Natural cytotoxic autoantibody against thymocytes in spontaneously hypertensive rats

A strain of SHR rats, which spontaneously develops hypertension and periarteritis nodosa, had a decreasing number of rosette-forming T cells in their thymuses and a progressive decline in cellular immune functions by aging. They were found to produce natural thymocytotoxic autoantibody (NTA) detected by a complement-dependent cytotoxicity test. This autoantibody occurred from 1 month of age and throughout life; the incidence was more than 60% of SHR rats at any age. Thymocytes from all six rat strains tested showed similarly high sensitivity to NTA but none of the strains tested produced NTA except the SHR strain. Rosette-forming thymocytes of WKA rats, which were separated by Ficoll gradient, showed much higher cytotoxic sensitivity to NTA than did whole thymocytes and nonrosetting thymocytes. The cytotoxicity of NTA was weak or negative for spleen cells, lymph node cells, bone marrow cells, and blood lymphocytes of WKA rats. However, the cytotoxic activity of NTA was completely absorbed with the thymus, spleen, lymph node cells, and brain homogenates and was partially absorbed with bone marrow cells, but not with liver and kidney homogenates. NTA in SHR rats was an IgM-globulin as determined by sensitivity to 2-ME treatment and by Sephadex G-200 column chromatography. These results suggest that NTA is responsible for the selective suppression of T-cell functions in SHR rats.     

Inhibition of erythroid cell growth by allogeneic murine lymphocytes. Evidence for a synergism between lymph node cells and thymocytes

Murine lymphoid cells from thymus and lymph nodes were tested for synergistic response in a graft-vs-host test. The test is based on the principle that allogeneic lymphocytes inhibit erythroid cell proliferation in the spleens of irradiated mice infused with syngeneic bone marrow cells.I was observed that mixtures of thymocytes and lymph node cells from the same parental strain yielded graft-vs-host responses in irradiated F₁-hybrids higher than expected by summing the responses of the two cell populations tested separately. A similar synergistic response was obtained using mixtures of thymocytes and lymph node cells obtained from the two parental strains of the hybrid, whereas such an effect was not detected using mixtures of lymph node cells or mixtures of thymocytes from the two parental strains. Nor could synergy be demonstrated between parental strain lymph node cells and thymocytes syngeneic with the bone marrow target cells. Thymocytes obtained from one parental strain which were injected into its irradiated F₁-hybrid transformed into a population of sensitized cells in the spleens of the recipients. This transformation was suppressed by the simultaneous injection of lymph node cells from the second parental strain. Since there is a synergistic immune response by such cell mixtures it is concluded that thymocytes may enhance the graft-vs-host response of lymph node cells. Parental strain thymocytes and lymph node cells, the latter being specifically immunologically tolerant to the bone marrow target cells, failed to give a synergistic response indicating that thymocytes do not transform unresponsive lymphocytes into responsive, but rather enhance the reactivity of existing, specifically responsive cells.The results thus show that thymocytes may enhance the response of lymph node cells in this specific graft-vs-host assay.     

The distribution of terminal deoxynucleotidyl transferase (TdT) among subsets of thymocytes in the rat.

Terminal deoxynucleotidyl transferase (TdT), a unique DNA-polymerizing enzyme,has been shown to be present in a moderately dense subpopulation of rat thymocytes separated on discontinuous Ficoll density gradients. This subpopulation has been characterized by using antigenic and functional markers to identify directly and quantify cortical and medullary thymocytes. The TdT-positive thymocytes are depleted by cortisone administration, lack responsiveness to phytohemagglutinin, concanavalin-A, and histocompatibility alloantigens, bear surface antigens characteristic of cortical thymocytes (bone marrow lymphocyte antigen) and lack surface antigens characteristic of medullary thymocytes (rat-masked thymocyte antigen and histocompatibility antigens). The results indicate that TdT is present exclusively (or in markedly higher concentrations) in a subset of cells which comprised about 65% of cortical thymocytes. Two other major subsets of cortical thymocytes were identified which appeared to be TdT-negative. A minor subset of very low density cortical thymocytes was also defined. These observations have provided insight into the possible pathways of thymocyte ontogeny.     

