of the T cell receptor for antigen.

Cloned populations of T cells have been used to derive clone-specific monoclonal antibodies. These antibodies influence the specific immunological functions of the cloned T cells, suggesting that these antibodies react with the T cell receptor for antigen. Immunoprecipitates have been obtained with some of these antibodies, and the characteristics of the polypeptides from different cells are surprising similar. At present, little is known about this molecular complex other than that the two polypeptides are disulfide-linked and have apparent molecular weights of about 45,000 daltons and 43,000 daltons. This molecular complex, at least in human T cells, is associated with, but not covalently linked with, another molecular complex (Leu4/T3). However, modern techniques of cellular and molecular immunology should provide information very soon about the actual role of these molecules in T cell recognition of antigen. Indeed, cDNA clones that may encode genes for one of the peptide chains have been obtained.

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PubMed1984

Control of cloned alloreactive T lymphocyte proliferative responses. A possible role for cell-surface-bound alloantigen.

Previous studies using flow cytofluorometry demonstrated that passively acquired alloantigens encoded by the I-A and H-2K/D regions of the MHC could be identified on the surface of murine cloned T lymphocytes. The present studies were designed to further characterize the nature of that bound alloantigenic material on cloned lines of alloreactive amplifier T cells (Th). Immune precipitation and polyacrylamide gel electrophoresis were used to substantiate that allogeneic I-A and H-2K/D antigens were present on he surface of these cloned Th. Using proteolytic digestion with papain, bound allogeneic I-A and H-2K/D was removed from the surface of a cloned Th designated L2. Allogeneic I-A or surface of a cloned Th designated L2. Allogeneic I-A or H-2K/D antigens were not reexpressed if papain digested cells were incubated overnight in culture medium without addition of fresh alloantigen. Further, cloned cells did not release detectable amounts of interleukin 2 (IL-2) in response to Con A stimulation immediately following enzymatic digestion. After overnight culture, papain-digested cells released amounts of IL-2 similar to untreated controls. The proliferative response of these cells measured by 3H-thymidine incorporation, however, was significantly less than that observed using undigested controls, unless alloantigen was provided in the form of fresh irradiated stimulating cells. The addition of partially purified preparations of monoclonal anti Ia antibodies to cultures of cloned Th resulted in marked reduction in incorporation of 3HTdR. These findings are consistent with the hypothesis that modulation of the Th proliferative response may occur at least partially through binding of allogeneic material to the cell surface.

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PubMed1984

Cell surface structures involved in T cell activation.

We have discussed four specific models which provide different kinds of information about the requirements for T cell activation. The first utilized a CTL clone designated L3, which is reactive specifically with Ld alloantigen, to study the involvement of the associative recognition structure Lyt-2 in cytolysis. The apparent requirements for activation of this CTL clone differ depending on whether the target cells bear specific alloantigen or are hybridoma cells which express on their cell surface a clonotypic antibody which reacts specifically with the L3 T cell receptor for antigen. When the antigen receptor reacts with alloantigen on the allogeneic target cell, cytolysis is inhibited by anti-Lyt-2 antibody. However, when the clonotypic antibody of the target cell reacts with the antigen receptor of the T cell, cytolysis is much less inhibited by anti-Lyt-2 antibody. The antigen receptor seems to be responsible for the specificity of both these interactions but the avidity of the interaction between CTL and target cell seems to differ in the two situations. Evidence that participation of the L3T4 associative recognition structure on HTL is less important for cloned T cells which have higher affinity antigen receptors was provided by the second model system which used cloned HTL selected for optimal responses to different concentrations of nominal antigen. Proliferative responses of those clones which responded to lower antigen concentrations were less readily inhibited by anti-L3T4 mAb. Evidence provided by these two model systems is consistent with the concept that associative recognition structures are of lesser importance for T cell activation for those T cells which have higher affinity antigen receptors. In the third model system, we have identified several monoclonal antibodies which augment proliferative response of cloned T cells to sub-optimal amounts of IL-2, probably by reacting with the antigen receptor or with the associated Leu-4/T3 structure. The reactivity patterns of these antibodies indicate that several different epitopes are being recognized. Some appear to be clonotypic although they do not block functional activity of the clone with which they react. Others react with all T clones which we have tested. Several of these react with a cell surface antigen which is expressed at about the same level as the clonotypic structures: these antibodies may react with the murine equivalent of the human Leu-4/T3 molecular complex. One of the "pan-T cell" antibodies which augments IL-2-induced T cell proliferation appears to react with Thy-1; this antibody is similar to one described recently by Gunter et al. (1984).(ABSTRACT TRUNCATED AT 400 WORDS)

