Trypanosoma gambiense: Immunity with thymic cell transfer in mice

EXPERIMENTAL PARASITOLOGY 39, 234-243

Trypanosoma

(19%)

gambiense: Immunity with Thymic Transfer in Mice

TAN TAKAYANACI Department

Cell

AND YOSHISADA NAKATAKE

of Medical Zoology, Medical School, Nagoya City University, Mizuho-ku, (Accepted

Nagoya,

for publication

&pan

June 25, 1975)

TAKAYANAGI, T., AND NAKATAKE, Y. 1976. Trypanosoma gambiense: Immunity with thymic cell transfer in mice. Experimental Parasitology 39, 234-243. This report deals with the enhanced agglutinin production and protection in thymectomized, lethally irradiated mice (TI-mice) with transferred thymic cells from mice immune to T. gambiense. Such mice, when sensitized with trypanosome antigen showed protection against experimental infection and also produced agglutinins. Thymic cells from cortisone-treated immune mice were able to induce the production of agglutinins in TI-mice subsequently injected with antigen. However, these agglutinin titers were very low. In bovine serum albumin gradient centrifugation experiments, agglutinin production could be efficiently induced by inoculation of TI-mice with a rather high density thymic cell subpopuIation taken from immune mice. Fractionated by Sephadex G-200, the agglutinins displayed a division into two parts, a first and second peak. The main agglutination reaction was seen in the first or macroglobulin peak. In the fractionation of serum by DEAE-cellulose column chromatography, agglutinins were eluted in two parts, the 0.0175 M and 0.4 M effluents. The agglutination by the 0.4 it4 effluent was much stronger than that of the 0.0175 M effluent, in agreement with the gel filtration results. The sera containing agglutinins were abb to enhance the phagocytosis of trypanosomes by cultured macrophages from the peritoneal cavity of norma and irradiated mice. Delay of parasitemia was evident in some of the TI-mice having detectable agglutinins. The delayed parasitemia resulted from antigenically altered trypanosomes which were able to withstand the lethal factors of TI-mice. Transplantation of thymic cells was considered to be responsible for agglutinins induced by the antigenic stimulation in TI-mice and for protection against experimental infection. INDEX DESCRIPTORS: Typunosoma gumbiense; Thymectomy; Thymic cell suspension; Cortisone; Subpopulation; Anti-mouse thymic cell serum; Macrophage; Protective test; Agglutination test; Phagocytic test; Serotype test; Immunity; Mice; Chromatography column.

Recently there have been published reports about the immunologically important roles of the thymus, one of which is that of providing T (thymus derived) cells which are known to be responsible for cell-mediated immunity. More recently, T cells have been found to act as helpers to B (bone marrow derived) cells in the production of humoral antibody to a variety of antigens (Miller 1971).

Reports on parasitic protozoa have dealt with the role of the thymus in acquired immunity in thymectomized animals or in an anti-thymocyte those administered serum. With Plasmodium berghei, thymectomized rats were found to show increased severity of infection and increased mortaIity ( Stechschulte 1969)) whiIe neonatally thymectomized hamsters developed parasitemia more rapidly and exhibited earlier 234

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@J 1976 by Academic

of reproduction in any

Press, Inc. form reserved

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and higher mortality (Chapman and Hanson 1971). By treating rats with antilymphocytic serum prior to and during infection with Typanosoma lewisi, the immune response was suppressed and high parasitemias persisted (Tawil and Dusanic 1971). Neonatally thymectomized mice developed an enhanced parasitemia and high mortality after infection with Typanosoma cruzi (Schmunis

et al

1971). Parasitemia

and

mortality were enhanced with Chagas’ disease in mice when they were thymectomized and subsequently treated with antithymocyte serum (Behbehani 1971). Roberson and Hanson (1973) reported markedly enhanced parasitemia, increased tissue stages ( amastigotes ), and higher mortality in antithymocyte serum-treated mice. The same authors also observed a greater parasitemia and increased mortality in neonatally thymectomized rats. The present paper reports the immunologic responses to infection by Trypanosomu gambiense in thymectomized, lethally irradiated recipients of passively transferred thymic cells sensitized with parasitic antigens in uiuo. MATERIALS