Augmentation of erythroid burst formation by the addition of thymocytes and other myelo-lymphoid cells

Bone marrow from barrier-sustained specific pathogen-free (SPF) CBA and C57BL/6 mice gave relatively low numbers of BFU-E colonies in methylcellulose culture, as compared to conventional mice. Addition of thymocytes to the marrow cultures increased the yield of BFU-E colonies more than fourfold in SPF mice but only 1.5-fold in conventional mice. Colony size was also increased. Increased yield of BFU-E colonies was also obtained by co-culture of bone marrow with lymph node cells or with bone marrow or spleen cells from 900R whole-body-irradiated mice. The effect appeared to be cellular rather than humoral. It was not reproduced by conditioned medium from thymus or pokeweed mitogen stimulated spleen cells. The helper effect of thymus cells was eliminated or reduced by freezing and thawing, or by 48 hours of incubation after irradiation. Treatment of bone marrow cells in vitro with anti-theta serum and complement did not decrease the number of BFU-E colonies. The putative helper cells appear not to be T cells, were non-adherent to the plastic culture dish, and were cortisone resistant and radioresistant. The low BFU-E colony yield from SPF mouse marrow is presumed to be largely the result of deficiency of these non-T helper cells in SPF bone marrow, rather than of BFU-E progenitor cells.     

Antigenic relationship between bone marrow lymphocytes cortical thymocytes and a subpopulation of peripheral T cells in the rat: description of a bone marrow lymphocyte antigen.

Populations of rat bone marrow lymphocytes (BML) consisting of approximately 90 percent, “tnull” cells were prepared by density gradient centrifugation, passage through a column of fine glass beads, and treatment with anti-T cell and anti-B cell serum plus complement. Antisera to these bone marrow lymphocytes were raised in rabbits. After absorption with RBC and peritoneal exudate cells, the anti-BML sera were found by immunofluorescence to react selectively with “null” cells in bone marrow, with cortical thymocytes, and with a cortisone-sensitive subset of T cells in blood and in spleen, possibly in red pulp. The antigen that is common to these cell types is designated the rat bone marrow lymphocyte antigen (RBMLA). Lymphocytes that are positive fur KBMLA are negative for another lymphocyte-specific heteroantigen, rat musked thymocyte antigen (RMTA). As shown previously, RMTA is present on medullary thymocytes and ou cortisone-resistant T cells in white pulp of spleen, paracortex of lymph node and thoracic duct lymph. It is postulated that two developmentally and functionally distinct lines of T cells exist in peripheral lymphoid tissues of the rat, one derived from cortical thymocytes and one derived from medullary thymocytes. It is further postulated that the “null” population of bone marrow lymphocytes contains the lymphopoietic stem cells from which these two lines of T cells originate.     

Suppression of cell-mediated immune responses after total lymphoid irradiation (TLI). I. Characterization of suppressor cells of the mixed lymphocyte reaction

Total lymphoid irradiation (TLI) was administered to (BALB/c X C57BL/6)F1 mice in eight daily doses of 200 rad (total 1600 rad). Spleen cells isolated from mice after treatment with TLI do not respond to alloantigens in vitro in a one-way mixed lymphocyte reaction (MLR), but normal reactivity recovers after approximately 2 mo. Radioresistant, antigen-nonspecific suppressor cells are documented in the spleens of TLI-treated mice immediately after radiotherapy, but suppressive capacity gradually disappears within 30 days. After TLI, the spleen is repopulated with large cells, the proportion of which is greatest at a time when theta-bearing cells are still depleted. Radioresistant suppression is mediated predominantly by the large cell subset and is thymus independent. Suppressor function can be abolished by lethal physicochemical procedures including formaldehyde fixation, multiple freeze-thawing, and heating to 56 degrees C, and it cannot be conferred by supernatants of TLI-suppressed MLR suspensions. Suppression cannot be overcome by adding various cell factors including T cell growth factor (TCGF) and lymphocyte-activating factor (LAF), nor is it affected by a prostaglandin inhibitor. Equally potent radioresistant suppressive activity is documented by co-culturing cells derived from other sources enriched in large, immature hematopoietic cells, including fetal liver cells and bone marrow cells obtained from normal and congenitally athymic mice. The presence of a large cell population and MLR suppressor function is also documented in the spleens of mice treated with single dose or fractionated doses of lethal whole body irradiation, followed by reconstitution with bone marrow cells obtained from normal mice. The data suggest that MLR suppressor cells, which are large, immature and predominantly radioresistant, can be induced after a short and well-tolerated TLI regimen.     