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PubMed1984

Enhancement of macrophage survival and complement production by factors from cloned T cells.

The addition of supernatants of some cloned mouse T cell lines to guinea pig peritoneal cells in tissue culture increased the production of the second (C2) and fourth (C4) components of complement as well as the enzyme beta-glucuronidase. Enumeration of cell populations demonstrated a simultaneous increase in cell survival. Supernatants that augmented functions of the cultured cells were obtained after alloantigen stimulation of a T cell line of C57BL/6 origin termed L2, but supernatants from a variant cell line derived from L2 and termed L2V had no effect. A delay of as little as 24 h in the addition of L2 T cell factors at the start of the cultures markedly diminished the effects on the cultured cells that were ordinarily observed 1-2 weeks later. These experiments suggest that, in this xenogenic system, complement-enhancing activity is part of a general stimulation of macrophages by cloned T cell factors.

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PubMed1984

Monoclonal antibody to L3T4 blocks the function of T cells specific for class 2 major histocompatibility complex antigens.

The ability of antibody to Lyt-2 and to the newly described L3T4 antigen to block the functions of helper T cells with specificity for allogeneic class 1 or class 2 MHC subregion antigens was determined. Anti-Lyt-2 blocked both allohelp delivered by unprimed T cells and IL 2 production by primed T cells when the response was directed to class 1 MHC antigens, but had no effect when the response was directed to class 2 MHC or Mls antigens. Anti-L3T4 had reciprocal activity. A control reagent, anti-LFA-1, blocked all responses tested regardless of specificity. This and other reports provide strong evidence that L3T4 is the murine equivalent of the human T cell markers Leu 3/OKT4. The ability of antibodies directed against Lyt-2 and L3T4 to block T cell function in a fashion determined by the class of MHC antigen recognized by the T cell further supports the hypotheses that Lyt-2 and L3T4 molecule in the mouse and Leu-2 and Leu-3 molecules in the human are involved in the interaction of the T cell with class-specific determinants on MHC molecules.

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PubMed1984

Comparison of allogeneic and self-restricted stimulation of lymphokine production by dual-reactive cloned T cells.

Murine T cell clones were derived that proliferated in response to stimulation by alloantigen or by ovalbumin (OVA) in the presence of irradiated syngeneic spleen cells. Two cloned cell lines of strain B10.BR (H-2k) proliferated in response to alloantigen encoded by I-Ab, whereas the response to OVA was restricted by an element encoded by I-Ak. A cloned cell line of strain B10.A (H-2a) proliferated in response to alloantigen encoded by I-As, whereas the response to OVA was restricted by an element encoded by I-Ak. Cloned cells were stimulated by alloantigen or by OVA to produce lymphokines and to incorporate thymidine. Culture supernatants were collected 24 hr later and were assayed for interleukin 2, colony stimulating factor, interferon, Ia-inducing activity, and interleukin 3; thymidine incorporation was measured 72 hr after stimulation. For each clone tested, stimulation by alloantigen or by OVA led to the production of an identical array of lymphokines. Likewise, the strength of stimulation by alloantigen was approximately equal in magnitude to the strength of stimulation by a particular concentration of OVA. Lymphokine production and thymidine incorporation were co-variant measures of the intensity of stimulation. These data, in combination with data linking OVA reactivity and alloreactivity to identical regions of the major histocompatibility complex, suggest that dual reactivity represents a cross-reaction between alloantigen and self determinants associated with nominal antigen.