AND

METHODS

Parasites. The Wellcome strain, antigenitally 0 type T. gambiense maintained in white mice (dd strain) by serial transfer at 3-day intervals, was used throughout the study. Animals. Female mice (dd strain) weighing approximately 18-20 g were used for all experiments. Wistar male rats weighing approximately 100-150 g were used for collection of parasites. Young adult male New Zealand white rabbits were used for preparation of mouse antithymic cell serum. Male golden hamsters weighing approximately 70-100 g were used as a source of complement. Immunization of mice. Female mice were inoculated intraperitoneally (ip ) with 1

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X 10’ living trypanosomes in 0.5 ml of saline. Seventy-two hours after inoculation, each mouse was injected ip with 1 ml of fresh normal human serum to cure completely the trypanosome infection. Mice were exsanguinated by heart puncture 5 days after immunization. Pooled serum had an agglutination titer of 164, and it was stored at -25 C for immune phagocytic testing and for serotype testing as the anti-0 type test serum. The thymus was removed for use as presensitized thymic cells (hereafter referred to as i-TC) in further experiments. On the other hand, normal thymic cells derived from untreated mice were used as control (referred to as n-TC). Adult thymectomy. Female mice were thymectomized about 4 weeks prior to irradiation. Thymectomy was performed under sodium nembutal (Abbott Laboratories) anesthesia. After a skin incision, the upper third of the sternum was divided by scissors, and the two thymus lobes were removed by suction through a Pasteur pipette, The skin was then closed with Aron Alpha A ( Sankyo) (a-cyanoachrylate monomer). Mice with a macroscopically visible thymic remnant were not used. Iwadiution. About 4 weeks after operation, thymectomized mice were exposed to 800R of y-irradiation from a @OCo irradiator at a dose rate of 49.5 rads/min. Thymic cell suspension. Cell suspensions from normal and immune mice were prepared in ice cold Hanks’ balanced salt solution. Thymuses from eight to @teen mice were freed carefully from adjacent tissues and gently pressed through a 60gauge stainless steel mesh into cold Hanks’ balanced salt solution. Cell suspensions were forced through an 18-gauge needle to break up aggregates. Thymic cells were washed three times in ice cold Hanks’ balanced salt solution by centrifugation at 200g for 5 min at 4 C. The cells were kept on ice at all times while processing. Cells were counted in a hemocytometer. The percentage of viable cells immediately prior

236

TAKAYANAGI

AND

to injection into animals was estimated by the use of trypan blue. Thymic cell transplantation. Thymectomized mice which were irradiated (TImice) were injected ip within 2 hr after whole-body irradiation (800R) with OS3.0 x lOa n-TC or i-TC suspended in 0.5 ml of Hanks’ balanced salt solution. Immunization of TI-mice. One milliliter of a lo/O protein concentration in the final supernatant obtained from the homogenate of trypanosome in Hanks’ balanced salt solution prepared as described below was injected ip into TI-mice within 1 hr after the transplantation ip of n-TC or i-TC. Cortisone treatment. The decortication of the thymus of immune mice was achieved by an intraperitoneal injection of 2.5 mg/20 g body wt of cortisone acetate ( Blomgren and Andersson 1969), and 2 days later the thymus was harvested. Gradient separation. Six milliliters of a thymic cell suspension containing 3.3 x lo1 cells/ml in Hanks’ balanced salt solution were layered upon 2 ml each of 35, 29, 26, 23, and 10% bovine albumin (Cohn Fraction V, Sigma) in the tissue culture medium RPMI-1640 from the bottom to top in Pyrex glass centrifuge tubes ( 1.5 x 10 cm). The tubes were centrifuged at 2000g for 20 min at 4 C. After centrifugation, five opaque bands were observed. The layers were designated as described by Raidt et al. (1968). The top layer (above 23% albumin) was designated layer A, second layer (above 26% ) was called layer B, third layer (above 29% ) was called layer C, fourth layer (above 35%) was called layer D, and fifth layer (sediment) was called layer E. These layers, formed on the boundary of the separating media, were so distinguishable that it is easy to pipette off any fraction, The cells were harvested by means of a Pasteur pipette and washed three times with an excess (40 ml) of ice cold Hanks’ balanced salt solution. Lymphocytes of each layer were classified into two groups by their cell size, namely, large lymphocyte (cell diameter