Intrathymic T cell differentiation in radiation bone marrow chimeras and its role in T cell emigration to the spleen. An immunohistochemical study

Immunohistochemical studies were made on the regeneration of T cells of host- and donor-type in the thymus and spleen of radiation bone marrow chimeras by using B10- and B10.BR-Thy-1 congenic mice. Both the thymic cortex and the medulla were first repopulated with thymocytes of irradiated host origin, restoring the normal histologic appearance by days 11 to 14, regardless of the H-2 compatibility between the donor and the host. In Thy-1 congenic chimeras, thymocytes of donor bone marrow origin, less than 100 cells in one thymic lobe, were first recognized at day 7, when the thymus involuted to the smallest size after the irradiation. The thymocytes of donor-type then proliferated exponentially, showing a slightly faster rate when higher doses of bone marrow cells were used for reconstitution, reaching a level of 100 million by day 17 and completely replacing the cortical thymocytes of host origin by day 21. The replacement of cortical thymocytes started from the subcapsular layer in a sporadic manner. The replacement of medullary thymocytes from host- to donor-type occurred gradually between days 21 and 35, after the replacement in the cortex was completed. In the spleen, about 1 million survived cells were recovered at day 3 after the irradiation, and approximately 60% of them were shown to be host-type T cells that were observed in the white pulp areas. The host-type T cells in the spleen increased gradually after day 10, due to the influx of host-type T cells from the regenerating thymus. Thus a pronounced increase of T cells of host-type was immunohistochemically observed in the splenic white pulp between days 21 and 28, when thymocytes of host-type were present mainly in the thymic medulla. These host-type T cells were shown to persist in the spleen for a long time, as long as 420 days after the treatment. Phenotypically, they were predominantly Lyt-1+2+ when examined at day 28, but 5 mo later, they were about 50% Lyt-1+2+ and 50% Lyt-1+2-. Donor-type T cells in the spleen began to appear at about day 14 in chimeras that were transplanted with a larger dose of bone marrow cells, whereas this was slightly delayed in those grafted with a smaller dose of bone marrow cells, starting at about day 28.(ABSTRACT TRUNCATED AT 400 WORDS)     

Detection of a cell surface antigen common to a mouse myeloma and a population of mouse thymocytes.

A new antigen termed PTA (plasmocytoma thymus antigen) is described which occurs on 70--80% of mouse thymocytes of all mouse strains tested and on an IgG 2b producing BALB/c plasma cell tumor. PTA is detectable on mouse spleen and lymph node cells. It is absent from liver, brain, bone marrow, and antibody plaque forming cells. PTA is not related to theta antigen and is not detectable on rat thymocytes.     

CFU-S and haematopoietic microenvironment in mice after fractionated irradiation. A study on spleen colonies.

The paper is aimed at evaluating the quantity and quality of the haematopoietic stem cells, CFU-S, in the bone marrow and the functional effectiveness of the haematopoietic microenvironment of the spleen in two time intervals after repeated exposure of mice to doses of 0.5 Gy gamma-rays once a week (total doses of 12 and 24 Gy). After irradiation, bone marrow was cross-transplanted between fractionatedly irradiated and control mice. The parameter evaluated were numbers of spleen colonies classified into size categories. The data obtained provide evidence for a significant damage to the CFU-S, concerning both their number and proliferation ability, after both total doses used. The functional effectiveness of the haematopoietic microenvironment of the spleen was impaired only in bone marrow recipients receiving a transplant after having been exposed to a total dose of 24 Gy; this dose combined with subsequent pre-transplantation irradiation resulted in a marked suppression of cell production within the spleen colonies formed from a normal bone marrow on the spleens of fractionatedly irradiated mice.     

Antigenic relationship between medullary thymocytes and a subpopulation of peripheral T cells in the rat: description of a masked antigen.