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PubMed1984

Antigen-reactive cloned helper T cells. I. Unresponsiveness to antigenic restimulation develops after stimulation of cloned helper T cells.

Murine helper T lymphocyte (HTL) clones reactive to ovalbumin (OVA) were maintained in continuous culture in vitro. Clones were propagated by weekly stimulation in the presence of irradiated splenic filler cells, antigen, and supernatant fluid (SF) containing IL 2. By varying the quantity of these reagents in cultures of HTL cells, the reactivity to antigen of the cloned cells was altered markedly. After stimulation by antigen or SF, HTL clones became profoundly unresponsive to antigenic restimulation. Cells remained unresponsive for 2 to 9 days after stimulation, depending on the culture conditions that were chosen for their maintenance. The addition of SF containing a high concentration of IL 2 prolonged the duration of unresponsiveness by 3 days, and the presence of a non-T splenic filler cell increased the period of unresponsiveness by an additional 4 days. The use of a high concentration of OVA in cell cultures also prolonged the time of unresponsiveness. The results described here demonstrate that the response to antigen of HTL cells is down-regulated after stimulation, and appears to be correlated with exposure to SF that contains IL 2.

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PubMed1984

Antigen-reactive cloned helper T cells. II. Exposure of murine cloned helper T cells to IL 2-containing supernatant induces unresponsiveness to antigenic restimulation and inhibits lymphokine production after antigenic stimulation.

Cloned murine helper T lymphocytes (HTL) reactive to alloantigen or to ovalbumin (OVA) become unresponsive to antigenic restimulation after exposure to antigen or to culture supernatant fluids (SF) containing multiple lymphokine activities. Unresponsiveness is manifest by a failure of antigen-stimulated cells to incorporate thymidine or to produce lymphokines after antigenic challenge. Antigen-unresponsive HTL, however, will incorporate thymidine when exposed to an exogenous source of interleukin 2 (IL 2). The duration of unresponsiveness to antigen is correlated with the concentration of IL 2 in SF to which the cloned HTL had been exposed. Chromatographic fractionation of IL 2-containing supernatant from EL-4 thymoma cells (EL-4 SF) yielded a pool of SF that was enriched for IL 2 activity. Exposure of HTL to lymphokines contained in this pool induced unresponsiveness to antigen that was comparable to that observed when HTL were exposed to unfractionated EL-4 SF. Unresponsiveness to antigen also developed after cloned HTL were stimulated with concanavalin A (Con A) or with OVA and syngeneic splenic filler cells. We have used monoclonal antibody (mAb) GK1.5 (anti-L3T4) to investigate the role of lymphokine production in the induction of unresponsiveness. This antibody did not inhibit IL 2-induced thymidine incorporation by cloned HTL, and did not inhibit the induction of unresponsiveness after exposure of cloned HTL to EL-4 SF. In the presence of mAb GK1.5, however, HTL that were stimulated with Con A or OVA did not become unresponsive to antigenic restimulation, an effect that was overcome by the addition of EL-4 SF. These results suggest that HTL become unresponsive to antigen after exposure to IL 2-containing SF, and that stimulation by antigen or Con A can induce the unresponsive state by virtue of stimulating lymphokine production.

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PubMed1984

Evidence implicating I region-restricted antigen presentation in alloantigen and nominal antigen recognition by a dual-reactive helper T lymphocyte clone.