NAKATAKE

larger than 10 pm) and small lymphocyte (smaller than 10 pm). Gel filtration chromatography. Gel filtration for separation of whole serum (2 ml) was performed at 4 C in a column, 3.0 by 100 cm, packed with Sephadex G-200 equilibrated in a solution of 0.1 M TrisHCI, pH 8.6, in 0.15 M NaCI, containing 0.1% NaN3. Elution was carried out with a flow rate of 15 ml/l-n. Four-milliliter fractions were collected and the protein distribution in the effluent was determined by measuring the optical density at 280 nm. DEAE-cellulose column chromatography. Five milliliters of whole serum were equilibrated by dialysis in 0.0175 M sodium phosphate buffer, pH 6.3, at 4 C. Then, it was fractionated on a DEAE-cellulose column (3.0 by 30 cm) containing 20 g of anion exchanger (Whatman DE52). Adsorbed proteins were removed by stepwise elution by increasing phosphate molarity to 0.4 M. Five-milliliter fractions were collected, and the protein distribution in the effluent was determined by measuring the optical density at 280 nm. Collection and separation of parasites. Wistar male rats were infected ip with 5 x lo7 parasites from a mouse infected 3 days earlier. The blood was collected 3 days after infection by heart puncture. Parasites free of host blood cell components were obtained by the method of Lanham and Godfrey (1970). The parasites were washed three times with 1% glucose phosphate buffer (GPB), ,J = 0.271, pH 7.5 and centrifuged at 800g for 10 min. They were used for agglutination tests, immune phagocytic tests, inoculations for challenge, and for making antigen solution. They were homogenized in a sonicator (20 kc) for 5 min. After centrifugation at 15,OOOg for 60 min, the supernatant was stored at -25 C until used as the antigen. Protein determination. The optical density was read at 280 nm with the aid of Toyo’s Uvicon 540 automatic recording spectrophotometer for gel filtration chromatography, DEAE-cellulose column chro-

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matography, and the determination of the protein concentration of the trypanosome antigen solution. Preparation of rabbit anti-mouse thymic cell serum. Anti-mouse thymic cell serum

was prepared by a slight modification of the method of Sutthiwan et al. ( 1969). Thymic cells were suspended at a concentration of 1 x log cells/ml in Hanks’ balanced salt solution for injection into New Zealand rabbits weighing 2-3 kg. Rabbits received three ip injections of 1 x log thymic cells at intervals of 2 weeks. One week after the final injection, the rabbits were exsanguinated by heart puncture, the bloods were allowed to clot, and the sera were collected and inactivated at 56 C for 30 min. The sera were completely adsorbed with the adherent cells of the spleen by the method of Shortman et al. ( 1971), filtrated through a Millipore filter (Millipore, pore size, 0.22 nm ), and held frozen at -25 C until used. Control normal rabbit serum was handled in the same manner. Cytotoxicity assay. 2.5 x lo5 thymic cells were incubated with 1 ml of anti-mouse thymic cell serum plus an equal volume of fresh golden hamster serum (or Hanks’ balanced salt solution plus fresh golden hamster serum) for 30 min at 37 C. Exclusion of trypan blue was evaluated within 30 min after the end of incubation, and cytotoxicity was expressed as the percentage of nonviable cells in each population. Anti-mouse thymic cell serum in the study had a titer of 1:32. Tissue culture of macrophages. Peritoneal exudates of mice were induced by the injection ip of 1 ml of lo/O sterile glycogen in Hanks’ balanced salt solution. After 4 days, the mice were killed rapidly with ether and the peritoneal exudates collected. Cells were cultured in Leighton tubes, each containing an 8 X 30 mm coverslip. Cell suspensions (2 X 10e) in volumes of 1 ml, consisting of the tissue culture medium 199, 20% calf serum, 50 units/ml penicillin and 50 rg/ml streptomycin, were dispensed