Using differentially absorbed rabbit antisera to rat thoracic duct cells, an antigen is described which normally is expressed on the surface of T cells in thoracic duct lymph and lymph node, but which exists in a masked form on medullary thymocytes and apparently not at all on cortical thymocytes. This antigen is termed the rat masked thymocyte antigen (RMTA). RMTA on medullary thymocytes can be unmasked mechanically by sectioning in a cryostat or enzymatically by treating with neuraminidase. Trypsin destroys or removes RMTA. Nearly all the T cells in thoracic duct lymph and lymph node are RMTA⁺, whereas only 58–66% of T cells in spleen are RMTA⁺. RMTA⁺ T cells, which are cortisone resistant, reside in the paracortex and periarteriolar sheath regions of lymph node and spleen. RMTA^? T cells, which are cortisone sensitive, appear to reside in the red pulp of spleen. The results suggest that (i) two antigenically distinct populations of T cells exist in the rat, RMTA⁺ and RMTA^? T cells, (ii) medullary thymocytes are the immediate precursors of RMTA⁺ T cells, and (iii) cortical thymocytes may be the immediate precursors of RMTA^? cells.     

A bicellular mechanism in the immune response to chemically defined antigens. 3. Interaction of thymus and bone marrow-derived cells

The role of thymus and bone marrow-derived cells in the in vitro response to the dinitrophenyl (DNP) determinant was studied using the millipore filter well technique for spleen organ cultures. Antibodies to DNP were assayed by the technique of inactivation of DNP-coupled T-4 bacteriophage. It was found that spleens of mice total-body irradiated at 750 R, treated with bone marrow and thymus cells after exposure and immunized against rabbit serum albumin (RSA) were able to produce antibodies to DNP when challenged in vitro with DNP-RSA. Such a response was not produced by spleen explants from x-irradiated mice treated with either thymus or bone marrow cells. Neither were antibodies to DNP produced by spleens of animals repopulated with thymus and bone marrow cells, but not immunized with the carrier. This carrier effect was manifested when the irradiated mice were treated with RSA and thymus cells 6–8 days before administration of the bone marrow cells. Yet, such an effect was not observed when the RSA and bone marrow cells were given 6–8 days before injection of the thymus cells. Thus, the thymus-derived cells appear to play the role of cells sensitive to the carrier (RSA), whereas the bone marrow seems to be involved in the production of antibodies.     

The immunocompetence of murine stromal cell-associated thymocytes

Thymocyte subpopulations that associate in vivo with distinct nonlymphoid cells of the thymus have been isolated, and their immunocompetence was analyzed. Previous studies have indicated that greater than 95% of such cells bear a surface antigen phenotype representative of immature thymocytes, and are among the earliest thymic compartments repopulated by bone marrow-derived cells after lethal and sub-lethal irradiation. Stromal cell-associated thymocytes may be activated in vivo because they proliferate well in vitro with no additional stimulus, and show little increase in proliferation with the addition of T cell mitogens or allogeneic spleen cells. Stromal cell-associated T cells contain cytotoxic T lymphocyte (CTL) precursors that are indistinguishable from mature peripheral T cells by the parameters of self tolerance, alloreactivity, H-2 restriction, and stringency of self H-2 preference. CTL precursor frequencies and the cytotoxic activity of cells further separated on the basis of high levels of Thy-1 expression argue against the possibility that stromal cell-associated CTL activity is due solely to contaminating mature lymphocytes. Our data suggest that stromal cell-associated thymocytes represent an intermediate subpopulation of thymocytes that is functionally mature and that expresses an immature surface phenotype. Furthermore, the imposition of self tolerance and MHC restriction specificity appears to be tightly associated with the acquisition of immunocompetence in these thymocyte subpopulations.     

Glycolytic inhibitor, 2-deoxy-D-glucose, does not enhance radiation-induced apoptosis in mouse thymocytes and splenocytes in vitro

Earlier studies have shown that 2-deoxy-D-glucose (2-DG), a glucose analogue and inhibitor of glycolytic ATP production selectively enhances radiation-induced damage in cancer cells by inhibiting the energy (ATP) dependent postirradiation DNA and cellular repair processes. A reduction in radiation induced cytogenetic damage has been reported in normal cells viz., peripheral blood lymphocytes and bone marrow cells. Since induction of apoptosis plays a major role in determining the radiosensitivity of some most sensitive normal cells including splenocytes and thymocytes, we investigated the effects of 2-DG on radiation induced apo tosis in these cells in vitro. Thymocytes and splenocytes isolated from normal Swiss albino mouse were irradiated with Co60 gamma-rays and analyzed for apoptosis at various post-irradiation times. 2-DG added at the time of irradiation was present till the termination of cultures. A time dependent, spontaneous apoptosis was evident in both the cell systems, with nearly 40% of the cells undergoing apoptosis at 12 hr of incubation. The dose response of radiation-induced apoptosis was essentially similar in both the cell systems and was dependent on the incubation time. More than 70% of the splenocytes and 60% of the thymocytes were apoptotic by 12 hr following an absorbed dose of 2 Gy. Presence of 2-DG marginally reduced the fraction of splenocytes undergoing apoptosis at all absorbed doses, while no change was observed in thymocytes. Presence of 2-DG did not significantly alter either the level or the rate of induction of spontaneous apoptosis in both these cell systems. These results are consistent with the earlier findings on radiation-induced cytogenetic damage in human PBL in vitro and mouse bone marrow cells and lend further support to the proposition that 2-DG does not enhance radiation damage in normal cells, while radiosensitizing the tumors and hence is an ideal adjuvant in the radiotherapy of tumors.     

