Dynamic Study Of T Cell Subsets In Melanoma Bearing Mice And Its Clinical Significance

Objective: Present study aims to dynamically monitor the changes ofsplenic T cell subsets in number during tumor growing of the mice, in order toevaluate the immune status of the patients with cancer, and in addition, toprovide the basis for predicting tumor recurrence, therapeutic response, and theoutcome of the patient’s.Methods: One hundred and twenty healthy mice with equal conditionswere brought into experiment, and6of them with tumor-free grouped asNormal Group. The remaining114mice were randomly divided equally intoTumor-bearing Group （Group A,57mice） and Blank Group （Group B,57mice）.On the day that a tumor was firstly found （D0）,5mice randomly selectedfrom group A were used to measure tumor-bearing curves. For the remainingmice of group A and group B, six mice were randomly selected respectivelyevery five days since D0（D0is inclusive）, the spleens were removed and cellsuspensions were prepared, and CD+3T, CD++4T, CD8T, CD+4T/CD+8T valueand naive T lymphocytes were analyzed by using flow cytometry （FACS）. Theexperiment did not stop until more than half of the tumor-bearing curveobservation group mice or remaining Group A mice had been found died.Results:1The dynamic changes of proportion of CD+3T lymphocytes:①Theproportions of CD+3T of Blank Group and Tumor-bearing Group onD0（U=4.500P<0.05）, D5（U=0.000P<0.05）, D10（U=0.000P<0.05）,D₁₅（U=0.000P<0.05）, D₂₀（U=0.000P<0.05） and D₂₅（U=0.000P<0.05） weresignificantly different, which meant the values of Blank Group weresignificantly higher than those of the Tumor-bearing Group;②In BlankGroup, the values of Normal, D0, D5, D10, D₁₅, D₂₀and D₂₅were significantlydifferent（F=4.960P=0.001）. Values of normal, D0, D5, D10, D₁₅were significantly different with those of D₂₀and D₂₅（F=4.960P<0.05）.However,the proportions of CD+3T of Normal, D0, D5, D10, D₁₅were not significantlydifferent （P>0.05）and either were those of D₂₀and D₂₅. Consequently,CD+3Tlymphocyte ratio （%）remained at basically unchanged64.7during D0and D₁₅,after D₁₅the rate dropped to53.4on D₂₅with a decline rate of17.6%;③ForTumor-bearing Group, any two of the values of Normal, D0, D5, D10, D₁₅,D₂₀,D₂₅were significantly different （F=3397.196P <0.05）,which meant the ratio（%） of Tumor-bearing Group reduced from61.6on D0to7.9on D₂₅with whena decline rate of87.2%and it decreased with time.2The dynamic changes of proportion of CD+4T lymphocytes:①BlankGroup and Tumor-bearing Group had no significant difference in theproportion on D0（t=-0.036P=0.972）.However, the proportions of CD+4T onD5, D10, D₁₅, D₂₀, D₂₅were significantly different （t=-14.933P<0.05, U=0.000P<0.05, t=-37.860P<0.05, U=0.000P<0.05, U=0.000P<0.05）, which meantthat the proportion of CD+4T lymphocytes of Blank Group was higher than thatof Tumor-bearing Group in overall;②In Blank Group, CD+4T lymphocytesdecreased over time. Overall difference exists in those values（F=3397.196P<0.05）. The values of D₂₀and D₂₅were significantly different （P <0.05）. D₂₀,D₂₅were both significantly different with the Normal, D0, D5, D10, D₁₅（P<0.05）. Any two of Normal, D0, D5, D10, D₁₅were not significantly different（P>0.05）. All of those results showed that ratio （%） of CD+4T lymphocyte inBlank Group remained at basically unchanged value of38.7during D0and D₁₅,after D₁₅the rate dropped to27.9on D₂₅with a decline rate of27.9%;③InTumor-bearing Group, Overall difference exists in those values（F=160.370P<0.05）. Normal, D0were both significantly different with D5, D10, D₁₅, D₂₀,D₂₅（P <0.05）; and the values of normal and D0were not significantly different（P>0.05）; which can be found that the value of Tumor-bearing Groupreduced from38.9on D0to3.1on D₂₅with a decline rate of92.0%.3The dynamic changes of proportion of CD+8T lymphocytes:①BlankGroup and Tumor-bearing Group had no significant difference in theproportion on D0（t=0.645P=0.533）.However, the proportions of CD+8T on D5, D10, D₁₅, D₂₀, D₂₅were significantly different （U=3.000P<0.05, U=0.000P<0.05, t=-8.131P<0.05, t=-16.059P<0.05, U=3.000P<0.05）, whichmeant that the proportion of CD+8T lymphocytes of Blank Group was higherthan that of Tumor-bearing Group in overall;②In Blank Group, theproportions of CD+8T in different time were not significantly different（F=0.434P=0.851）,which meant that the ratio （%） of CD+8T lymphocyte remainedrelatively stable and it dropped from21.6on D0to20.5on D₂₅with a declinerate of5.1%;③In Tumor-bearing Group, difference exists in those proportionsin overall（F=76.815P<0.05）. Those values of Normal and D0were bothsignificantly