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into each culture tube. The tubes were then allowed to stand at 37 C for 60 min and then shaken vigorously before withdrawing the medium. The cell layer was next washed once with 2 ml of the medium and then overlayed with 0.8 ml of the medium for incubation. Test for protective ability. Separated trypanosomes (1 x 104) in 0.1 ml GPB were given ip to the mice, which were examined for the presence or absence of the parasites every 24 hr by wet mount. If no parasites were observed by the Day 8 after infection, then protection was considered to be complete. AggEutimtion test. Five-tenths milliliter of the different dilutions of the sera were each mixed with equal volumes of GPB containing 1 X 10s separated trypanosomes. After 10 min, the agglutination titer was determined as the highest dilution of antiserum in which agglutination occurred. Observations were done under the microscope at a magnification of 200 X. Immune phagocytic test. The culture medium was removed with a sterile pipette and then 0.25 ml of parasite suspension, 2 X 10’ cells/ml in final concentration, was added. After 1 min, 0.25 ml of test serum was added. After mixing thoroughly, the culture tube was incubated at 37 C for 5 min, and then vigorously washed twice in 2 ml of GPB to remove excess parasites. Observations were made microscopically at a magnification of 400 X. Staining. Coverslips were &ed in methanol for 10 min and stained in Giemsa. Serotype test. Mice showing parasitemia were killed rapidly by ether, and the mice were exanguinated by heart puncture with a heparin-rinsed syringe and needle. The blood was centrifuged at 8OOgfor 10 min to separate the plasma from the parasites. The plasma was used for determinations of the agglutination titer. The isolated parasites were used to determine their antigenic type with the test antiserum. The overall design of the experiment is shown in Fig. 1.

2.38

TAKAYANACI

(2)

Immunized

(3)

Immunized

No parasitemia

FIG.

at

AND

8th

day

1. Protection with Transplantation

Thymic

Cell

Thymic cells from immune mice (I-TC) were transferred ip to thymectomized irradiated mice (TI-mice). Within 1 hr after the transfer, the TI-mice were injected ip with 1 ml of lo/O parasitic antigen solution. Five days later, the mice were challenged with 1 x lo4 living parasites, and protection was evident (Table I). TABLE

Thymic cells inoculated (no.1

examination

Protection

1. Design of the experiment with Trypanosoma gumbiense RESULTS

Protection

NAKATAKE

However, in TI-mice receiving thymic cells transplanted from normal mice (nTC) and an injection of the antigen, protection was not achieved against the challenge of parasites on Day 5 after the transfer. Similarly, TI-mice recipients of i-TC without the injection of the antigen failed to show any protection. II. Production of Agglutinins by TZ-Mice with Thymic Cell Transplantation In TI-mice with transferred i-TC and stimulated with parasitic antigen it was I

and Delay of Parasitemia in Thymectomized Irradiated Recipients of Thymic Cells against Experimental Injection with Trypanosoma gambiense Source of thymic cellso

Inoculation of antigen”

Mice Mice used protected (no.) (no.)

Mice showing parasitemia (no.) Day (after inoculation of parasites) 1

3

x 108

2 x 108 1 x 108 0.5 x 108

in mice.

i-TC i-TC n-TC C-1 C-1 i-TC i-TC i-TC

Antigen C-J Antigen Antigen C-1 Antigen Antigen Antigen

10 10 10 10 10 10 10 10

7 0 0 0 0 8 6 0

a The absence of thymic cells and/or antigen is indicated by (- ).

2

345678 1

2

1 7

2 3 1

10 10 10 10 2

1 -

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shown that agglutinins were produced and released into their sera. Three mice were bled every 24 hr, the sera were pooled, and the titer determined. The results are presented in Fig. 2. First appearance of the agglutinins was on Day 3 after the transfer of i-TC with the antigen, and agglutinin titers increased rapidly thereafter. Most mice capable of producing agglutinins were found to be protected or partially protected (delay of parasitemia) when subsequently challenged. On the other hand, no agglutinin was noted in sera from TI-mice with transplanted n-TC and the antigen. In the sera of TI-mice receiving i-TC without the antigen, agglutinins were also not detected.

III. Production of Agglutinins by TI-Mice with Thymic Cell Subpopulation Transfer Thymic cells (i-TC ) (2 x 108) were centrifuged at 2OOOgfor 20 min in a bovine serum albumin gradient and divided into five layers, namely, A, B, C, D, and E from the top to bottom of the centrifuge tube. The relative proportions of the separated lymphocyte populations in each layer with regard to the original cell population is as follows: layer A, LO-1.5%; layer B, 1.4ACGLUTlNlN TlTER 32 /-'

16 a I

4 i

2

.---' / .-.--0-0-0--0-o-

0 I-

12345678

FIG. 2. Changes in the agglutination titer of mouse antiserum against Typanosoma gambknse. ( 0-0) agglutination titer of serum from 8OOR, thymectomized recipients of I-TC (1 X 108 cells) and antigen. (O-O) agglutination titer of serum from 800R, thymectomized recipients with n-TC (1 x 108) and antigen, and i-TC (1 X 108) without antigen.