The distribution of adenosine deaminase among lymphocyte populations in the rat.

Adenosine deaminase (ADA) activity was determined in young rat lymphocyte populations. The ADA-specific activity (per 10(8) cells and per milligram protein) was 3- to 10-fold higher in thymocytes than in lymphocytes from thoracic duct, lymph node, spleen, and bone marrow. The high ADA activity in thymocytes appeared to be preferentially associated with cortical thymocytes. Enrichment or depletion of cortical thymocytes by density gradient centrifugation, cortisone treatment, or selective lysis with anti-Thy-1 plus complement resulted in parallel increases or decreases in ADA levles. These results also suggested that medullary thymocytes have ADA levels similar to those of peripheral lymphocytes. "Immature" cortical thymocytes and thymocyte progenitors appeared to have low ADA activity; low enzyme levels were found in fetal thymus at 16 days of embryonic life, in the early phases of thymus regeneration, and in a "null" cell population isolated from bone marrow. This study demonstrates that ADA activity varies markedly during T lymphocyte differentiation and suggests that fundamental differences in nucleotide metabolism may exist in T cells at different stages of development.     

Modulation of antibody synthesis by cholera exotoxin: Influence on helper thymocytes

This report demonstrates that a major biological influence of cholera exotoxin (CT) on antibody formation to sheep erythrocytes is mediated by the toxin's influence on the helper function of thymic lymphocytes. Hemolytic plaque-forming cell (PFC) responses for lethally X-irradiated mice repopulated with isologous thymus-marrow cell suspensions were stimulated three- to fivefold when 1.0 μg CT was administered at cell transfer. For mice given marrow cells only, CT did not elevate the PFC response; however, CT stimulated as many PFC in spleens of mice given marrow and 1 × 10⁷ thymus cells as mice given marrow and 4 × 10⁷ thymus cells. In other transfer experiments, depressed PFC responses were observed when irradiated mice were given marrow and thymus cells from donor mice inoculated with CT 24 hr prior to cell preparation and infusion, or given marrow cells from normal mice and thymus cells from CT-treated mice. In contrast, mice given marrow from CT-treated mice and thymus cells from normal mice responded as well as mice repopulated with normal thymus-marrow cell suspensions.     

Strain differences in the early development of the thymus-dependent cells: precocity of T lineage cells in AKR mice as compared to those in C3H mice

Early development of T lineage cells were compared between AKR and C3H mice by using two experimental strategies--neonatal thymectomy (NTx) and bone marrow transplantation (BMT)--between these two strains of mice. After NTx, AKR mice developed less wasting disease and showed better maintenance of several T cell functions. In addition, the response of neonatal spleen cells to PHA and ConA was much greater in AKR mice than in C3H mice. Further, when AKR mice were used as recipients of BMT, cell numbers recovered from thymuses between 2 and 7 weeks after reconstitution were consistently much greater (about 10 times greater) than those from chimeras where C3H mice were used as recipients, regardless of the donor strains of bone marrow cells. However, 4 weeks after BMT the proliferative responses to ConA were consistently higher in the donor-derived thymocytes from chimeras where AKR mice were used as bone marrow donors than in those from chimeras in which C3H were donors. The present findings suggest that these differences may be attributed to characteristics of recipient microenvironment (e.g., thymic stroma) which maintain developing thymocytes and supply them to the peripheral lymphoid tissue. Alternatively the differences may to some degree also be attributable to characteristics of the thymic progenitors themselves, which may determine the rates of maturation of thymocyte functions.     