different with those of D5, D10, D₁₅, D₂₀, D₂₅（P <0.05）. D5, D10were both significantly different with D₁₅, D₂₀, D₂₅（P <0.05）.However, thevalues of Normal and D0were not significantly different （P>0.05）, so eitherwere the values of D5and D10, and the values of D₁₅, D₂₀and D₂₅. Theproportion of CD+8T lymphocytes reduced from22.2on D0to5.2on D₂₅witha decline rate of76.6%.4The dynamic changes of the ratio of CD+4T/CD+8T:①Blank Group andTumor-bearing Group had no significant difference in the ratio ofCD+4T/CD+8T of D0（U=12.500P=0.363）.However, the ratio of D5, D10, D₁₅,D₂₀, D₂₅were significantly different （U=0.000P<0.05, t=-16.971P<0.05, t=-16.483P<0.05, t=-12.562P<0.05, U=0.000P<0.05）, which meant that theratio of Blank Group was higher than that of Tumor-bearing Group in overall;②In Blank Group, overall difference exists in those values（P<0.05）. Thevalues of CD++4T/CD8T of Normal, D0, D5, D10, D₁₅were all significantlydifferent with those on D₂₀, D₂₅（P <0.05）. And the values of D₂₀and D₂₅weresignificantly different（P<0.05）. However, no difference exists in those valuesof Normal, D0, D5, D10and D₁₅. It also can be found that the ratio remained atbasically unchanged value of1.8during D0and D₁₅, after D₁₅the rate droppedto1.4on D₂₅with a decline rate of22.2%;③In Tumor-bearing Group, overalldifference exists in those values in overall （F=97.348P<0.05）. The ratio ofNormal and D0were significantly different with those of D5, D10, D₁₅, D₂₀, D₂₅（P<0.05）. However, those of Normal and D0were not significantly different （P>0.05）, and either were the values of D5, D10, D₁₅, D₂₀and D₂₅. It can befound that the ratio decreased sharply over time; and it reduced from1.8on D0to0.6on D₂₅.5The dynamic changes of proportion of naive T cells:①Blank Groupand Tumor-bearing Group had no significant difference in the proportion on D0（t=-0.200P=0.846）.However, the proportions of naive T cells on D5, D10, D₁₅,D₂₀, D₂₅were significantly different （U=0.000P<0.05, U=0.000P<0.05,t=-18.543P<0.05, U=0.000P<0.05, U=0.000P<0.05）, which meant that theproportion of naive T cells of Blank Group was higher than that ofTumor-bearing Group in overall;②In Blank Group, the values weresignificantly different in overall（F=8.865P<0.05）. Those of Normal, D0, D5,D10, D₁₅were all significantly different with those of D₂₀, D₂₅（P <0.05）. Thevalues of D₂₀and D₂₅were significantly different （P<0.05）. However, thevalues of Normal, D0, D5, D10were not significantly different（P>0.05）. Theresult was that the ratio （%） of Naive T cells dropped from34.1on D0to32.2on D₂₅with a small decline rate of5.4%.Then the ratio （%） dropped to21.7onD₂₅with a decline rate of32.6%;③In Tumor-bearing Group, any two of thevalues of D0,D5, D10, D₁₅, D₂₀, D₂₅were significantly different （F=178.995P<0.05）,and the ratio （%） of Naive T cells reduced from34.1on D0to2.5onD₂₅with a decline rate of92.7%.Conclusion:1For Tumor-bearing Group, with the passage of time (D0to D₂₅), theproportions of CD++3T, CD+4T, CD8T in spleen lymphocyte suspensiondecreased. And the sort of decline rate was as follows: CD+4T lymphocytes>CD+3T lymphocytes> CD+8T lymphocytes; The value of CD+4T/CD+8Tdropped obviously, which is the performance of gradually growth of tumor andthe gradually decreased immune function; And the ratio of naive T cell alsodeclined substantially.2For Blank Group, with the passage of time (D0to D₂₅), the proportionsof CD+3T, CD+4T, CD+8T in spleen lymphocyte suspension remained relativelystable at first, then decreased. D₁₅was the turning point time on which these proportions changed from stable to decline. And the sort of decline rate was asfollows: CD+4T lymphocytes> CD+3T lymphocytes> CD+8T lymphocytes; Thevalue of CD+4T/CD+8T remained relatively stable at first, after D₁₅decreasedobviously; And the proportion of naive T cell also remained relatively stable atfirst, after D₁₅decreased obviously.3For Blank Group and Tumor-bearing Group, the proportions of CD+3T,CD+4T, CD+8T and naive T cell in spleen lymphocyte suspension and the ratioof CD+4T/CD+8T decreased with time. However, the decline rates of thosevalues in Blank Group were smaller than those of Tumor-bearing Group. Thoseresults indicate that both the immune functions of Blank Group andTumor-bearing Group decline with age, but Blank Group with a lower declinedegree than that of Tumor-bearing Group.4Due to the accurately detecting of T lymphocyte subsets in peripheralblood by Flow cytometry （FACS）, the technology can provide individualizedguidance for clinical adoptive immunotherapy of tumors and can be widelyused in clinical medicine.