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AGGLUTININ TlTER 32

a

12

3

4

5

6

7

8

TIME IN DAYS

FIG. 3. Changes in the agglutination titer of mouse antiserum against Typanosoma gumbknse from 80OR, thymectomized recipients of different i-TC subpopulations and antigen. (0-e) original population (200 x 10’ cells), (0-O) layer A (2 X 10’) (+-+) layer B (6 X 108) and layer C (48 X lOe), (X-X) layer D (80 X lo’), and (“-*) layer E (64 X IO’).

2.0%; layer C, 22.0-28.5%; layer D, 37S 41.2%; and layer E, 32.3-33.1%. In layer A 80% of the cells were greater than 10 pm in diameter and 20% were small lymphocytes. Seventy-five percent of layer B was composed of cells having diameters of over 10 w, and 25% were small lymphocytes. Ninety percent of layer C was made up of small lymphocytes of less than 10 pm in diameter, the remainder being large lymphocytes. More than 98% of layers D and E were small lymphocytes. The cells of each layer were transplanted to TI-mice which were sensitized with parasitic antigen in uiuo. The cells of layer D were very effective in producing agglutinins (Fig. 3). Rather weak agglutinin activities were observed in recipients of cells from the B, C, and E layers. However, layer A was not effective in producing detectable agglutinins.

Iv. Production of Agglutinins by T&Mice with Cortisone-Treated Thymic Cell

TTan.sfeT Immune mice were treated with co16 sone acetate 2 days before removal of the thymus. The mean number of cells from untreated mice was 224 x 10E/mouse in a

240

TAKAYANAGI

1G

NAKATAKE

/-’

84.

/

2 0

AND

i ./* .-.-o---o-/ 1

2

3

o-o-o 4

5

6

7

8 10

FIG. 4. Changes in the agglutination titer of mouse antiserum against Trypanosoma gambiense from 800R, thymectomized recipients of cortisone resistant cells and antigen. ( l -• ) untreated i-TC (I X lo*), (O-O) cortisone resistant cells (0.1 x 108).

volume analysis as determined by hemocytometer count. A marked depletion of the total cell number to 16 x lo6 was observed after treatment with 2.5 mg/20 g body wt of cortisone acetate. Thymic cells resistant to cortisone acetate were transferred into TI-mice with the antigen. It was found that these cortisone-resistant thymic cells were able to induce the production of low levels of agglutinins by recipients (Fig. 4). V. Fractionation

of Agglutinins

Fractionation of the sera which showed positive agghrtination titer was performed by means of gel filtration (Fig. 5) and

I20

30

40

50

60

70

FRACTlON

FIG. 6. Fractionation of mouse serum by DEAEcellulose column chromatography. Fraction I was eked with 0.0175 M phosphate buffer, pH 6.3; II, 0.04 M, pH 5.9; III, 0.1 M, pH 5.8; IV, 0.4 M, pH 5.3. (O-O), O.D. measurement of fractions. Agglutination titer is shown as AT.

DEAE-cellulose column chromatography (Fig. 6). Results of gel filtration indicated agglutinin titers observable in the form of bimodal peaks. A higher agglutinin titer was demonstrated in the first peak which contains macroglobulins. A weak agglutinin titer was observed in the second peak. With DEAE-cellulose column chromatography, Fraction I showed an agglutinin titer, but it was rather low. Another fraction showing agglutinin activity is Fraction IV. This fraction showed higher activity. VI. Promoting E&et Immunophagocytosis

of Agglutinins

for

The sera were tested for phagocytosis promoting effects (Fig. 7). They markedly enhanced phagocytosis in cultured peritoneal macrophage both from normal mice and 800R-irradiated ones. VII. Appearance

20

I!0 inAcTIoN

FIG. 5. Gel filtration profiles and agglutination titers of mouse serum from 800R, thymectomized recipients of i-TC (I X IO’) and antigen 5 days after the transfer. (-), O.D. measurement of fractions, (O-O), agglutinin titer of fractions.

of Variant

Serotype

The parasites from incompletely protected TI-mice (a delay of parasitemia) with transferred i-TC and the antigen were examined for their serological characteristics. Almost all parasites resisting the agglutinin effects were found to have changed their antigenic types. In plasmas from these mice showing a delay of parasitemia, ag-