Autoreactive antibody-forming cells directed against thymocytes and thymus-derived lymphocytes.

Antibody-forming cells with specificities against syngeneic and allogeneic thymocytes are detected in the spleens of normal mice after activation in vitro or in vivo with lipopolysaccharide (LPS). The activity of such cells was measured in a complement-dependent plaque assay employing trypan blue dye to assess zones of lysis. Plaques were rarely seen in the absence of LPS treatment. Anti-immunoglobulin added to the plaque assay abrogated the appearance of plaques, but the addition of LPS had no effect. Furthermore, plaque formation was 2-mercaptoethanol sensitive indicating that the antibody responsible was of the IgM class. Plaque forming cells (PFC) were also detected against syngeneic and allogeneic lymph node cells and to a much lesser extent against splenocytes. The numbers of PFC found against syngeneic, allogeneic, or a mixture of thymocytes was similar and ranged from 1000 to 3000 PFC/10(8) viable spleen cells tested. All murine strains tested, including congenitally athymic nude mice, exhibited anti-thymocyte PFC after LPS activation. C3H/HeJ mice, genetically unresponsive to LPS, did not respond mitogenically to LPS and no anti-thymocyte plaques were observed. These findings suggest that clones of autoreactive B cells are present in normal mice and can be activated by LPS.     

Opposing reactivities of subpopulations of T lymphocytes toward syngeneic tumor cells: separation of early thymocytes.

The present communication is a continuation of earlier studies which indicated that interaction between syngeneic tumors and those lymphocytes in the early stages of thymic processing can result in enhanced tumor growth in vivo. The thymocytes involved in this tumor enhancement were found previously in the rapidly dividing subpopulation of subcapsular cortical thymocytes, both in the untreated thymus and in the thymus undergoing repopulation after cortisone depletion. In the present experiments we have isolated this small subpopulation of early thymocytes. After cortisone injection such cells could be separated from the medullary cortisone-resistant thymocytes since the latter cells exhibit a high level of surface H-2 antigens and were thus lysed preferentially by anti-H-2 serum and complement. The repopulating subcapsular early thymocytes, which were resistant to this treatment, were incapable of responding to PHA while their basal proliferation rate was undiminished, and the majority of the cells were found to be dividing. When such low H-2 early thymocytes were injected together with three different tumors into syngeneic mice their tumor-enhancing activity was evident. It is clear that such early thymocytes are not devoid of biologic reactivity and their release from the thymus could have decisive results.     

A chemotactic factor for rat thymocytes may regulate T-lymphocyte migration toward the thymic microenvironment

Using a modified Boyden chamber assay, extracts or culture supernatants of rat thymic stromal cells, or thymocytes were examined by chemotactic activity to rat leukocytes. Rat thymocytes responded chemotactically to the aqueous extract as well as to culture supernatants of thymic stromal cells. However, neither the extract and culture medium from concanavalin A-stimulated thymocytes nor any component of rat serum has shown such an activity. The thymic extract was fractionated into three molecular species with chemotactic activity for thymocytes. The thymocyte chemotactic factor(s) (TCFs) in the extract was distinct from known lymphocytic chemotactic factors, such as interleukin-1 (IL-1), IL-2, C5a, and the culture supernatant of stimulated thymocytes. In vitro, TCFs could attract, in addition to thymocytes, bone marrow cells, fetal liver cells, and nylon-wool nonadherent lymphocytes from peripheral blood and spleen. Lymph node cells, neutrophils, macrophages, and B cells from peripheral blood could not respond to TCFs. Thymocytes also responded to the extract of splenic stromal cells. Unlike the thymic extract, however, the splenic extract was chemotactically active for lymphocytes from lymph nodes but not for bone marrow cells. These results indicate that thymic stromal cells secrete a chemotactic factor(s) for a relatively immature type of T-lineage cells, which may by a thymus-homing progenitor T cell, while spleen may contain an attractant for a relatively mature type of T-lineage cells.     

Viability of rat thymocytes as affected by radiation and combined radiation-heat damage

A share of damaged (nonviable) cells of rat thymus was quantitatively estimated on days 1 and 3 following irradiation with doses of 1, 3 and 6 Gy and a combined treatment with radiation and heat. The percentage of nonviable thymocytes was higher after the combined treatment than after the effect of radiation alone delivered in equivalent doses.