A helper T lymphocyte clone, designated A10, was generated from spleen cells of a B10.A mouse and demonstrated reactivity to both the nominal protein antigen hen egg ovalbumin (OVA) presented by I-Ak-bearing antigen-presenting cells (APC) and irradiated I-As-bearing spleen cells in the absence of OVA. A stimulatory signal was delivered to the cloned cells by syngeneic spleen cells that were exposed to OVA and then fixed with paraformaldehyde. However, paraformaldehyde-fixed allogeneic spleen cells that bear the I-As determinant recognized by a monoclonal antibody (mAb) were unable, by themselves, to stimulate the A10 cells. The inability of fixed allogeneic spleen cells to stimulate was rectified by the addition of irradiated I-Ak-positive spleen cells, suggesting that at least in this situation, alloantigen must be presented to the alloreactive A10 cells. Further evidence supporting this proposal for class II-restricted presentation of alloantigen included the observations that 1) irradiated I-Ak-negative spleen cells were unable to present the fixed H-2s spleen cells, 2) the anti-I-As mAb blocked the stimulatory signals delivered by irradiated H-2s spleen cells and by fixed H-2s spleen cells plus irradiated syngeneic spleen cells, 3) some anti-I-Ak mAb preparations were able to inhibit stimulation by OVA and by fixed H-2s spleen cells plus irradiated syngeneic spleen cells, and 4) an anti-L3T4a mAb effectively blocked all three stimulatory signals. These data suggested that alloreactivity can be mediated by an antigen-presentation process similar to that proposed for nominal peptide antigen presentation, and that alloantigen in the form of paraformaldehyde-fixed allogeneic spleen cells is recognized in the context of self-determinants before a stimulatory signal can be delivered.

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PubMed1984

Effects of monoclonal antibodies directed against murine T lymphocyte cell surface antigens on lymphokine production by cloned T lymphocytes reactive with class I MHC or Mls alloantigens.

Monoclonal antibodies recognizing murine T lymphocyte cell surface structures implicated in T lymphocyte-mediated cytolysis, including Lyt-2, L3T4, LFA-1, and a cytolytic T lymphocyte (CTL) clonotypic determinant, were used as probes to investigate the role of these structures in lymphokine production by T cell clones induced by antigen or lectin. The clone-specific antibody 384.5 bound to and inhibited antigen-induced lymphokine production by the L3 CTL clone, but did not affect lymphokine production by other T cell clones. Antibodies against the T cell surface structures Lyt-2 or L3T4, which are expressed by mutually exclusive T cell subsets, inhibited antigen-induced lymphokine production by class I major histocompatibility complex (MHC) antigen-reactive CTL clones or an M1s-reactive helper T lymphocyte (HTL) clone, respectively. Antibody against the broadly distributed LFA-1 molecule inhibited antigen-induced lymphokine production by all of the clones tested. Lectin-induced lymphokine production by cloned T cells was not inhibited by the clonotypic antibody, anti-Lyt-2, or anti-LFA-1; slight inhibition of the HTL clone was observed with the anti-L3T4 antibody. None of these structures appear to be uniquely involved with a particular functional response. Our results suggest that each of these structures is involved with the interactions between the effector cell and the stimulating cell leading to lymphokine production.

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PubMed1984

TRF requirements for in vitro PFC responses to SRBC and R36a. I. TRF is distinct from IL 2 but indistinguishable from polyclonal BCSF.

In vitro PFC responses to the thymus-independent (TI) antigen Streptococcus pneumoniae R36a require T cell replacing factor(s) (TRF). This requirement for TRF is as significant as for the thymus-dependent (TD) antigen SRBC. TRF is shown to be distinct from IL 2 by the following observations: 1) culture supernatants from the cloned T cell line L2, collected over an 8-day period after allogeneic stimulation, transiently contain IL 2 activity but maintain high levels of TRF activity throughout 192 hr; 2) L2V, a variant subclone of L2, produces much higher levels of TRF activity than the parental line but no detectable IL 2 activity; 3) the addition of IL2+, TRF- supernatants from the T cell hybridoma FS6-14.13 does not affect the L2V SF-driven PFC responses to R36a or SRBC; and 4) the addition of contaminating T cells to cultures containing T cell-depleted spleen cells, L2V SF, and antigen does not affect the PFC response. TRF does appear to be indistinguishable from polyclonal B cell stimulating factor (BCSF), which stimulates polyclonal PFC responses in the absence of antigen, mitogen, or anti-Ig. The TRF and BCSF activities of L2V SF could not be separated by ion-exchange, hydrophobic-interaction, and gel-filtration chromatography. TRF and BCSF have an apparent m.w. of approximately 40,000.

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