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lated with i-TC and then sensitized with the parasitic antigen. At the same time, protection and delayed parasitemia were observed in the mice. The delayed parasitemia might be caused by antigenically changed trypanosomes. In bovine serum albumin gradient centrifugations of i-TC, agglutinin was found to be produced more efficiently in recipients of rather high density subpopulations of thymic cells. Subpopulations of lowdensity cells were rather less effective in 2 4 8 16 32 inducing agglutinin production. However, the TI-mice with transplanted II~LUTION n-TC, which were then sensitized with FIG. 7. Phagocytic index of macrophagesin the antigen, were not able to produce detectpresence of serum (agglutination titer 1:32) from 800R, thymectomized recipients of i-TC ( 1 X lo*) able agglutinins. Moreover, TI-mice receivand antigen 5 days after transfer. (O-O) macroing i-TC without antigen could not induce phages from normal mice, (O.-O ) macrophages from 800R-irradiated mice. PI (phagocytic index) production of the agglutinins. In the cortisone acetate experiments, TIis defined as number of macrophageswith parasites X lOO/ total number of macrophages ob- mice transplanted with cortisone-resistant served. i-TC and subsequently sensitized were able to induce the production of detectable agglutinins which were of very low acglutinating activities still remained against cells having a 0 type parasites. At the same time, these tivity, Cortisone-resistant low content of thymus antigen are said to parasites did not agglutinate with anti-0 be immunologically mature and are retype test serum. The parasites which sponsible for graft versus host reactions appeared in the mice not showing any delay in viva and for alloantigen responses in of parasitemia did not change their types. vitro (Konda and Smith 1972). In cell-to-cell interactions for the producVIII. The Treatment of i-TC with Antition of antibody, thymic cells do not proThymic Cell Rabbit Serum duce antibodies directly in the immune The i-TC were mixed with anti-mouse response (Friedman 1964, Miller et al. thymic cell rabbit serum plus fresh golden 1965). They fail to produce antibodies hamster serum and then incubated at 37 C against sheep erythrocytes when injected for 30 min. The i-TC were washed by into a lethally irradiated recipient (Kencentrifugation at 2OOg for 10 min. After nedy et al. 1965) in spite of the fact that washing, the I-TC were injected ip into they respond to antigenic stimulation by TI-mice and the parasitic antigen was also an intensive mitotic activity (Davies et al. injected. Phenomena of agglutinin produc1967, Gershon et al. 1968). The thymic cells tion and protection which normally oc- nevertheless have the capacity of inducing curred after transferring i-TC could not be antibody formation by bone marrowobserved after the application of the antiderived cells (Mitchell and Miller 1968). serum. The interaction of thymus-derived cells and bone marrow-derived cells which results in DISCUSSION the production of antibody has been well In these experiments, the detectable ag- demonstrated (Claman and Chaperon 1969, Miller et al. 1971, Playfair 1971). glutinins were observed in TI-mice inocu-

01,

, ,”

212

TAKAYANAGI AND NAKATAKE

From the results, it is possible to infer parental population. Thus, it could be that that transferred thymic cells from rather trypanosomes in delayed parasitemia in the high-density subpopulations from immune TI-mice that had detectable agglutinins mice may be much better equipped to might appear by changing their antigenic recognize the antigen following in viva types through an antigenic transformation sensitization. Such valid information could or the selection of antigenic mutants. be then efficiently transmitted to some antibody-forming cells. Still, if such detectACKNOWLEDGMENT able agglutinin in sera of TI-mice injected with i-TC and antigen represents the presThe authors wish to express their gratitude to ence of antibody-forming cells, there is the Professor Sigefusa Sat0 for many facilities propossibility that either the inoculum must vided during this experiment. The authors are also grateful to Professor Sadayuki Sakuma and contain cells potentially capable of producDr. Mitsuaki Mizoguchi, Department of Radiology, ing agglutinin or that the host must provide for their valuable suggestions and help in preantibody-forming precursor cells which are paring irradiated mice. extremely radio-resistant. The sera having the agglutinins from TIREFERENCES mice with transferred i-TC and the antigen exhibited a phagocytosis-promoting effect BEHBEHANI, M. K. 1971. Trypanosoma (Schizotrypanum) cruzi infections in X-irradiated and on cultured peritoneal macrophages both in thymectomized mice. Transactions of the from normal and irradiated mice. PhagoRoyal Society of Tropical Medicine and Hycytosis by macrophages of homologous giene 65, 265. parasites was greatly enhanced by specific BLOMGREN, H., AND ANDERSON, B. 1969. Evidence antisera from mice immunized with tryfor a small pool of immunocompetent cells in et al. panosomal antigen (Takayanagi the mouse thymus. Experimental Cell Research 57, 185-192. 1974). In trypanosome infections, Brown BROWN, W. H. 1915. Concerning changes in the (1915) considered phagocytosis an essential biological properties of Trypanosom~ lewisi mechanism in relieving the host of infection produced by experimental means, with special with T. lewisi. Lange and Lysenko (1960) reference to virulence. Journal of Experimental also stated that a phagocytosis-enhancing Medicine 21, 345-364. CHAPMAN, W. L., AND HANSON, W. L. 1971. antibody may be of significance in the rapid PEusmodium berghei infection in neonatally clearing of parasites injected into the cirthymectomized hamsters. Journal of Parasitology culating blood of immune rats. It might be 57, 24-28. considered that phagocytosis by macroCLA;LIAN, H. N., AND CHAPERON, E. A. 1969. phages could play a considerable role in Immunologic complementation between thymus and marrow cells-A model for the two cell the protection of TI-mice having agglutheory of immunocompetence. Transpkzntation tinins against experimental infection. Reuiew 1, 92-113. Trypanosomes are able to change freDAVIES, A. J. S., LEUCHARS, E., WALLIS, V. quently their antigenic types under an unMARCHANT, R., AND ELLIOTT, E. V. 1967. The favorable condition such as the presence failure of thymus-derived cells to produce antibody. Transplantation 5, 222-231. of specific antibodies. lnoki et al. (1956) FRIEDMAN, H. 1964. Distribution of antibody and Osaki (1959) reported that individual plaque forming cells in various tissues of several trypanosomes are capable of undergoing strains of mice injected with sheep erythrocytes. antigenic transfomration or changes in the Proceedings of the Society for Experimental Biology and Medicine 117, 526-530. presence of an antibody. Seed and Gam GERSHON, R. K., WALLS, V., DAVIES, A. J. S., (1966) reported that relapses in trypanoAND LEUCHARS, E. 1968. Inactivation of thymus somiasis are due to the selection of anticells after multiple injections of antigen. Nature from a heterogeneous genie mutants 218, 380-381.

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INOKI, S., OSAKI, H., AND NAKABAYASHI, T. 1956. Studies on the immunological variation in Trypanosoma gambiense. II. Verifications of the new variation system by Ehrlich’s and in oitro methods. Medical Journal of Osaka University

7, m-173. KENNEDY, J. C., SIMINOVITCH, L., TILL, J. E., AND MCCULLOCH, E. A. 1965. A transplantation assay for mouse cells responsive to antigenic stimulation by sheep erythrocytes. Proceedings of the Society for Experimental Biology and Medicine 120, 868-873. KONDA, S., AND SMITH, R. T. 1972. Functional and antigenic heterogeneity of thymus cell subFederation Proceedings 31, 775. populations. LANGE, D. E., AND LYSENKO, M. G. 1960. In vitro phagocytosis of Trypanosoma Zewisi by rat exudative cells. Experimental Parasitology 10, 3942. LANHAM, S. M., AND GODFREY, D. G. 1970. Isolation of sahvarian trypanosomes from man and other mammals using DEAE-cellulose. Experimental Parasitology 28, 521-534. MILLER, J. F. A. P., DE BURGH, P. M., AND GRANT, G. A. 1965. Thymus and the production of antibody-plaque-forming cells. Nature 208, 1332 1334. MILLER, J. F. A. P. 1971. The thymus and the immune system. VOX Sanguinis 20, 481491. MILLER, J. F. A. P., BASTEN, A., SPRENT, J., AND CHEERS, C. 1971. Interaction between lymphocytes in immune responses. Cellular Zmmunology 2, 469495. MITCHELL, G. F., AND MILLER, J. F. A. P. 1968. Immunological activity of thymus and thoracicProceedings of the National duct lymphocytes. Academy of Sciences of the United States of America 59, 296-303. OSAKI, H. 1959. Studies on the immunological variation in Trypanosoma gambiense (Serotypes and the mode of relapse). B&en’s Journal 2, 113-127. PLAYFAIR, J. H. L. 1971. Cell cooperation in the immune response. Clinical Experimental Zmmunology 8, 839-856.

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